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EFFECT OF DRUGS ON ION TRANSPORT
AND AQUEOUS HUMOR FORMATION
LAI. CHAND GARG /' --
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
The author expresses his sincere gratitude to Dr. W.
Walter Oppelt, Chairman of the Supervisory Committee, for his valu-
able guidance in research work and help in the preparation of this
manuscript. To Dr. Thomas H. Maren, Chairman of the Department
of Pharmacology and Therapeutics, and to the other members of his
committee, Dr. Kenneth C. Leibman, Dr. Sidney Cassin, and Dr.
Melvin L. Rubin, go special thanks for their helpful suggestions and
for the time and effort they spent for him.
Thanks are also due to Dr. Herbert E. Kaufman, Chairman
of the Department of Ophthalmology, for his participation and helpful
suggestions in the preparation of this manuscript.
The author extends his appreciation to the other faculty
members of the Department of Pharmacology and Therapeutics, Dr.
David M. Travis, Dr. Thomas F. Muther, Dr. Lauretta E. Fox, Dr.
John W. Cramer, Dr. C. Van Breemen, Dr. Roger F. Palmer, Dr.
Betty P. Vogh, and Dr. Aaron H. Anton, for their participation and
helpful suggestions during the formal and informal discussions per-
taining to this investigation.
Sincere thanks are extended to Mrs. Bobbie Ditto for
typing this manuscript and Miss Sharon Chapman and Miss Pat Powers
for reading the manuscript and suggesting some changes.
The work was supported by grants from Fight for Sight
and NIH Grant GM-01764-01.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .... . .. ii
LIST OF TABLES . .......... vi
LIST OF FIGURES. . ....... viii
INTRODUCTION ........ .......... 1
Aqueous Humor Formation . .. 1
Cation Transport ................... 6
Anion Transport . . .. 7
Outflow Facility. . ... ..... 13
METHODS. . .. . ... 17
Composition of AH-Like Buffer ............ 17
Determination of AH Formation Rate . 17
Determination of Transport of Na22 from Plasma to AH. 23
Determination of Transport of C136 from Plasma to AH. 24
Determination of Outflow Facility . 25
Determination of Effect of Ouabain . 26
Determination of Effect of Acetazolamide. . .27
Comparison of Loss of Inulin and Na22 or C136 from Eye.
Chambers . . 27
Statistical Evaluation of Results . 28
RESULTS . . . 30
Effect of Intravenous Ouabain . 30
Effect of Intracameral Ouabain . 33
Effect of Intravenous Acetazolamide . 39
Effect of Intracameral Acetazolamide . 42
Comparison of Loss of Inulin, Na22 or Cl36 from Eye
Chambers ..................... 48
DISCUSSION .. . . 54
Methodology. . . 54
AH Formation. ............. 54
Transport of Ions from Plasma to AH. ... 57
Advantages . . 58
Disadvantages. ... . 59
Results . . 59
Ouabain . . 59
Acetazolamide . . 62
Mechanism of AH Formation and the Effect of Drugs. 66
Proposed Mechanism of AH Formation . 69
SUMMARY ........... ........ 80
REFERENCES . . 83
BIOGRAPHICAL SKETCH .................. 89
LIST OF TABLES
1. Anionic Composition of AH and Effect of Carbonic
Anhydrase Inhibition ................. 9
2. Cat AH-Like Buffer . . 18
3. Effect of I. V. Ouabain on AH Formation Rate 31
4. Effect of I.V. Ouabain on Transport of Na22 and Cl36
from Plasma to AH ...... 32
5. Effect of I.V. Ouabain on Inulin Recovery in Outflow
Perfusate . . 34
6. Effect of Intracameral Ouabain on AH Formation Rate 35
7. Effect of Intracameral Ouabain on Transport of Nazz and
C136 from Plasma to AH ............... 37
8. Effect of Intracameral Ouabain on Inulin Recovery in
Outflow Perfusate ............... 38
9. Effect of I.V. Acetazolamide on AH Formation Rate 40
10. Effect 3f I.V. Acetazolamide on Transport of Na and
Cl from Plasma to AH ...... ..... .. 41
11. Effect of I.V. Acetazolamide on Inulin Recovery in
Outflow Perfusate ............... 43
12. Effect of Intracameral Acetazolamide on AH Formation
Rate . . 44
13. Effect of Intracameral Acetazolamide on Transport of
Na22 and Cl from Plasma to AH'. .. 46
14. Effect of Intracameral Acetazolamide on Inulin Recovery
in Outflow Perfusate ............... 47
15. Comparison of Inulin-C14 or Inulin-H and Na or
Cl36 Recovery in Outflow Perfusate. . 53
LIST OF FIGURES
1. Diagrammatic representation of the current of AH from
the ciliary body to the canal of Schlemm 4
2. Accumulation of HCOG in AH from blood CO2 and its in-
hibition by acetazolamide . 12
3. Diagrammatic representation of the experimental
preparation . . 22
4. Disappearance of inulin-C and Na from eye chambers. 50
5. Disappearance of inulin-H3 and C136 from eye chambers. 52
6. Proposed mechanism of ion transport from plasma to AH 71
Aqueous Humor Formation
Aqueous humor (AH) formation and its modification by drugs
have been described by numerous authors, such as Becker (1959),
Barany (1963), Davson (1962, 1963), Kinsey and Reddy (1964), Maren
(1967) and Grant (1969).
Most investigations have been done on rabbits but increas-
ing use is now being made of cats, dogs and monkeys. Therefore,
observations on different species should be compared and cautiously
interpreted. Caution is also indicated in comparing and interpreting
results obtained from anterior or posterior chamber fluids.
AH differs from the plasma mainly because of its very low
concentration of protein. In man, AH contains about 15 mg of protein
per 100 ml, while the plasma contains about 7000 mg of protein per
100 ml. Duke-Elder (1932) postulated that AH was a filtrate of plasma.
Later on, more careful studies of the individual constituents of AH
(reviewed by Davson, 1962) showed that AH is not a simple dialysate of
plasma. Now it is well accepted that AH is a specialized fluid.
Because of the importance of chronic simple glaucoma,
which causes damage by a slight increase in intra-ocular pressure and
is able to cause irreversible blindness, interest has been focused on
various aspects of AH dynamics such as intra-ocular pressure, rate
of AH formation and resistance to its flow from eye to plasma.
AH formation may be considered to consist of three pro-
cesses: (a) diffusion from plasma (b) ultrafiltration across blood-
aqueous barrier in ciliary body and (c) specialized secretion, possibly
involving the active transport of at least one constituent (possibly Na+,
Cl- or HCO3 ion) by the epithelial covering of ciliary processes.
AH, so formed, fills the posterior chamber and passes be-
tween the lens and the back side of the iris and enters the anterior
chamber through the pupil (Fig. 1). As the newly-formed posterior
AH is not in equilibrium with plasma, its composition is modified dur-
ing its passage to the anterior chamber, due to diffusional exchanges
with blood in the iris capillaries. In the anterior chamber, AH com-
position may be further modified due to diffusional exchanges with the
metabolic products of the cornea. From the anterior chamber, AH
leaves by bulk flow through the minute channels or pores in the tra-
becular meshwork, passing into the venous canal of Schlemm in
primates or corresponding veins in lower animals, and then through
the scleral channels out of the eye into the general circulation.
The actual mechanism involved in the special secretary
process of AH formation is not known. However, it is known that at
least two enzymes are present in ciliary body which are likely to be
involved in AH formation. Carbonic anhydrase is found in ciliary pro-
cesses of all vetebrates so far examined (Maren, 1967). All
t~m e- 43
investigators agree that after intravenous injection of acetazolamide,
the intra-ocular pressure is lowered. Under these conditions, the
carbonic anhydrase activity of ciliary processes is inhibited
(Ballintine and Maren, 1955). Maren (1967) has reviewed and criti-
cally analyzed the arguments both in favor and against the hypothesis
that acetazolamide decreases intra-ocular pressure by reducing the
rate of AH formation. Maren, Becker, Kinsey and many other in-
vestigators, have produced ample evidence to suggest that acetazola-
mide decreases intra-ocular pressure by reducing the rate of AH
formation (Maren, 1967). However, Davson (1962) argues that car-
bonic anhydrase inhibition causes a general "poisoning" of active
transport and later causes a reduction in resistance to flow and/or
episcleral venous pressure. Macri and Brown (1961), on the basis of
the effect of acetazolamide on ciliary artery, feel that a fall in intra-
ocular pressure produced by acetazolamide is due to vasoconstriction
of the ciliary artery. Macri et al. (1965) did not find any decrease in
AH formation, as measured by inulin turnover in cat eye.
Bonting et al. (1961) reported that the human ciliary body
has 26 times more Na-K-activated ATPase specific activity than does
the human erythrocyte. A similar specific activity of Na-K-activated
ATPase was present in the cat ciliary body. On this basis, they
suggested that Na-K-activated ATPase may be involved in the forma-
tion of AH. The Na-K-activated ATPase of ciliary body was sensitive
to 50% inhibition by 3 x 10-7M ouabain and complete inhibition by
10"4M ouabain. Simon et al.(196Z) found a 70% decrease in AH inflow
after 66 pig/kg intravenous ouabain, as measured by the slope of a
pressure recovery curve after drainage of AH. Becker (1963) did not
find any reduction in intra-ocular pressure after intravenous ouabain
administration to rabbits in maximum tolerated doses (250 pg/kg).
Langham and Eakin (1964) also did not find any change in intra-ocular
pressure, AH formation and outflow facility when-measured by tono-
metry in a single determination 20 minutes after infusion of various
doses of ouabain through the lingual artery in rabbits and cats. On the
other hand, Macri et al. (1966) reported that ouabain produced a dose-
related inhibition in turnover rates of intracamerally administered
inulin. Recently, Oppelt and White (1968) found that intravenous
ouabain significantly decreased AH formation in the cat, as determined
by continuous posterior-anterior chamber perfusion of AH-like buffer
containing trace quantities of inulin-C14.
In view of all these contradictory results, it was thought
worthwhile to approach the mechanism of AH formation by studying the
effect of ouabain and acetazolamide on the movement of two major ions
(Na+ and Cl-) from plasma to AH.
Na+ is the chief cation both in plasma and AH and its move-
ment from plasma to AH is of considerable interest. In all species
examined there is an excess of Na+ in AH over that of a plasma
dialysate. In a study of the effect of metabolic inhibitors on AH forma-
tion rate in rabbits, Cole (1960) reported that influx of Na as de-
termined by analyzing effluent AH collected after obstructing drainage
channels by paraffin, was reduced approximately 70-75% following
administration of dinitrophenol (DNP) or fluoroacetamide and approxi-
mately 60% following administration of strophanthin-G (ouabain).
From the analysis of AH turnover rates, in vivo, Kinsey (1960) con-
cluded that approximately two-thirds of AH Na+ enters the posterior
chamber of the rabbit by secretion. Holland and Stockwell (1967)
studied Na+ transport across the isolated ciliary body of the cat. They
reported that, in vitro, cat ciliary body transports Na+ inwardly in a
short-circuited state (absence of electrochemical potential difference).
This is consistent with the hypothesis of active sodium transport. How-
ever, they did not study the sensitivity of the Na+ transport pump to any
metabolic inhibitor or ocular hypotensive drug. Moreover, Davson
and co-workers (1957, 1960) did not find any significant decrease in
AH Na+ turnover rate following large doses of intravenous acetazola-
mide to rabbits, cats, dogs and monkeys. This indicates the need for
re-examination of Na+ transport after administration of acetazolamide
Cl" and HCO3 are two major ions of plasma and AH. The
anionic composition of AH of various species and the effect of car-
bonic anhydrase inhibition is given in Table 1. AH/plasma water
ratios of HCO3 were greater in the posterior AH of all the species
examined and the anterior AH of rabbit, guinea pig, dog, cat and dog-
fish than that expected of Gibbs-Donnan equilibrium. Gibbs-Donnan
equilibrium requires a ratio of 1. 04 for univalent anions (Davson,
1962). After the complete inhibition of carbonic anhydrase, the most
striking drop occurred in AH/plasma water HCO3 ratio in the posterior
chamber of the rabbit. The change was independent of plasma HCO3
However, the results are not so .clear in other species.
Table 1 shows that acetazolamide causes a significant decrease in
AH/plasma water HCO3 ratio in dogfish. The dogfish has no carbonic
anhydrase in lens and kidney (Maren, 1959). The overall effect of
acetazolamide was an elevation of plasma HCO concentration and
plasma pCOZ (Maren, 1962). This was due to inhibition of carbonic
anhydrase in erythrocytes and gills. After acetazolamide administra-
tion, although there was a slight increase in AH HCO3 concentration,
AH/plasma water HCO3 ratio showed a significant decrease mainly
due to the increase in plasma HCO3 concentration. Similarly, the
large decrease in the AH/plasma water HCO3 ratio after acetazola-
mide administration in lake trout (Table 1) was due to the increase in
plasma HCO3 concentration and plasma pCO2, whereas the actual
".4 I-- 0
M l U
wir u u
+. (U ii -
T) 0 o!
decrease in AH HCO3 concentration was quite small (Hoffert and
Fromm, 1966). Thus, it is difficult to interpret the changes in
AH/plasma water HCO3 ratio when plasma HCGO concentration is
A large change in HCO3 concentration occurring at the
site of AH formation may be reflected by only a small change or even
no change in posterior or anterior AH. Wistrand et al. (1961) found a
small decrease in posterior AH/plasma water HCO3 ratio after com-
plete carbonic anhydrase inhibition in the dog. A negligible change
was seen in HCO3 concentration of anterior AH of the dog. The
changes in HCO3 concentration in anterior or posterior AH do not
reveal the actual changes at the site of AH formation.
Kinsey and Reddy (1959) approached this problem differently.
They determined the accumulation of C1402 and HC1403 (total C1402)
in posterior and anterior AH after parenteral administration of HC140O
in rabbits. They found that within less than one minute, after HC1403
injection, total C1402 in posterior AH exceeded that of plasma by
approximately Z00% (Fig. ZA). When 50 mg/kg acetazolamide was
given 15 minutes prior to HC1403 injection, there was a marked de-
lay in accumulation of C1402 in posterior AH (Fig. 2B). This shows
that catalytic HCO3 accumulation is an important step in AH forma-
tion in the rabbit. This may be true in other species also, but similar
studies have not been done on other species.
Fig. 2. --Accumulation of HCO3 in AH from blood CO2
and its inhibition by a'cetazolamide (from
Kinsey and Reddy, 1959).
0 I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22
TOTAL C40Oz (AFTER ACETAZOLAMIDE)
N,, .,* 3,*"
POSTERIOR '*. =
.oO~~o*0 0o 0 0*
0 1 2 3 4 5
3 4* ,* X0 x
o0 000 00 0 0 0 0
6 7 8 9 10 11 12 13 14 15 16 17
- '* *. POSTERIOR.
'. ..* *
PLASMA lka l x
.- "e* .. .; :.... .
TE IO**R* 0 a 0
Furthermore, on the basis of a mathematical analysis
of AH secretion in the rabbit, Kinsey and Reddy (1964) suggested that
the primary AH, as formed in the ciliary body cells bordering the post-
erior chamber, contains about 90 mM HCO3 while the HCO3 con-
centration in anterior AH is only 30 mM. This again indicates that
analysis of ionic composition of anterior or even posterior AH is not
a very reliable index of events occurring at the site of AH formation.
The same arguments also apply to the study of changes in
Cl" concentration of AH. Table 1 shows that the changes in the AH/
plasma water Cl- ratio after complete inhibition of carbonic anhydrase,
in all species, are small. If the decrease in Cl- or other ion trans-
port by any drug is accompanied by iso-osmotic decrease in water
transport, very little change in concentration of that ion may be de-
tected by analyzing the anterior or posterior AH ionic composition.
Therefore, the absence of change in Cl" or any other ion concentration
in AH after a drug administration, does not necessarily indicate the
lack of effect of the drug on the transport of this ion. Thus, it was
thought to be of interest to study the effect of acetazolamide and
ouabain on transport of C1- from plasma to AH in cat by a direct post-
erior-anterior chamber perfusion method (Oppelt, 1967) where even
iso-osmotic changes in ion transport from plasma to AH would be de-
Moses and Bruno (1950) and Grant (1950) used tonographic
measurements for determining outflow facility. In this method, a
weight is placed on the cornea. This raises intra-ocular pressure
and causes an increased flow of AH out of the eye. Because of the in-
creased outflow, the intra-ocular pressure falls after a time back to
its original value. The rate of return of intra-ocular pressure to the
original value enables one to calculate the outflow facility. Accordingly,
the outflow facility has been defined as the volume of AH expelled out of
the eye through the normal outflow channels, into the venous circulation
in a unit time when the intraocular pressure is raised 1 mm above
normal (Davson, 1962).
The tonographic procedure has been used extensively, both
clinically and experimentally, for determination of intra-ocular
pressure and the outflow facility of the eye. Sears (1960), Davson
(1962) and Barany (1964) doubted the validity of the assumptions made
in tonography to calculate the outflow facility. It has been assumed
that episcleral venous pressure is the same both with and without the
weight on the eye, which may not be the case. Secondly, the volume
change induced by placing the weight on the eye may not be entirely due
to expression of ocular fluid through the chamber angle but it may be
partly due to expulsion of scleral fluids and blood. A third assumption
is that AH formation rate is constant both with and without weight on
the eye. Macri (1967) reported that increased intra-ocular pressure
can inhibit AH formation rate.
Sears (1960) determined the outflow facility in rabbit eye
by measuring the increase in pressure produced by constant rate in-
fusions into the anterior chamber. Barany (1962, 1964, 1968) used the
constant pressure infusion method, and its various modifications, for
determination of outflow facility in primates. Both constant rate and
constant pressure infusion methods are based on the assumption that
both outflow facility and AH formation rate are independent of pressure
changes. Walinder and Bill (1969b) have shown that increase intra-
ocular pressure can inhibit AH formation rate.
Oppelt et al. (1969) described a continuous posterior-
anterior chamber perfusion method for determination of outflow facility.
In this method both pressure and rate of perfusion were held constant.
The perfusion fluid contained inulin-C14. Some of the infused inulin
leaves the eye by bulk flow through the normal outflow channels and
the remainder comes out through the outflow cannula. It can be
reasoned that the total amount of inulin collected through the outflow
cannula during each time period is a function of the resistance in the
outflow channels of the eye. As the pressure and rate of perfusion are
held constant, it may be assumed that any change in total amount of
inulin collected indicates the change in resistance in the outflow
channels of the eye. Therefore, it was of interest to study the effect of
ouabain and acetazolamide on the outflow facility by the inulin recovery
In the present system, the intra-ocular pressure was held
constant; therefore, the term "outflow facility" is re-defined as the
volume of AH expelled out of the eye by bulk flow through the normal
channels of the eye into the venous circulation in a unit time at a
constant intra-ocular pressure.
The methods used in all these experiments are based on the
initial studies on posterior-anterior chamber perfusion for determina-
tion of AH formation rate, as described by Oppelt (1967). In the pres-
ent study, the methods were extended to determine the outflow facility
and transport of ions from plasma to AH. Excluding a few special ex-
periments, the rate of AH formation and outflow facility was determined
in all experiments. In one-half the number of experiments at each dose
level of each drug, the transport of Naz from plasma to AH was de-
termined, while in the other half the transport of Cl36 from plasma
into the eye was determined.
Composition of AH-Like Buffer
The ionic composition of anterior AH of 6 cat eyes was de-
termined. AH-like buffer corresponding to the cat AH in ionic com-
position (Table 2) was prepared and used in all experiments. Heparin
(10 units/ml) was added to the AH-like buffer to prevent clotting of the
perfusate around the outflow cannula. The effect of perfusion on the
integrity of the blood-aqueous barrier is discussed below.
Determination of AH Formation Rate
The posterior-anterior chamber perfusion method, as
described by Oppelt (1967), was used for determination of AH formation
Cat AH-Like Buffer
5% CO2 (95% Oz)
4. 3 meq/L
2. 5 meq/L
1. 6 meq/L
Sufficient to bring pH to 7. 4
rate. It is similar to the ventricular perfusion method of determining
CSF production rate, as introduced by Pappenheimer and Heisey
(1962, 1963) and later used by Oppelt et al. (1964). Estimation of AH
formation rate is based on the determination of dilution of inulin as it
passes through the eye chambers. The same technique and its slight
modification were recently used by Macri (1967) and Wallinder and
Bill (1969), essentially confirming the validity of this approach to the
study of AH physiology.
Male and female cats of mixed breed, weighing 2 to 4 kilo-
grams were anesthetized with 30 mg per kilogram intrahepatic pento-
barbital. If needed, additional pentobarbital was given I. V. through a
cannula in the femoral vein to maintain anesthesia at a steady level.
The animal's head was mounted in a sterotaxic head holder. One side
of the post-limbal conjunctiva was grasped with a small hemostat to
stabilize the eye. The anterior chamber was cannulated with a hubless
24-gauge needle, using a drilling motion by hand or a needle gun, as
described by Sears (1960). Prior to cannulation, the other end of the
needle was attached by a polyethylene tubing to a 20 cc glass syringe
filled with buffer. The needle was advanced through the pupil, behind
and parallel to the iris, into the posterior chamber. The hemostat
was then removed from the eye and the intra-ocular pressure was
measured with a Sanborn pressure transducer previously attached to
the polyethylene tubing of the posterior chamber needle through a three-
way stopcock. The eye was again fixed with the hemostat and the tip
of another hubless needle attached to polyethylene tubing at its other
end was similarly introduced into the anterior chamber (Fig. 3).
Infusion of AH-like buffer containing trace quantities (1 mg or
2 pc per 100 ml) of inulin-C14 or inulin-H3 was then started through
the posterior chamber needle and the outflow perfusate was collected
from the anterior chamber into a stoppered test tube. The height of
the outflow tube was so adjusted that the pressure in the system
corresponded to the initially measured intra-ocular pressure. Con-
tinuous posterior to anterior chamber perfusion was thus accomplished
by using a Harvard pump model 600-919/920. For the first 10 minutes
the perfusion rate was set at 194 pl per minute, then it was reduced to
97 p.l per minute and run for another 10 minutes. Finally, the per-
fusion rate was set at 38. 8 al per minute and run for another 10 min-
utes. The rapid perfusion rates were used to wash out the original AH
and bring the chambers quickly into an equilibrium with the perfusate.
After the initial equilibration period of 30 minutes, the perfusion was
maintained at a rate of 38. 8 p.1 per minute for the six-hour experimental
period. The outflow perfusate was collected in half-hour periods. The
volume of each half-hourly collected perfusate was determined by know-
ing the difference in weights of collecting test tubes before and after
each collection. Inulin radioactivity was determined, using a Beckman
Liquid Scintillation Counter. In most experiments the original AH
was washed out of the eye by the buffer during the first 30 minutes of
equilibration. This was judged by the reasonably constant value of
w L. \
inulin concentration in each half-hourly collected outflow sample dur-
ing the control period. However, in a few experiments the original
AH probably was not completely washed out of the eye within the first
30 minutes of equilibration period. This could be noted by the pre-
sence of a very low concentration of inulin in the first half-hour out-
flow sample, as compared to that in the later samples of the control
period. This may be due to the difference in the rate of mixing of the
perfusate with the original AH. In such experiments the value of in-
ulin concentration in the first half-hour sample was discarded and not
used for computation of results. AH formation rates were calculated
using the following formula:
Formation Rate (pl/min) =i C) r
where Ci and Co are inulin concentrations in inflow and outflow per-
fusate, respectively and r is the rate of perfusion of the eye in ul per
The outflow perfusate samples were checked for total pro-
tein content, as previously described and any experiment in which total
protein concentration exceeded 300 mg per 100 ml of the outflow per-
fusate was discarded as previous work suggested that AH formation
rate was variable under such conditions (Oppelt, 1967).
Determination of Transport of Na22 from Plasma to AH
In one-half the number of experiments at each dose level of
each drug a single intravenous injection of 10 p.c of Na22 (as NaCl) was
given to the cat at 30 minutes before the beginning of the control
*period of each experiment. One ml blood samples were taken from
the polyethylene catheter inserted into the femoral artery at one-hourly
intervals. The blood samples were centrifuged and the radioactivity
of NaZ2 in plasma was determined in a Nuclear Chicago Gamma Radi-
ation Counter. Radioactivity due to Na22 in outflow perfusate samples
was similarly determined. As the plasma concentration of Na22 was
reasonably steady during the six-hour experimental period, the rate
of appearance of this isotope in aqueous outflow perfusate represents
the rate of movement of Na from plasma to AH. Correction for the
amount of Na22 lost through normal outflow channels of the eye was
made by knowing the quantity of inulin lost through these channels and
assuming that Na22 was lost in the same proportion as inulin (see
below). The quantity of inulin lost was calculated by measuring the
quantity of inulin recovered in the outlfow perfusate, as described
later for determination of outflow facility. Thus, the total transport
of Na22 from plasma into the eye chambers was calculated, taking into
consideration the concentration found in the outflow perfusate and add-
ing to it the quantity lost through the normal outflow channels of the
Determination of Transport of C136 from Plasma to AH
The methods followed for C136 transport experiments were
similar to those for Na transport. However, in these experiments
inulin-H3 was used in place of inulin-C14 in AH-like buffer. A single
intravenous injection of 10 p c of C136 (as NaCI) was given to the cat at
the beginning of the experiment. Radioactivity due to C136 in plasma
and aqueous outflow perfusate samples was determined, using a Beck-
man Liquid Scintillation Counter.
Determination of Outflow Facility
Outflow facility has been defined in the introduction as the
volume of AH expelled out of the eye by bulk flow through the normal
outflow channels into the venous circulation in a unit time at a constant
intra-ocular pressure. In the present system, where the intra-ocular
pressure and rate of perfusion are kept constant, some of the infused
inulin will leave the eye by bulk flow through the normal outflow chan-
nels of the eye and the remainder will come out through the outflow
cannula. The total amount of inulin collected through the outflow
cannula is a function of the resistance in the outflow channels of the
eye. Therefore, any change in total amount of inulin recovered in the
perfusate should represent a change in the resistance in the outflow
channels. Thus, one should recover a greater portion of infused in-
ulin when there is a decrease in outflow facility and a smaller portion
when there is an increase in outflow facility. Therefore, in all experi-
ments the total amount of inulin recovered in each half-hour period of
outflow perfusate was determined and compared to the total amount of
inulin infused during this period. Inulin recovery was calculated for
each half-hour period as follows:
% infused inulin recovered =
(Outflow Vol. in ml/30 min)x(Outflow Inulin-C14 counts/min/ml)
(Inflow Vol. in ml/30 min)x(Inflow inulin-C1l4 counts/min/ml x 100
It may be pointed out here that any dilution of inulin by newly
formed AH will not interfere with the calculation of total recovery of
inulin. The calculations for changes in total amount of inulin recovery
are different from the calculations of changes in inulin dilution which
are used in determination of AH formation rate. As the rate of per-
fusion, inulin concentration in the perfusate, and rate of outflow are
known and the rate of AH formation can be calculated, it could be
possible to estimate the outflow facility'in terms of volume of fluid
expelled out of the eye by bulk flow per unit time. However, the pres-
ent study was designed to find out the changes in outflow facility pro-
duced by the drugs rather than absolute values of the outflow facility.
Therefore, a more simple and direct estimation of a change in outflow
facility was made by simply noting the change in the percent of total
amount of inulin recovered through the outflow cannula.
Determination of Effect of Ouabain
To study the effect of various doses of ouabain, a two to
three hour control period was run in each cat. Ouabain, either 67, 34
or 10 [ig per kilogram, was then administered I.V. in a single dose.
The AH formation rate, concentration of Na22 or C136 in plasma and
perfusate,and outflow facility were then determined for another three
In another group of experiments buffer containing inulin-C14
or inulin-H3 was divided into two portions prior to the beginning of the
experiment. One portion was used during the control period, while
ouabain, to make a final concentration ranging from 10-10 to 10-4M
was added to the second portion. After the control period, the buffer
containing ouabain was substituted for the control buffer and perfusion
with this buffer was continued for a period of another three hours. The
changes in AH formation rate, concentration of Na or Cl in outflow
perfusate, and outflow facility were determined.
Determination of Effect of Acetazolamide
In these experiments, after a two to three hour control per-
iod, acetazolamide, either 30, 10, 5, 1, or 0. 3 mg per kilogram, was
given intravenously in a single dose. The AH formation rate, move-
ment of Na22 or C136 from plasma to AH, and outflow facility were
determined for another three hours. In some experiments acetazola-
mide was perfused through the eye chambers in concentrations of 10-4
to 10-8M, as described for ouabain, and its effect on AH formation
rate, transport of Na22 or C136, and outflow facility was determined.
Comparison of Loss of Inulin and Na22 or C136 from Eye Chambers
Another group of experiments was conducted to investigate
the validity of the assumption that the fraction of Na22 lost through
bulk flow and diffusion was similar to that of inulin. AH-like buffer was
divided into two portions prior to the beginning of the experiment. In
one portion, trace quantities of both Na and inulin-C were added
and eye perfusion was begun, as described previously. After three
hours, the second portion of AH-like buffer (not containing Na or
inulin-C14) was perfused for another three hours, at the same rate of
perfusion. From the measurement of radioactivity in inflow and out-
flow perfusate samples, the fraction of infused inulin-C14 recovered
in each half-hour period was determined and compared to that of Na22
recovered during the same period. If Na22 were lost to the same ex-
tent as inulin-C14, the recovery fraction in both cases would be the
same. Secondly, if a significant quantity of Na22 were lost by diffusion
into the vitreous humor during the first three hours of radioactive in-
fusion, then it would probably diffuse out during the period of non-
radioactive infusion and the rate of disappearance of Na22 from the
outflow perfusate would be different from that of inulin-C14. Similar
experiments were done using C136 and inulin-H3.
Statistical Evaluation of Results
In the case of AH formation rates, which are calculated in
p1l/minute (absolute numbers), the results, are expressed as arithmetic
mean standard error ot the mean (S.E.). The percent recovery of
inulin in outflow perfusate was calculated for each experiment. Unlike
the absolute numbers, experimental errors in percentages are not
normally distributed but tend to be binomial in form. When percent
values are transformed into arcsine values (i. e. the angle whose sine
is the square root of the percentage), the transformed data are
approximately normally distributed (Snedecor, 1956). Therefore, the
average value (and S. E.) of percent inulin recovery was calculated
using arcsine transformations. However, the final values expressed
are the average percent value S. E., converted back from the final
Similarly, the percent change from the control in AH forma-
tion rate, transport of Na and C136, and inulin recovery were calcu-
lated for each experiment. Here again, the average value, S. E. and
the P value were calculated using arcsine transformations. The final
values expressed are the average percent changes S. E., converted
back from the final arcsine values. The P value was calculated by
applying the student "t" test (of significance) to arcsine transformation
values. Changes with a P value less than 0. 01 are considered signi-
ficant and a P value of more than 0. 01 is considered non-significant.
A P value less than 0. 001 is considered highly significant.
Effect of Intravenous Ouabain
The results of I.V. ouabain on AH formation rate are shown
in Table 3. A highly significant reduction in AH formation rate was
noted after 67 ig/kg of I.V. ouabain. The onset of effect on AH forma-
tion rate could be seen during the first half-hour period after the drug
injection. The effect reached its maximum during the second half-
hour period and was sustained for a further two hours of the experi-
mental period. No significant changes in blood pressure or heart rate
were seen. The lower doses, 34 or 10 pg/kg, of I.V. ouabain, did not
produce any significant change in AH formation rate.
Table 4 shows the effect of I.V. ouabain on transport of Na22
and Cl36 from plasma to AH. In half of the experiments, the transport
of Na22 from plasma to AH was determined at each dose level of
ouabain, while C136 transport was measured in the other half. A dose
of 67 p.g/kg intravenous ouabain produced a highly significant reduction
in Na22 and C136 transport from plasma to AH without any appreciable
change in plasma concentrations of Na22 (-1.9%) or C136 (-1.7%).
Quantitatively, the reduction in Na22 transport was about 75% of the
reduction in AH formation rate, while the reduction in C136 transport
was about 50% of the reduction in AH formation rate. The onset of
N ~ 0
N N -4
-4 -4 -4
Q) C! k
X t U l
-4 Q U
0- a4 A
0 0 0
V A A
-H +-H -
0 0 0
I I +
0 ) a
o 0 0
V A A
-+1 + -H
% 0 n
effect on transport of Na22 was seen in the first half-hour period and
that for Cl36 was usually seen in the second half-hour period after the
injection of ouabain. The effects reached their maximum in the second
and third half-hour periods, respectively, and were sustained up to the
end of the experiment. The lower doses, 34 or 10 pg/kg of I.V. oua-
bain, did not produce any significant change in Na or Cl. transport
from plasma to AH.
Table 5 shows the effect of I. V. ouabain on inulin recovery
which is an indication of outflow facility. None of the doses of the
ouabain tested in these experiments produced any significant change in
percentage of inulin recovery. This suggests that there was no change
in outflow facility in spite of significant reduction in AH formation rate
and transport of Na22 and Cl36 from plasma to AH.
Effect of Intracameral Ouabain
The changes produced in AH formation by intracameral per-
fusion of various concentrations of ouabain are shown in Table 6. An
intracameral concentration of 10-4M ouabain produced a highly signi-
ficant reduction in AH formation rate. The pupil was widely dilated
within the first half-hour after ouabain perfusion. This may be due to
the local effect of the high concentration of ouabain on the iris. The
effect on AH formation rate was seen in the first half-hour period,
after ouabain perfusion, reached a maximum during the second half-
hour period and was sustained up to the end of the experiment. The
intracameral perfusion of lower concentrations, 10-6 and 10-10M of
A A A
r- '1 0
-.0 V ) -
-4 N -4
*-1 r-1 r-I
I I I
0 0 0
N N 00
ouabain, did not produce a significant !change in AH formation rate.
Table 7 shows the effect of intracameral perfusion of oua-
bain on transport of Na22 and C136 from plasma to AH. There was a
significant reduction in transport of Na22 and Cl36 from plasma to AH,
after intracameral perfusion of 10-4M ouabain, without any significant
changes in plasma concentrations of Na or C136. Quantitatively, the
reduction in Na22 transport was roughly parallel to decrease in AH
formation rate, while reduction in C136 transport was about 50% of the
decrease in AH formation rate. The onset of effect on Na transport
was seen in the first half-hour period, and on Cl transport it was
usually seen in the second half-hour period, after the perfusion of
ouabain. The effects reached a maximum in the second and third half-
hour periods, respectively, and were sustained up to the end of the
experiment. No significant effect on blood pressure or heart rate was
seen after perfusion of 10-4 M ouabain, intracamerally. The intra-
cameral perfusion of 10-6 and 10-10M ouabain did not produce signifi-
cant change in Na2 and C136 transport from plasma to AH. The very
low concentration of 10 M ouabain perfusion was tested to see
whether the low concentrations of ouabain produces any stimulatory
effect on ion movement, or AH formation, as reported in CSF pro-
duction (Oppelt and Palmer, 1966). In the eye, such stimulatory effect
on AH formation or Na22 or C136 transport was not observed.
Table 8 shows that there was no significant change in inulin
recovery after ouabain perfusion. This suggests that the outflow
~ '0 r-4
I I I
0 0 0
1-4 -4 1-1
O N N
V A A
o 0 0
-H -H -H
V A A
o LA e-
0 -4 *
e .ri (
04 4j U,
C U (1
0 C) C
A A A
N N 00
1- 4 -4
facility was not changed after perfusing 10-4M ouabain intracamerally,
in spite of significant reduction in AH formation rate and of transport
of Na22 and C136 from plasma to AH.
Effect of Intravenous Acetazolamide
The results of various doses of I. V. acetazolamide on AH
formation rate are shown in Table 9. The maximum reduction of about
45% in AH formation rate was obtained after 10 mg/kg I.V. acetazola-
mide; no further reduction was noted after 30 mg/kg. The lower doses,
5 or 1 mg/kg I.V. acetazolamide, produced a smaller, but statistically
significant reduction in AH formation rate. There was no change after
0.3 mg/kg intravenous acetazolamide.' The onset of effect was seen
during the first half-hour period and reached its maximum during the
second half-hour period, after drug administration.
Table 10 shows the effect of I.V. acetazolamide on transport
of Na22 and C136 from plasma to AH. A dose of 10 mg/kg I. V. aceta-
zolamide produced a maximum reduction in transport of Na22 of about
38% and in that of Cl36 of about 22%. The administration of 30 mg/kg
I.V. acetazolamide did not produce a greater effect either on Na22 or
Cl36 transport from plasma to AH. The lower doses, 5 or 1 mg/kg
I.V. acetazolamide, produced a smaller but statistically significant
decrease in transport of Na as well as Cl36 from plasma to AH.
Quantitatively the reduction in Na22 transport was approximately
parallel to the reduction in AH formation rate, while reduction in C136
transport was about 50% of the reduction in AH formation rate, at all
O 0 0 0
V V v V
-H -H -H -H
00 C00 0 0 Ln
o 0 0 r-1 0
-+1 -H -H .-H -H
N o f sN 1-10
CO 00 CO -
+1 + + + +
M 0 C0
10 0 LO n
-4 0 N N
+4 -4 V-H -4
u < >
a) 4- J
--4 -4 --
0 0 0 0
0 0 0 0 4
V v v V A
o C C
-H +-H -+t -H +-H
I I I I I
o o o o
0 0 0 0 0
C; C; c r4
o0 0 0 0 0
-H -H -H+1 -H -H
o0 .0 V' .-4 O
* I I I
o0 : Lf ,0 'c0
o o n L ID
V V V
dose levels of the drug. The onset of effect on transport of Na 22was
observed in the first half-hour period, and that of C136 in the second
half-hour period, after acetazolamide administration. The effects
reached a maximum in the second and third half-hour periods, re-
spectively. A dose of 0.3 mg/kg I.V. acetazolamide did not produce
any significant change in NaZ2 or C136 transport.
Table 11 shows the effect of various doses of I.V. acetazo-
lamide on inulin recovery. In spite of a significant reduction in AH
formation rate and of Na22 and Cl36 transport, there was only a small
increase in inulin recovery, indicating a slight decrease in outflow
facility. The lower doses, 5 or 1 mg/kg I.V. acetazolamide, did not
produce any significant change in inulin recovery, in spite of significant
reduction in AH formation rate and transport of Na22 and C136 from
plasma to AH.
Effect of Intracameral Acetazolamide
The changes produced in AH formation after intracameral
perfusion of various concentrations of acetazolamide are shown in
Table 12. Intracameral perfusion of 10-4M acetazolamide produced
significant reduction in AH formation rate. The effect on AH forma-
tion rate was observed in the first half-hour period, reached maximum
during the second half-hour period and was sustained as long as the
drug perfusion was continued. In contrast to the effect of 10-4M
intracameral ouabain, dilatation of the pupil was not seen after
O O O O O
A A A A A
o o o o o
-H -H -H -H
%- N 00 CO .-0
" N ( N- 4
+ + + + 0
-H -H -H -+1 -H.
0m N-l 0' N N
-+1 -H -H -H -H
O-i O 0 C N
-4 i-4 i-4 -4
perfusion of 10-4M intracameral acetazolamide. The intracameral
perfusion of lower concentrations of acetazolamide did not produce
a significant change in AH formation rate.
Table 13 shows the effect of perfusing acetazolamide intra-
camerally on transport of Na22 and Cl36 from plasma to AH. After
10-4M acetazolamide there was a significant reduction in transport of
Na and Cl from plasma to AH, without any significant change in
Na22 (-3.0%) or Cl (-2.0%) concentration in plasma. Quantitatively,
the reduction in Na22 transport was about 75% of the reduction in AH
formation rate, while the reduction in C1 transport was about 50% of
the decrease in AH formation rate. The onset of effect on Na trans-
port was seen in the first half-hour period and on Cl transport, the
effect was usually observed in the second half-hour period, after aceta-
zolamide perfusion. The effects reached maximum in the second and
third half-hour periods, respectively, and were sustained as long as
the drug perfusion was continued. The lower concentrations of aceta-
zolamide did not produce any changes in Na22 and Cl36 transport from
plasma to AH.
Table 14 shows the effect of intracameral acetazolamide on
inulin recovery in outflow perfusate. In spite of the large reduction in
AH formation rate and Na22 and C136 transport, there was only a 6%
increase in inulin recovery after the intracameral perfusion of 10-4M
acetazolamide, indicating a very small effect on outflow facility. The
o N I(
o o o >
E -H -H
o 0 00 0 0 -
0 a to
0 .0 o
0 0000 m
a a k k |
W 1- ) U
N oZ o 0
0o U ) o
Cr Q) ,
0 0 C 0 < U)
-i 0. -4
o o x bo m
CdP4 V A A ) 10
So o 0 0 d>
x 1o u z
-H + C
in C V) 5 SC
0 N0 U) -
2 M + 0
0 P0 bO t
d0 0 Q
C) 0 <
C .5 .
4 cd (
* No 'C
g- t ^
o so n
ff a >
0'1 C (.
intracameral perfusion of lower concentrations, 10-6 and 10-8M
acetazolamide, did not produce any significant change in inulin
Comparison of Loss of Inulin, Na22 and C136 from Eye Chambers
When a perfusion of radioactive AH-like buffer (containing
trace quantities of inulin-C14) was followed by non-radioactive AH-
like buffer in six eyes, the rate of disappearance of inulin-C14 and
Na22 from the eye was found to be very similar (F ig. 4). The same
is true of Cl (Fig. 5).
In experiments where inulin-C14 and Na22 were perfused
through the eyes simultaneously, a similar percentage of each isotope
was recovered in the outflow perfusate. The same was true of C136
(Table 15). This suggests the validity of using inulin loss from the
22 and C136 loss.
perfusate as an index of Na and Cl loss.
I I I I I
0 0 0 0
("lI I'O/Id3) 31VSnud3d MO'IlnO NI AlIAIiOVOIOV8
RADIOACTIVITY IN OUTFLOW PERFUSATE (CPM/QIML)
n "o "& "o
01 0 0 2 S
o o o o
I I I t I I
.J > 0>
( I CI
0 0 (
The posterior-anterior chamber perfusion method, used in
the present studies for determination of AH formation, involves the
introduction of AH-like perfusate, containing inulin, into the posterior
chamber. During its passage through the eye, the perfusate.is mixed
with newly-formed AH which lowers the inulin concentration of the per-
fusate. A part of the perfusate will then pass into the blood through the
normal outflow channels of the eye and the other part will exit through
the outflow cannula. Knowing the rate of infusion, inulin concentration
in inflow and outflow perfusate, it is possible to calculate the net AH
formation rates. Oppelt (1967) discussed in detail the validity of the
various assumptions made in this system.
The first assumption made is that the puncture of the
cornea and perfusion of AH-like buffer does not alter the normal physio-
logy of the eye to such an extent as to interfere with the normal AH
formation or outflow facility. The present technique can be expected
to disturb the eye to a varying degree. As long as the puncture of the
cornea was clean, there was no leakage of AH and no obvious injury
to intra-ocular tissue; the only disturbance seen was an increase in
protein concentration in outflow perfusate. However, any experiment
in which total protein concentration exceeded 300 mg per 100 ml of
outflow perfusate, was discarded. Although 300 mg/100 ml is quite a
large concentration as compared to the normal protein concentration of
15-50 mg/100 ml of AH, it is only 4% of the plasma protein concentra-
tion which is about 7000 mg/100 ml. Moreover, the AH turnover rate,
obtained by using this technique was 1. 4% per minute (Oppelt, 1967).
This is identical to the value estimated in cats by Davson and Spaziani
(1960), using turnover rates of Na24 and close to the value of 1. 5% per
minute reported by Barany (1955). This may be taken as evidence that
the normal physiology of the eye is not disturbed enough to affect AH
formation rate significantly. Furthermore, the present study was de-
signed to study the effect of ouabain and acetazolamide on AH dynamics.
Therefore, even if the blood-aqueous barrier was disturbed to some
extent, the administration of the drugs did not make any further change
in the blood-aqueous barrier, as was judged by absence of any further
increase in protein concentration of outflow perfusate. Thus, the change
in AH dynamics after drug administration was due to the effect of the
drug and not due to the change in blood-aqueous barrier. Some confi-
dence regarding the validity of the present method can be obtained from
the fact that the present technique and its slight modification have re-
cently been used for determination of AH formation rate by various
other workers (Wallinder and Bill, 1969a, 1969b; Macri, 1967).
The second assumption in this system is that inulin acts as
an inert molecule in the eye. It does not bind to the ocular tissues, is
not metabolized in the eye, does not diffuse into the ocular tissues to a
significant degree, and is not transported out of the eye. Oppelt (1967)
showed that infused inulin could be recovered up to 100% when the outflow
perfusate was collected at abnormally low intra-ocular pressure. This
suggests that inulin does indeed act as an inert molecule in the eye.
Moreover, if any of the above possibilities were occurring, the inulin
concentration in the outflow perfusate would be smaller than that which
is due to dilution of newly-formed AH. This would cause an overesti-
mation of AH formation rate. However, the AH turnover rates deter-
mined by using this method were comparable to those obtained by other
methods (Oppelt, 1967), indicating that inulin is not lost to any signifi-
cant extent by any of the above-mentioned possibilities. Furthermore,
inulin behaves as an inert substance in kidney (Pitts, 1965) and there is
no evidence of its metabolism, active transport, binding or passive
diffusion in any other mammalian tissue.
The third assumption is that the inulin containing inflow per-
fusate is adequately mixed with the newly-formed AH. If this does not
occur, one would expect irregular and constantly changing inulin con-
centrations in outflow perfusate. This was not the case in experimental
periods of up to six hours (Oppelt, 1967) Moreover, when the radio-
active perfusion (containing inulin-C14) was followed by non-radioactive
perfusion, inulin from the outflow perfusate disappeared rapidly,
without any indication of reappearance (Fig. 4 and 5), which one would
expect, if it were not completely mixed. Furthermore, when fluorescein
was added to the inflow perfusate, the dye mixed immediately and com-
pletely in the whole eye fluid after starting the perfusion, as was ex-
amined visually. Recently, Wallinder and Bill (1969a) used an anterior
chamber perfusion method for determination of AH formation rate in
monkeys. They showed that there was a complete mixing of the buffer
with AH formed in the eye, as long as the cannulas were 4 mm. or more
apart and the bevels did not face each other. In the present experiments,
the cannulas were more than 4 mm. apart and the bevels did not face
each other. In addition, the inflow cannula in the present system was
behind the iris in the posterior chamber. All these evidences suggest
that there was an adequate mixing of the inulin with the newly-formed
Transport of Ions from Plasma to AH
In the present experiments the posterior-anterior perfusion
technique has been used for the study of the transport of Na22 and C136
ions from plasma to AH. If the plasma concentrations of Na22 or C136
are maintained at a steady level, the appearance of these isotopes in
the outflow perfusate of the system would represent the transport of Na22
or C136 from plasma to AH. Of course, some NaZ2 or C136 would be
lost through the normal outflow channels of the eye, which can also be
accounted for by measuring the total quantity of inulin recovered in out-
flow perfusate, calculating the quantity of inulin lost through the normal
outflow channels and assuming that Na22 and C136 are lost to the same
extent as inulin (Table 15). Thus, in the present studies, the total
transport of Na22 and Cl36 from plasma to AH was computed by taking
into consideration the concentration found in the outflow perfusate and
adding to it the quantity lost through the normal outflow channels of the
eye. It should be noted that the same calculations of inulin recovery
were used to determine the outflow facility.
One assumption in using this system for the study of the ion
transport from plasma to AH is that the newly transported ion is mixed
properly with the inflow perfusate. The arguments mentioned for proper
mixing of newly-formed AH with the inflow perfusate also hold true for
mixing of newly-transported ions (from plasma to AH) with inflow per-
The advantages of using the posterior-anterior chamber
perfusion technique for determining drug effects on AH formation rate,
transport of ions from plasma to AH and outflow facility are as follows:
(a) The technique allows one to measure AH formation rate
directly, independent of changes in intra-ocular pressure.
(b) One can detect changes occurring in ion transport at
the AH secretion site by sampling outflow perfusate instead of taking
AH samples from anterior and posterior chambers and extrapolating
the results to the secretion site by elaborate kinetic analysis.
(c) In this system it is possible to detect even iso-osmotic
changes in ion transport from plasma to AH which are not detected by
analyzing ionic composition of anterior or posterior AH. It should be
noted that all previous in vivo studies on ion secretion were done in a
manner that would make detection of such iso-osmotic changes im-
(d) The changed in the outflow facility can be determined at
a constant pressure and constant rate of perfusion.
(3) The effect of a drug on AH formation rate, transport
of ion from plasma to AH and outflow facility of the eye can be deter-
(f) In determination of changes in AH dynamics after admini-
stration of a drug, each eye can be used as its own control.
(a) Two needles are to be inserted into the eye; therefore,
the method cannot be used clinically.
(b) The technique does disturb the blood-aqueous barrier
to a certain extent, as seen by an increase.in protein concentration of
(c) If the primary effect of the drug is on intra-ocular
pressure, it would not be detected by this method.
The direct measurement of AH formation rates in cats
indicates that a high dose (67 pg/kg) of I.V. ouabain inhibits the rate
of AH formation by about 40%. The results are in agreement with those
of Simon et al. (1962) and Macri et al. (1966) and also confirm the pre-
vious results from this laboratory (Oppelt and White, 1968). However,
they do not agree with those of Langham and Eakin (1964), who used
more indirect methods for measurement of AH formation rate. The
difference may also be due to the fact that they made only one determina-
tion of AH dynamics, 20 minutes after injection of the drug. Becker
(1963) did not find any change in intra-ocular pressure in rabbits after
a dose of 250 p.g/kg intravenous ouabain. This may be due to'species
The 40% decrease in AH formation rate in cat eyes after
a high dose of I.V. ouabain was accompanied by about a 30% decrease in
transport of Na+ from plasma to AH. The results are qualitatively in
agreement with those of Cole (1961), who reported a 60% decrease in
Na+ influx in rabbit eye after administration of ouabain. The quantita-
tive difference may be due to species difference. The 40% decrease in
AH formation rate in the cat after high intravenous ouabain was
accompanied by a 20% decrease in transport of Cl- from plasma to AH.
The results are in agreement with those of Cole (1960), who found a
reduction in Cl- influx which was less pronounced and occurred more
slowly than that of Na+, after giving DNP or fluoroacetamide.
In spite of significant changes in AH formation rate and
in transport of Na+ and Cl" from plasma to AH after a high dose of I. V.
ouabain, there was no significant change in outflow facility. This is in
agreement with the results of Simon et al. (1962), who found no signifi-
cant change in outflow facility after oral administration of digoxin to man.
Intracameral perfusion of 10-4M ouabain produced about
a 40% decrease in AH formation rate. Langham and Eakin (1964) did not
find any change in intra-ocular dynamics after direct infusion of 10-5M
ouabain into the posterior chamber of the rabbit eye, at a rate of 3 p.1/
minute, for 30 minutes. The absence of effect in their studies may be
due to the low dose used and species difference. Waltzman and Jackson
(1964) found a decrease in intra-ocular pressure after subconjunctival
injections of ouabain in rabbits. Becker' (1963), and Bonting and Becker
(1964) reported that intravitreous injection of ouabain produced a signi-
ficant decrease in intra-ocular pressure in rabbit. The maximum effect
occurred four to five days after injection. These reports indicate that
ouabain, if allowed to concentrate locally in the eye, can produce
changes in AH dynamics. The present results are in agreement with
The 40% decrease in AH formation rate in cat after 10-4M
intracameral perfusion of ouabain was accompanied by a parallel de-
crease in transport of Na+ and a 20% decrease in Cl transport from
plasma to AH without any significant change in outflow facility.
After both intracameral and I.V. ouabain, the reduction
in Cl" transport was about 50% of the reduction in Na22 transport. The
difference may possibly be due to a reduction of HCO3 transfer into
the AH. However, this possibility has not been investigated.
Ac eta zolamide
The direct measurement of AH formation rate in cats
indicates that a dose of 10 mg/kg or more I.V. acetazolamide inhibits
AH formation rate by about 45%. The results are in agreement with
those of Wistrand (1959), who reported a 35% decrease in outflow
pressure (intra-ocular minus episcleral venous pressure) in rabbits,
with doses above 10 mg/kg of acetazolamide, and those of Becker (1959),
who reported a 58% decrease of aqueous flow in rabbits after 100 mg/kg
of intravenous acetazolamide, and a 52% decrease in aqueous flow in
man, after 500 mg oral acetazolamide.
Recently, Walinder and Bill (1969b) obtained an 80% de-
crease in AH formation rate, as calculated from dilution of inulin per-
fused through the anterior chamber of monkeys, after 100 mg/kg
acetazolamide. After 10 mg/kg acetazolamide, AH formation rate was
reduced by 30%. These results confirm the present results in cats.
The quantitative difference may be due to species variation. However,
the results do not agree with those of Macri et al. (1965), who did not
find any change in AH turnover rates after 100 mg/kg intravenous
acetazolamide and only a small decrease after 250 mg/kg intravenous
acetazolamide in the cat. They did not always find a decrease in
intra-ocular pressure after acetazolamide doses which produced a
decrease in AH turnover rates. They did not use acetazolamide in
usual therapeutic doses.
The 45% decrease in AH formation rate in cat eyes after
10 mg/kg or more of intravenously administered acetazolamide was
accompanied by an approximately parallel decrease in transport of Na+
from plasma to AH. Becker (1959) studied the turnover rates of Na+
by determining the rate of accumulation in AH of systemically injected
Na2. He did find a lag in the rate of accumulation of Na in both
anterior and posterior chamber after injecting acetazolamide into
rabbits. He dismissed this difference as experimental error. More-
over, Becker (1959) pointed out that the handicaps of the method were
the large fraction of Na+ entering the eye by diffusion and the failure
of carbonic anhydrase inhibition to alter the steady state concentration
significantly. In the present experiments, the changes in transport of
Na+ from plasma to AH could be detected, even if the changes were
iso-osmotic, without much alteration in relative Na concentration.
Davson and Luck (1957) did not find any change in AH/plasma water
ratio of Na24 in rabbits after 100 mg/kg of intravenous acetazolamide.
Similarly, they did not find any change in turnover rates of Na24 in dog
after giving a large dose of intravenous acetazolamide. In cats they
found a very small fall in Na+ concentration in AH, by 0. 8 and 1. 3% in
two experiments, while Na+ concentration "in plasma stayed constant or
was slightly increased. In the third experiment both AH and plasma
concentration of Na+ fell slightly. If the decrease in Na+ transport is
approximately parallel or iso-osmotic with decrease in AH formation,
one would not expect any significant change in AH/plasma water ratio
or AH concentration of Na+ after administration of acetazolamide.
The 45% decrease in AH formation rate in the cat after
10 mg/kg or more of intravenously administered acetazolamide was
accompanied by a 23% decrease in transport of Cl- from plasma-to AH.
In spite of the very large decrease in AH formation rate,
and in transport of Na and Cl- from plasma toAH after 30 mg/kg or
more I.V. acetazolamide in the cat, there was a small decrease in
outflow facility, as determined by inulin recovery in the outflow per-
fusate. The results are in agreement with those of Linner (1966), who
reported that acetazolamide produced a decrease in rate of aqueous
flow followed by a compensatory increase in outflow resistance (or
decrease in outflow facility) in humans. The reduction in flow was
dominating and the net result was a fall in intra-ocular pressure. The
present results are also in agreement with those of Sears (1960), who
found an increase in outflow resistance and a decrease in aqueous in-
flow after administration of 100 mg/kg intravenous acetazolamide to
rabbits. However, Galin and Harris (1966) are of the view that although
acetazolamide primarily reduces aqueous flow, it lowered intra-ocular
pressure in certain glaucomatous patients by improving outflow facility,
as determined by tonography.
An intracameral perfusion of 10-4M acetazolamide in the
cat produced a 36% decrease in AH formation rate, 27% decrease in
transport of Na+ and 18% decrease in movement of Cl" from plasma
to AH. There was a decrease in outflow facility of about 6%. The
intracameral perfusion of lower concentrations, 10-6 and 10-8M
acetazolamide, did not produce any significant change in these para-
meters. The inhibitory effect of 10-4M intracameral acetazolamide on
AH formation rate and transport of Na+ and Cl" ions is a surprising
finding, as many workers did not find any effect of locally applied
acetazolamide. The lack of effect of locally applied acetazolamide may
be explained as follows:
(a) Acetazolamide may not be absorbed through the cornea
in sufficient quantity to produce a concentration of 10-4M in and around
the eye chambers.
(b) Even if the acetazolamide or any other carbonic an-
hydrase inhibitor, after local application to the eye, is absorbed in
sufficient quantity into the eye, it may be removed from the chambers
at such a rapid rate that 10-4M concentration is never attained in the
(c) The effect of intracameral acetazolamide may be
partly due to its local effect on the blood-aqueous barrier.
After both intravenous and intracameral (10-4M) acetazola-
mide, the reduction in Cl" transport from plasma to AH was about 50%
of the reduction in Na+ transport. This difference is probably due to the
reduction in HCO3 accumulation into AH. In fact, such an effect of
acetazolamide has been reported in rabbit and many other species
Mechanism of AH Formation and the Effect of Drugs
As mentioned in the introduction, AH formation may be
considered as consisting of three processes:
(a) Diffusion from plasma
(b) Ultrafiltration across blood-aqueous barrier
(c) Specialized secretion, possibly involving active trans-
port of at least one constituent (possibly Na+, Cl" or HCO5 ion), by the
epithelial covering of the ciliary process.
Any change in AH formation rate and movement of ions
after administration of a drug could be'explained in terms of one or
more of these processes.
Cole (1960) obtained a 75% inhibition of Na+ influx after
the injection of DNP or fluoroacetamide into the carotid artery of
rabbits. This may be considered as an actively secreted fraction be-
cause of its energy dependence. The remaining 25% of Na+ may be
accumulated in AH by ultrafiltration, as the contribution by passive
diffusion should be very little, due to the absence of a concentration
gradient between plasma and AH. The ultrafiltration fraction could
possibly be abolished by decreasing arterial pressure in the animal.
In fact, Barany (1947) found up to about 28% decrease in Na24 accumu-
lation in rabbit AH when the arterial pressure of one eye was reduced
by unilateral clamping with a carotid loop. Since the blood-aqueous
barrier is readily permeable to water, entry of solutes and water
approximates iso-osmotic transfer. Cole (1961) reported that not only
the influx of Na+ and water was reduced by DNP (70-75%0) and ouabain
(60%), but also the potential difference across the blood-aqueous
Through analysis of the rates of accumulation of Na24 and
Cl36 in the posterior chamber, Kinsey (1960) mathematically analyzed
the mode of entry of ions from plasma to posterior chamber. He con-
cluded that approximately two-thirds of AH Na+ enters the posterior
chamber of rabbits by unidirectional secretion.
Holland and Stockwell (1967) studied the Na+ transport in
ciliary body in vitro. They found that, in vitro, the ciliary body
transports Na ions inwardly (AH side) against an electrochemical
gradient. This is in agreement with the hypothesis of active transport.
In the present study, it has been shown that ouabain, in
doses of 67 p.g/kg I.V. or 10-4M intracamerally, produces about a
40Coreduction in Na transport from plasma to AH, along with an
approximately parallel reduction in AH formation rate.
The above observations suggest that a Na pumping mech-
anism participates in AH secretion. If this is the case, the question
arises what type of pump is present in the ciliary body. Ussing (1964)
described the following three general types of sodium pumps:
Sodium-potassium exchange pump. --This has been demon-
strated to be present in cell membranes of erythrocytes, nerve and
muscle. It is dependent on Na-K-activated ATPase,present in
membranes. It helps to maintain the resting potential of the cells by
pumping Na+ out and K+ into the cell. The pump is sensitive to inhibi-
tion by ouabain.
Electrogenic sodium pump. --Sodium is transported without
any concomitant transport of another ion of the same or opposite charge.
It has been reported to be present in the toad bladder (Essig and Leaf,
1963), where Na+ transport proceeds from the lumen to the serosal
side in the presence of choline -Ringer bathing medium (without K+) on
the serosal side. Whether or not the electrogenic Na+ pump is sensi-
tive to inhibition by ouabain has not been investigated.
Sodium-anion pump. --Na+ is transported together with an
anion. In this system sodium and anion pumps may be completely in-
dependent or coupled. The rat intestine and gall bladder of carp and
rabbit transports Na+ actively, but they do not develop enough potential
difference to account for the transport of anion associated with Na+.
Therefore, it has been suggested that in addition to active Na+ trans-
port, these organs also possess an active anion transport mechanism
(Ussing, 1964). Other evidence in various systems for such a sodium-
anion pump is discussed later. Macrobbie and Ussing (1961) showed
that ouabain inhibited the perme. ity of frog skin to water and Cl .
Coopersteine (1959) reported that ouabain inhibited Cl" transport into
the frog stomach. In the present studies, ouabain reduced both Na+
and Cl- transport from plasma to AH. Therefore, the ouabain effect
is not specific to Na-K-exchange systems, but could also inhibit other
Proposed Mechanism of AH Formation
Although the mechanism of AH formation is far from clear,
on the basis of the results of the present experiments and other avail-
able evidence, the following scheme of ion secretion is proposed
(Fig. 6). Mechanisms for concentrating at least three ions (Na Cl"
and HCO3 ) are present, possibly on the epithelial surface of the
ciliary process cell. The ratio of secretion of one ion to the other
differs in various species and so does the composition of AH.
Na+ concentrating mechanism
In the present experiments, 67 pg/kg I.V. ouabain and
10-4M intracameral ouabain produced an approximately 40% reduction
in transport of Na+ from plasma toAH along with a parallel decrease in
AH formation rate. This suggests that ouabain sensitive Na+ concen-
trating mechanism is involved in AH formation. Other evidence for the
presence of a Na concentrating mechanism in the eye has been dis-
It has been proposed that Na-K-activated ATPase is in-
volved in the Na+ concentrating mechanism of AH formation (Bonting
et al., 1961). In the present experiments, a maximum dose of 67 p.g/kg
ouabain was used I.V. Assuming that ouabain is uniformly distributed
in total body water, 67 pg/kg I.V. ouabain would produce a concentration
of 10-7M in the ciliary processes. 10-7M ouabain would certainly
produce about 50% inhibition of Na-K-activated ATPase. Similarly,
10-4M intracameral ouabain (assuming that 10-4M concentration is
reached in ciliary processes) would produce 100% inhibition of Na-K-
activated ATPase. However, Na-K-activated ATPase helps to main-
tain the resting potential of cells by exchanging intracellular Na+ with
extracellular K+. If a Na-K exchange system were involved in the
formation of AH, then a high Na concentration in AH should be accom-
panied by a low K+ concentration, relative to plasma dialysate. How-
ever, this is not the case. On the other hand, AH has an excess of
HCO3 and/or Cl along with an excess of Na Moreover, after in-
jection of DNP and fluoroacetamide into the carotid artery of the rabbit,
Cole (1960) found a decrease both in Na+ and K+ influx from plasma to
AH. If the Na-K exchange were involved, then a decrease in Na+ in-
flux should be accompanied by a decrease in K+ outflux, thus raising
the K+ concentration in AH. However, this was not the case. This
suggests that the Na+ concentrating mechanism present in the eye does
not involve Na-K exchange but a different system for AH formation.
It would be of interest to mention here that K+ concentration
in CSF is significantly lower than that in plasma. This suggests that
the Na-K exchange system may have some role in CSF formation. In
addition, there are significant differences in the way ouabain affects
the CSF. 10-6M ouabain perfused intraventricularly in the cat produced
about a 50% reduction in CSF production rate (Vates et al., 1964),
while in the present experiments 10-6M intracameral ouabain had no
effect on AH formation rate. Intraventricular perfusion of 10"10M
ouabain produced about 45% stimulation in CSF production rate (Oppelt
and Palmer, 1966). In the present experiments, 10-10M intracameral
ouabain produced no such stimulation of AH formation rate. These
results suggest that the effect of ouabain on CSF production may be
mediated through Na-K-activated ATPase, while its effect on AH
formation is probably mediated through another mechanism;
Moreover, if Na-K exchange were involved in AH forma-
tion, there should be little or no change in anion (Cl or HCO3 ) con-
centration or influx accompanying the changes in Na+ influx. However,
Cole (1960) found a 50% reduction in Cl- influx along with a 70% re-
duction in Na+ influx after administration of DNP or fluoroacetamide
to rabbits. In the present study, in addition to a 40% reduction in Na+
transport, high doses of ouabain produced a 20% reduction in Cl- trans-
port from plasma to AH. The quantitative difference between reduction
in Na+ and Cl" transport is probably due to the decrease in accumula-
tion of HCO3 However, this possibility has not been investigated.
Holland and Stockwell (1967) measured membrane potential or short-
circuit current (SCC) concurrently with Na+ influx and outflux across
cat ciliary body in vitro. They found that in non-short-circuited mem-
brane, where membrane electromotive force assisted Na+ influx, Na+
influx was two to three times larger than that which could be explained
by SCC. This suggests that cat ciliary body transports some anion
along with Na+. All these results cannot be explained by a Na-K-
exchange system involving Na-K-activated ATPase.
However, the present results and the results of previous
workers can be explained by the presence of a Na+ and Cl" pumping
mechanism in ciliary processes. The transport of Na+ and Cl" may
be coupled or independent. No molecular (enzymic) model is known for
Cl concentrating mechanism
In the present experiments 67 p.g/kg I.V. and 10-4M intra-
cameral ouabain produced approximately a 20% reduction in transport
of Cl- from plasma to AH. This suggests that Cl mechanism is in-
volved in AH formation. Cl- is generally considered to move passively
through animal cells. Evidence is now accumulating to indicate that
certain tissues actively transport Cl- (Jorgensen et al., 1954;
Zadunaisky et al., 1964; Hogben, 1955; Romeau and Maetz, 1964;
Wheeler, 1963; Lasansky and deFish, 1960; Hogben et al., 1960;
Recently Cole (1969) produced an evidence for the presence
of an active transport of Cl in the ciliary epithelium of the rabbit.
He studied Cl- transport in the isolated ciliary body of the rabbit. He
found negative values for SCC when ciliary body was in Na- free med-
ium containing Cl-, indicating active transport of Cl~. SCC was
temperature dependent in Cl~ containing medium but not in mannitol
medium. This suggests the presence of a metabolically driven trans-
port system for Cl~. Thiocyanate inhibited the increased SCC in NaC1
medium, which means that it either stimulated Na+ transport or in-
hibited Cl- transport. However, similar concentrations of thiocyanate
had no effect on SCC in Cl" free median. This s~o~ that thiocyanate
blocked Cl transport. All his experiments indicate that active trans-
port of Cl- occurs across the ciliary epithelium from the stromal to
the posterior chamber side. After mathematical- analysis of the mode
of entry of various ions into rabbit AH, Kinsey (1960) concluded that
approximately one-third of Cl- enters the posterior chamber of the
rabbit by secretion.
Cl" concentrating mechanism in certain tissues is sensitive
to inhibition by ouabain (Coopersteine, 1959 ; Zadunaisky et al., 1964).
In the. present experiments high doses of ouabain produced a significant
reduction in transport of C1 from plasma to AH.
The above-mentioned evidence suggests that Cl transport
mechanism is involved in AH formation. The mechanism is sensitive
to inhibition by ouabain. This may be due to the primary effect of
ouabain on the Cl" transport mechanism or it may be secondary to its
effect on the Na transport mechanism. However, the molecular
(enzymic) mechanism for transport of Cl- is not known.
Cl- secretion (with or without Na+) has been shown to be
sensitive to inhibition by carbonic anhydrase inhibitors in various
systems. In from stomach, acetazolamide, in combination with
histamine, decreased electromotive force (emf) and inhibited,
partially, the Cl~ transport, while H+ secretion continued at an
almost normal rate. On the other hand, CL 8490, a non-carbonic
anhydrase inhibitor, had no effect on gastric emf (Durbin and Heinz,
1958). Nechay,et al. (1960) showed that carbonic anhydrase inhibition
reduced NaCl secretion in avian glands in sea gulls in which an I.V.
saline infusion was maintained. Wheller et al. (1969) reported that
carbonic anhydrase inhibitors decrease the absorption of NaC1 in
isolated gall bladder preparations, in absence of NaHCO3 in the bath-
ing solution. Recently, Broder and Maren (1969) found a decrease
in Cl~ transport from plasma to CSF after administration of acetazo-
lamide to the cat. Maren et al. (1969) found some decrease in Cl"
secretion in CSF of the dogfish.
In the present experiments 10 mg/kg or more of I.V.
acetazolamide produced a 20% decrease in transport of Cl" from plasma
to AH. This suggests that carbonic anhydrase is involved in secretion
of Cl" into AH, possibly by setting the intracellular pH at an optimal
point. The mechanism of this effect is still a matter of speculation.
HCO3 concentrating mechanism
HCO3 secretion or absorption has been demonstrated in
a variety of vertebrate tissues. The enzymic mechanism for HCO3
secretion or absorption involves catalytic hydration of COC in the
presence of carbonic anhydrase. Maren (1967) has written an exhaus-
tive review on the physiology and pharmacology of HCO3 secretion
and absorption in various animal tissues, both in the presence and
absence of carbonic anhydrase inhibitors.
In the eye, HCO3 produced by COZ accumulates in the
posterior AH bordering the ciliary cells (Fig. 6). As shown by
Kinsey and Reddy (1959) and analyzed by Maren (1967), the rate of
COZ hydration and HCO3 accumulation in posterior AH depends on the
presence of carbonic anhydrase (Fig. 2). The residual H+ may diffuse
back into the plasma. HCO3 accumulation in the posterior chamber is
accompanied by transport of Na+ (possibly active, as discussed above),
as shown in Fig. 6.
When acetazolamide decreases accumulation of HCO3 into
the AH (not measured in the present experiments), the secretion of an
accompanying cation would also decrease. In fact, the present results
show a significant decrease in Na+ transport from plasma to AH, after
acetazolamide administration. If the osmolarity of the AH is to be
maintained, there should be a concurrent decrease in water transport
along with a decrease in Na+ transport. In the present studies, it was
observed that the decrease in Na+ transport from plasma to AH was
approximately parallel to the decrease in AH formation rate.
It would be of interest to mention here that a mechanism of
HCO3 accumulation similar to that of AH is also present in CSF.
Recently, Broder and Maren (1969) found that acetazolamide decreased
HCO3 accumulation in CSF of the cat. Maren et al. (1969) also
observed decreased HCO3 accumulation in dogfish CSF, after aceta-
zolamide administration. Quantitatively, the decrease in HC03
accumulation after acetazolamide was less in dogfish CSF than in cat
However, Davson and Luck (1957), from their studies in
rabbits and other species, concluded that after acetazolamide admini-
stration, AH composition closely approaches the composition of a
plasma dialysate of that species. They interpreted this to mean that
acetazolamide "poisons the system". If it is assumed that a portion of
AH is produced by ultrafiltration and the remaining portion by special
secretary processes, their results can be interpreted as follows.
Acetazolamide inhibits the special secretary process part of AH forma-
tion by decreasing accumulation of HCO3 and possibly also Cl, accom-
panied by a decrease in Na+ and water transport, while the ultrafiltra-
tion part functions as usual or is increased because of lowered intra-
ocular pressure. If this is the effect of acetazolamide, only a small
variable or no change in ionic composition of AH would be detected,
although significant decrease in AH formation with iso-osmotic changes
in ion secretion may be occurring. Such changes could not be noted
in their experiments. However, posterior-anterior chamber perfus-
ion, as used in the present experiments, would detect such iso-osmotic
changes in ion secretion readily. In fact, present results have shown
that in cat, acetazolamide decreases transport of Na+ from plasma to
AH. This effect may be secondary to or coupled to a decrease in
HCO3 accumulation in AH (not studied in the present experiments).
On the basis of the evidence presented above, it is sug-
gested that AH formation involves the secretion of all the three major
ions, Na Cl" and HCO3 (Fig. 6).
Relationship between cation and anion secretion
The present results and the results of other workers
(referred above) can be explained by the presence of Na+, Cl- and
HCO3 concentrating mechanisms in the eye, which are involved in the
formation of AH. The question areises whether these mechanisms are
independent or interdependent. It is difficult to decide this point be-
cause of insufficient data. However, the present results are consis-
tent with the idea that Na+ and Cl- mechanisms are coupled because
when transport of one ion was inhibited, the transport of another ion
was also decreased. Additional evidence for this concept has been
In summary, it is proposed that active transport of Na+
is involved in AH formation. This is accompanied in part by transport
of Cl" (possibly coupled) and in part by accumulation of HCO3 due to
catalyzed hydration of COZ (Fig. 6).
1. The effect of I.V. and intracameral ouabain and
acetazolamide on aqueous humor (AH) formation rate, transport of Na22
and C136 from plasma to AH, and outflow facility was studied in
anesthetized cats. AH formation rate was determined by dilution of
inulin-C14 or inulin-H3 in AH-like buffer which was perfused continu-
ously from the posterior to the anterior chamber. The change in the
transport of ions was determined by injecting Na or Cl I.V. and
determining the change in the rate of their appearance in the outflow
perfusate. The change in outflow facility was estimated by measuring
the change in total quantity of inulin recovered in outflow perfusate.
2. After 67 pg/kg I.V. ouabain, there was about a 40%
decrease in AH formation rate, a 30% reduction in transport of Na22
and a 20% reduction in transport of C136 from plasma to AH, without
any significant change in outflow facility. The lower doses, 34 .g/kg
and 10 pg/kg, produced irregular and non-significant changes in these
3. When 10-4M ouabain containing buffer was perfused
intracamerally, there was about 40% reduction in AH formation rate
and transport of Na2z from plasma to AH, along with a 20% reduction
in Cl36 transport, without any significant change in outlfow facility.
The intracameral perfusion of lower concentrations, 10-6 and 10-10M
ouabain, produced no significant changes in any of these parameters.
4. After a single I.V. injection of 10 mg/kg or 30 mg/kg
acetazolamide, there was about 45% decrease in AH formation rate,
along with a 38% inhibition in transport of Na22 and 23% inhibition of
C136 transport from plasma to AH, with a slight decrease in outflow
facility. Lower doses of I. V. acetazolamide had a smaller but signi-
ficant effect on AH formation rate and transport of Na22 and C136 from
plasma to AH. Doses less than 1 mg/kg I.V. acetazolamide did not
produce any significant change in any of the parameters tested.
5. After intracameral perfusion of 10-4M acetazolamide,
there was a 36% reduction in AH formation rate, 27% reduction in
transport of Na22 and 18% reduction in transport of C136 from plasma
to AH, with a 6% decrease in outflow facility. The perfusion of lower
concentrations, 10-6 or 10-8M intracameral acetazolamide, did not
produce any significant change in either AH formation rate, ion trans-
port or outflow facility.
6. Intracameral perfusion of Na2z or C136 along with
inulin-C14 or inulin-H3 in AH-like buffer, followed by non-radioactive
buffer, showed that Na22 and Cl36 disappeared from the outflow per-
fusate as rapidly as inulin. When inulin and NaZ2 or C136 were per-
fused intracamerally, there was no significant difference between %
recovery of inulin and Naz or Cl in outflow perfusate. This indi-
cates that the loss of Na22 or Cl36 by diffusion into the eye was similar
to that of inulin, therefore probably insignificant.
7. Analysis of the data indicates that the inhibitory effect
of ouabain on AH formation and ion transport is probably mediated
through inhibition of a sodium chloride pumping mechanism present in
the ciliary body, and not through inhibition of the Na-K-exchange pump.
8. The inhibitory effect of acetazolamide on AH formation
rate and Na transport may be partly due to a reduction of HCO3
accumulation caused by inhibition of carbonic anhydrase present in
ciliary body and to a direct effect on Cl" transport.
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Lal Chand Garg was born January 22, 1933, at Malerkotla,
India. In August, 1948, he was graduated from the State High School,
Barnala, India. In April, 1954, he received the degree of Bachelor
of Pharmacy from Panjab University, India. In June, 1956, he re-
ceived the degree of Master of Pharmacy from Panjab University,
India. From 1956 until 1963 he served at the College of Veterinary
Science, Mhow, India, as an instructor and later on as an Assistant
Professor of Pharmacology. In 1963 he joined the Department of
Pharmacy, Panjab University, Chandigarh, India and worked as a
Lecturer in Pharmacology until August, 1965. In September, 1965, he
came to the United States and enrolled in the Graduate School of the
University of Florida. He worked as a graduate assistant in the
Department of Pharmacology and Therapeutics until the present time
and has pursued his work towards the degree of Doctor of Philosophy.
Lal Chand Garg is married to the former Shakuntla Devi
Goyal and is the father of two children. He is past Secretory of the
India Club, University of Florida, and Panjab University Pharma-
ceutical Society, India.
This dissertation was prepared under the direction of
the chairman of the candidate's supervisory committee and has been
approved by all members of that committee. It was submitted to the
Dean of the College of Medicine and to the Graduate Council, and was
approved as partial fulfillment of the requirements for the degree of
Doctor of Philosophy.
Dean. Coll6e of icine
Dean, Graduate School
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