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The Effect of alterations of the components of the bicarbonate buffer system on skeletal muscle contraction, potassium content and resting potential

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Title:
The Effect of alterations of the components of the bicarbonate buffer system on skeletal muscle contraction, potassium content and resting potential
Creator:
Fretthold, David Walter, 1948-
Publication Date:
Language:
English
Physical Description:
viii, 82 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Bicarbonates ( jstor )
Carbon dioxide ( jstor )
Membrane potential ( jstor )
Muscle contraction ( jstor )
Muscles ( jstor )
pH ( jstor )
Potassium ( jstor )
Rats ( jstor )
Skeletal muscle ( jstor )
Sodium ( jstor )
Bicarbonates ( mesh )
Carbon Dioxide ( mesh )
Department of Pharmacology and Therapeutics thesis Ph.D ( mesh )
Diaphragm -- physiology ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pharmacology and Therapeutics -- UF ( mesh )
Hydrogen-Ion Concentration ( mesh )
Muscle Contraction -- physiology ( mesh )
Muscle, Skeletal -- physiology ( mesh )
Rats -- physiology ( mesh )
Research ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1977.
Bibliography:
Bibliography: leaves 76-81.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by David W. Fretthold.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
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25665249 ( OCLC )
AEK6249 ( NOTIS )
AA00006117_00001 ( sobekcm )

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Full Text










THE EFFECT OF ALTERATIONS OF THE COMPONENTS OF THE
BICARBONATE BUFFER SYSTEM ON SKELETAL MUSCLE CONTRACTION,
POTASSIUM CONTENT AND RESTING POTENTIAL
















By

David W: Fretthold
















A DISSERTATION PRES.;;L'U'D TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY




UNIVERSITY OF FLORIDA


1977




THE EFFECT OF ALTERATIONS OF THE COMPONENTS OF THE
BICARBONATE BUFFER SYSTEM ON SKELETAL MUSCLE CONTRACTION,
POTASSIUM CONTENT AND RESTING POTENTIAL
David
By
V, X.
W. Fretthold
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGMENTS
I wish first and foremost to express my sincere gratitude
to Dr. Lai C. Garg for his help and understanding. His
patience and encouragement has been invaluable to me.
I am also grateful for the advice and attention of Dr.
Maren and the members of my supervisory committee, Dr. Betty
Vogh, Dr. C.Y. Chiou, Dr. William Kem, and Dr. Philip Posner.
I thank Dr. John Munson, Dr. Vernon Senterfitt, and Mr.
Norman Lawrence for generously providing much of the equip
ment used in these experiments, and Mr. Butch Landsiedel,
who was a great help in maintaining the experimental animals.
I am also grateful to Ms. Mary Pavlovski, Ms. Joyce
Simon, and Ms. Debbie Werther for help in preparation of
this manuscript.
My sincere thanks is also extended to Ms. Jan Nichols,
and Bills Elmquist and Link for their friendship and cooperation.
Finally, I would like to thank the other members of the
Department of Pharmacology for their good company over the
years.
This research was supported by a traineeship from the
National Institute of Health (GM-00760) and by a research
grant from the National Institute of Health (GM-16934).
li


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES 1V
LIST OF FIGURES v
ABSTRACT vi
INTRODUCTION 1
Objective 1
Background 2
RATIONAL 14
SPECIFIC AIMS 16
MATERIALS AND METHODS 17
Buffer Composition 17
Experimental Animals ..... 18
Rat Hemidiaphragm Preparation 20
Contraction Experiments 21
Intracellular pH and Tissue Cation
Determinations 23
Potential Measurements 27
RESULTS 29
Contraction Studies 29
Tissue Analysis 47
Resting Potential Measurement 57
DISCUSSION 63
CONCLUSION 74
BIBLIOGRAPHY 76
BIOGRAPHICAL, SKETCH 8 2


LIST OF TABLES
TABLE Page
1 BUFFER SERIES 19
2 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
C02 CONCENTRATIONS 50
3 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
hco3~ CONCENTRATIONS 51
4 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN CONSTANT
pH BUFFERS 52
5 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM CONTROL RATS, IN CONSTANT pH BUFFERS . 53
6 COMPARISON OF ICF ACID-BASE AND EX
CHANGES IN HEMIDIAPHRAGMS IN VARIOUS
BUFFERS 54
IV


LIST OF FIGURES
FIGURE Page
1 Effect of d-tubocurarine on directly
and indirectly stimulated hemidiaphragm
muscles 30
2 Contraction of control muscles in 5 mM
potassium as a function of pH 33
3 Contraction of control muscles in 0.5 mM
potassium as a function of pH 35
4 Contraction of potassium-deficient
muscles in 0.5 mM potassium as a
function of pH 37
5 Contraction of control muscles in 5 mM
potassium, constant pH buffers, vs CC>2
volumes percent and HCO^- 39
6 Contraction of control muscles in 0.5 mM
potassium, constant pH buffers, vs CC^
volumes percent and HCO^ ........ 41
7 Contraction of potassium-deficient
muscles in 0.5 mM potassium, constant
pH buffers, vs CO volumes percent and
HC03~ 43
8 Effect of alterations in HCOg- and CC>2,
at constant pH, on hemidiaphragm
contraction 55
9 Effect of alterations in HCO3 CO^,
and potassium concentration on resting
potential of potassium-deficient rat
hemidiaphragm muscles 58
10 Effect of alterations in HCO^ CO~,
and potassium concentration on resting
membrane potential of control rat
hemidiaphragm muscles ..... 60
v


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF ALTERATIONS OF THE COMPONENTS OF THE
BICARBONATE BUFFER SYSTEM ON SKELETAL MUSCLE CONTRACTION,
POTASSIUM CONTENT AND RESTING POTENTIAL
By
David W. Frett.hold
Chairperson: Dr. Lai C. Garg
Department: Pharmacology and Therapeutics
The purpose of this investigation was to examine the
effect of alterations in bicarbonate concentration and
carbon dioxide tension on skeletal muscle contractility and
to elucidate a mechanism for these effects. Hemidiaphragm
muscles from control rats and rats fed potassium-deficient
chow were examined. Particular attention was focused on
this potassium-depleted muscle because it has been reported
to exhibit some of the same properties as muscle in patients
suffering from the hereditary disease hypokalemic periodic
paralysis. Patients with this disease are protected from
attacks of skeletal muscle paralysis by chronic doses of
ammonium chloride or carbonic anhydrase inhibitors, agents
that produce alterations in the bicarbonate buffer system.
The in vitro situation offers the opportunity to control
carefully all three elements of the buffer system: pH, bicar
bonate, and carbon dioxide tension. Therefore, all muscle
vi


studies were conducted .in vitro. One of these, three elements
was kept constant while the other two were systematically
varied.
When pH was allowed to vary, stimulated muscles con
tracted with more force in high pH buffers than in low pH
buffers. This was true when the pH change was effected by
an elevated buffer bicarbonate concentration or by a reduced
carbon dioxide tension. This effect was always more dramatic
in muscles taken from potassium-depleted animals. With these
muscles, a change in pH from 7.7 to 7.1 was accompanied by
a 65% reduction in contractile force. This effect showed a
rapid onset, was reversible, persisted until the buffer
composition was changed, and was independent of synaptic
transmission. When pH was kept constant but buffer bicar
bonate and carbon dioxide tension were increased together
at the same ratio, there was an accompanying increase in
muscle contractile force. This relationship was also isore
pronounced in potassium-deficient rat muscles.
Intracellular pH and intracellular potassium deter
minations were performed on diaphragm muscles that had been
contracting in different bicarbonate buffers. Radio-labeled
dimethyloxazolidinedione (DMO) was used for these intracellu
lar pH determinations. Muscles bathed in acidic buffers
were found to have lower intracellular pH levels and lower
intracellular potassium concentrations than muscles bathed
in more alkaline buffers. Analysis of muscles bathed in
high bicarbonate, high carbon dioxide buffer revealed that
these muscles had a lower intracellular pH and higher
vii


potassium content than muscles bathed in low bicarbonate,
low carbon dioxide buffer, although both buffers had the
same pH.
Resting membrane potentials, recorded in diaphragm
muscles bathed in different bicarbonate buffers, were not
affected by changes in buffer bicarbonate concentration
or buffer carbon dioxide tension. Changes in the buffer
potassium concentration over ranges which did not affect
contractile response were found to alter the recorded
resting potentials.
The changes in contractility showed a positive corre
lation with the intracellular muscle potassium concentration
and with the ratio of intracellular to extracellular proton
concentrations. Changes in muscle contractility did not
correlate with changes in intramuscular pH or with changes
in the resting membrane potential. It was speculated that
the benefits which carbonic anhydrase inhibitors afford
victims of hypokalemic periodic paralysis may be derived
from a more favorable distribution of potassium which results
from reductions in serum bicarbonate as well as the accom
panying acidosis.
vm


INTRODUCTION
Objective
Patients suffering from the disease familial hypokal
emic periodic paralysis are protected from attacks of muscle
weakness by acetazolamide, a potent carbonic anhydrase in
hibitor (Resnick et al., 1968). It is paradoxical that a
diuretic which causes potassium loss can prevent the attacks
of muscle weakness which are associated with a fall in serum
potassium. Since no carbonic anhydrase has been found in
muscle (Maren, 1967), it seems unlikely that the benefits
afforded victims of this malady are derived from action of
the drug directly on muscle. Griggs (1970) suggested that
metabolic acidosis, brought about by inhibition of renal
carbonic anhydrase, was responsible for the beneficial
effects of this drug. Jarrell et al. (1976) demonstrated
that patients given acidifying doses of ammonium chloride
also were protected from periodic attacks. Further,
Viskoper et al. (1973) were able to precipitate an attack
of paralysis in a patient by infusing sodium bicarbonate which
produced a metabolic alkalosis. Treatment v/ith acetazolamide
appeiirs to prevent hypokalemia and muscle paralysis as a
consequence of alterations effected in the bicarbonate buffering
system. These alterations include not only a decrease in the
plasma pH, but also decreases in plasma bicarbonate and carbon
1


2
dioxide concentrations. Precisely how this new acid-base
equilibrium prevents attacks of periodic paralysis is not
clear. Therefore, the objective of this investigation is to
determine in vitro the influence of changes in components of
the bicarbonate buffer system on skeletal muscle contraction,
potassium content, pH, and resting membrane potential.
Background
Bicarbonate Buffer System
The bicarbonate buffer system is quantitatively the
most important extracellular buffer in the body and is unique
because of its intimate link with carbon dioxide. Although
the ionization constant (pKa) for the first proton dissoci
ation of carbonic acid is 3.6, the buffer system has a
considerable capacity at physiological pH because of the
constant 400:1 ratio of dissolved carbon dioxide to carbonic
acid. The Henderson-Hasselbalch equation can be modified,
substituting the concentration of dissolved carbon dioxide
divided by 400 for the carbonic acid concentration. Rear
ranging the terms yields the familiar form of the equation
which expresses the pH as a function of bicarbonate concen
tration and dissolved carbon dioxide, with an apparent pKa
value of 6.1 (Hills, 1973); pH=6 1+log In vivo, regula
tory mechanisms control the ventilation rate and consequently
the carbon dioxide concentration in blood, thus controlling the


3
concentration of the proton donor, carbonic acid. Extracel
lular pH is maintained between 7.1 and 7.6, the range generally
considered compatible with life. Even during strenuous
exercise, when the carbon dioxide evolution rate can increase
to as much as thirty times the rate at rest, extracellular
pH falls no more than 0.1 unit.
Relation Between Intracellular and Extracellular pH and
Hiearbonate of Muscle Cells
A variety of techniques have been employed to determine
the intracellular pH of muscle. Early methods used to measure
intracellular pH and the theoretical and practical diffi
culties associated with these methods are discussed in a review
by Caldwell (1956). Waddell and Butler (1959) first used the
weak acid, dimethyloxazolidinedione (DM0), a metabolite of the
antiepileptic agent trimethadione, to measure the intracellular
pH of dog muscles in vivo. Using bicarbonate-specific micro
electrodes, Khuri et al. (1974, 1976) determined the intra
cellular bicarbonate activity of skeletal muscles, and calcu
lated equilibrium potentials for bicarbonate of between -20
millivolts and -44 millivolts under different conditions in
r intracellular pH to be between 6.6 and 7.4, and have concluded
that proton and bicarbonate gradients must exist across muscle
cell membranes which are not in Nernstian equilibrium with the
resting muscle membrane potential, although the membrane is
permeable to Lhese ions (Adler et al., 1965). In order for such
gradients to be maintained, either hydrogen ions would have to


4
be transported out of cells or bicarbonate ions would have to
be transported into cells (Adler et al., 1972; Heisler, 1975;
Khuri et al., 1974; Lai et al., 1973).
Numerous investigations have been undertaken to determine
the relation of intracellular pH to changes in extracel
lular pH. This was the objective of Wfiddell and Butler (1959)
in their DMO study in dogs. They altered the blood carbon
dioxide tension and bicarbonate concentration and concluded,
from skeletal muscle biopsies, that the greatest changes they
observed in intracellular pH resulted from changes in carbon
dioxide tension. Irvine and Dow (1966) found that metabolic
acidosis in rats had no effect on skeletal muscle .intracel
lular pH. Kim and Brown (1968) showed that skeletal muscle
intracellular pH in dogs fell when carbon dioxide tension
was increased along with bicarbonate concentration, and rose
when animals were hyperventilated during HC1 infusion, even
though the plasma pH remained constant. Similar results have
been obtained in vitro; respiratory acidosis produced larger
alterations in muscle; intracellular pH than metabolic acidosis
(Heisler, 1975; Adler et al., 1965). Both these authors
found that rat diaphragm muscles were most effectively buffered
when extracellular pH ranged between 7.1 and 7.4. They
reported that when extracellular pH varied from 7.1 to 7.4
during simulated respiratory acidosis or alkalosis, intra
cellular pH, measured with DMO, varied from 7.0 to about 7.28.
Outside this extracellular pH range, intracellular pH changed
about as much as extracellular pH.


5
Electrolyte Shifts and Acid-Base Disturbances
A close relationship exists between tissue and plasma
electrolyte concentrations and acid-base balance. Potassium
depletion results in increases in serum bicarbonate (Heppel,
1939), whereas sodium depletion leads to a fall in serum
bicarbonate (Darrow et al., 1948). Acidosis produces ele
vations in total body potassium, and decreases in total body
sodium (Cooke et al., 1952). All of the above studies were
chronic experiments involving chronic alterations in diet and
acid-base balance and the changes largely result from renal
electrolyte regulation. A reciprocal relation exists for
hydrogen and potassium ion excretion in the distal tubule,
and reabsorption of bicarbonate in the proximal tubule is
coupled to the reabsorption of sodium (Pitts, 1963).
In a series of experiments on nephrectomized dogs,
Pitts and co-workers (1963) measured serum ion concentra
tions and demonstrated that considerable ion exchange
occurs between intracellular and extracellular fluid in
acute episodes of respiratory and metabolic acidosis and
alkalosis. Sodium and potassium exchange for protons, and
chloride exchanges for bicarbonate. Other authors have
analyzed muscle taken from animals during acid-base distur
bances and have noted positive correlations between hydrogen
and potassium ion gradients across muscle membranes (Brown
and Goott, 1963; Grantham and Schloerb, 1965; Irvine and Dow,
1966). In various types of acid-base alterations, intra
muscular potassium concentration changed in the direction
which tended to keep the transmembrane ratios of potassium


6
and hydrogen ions equal, although true equality was never
attained (Brown and Goott, 1963). In vitro experiments on
rat diaphragms by Adler et al. (1965) and on frog muscles by
Fenn and Cobb (1934) have shown a positive correlation between
changes in intramuscular potassium and intramuscular pH.
In vitro potassium depletion in rat diaphragm has been shown
to lower intracellular pH (Adler et al., 1972). Skeletal
muscles depleted of potassium contain elevated concentrations
of sodium (Cooke et al., 1952).
Mechanism of Ion Exchange.
Isotope exchange studies with isolated frog skeletal
muscle have shown that a decrease in extracellular pH causes
an increase in the potassium efflux rate, a decrease in the
potassium uptake rate, and a bi-directional depression of
sodium exchange (Voile, 1972). This type of membrane beha
vior could explain the potassium shift seen in acidosis
(Voile, 1972). Keynes and Swan (1959) reported that sodium
efflux from isolated frog muscle varied approximately with
the cube of the internal sodium concentration. Keynes (1965)
found that sodium efflux depended on the square of the internal
sodium concentration in acidosis which was produced by an
increase in carbon dioxide tension. Whether these observa
tions represent interactions of ion pumps, substitution of
protons for other cations, or suppression of pumping activity
is not known.


7
Muscle Cell Potential and pH, Bicarbonate Concentration,
and Carbon Dioxide Tension
Resting muscle membrane potential depends on the ion
distribution across the membrane and the membrane permeability
to these ions. The Goldman equation describes this relation
ship quantitatively:
-RT VK+)i + PNa(Na+)i + PCl(C1">o
E = In T
m ~ P (K ) + P (Na ) + P_n (Cl ) .
K o Na o Cl i
where Em is membrane potential, P is the membrane permeabil
ity coefficient for a particular ion, R is the universal gas
constant, T is temperature in degrees kelvin, and F is Fara
day's constant (Hodgkin and Horowicz, 1959). In skeletal
muscle, chloride is distributed in agreement with Em, and the
. +
contribution of the term P (Na l.s small compared to the
contribution of the term P (K+).. Therefore, these terms
IN. 1
may be neglected without appreciably altering the validity
of the equation (Bilbrey et al., 1973). Bilbrey et al. (1973)
found that resting muscle potentials, recorded in vivo in
dogs and rats, agreed with this equation when 0.01 was used
for the ratio of the permeability coefficients of sodium to
potassium. Any factor which influences a change in any of
the parameters in the equation would affect the membrane
potential. Studies on the effect of pH and carbon dioxide
tension on transmembrane potential have yielded conflicting
results. In general, an increase in carbon dioxide tension
has been shown to cause a decrease in Em when Em is initially
high and an increase when Ej^ is initially low (Shanes, 1958).


8
In vivo studies by Williams et al. (1971) showed that a fall
in blood pH induced by an elevation in pC02 causes a slow
depolarization of skeletal muscle in rat. This depolarization
could be explained by the observed redistribution of ions,
principally an elevation in plasma potassium. Iri vitro alter
ations in pH have been shown to affect membrane potential in
frog muscle; the effect has been attributed to a decreased
chloride permeability in acidosis (Brooks and Hutter, 1962;
Hutter and Warner, 1967) ; when the external potassium concen
tration was 2.5 mM, acidosis hyperpolarized isolated frog
skeletal muscle by a few millivolts, but when the external
potassium concentrations was elevated to 10 mM, acidosis
depolarized the muscle by a few millivolts (Mainwood and Lee,
1967). It has also been reported in rat skeletal muscle that
bicarbonate has a permeability coefficient approximately 0.1
that of chloride (Hugeunin, 1975).
Muscle Contraction
The first in a series of events leading to muscle con
traction is usually the propagation of an action potential
to the pre-synaptic ending of the motor nerve, and the sub
sequent release of acetylcholine from the nerve ending.
Acetylcholine diffuses across the synaptic cleft and inter
acts with the post-synaptic membrane on the muscle, causing
a temporary local increase in permeability to sodium and
potassium. As a result of this permeability increase, the


9
membrane becomes locally depolarized. If the muscle membrane
depolarizes beyond a critical threshold, a propagated action
potential results. Depolarization of the muscle membrane
initiates the liberation of calcium from sarcoplasmic retic
ulum, the primary source of activator calcium in skeletal
muscle. Movement of calcium to the contractile apparatus of
the muscle links the excitation process to the contractile
process. At low intramuscular calcium concentrations (less
6
than 10 M), the muscle fiber protein, actin, is bound to
the regulator proteins, tropomyosin and troponin, and actin
remains in an inactive state. After calcium release, calcium
binds to troponin, and actin complexes with myosin, the other
primary contractile protein. Several subsequent steps result
in the hydrolysis of ATP by myosin ATPase and the shortening
of the sarcomere. Calcium is then actively pumped back into
the sarcoplasmic reticulum, and the formation of actin-myosin
contractile complexes is inhibited and contraction ceases.
For a more complete treatment of this subject, see Tonomura
(1973) and V7eber and Murray (1973).
Effect of Acidosis on Muscle Contraction
As an interesting corollary of studies on the effect of
pH on the neuromuscular blocking agents, Baraka (1964) and
Crul-Sluijter and Crul (1974) observed a slight depressive
effect of acidosis alone on skeletal muscle twitch response.
Baraka (1964) conducted his experiments on humans i_n vivo and
interpretation of his results is complicated because acidosis
also effects elevations in serum sodium and potassium


10
concentrations and in levels of circulating epinephrine and
norepinephrine (Sechzer et al., 1960). However, experiments
by Crul-Sluijter and Crul (1974) with in vitro rat diaphragm
preparations show that muscles contract with slightly less
force when the bath fluid is made acidic by decreasing the
bicarbonate concentration. The mechanism of this effect
has not been determined, and is is not clear whether the
depression is dependent on the change in pH or the change in
the bicarbonate concentration.
Potassium Deficiency with Reference to Skeletal Muscle
Potassium deficiency is manifested differently in dif
ferent species. In both dog and rat, the decrease in intra
muscular potassium concentration is relatively less than the
decrease in extracellular potassium concentration (Bilbrey et
al. 1973). In man, serum potassium concentrations may remain
near normal despite severe potassium loss from muscles (Rettori
et al., 1972). Potassium deficiency in rats causes character
istic myocardial and renal damage (Welt et al., 1960), but no
skeletal muscle paralysis develops, even in severe potassium
deficiency (Smith et al., 1950). Resting muscle membrane
potentials in potassium-deficient rats were found to be in
agreement with values predicted by the Goldman equation (Bilbrey
et al. 1973). In both dog and man, progressive skeletal
muscle weakness and structural degeneration develop (Chanpion
et al., 1972; Smith et al., 1950). Bilbrey et al. (1973) have
attributed muscle weakness in severely potassium-deficient
dogs to an abnormally low resting membrane potential. They


11
suggested that these low resting potentials resulted because
the membrane permeability to sodium relative to the permeability
to potassium had increased, although they did not test this
hypothesis. Potassium release from exercised muscle, which
is thought to mediate increased local blood flow, has been
demonstrated to be severely reduced in potassium-deficient
dog (Knochel and Schlein, 1972). Muscle necrosis and associ
ated myoglobinuria following exercise in potassium-depleted
dog and man have been attributed to failure of these muscles
to show the normal hyperemic response to exercise.
Muscle Paralysis and Potassium Distribution
There are several conditions in which transitory muscular
paralysis is associated with abnormal potassium distribution.
Two such kinds of paralysis seen in humans are hyperkalemic
familial periodic paralysis, also known as adynamia episdica
hereditaria, and hypokalemic periodic paralysis. In hyper
kalemic periodic paralysis, attacks of muscle weakness are
associated with elevations in serum potassium (Gamstorp et al.,
1957). Acetazolamide and hydrochlorothiazide are used to
control this disease, presumably acting by lowering the serum
potassium through kaluresis (McArdle, 1969).
Acetazolamide, a potent carbonic anhydrase inhibitor and
potassium wasting diuretic, has been found to be the most effec
tive treatment available for hypokalemic periodic paralysis
(Griggs et al., 1970). In hypokalemic periodic paralysis,
attacks are associated with a fall in the serum potassium
concentration (Biemond and Daniels, 1934). During attacks,


12
potassium has been shown to move from serum into skeletal
muscle (Ionasescu and Merculiev, 1962) and serum potassium
concentrations fall to 2.5 mM or lower (Vroorn et al., 1975).
During attacks there is a depression of muscle electrical
activity (Viskoper et al., 1973) and muscles have been reported
to be depolarized rather than hyperpolarized as would be
expected on the basis of the abnormal potassium distribution
(Shy et al., 1961; Creutzfeldt et al., 1963). These obser
vations are consistent with the theory that muscles are paral
yzed by a depolarization block resulting because of a decrease
in the potassium permeability of the muscle membrane. The
sodium contribution to the resting potential becomes relatively
more important, and depolarization results. During depolar
ization, the muscle membrane sodium-potassium ATPase activity
increases and the serum potassium concentration continues to
fall (Otsuka and Ohtsuki, 1970). Although increasing the
extracellular fluid potassium concentration might be expected
to further depoleirize the muscle membranes, potassium chloride
infusion terminates the paralysis (Aitken et al., 1937).
Diaphragm muscles isolated from potassium-deficient rats
have several characteristics in common with muscle in hypo
kalemic periodic paralysis patients, although these rats do
not suffer paralysis in vivo (Smith et. al., 1950). Muscles
in periodic paralysis patients and in potassium-deficient
rats contain elevated intracellular sodium and reduced intra
cellular potassium (Hoffmann and Smith, 1970; Offerijns et
al., 1958). Offerijns et al. (1958) reported that insulin
caused flacid paralysis in the hemidiaphragm preparation in


13
conjunction with low potassium in the buffering medium.
Insulin, which lowers serum potassium more in hypokalemic
periodic paralysis patients than in normal persons, also
induces severe attacks of paralysis in these patients (Vroom
et al., 1975), although it is not clear whether insulin is
essential in all episodes. In potassium-deficient rat diaphragm
muscle paralysis, as in hypokalemic periodic paralysis, the
resting muscles have been reported to be depolarized while
the extracellular potassium concentration remains reduced;
paradoxically normal contractile function could be restored
in both cases by elevating the extracellular potassium
concentration (Gordon et al., 1970; Ohtsuka and Ohtsuki, 1970).


RATIONALE
Changes in pH, with accompanying changes in either
carbon dioxide tension or bicarbonate concentration, affect
several important, skeletal muscle properties. These proper
ties include ion distribution, cell permeability, cell poten
tial, and contractile force. Each of these factors plays a
vital role in muscle performance, but the influence of the
bicarbonate buffer system on these properties has never been
extensively investigated. Therefore, we measured skeletal
muscle contractile force, cation composition, and resting
membrane potential in in vitro experiments using three dif
ferent bicarbonate buffer series. One of these series had
buffers with different carbon dioxide concentrations, simu
lating the changes seen in respiratory acid-base disturbances.
One series had buffers with different bicarbonate concentra
tions, simulating metabolic acid-base disturbances. The
third series had buffers with different concentrations of
bicarbonate equilibrated with different carbon dioxide
mixtures, but with the same ratio of HCO^iCC^, and hence,
with the same pH. Through this approach we hoped to deter
mine whether bicarbonate and carbon dioxide have any specific
effects on skeletal muscle apart from changes in pH.
Rat diaphragm muscle from both normal and potassium-
depleted rats was studied in these experiments. Potassium-
14


15
deficient rat diaphragm muscle was selected for investigation
since it previously has been shown to be similar in many
respects to skeletal muscle of hypokalemic periodic paralysis
patients. Further, the rat diaphragm muscle is less than on
millimeter thick, and can easily be oxygenated in vitro. The
main objective was to determine whether potassium-depleted rat
muscle shares with hypokalemic periodic paralysis muscle a
sensitivity to changes in the bicarbonate buffer system. If
such a bicarbonate-carbon dioxide sensitivity were found, per
haps an investigation of the mechanism would provide a better
understanding of the influence of acid-base balance upon skeletal
muscle and aid in elucidation of the effect of carbonic anhydrase
inhibitors upon hypokalemic periodic paralysis.


SPECIFIC AIMS
The specific aims of this investigation are to determine
in both normal and potassium-depleted rat muscle:
1. the effect of alterations in bicarbonate concentration,
carbon dioxide tension and pH on contractility
2. the effect of alterations in bicarbonate concentration,
carbon dioxide tension, and pH on muscle potassium con
tent
3. the effect of alterations in bicarbonate concentration,
carbon dioxide tension, and pH on muscle intracellular
PH
4. the effect of alterations in bicarbonate concentration,
resting carbon dioxide tension, and pH on membrane
potential
5. the effect of variations in the extracellular potassium
concentration on resting muscle membrane potential
16


MATERIALS AND METHODS
Buffer Composition
Muscle experiments were performed in Tyrode solution of
the following composition; NaCl, 126 mM; KC1, 5 mM; CaCl2,
3 mM; MgCl2, 1-6 mM; dextrose, 11.1 mM; Na2HPO^, 0.677 mM;
Nali2P0^, 0.17 2 mM; and NallCO^, 16 mM. Low bicarbonate solu
tions were made by substituting 8 millimoles per liter of
sodium chloride for 8 millimoles per liter of sodium bicar
bonate. High bicarbonate solutions were made by substituting
16 millimoles per liter of sodium bicarbonate for 16 milli
moles per liter of sodium chloride. In one series of
experiments, the sodium chloride concentration was kept con
stant at 118 mM, and 0.5 millimoles sodium sulfate plus 0.5
millimoles sucrose were substituted for each millimole of
sodium bicarbonate in reduced bicarbonate solutions. Low
potassium solutions contained 0.5 mM potassium chloride plus
an additional 4.5 millimoles per liter of sodium chloride.
Elevated potassium solutions were prepared by substituting
potassium chloride millimole for millimole for sodium chloride.
Solutions were gassed with mixtures of oxygen and carbon
dioxide. The gas mixtures contained oxygen with either 2.5%,
5%, or 10% carbon dioxide by volume. The mixture with 2.5%
carbon dioxide was obtained from and certified by Matheson
17


18
Products, and the mixtures with 5% and 10% carbon dioxide
v;ere obtained from Liquid Air Company.
Muscle experiments were performed in one of the three
series of buffers illustrated in Table 1. The pH of the buffer
solutions was measured near the tissue site in all experi
ments. The pH was measured with an Arthur Thomas Company
combination electrode and a Beckman pH meter. It was neces
sary to carefully regulate the bubble rate of the gas, and
to compensate for cylinder variations in gas mixtures by
varying the bicarbonate concentrations slightly when dealing
with the constant pH buffers (series three buffers). Bicar
bonate concentrations other than those shown in Table 1 were
used, although none of these values varied more than 25% from
the values expressed in the table. The bicarbonate concen
trations illustrated in Table 1 represent approximate concen
trations as well as approximate carbon dioxide volumes percent
of the gases.
Experimental Animals
Male Sprague-Dawley rats, 100-150 g, obtained from Flow
Labs, were housed individually in wire cages. They were fed
potassium-deficient rat chow, completely supplemented with
vitamins and minerals, obtained from Nutritional Biochemicals.
Rats were maintained on this diet for 5 to 6 weeks prior to
the experiment. At the time of the experiment, these rats
weighed between 200 g and 275 g. Male Sprague-Dawley rats,
weighing 200-275 g, obtained from Flow Labs, were used as
control rats. These animals were fed standard rat chow.


19
TABLE 1
BUFFER
SERIES
HCO3 / C02
(mM)/(Vol. %)
A
B
C
SERIES 1 Constant HC03
(respiratory changes)
16/2.5
16/5
16/10
SERIES 2 Constant CC>2
(metabolic changes)
8/5
16/5
32/5
SERIES 3 Constant pH
(compensated changes)
8/2.5
16/5
32/10


20
Rat Hemidiaphragm Preparation
The rat phrenic nerve hemidiaphragm in vitro preparation
*
was originally described by Bulbring (1946). This tissue was
chosen for these experiments because it is a thin skeletcil
muscle which is easily oxygenated and supplied with nutrients.
The preparation is stable over several hours if left undisturbed.
It contains mostly fast muscle fibers and can be dissected
out with the phrenic nerve for indirect stimulation or without
the nerve for direct stimulation.
Surgery was performed on rats during ether anesthesia.
The abdominal cavity was opened by a ventral incision and the
intestines were pushed aside to expose the dorsal aorta.
Arterial blood was drawn into a heparinized syringe and plasma
was separated by centrifugation for sodium and potassium
determinations, performed on an Instrumentation Laboratory
343 flame photometer, against a lithium standard. Animals
were then decapitated and the skin removed from the chest.
The thoracic wall was cut to expose the rostral surface of
the diaphragm and the phrenic nerves. When the phrenic nerve
was to be isolated with the hemidiaphragm, it was tied close
to the thymus and cut, then carefully dissected away from
any attaching tissues. The diaphragm was cut down the mid
line, and the ribs and tissues connecting each hemidiaphragm
were cut away. The hemidiaphrgms were then removed from the
animal and placed in oxygenated Tyrode solution where the
remainder of the surgery was performed. Extraneous tissues
were then cut away from the hemidiaphragm leaving a section


21
of the ribs attached to the muscle tendons. A long piece of
silk thread was tied to the central tendon and two loops of
silk thread were tied to each edge of the rib segment. For
direct stimulation, two teflon-coated stainless steel wire
electrodes were tied to the muscle. The preparation was
mounted by the two loops to a stationary acrylic hook at
the bottom of a cylindrical glass bath containing 45 ml of
Tyrode solution bubbled with the appropriate gas mixture at
37 C. The silk thread attached to the central tendon was
tied to a Myograph-B transducer at one gram of resting tension.
For indirect stimulation experiments, the phrenic nerve was
passed through two silver ring electrodes imbedded in insulating
dental acrylic. Muscles were stimulated with slightly more
than enough voltage to elicit a maximum contractile response.
The muscles were stimulated at five second intervals, with
square wave monophasic pulses, for 5 milliseconds duration
for direct stimulation or for 0.5 milliseconds for indirect
stimulation. Contractions were recorded on a Narco Bio-Systems
Physiograph equipped with a low-pass filter set at 10 cycles
per second.
Contraction Experiments
Experiments were performed on paired hemidiaphragms from
the same rat in the same series of buffers (see Table 1) In
contraction experiments, both hemidiaphragms were first equi
librated and stimulated in Tyrode solution containing 16 mM


22
bicarbonate bubbled with 95% oxygen, and 5% carbon dioxide.
During this stabilization period the buffer was changed every
twenty minutes. The stabilization period lasted until a
stable base line and a stable peak contraction height were
obtained, usually about one hour. Subsequently, one of the
paired hercidiaphragms was bathed and stimulated first in
buffer A, then buffer B, then buffer C, then again in buffer
A, while the companion hemidiaphragm was bathed and stimulated
first in C, then in B, then in A, then again in C (see Table
1). Muscles remained in each buffer for 10 minutes, then
the bath was drained and fresh buffer of the same type was
added for 20 minutes. Thus the contraction experiments lasted
2 hours after the stabilization period. If a muscle showed
signs of deterioration, that muscle was eliminated from
consideration. Contractile responses on the physiograph
trace were measured 15 minutes after each of the four buffer
changes. The first and the last contractile responses were
averaged to obtain one value for a response in that buffer.
For statistical comparison, individual muscle contractions
in each buffer were expressed as a percent of the maximum stable
contraction recorded for that hemidiaphragm, at any time.
Diaphragms from potassium-deficient rats were studied
in all three series of buffers in Tyrode solution containing
0.5 inM potassium. These muscles were stimulated directly
with stainless steel electrodes. Potassium-deficient rat
diaphragms were also examined in series three, (constant pH
buffers, containing 0.5 mM potassium) stimulated indirectly
via the phrenic nerve. Diaphragms from control rats were


studied in all three series of buffers. Eemidiaphragms were
stimulated directly in these experiments which were conducted
in both 5 raM potassium Tyrode solution and in 0.5 inM potassium
Tyroae solution.
The effect of d-tubocurarine on directly stimulated and
indirectly stimulated muscle response was examined in several
pairs of hemidiaphragms. The paralyzing effect of regular
insulin (Lilly) on potassium-deficient diaphragm muscle was
also tested at the end of several contraction experiments.
Intracellular pH and Tissue Cation Determinations
Intracellular pH was determined on isolated rat hemi
diaphragms according to the method of Adler et al. (1965).
Paired hemidiaphragms were isolated, mounted, and stimulated
in the manner described in the preceding section. Muscles
were equilibrated for at least one hour in 16 mM bicarbonate
buffer, bubbled with a 95% oxygen, 5% carbon dioxide gas
mixture. After this equilibration period, buffer A of a
particular series was placed in the bath containing one
hemidiaphragm and buffer C of that same series was added to
the bath containing the companion hemidiaphragm. Fresh
buffer was supplied after 10 minutes. After 20 minutes,
the buffers were switched, and contractions were recorded
for 30 minutes; the buffer was renewed after 10 minutes,
as usual. After 30 minutes, these same buffers containing
3
200 microcunes per liter of H labeled mannitol and 50
14
microcunes per liter of C labeled DMO, both obtained
from New England Nuclear, were added to the appropriate organ


baths. The purity of these radiolabeled compounds was
verified with paper chromatography. These buffers were
renewed after 30 minutes. After 30 minutes 2 ml samples of
the bath solutions were taken for analysis and the experiment
was terminated. Thus each muscle was equilibrated and stimu
lated for one hour in buffer B, then for 1/2 hour in either
buffer A or C, then for 1 1/2 hours in either C or B, so that
each muscle was tested in buffers A, B, and C, one ending in
buffer C and one ending buffer A. During the last hour,
each buffer contained radiolabeled DM0 and radiolabeled
mannitol. If either hemidiaphragm showed signs of deteriora
tion, the experiment was terminated and the results discarded.
After completion of the contraction phase of the experi
ment, the muscles were removed and cut away from the attaching
ribs. The tendon was cut away and each muscle was cut into
two parts. One piece of each muscle was placed into a tared
polyethylene vial. Both vessels were weighed. The piece of
muscle in the crucible was dried overnight at 100 C and
reweighed, then ashed overnight at 600 C in a muffle oven.
The ash was then dissolved in concentrated HCl and the calcium
content of the ash was determined on a Perkin-Elmer atomic
absorption spectrometer against a lanthanum standard. Distil
led water, 100 times the weight of the tissue, was added to
the polyethylene vial to leach out the ions and the isotopi-
cally labeled DMO and mannitol (Lipicky and Bryant, 1966).
After an overnight soak, the sodium and potassium concentrations


25
14 3 ...
and the C and H activities of the fluid was determined.
Sodium and potassium determinations were performed on an
Instrumentation Laboratory 343 flame photometer against a
lithium standard. Radioactivity of the solutions was
determined in a Beckman model LS 200 liquid scintillation
counter. The buffers were analyzed for radioactivity and
3
sodium and potassium content m a similar manner. The H
14
counts were corrected for C spectral overlap according to
the formula A^ = R. (E x A ) where A. is the corrected
tic t
3 3
H counts per minute, R^ is the uncorrected h counts per
minute over background, E is the counting efficiency of
14 3
a pure C sample on the H channel relative to the counting
efficiency of that same pure sample on the 14c channel, and
A^ is the counts per minute over background of the mixed
14
sample on the C channel. The extracellular space in ml of
each muscle was estimated as the mannitol space of that
muscle, determined by the formula:
counts per minute of that muscle
n counts per minute of 1 ml of medium
The intracellular space was estimated as the tissue water
weight lost in drying minus the estimated extracellular water
weight. Intracellular sodium and potassium contents were
determined from the sodium and potassium concentration of the
solutions in which the muscles had been soaked, minus the
sodium and potassium that was calculated to have been contri
buted by extracellular fluid. Intracellular pH was calculated
from the formula:


26
/C. (V. + V ) V .
pH p.Ka + log!^ x 2 x [10(p~ ~ pHe + 1] -
\ e i i
where pK is the ionization constant of DMO (6.13), C is the
a t
concentration of DMO in the water of the entire tissue, C
e
is the concentration of DMO in the extracellular fluid, V
e
is the volume of the extracellular fluid, V. is the volume
i
of the intracellular fluid, and pHe is the extracellular pH
of the buffering medium (Waddell and Butler, 1959).
The mean intracellular muscle bicarbonate concentrations
were calculated according to the Henderson-Hasselbalch equa
tion, assuming an apparent pK value of 6.13 for carbonic
3.
acid in muscle. The solubility coefficient of carbon dioxide
in intracellular fluid was taken to be 0.035 millimoles per
liter per millimeter mercury pressure (Brown and Goott, 1963).
The partial pressure of carbon dioxide in the buffer was
determined from the measured pH and the Henderson-Hasselbalch
equation, using 0.0241 millimoles per liter per millimeter
of mercury pressure for the solubility coefficient of carbon
dioxide in the buffer (Edsall and Wyman, 1958).
Intracellular pH and cation determinations were made on
diaphragm muscle from potassium-deficient rats in all three
series of buffers in 0.5 mM potassium. Similar determina
tions were made on muscle from control rats in series 3,
constant pH buffers in 5 mM potassium. Contractile responses
were measured at the end of each experiment and expressed as
a percent of the maximum response recorded from that muscle.


27
Potential Measurements
Diaphragms were rapidly dissected out of rats as
previously described. Strips of hemidiaphragm were placed
in plexiglass beaker containing 16 mM bicarbonate buffer
bubbled with 95% oxygen, 5% carbon dioxide. After 30
minutes, the strips were transferred to a 10 ml plexiglass
chamber where they were perfused with Tyrode solution at a
rate of approximately 10 ml per minute. Solutions were kept
at 35 C and were bubbled with the appropriate gas mixture.
The muscle stri.ps were pinned by the adjoining tendons to a
silicone-resin mat at the bottom of the chamber. Resting
membrane potentials were recorded v/ith standard microelectrode
techniques, using glass micropipettes filled with 3 M potas
sium chloride. The electrodes had a tip resistance of 5-10
megohms. Prepulled microelectrodes with a 1 micron tip con
taining a glass fiber were obtained from Fredrick Haer and
Company. These electrodes were filled by capillary action,
and examined under a microscope. Potential signals were
amplified on an Electronics for Life Science wide band elec
trometer equipped with a circuit for monitoring the electrode
tip resistance. The signal from the electrometer was displayed
on a Tektronic type 564B oscilloscope. The criteria followed
for judging whether a potential was a valid resting potential
were:
1. a resting potential had to be more negative than
-40 millivolts
2. a resting potential had to be stable within 5
millivolts for one minute


28
Potential measurements were made on potassium-deficient
and control diaphragm muscles bathed in buffers from series
1 and 2, containing 0.5 mM potassium. After the muscles
were transferred to the chambers, the chambers were perfused
with either buffer A or C of the series. After 20 minutes,
6 resting potentials were recorded. The chambers were then
perfused with the other buffer of that series, and after 20
minutes, 6 potentials were again recorded.
Resting potentials of diaphragm muscles from control and
from potassium-deficient rats were also determined in 16 mM
bicarbonate buffer bubbled with 95% oxygen, 5% carbon dioxide,
but containing different concentrations of potassium. These
potassium concentrations were 20 mM, 10 mil, 5 mM, 2.5 mM,
and 1.25 mM. Equilibration time in these experiments was
5 minutes, since it was reasoned that the extracellular fluid
potassium will equilibrate in this time. Six potentials
were recorded in each muscle at each potassium concentration.
To eliminate bias during the potential recording process,
the buffers were presented to the investigator in a sequence
unknown to him. The buffer sequence was revealed after
completion of the experiment.


RESULTS
Contraction Studies
Action of d-tubocurarine on Directly and Indirectly Stimu
lated Muscle
When hemidiaphragms were stimulated directly or in
directly, a stable base line and peak contraction height
were usually evident on the physiograph trace after a short
equilibration period. Figure 1 shows the result of the
addition of blocking doses of d-tubocurarine on paired hemi-
diaphragms, one stimulated directly via electrodes tied
adjacent to the muscle fibers and one stimulated via the
phrenic nerve. It can be seen that d-tubocurarine, at a
-4
bath concentration of 10 M, had no effect on the contractile
response of the directly stimulated muscle. A lesser
concentration, 10 ^ M, almost completely blocked the contrac
tile response of the indirectly stimulated muscle within
10 minutes. When the bath was flushed and normal Tyrode
solution was again added, contractile responses to phrenic
nerve stimulation began to return immediately.
29


Figure 1. Effect of d-tubocurarine (DTC) on directly stimulated
hemidiaphragm muscle (top trace), and on indirectly
stimulated muscle (bottom trace). Doses were 10-4 m
and 10-^ M respectively. Marked intervals correspond
to 1 minute.


\
1_1 I I 1 I I I I I I I I I I I I I 1 1 I t i L-l_l J L- t '
figure 1


32
Effect of Variations in Bicarbonate Buffer Components on
Contractile Response
Rat diaphragm muscle responded to changes in bath solu
tion pH by contracting with significantly'*" less force in
more acidic buffers. This was true when acidosis resulted
from an increase in the carbon dioxide concentration in the
Tyrode solution (series one buffers) or from a reduction in
the bicarbonate concentration in the Tyrode solution (series
two buffers). Figures 2 and 3 show the contractile response
of hemidiaphragm muscles from control rats plotted as a
function of the pH in the Tyrode solutions. The data in
Figure 2 represent muscle response in 5 mM potassium buffers,
and the data in Figure 3 represent muscle response in 0.5 mM
potassium buffers. It can be seen that reducing the pH of
the bath fluid from 7.8 to 7.1 resulted in approximately a
20% reduction in contractile response. Figure 4 shows the
effect of alterations in bath solution pH on contractile re-
. 2
sponse of diaphragm muscle from potassium-deficient rats.
These hemidiaphragms, bathed in Tyrode solution containing
0.5 mM potassium, showed approximately a 65% reduction in
^Differences described as significant in this paragraph have
a p value of less than 0.05 in the F-test.
2
Serum potassium concentrations of rats on potassium-deficient
rat chow diets averaged 1.78 mM with a standard error of 0.076
mM; n=18. When hemidiaphragms from several potassium-defi
cient rats were challenged with insulin, 20 units per liter,
the contractile response to direct stimulation was reduced
considerably; contractile response was restored when the bath
potassium concentration was elevated from 0.5 mM to 5 mM,
behavior characteristic of potassium-deficient rat diaphragm.


Figure 2.
Contraction of control muscles in 5 roM
potassium is plotted as a function of the
pH of the bathing fluid. Filled circles show
values obtained in exp ments done in series
1, constant HCO^~ buffo Empty circles show
values obtained'in series 2, constant CO^
buffers. The bars represent standard errors.
Responses within both experiments differed signif
cantly at p=0.05, in the F-test. n~6, for each
point. For further details, see Methods, page 22


34
CONTRACTION vs pH
100 i
¡2
O
75 -
O
<
cr
y-
o
o
| 50-
X
<
2
K-
2.'
IjJ
CJ>
tr
.I
¡
r 1 T-
7.1 7.2 7.3
i
7.4
7.5
PH
~i r~
7.6 7.7
7.8
FIGURE 2


Figure 3. Contraction of control muscles in 0.5 mM
potassium is plotted as a function of the
pH of the bathing fluid. Filled circles
show the values obtained in experiments
done in series 1, constant HCCt ~ buffers.
Empty circles show values obtained in serie
2, constant CO2 buffers. The bars represen
standard errors. Responses within both exper
ments differed significantly at p=0.05,
in the F-test. n-6, for each point. For
further details, see page 22.
(T U)


36
CONTRACTION vs pH
IOO
o
1
o
<
or
\-
2
o
o
z>
X
<
2
75
50 -
h-
2:
UJ
O
nr
UJ
Q-
i r
7.2 7.3
p
I
i 1
7.4 7.5
-T r
7.6 7.7
pH
7.8
FIGURE 3


Figure 4.
Contraction of potassium-deficient muscles
in 0.5 mM potassium is plotted as a function
of the pH of the bathing fluid. Filled circles
show the values obtained in experiments done
in series 1, constant HCO3" buffers. Empty
circles show values obtained in series 2,
constant CO2 buffers. The bars represent
standard errors. Responses within both experi
ments dif fered significantly at. p~0.05, in the
F-test. n-6, for each point. For further
details, see page 22.


38
CONTRACTION vs pH
FIGURE 4


Figure 5. Contraction of control muscles in 5 mM
potassium is plotted as a function of
the CO^ volumes percent bubbling the
buffer and the HCO^ concentration of
the buffer. The values shown were obtained
in series 3, constant pH buffers. The bars
represent standard errors. Responses did
not differ significantly at p=0.10, in
the F-test. n=6, for each point. For further
details, see page 22.


PERCENT MAXIMUM CONTRACTION
40
CONTRACTION vs C02 AND HCO3 AT CONSTANT pH
HCO¡ [mM]
8 16 32
C02 [Vol.%]
FIGURE 5


Figure 6.
Contraction of control muscles in 0.5 mM
potassium is plotted as a function of
the CO^ volumes percent bubbling the
buffer and the HCO^ concentration of
the buffer. The values shown were obtained
in series 3, constant pH buffers. The bars
represent standard errors. Responses did
not differ significantly at p-0.10, in
the F-test. n-6 for each point. Fr further
details, see page 22.


42
100
9 75
h-
o
<
Or:
h
O
o
s
Z>
5
x
<
t-
z
LJ
O
cr
LU
D.
50
25
CONTRACTION vs C02 AND HCO3 AT CONSTANT pH
HCO; [mM]
8 IG 32

2 5
r~
10
C02 [V0!.%]
FIGURE 6


Figure 7. Contraction of potassium-deficient muscles
in 0.5 mM potassium is plotted as a function
of the CO2 volumes percent bubbling the
buffer and the HCO3- concentration of the
buffer. The values shown were obtained in
series 3, constant pH buffers. Filled circles
show values obtained in experiments when
muscles were stimulated directly, as usual.
Empty circles show values obtained when the
muscles were stimulated indirectly, via the
phrenic nerve. Triangles show values obtained
when the muscles_were stimulated directly in
buffers with SO.- and sucrose substituted for
HC03_ in the reduced HC03_ solutions. Muscle
performance in SO.- and sucrose buffers was
generally poor. The bars represent standard
errors. Responses within all three experiments
differed significantly at p=0.05, in the F-test.
n=6, for each point. For further details, see
page 22.


PERCENT MAXIMUM CONTRACTION
44
CONTRACTION vs C02 AND HCOj AT CONSTANT pH
HCOj [mM]
8 16 32
FIGURE 7


45
contractile response when the pH of the bath solution was
reduced from 7.8 to 7.1. This was a quantitatively greater
response than that which was seen in the control rat muscles.
In all of these experiments, the magnitude of the change in
muscle contractility seemed independent of whether the change
in pH was effected by respiratory of metabolic changes.
Figures 5 through 7 show results of contraction experiments
done in series three, (constant pH buffers). In these experi
ments, contraction of hemidiaphragm muscles was recorded in
buffers containing different bicarbonate concentrations
bubbled with different carbon dioxide mixtures, but with the
same ratio of bicarbonate to carbon dioxide. Figures 5 and
6 show contraction of hemidiaphragms from control rats plotted
as a function of both carbon dioxide volumes percent in the
gas mixture bubbling the buffer solution and the bicarbonate
concentration of the solution. The points in Figure 5
represent the responses recorded in muscles in Tyrode solution
containing 5 mM potassium; those in Figure 6 represent
responses exhibited by the muscles in 0.5 mM potassium Tyrode
solution. There was no statistically significant difference
in the responses exhibited by the muscles in either experi
ment, although there appeared to be a trend toward more
vigorous contractions in buffers containing elevated bicar
bonate concentrations and elevated carbon dioxide tensions.
This trend reappeared in experiments done on hemidiaphragm
muscles taken from potassium-deficient animals. Figure


46
7 shows the results of experiments which were done on hemi-
diaphragms from potassium-deficient rats in series three,
(constant pH buffers). These buffers contained 0.5 mM
potassium. In figure 7, the results of three different
experiments are shown. Again, contractile response is plotted
as a function of both buffer bicarbonate concentration and
carbon dioxide volumes percent of the gas mixture. One set
of points represents the response of muscles to direct stimu
lation; one set of points represents the response of muscles
to indirect stimulation via the phrenic nerve; and one set of
points represents the response of muscles to direct stimulation
when sulfate and sucrose rather than chloride were sxibstituted
for bicarbonate in the reduced bicarbonate solutions. In all
three experiments, the contractile responses were significantly
greater in the high bicarbonate, high carbon dioxide buffers
than in low bicarbonate, low carbon dioxide buffers.
In summary, muscle contractile force was always greater
in buffers with high pH levels or high bicarbonate concen
trations. Muscle contractile force increased with increasing
carbon dioxide tension in constant pH experiments, but
decreased with increasing carbon dioxide tension in constant
bicarbonate experiments. These relationships were always
more pronounced in potassium-deficient rat muscles than in
muscles from control rats.
Changing the potassium concentration from 0.5 mM to 5 mM
had no perceptible effect on contraction of either potassium-
deficient rat muscle or control rat muscle. There was no


47
significant difference in contractile force per gram of
muscle between control rat muscles in 5 mM potassium and
potassium-deficient rat muscle in 0.5 mM potassium. For
3.0 control and 10 potassium-deficient rat muscles, these
values averaged 40.7 grams of force per gram of tissue and
33.5 grams of force per gram of tissue respectively, with a
pooled standard deviation of 15.6 grams of force per gram of
muscle tissue. These values were recorded in 32 mM bicarbon
ate buffers bubbled with a 95% oxygen, 5% carbon dioxide
mixture.
Tissue Analysis
Effect of Bicarbonate and Carbon Dioxide Tension Changes
on Muscle Water and Calcium Content
Changes in bicarbonate concentration and in carbon dioxide
4
tension had no significant effect on total muscle water or
extracellular fluid volume. There was no significant dif
ference between the means of these values obtained from
potassium-deficient animals and the means obtained from con
trol animals. For all experiments, 78% of muscle wet weight
The difference between these two means has a p value greater
than 0.05 in the Student's t-test.
4
Differences described as significant m this section, unless
otherwise noted, have a p value less than 0.05 in the paired
t--test.


48
was water, and 36% of muscle wet weight was extracellular
fluid The standard deviations of these two values were
2% and 5%, respectively.
Buffer changes had no significant effect on intracellular
calcium content of muscles, nor was there any significant
difference between the mean of this value determined in control
rats and the mean of this value determined in potassium-
deficient rats. For all tissues, the single mean and the stan
dard deviation were 3.1 mM and 1.4 mM, respectively.
Effect of Buffer Bicarbonate and Carbon Dioxide Tension
Changes on Muscle Intracellular pH and Potassium Content
A decrease in extracellular pH caused by an increase in
buffer carbon dioxide tension or by a decrease in buffer bi
carbonate concentration, was accompanied by a significantly
lower intramuscular pH. Changes in buffer carbon dioxide
resulted in greater changes in intracellular pH than changes in
buffer bicarbonate. The muscles in the more acidic buffers
also contained less intracellular potassium than those in
^Because of the consistency of the measured tissue water
weights, it was reasoned that the variation in the mannitol
space measurements did not reflect true variations in the
extracellular fluid volume, for any change in extracellular
fluid volume would necessitate an exactly opposite change in
the intracellular fluid volume. Therefore, in calculations
requiring a value for the extracellular fluid volume, the
average 36% of muscle wet weight was used to approximate this
value.


49
alkaline buffers. Muscle twitch tension, measured at the
end of the experiment after 1 1/2 hours total time in the
last buffer, was always significantly greater in the more
alkaline buffers. These results, obtained in potassium-
deficient rat muscle, are shown in Tables 2 and 3.
The results of tissue analysis on potassium-deficient
and control rat muscles in series 3, constant pH buffers,
are shown in Tables 4 and 5. Muscles bathed in high bicar
bonate, high carbon dioxide buffers were significantly more
acidotic than companion muscles bathed in low bicarbonate,
low carbon dioxide buffers. Muscles in the high bicarbonate,
high carbon dioxide buffers also contained significantly
more potassium than the hemidiaphragm muscles in low bicar
bonate, low carbon dioxide buffers. Contraction, measured
after 1 1/2 hours of equilibration, was always significantly
greater in the high bicarbonate, high carbon dioxide buffers.
Figure 8 is the physiograph trace recording of contractile
responses of paired hemidiaphragm muscles from a potassium-
deficient rat. The trace shows the response of the muscles
before and after a change in the buffer. It can be seen
that changing from a low bicarbonate, low carbon dioxide
buffer to a high bicarbonate, high carbon dioxide buffer
resulted in a stronger muscle response in one of the muscles
while changing the buffers in the opposite direction resulted
in a weaker contractile response from the companion muscle.
Both buffers had a pH of 7.39.
Table 6 summarizes the pertinent data shown on Tables
2 through 5. Shown in this table are the intracellular


TABLE 2
TISSUE ANALYSIS
Kt DEFICIENT RATS, IN
OF KEMIDIAPK
DIFFERENT CO
RAGAS, PROM
- CONCENT!wATIONS
HCO ~
j
Buffer Series
(Vol.
CO.
a
1
PHC
phR
HCO
- b
i
k"
o
Kr. (mM)3
i Maximum3
Contraccin
16 mM
2.5
7,64
7.36.07 n-5
c
11 6
mM
0 5 mM
12 3 15 n=5
a
885.5 n=5
c, e
16 mM
10
7.05
o.9 4 .0 3 n=5
17.2
mM
0.5 mM
103+9.1 n=5
575.9 n=5
a ,
data presented as means S.E.
calculated from mean pH.
J.
c
significant difference at p=0.Q5 m paired t~test
no significant airference at p=0.05 m paired t-test
Q ,
these values were determined at the end of the experiment and reflect
a gradual waning of contractile response over me three hour experi
mental period
Cl
*>


TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
K -DEFICIENT RATS, IN DIFFERENT HC03- CONCENTRATIONS
HCO3-
9 mM
3 2 mM
Buffer Series 2
(Vol.%)
C02 pHc pHia
5 7.12 6.63.09 n=6
c
5 7.62 6.77.17 n=6
4.0 mM
6.3 mM
0.5 mM
0.5 mM
% Maximuma
K ^ (mM)a Contraction
946.5 n=l 517.2 n=5
c c,d
1154.1 n=7 7 05 .4 n=5
data presented as means S.E.
calculated from mean pHj_
Q
significant difference at p=0.05 m paired t-test
these values were determined at the end of the experiment and reflect
a gradual waning of contractile response over the three hour experi
mental period


TABLE 4
TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
K+-DEFICIENT RAT'S, IN CONSTANT pH BUFFERS
Buffer Series 3
(Vol. %)
HC03 C02 pH0
% Maximum3
Contraction
8 mM
2.5
6.95.il n=8 4.0 mM
0.5 mM
91*9.1 n=8 396.1 n=8
32 mM
10
7.39
6.6 8 15 n= 8 3.7 mM
0.5 mM 1156.4 n=3
c,a
78x3.8 n=8
^data presented as means S.E.
calculated from mean pH
c *
significant difference at p=0.05 m paired t-test
these values were determined at the end of the experiment and reflect
a gradual waning of contractile response over the three hour experi
mental period


TABLE 5
TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
CONTROL RATS, IN CONSTANT pH BUFFERS
Buffer Series 3
HCO,
->
8 mM
32 mM
£
data presented as mean S.E.
^calculated from mean pH^
c .
significant difference at p=0.05 in paired t-test
these values were determined at the end of rhe experiment and reflect
a gradual waning of contractile response over the three hour experi
mental period
(Vol. %)
C0
PH
tt a
PH
HCO
- n
3 i
K
+
K+. (mM)a
% Maximum
Contraction
2.5 7.39 7.28.02 n=9 8.6 mM
c
5 mil
14511 n=9
c
705.2 n=9
c, d
10
7.39 7.19.03 n=9 28 mM
o mM
17013 n=9 81-4.5 n=9
U>


TAELE 6
Buffer
Series
1
2
3
COMPARISON OF ICF ACID-BASE AND K+
CHANGES IN HEMIDIAPHRAGMS IN VARIOUS BUFFERS
(Vol. %)
co2
HC03 o
PH
Hi/H+0
K\
% Maximum
Contraction
2.5
K+ Deficient
16 mM 7.36
1.91
128
mM
88
10
16 mM
6.94
1.29
108
mM
57
5
9 mM
6.63
3.09
94
mM
51
5
32 mM
6.77
7.08
115
mM
70
2.5
8 mM
6.95
2.75 ,
91
mM
39
10
32 mM
6.68
5.13
115
mM
78
2.5
Control
8 mM
7.28
1.29
145
mM
70
10
32 mM
7.19
1.58
170
mM
81
Ln
4^


Figure 8.
Effect of change in buffer of directly stimulated
potassium-deficient rat hemidiaphragm muscles.
Buffers contained 8 mM bicarbonate or 32 mM bicar
bonate bubbled with 2.5 volumes percent or 10
volumes percent carbon dioxide with oxygen. The
pH of both buffers was 7.39. Buffers contained
0.5 mM potassium. Marked time intervals correspond
to 1 minute.


32 _8_
10 | 2.5
r i i i i [ f i i i i i h-ttttt i i i i i f i i n-rrr i inn
figure 8


57
potassium concentrations of the muscles, the contractile
forces, and the ratios of intracellular to extracellular
proton concentrations. It can be seen that in all four
experimental pairs, the muscles which contracted with the
greatest amount of force always contained the higher intra
cellular potassium concentration and had a higher ratio
of intracellular to extracellular protons.
Resting Potential Measurements
Changing the bicarbonate concentration in the Tyrode
solution from 8 mM to 32 mM had no significant effect on the
resting potential of diaphragm muscles from control or from
potassium-deficient rats. Changing the carbon dioxide mixture
from 2.5% to 10% also had no significant effect on resting
potential. These potentials were recorded in muscles bathed in
0.5 mM potassium buffers. These data are illustrated
graphically in Figures 9 and 10 for potassium-deficient and
control rat muscles. These graphs also show resting potentials
recorded in muscles bathed in different potassium concentrations
ranging from 0.5 mM to 20 mM. The recorded potentials are plot
ted as a function of the ratio of the intracellular potassium con
centration to the extracellular (buffer) potassium concentra
tion. The intracellular potassium concentration of potassium-
deficient. muscles was taken from the average of the values in
Tables 2 through 4. The intracellular potassium concentration
of control rat muscle was taken from the average of the val
ues in Table 5. The horizontal bars extending from two of


Figure 9. Resting membrane potentials recorded in potassium-
deficient rat hemidiaphragm muscles are plotted as a
function of the ratio of intracellular to extracellular
potassium concentration. Intracellular potassium
concentration was estimated to be 107 mM. The hori
zontal bars represent the error that would be introduced
if the estimated intracellular potassium concentration
was incorrect by 20 mM. Vertical bars represent standard
errors. The curved line is the solution to the reduced
Goldman equation:
Em = -61.5 log (K+) i
(K+)q + .01 x (Na+)0
The straight line is the solution to the Ne.rnst equation,
(K+)i
Em = -61.5 log
(K+)0
n=24.
The four symbols on the extreme left represent potentials
recorded in buffers containing, from top to bottom,
8 mM HCO3" 32 mM HCO3- 16 mM HCO3- 16 mM HC03
5% C02 5% C02 2.5% C02 &nd 10% C02 '
For these points only, n=12.


-1 OOmV -
Em
-90mV -
-8 OmV -
- 7 0 mV -
- 6 OmV-
-50mV-
5
320
160
FIGURE 9


Figure 10.
Resting membrane potentials recorded in control rat
hemidiaphragm muscles are plotted as a function of the
ratio of the intracellular to the extracellular potassium
concentration. Intracellular potassium concentration was
estimated to be 155 mM. Bars represent standard errors.
The curved line is the solution to the reduced Goldman
equation:
(K+)i
Em = -61.5 log
(K+)o + .01 (Na+)o
The straight line is the solution to the Nernst equation,
E = -61.5 log JJLii
(K+)0
n=24.
The four symbols on the extreme left represent potentials
in from top to bottom, 32 mM HCO3- 16 mM HCC>3_ 16 mM HCO3
5% C02 2.5% C02 10% C02"
and ^ ^ HCO3 For these points only, n=12.
5% C02


-lOOmV
-90mV-
-80mV
- 7 0 mV -
-GOmV-
-50rrV-
FIGURE 10


62
the points in Figure 9 represent the error that would be
introduced if the estimated intracellular potassium concen
tration were incorrect by as much as 20 mM. The straight
line and the smooth curve drawn on Figures 9 and 10 are,
respectively, the relations described by the Nernst equation,
and the reduced Goldman equation using 0.01 for the ratio of
the permeability coefficients of sodium to potassium (Bilbrey
et al., 1973). It can be seen that the recorded potentials
do increase with decreasing extracellular potassium concen
tration, but not to the extent predicted by either the
Nernst equation of the reduced Goldman equation. The poten
tials recorded in potassium-deficient rat muscles best fit
the reduced Goldman equation when 0.022 was used for the
ratio of the permeability of sodium to potassium. The data
recorded in control rat muscles fit the equation best when
0.027 was used for the permeability coefficient ratio. All
of the potential measurements shown were recorded with one
of 3 microelectrodes on muscles from 2 potassium-deficient
rats and 2 control rats.


DISCUSSION
The data presented in this work are the first to be
reported correlating changes in muscle contraction brought
about by alterations in the bicarbonate buffer system with
changes in muscle ion composition. Other authors have reported
that acidosis caused depression of muscle contraction, but
this depression was slight in the preparations studied and
the authors offered no explanation for this effect (Crul-
Sluijter and Crul, 1974; Baraka, 1964). By analyzing
muscles that had been contracting in different bicarbonate
buffers, we hoped to determine specifically which components
of the buffer system affected contraction and to elucidate
the mechanism of this effect.
In the present study, acidosis mildly depressed the
contractile response of control muscles. The degree of
this depression was independent of the nature of the acidosis,
whether caused by increased carbon dioxide tension (respira
tory acidosis) or decreased external bicarbonate concentration
(metabolic acidosis). These results, obtained in directly
stimulated muscles, agree quantitatively with those of
Crul-Sluijter (1974) who reported a 4% depression of phrenic
nerve stimulated muscle contraction when HC1 lowered the bath
solution pH from 7.5 to 7.1. Over the same pH range, the
63


64
data in the present study show an 8% decrease in contractile
response, which is within experimental error of the value
reported by Crul-Sluijter,
Acidosis suppressed the contractility of muscle isolated
from potassium-deficient animals to a much greater extent
than muscles from control rats. These results are the first
to be reported showing the increased sensitivity of potassium-
depleted skeletal muscle to pH changes, A change in the
buffer pH from 7.75 to 7.1 was accompanied by a 60% fall in
the contractile response of this tissue compared with the
20% fall in the contactile response of muscle from control-
dieted rats when pH changed over this same range. The increased
sensitivity of this tissue to pH changes was not a consequence
of the low potassium concentration in the buffer (0.5 mM)
since control muscle contracting in 0.5 mM potassium buffer
did not display any increase in sensitivity to bath pH
changes, compared to control muscle in 5 mM potassium buffers.
Analysis of potassium-deficient muscles that had been
contracting in buffers with different pH levels revealed
that muscles in acidic buffers had lower intracellular pH
values than paired muscles that had been in more alkaline
buffers. The difference in intramuscular pH was greater in
response to simulated respiratory changes than in response
to simulated metabolic changes, although the extracellular
pH changes were similar. These results are qualitatively
similar to those obtained by Adler (1965) in diaphragm
muscles from normal rats. Mean intracellular muscle pH val
ues, determined in 16 mM bicarbonate solutions bubbled with


65
oxygen with 2.5 volumes percent carbon dioxide or 10 volumes
percent carbon dioxide, both fell on the curve Adler obtained
for intracellular pH values of control muscles. The two
mean intracellular muscle pH values, obtained for muscles in
buffers containing 8 mM bicarbonate and 32 mM bicarbonate
both bubbled with 5% carbon dioxide, 95% oxygen, were lower
than the two values obtained in the constant bicarbonate
buffers. The results of Adler (1972) on control rat muscles
depleted of potassium in vitro indicate that potassium-
depletion tends to reduce the muscle intracellular pH. There
fore, this would predict that the intracellular pH of in
vivo potassium-depleted muscle would be lower than the
values obtained in control animals. Considering this
evidence, the intracellular pH values we obtained in the
constant bicarbonate experiments were high, whereas, those
obtained in the constant carbon dioxide experiments were low
compared to Adler's values for intracellular pH of in vitro
potassium-depleted rat muscles. From the data in Tables 2
and 3, one can see that the muscles used in the constant
carbon dioxide buffer experiments were somewhat more potassium-
deficient than the muscles used in the constant bicarbonate
buffer experiments. Therefore, the muscles used in the con
stant carbon dioxide experiments probably were also more
acidotic at the beginning of the experiments. This would
have contributed to the disparity between these intracellular
pH values. This disparity does not detract from the essential
feature which is the qualitative relationship between bicar
bonate concentration and intracellular pH, and the relation
ship between carbon dioxide tension and intracellular pH.


66
Intracellular pH was found to be lower in muscles exposed to
buffers with low bicarbonate concentrations compared to paired
muscles in high bicarbonate buffers. Muscles in high carbon
dioxide buffers were found to have a lower intracellular pH
that paired muscles exposed to low carbon dioxide buffers.
Determination of muscle potassium content, performed on
the same muscles used for intracellular pH determinations,
showed higher intracellular potassium concentrations in
muscles that had been in alkaline buffers compared to the
intracellular potassium concentrations of paired muscles
that had been in acidic buffers. The differences in both
experiments was approximately 20 mM. Adler et al. (1965)
also reported that muscles bathed in alkaline buffers had a
higher intracellular potassium content than muscles bathed in
acidic buffers. But the differences reported in his data
were not as great as the differences found in the present
study. The muscles in Adler's experiments were not contracting,
while the muscles in our experiments were contracting. This
difference may have been important, since Lade and Brown (1963)
determined that i_n vivo decreases in muscle intracellular
potassium concentration in response to acidosis took place
faster when muscles were stimulated to contract. This was
probably because contraction increases the rate of loss of
potassium from skeletal muscle. The range of the intracellular
potassium concentrations determined in potassium-deficient
muscles, 90 mM to 130 mM, shows good agreement with the value
of 97 mM reported by Bilbrey et al. (1973), determined on
skeletal muscle from potassium-depleted rats.


67
Muscles exposed to high bicarbonate, high carbon dioxide
buffers contracted with greater force than muscles bathed
in low bicarbonate, low carbon dioxide buffers, even though
these buffers had the same pH. This relationship was more
pronounced in potassium-deficient muscles than in control
rat muscles. This effect was independent of variations in
buffer chloride concentration, and was seen in directly and
indirectly stimulated muscles alike.
Tissue analysis revealed that muscles bathed in the
high bicarbonate, high carbon dioxide buffers had a lower
intracellular pH and a higher intracellular potassium con
tent than muscles in low bicarbonate, low carbon dioxide
buffers. Kim and Brown (1968) obtained similar results in
in vivo experiments on dogs. They recorded a decrease in
intracellular muscle pH when serum bicarbonate concentration
and carbon dioxide tension were elevated simultaneously.
They also detected an increase in extracellular potassium
but did not seek the source of this potassium. In their
experiments, the intracellular pH declined from 6.97 to
6.87 while the extracellular bicarbonate concentration rose
from 25 mM to 65 mM, extracellular pH remained constant. The
calculated change in intracellular bicarbonate was from 12 mM
to 24 mM. In the present constant pH experiments with control
rat muscle, intracellular pH declined from 7.28 to 7.19
while extracellular bicarbonate rose from 8 mM to 32 mM.
This represents an increase in the calculated intracellular
bicarbonate from 9 mM to 28 mM. In potassium-deficient rat


68
muscles subjected to the same treatment, intracellular pH
declined from 6.95 to 6.68. The calculated intracellular
bicarbonate rose from 4 mM to only 8.7 mM. These data sug
gest that potassium-deficient muscles are less well buffered
than control muscles. Whether this means that less intra
cellular buffer is available for titration or that bicar
bonate or proton flux is restricted in this tissue is not
defined by these results.
The results of these constant pH experiments are pivotal.
Comparing the results of these contraction experiments with
the results of experiments conducted in non-constant pH
buffers, it is clear that contractile force always increased
with increasing extracellular bicarbonate concentrations and
with increasing extracellular pH. However, contractile force
increased with increasing carbon dioxide tension in the con
stant pH experiments, but decreased with increasing carbon
dioxide tension in constant bicarbonate experiments. These
relationships were always more pronounced in potassium-
deficient rat muscle experiments than in control muscle
experiments.
The paired data sets obtained from tissue analysis,
summarized in Table 6, indicate that all buffer changes which
effected an increase in muscle contractile force also effected
an elevation in intracellular potassium. In all four experi
ments the difference in intracellular potassium concentration
caused by the buffer change was about 20 mM. No correlation
between contraction and intracellular pH was observed, however,


69
the ratio of the intracellular to the extracellular proton
concentrations was always higher in the muscles with the
higher intracellular potassium concentration. Despite the
decrease in intracellular pH when extracellular pH was reduced,
the ratio of intracellular to extracellular proton concen
trations always fell when extracellular pH was reduced. Other
authors have reported a similar relationship between potassium
and hydrogen ion gradients across muscle cell membranes
(Brown and Goott, 1963; Fenn and Cobb, 1934; Lade and Brown,
1963; Irvine and Dow, 1966). These results are consistent
with the concept that potassium ions exchange with hydrogen
ions across muscle membranes in the direction which would
tend to equalize K.+/K + and H.+/H,+.
The redistribution of potassium brought about by the
buffer changes may be responsible for the observed changes
in muscle contraction. One possible mechanism whereby
increased intracellular potassium concentrations might effect
a change in muscle contractile force is by increasing the
resting muscle membrane potential. However, the data in the
present study do not support this hypothesis. Buffer changes
brought about alterations in the intracellular potassium
concentration of at least 20 millimoles per liter in all
experiments measuring this value. As a result, the resting
membrane potential would have been shifted as predicted by
the Goldman equation. For potassium-deficient muscles, based
on the potassium concentrations measured in Tables 2 to 4,
the predicted shift in resting potential would be about 6


70
millivolts. In experiments recording resting muscle membrane
potentials, no statistically significant shift in potential
was observed in response to buffer changes of any sort.
However, because our standard errors were around 7 millivolts,
a shift in resting potential consistent with a 20 mM change
in intramuscular potassium might not have been detected. If
the average resting potential of all the muscle fibers was
increased by even a few millivolts, the maximum contractile
force of the muscle might be noteably increased, but the
observation that changing the extracellular potassium concen
tration from 0.5 mM to 5 mM resulted in no change in muscle
contraction argues against this theory. With a change in bath
potassium of this magnitude, we measured approximately a 15
millivolt change in the resting membrane potential. Otsuka
and Ohtsuki (1970) reported a 15 millivolt shift in resting
rat diaphragm muscle membrane potential when the bath potas
sium concentration was changed from 4 mM to 0.5 mM, but saw
no change in twitch tension. If a 15 millivolt shift in
resting potential brought about by a change in the bath
potassium concentration produced no detectable change in the
contractile force, one must reject the hypothesis that a
resting potential shift of 6 millivolts, effected by a change
in the intracellular potassium concentration as a result of
buffer alterations, could be responsible for the observed
changes in muscle contractile force.
A change in the intracellular potassium concentration
might affect muscle contraction by another mechanism. The


71
movement of calcium from the sarcoplasmic reticulum to the
site which triggers muscle contraction is probably coupled
to the movement of ions in the opposite direction. Potassium
and magnesium are the likely candidates for this role since
active transport of calcium into isolated sarcoplasmic retic
ulum has been shown to be coupled to magnesium and potassium
ion transport out of the sarcoplasmic reticulum (Kanazawa
et al. 1971) Morad and Orkand (1971) have suggested
that potassium exchanges for activator calcium during contrac
tion of cardiac muscle from frogs. If during muscle excita
tion intracellular potassium exchanges for calcium across
the sarcoplasmic reticulum, a higher intracellular potassium
concentration may facilitate this calcium movement and
increase the force of the skeletal muscle contraction. This
mechanism could explain why the buffer changes were more
influential on contraction of potassium-deficient rat muscle.
The observed changes in intracellular potassium concentration
were proportionately greater in this tissue than in control
muscle.
The changes in intracellular potassium concentration
might be linked to changes in intracellular sodium concen
tration. Muscles containing less intracellular sodium might
depolarize more rapidly than muscles containing more intra
cellular sodium. The rate of depolarization, which is in
part determined by the driving force for sodium entry into
cells, has been shown to influence skeletal muscle contraction
(Taylor et al., 1972; Sandow, 1973). Unfortunately, it is


72
difficult to obtain reliable data for intracellular sodium
concentrations. A better method to test this hypothesis of
sodium involvement would be to record the action potentials
of muscles in different buffers. Ideally, the action poten
tials and contractions could be recorded simultaneously. A
more elaborate system could be devised to record intracellular
bicarbonate activity (Khuri et al., 1974) and intracellular
potassium activity as well. Experiments having this design
would yield data concerning the time sequence of these events
which would aid in separating cause and effect, important
information that could not be obtained in the present study.
When resting membrane potentials were recorded in mus
cles bathed in different potassium concentrations, a smooth
curve relating potential and potassium distribution was
generated in which resting membrane potential continues to
become more negative as extracellular potassim decreases.
This is consistent with the findings of Otsuka and Ohtsuki
(1970) in potassium-deficient rat diaphragm. Our recorded
potential values compare favorably with the values reported by
these workers. The potentials reported in the present study
were not as great as the potentials the reduced Goldman equation
predicts, using the value of 0.01 for the ratio of the perme
abilities of sodium to potassium. Bilbrey et al. (1973)
reported that resting thigh-muscle potentials, recorded in
vivo in dogs and rats, agreed with the values predicted by
this equation. The values in the present study, recorded


/ o
in vitro, fit. curves described by the reduced Goldman equation
with a higher value for the ratio of sodium to potassium
permeability coefficients.


CONCLUSION
Elevations in buffer bicarbonate concentration or in
buffer pH effected an increase in rat diaphragm muscle
contractility. Elevations in carbon dioxide tension were
accompanied by increases in muscle contractility when buffer
bicarbonate was elevated along with and at the same ratio
as carbon dioxide tension. Elevations in carbon dioxide
tension brought about decreases in muscle contractility in
buffers with a constant bicarbonate concentration. Hemi-
diaphragm muscles from rats made potassium-deficient by
diet showed a greater sensitivity to these buffer changes
than muscles from control rats. Increases in muscle
contractility consistently showed a positive correlation
with increases in intracellular potassium concentration,
but showed no consistent correlation with changes in intra
cellular pH. Changes in contractility and intramuscular
potassium concentration did show a positive correlation
with changes in the ratio of intracellular to extracellular
proton concentration. Buffer changes did not effect any
measurable alterations in resting muscle membrane
potentials. Changes in the extracellular potassium concen
tration, which affected resting potentials, did not affect
muscle contractility.


75
In contrast to results obtained in potassium-deficient
and control rat muscles, contractility of skeletal muscles
in patients with familial hypokalemic periodic paralysis
show an unusual sensitivity to changes in extracellular
potassium concentration. Gordon et al. (1970) proposed that
hypokalemia renders muscle in these individuals inexcitable
by causing a depolarization block. He suggested that depolar
ization results during hypokalemia because of a decrease in
the membrane permeability for potassium. Reductions in
serum bicarbonate and pH, resulting from administration of
ammonium chloride or administration of acetazolamide and
the subsequent renal loss of bicarbonate, prevent muscle
weakness and bouts of hypokalemia (Vroom et al., 1975;
Jarrell et al., 1976).
Reductions in extracellular pH and bicarbonate concen
tration result in improved muscle performance in hypokalemic
periodic paralysis, but result in poorer muscle performance
in rat muscles. However, the effect in both tissues appears to
be related to changes in transmembrane potassium distribution.
The results of the present study predict that both acidosis
and low serum bicarbonate levels would promote a lower
ratio of intracellular to extracellular potassium concentra
tions, but this value has never been determined in hypokalemic
periodic paralysis patients treated with acetazolamide. Whether
acid-base changes benefit victims of periodic paralysis by
effecting a more favorable steady state potassium balance or
by abolishing some other pathological change is not clear.


BIBLIOGRAPHY
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BIOGRAPHICAL SKETCH
David Walter Fretthold was born on February 28, 1948,
in Fairview Park, Ohio. He attended school in Rocky River,
Ohio, graduating from Lutheran High School West in 1966.
His education continued at Miami University in Oxford,
Ohio, where he received the Bachelor of Arts and Science
degree in Zoology in 1970. In 1972 he began graduate study
at the University of Florida, leading to the degree, of
Doctor of Philosophy in the Department of Pharmacology
and Therapeutics.
82


I certify that I have read this study and that in ray
opinion it conforms to acceptable standax'ds of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
L*_X C Gt'L-Oj*
Lai C. Garg, Chairperson
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Betty P, Vogh 0
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
WilTTam R Kem
Assistant Professor of
Pharmacology


rt- t-3 Cu T
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
resentation and is fully adequate, in scope and quality,
s a dissertation for the degree of Doctor of Philosophy.
n 0
>
Philip Posner
Assistant Professor of
Physiology
lis dissertation was submitted to
he College of Medicine and to the
accepted as partial fulfillment of
degree of Doctor of Philosophy.
the Graduate Faculty of
Graduate Council, and was
the requirements for the
March, 1977
Dean, College of Mpdicine
Dean, Graduate School


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THE EFFECT OF ALTERATIONS OF THE COMPONENTS OF THE
BICARBONATE BUFFER SYSTEM ON SKELETAL MUSCLE CONTRACTION,
POTASSIUM CONTENT AND RESTING POTENTIAL
David
By
W. Fretthold
A DISSERTATION PRESENTED TO THE
GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

ACKNOWLEDGMENTS
I wish first and foremost to express my sincere gratitude
to Dr. Lai C. Garg for his help and understanding. His
patience and encouragement has been invaluable to me.
I am also grateful for the advice and attention of Dr.
Maren and the members of my supervisory committee, Dr. Betty
Vogh, Dr. C.Y. Chiou, Dr. William Kem, and Dr. Philip Posner.
I thank Dr. John Munson, Dr. Vernon Senterfitt, and Mr.
Norman Lawrence for generously providing much of the equip¬
ment used in these experiments, and Mr. Butch Landsiedel,
who was a great help in maintaining the experimental animals.
I am also grateful to Ms. Mary Pavlovski, Ms. Joyce
Simon, and Ms. Debbie Werther for help in preparation of
this manuscript.
My sincere thanks is also extended to Ms. Jan Nichols,
and Bills Elmquist and Link for their friendship and cooperation.
Finally, I would like to thank the other members of the
Department of Pharmacology for their good company over the
years.
This research was supported by a traineeship from the
National Institute of Health (GM-00760) and by a research
grant from the National Institute of Health (GM-16934).
li

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES 1V
LIST OF FIGURES v
ABSTRACT vi
INTRODUCTION 1
Objective 1
Background 2
RATIONAL 14
SPECIFIC AIMS 16
MATERIALS AND METHODS 17
Buffer Composition 17
Experimental Animals ..... 18
Rat Hemidiaphragm Preparation 20
Contraction Experiments 21
Intracellular pH and Tissue Cation
Determinations 23
Potential Measurements 27
RESULTS 29
Contraction Studies 29
Tissue Analysis 47
Resting Potential Measurement 57
DISCUSSION 63
CONCLUSION 74
BIBLIOGRAPHY 76
BIOGRAPHICAL, SKETCH 8 2

LIST OF TABLES
TABLE Page
1 BUFFER SERIES 19
2 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
C02 CONCENTRATIONS 50
3 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
hco3~ CONCENTRATIONS 51
4 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN CONSTANT
pH BUFFERS 52
5 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM CONTROL RATS, IN CONSTANT pH BUFFERS . . 53
6 COMPARISON OF ICF ACID-BASE AND EX¬
CHANGES IN HEMIDIAPHRAGMS IN VARIOUS
BUFFERS 54
IV

LIST OF FIGURES
FIGURE Page
1 Effect of d-tubocurarine on directly
and indirectly stimulated hemidiaphragm
muscles 30
2 Contraction of control muscles in 5 mM
potassium as a function of pH 33
3 Contraction of control muscles in 0.5 mM
potassium as a function of pH 35
4 Contraction of potassium-deficient
muscles in 0.5 mM potassium as a
function of pH 37
5 Contraction of control muscles in 5 mM
potassium, constant pH buffers, vs CC>2
volumes percent and HCO^- 39
6 Contraction of control muscles in 0.5 mM
potassium, constant pH buffers, vs CC^
volumes percent and HCO^ ........ 41
7 Contraction of potassium-deficient
muscles in 0.5 mM potassium, constant
pH buffers, vs CO„ volumes percent and
HC03~ 43
8 Effect of alterations in HCOg- and CC>2,
at constant pH, on hemidiaphragm
contraction 55
9 Effect of alterations in HCO3 , CO^,
and potassium concentration on resting
potential of potassium-deficient rat
hemidiaphragm muscles . 58
10 Effect of alterations in HCO^ , CO~,
and potassium concentration on resting
membrane potential of control rat
hemidiaphragm muscles ..... 60
v

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF ALTERATIONS OF THE COMPONENTS OF THE
BICARBONATE BUFFER SYSTEM ON SKELETAL MUSCLE CONTRACTION,
POTASSIUM CONTENT AND RESTING POTENTIAL
By
David W. Fretthold
Chairperson: Dr. Lai C. Garg
Department: Pharmacology and Therapeutics
The purpose of this investigation was to examine the
effect of alterations in bicarbonate concentration and
carbon dioxide tension on skeletal muscle contractility and
to elucidate a mechanism for these effects. Hemidiaphragm
muscles from control rats and rats fed potassium-deficient
chow were examined. Particular attention was focused on
this potassium-depleted muscle because it has been reported
to exhibit some of the same properties as muscle in patients
suffering from the hereditary disease hypokalemic periodic
paralysis. Patients with this disease are protected from
attacks of skeletal muscle paralysis by chronic doses of
ammonium chloride or carbonic anhydrase inhibitors, agents
that produce alterations in the bicarbonate buffer system.
The in vitro situation offers the opportunity to control
carefully all three elements of the buffer system: pH, bicar¬
bonate, and carbon dioxide tension. Therefore, all muscle
vi

studies were conducted .in vitro. One of these, three elements
was kept constant while the other two were systematically
varied.
When pH was allowed to vary, stimulated muscles con¬
tracted with more force in high pH buffers than in low pH
buffers. This was true when the pH change was effected by
an elevated buffer bicarbonate concentration or by a reduced
carbon dioxide tension. This effect was always more dramatic
in muscles taken from potassium-depleted animals. With these
muscles, a change in pH from 7.7 to 7.1 was accompanied by
a 65% reduction in contractile force. This effect showed a
rapid onset, was reversible, persisted until the buffer
composition was changed, and was independent of synaptic
transmission. When pH was kept constant but buffer bicar¬
bonate and carbon dioxide tension were increased together
at the same ratio, there was an accompanying increase in
muscle contractile force. This relationship was also Bore
pronounced in potassium-deficient rat muscles.
Intracellular pH and intracellular potassium deter¬
minations were performed on diaphragm muscles that had been
contracting in different bicarbonate buffers. Radio-labeled
dimethyloxazolidinedione (DMO) v/as used for these intracellu¬
lar pH determinations. Muscles bathed in acidic buffers
were found to have lower intracellular pH levels and lower
intracellular potassium concentrations than muscles bathed
in more alkaline buffers. Analysis of muscles bathed in
high bicarbonate, high carbon dioxide buffer revealed that
these muscles had a lower intracellular pH and higher
vii

potassium content than muscles bathed in low bicarbonate,
low carbon dioxide buffer, although both buffers had the
same pH.
Resting membrane potentials, recorded in diaphragm
muscles bathed in different bicarbonate buffers, were not
affected by changes in buffer bicarbonate concentration
or buffer carbon dioxide tension. Changes in the buffer
potassium concentration over ranges which did not affect
contractile response were found to alter the recorded
resting potentials.
The changes in contractility showed a positive corre¬
lation with the intracellular muscle potassium concentration
and with the ratio of intracellular to extracellular proton
concentrations. Changes in muscle contractility did not
correlate with changes in intramuscular pH or with changes
in the resting membrane potential. It was speculated that
the benefits which carbonic anhydrase inhibitors afford
victims of hypokalemic periodic paralysis may be derived
from a more favorable distribution of potassium which results
from reductions in serum bicarbonate as well as the accom¬
panying acidosis.
vm

INTRODUCTION
Objective
Patients suffering from the disease familial hypokal¬
emic periodic paralysis are protected from attacks of muscle
weakness by acetazolamide, a potent carbonic anhydrase in¬
hibitor (Resnick et al., 1968). It is paradoxical that a
diuretic which causes potassium loss can prevent the attacks
of muscle weakness which are associated with a fall in serum
potassium. Since no carbonic anhydrase has been found in
muscle (Maren, 1967), it seems unlikely that the benefits
afforded victims of this malady are derived from action of
the drug directly on muscle. Griggs (1970) suggested that
metabolic acidosis, brought about by inhibition of renal
carbonic anhydrase, was responsible for the beneficial
effects of this drug. Jarrell et al. (1976) demonstrated
that patients given acidifying doses of ammonium chloride
also were protected from periodic attacks. Further,
Viskoper et al. (1973) were able to precipitate an attack
of paralysis in a patient by infusing sodium bicarbonate which
produced a metabolic alkalosis. Treatment v/ith acetazolamide
appeiirs to prevent hypokalemia and muscle paralysis as a
consequence of alterations effected in the bicarbonate buffering
system. These alterations include not only a decrease in the
plasma pH, but also decreases in plasma bicarbonate and carbon
1

2
dioxide concentrations. Precisely how this new acid-base
equilibrium prevents attacks of periodic paralysis is not
clear. Therefore, the objective of this investigation is to
determine in vitro the influence of changes in components of
the bicarbonate buffer system on skeletal muscle contraction,
potassium content, pH, and resting membrane potential.
Background
Bicarbonate Buffer System
The bicarbonate buffer system is quantitatively the
most important extracellular buffer in the body and is unique
because of its intimate link with carbon dioxide. Although
the ionization constant (pKa) for the first proton dissoci¬
ation of carbonic acid is 3.6, the buffer system has a
considerable capacity at physiological pH because of the
constant 400:1 ratio of dissolved carbon dioxide to carbonic
acid. The Henderson-Hasselbalch equation can be modified,
substituting the concentration of dissolved carbon dioxide
divided by 400 for the carbonic acid concentration. Rear¬
ranging the terms yields the familiar form of the equation
which expresses the pH as a function of bicarbonate concen¬
tration and dissolved carbon dioxide, with an apparent pKa
value of 6.1 (Hills, 1973); pH=6.1+log • In vivo, regula¬
tory mechanisms control the ventilation rate and consequently
the carbon dioxide concentration in blood, thus controlling the

3
concentration of the proton donor, carbonic acid. Extracel¬
lular pH is maintained between 7.1 and 7.6, the range generally
considered compatible with life. Even during strenuous
exercise, when the carbon dioxide evolution rate can increase
to as much as thirty times the rate at rest, extracellular
pH falls no more than 0.1 unit.
Relation Between Intracellular and Extracellular pH and
Bicarbonate of Muscle Cells
A variety of techniques have been employed to determine
the intracellular pH of muscle. Early methods used to measure
intracellular pH and the theoretical and practical diffi¬
culties associated with these methods are discussed in a review
by Caldwell (1956). Waddell and Butler (1959) first used the
weak acid, dimethyloxazolidinedione (DMO), a metabolite of the
antiepileptic agent trimethadione, to measure the intracellular
pH of dog muscles in vivo. Using bicarbonate-specific micro¬
electrodes, Khuri et al. (1974, 1976) determined the intra¬
cellular bicarbonate activity of skeletal muscles, and calcu¬
lated equilibrium potentials for bicarbonate of between -20
millivolts and -44 millivolts under different conditions in
r intracellular pH to be between 6.6 and 7.4, and have concluded
that proton and bicarbonate gradients must exist across muscle
cell membranes which are not in Nernstian equilibrium with the
resting muscle membrane potential, although the membrane is
permeable to Lhese ions (Adler et al., 1965). In order for such
gradients to be maintained, either hydrogen ions would have to

4
be transported out of cells or bicarbonate ions would have to
be transported into cells (Adler et al., 1972; Heisler, 1975;
Khuri et al-, 1974; Lai et al., 1973).
Numerous investigations have been undertaken to determine
the relation of intracellular pH to changes in extracel¬
lular pH. This was the objective of Wfiddell and Butler (1959)
in their DMO study in dogs. They altered the blood carbon
dioxide tension and bicarbonate concentration and concluded,
from skeletal muscle biopsies, that the greatest changes they
observed in intracellular pH resulted from changes in carbon
dioxide tension. Irvine and Dow (1966) found that metabolic
acidosis in rats had no effect on skeletal muscle .intracel¬
lular pH. Kim and Brown (1968) showed that skeletal muscle
intracellular pH in dogs fell when carbon dioxide tension
was increased along with bicarbonate concentration, and rose
when animals were hyperventilated during HC1 infusion, even
though the plasma pH remained constant. Similar results have
been obtained in vitro; respiratory acidosis produced larger
alterations in muscle; intracellular pH than metabolic acidosis
(Heisler, 1975; Adler et al., 1965). Both these authors
found that rat diaphragm muscles were most effectively buffered
when extracellular pH ranged between 7.1 and 7.4. They
reported that when extracellular pH varied from 7.1 to 7.4
during simulated respiratory acidosis or alkalosis, intra¬
cellular pH, measured with DMO, varied from 7.0 to about 7.28.
Outside this extracellular pH range, intracellular pH changed
about as much as extracellular pH.

5
Electrolyte Shifts and Acid-Base Disturbances
A close relationship exists between tissue and plasma
electrolyte concentrations and acid-base balance. Potassium
depletion results in increases in serum bicarbonate (Heppel,
1939), whereas sodium depletion leads to a fall in serum
bicarbonate (Darrow et al., 1948). Acidosis produces ele¬
vations in total body potassium, and decreases in total body
sodium (Cooke et al., 1952). All of the above studies were
chronic experiments involving chronic alterations in diet and
acid-base balance and the changes largely result from renal
electrolyte regulation. A reciprocal relation exists for
hydrogen and potassium ion excretion in the distal tubule,
and reabsorption of bicarbonate in the proximal tubule is
coupled to the reabsorption of sodium (Pitts, 1963).
In a series of experiments on nephrectomized dogs,
Pitts and co-workers (1963) measured serum ion concentra¬
tions and demonstrated that considerable ion exchange
occurs between intracellular and extracellular fluid in
acute episodes of respiratory and metabolic acidosis and
alkalosis. Sodium and potassium exchange for protons, and
chloride exchanges for bicarbonate. Other authors have
analyzed muscle taken from animals during acid-base distur¬
bances and have noted positive correlations between hydrogen
and potassium ion gradients across muscle membranes (Brown
and Goott, 1963; Grantham and Schloerb, 1965; Irvine and Dow,
1966). In various types of acid-base alterations, intra¬
muscular potassium concentration changed in the direction
which tended to keep the transmembrane ratios of potassium

6
and hydrogen ions equal, although true equality was never
attained (Brown and Goott, 1963). In vitro experiments on
rat diaphragms by Adler et al. (1965) and on frog muscles by
Fenn and Cobb (1934) have shown a positive correlation between
changes in intramuscular potassium and intramuscular pH.
In vitro potassium depletion in rat diaphragm has been shown
to lower intracellular pH (Adler et al., 1972). Skeletal
muscles depleted of potassium contain elevated concentrations
of sodium (Cooke et al., 1952).
Mechanism of Ion Exchange.
Isotope exchange studies with isolated frog skeletal
muscle have shown that a decrease in extracellular pH causes
an increase in the potassium efflux rate, a decrease in the
potassium uptake rate, and a bi-directional depression of
sodium exchange (Voile, 1972). This type of membrane beha¬
vior could explain the potassium shift seen in acidosis
(Voile, 1972). Keynes and Swan (1959) reported that sodium
efflux from isolated frog muscle varied approximately with
the cube of the internal sodium concentration. Keynes (1965)
found that sodium efflux depended on the square of the internal
sodium concentration in acidosis which was produced by an
increase in carbon dioxide tension. Whether these observa¬
tions represent interactions of ion pumps, substitution of
protons for other cations, or suppression of pumping activity
is not known.

7
Muscle Cell Potential and pH, Bicarbonate Concentration,
and Carbon Dioxide Tension
Resting muscle membrane potential depends on the ion
distribution across the membrane and the membrane permeability
to these ions. The Goldman equation describes this relation¬
ship quantitatively:
(Cl )
o
E
m
(Cl )±
where Em is membrane potential, P is the membrane permeabil¬
ity coefficient for a particular ion, R is the universal gas
constant, T is temperature in degrees kelvin, and F is Fara¬
day's constant (Hodgkin and Horowicz, 1959). In skeletal
muscle, chloride is distributed in agreement with Em, and the
contribution of the term PNa(Na is small compared to the
contribution of the term P^(K+).. Therefore, these terms
may be neglected without appreciably altering the validity
of the equation (Bilbrey et al., 1973). Bilbrey et al. (1973)
found that resting muscle potentials, recorded in vivo in
dogs and rats, agreed with this equation when 0.01 was used
for the ratio of the permeability coefficients of sodium to
potassium. Any factor which influences a change in any of
the parameters in the equation would affect the membrane
potential. Studies on the effect of pH and carbon dioxide
tension on transmembrane potential have yielded conflicting
results. In generalf an increase in carbon dioxide tension
has been shown to cause a decrease in Em when Em is initially
high and an increase when Em is initially low (Shanes, 1958).

8
In vivo studies by Williams et al. (1971) showed that a fall
in blood pH induced by an elevation in pC02 causes a slow
depolarization of skeletal muscle in rat. This depolarization
could be explained by the observed redistribution of ions,
principally an elevation in plasma potassium. Iri vitro alter¬
ations in pH have been shown to affect membrane potential in
frog muscle; the effect has been attributed to a decreased
chloride permeability in acidosis (Brooks and Hutter, 1962;
Hutter and Warner, 1967); when the external potassium concen¬
tration was 2.5 mM, acidosis hyperpolarized isolated frog
skeletal muscle by a few millivolts, but when the external
potassium concentrations was elevated to 10 mM, acidosis
depolarized the muscle by a few millivolts (Mainwood and Lee,
1967). It has also been reported in rat skeletal muscle that
bicarbonate has a permeability coefficient approximately 0.1
that of chloride (Hugeunin, 1975).
Muscle Contraction
The first in a series of events leading to muscle con¬
traction is usually the propagation of an action potential
to the pre-synaptic ending of the motor nerve, and the sub¬
sequent release of acetylcholine from the nerve ending.
Acetylcholine diffuses across the synaptic cleft and inter¬
acts with the post-synaptic membrane on the muscle, causing
a temporary local increase in permeability to sodium and
potassium. As a result of this permeability increase, the

9
membrane becomes locally depolarized. If the muscle membrane
depolarizes beyond a critical threshold, a propagated action
potential results. Depolarization of the muscle membrane
initiates the liberation of calcium from sarcoplasmic retic¬
ulum, the primary source of activator calcium in skeletal
muscle. Movement of calcium to the contractile apparatus of
the muscle links the excitation process to the contractile
process. At low intramuscular calcium concentrations (less
— 6
than 10 M), the muscle fiber protein, actin, is bound to
the regulator proteins, tropomyosin and troponin, and actin
remains in an inactive state. After calcium release, calcium
binds to troponin, and actin complexes with myosin, the other
primary contractile protein. Several subsequent steps result
in the hydrolysis of ATP by myosin ATPase and the shortening
of the sarcomere. Calcium is then actively pumped back into
the sarcoplasmic reticulum, and the formation of actin-myosin
contractile complexes is inhibited and contraction ceases.
For a more complete treatment of this subject, see Tonomura
(1973) and V7eber and Murray (1973).
Effect of Acidosis on Muscle Contraction
As an interesting corollary of studies on the effect of
pH on the neuromuscular blocking agents, Baraka (1964) and
Crul-Sluijter and Crul (1974) observed a slight depressive
effect of acidosis alone on skeletal muscle twitch response.
Baraka (1964) conducted his experiments on humans i_n vivo and
interpretation of his results is complicated because acidosis
also effects elevations in serum sodium and potassium

10
concentrations and in levels of circulating epinephrine and
norepinephrine (Sechzer et al., 1960). However, experiments
by Crul-Sluijter and Crul (1974) with in vitro rat diaphragm
preparations show that muscles contract with slightly less
force when the bath fluid is made acidic by decreasing the
bicarbonate concentration. The mechanism of this effect
has not been determined, and is is not clear whether the
depression is dependent on the change in pH or the change in
the bicarbonate concentration.
Potassium Deficiency with Reference to Skeletal Muscle
Potassium deficiency is manifested differently in dif¬
ferent species. In both dog and rat, the decrease in intra¬
muscular potassium concentration is relatively less than the
decrease in extracellular potassium concentration (Bilbrey et
al. , 1973). In man, serum potassium concentrations may remain
near normal despite severe potassium loss from muscles (Rettori
et al., 1972). Potassium deficiency in rats causes character¬
istic myocardial and renal damage (Welt et al., 1960), but no
skeletal muscle paralysis develops, even in severe potassium
deficiency (Smith et al., 1950). Resting muscle membrane
potentials in potassium-deficient rats were found to be in
agreement with values predicted by the Goldman equation (Bilbrey
et al. , 1973). In both dog and man, progressive skeletal
muscle weakness and structural degeneration develop (Chanpion
et al., 1972; Smith et al. , 1950). Bilbrey et al. (1973) have
attributed muscle weakness in severely potassium-deficient
dogs to an abnormally low resting membrane potential. They

11
suggested that these low resting potentials resulted because
the membrane permeability to sodium relative to the permeability
to potassium had increased, although they did not test this
hypothesis. Potassium release from exercised muscle, which
is thought to mediate increased local blood flow, has been
demonstrated to be severely reduced in potassium-deficient
dog (Knochel and Schle.in, 1972). Muscle necrosis and associ¬
ated myoglobinuria following exercise in potassium-depleted
dog and man have been attributed to failure of these muscles
to show the normal hyperemic response to exercise.
Muscle Paralysis and Potassium Distribution
There are several conditions in which transitory muscular
paralysis is associated with abnormal potassium distribution.
Two such kinds of paralysis seen in humans are hyperkalemic
familial periodic paralysis, also known as adynamia episódica
hereditaria, and hypokalemic periodic paralysis. In hyper¬
kalemic periodic paralysis, attacks of muscle weakness are
associated with elevations in serum potassium (Gamstorp et al.,
1957). Acetazolamide and hydrochlorothiazide are used to
control this disease, presumably acting by lowering the serum
potassium through kaluresis (McArdle, 1969).
Acetazolamide, a potent carbonic anhydrase inhibitor and
potassium wasting diuretic, has been found to be the most effec¬
tive treatment available for hypokalemic periodic paralysis
(Griggs et al., 1970). In hypokalemic periodic paralysis,
attacks are associated with a fall in the serum potassium
concentration (Biemond and Daniels, 1934). During attacks,

12
potassium has been shown to move from serum into skeletal
muscle (Ionasescu and Merculiev, 1962) , and serum potassium
concentrations fall to 2.5 mM or lower (Vroorn et al., 1975).
During attacks there is a depression of muscle electrical
activity (Viskoper et al., 1973) and muscles have been reported
to be depolarized rather than hyperpolarized as would be
expected on the basis of the abnormal potassium distribution
(Shy et al., 1961; Creutzfeldt et al., 1963). These obser¬
vations are consistent with the theory that muscles are paral¬
yzed by a depolarization block resulting because of a decrease
in the potassium permeability of the muscle membrane. The
sodium contribution to the resting potential becomes relatively
more important, and depolarization results. During depolar¬
ization, the muscle membrane sodium-potassium ATPase activity
increases and the serum potassium concentration continues to
fall (Otsuka and Ohtsuki, 1970). Although increasing the
extracellular fluid potassium concentration might be expected
to further depolarize the muscle membranes, potassium chloride
infusion terminates the paralysis (Aitken et al., 1937).
Diaphragm muscles isolated from potassium-deficient rats
have several characteristics in common with muscle in hypo¬
kalemic periodic paralysis patients, although these rats do
not suffer paralysis in vivo (Smith et. al., 1950). Muscles
in periodic paralysis patients and in potassium-deficient
rats contain elevated intracellular sodium and reduced intra¬
cellular potassium (Hoffmann and Smith, 1970; Offerijns et
al., 1958). Offerijns et al. (1958) reported that insulin
caused flacid paralysis in the hemidiaphragm preparation in

13
conjunction with low potassium in the buffering medium.
Insulin, which lowers serum potassium more in hypokalemic
periodic paralysis patients than in normal persons, also
induces severe attacks of paralysis in these patients (Vroom
et al., 1975), although it is not clear whether insulin is
essential in all episodes. In potassium-deficient rat diaphragm
muscle paralysis, as in hypokalemic periodic paralysis, the
resting muscles have been reported to be depolarized while
the extracellular potassium concentration remains reduced;
paradoxically normal contractile function could be restored
in both cases by elevating the extracellular potassium
concentration (Gordon et al., 1970; Ohtsuka and Ohtsuki, 1970).

RATIONALE
Changes in pH, with accompanying changes in either
carbon dioxide tension or bicarbonate concentration, affect
several important, skeletal muscle properties. These proper¬
ties include ion distribution, cell permeability, cell poten¬
tial, and contractile force. Each of these factors plays a
vital role in muscle performance, but the influence of the
bicarbonate buffer system on these properties has never been
extensively investigated. Therefore, we measured skeletal
muscle contractile force, cation composition, and resting
membrane potential in in vitro experiments using three dif¬
ferent bicarbonate buffer series. One of these series had
buffers with different carbon dioxide concentrations, simu¬
lating the changes seen in respiratory acid-base disturbances.
One series had buffers with different bicarbonate concentra¬
tions, simulating metabolic acid-base disturbances. The
third series had buffers with different concentrations of
bicarbonate equilibrated with different carbon dioxide
mixtures, but with the same ratio of HCO^iCC^, and hence,
with the same pH. Through this approach we hoped to deter¬
mine whether bicarbonate and carbon dioxide have any specific
effects on skeletal muscle apart from changes in pH.
Rat diaphragm muscle from both normal and potassium-
depleted rats was studied in these experiments. Potassium-
14

15
deficient rat diaphragm muscle was selected for investigation
since it previously has been shown to be similar in many
respects to skeletal muscle of hypokalemic periodic paralysis
patients. Further, the rat diaphragm muscle is less than on
millimeter thick, and can easily be oxygenated in vitro. The
main objective was to determine whether potassium-depleted rat
muscle shares with hypokalemic periodic paralysis muscle a
sensitivity to changes in the bicarbonate buffer system. If
such a bicarbonate-carbon dioxide sensitivity were found, per¬
haps an investigation of the mechanism would provide a better
understanding of the influence of acid-base balance upon skeletal
muscle and aid in elucidation of the effect of carbonic anhydrase
inhibitors upon hypokalemic periodic paralysis.

SPECIFIC AIMS
The specific aims of this investigation are to determine
in both normal and potassium-depleted rat muscle:
1. the effect of alterations in bicarbonate concentration,
carbon dioxide tension and pH on contractility
2. the effect of alterations in bicarbonate concentration,
carbon dioxide tension, and pH on muscle potassium con¬
tent
3. the effect of alterations in bicarbonate concentration,
carbon dioxide tension, and pH on muscle intracellular
PH
4. the effect of alterations in bicarbonate concentration,
resting carbon dioxide tension, and pH on membrane
potential
5. the effect of variations in the extracellular potassium
concentration on resting muscle membrane potential
16

MATERIALS AND METHODS
Buffer Composition
Muscle experiments were performed in Tyrode solution of
the following composition; NaCl, 126 mM; KC1, 5 mM; CaCl2,
3 mM; MgCl2, 1-6 mM; dextrose, 11.1 mM; Na2HPO^, 0.677 mM;
NaII2P0^, 0.17 2 mM; and NallCO^, 16 mM. Low bicarbonate solu¬
tions were made by substituting 8 millimoles per liter of
sodium chloride for 8 millimoles per liter of sodium bicar¬
bonate. High bicarbonate solutions were made by substituting
16 millimoles per liter of sodium bicarbonate for 16 milli¬
moles per liter of sodium chloride. In one series of
experiments, the sodium chloride concentration was kept con¬
stant at 118 mM, and 0.5 millimoles sodium sulfate plus 0.5
millimoles sucrose were substituted for each millimole of
sodium bicarbonate in reduced bicarbonate solutions. Low
potassium solutions contained 0.5 mM potassium chloride plus
an additional 4.5 millimoles per liter of sodium chloride.
Elevated potassium solutions were prepared by substituting
potassium chloride millimole for millimole for sodium chloride.
Solutions were gassed with mixtures of oxygen and carbon
dioxide. The gas mixtures contained oxygen with either 2.5%,
5%, or 10% carbon dioxide by volume. The mixture with 2.5%
carbon dioxide was obtained from and certified by Matheson
17

18
Products, and the mixtures with 5% and 10% carbon dioxide
v?ere obtained from Liquid Air Company.
Muscle experiments were performed in one of the three
series of buffers illustrated in Table 1. The pH of the buffer
solutions was measured near the tissue site in all experi¬
ments. The pH was measured with an Arthur Thomas Company
combination electrode and a Beckman pH meter. It was neces¬
sary to carefully regulate the bubble rate of the gas, and
to compensate for cylinder variations in gas mixtures by
varying the bicarbonate concentrations slightly when dealing
with the constant pH buffers (series three buffers). Bicar¬
bonate concentrations other than those shown in Table 1 were
used, although none of these values varied more than 25% from
the values expressed in the table. The bicarbonate concen¬
trations illustrated in Table 1 represent approximate concen¬
trations as well as approximate carbon dioxide volumes percent
of the gases.
Experimental Animals
Male Sprague-Dawley rats, 100-150 g, obtained from Flow
Labs, were housed individually in wire cages. They were fed
potassium-deficient rat chow, completely supplemented with
vitamins and minerals, obtained from Nutritional Biochemicals.
Rats were maintained on this diet for 5 to 6 weeks prior to
the experiment. At the time of the experiment, these rats
weighed between 200 g ¿uid 275 g. Male Sprague-Dawley rats,
weighing 200-275 g, obtained from Flow Labs, were used as
control rats. These animals were fed standard rat chow.

19
TABLE 1
BUFFER
SERIES
HC03 / CO2
(mM)/(Vol. %)
A
B
C
SERIES 1 - Constant HC03“
(respiratory changes)
16/2.5
16/5
16/10
SERIES 2 - Constant CC>2
(metabolic changes)
8/5
16/5
32/5
SERIES 3 - Constant pH
(compensated changes)
8/2.5
16/5
32/10

20
Rat Hemldiaphragm Preparation
The rat phrenic nerve hemidiaphragm in vitro preparation
*
was originally described by Biilbring (1946) . This tissue was
chosen for these experiments because it is a thin skeletcil
muscle which is easily oxygenated and supplied with nutrients.
The preparation is stable over several hours if left undisturbed.
It contains mostly fast muscle fibers and can be dissected
out with the phrenic nerve for indirect stimulation or without
the nerve for direct stimulation.
Surgery was performed on rats during ether anesthesia.
The abdominal cavity was opened by a ventral incision and the
intestines were pushed aside to expose the dorsal aorta.
Arteria], blood was drawn into a heparinized syringe and plasma
was separated by centrifugation for sodium and potassium
determinations, performed on an Instrumentation Laboratory
343 flame photometer, against a lithium standard. Animals
were then decapitated and the skin removed from the chest.
The thoracic wall was cut to expose the rostral surface of
the diaphragm and the phrenic nerves. When the phrenic nerve
was to be isolated with the hemidiaphragm, it was tied close
to the thymus and cut, then carefully dissected away from
any attaching tissues. The diaphragm was cut down the mid¬
line, and the ribs and tissues connecting each hemidiaphragm
were cut away. The hemidiaphrágms were then removed from the
animal and placed in oxygenated Tyrode solution where the
remainder of the surgery was performed. Extraneous tissues
were then cut away from the hemidiaphragm leaving a section

21
of the ribs attached to the muscle tendons. A long piece of
silk thread was tied to the central tendon and two loops of
silk thread were tied to each edge of the rib segment. For
direct stimulation, two teflon-coated stainless steel wire
electrodes were tied to the muscle. The preparation was
mounted by the two loops to a stationary acrylic hook at
the bottom of a cylindrical glass bath containing 45 ml of
Tyrode solution bubbled with the appropriate gas mixture at
37° C. The silk thread attached to the central tendon was
tied to a Myograph-B transducer at one gram of resting tension.
For indirect stimulation experiments, the phrenic nerve was
passed through two silver ring electrodes imbedded in insulating
dental acrylic. Muscles were stimulated with slightly more
than enough voltage to elicit a maximum contractile response.
The muscles were stimulated at five second intervals, with
square wave monophasic pulses, for 5 milliseconds duration
for direct stimulation or for 0.5 milliseconds for indirect
stimulation. Contractions were recorded on a Narco Bio-Systems
Physiograph equipped with a low-pass filter set at 10 cycles
per second.
Contraction Experiments
Experiments were performed on paired hemidiaphragms from
the same rat in the same series of buffers (see Table 1) . In
contraction experiments, both hemidiaphragms were first equi¬
librated and stimulated in Tyrode solution containing 16 mM

22
bicarbonate bubbled with 95% oxygen, and 5% carbon dioxide.
During this stabilization period the buffer was changed every
twenty minutes. The stabilization period lasted until a
stable base line and a stable peak contraction height were
obtained, usually about one hour. Subsequently, one of the
paired hercidiaphragms was bathed and stimulated first in
buffer A, then buffer B, then buffer C, then again in buffer
A, while the companion hemidiaphragm was bathed and stimulated
first in C, then in B, then in A, then again in C (see Table
1). Muscles remained in each buffer for 10 minutes, then
the bath was drained and fresh buffer of the same type was
added for 20 minutes. Thus the contraction experiments lasted
2 hours after the stabilization period. If a muscle showed
signs of deterioration, that muscle was eliminated from
consideration. Contractile responses on the physiograph
trace were measured 15 minutes after each of the four buffer
changes. The first and the last contractile responses were
averaged to obtain one value for a response in that buffer.
For statistical comparison, individual muscle contractions
in each buffer were expressed as a percent of the maximum stable
contraction recorded for that hemidiaphragm, at any time.
Diaphragms from potassium-deficient rats were studied
in all three series of buffers in Tyrode solution containing
0.5 inM potassium. These muscles were stimulated directly
with stainless steel electrodes. Potassium-deficient rat
diaphragms were also examined in series three, (constant pH
buffers, containing 0.5 mM potassium) stimulated indirectly
via the phrenic nerve. Diaphragms from control rats were

studied in all three series of buffers. Hemidiaphragms were
stimulated directly in these experiments which were conducted
in both 5 raM potassium Tyrode solution and in 0.5 inM potassium
Tyroae solution.
The effect of d-tubocurarine on directly stimulated and
indirectly stimulated muscle response was examined in several
pairs of hemidiaphragms. The paralyzing effect of regular
insulin (Lilly) on potassium-deficient diaphragm muscle was
also tested at the end of several contraction experiments.
Intracellular pH and Tissue Cation Determinations
Intracellular pH was determined on isolated rat hemi¬
diaphragms according to the method of Adler et al. (1965).
Paired hemidiaphragms were isolated, mounted, and stimulated
in the manner described in the preceding section. Muscles
were equilibrated for at least one hour in 16 mM bicarbonate
buffer, bubbled with a 95% oxygen, 5% carbon dioxide gas
mixture. After this equilibration period, buffer A of a
particular series was placed in the bath containing one
hemidiaphragm and buffer C of that same series was added to
the bath containing the companion hemidiaphragm. Fresh
buffer was supplied after 10 minutes. After 20 minutes,
the buffers were switched, and contractions were recorded
for 30 minutes; the buffer was renewed after 10 minutes,
as usual. After 30 minutes, these same buffers containing
3
200 microcunes per liter of H labeled mannitol and 50
14
microcunes per liter of C labeled DMO, both obtained
from New England Nuclear, were added to the appropriate organ

baths. The purity of these radiolabeled compounds was
verified with paper chromatography. These buffers were
renewed after 30 minutes. After 30 minutes 2 ml samples of
the bath solutions were taken for analysis and the experiment
was terminated. Thus each muscle was equilibrated and stimu¬
lated for one hour in buffer E, then for 1/2 hour in either
buffer A or C, then for 1 1/2 hours in either C or B, so that
each muscle was tested in buffers A, B, and C, one ending in
buffer C and one ending buffer A. During the last hour,
each buffer contained radiolabeled DM0 and radiolabeled
mannitol. If either hemidiaphragm showed signs of deteriora¬
tion, the experiment was terminated and the results discarded.
After completion of the contraction phase of the experi¬
ment, the muscles were removed and cut away from the attaching
ribs. The tendon v/as cut away and each muscle was cut into
two parts. One piece of each muscle v/as placed into a tared
polyethylene vial. Both vessels were weighed. The piece of
muscle in the crucible was dried overnight at 100° C and
reweighed, then ashed overnight at 600° C in a muffle oven.
The ash v/as then dissolved in concentrated HCl and the calcium
content of the ash v/as determined on a Perkin-Elmer atomic
absorption spectrometer against a lanthanum standard. Distil¬
led water, 100 times the weight of the tissue, was added to
the polyethylene vial to leach out the ions and the isotopi-
cally labeled DMO and mannitol (Lipicky and Bryant, 1966).
After an overnight soak, the sodium and potassium concentrations

25
14 3 ...
and the C and H activities of the fluid was determined.
Sodium and potassium determinations were performed on an
Instrumentation Laboratory 343 flame photometer against a
lithium standard. Radioactivity of the solutions was
determined in a Beckman model LS 200 liquid scintillation
counter. The buffers were analyzed for radioactivity and
3
sodium and potassium content m a similar manner. The H
14
counts were corrected for C spectral overlap according to
the formula = R. - (E x A ) where A. is the corrected
tic t
3 . 3
H counts per minute, R^ is the uncorrected h counts per
minute over background, E is the counting efficiency of
14 3
a pure ' C sample on the H channel relative to the counting
efficiency of that same pure sample on the 14c channel, and
A^ is the counts per minute over background of the mixed
14
sample on the C channel. The extracellular space in ml of
each muscle was estimated as the mannitol space of that
muscle, determined by the formula:
counts per minute of that muscle
n counts per minute of 1 ml of medium
The intracellular space was estimated as the tissue water
weight lost in drying minus the estimated extracellular water
weight. Intracellular sodium and potassium contents were
determined from the sodium and potassium concentration of the
solutions in which the muscles had been soaked, minus the
sodium and potassium that was calculated to have been contri¬
buted by extracellular fluid. Intracellular pH was calculated
from the formula:

26
/C. (V. + V ) V . „ „ .
pH± - p.Ka + log(ó^ ^ 2 x [10(p~ ~ pHe + 1] -
\ e i i
where pK is the ionization constant of DMO (6.13), C is the
a t
concentration of DMO in the water of the entire tissue, C
e
is the concentration of DMO in the extracellular fluid, V
e
is the volume of the extracellular fluid, V. is the volume
i
of the intracellular fluid, and pHg is the extracellular pH
of the buffering medium (Waddell and Butler, 1959).
The mean intracellular muscle bicarbonate concentrations
were calculated according to the Henderson-Hasselbalch equa¬
tion, assuming an apparent pK value of 6.13 for carbonic
3.
acid in muscle. The solubility coefficient of carbon dioxide
in intracellular fluid was taken to be 0.035 millimoles per
liter per millimeter mercury pressure (Brown and Goott, 1963) .
The partial pressure of carbon dioxide in the buffer was
determined from the measured pH and the Henderson-Hasselbalch
equation, using 0.0241 millimoles per liter per millimeter
of mercury pressure for the solubility coefficient of carbon
dioxide in the buffer (Edsall and Wyman, 1958).
Intracellular pH and cation determinations were made on
diaphragm muscle from potassium-deficient rats in all three
series of buffers in 0.5 mM potassium. Similar determina¬
tions were made on muscle from control rats in series 3,
constant pH buffers in 5 mM potassium. Contractile responses
were measured at the end of each experiment and expressed as
a percent of the maximum response recorded from that muscle.

27
Potential Measurements
Diaphragms were rapidly dissected out of rats as
previously described. Strips of hemidiaphragm were placed
in plexiglass beaker containing 16 mM bicarbonate buffer
bubbled with 95% oxygen, 5% carbon dioxide. After 30
minutes, the strips were transferred to a 10 ml plexiglass
chamber where they were perfused with Tyrode solution at a
rate of approximately 10 ml per minute. Solutions were kept
at 35° C and were bubbled with the appropriate gas mixture.
The muscle stri.ps were pinned by the adjoining tendons to a
silicone-resin mat at the bottom of the chamber. Resting
membrane potentials we re recorded v/ith standard microelectrode
techniques, using glass micropipettes filled with 3 M potas¬
sium chloride. The electrodes had a tip resistance of 5-10
megohms. Prepulled microelectrodes with a 1 micron tip con¬
taining a glass fiber were obtained from Fredrick Haer and
Company. These electrodes were filled by capillary action,
and examined under a microscope. Potential signals were
amplified on an Electronics for Life Science wide band elec¬
trometer equipped with a circuit for monitoring the electrode
tip resistance. The signal from the electrometer was displayed
on a Tektronic type 564B oscilloscope. The criteria followed
for judging whether a potential was a valid resting potential
were:
1. a resting potential had to be more negative than
-40 millivolts
2. a resting potential had to be stable within 5
millivolts for one minute

28
Potential measurements were made on potassium-deficient
and control diaphragm muscles bathed in buffers from series
1 and 2, containing 0.5 mM potassium. After the muscles
were transferred to the chambers, the chambers were perfused
with either buffer A or C of the series. After 20 minutes,
6 resting potentials were recorded. The chambers were then
perfused with the other buffer of that series, and after 20
minutes, 6 potentials were again recorded.
Resting potentials of diaphragm muscles from control and
from potassium-deficient rats were also determined in 16 mM
bicarbonate buffer bubbled with 95% oxygen, 5% carbon dioxide,
but containing different concentrations of potassium. These
potassium concentrations were 20 mM, 10 mil, 5 mi-1, 2.5 mM,
and 1.25 mM. Equilibration time in these experiments was
5 minutes, since it was reasoned that the extracellular fluid
potassium will equilibrate in this time. Six potentials
were recorded in each muscle at each potassium concentration.
To eliminate bias during the potential recording process,
the buffers were presented to the investigator in a sequence
unknown to him. The buffer sequence was revealed after
completion of the experiment.

RESULTS
Contraction Studies
Action of d-tubocurarine on Directly and Indirectly Stimu¬
lated Muscle
When hemidiaphragms were stimulated directly or in¬
directly, a stable base line and peak contraction height
were usually evident on the physiograph trace after a short
equilibration period. Figure 1 shows the result of the
addition of blocking doses of d-tubocurarine on paired hemi-
diaphragms, one stimulated directly via electrodes tied
adjacent to the muscle fibers and one stimulated via the
phrenic nerve. It can be seen that d-tubocurarine, at a
-4
bath concentration of 10 M, had no effect on the contractile
response of the directly stimulated muscle. A lesser
— 6
concentration, 10 M, almost completely blocked the contrac¬
tile response of the indirectly stimulated muscle within
10 minutes. When the bath was flushed and normal Tyrode
solution was again added, contractile responses to phrenic
nerve stimulation began to return immediately.
29

Figure 1. Effect of d-tubocurarine (DTC) on directly stimulated
hemidiaphragm muscle (top trace), and on indirectly
stimulated muscle (bottom trace). Doses were 10-4 m
and 10-^ M respectively. Marked intervals correspond
to 1 minute.

\
' ’ I t 1 ! I I I I I 1 I I I I I I I t i . ! t I ■ t I \ I
figure 1

32
Effect of Variations in Bicarbonate Buffer Components on
Contractile Response
Rat diaphragm muscle responded to changes in bath solu¬
tion pH by contracting with significantly'*" less force in
more acidic buffers. This was true when acidosis resulted
from an increase in the carbon dioxide concentration in the
Tyrode solution (series one buffers) or from a reduction in
the bicarbonate concentration in the Tyrode solution (series
two buffers). Figures 2 and 3 show the contractile response
of hemidiaphragm muscles from control rats plotted as a
function of the pH in the Tyrode solutions. The data in
Figure 2 represent muscle response in 5 mM potassium buffers,
and the data in Figure 3 represent muscle response in 0.5 mM
potassium buffers. It can be seen that reducing the pH of
the bath fluid from 7.8 to 7.1 resulted in approximately a
20% reduction in contractile response. Figure 4 shows the
effect of alterations in bath solution pH on contractile re-
. . 2
sponse of diaphragm muscle from potassium-deficient rats.
These hemidiaphragms, bathed in Tyrode solution containing
0.5 mM potassium, showed approximately a 65% reduction in
^Differences described as significant in this paragraph have
a p value of less than 0.05 in the F-test.
2
Serum potassium concentrations of rats on potassium-deficient
rat chow diets averaged 1.78 mM with a standard error of 0.076
mM; n=18. When hemidiaphragms from several potassium-defi¬
cient rats were challenged with insulin, 20 units per liter,
the contractile response to direct stimulation was reduced
considerably; contractile response was restored when the bath
potassium concentration was elevated from 0.5 mM to 5 mM,
behavior characteristic of potassium-deficient rat diaphragm.

Figure 2.
Contraction of control muscles in 5 roM
potassium is plotted as a function of the
pH of the bathing fluid. Filled circles show
values obtained in exp ments done in series
1, constant HCO^~ buffe s. Empty circles show
values obtained'in series 2, constant CO^
buffers. The bars represent standard errors.
Responses within both experiments differed signif
cantly at p=0.05, in the F-test. n=6, for each
point. For further details, see Methods, page 22

34
CONTRACTION vs pH
100
o
75 -
O
<
cc
y-
o
o
| 50-
X
<
2
h-
z:
ijj
o
tr
Ü.I
¡
—r 1 —r-
7.1 7.2 7.3
—i—
7.4
7.5
PH
"i r~
7.6 7.7
7.8
FIGURE 2

Figure 3. Contraction of control muscles in 0.5 mM
potassium is plotted as a function of the
pH of the bathing fluid. Filled circles
show the values obtained in experiments
done in series 1, constant UCO^~ buffers.
Empty circles show values obtained in serie
2, constant CO2 buffers. The bars represen
standard errors. Responses within both exper
ments differed significantly at p=0.05,
in the F-test. n-6, for each point. For
further details, see page 22.
(T 0)

36
CONTRACTION vs pH
IOO
2 75
H*
o
<
cir
t-
z:
a
o
s
z>
X
<
s
h-
Z2L
1¿J
O
nr
UJ
Q-
50 -
25 -
I
~T~
7.1
—i—
7.2
—i—
7.3
—i—
7.4
—i—
7.5
—i—
7.6
7.7
pH
7.8
FIGURE 3

Figure 4.
Contraction of potassium-deficient muscles
in 0.5 mM potassium is plotted as a function
of the pH of the bathing fluid. Filled circles
show the values obtained in experiments done
in series 1, constant HCO3" buffers. Empty
circles show values obtained in series 2,
constant CO2 buffers. The bars represent
standard errors. Responses within both experi¬
ments dif fered significantly at. p~0.05, in the
F-test. n-6, for each point. For further
details, see page 22.

38
CONTRACTION vs pH
FIGURE 4

Figure 5. Contraction of control muscles in 5 mM
potassium is plotted as a function of
the CO^ volumes percent bubbling the
buffer and the HCO^ concentration of
the buffer. The values shown were obtained
in series 3, constant pH buffers. The bars
represent standard errors. Responses did
not differ significantly at p=0.10, in
the F-test. n=6, for each point. For further
details, see page 22.

PERCENT MAXIMUM CONTRACTION
40
CONTRACTION vs C02 AND HCOj AT CONSTANT pH
HCO¡ [mM]
8 16 32
C02 [Vol.%]
FIGURE 5

Figure 6.
Contraction of control muscles in 0.5 mM
potassium is plotted as a function of
the CO^ volumes percent bubbling the
buffer and the HCO^ concentration of
the buffer. The values shown were obtained
in series 3, constant pH buffers. The bars
represent standard errors. Responses did
not differ significantly at p-0.10, in
the F-test. n=6, for each point. F°r further
details, see page 22.

42
100
9 75
h-
o
<
Or:
h
O
o
s
Z3
5
x
<
2
t-
z
liJ
o
cc
UJ
D.
50
25
CONTRACTION vs C02 AND HCO3 AT CONSTANT pH
HCO; [mM]
8 IG 32
ó
2 5
r~
10
C02 [V0!.%]
FIGURE 6

Figure 7. Contraction of potassium-deficient muscles
in 0.5 mM potassium is plotted as a function
of the CO2 volumes percent bubbling the
buffer and the HCO3- concentration of the
buffer. The values shown were obtained in
series 3, constant pH buffers. Filled circles
show values obtained in experiments when
muscles were stimulated directly, as usual.
Empty circles show values obtained when the
muscles were stimulated indirectly, via the
phrenic nerve. Triangles show values obtained
when the muscles_were stimulated directly in
buffers with SO.- and sucrose substituted for
HC03_ in the reduced HC03_ solutions. Muscle
performance in SO^- and sucrose buffers was
generally poor. The bars represent standard
errors. Responses within all three experiments
differed significantly at p=0.05, in the F-test.
n=6, for each point. For further details, see
page 22.

PERCENT MAXIMUM CONTRACTION
44
CONTRACTION vs C02 AND HCOj AT CONSTANT pH
HCOj [mM]
8 16 32
FIGURE 7

45
contractile response when the pH of the bath solution was
reduced from 7.8 to 7.1. This was a quantitatively greater
response than that which was seen in the control rat muscles.
In all of these experiments, the magnitude of the change in
muscle contractility seemed independent of whether the change
in pH was effected by respiratory of metabolic changes.
Figures 5 through 7 show results of contraction experiments
done in series three, (constant pH buffers). In these experi¬
ments, contraction of hemidiaphragm muscles was recorded in
buffers containing different bicarbonate concentrations
bubbled with different carbon dioxide mixtures, but with the
same ratio of bicarbonate to carbon dioxide. Figures 5 and
6 show contraction of hemidiaphragms from control rats plotted
as a function of both carbon dioxide volumes percent in the
gas mixture bubbling the buffer solution and the bicarbonate
concentration of the solution. The points in Figure 5
represent the responses recorded in muscles in Tyrode solution
containing 5 mM potassium; those in Figure 6 represent
responses exhibited by the muscles in 0.5 mM potassium Tyrode
solution. There was no statistically significant difference
in the responses exhibited by the muscles in either experi¬
ment, although there appeared to be a trend toward more
vigorous contractions in buffers containing elevated bicar¬
bonate concentrations and elevated carbon dioxide tensions.
This trend reappeared in experiments done on hemidiaphragm
muscles taken from potassium-deficient animals. Figure

46
7 shows the results of experiments which were done on hemi-
diaphragms from potassium-deficient rats in series three,
(constant pH buffers). These buffers contained 0.5 mM
potassium. In figure 7, the results of three different
experiments are shown. Again, contractile response is plotted
as a function of both buffer bicarbonate concentration and
carbon dioxide volumes percent of the gas mixture. One set
of points represents the response of muscles to direct stimu¬
lation; one set of points represents the response of muscles
to indirect stimulation via the phrenic nerve; and one set of
points represents the response of muscles to direct stimulation
when sulfate and sucrose rather than chloride were substituted
for bicarbonate in the reduced bicarbonate solutions. In all
three experiments, the contractile responses were significantly
greater in the high bicarbonate, high carbon dioxide buffers
than in low bicarbonate, low carbon dioxide buffers.
In summary, muscle contractile force was always greater
in buffers with high pH levels or high bicarbonate concen¬
trations. Muscle contractile force increased with increasing
carbon dioxide tension in constant pH experiments, but
decreased with increasing carbon dioxide tension in constant
bicarbonate experiments. These relationships were always
more pronounced in potassium-deficient rat muscles than in
muscles from control rats.
Changing the potassium concentration from 0.5 mM to 5 mM
had no perceptible effect on contraction of either potassium-
deficient rat muscle or control rat muscle. There was no

47
significant difference in contractile force per gram of
muscle between control rat muscles in 5 mM potassium and
potassium-deficient rat muscle in 0.5 mM potassium. For
3.0 control and 10 potassium-deficient rat muscles, these
values averaged 40.7 grams of force per gram of tissue and
33.5 grams of force per gram of tissue respectively, with a
pooled standard deviation of 15.6 grams of force per gram of
muscle tissue. These values were recorded in 32 mM bicarbon¬
ate buffers bubbled with a 95% oxygen, 5% carbon dioxide
mixture.
Tissue Analysis
Effect of Bicarbonate and Carbon Dioxide Tension Changes
on Muscle Water and Calcium Content
Changes in bicarbonate concentration and in carbon dioxide
4
tension had no significant effect on total muscle water or
extracellular fluid volume. There was no significant dif¬
ference between the means of these values obtained from
potassium-deficient animals and the means obtained from con¬
trol animals. For all experiments, 78% of muscle wet weight
The difference between these two means has a p value greater
than 0.05 in the Student's t-test.
4
Differences described as significant m this section, unless
otherwise noted, have a p value less than 0.05 in the paired
t--test.

48
was water, and 36% of muscle wet weight was extracellular
fluid . The standard deviations of these two values were
2% and 5%, respectively.
Buffer changes had no significant effect on intracellular
calcium content of muscles, nor was there any significant
difference between the mean of this value determined in control
rats and the mean of this value determined in potassium-
deficient rats. For all tissues, the single mean and the stan¬
dard deviation were 3.1 mM and 1.4 mM, respectively.
Effect of Buffer Bicarbonate and Carbon Dioxide Tension
Changes on Muscle Intracellular pH and Potassium Content
A decrease in extracellular pH caused by an increase in
buffer carbon dioxide tension or by a decrease in buffer bi¬
carbonate concentration, was accompanied by a significantly
lower intramuscular pH. Changes in buffer carbon dioxide
resulted in greater changes in intracellular pH than changes in
buffer bicarbonate. The muscles in the more acidic buffers
also contained less intracellular potassium than those in
^Because of the consistency of the measured tissue water
weights, it was reasoned that the variation in the mannitol
space measurements did not reflect true variations in the
extracellular fluid volume, for any change in extracellular
fluid volume would necessitate an exactly opposite change in
the intracellular fluid volume. Therefore, in calculations
requiring a value for the extracellular fluid volume, the
average 36% of muscle wet weight was used to approximate this
value.

49
alkaline buffers. Muscle twitch tension, measured at the
end of the experiment after 1 1/2 hours total time in the
last buffer, was always significantly greater in the more
alkaline buffers. These results, obtained in potassium-
deficient rat muscle, are shown in Tables 2 and 3.
The results of tissue analysis on potassium-deficient
and control rat muscles in series 3, constant pH buffers,
are shown in Tables 4 and 5. Muscles bathed in high bicar¬
bonate, high carbon dioxide buffers were significantly more
acidotic than companion muscles bathed in low bicarbonate,
low carbon dioxide buffers. Muscles in the high bicarbonate,
high carbon dioxide buffers also contained significantly
more potassium than the hemidiaphragm muscles in low bicar¬
bonate, low carbon dioxide buffers. Contraction, measured
after 1 1/2 hours of equilibration, was always significantly
greater in the high bicarbonate, high carbon dioxide buffers.
Figure 8 is the physiograph trace recording of contractile
responses of paired hemidiaphragm muscles from a potassium-
deficient rat. The trace shows the response of the muscles
before and after a change in the buffer. It can be seen
that changing from a low bicarbonate, low carbon dioxide
buffer to a high bicarbonate, high carbon dioxide buffer
resulted in a stronger muscle response in one of the muscles
while changing the buffers in the opposite direction resulted
in a weaker contractile response from the companion muscle.
Both buffers had a pH of 7.39.
Table 6 summarizes the pertinent data shown on Tables
2 through 5. Shown in this table are the intracellular

TABLE 2
TISSUE
K+ DEFICIENT
ANALYSIS
RATS, IN
OF HEMIDIAPK
DIFFERENT CO
RAGAS, FROM
- CONCENT!JvfIGNS
Buffer Series
1
HCO ”
j
(Vol.
CO.
PHC
phR
HCO^
- b
i
k"
o
16 mM
2.5
7,64
7.36±.07
n=5
11.6
inM
0 - 5 mM
c
16 mM
10
7.05
o.94+.03
— n
l x —
17.2
iVU'i
0.5 mM
k"1* .
(mM)
% Maximum3,
Contraction
123+15 n=5 88±5.5 n=5
d c, e
103±9.1 n=5 57±5.9 n=5
a ,
c .
Q ,
F i n :
as means ±
S.E.
. mean pH.
* J.
ference at
p=0.05 m
pairod t-test
difference
at p=0.05
in paired t-test
re determi
ned at the
end of the experiment
l-n
*—>
a gradual waning of contractile response over one three hour experi¬
mental period

TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
K‘-DEFICIENT RATS, IN DIFFERENT HCO3- CONCENTRATIONS
HCO3-
9 mM
3 2 mM
Buffer Series 2
(Vol.%)
C02 PH0 pH±a HC03"iD
5
7.12 6.63±.09 n=6 4.0 mM
5
c
7.62 6.77±.17 n= 6 6.3 mM
0.5 mM
0.5 mM
% Maximuma
K ^ (mM)a Contraction
94±6.5 n=7 51±7.2 r.=5
c c,d
115±4.1 n=7 7 0±5 .4 n=5
data presented as means ± S.E.
calculated from mean pHj_
significant difference at p=0.05 m paired t-test
these values were determined at the end of the experiment and reflect
a gradual waning of contractile response over the three hour experi¬
mental period

TABLE 4
TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
K+-DEFICIENT RAT'S, IN CONSTANT pH BUFFERS
Buffer Series 3
HC03
(Vol. %)
}2
uOt pH
o
8 mM
2.5 7.3Í
32 mM
10
7.39
PHia HC°3'ib
6.95±.11 n=8 4.0 mM
c
6.6 8 ± , 15 n= 8 8.7 xrM
% Máximum3
Contraction
0.5 mM 91±9.1 n=8
39±6.1 n=8
c
c,d
0.5 mM 115±6.4 n=3
78±3.8 n-8
^data presented as means ± S.E.
calculated from mean pH¿
C
significant difference at p=0.05 m paired t-test
these values were determined at the end of the experiment and reflect
a gradual waning of contractile response over the three hour experi¬
mental period

TABLE 5
TISSUE ANALYSIS OF HEMIDIAPHRAGMS, FROM
CONTROL RATS, IN CONSTANT pH BUFFERS
Buffer Series 3
HCO,“
o
8 mM
32 mM
£
data presented as mean ± S.E.
^calculated from mean pH^
c .
significant difference at p=0.05 in paired t-test
these values were determined at the end of rhe experiment and reflect
a gradual waning of contractile response over the three hour experi¬
mental period
(Vol. %)
C0„
PH
tt a
PH±
HCO
- D
3 i
K
+
K+i (mM)a
% Maximum
Contraction
2.5 7.39 7.28±.02 n=9 8.6 mM
c
5 mil
145111 n=9
c
70±5.2 n=9
c, d
10
7.39 7.191.03 n=9 28 mM
o mM
170H3 n=9 8H4.5 n=9
U>

TAELE 6
Buffer
Series
1
2
3
COMPARISON OF ICF ACID-BASE AND K+
CHANGES IN HEMIDIAPHRAGMS IN VARIOUS BUFFERS
(Vol. %)
co2
hco3"0
PHí
Hi/H+0
K+i
% Maximum
Contraction
2.5
K+ Deficient
16 mM 7.36
1.91
128
mM
88
10
16 mM
6.94
1.29
108
mM
57
5
9 mM
6.63
3.09
94
mM
51
5
32 mM
6.77
7.08
115
mM
70
2.5
8 mM
6.95
2.75 ,
91
mM
39
10
32 mM
6.68
5.13
115
mM
78
2.5
Control
8 mM
7.28
1.29
145
mM
70
10
32 mM
7.19
1.58
170
mM
81
Ln

Figure 8.
Effect of change in buffer of directly stimulated
potassium-deficient rat hemidiaphragm muscles.
Buffers contained 8 mM bicarbonate or 32 mM bicar¬
bonate bubbled with 2.5 volumes percent or 10
volumes percent carbon dioxide with oxygen. The
pH of both buffers was 7.39. Buffers contained
0.5 mM potassium. Marked time intervals correspond
to 1 minute.

32. JL
10 | 2.5
i ii i i f [ i i i i i i i i ! r i i rHrTT i i i i n
figuro 3

57
potassium concentrations of the muscles, the contractile
forces, and the ratios of intracellular to extracellular
proton concentrations. It can be seen that in all four
experimental pairs, the muscles which contracted with the
greatest amount of force always contained the higher intra¬
cellular potassium concentration and had a higher ratio
of intracellular to extracellular protons.
Resting Potential Measurements
Changing the bicarbonate concentration in the Tyrode
solution from 8 mM to 32 mM had no significant effect on the
resting potential of diaphragm muscles from control or from
potassium-deficient rats. Changing the carbon dioxide mixture
from 2.5% to 10% also had no significant effect on resting
potential. These potentials were recorded in muscles bathed in
0.5 mM potassium buffers. These data are illustrated
graphically in Figures 9 and 10 for potassium-deficient and
control rat muscles. These graphs also show resting potentials
recorded in muscles bathed in different potassium concentrations
ranging from 0.5 mM to 20 mM. The recorded potentials are plot¬
ted as a function of the ratio of the intracellular potassium con¬
centration to the extracellular (buffer) potassium concentra¬
tion. The intracellular potassium concentration of potassium-
deficient muscles was taken from the average of the values in
Tables 2 through 4. The intracellular potassium concentration
of control rat muscle was taken from the average of the val¬
ues in Table 5. The horizontal bars extending from two of

Figure 9. Resting membrane potentials recorded in potassium-
deficient rat hemidiaphragm muscles are plotted as a
function of the ratio of intracellular to extracellular
potassium concentration. Intracellular potassium
concentration was estimated to be 107 mM. The hori¬
zontal bars represent the error that would be introduced
if the estimated intracellular potassium concentration
was incorrect by 20 mM. Vertical bars represent standard
errors. The curved line is the solution to the reduced
Goldman equation:
Em = -61.5 log (K+) i
(K+)q + .01 x (Na+)0
The straight line is the solution to the Ne.rnst equation,
(K+)i
Em = -61.5 log
(K+)0
n=24.
The four symbols on the extreme left represent potentials
recorded in buffers containing, from top to bottom,
8 mM HCQ3" 32 mM HC03~ 16 mM HC03“ 16 mM HC03“
5% C02 ' 5% C02 ' 2.5% C02 ' and 10% C02 '
For these points only, n=12.

-1 OOmV -
Em
-90mV -
-8 OmV -
- 7 0 mV -
- 6 OmV-
-50mV-
5
320
160
FIGURE 9

Figure 10.
Resting membrane potentials recorded in control rat
hemidiaphragm muscles are plotted as a function of the
ratio of the intracellular to the extracellular potassium
concentration. Intracellular potassium concentration was
estimated to be 155 mM. Bars represent standard errors.
The curved line is the solution to the reduced Goldman
equation:
(K+)i
Em = -61.5 log
(K+)o + .01 (Na+)o
The straight line is the solution to the Nernst equation,
E„ = -61.5 log JJLii
(K+)o
n=24.
The four symbols on the extreme left represent potentials
in from top to bottom, 32 mM HCO3- 16 mM HCC>3_ 16 mM HCO3
5% C02 2.5% C02 ' 10% C02"
and ^ ^ HCO3 . For these points only, n=12.
5% C02

-lOOmV
-90mV-
-80mV
- 7 0 mV -
-GOmV-
-50rrV-
FIGURE 10

62
the points in Figure 9 represent the error that would be
introduced if the estimated intracellular potassium concen¬
tration were incorrect by as much as 20 mM. The straight
line and the smooth curve drawn on Figures 9 and 10 are,
respectively, the relations described by the Nernst equation,
and the reduced Goldman equation using 0.01 for the ratio of
the permeability coefficients of sodium to potassium (Bilbrey
et al., 1973). It can be seen that the recorded potentials
do increase with decreasing extracellular potassium concen¬
tration, but not to the extent predicted by either the
Nernst equation of the reduced Goldman equation. The poten¬
tials recorded in potassium-deficient rat muscles best fit
the reduced Goldman equation when 0.022 was used for the
ratio of the permeability of sodium to potassium. The data
recorded in control rat muscles fit the equation best when
0.027 was used for the permeability coefficient ratio. All
of the potential measurements shown were recorded with one
of 3 microelectrodes on muscles from 2 potassium-deficient
rats and 2 control rats.

DISCUSSION
The data presented in this work are the first to be
reported correlating changes in muscle contraction brought
about by alterations in the bicarbonate buffer system with
changes in muscle ion composition. Other authors have reported
that acidosis caused depression of muscle contraction, but
this depression was slight in the preparations studied and
the authors offered no explanation for this effect (Crul-
Sluijter and Crul, 1974; Baraka, 1964). By analyzing
muscles that had been contracting in different bicarbonate
buffers, we hoped to determine specifically which components
of the buffer system affected contraction and to elucidate
the mechanism of this effect.
In the present study, acidosis mildly depressed the
contractile response of control muscles. The degree of
this depression was independent of the nature of the acidosis,
whether caused by increased carbon dioxide tension (respira¬
tory acidosis) or decreased external bicarbonate concentration
(metabolic acidosis). These results, obtained in directly
stimulated muscles, agree quantitatively with those of
Crul-Sluijter (1974) who reported a 4% depression of phrenic
nerve stimulated muscle contraction when HC1 lowered the bath
solution pH from 7.5 to 7.1. Over the same pH range, the
63

64
data in the present study show an 8% decrease in contractile
response, which is within experimental error of the value
reported by Crul-Sluijter,
Acidosis suppressed the contractility of muscle isolated
from potassium-deficient animals to a much greater extent
than muscles from control rats. These results are the first
to be reported showing the increased sensitivity of potassium-
depleted skeletal muscle to pH changes, A change in the
buffer pH from 7.75 to 7.1 was accompanied by a 60% fall in
the contractile response of this tissue compared with the
20% fall in the contactile response of muscle from control-
dieted rats when pH changed over this same range. The increased
sensitivity of this tissue to pH changes was not a consequence
of the low potassium concentration in the buffer (0.5 mM)
since control muscle contracting in 0.5 mM potassium buffer
did not display any increase in sensitivity to bath pH
changes, compared to control muscle in 5 mM potassium buffers.
Analysis of potassium-deficient muscles that had been
contracting in buffers with different pH levels revealed
that muscles in acidic buffers had lower intracellular pH
values than paired muscles that had been in more alkaline
buffers. The difference in intramuscular pH was greater in
response to simulated respiratory changes than in response
to simulated metabolic changes, although the extracellular
pH changes were similar. These results are qualitatively
similar to those obtained by Adler (1965) in diaphragm
muscles from normal rats. Mean intracellular muscle pH val¬
ues, determined in 16 mM bicarbonate solutions bubbled with

65
oxygen with 2.5 volumes percent carbon dioxide or 10 volumes
percent carbon dioxide, both fell on the curve Adler obtained
for intracellular pH values of control muscles. The two
mean intracellular muscle pH values, obtained for muscles in
buffers containing 8 mM bicarbonate and 32 mM bicarbonate
both bubbled with 5% carbon dioxide, 95% oxygen, were lower
than the two values obtained in the constant bicarbonate
buffers. The results of Adler (1972) on control rat muscles
depleted of potassium in vitro indicate that potassium-
depletion tends to reduce the muscle intracellular pH. There¬
fore, this would predict that the intracellular pH of in
vivo potassium-depleted muscle would be lower than the
values obtained in control animals. Considering this
evidence, the intracellular pH values we obtained in the
constant bicarbonate experiments were high, whereas, those
obtained in the constant carbon dioxide experiments were low
compared to Adler's values for intracellular pH of in vitro
potassium-depleted rat muscles. From the data in Tables 2
and 3, one can see that the muscles used in the constant
carbon dioxide buffer experiments were somewhat more potassium-
deficient than the muscles used in the constant bicarbonate
buffer experiments. Therefore, the muscles used in the con¬
stant carbon dioxide experiments probably were also more
acidotic at the beginning of the experiments. This would
have contributed to the disparity between these intracellular
pH values. This disparity does not detract from the essential
feature which is the qualitative relationship between bicar¬
bonate concentration and intracellular pH, and the relation¬
ship between carbon dioxide tension and intracellular pH.

66
Intracellular pH was found to be lower in muscles exposed to
buffers with low bicarbonate concentrations compared to paired
muscles in high biccirbonate buffers. Muscles in high carbon
dioxide buffers were found to have a lower intracellular pH
that paired muscles exposed to low carbon dioxide buffers.
Determination of muscle potassium content, performed on
the same muscles used for intracellular pH determinations,
showed higher intracellular potassium concentrations in
muscles that had been in alkaline buffers compared to the
intracellular potassium concentrations of paired muscles
that had been in acidic buffers. The differences in both
experiments was approximately 20 mM. Adler et al. (1965)
also reported that muscles bathed in alkaline buffers had a
higher intracellular potassium content than muscles bathed in
acidic buffers. But the differences reported in his data
were not as great as the differences found in the present
study. The muscles in Adler's experiments were not contracting,
while the muscles in our experiments were contracting. This
difference may have been important, since Lade and Brown (1963)
determined that i_n vivo decreases in muscle intracellular
potassium concentration in response to acidosis took place
faster when muscles were stimulated to contract. This was
probably because contraction increases the rate of loss of
potassium from skeletal muscle. The range of the intracellular
potassium concentrations determined in potassium-deficient
muscles, 90 mM to 130 mM, shows good agreement with the value
of 97 mM reported by Bilbrey et al. (1973), determined on
skeletal muscle from potassium-depleted rats.

67
Muscles exposed to high bicarbonate, high carbon dioxide
buffers contracted with greater force than muscles bathed
in low bicarbonate, low carbon dioxide buffers, even though
these buffers had the same pH. This relationship was more
pronounced in potassium-deficient muscles than in control
rat muscles. This effect was independent of variations in
buffer chloride concentration, and was seen in directly and
indirectly stimulated muscles alike.
Tissue analysis revealed that muscles bathed in the
high bicarbonate, high carbon dioxide buffers had a lower
intracellular pH and a higher intracellular potassium con¬
tent than muscles in low bicarbonate, low carbon dioxide
buffers. Kim and Brown (1968) obtained similar results in
in vivo experiments on dogs. They recorded a decrease in
intracellular muscle pH when serum bicarbonate concentration
and carbon dioxide tension were elevated simultaneously.
They also detected an increase in extracellular potassium
but did not seek the source of this potassium. In their
experiments, the intracellular pH declined from 6.97 to
6.87 while the extracellular bicarbonate concentration rose
from 25 mM to 65 mM, extracellular pH remained constant. The
calculated change in intracellular bicarbonate was from 12 mM
to 24 mM. In the present constant pH experiments with control
rat muscle, intracellular pH declined from 7.28 to 7.19
while extracellular bicarbonate rose from 8 mM to 32 mM.
This represents an increase in the calculated intracellular
bicarbonate from 9 mM to 28 mM. In potassium-deficient rat

68
muscles subjected to the same treatment, intracellular pH
declined from 6.95 to 6.68. The calculated intracellular
bicarbonate rose from 4 mM to only 8.7 mM. These data sug¬
gest that potassium-deficient muscles are less well buffered
than control muscles. Whether this means that less intra¬
cellular buffer is available for titration or that bicar¬
bonate or proton flux is restricted in this tissue is not
defined by these results.
The results of these constant pH experiments are pivotal.
Comparing the results of these contraction experiments with
the results of experiments conducted in non-constant pH
buffers, it is clear that contractile force always increased
with increasing extracellular bicarbonate concentrations and
with increasing extracellular pH. However, contractile force
increased with increasing carbon dioxide tension in the con¬
stant pH experiments, but decreased with increasing carbon
dioxide tension in constant bicarbonate experiments. These
relationships were always more pronounced in potassium-
deficient rat muscle experiments than in control muscle
experiments.
The paired data sets obtained from tissue analysis,
summarized in Table 6, indicate that all buffer changes which
effected an increase in muscle contractile force also effected
an elevation in intracellular potassium. In all four experi¬
ments the difference in intracellular potassium concentration
caused by the buffer change was about 20 mM. No correlation
between contraction and intracellular pH was observed, however,

69
the ratio of the intracellular to the extracellular proton
concentrations was always higher in the muscles with the
higher intracellular potassium concentration. Despite the
decrease in intracellular pH when extracellular pH was reduced,
the ratio of intracellular to extracellular proton concen¬
trations always fell when extracellular pH was reduced. Other
authors have reported a similar relationship between potassium
and hydrogen ion gradients across muscle cell membranes
(Brown and Goott, 1963; Fenn and Cobb, 1934; Lade and Brown,
1963; Irvine and Dow, 1966). These results are consistent
with the concept that potassium ions exchange with hydrogen
ions across muscle membranes in the direction which would
tend to equalize K.+/K + and H.+/H,+.
The redistribution of potassium brought about by the
buffer changes may be responsible for the observed changes
in muscle contraction. One possible mechanism whereby
increased intracellular potassium concentrations might effect
a change in muscle contractile force is by increasing the
resting muscle membrane potential. However, the data in the
present study do not support this hypothesis. Buffer changes
brought about alterations in the intracellular potassium
concentration of at least 20 millimoles per liter in all
experiments measuring this value. As a result, the resting
membrane potential would have been shifted as predicted by
the Goldman equation. For potassium-deficient muscles, based
on the potassium concentrations measured in Tables 2 to 4,
the predicted shift in resting potential would be about 6

70
millivolts. In experiments recording resting muscle membrane
potentials, no statistically significant shift in potential
was observed in response to buffer changes of any sort.
However, because our standard errors were around 7 millivolts,
a shift in resting potential consistent with a 20 mM change
in intramuscular potassium might not have been detected. If
the average resting potential of all the muscle fibers was
increased by even a few millivolts, the maximum contractile
force of the muscle might be noteably increased, but the
observation that changing the extracellular potassium concen¬
tration from 0.5 mM to 5 mM resulted in no change in muscle
contraction argues against this theory. With a change in bath
potassium of this magnitude, we measured approximately a 15
millivolt change in the resting membrane potential. Otsuka
and Ohtsuki (1970) reported a 15 millivolt shift in resting
rat diaphragm muscle membrane potential when the bath potas¬
sium concentration was changed from 4 mM to 0.5 mM, but saw
no change in twitch tension. If a 15 millivolt shift in
resting potential brought about by a change in the bath
potassium concentration produced no detectable change in the
contractile force, one must reject the hypothesis that a
resting potential shift of 6 millivolts, effected by a change
in the intracellular potassium concentration as a result of
buffer alterations, could be responsible for the observed
changes in muscle contractile force.
A change in the intracellular potassium concentration
might affect muscle contraction by another mechanism. The

71
movement of calcium from the sarcoplasmic reticulum to the
site which triggers muscle contraction is probably coupled
to the movement of ions in the opposite direction. Potassium
and magnesium are the likely candidates for this role since
active transport of calcium into isolated sarcoplasmic retic¬
ulum has been shown to be coupled to magnesium and potassium
ion transport out of the sarcoplasmic reticulum (Kanazawa
et al., 1971). Morad and Orkand (1971) have suggested
that potassium exchanges for activator calcium during contrac¬
tion of cardiac muscle from frogs. If during muscle excita¬
tion intracellular potassium exchanges for calcium across
the sarcoplasmic reticulum, a higher intracellular potassium
concentration may facilitate this calcium movement and
increase the force of the skeletal muscle contraction. This
mechanism could explain why the buffer changes were more
influential on contraction of potassium-deficient rat muscle.
The observed changes in intracellular potassium concentration
were proportionately greater in this tissue than in control
muscle.
The changes in intracellular potassium concentration
might be linked to changes in intracellular sodium concen¬
tration. Muscles containing less intracellular sodium might
depolarize more rapidly than muscles containing more intra¬
cellular sodium. The rate of depolarization, which is in
part determined by the driving force for sodium entry into
cells, has been shown to influence skeletal muscle contraction
(Taylor et al., 1972; Sandow, 1973). Unfortunately, it is

72
difficult to obtain reliable data for intracellular sodium
concentrations. A better method to test this hypothesis of
sodium involvement would be to record the action potentials
of muscles in different buffers. Ideally, the action poten¬
tials and contractions could be recorded simultaneously. A
more elaborate system could be devised to record intracellular
bicarbonate activity (Khuri et al., 1974) and intracellular
potassium activity as well. Experiments having this design
would yield data concerning the time sequence of these events
which would aid in separating cause and effect, important
information that could not be obtained in the present study.
When resting membrane potentials were recorded in mus¬
cles bathed in different potassium concentrations, a smooth
curve relating potential and potassium distribution was
generated in which resting membrane potential continues to
become more negative as extracellular potassim decreases.
This is consistent with the findings of Otsuka and Ohtsuki
(1970) in potassium-deficient rat diaphragm. Our recorded
potential values compare favorably with the values reported by
these workers. The potentials reported in the present study
were not as great as the potentials the reduced Goldman equation
predicts, using the value of 0.01 for the ratio of the perme¬
abilities of sodium to potassium. Bilbrey et al. , (1973)
reported that resting thigh-muscle potentials, recorded in
vivo in dogs and rats, agreed with the values predicted by
this equation. The values in the present study, recorded

/ o
in vitro, fit. curves described by the reduced Goldman equation
with a higher value for the ratio of sodium to potassium
permeability coefficients.

CONCLUSION
Elevations in buffer bicarbonate concentration or in
buffer pH effected an increase in rat diaphragm muscle
contractility. Elevations in carbon dioxide tension were
accompanied by increases in muscle contractility when buffer
bicarbonate was elevated along with and at the same ratio
as carbon dioxide tension. Elevations in carbon dioxide
tension brought about decreases in muscle contractility in
buffers with a constant bicarbonate concentration. Hemi-
diaphragm muscles from rats made potassium-deficient by
diet showed a greater sensitivity to these buffer changes
than muscles from control rats. Increases in muscle
contractility consistently showed a positive correlation
with increases in intracellular potassium concentration,
but showed no consistent correlation with changes in intra¬
cellular pH. Changes in contractility and intramuscular
potassium concentration did show a positive correlation
with changes in the ratio of intracellular to extracellular
proton concentration. Buffer changes did not effect any
measurable alterations in resting muscle membrane
potentials. Changes in the extracellular potassium concen¬
tration, which affected resting potentials, did not affect
muscle contractility.

75
In contrast to results obtained in potassium-deficient
and control rat muscles, contractility of skeletal muscles
in patients with familial hypokalemic periodic paralysis
show an unusual sensitivity to changes in extracellular
potassium concentration. Gordon et al. (1970) proposed that
hypokalemia renders muscle in these individuals inexcitable
by causing a depolarization block. He suggested that depolar¬
ization results during hypokalemia because of a decrease in
the membrane permeability for potassium. Reductions in
serum bicarbonate and pH, resulting from administration of
ammonium chloride or administration of acetazolamide and
the subsequent renal loss of bicarbonate, prevent muscle
weakness and bouts of hypokalemia (Vroom et al., 1975;
Jarrell et al., 1976).
Reductions in extracellular pH and bicarbonate concen¬
tration result in improved muscle performance in hypokalemic
periodic paralysis, but result in poorer muscle performance
in rat muscles. However, the effect in both tissues appears to
be related to changes in transmembrane potassium distribution.
The results of the present study predict that both acidosis
and low serum bicarbonate levels would promote a lower
ratio of intracellular to extracellular potassium concentra¬
tions, but this value has never been determined in hypokalemic
periodic paralysis patients treated with acetazolamide. Whether
acid-base changes benefit victims of periodic paralysis by
effecting a more favorable steady state potassium balance or
by abolishing some other pathological change is not clear.

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BIOGRAPHICAL SKETCH
David Walter Fretthold was born on February 28, 1948,
in Fairview Park, Ohio. He attended school in Rocky River,
Ohio, graduating from Lutheran High School West in 1966.
His education continued at Miami University in Oxford,
Ohio, where he received the Bachelor of Arts and Science
degree in Zoology in 1970. In 1972 he began graduate study
at the University of Florida, leading to the degree, of
Doctor of Philosophy in the Department of Pharmacology
and Therapeutics.
82

I certify that I have read this study and that in ray
opinion it conforms to acceptable standax'ds of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
L*_X C . Gt'L-Oj*
Lai C. Garg, Chairperson
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Betty P, Vogh 0 ^
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Associate Professor of
Pharmacology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
WilTTam R’ Kem
Assistant Professor of
Pharmacology

r+ k3 Cu hO
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
resentation and is fully adequate, in scope and quality,
s a dissertation for the degree of Doctor of Philosophy.
n 0
Ü>
Philip Posner
Assistant Professor of
Physiology
•lis dissertation was submitted to
he College of Medicine and to the
accepted as partial fulfillment of
degree of Doctor of Philosophy.
the Graduate Faculty of
Graduate Council, and was
the requirements for the
March, 1977
Dean, College of Mpdicine
Dean, Graduate School

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