The Effect of alterations of the components of the bicarbonate buffer system on skeletal muscle contraction, potassium c...

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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
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viii, 82 leaves : ill. ; 29 cm.
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English
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Fretthold, David Walter, 1948-
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Research   ( mesh )
Muscle Contraction -- physiology   ( mesh )
Muscle, Skeletal -- physiology   ( mesh )
Bicarbonates   ( mesh )
Carbon Dioxide   ( mesh )
Hydrogen-Ion Concentration   ( mesh )
Diaphragm -- physiology   ( mesh )
Rats -- physiology   ( mesh )
Department of Pharmacology and Therapeutics thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pharmacology and Therapeutics -- UF   ( mesh )
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Notes

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

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University of Florida
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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















ACKNOWLEDGMENTS


I wish first and foremost to express my sincere gratitude

to Dr. Lal 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 Ker, 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).









TABLE OF CONTENTS




Page

ACKNOWLEDGMENTS . . ii

LIST OF TABLES . .. iv

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


S . 82









LIST OF TABLES


Page


TABLE

1 BUFFER SERIES . .

2 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
CO2 CONCENTRATIONS .


. 19


3 TISSUE ANALYSIS OF HEMIDIAPHRAGMS,
FROM K+-DEFICIENT RATS, IN DIFFERENT
HCO 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 K+
CHANGES IN HEMIDIAPHRAGMS IN VARIOUS
BUFFERS . .. 54









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 CO2
volumes percent and HCO3~ ...... 39

6 Contraction of control muscles in 0.5 mM
potassium, constant pH buffers, vs CO2
volumes percent and HCO3 .. 41

7 Contraction of potassium-deficient
muscles in 0.5 mM potassium, constant
pH buffers, vs CO2 volumes percent and
HCO . 43

8 Effect of alterations in HC03- and CO2,
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 HCO3, CO ,
and potassium concentration on resting
membrane potential of control rat
hemidiaphragm muscles .. 60












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. Lal 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







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 more

pronounced in potassium-deficient rat muscles.

Intracellular p1H 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.


viii











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,

Vislkoper et al. (1973) were able to precipitate an attack

of paralysis in a patient by infusing sodium bicarbonate which

produced a metabolic alkalosis. Treatment with acetazolamide

appears 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

pla~mna pH, but also decreases in plasma bicarbonate and carbon







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 (HC03-) In vivo, regula-
(C02)
tory mechanisms control the ventilation rate and consequently

the carbon dioxide concentration in blood, thus controlling the








concentration of the proton donor, carbonic acid. Extracel-

Jular; pH is maintained between 7.1 and 7.6, the range generally

considered colmatible w ith life. Even during strenuous

exercise, when the carbon :dioxide evoluLion rate can increase

to as much as thirty times the rate at rest, extracellular

p1l falls no more thcn 0.1 unit.

Relation Between In trLacel].1-ular and Extracel ular pl and
BicarbonaLte of iMuscl e (Cells

A variety of techniques have been employed to determine

the intracell.ul.:ar pIi of muscle. Early methods used to measure

intracellular pHl and the theoretical and practical, diffi-

cultLies associated with these methods are discussed in a review

by Caldwell (1956). Waddell and Butler (1959) first used the

weak acid, di ilthy]oxazolidinedione (DINO), a metcabolite of the

antiopileptic agent trimethadione, to measure the intraccllullar

pll of dog muscles in vivo. Using bicarbonate-specific micro-

electrodes, Khuri et al. (1974, 1976) determined the intra-

ce.llul ar bicarbonate activity of skeletal muscles, and calcu-

lated equilibrium potentials for bicarbonate of between -20

m:i llivolts and -44 millivolts u-nd .er different conditions in

rat skleta .uscles. These authors and others have determined

introace]llular piI to be 1btcween G.6 and 7.4, and have concluded

tha t proton and bicarbonate gradicxts m.i;st exist across muscle

cell mmcb,,rnbrr< ,... which are not in Nirna tan equilibrium with the

res, ting muscle mbrano poet. a although the membrane) is

p e; able to La L e ;i.n (Ad I.c:: ut al 1965) In order for such

gradients L c to iie mai tLaind, either hyd,-roen ions would have to









be transported out of cells or bicarbonate ions would have to

be transported into cells (Adler et al., 1972; IIeisler, 1975;

Khuri et al., 1974; Lai et al., 1973).

Numerous investigations have been undertaken to determine

the rilation of intracellular pH to changes in extracel-

lular pH. This was the objective of Waddell 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-

lul l- pH. Kim and Brown (1968) showed that skeletal muscle

intracellular pHl in dogs fell when carbon dioxide tension

was increased along with bicarbonate concentration, and rose

when animals were hyperventilatl-d during HC1 infusion, even

though. the plasma pHl remained constant. Similar results have

been obtained in vitro; respiratory acidosis produced larger

altr rtion:s in muscle intracellular pH than metabolic acidosis

(Hi n d1r, 1975; Adler et al., 1965). Both these authors

found that rat diaphragm muscles were most effectively buffered

when c(xtracellular pH ranged between 7.1 and 7.4. They

Irepo*c Led Lhat when extracel luar pll varied from 7.1 to 7.4

duri.i! simulated respiratory acidoacis or alkalosis, intra-

cel i.lar] pH, measured with )DMO, varied] from 7.0 to about. 7.28.

Outside this extrac ellulacr pll range, in t racellultar pH changed

about as much as extracellular pH.








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









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 (Volle, 1972). This type of membrane beha-

vior could explain the potassium shift seen in acidosis

(Volle, 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.








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 n PK(K+)i + PNa(Na+)i + P l(C-)
SPK(K)o + PNa(Na+) + PC(C-)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 E,, and the

contribution of the term PNa(Na )i 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 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 Em is initially low (Shanes, 1958).








In vivo studies by Williams et al. (1971) showed that a fall

in blood pH induced by an elevation in pCO2 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. In 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








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

than 106 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 Weber 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 in vivo and

interpretation of his results is complicated because acidosis

also effects elevations in serum sodium and potassium








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

a]., 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

t a. 1973). In both dog and man, progressive skeletal

muscle weakness and structural degeneration develop (Chanpion

eA. al., 1972; Smith 0it al. 1950) Bilbrey et al. (1973) have

att ributed muscle weakness in severely potassiu!m-deficient

docjg to an abnormally low resting membrane potential. They








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 episodica

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). Acetazolami.de 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

potas.Lium wasting diuretic, has been found to be the most effec-

tiver treatment available for hypokalemic periodic paralysis

((riggs et al., 1970). In hypokalem(ic periodic paralysis,

attacks are associated with a fall in the serum potassium

concentration (Biemond and Daniels, 1934). During attacks,








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 (Vroom 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, 1.970). 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 pe'ri odic paralysis patients and in potassium-deficient

ra~s contain elevated intracellular sodium and reduced intra-

cellular potcasrium (IIoffmann and Smit.li, 1970; Offerijns et

al., 1958). Offerijns et al. (1958) reported that insulin

can ed fjacid paralysis in the hemidi a}phragm preparation in








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 ;CO2, 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-








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













MATERIALS AND METHODS


Buffer Composition


Muscle experiments were performed in Tyrode solution of

the following composition: NaCI, 126 mM; KC1, 5 mM; CaCl2,

3 rmM; MgCl2, 1.6 mM; dextrose, 11.1 mM; Na2HPO4, 0.677 mM;

NaH2PO4, 0.172 mMl; and NaHCO3, 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









Products, and the mixtures with 5% and 10% carbon dioxide

were 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

vitamin-; and minerals, obtained from Nutritional Biochemicals.

Rat.r were maintained on this diet for 5 to 6 weeks prior to

the print. the t he time of the experiment, these rats

we il'-d between 200 g and 275 q. Male Sprague-Dawley rats,

weighing 200-275 g, obtained from Flow Labs, were used as

control rats. These animals were fed standard rat chow.
























TABLE 1

BUFFER SERIES



HCO3/ CO2
(mM)/Vol.%)

A B C

SERIES 1 Constant HCO3- 16/2.5 16/5 16/10
(respiratory changes)

SERIES 2 Constant CO2 8/5 16/5 32/5
(metabolic changes)

SERIES 3 Constant pH 8/2.5 16/5 32/10
(compensated changes)









Rat I emidiaphra m Preparation

The rat phrenic nerve hemid.i.aphragm in vitro preparation

was originally described by Bi.)'ring (1946). This tissue was

chosen for these experiments because it is a thin skeletal

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 centrifuga tion for sodium and potassium

detemi nations, performed on an Instrumentation Laboratory

343 flame photometer, against a lithium standard. Animals

were then decapitated and the skin removed from the chest.

The tlho.acic wall was cut to expose the rostral surface of

the diaphragm and the phrenic nerves. W1~i the phrenic nerve

was to be isolated with the hemn.diaphragm, it was tied close

to the thymus and cut, then carefully dissected away from

any attaching tissues. The diaphragm was cut down the nid-

lin., and the ribs and tissues connecting each hemidiaphr:agm

were cut away. The hemnA faphdms wore then r-cmoved from the

anim.l:i1. and placed in oxygenai:-te Tyr(ode solution where the

remainder of the surgery wais performed. Extraneous tissues

wcr,-- then cut away from the hemidiaphragm leaving a section









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

370 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








bicarbonate bubbled with 951 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 hemidiaphragmns 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 mM pot-assi;urn. These muscles were stimulated directly

wit-I stains ;;s sr-eel electrodes. Potassium-deficient rat

di.aiph-tagms were also examined in series three, (constant pH

buffers, containing 0.5 rmM potassium) stimulated indirectly

viI the phronirc nerve. Diaphrangms from control rats were








studied in all three series of buffers. Hemidiaphragms were

stimulated directly in these experiments which were conducted

in both 5 mM4 potassium Tyrode solution and in 0.5 mM potassium

Tyrode 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

200 microcuries per liter of 3H labeled mannitol and 50
14
microcuries per liter of 1C labeled DMO, both obtained

from New England Nuclear, were added to the appropriate organ


L-/









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 DMO 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 1000 C and

reweighed, then ashed overnight at 6000 C in a muffle oven.

The ash was then dissolved in concentrated HC1 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








and the 14C and 3H 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

sodium and potassium content in a similar manner. The 3H
14
counts were corrected for 1C spectral overlap according to

the formula At = R (E x Ac) where At is the corrected

H counts per minute, R1 is the uncorrected H counts per

minute over background, E is the counting efficiency of
14 3
a pure 1C sample on the H channel relative to the counting

efficiency of that same pure sample on the 14C channel, and

Ac is the counts per minute over background of the mixed
14
sample on the 1C channel. The extracellular space in ml of

each muscle was estimated as the mannitol space of that

muscle, determined by the formula:

3H counts per minute of that muscle
H 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:










Ca (V. + V ) V
pH. = pK + log x [10 (pK PHe) + ] -
e Vi i

where pKa is the ionization constant of DMO (6.13), Ct is the

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
1
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 pKa value of 6.13 for carbonic

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.








Potential Measurements

Diaphragms were rapidly dissected out of rats as

previously described. Strips of hemidiaphragm were placed

in plexiglass beaker containing 16 nrl 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 350 C and were bubbled with the appropriate gas mixture.

The wuiscle strips were pinned by the adjoining tendons to a

silicone-resin mat at the bottom of the chamber. Resting

membrane potentials were recorded with standard microelectrode

techniquLes, using glass micropipettes filled with 3 M potas-

sium chloride. The electrodes had a tip resistance of 5-10

megoh:s. Prepulled microelectrodes with a I micron tip con-

tairing 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

a-rtplri t ied on an Electronics for Life Science wide band elec-

tror'-.ter equipped with a circuit for monitoring the electrode

tip resistance. The signal frjo the electromelc-r was displayed

on a TOktroric type 5641 oscilloscope. The criteria followed

for judging whether a potential was a valid resting potentLal

were:

1. a rest ing pot en tial had to be more negative than
-40 millivolts

2. a resting potential had to be stable within 5
millivolts for one minute








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 potassiumi-deficient rats were also determined in 16 ni

bicarbonate buffer bubbled with 95% oxygen, 5% carbon dioxide,

but containing different concentrations of potassium. These

potassium concentrations were 20 mM, 10 mM, 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 w2re 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, 10-6 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.



























4J> 0
rdH -4,T
r--4 J I
c o a o
6 ,H 0


,--4
4) C) 0O

- 0-4


0 .- > U
Cr-4 3)









C)


U 0 aE
0 Or O
o -to




















0-0 4-*
CJ 4- C) A















Ou ro -
S-Ph r
S0 to
3 U 4.













^ (1) (P
M ^! f0
































0'o~
,u


C2

ta-als


-pr









Effect of Variations in Bicarbonate Buffer Components on
Contractile Response

Rat diaphragm muscle responded to changes in bath solu-

tion pll 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-

sponse of diaphragm muscle from potassium-deficient2 rats.

These hemidiaphragms, bathed inTyrode 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.

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 mM
potassium is plot ted as a function of the
pH of the bathing fluid. Filled circles show
values obtained in exi ':aents done in series
1, constant HCO buffi Empty circles show
values obtained in ser.:.. 2, constant CO2
buffers. The bars repro:;nt standard errors.
Responses within both exper-imnts differed signifi-
cantly at p=0.05, in the F-test. n-6, for each
point. For further details, see Methods, page 22.













CONTRACTION vs pH


100


75


50-








25-


7.1 7.2


7.3 7.4


7.6 7.7


pH
FIGURE 2


- I r 1






























Figure 3. Contraction of control muscles in 0.5 inM
potassium is plotted as a function of the
pH of the bathing fluid. Filled circles
show the values obi.ai.ned; in experiments
done in series 1, conCstrl; t iJCO3.' buffers.
l..i:y circles slow values obta. ned in series
2, constant CO2 offers. Tlhe bars represent
standard errors. Responses within both experi-
ments differed s rjnificantly at p=:0.05,
in the F-test. n-G, for each point. For
further d&-tailz;K, see page 22.












CONTRACTION vs pH


7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8


pH

FIGURE 3


100






















ur'; 2e 4.


Conracict ~:ni- of pot.ass urn-def cient musceoc
in 0.5 mra M poLtiss lum: is plotted as a func ion
of the ph of the bathing fliui.. Filled circles
show the values obtained in e::pe-riiments done
in series 1, cr:nstant HC:O3- buffeirs. Inpty
circles show values obtained in csries 2,
constant CO2 buffers. The bahis repr~eent
standard errors. Responses within both experi-
ments differed significantly at p C .05, in ithe
F-test. n-6, for each point. For further
details, sce page 22.









CONTRACTION vs pH







t


7.1 7.2 7.3 7.4


I I I I


pH
FIGURE 4


100.


75-




50-




25-































Figure 5.


Contraction of control muscles in 5 mM
potassium is plotted as a function of
the CO2 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.
















CONTRACTION vs CO2 AND HCO3 AT CONSTANT pH

HCOg [mM]

8 16 32
'


75








50 -


CO2 [Vol.%]


FIGURE 5


100


























Figure 6.


Contraction of control muscles in 0.5 mIr
potassium is plotted as a function of
the CO, volumes percent bubbling the
buffer and the HCO3 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=0.1.0, in
the F-test. n=6, for each point. For further
details, see page 22.
















CONTRACTION vs COz AND HCO3 AT CONSTANT pH


HCO, [mM]


CO2 [Vol.%]


FIGURE 6


100


| L


- --------~ L


- --~---~-






















Figure 7. Contraction of potassium-deficient muscles
in 0.5 rmM 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 obtain d 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
HCO3 in the reduced HC03- solutions. Muscle
performance in SO0~ and sucrose buffers was
generally poor. The bars represent standard
errors. Responses within all three cx:perim.ents
differed significantly at p=0.05, in the F-test.
n=6, for each point. For further details, see
page 22.














CONTRACTION vs COz AND HCO; AT CONSTANT pH

HCO; [mM]
8 16 32
T-- -

T


I I I
2.5 5 I0
CO2 [VolO/%]


FIGURE 7


100


75







50-







25 -









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









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 represen-s 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 p1l 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







significant3 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

10 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 rmM 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

tension had no significant4 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.

Differences described as significant in this section, unless
otherwise noted, have a p value less than 0.05 in the paired
t--test.








was water, and 36% of muscle wet weight was extracellular

fluid5. 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 p1H 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




5Because 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.








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 pIT of 7.39.

Table 6 summarizes the pertinent data shown on Tables

2 through 5. Shown in this table are the intracellular

















:1,
i-i O J

'-4
*o



(.1 '-.





n i



>9


'2
C)i
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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



























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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








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








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.








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 in'racellular 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 in 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

pot;ass nm 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 potassiulm-depleted rats.








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 mrM. In potassium-deficient rat










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,







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









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 notably 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









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








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 potassium 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

wocr 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

tli.s equation. The values in the present study, recorded




/ J



in vitro, fi t curves described by the reduced Goldman equation

.it.h aI higqbr value for the ratio of sodium to potassium

permcfeabili ti y coef ficient-s.














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.








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.
















BiBL IOGRAPJ HY


Ad'ler, S., Roy, A. and Relman, A,S. Intracellular acid-
base regulation. I. The response of muscle cells
to changes in CO2 tension or extracel.lular bicar-
bonate concentration. J. Clin. Invest., 44: P,
1965.

Adiler, S., Zett, B. and Anderson, B. The effect of acute
potassium depletion on muscle cell pH in vitro.
Kidney Int., 2: 159, 1972.

Ai tken, R.S., Allott, E.N. and Castleden, L.I. Observa-
tions on a case of familial periodic paralysis.
Clin. Sci., 3: 47, 1937.

Earaku.t, iAnis. The influence of carbon dioxide on the
neurol.uscular block caused by tubocurarine c,"l.oride
in th<-e human subject. Brit. J. Anaesth., 36: 272,
1964. .

Riednmond, A. and Daniels, A. Familial periodic paralysis
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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.










E 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.


L.4j C 6 u^
Lal C. Garg, ChairpeYson
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 V
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.



C.Y. Chiou
Associate Professor of
Pharmacology



I certify that I have read this study and thai 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.



iTll-iam R. Kern
Assistant 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.


Q- ______ ________
Philip Posner
Assistant Professor of
Physiology





This dissertation was submitted to the Graduate Faculty of
the College of Medicine and to the Graduate Council, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.

March, 1977



Dean, College of :--idicine


Dean, Graduate School











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UNIVERSITY OF FLORIDA

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