Sodium ion transport of the
ciliary body in vitro
Monte G. Holland and Marion Stockwell
Sodium ion influx and outflux were measured sinmltaneously, with two radioactive isotopes,
in the cat ciliary body isolated in vitro. Measurements of membrane potential or short-circuit
current were made concurrently with the isotope studies. In the non-short-circuited membrane,
where the membrane electromotive force assisted sodium influx, the sodium ion current was
two to three times larger than the estimated average short-circuit current, implying the coexistence of an anion transport. In the continuously short-circuited membrane, in tohich the
membrane electromotive force was eliminated, a net sodium ion influx persisted and was approximately equal to the short-circuit current. Thus, the in vitro cat ciliary body membrane
appears to function as a mixed sodium-anion pump in the non-short-circuited state, but mainly
as a sodium ion pump ivhen short circuited.
that approximately two thirds of aqueous
humor sodium enters the posterior chamber
by secretion.7 However, absence of a
diminished aqueous humor sodium turnover rate following acetazolamide administration8 highlights the need for a more
direct determination of sodium ion flux.
Using two radioisotopes of sodium to
measure influx and outflux simultaneously
through the isolated, surviving frog skin,
Levi and Ussing9 showed unequivocally
that a net influx of sodium existed in the
absence of electromotive or concentration
gradients, thereby directly demonstrating
an active transport of sodium. They also
demonstrated that the electric current
generated by the short-circuited membrane
was equal to the isotopically measured
sodium ion current.
Similar techniques have been applied
in the present investigation. The isolated,
surviving ciliary body has been prepared
in vitro as a membrane separating reservoirs filled with identical fluids. While
measuring the transciliary membrane potential or short-circuit current, —Na and 2lNa
.he demonstration of a sodium-potassium-activated adenosine triphosphatase
(transport-ATPase) in the ciliary epithelium1' - and the observation that inhibition
of this enzyme with ouabain lowers intraocular pressure3 and diminishes aqueous
humor flow,'1 suggest that active transport
of sodium ions by the ciliary epithelium
plays an important role in aqueous humor
formation. Previous investigations have
established that aqueous humor sodium
ion concentration exceeds that required for
dialysis equilibrium with plasma,5'G and
from the analysis of aqueous humor turnover rates in vivo it has been deduced
From the Department of Ophthalmology, Tulane
University School of Medicine, New Orleans,
La.
This investigation was supported by Research
Crant B2212 and Career Development Grant
l-KB-NB-22,651 from the National Institutes of
Health, United States Public Health Service,
and also in part by National Institutes of Health
Institutional Research Grant 29784.
401
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Investigative Ophthalmology
August 1967
402 Holland and Stockwell
to current source
to electrometer
to current
source
(4) polyethylene
Kreba-Agar bridges
to oxygen
Calomel celb (4)
baffle
to constant temperature
water b a t h
a. b. c.
Fig. 1. Left: Method used to mount the pars plicata of the ciliary body as a membrane. Nylon
gauze mesh (a) is used to support the ciliary body membrane (b) over an elevated collar which
surrounds a central aperture 3.6 mm. in diameter; c is a plastic ring used to clamp the tissue
in the manner of an embroidery hoop. Silicone grease is used around the collar to achieve a
good seal. Right: Chamber showing mounted membrane immersed in, a constant temperature
bath and containing agar-salt bridges for potential and short-circuit current measurements.
Bridges used for potential measurement are positioned close to the membrane. Reservoir fluids
are kept well mixed and oxygenated by small bubbles from porous glass gas dispersion tubes.
Each reservoir contains 3 ml. of Krebs' III medium with isotopically labeled sodium.
isotopes were utilized to determine influx
and outflux simultaneously through the
membrane. By these methods sodium ion
transport has been studied in inert cellulose
membranes, in non-short-circuited, and in
short-circuited living ciliary body membranes.
Methods of procedure
Surgical technique of isolating the membrane.
Mature cats are anesthetized with pentobarbital.
An eye is enucleated, bisected equatorially, and
the vitreous humor removed from the anterior
half. The zonular fibers are cut under direct visualization using 8x magnification and the lens
removed in capsule. The anterior segment is sectioned meridionally and a cyclodialysis done while
maintaining the tissue under Krebs' III solution.10
The opposite eye of the animal is used if the
first preparation is unsatisfactory or if more than
one membrane is to be studied simultaneously. On
the average, 12 to 15 minutes elapse between
enucleation and the beginning of an experiment.
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Mounting the membrane. The isolated ciliary
body is placed over the end of a special chamber
machined from methacrylate (Fig. 1), and is
supported by nylon gauze overlying an elevated
collar which surrounds an opening 3.6 mm. in
diameter. After positioning the pars plicata over
the collar, a closely fitting methacrylate ring is
applied which clamps the membrane tightly in
the manner of an embroidery hoop. Excess tissue
is excised and silicone grease spread around the
base of the ring to achieve a water-tight seal.
The Krebs' medium used in the reservoirs is prepared in two stages so that when> the sodium
chloride-isotope solutions are added to each side,
the final composition is that of a Krebs' III
medium. The final salt concentration for each
reservoir solution is 0.154 molar in a volume of
3 ml. Considerable care is taken to adjust the
sodium content so that it will be equal in the
two reservoirs within analytical tolerances.* ImO22
Na was obtained from Abbott Laboratories in a carrier
of aqueous NaCl with its concentration in milligrams
per
milliliter specified to the second decimal place. :lNa was
obtained from the Oak Ridge Laboratories of Union
Carbide in a carrier of aqueous NaCl with its concentration in milligrams per milliliter specified to the third
decimal place.
Volume 6
Number 4
Sodium ion transport in. ciliary body 403
mediately after mounting the membrane the two
parts of the chamber are joined together, sealed
with silicone grease, and equal volumes of sodium
chloride deficient Krebs' solution are added to
each reservoir simultaneously. The reservoir with
the mounted membrane (left side of Fig. 1) contains fluid which bathes the stromal surface, and
is designated as the outside reservoir; the opposite reservoir, containing fluid which bathes the
epithelial or posterior chamber surface of the ciliary body, is referred to as the inside reservoir.
The chamber is placed in a constant temperature
bath which maintains the reservoir fluid at 30°
± 0.1° C.
Experimental procedure for measuring membrane potential, isotope flux across the membrane,
and short-circuit current (SCC). Krebs-agar
bridges are set at zero potential in the inner reservoir. Then the transciliary membrane potential is
measured, keeping the bridges close to the membrane (3 to 5 mm. apart). If the potential measurement indicates a satisfactory preparation, i.e.,
at least 1.5 mv., the isotopic sodium chloride
solutions are added to each side, completing the
Krebs' medium. Thus, the final solutions bathing
the opposite sides of the ciliary body membrane
are identical chemically, except that one side
contains 22Na and the other a 24Na isotope. The
isotope used for sodium influx measurement is
systematically alternated in the series of experiments. A concentration of approximately 0.04 me.
per milliliter is used for 22Na and 1.0 me. per
milliliter for 24Na.
Reservoir solutions are kept well oxygenated
and mixed by small oxygen bubbles from gas
dispersion tubes made of a porous glass frit.
The preparation is checked carefully to ensure
adequate mixing and good oxygenation, and to
see that no bubbles touch the membrane. A 50 /iiL
sample is withdrawn from each reservoir every
hour during the five hours of the experiment.
Suitable standards are prepared for each isotope
to measure counting efficiency and to make appropriate background counts. The 24Na samples and
standards are counted immediately by means of
a pulse-height analyzer to eliminate 22Na radiation. Samples and standards for both 22Na and
24
Na are counted in duplicate for a minimum of
10,000 counts. 22Na samples and standards are
counted two weeks after the experiment when
virtually all of the 24Na has disappeared by radioactive decay. Corrections for background radiation and decay loss are applied to both 24Na and
22
Na data.
The SCC is measured according to the method
of Ussing and Zerahn,11 in which the current
required to reduce the membrane potential to
zero is determined by applying an external electromotive force in a series circuit. In those experiments in which the membrane is maintained con-
tinuously in the short-circuited condition, the
current is recorded at 5 to 10 minute intervals and
the mean value calculated for the entire experimental period. In experiments in which sodium
flux is investigated in the non-short-circuited
membrane, SCC measurements are taken only at
the end of the experiment. These terminal SCC
measurements, when compared with the average
SCC of the continuously short-circuited membranes, are usually less than 10 per cent lower.
Data preparation. For each isotope, the experimental data are in the form of counts per minute
per 50 lambda sample for each of the 5 hours
of the experiment, with a similar set of data for
standards. All subsequent data processing, including corrections for decay loss, is done with
an IBM 1410 digital computer.
Methods of analysis. Two methods are used to
calculate the flux of sodium through the membrane.
In the first, the total mass of sodium transported in each hour of the experiment is calculated from the concentration of the isotope that
has passed through the membrane. Appropriate
volume corrections are made for fluid removed
in each sample, and the 5 hour values are averaged. The results are expressed as micromoles of
sodium per hour per square centimeter of stromal
surface area. The latter is chosen as an appropriate reference area because the plicated surface
area cannot be measured. The net flux, influx
minus outflux, is computed, as well as the flux
ratio, outflux/influx.
In the second method, the Fick differential
equation for unidirectional diffusion is utilized:
dm/dt = DA dC/dx,
'(1.)
where dm/dt represents the mass flow through
area, A, of the membrane whose diffusion coefficient is D; dC/dx represents the concentration
gradient of mass, m, in the x direction. By using
the initial condition that at time, t = 0, dm/dt =
0 (i.e., no diffusion has occurred until the experiment starts) and by using the following boundary
conditions, the equation can be solved by separation of variables. The boundary conditions used
are:
(Co - Ci)/L = dC/dx
(i.e., the concentration difference between outside
and inside reservoirs, divided by membrane thickness [L], is an approximation of the gradient);
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m t = nil + m 0
(i.e., the total mass is constant with time and
equal to the sum of the mass in each reservoir);
D is constant, and the volume of each reservoir
is equal and constant. The solution under these
assumptions, for diffusion into the inside reservoir,
is as follows:
- In (Co - 2Ci) = (2DA/VL)t - In Co, (2)
where Co (a constant) is the concentration of isotope in the outside chamber at the beginning of
Investigative Ophthalmology
August 1967
404 Holland and Stockwell
the experiment, and Ci its concentration in the
inside chamber at any time, t. D is the diffusion
coefficient for influx; A, the effective membrane
area; V, the volume of the reservoirs; and L, the
effective membrane thickness. A similar equation
applies to diffusion into the outside reservoir.
The digital computer is programmed to determine the influx and outflux slopes of Equation (2)
by the method of least squares. Since all terms
in the slope are identical for the two directions,
except perhaps for the apparent diffusion coefficients, the ratio of slopes will represent the diffusion coefficient ratio. This is computed as s,,/si,
i.e., outflux slope divided by influx slope. If the
membrane is inert and sodium is moved only by
diffusion, this ratio must be unity. If sodium
transport occurs by other mechanisms, the ratio
may be different from unity and can be related
to the amount of nondiffusional transport. That
the experimental data are well approximated by
this equation can be seen by inspecting Fig. 2,
where the least squares line and experimental
data from a living membrane are represented.
Since a constant reservoir fluid volume is one
of the assumptions used in the formulation, a
transformation of the independent variable, t-H/V,
was made so that the volume changes from
sample removal could be introduced as a variable.
As would be predicted, the slope ratio remained
unchanged. Volume diminution from sampling is
C.56I
small (a total of 6.6 per cent) and equal in
each reservoir.
Results
Cellulose membranes. To validate the
experimental procedures and establish the
characteristics of passive sodium movement
through an inert membrane under these
experimental conditions, sixteen experiments were done with dialysis tubing membranes.* Results are summarized in Table
I, where the outflux/influx ratios obtained
from mass flow calculations and the slope
ratios obtained from the diffusion equation
are given. The means of the ratios derived
from both calculations are very close to
unity, which is the theoretically expected
value for flux equality. The dagger in
Column 1 of this table indicates those
experiments in which the 2JNa isotope was
used to measure outflux. Although these
latter flux ratios showed a variation about
unity, there was a slight tendency for them
°Curtin Co., cellulose tubing-transparent, seamless, diameter %", thickness, 0.00072", pore radius 24 Angstroms.
4.089,
2
3
Time in Hours
Na INFLUX
Z
3
Time in Hours
Na OUTFLUX
Fig. 2. Double isotope experiment measuring influx and outflux simultaneously in the living,
isolated cat ciliary body membrane. Ordinate values are obtained from a solution of the differential equation for unidirectional diffusion. Experimentally measured' quantities' are the isotope
concentration in the source reservoir at zero time (Co), and the concentration in the receiving
reservoir (d) at later times. The least squares line is shown with the experimental data. The
average point is indicated by a square and the least squares intercept by a triangle. The flux
ratio, outflux/influx, for this experiment is 0.80.
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Volume 6
Number 4
Sodium ion transport in ciliary body 405
Table I. Summary of simultaneous sodium
ion flux measurements in sixteen inert
cellulose membranes
experiments in which sodium ion influx
and outflux were measured simultaneously.
In Column 5 it can be seen that the outflux/influx ratios are obviously less than
unity. The null hypothesis and pooled
sample t test were used to test the difference between this mean and the mean flux
ratio of the inert membrane for the level
of statistical significance. The difference
was highly significant (p < < 0.001). The
result was similar when the slope ratios
were tested in this manner. The Wilcoxon
rank sum test, a nonparametric statistic not
requiring the normality assumption, gave
a similar result with approximately the
same confidence level.
Columns 7 and 8 of Table II list the
average sodium ion current determined
by isotopic methods and the SCC measured at the termination of the experiment.
In the following series of experiments in
which the membrane was continuously
short circuited, it was observed that the
terminal SCC was not more than 10 percent lower than the average value of the
SCC for the 5 hour period. To make a
better estimate of the 5 hour average SCC
for the present series of experiments, the
terminal values were increased by 10 per
cent, which increased the mean of Column
8 to 0.062. When this mean was compared
with the mean of the average sodium ion
current for the same period, using the null
hypothesis and a paired sample t test, it
was found to be significantly different
(0.001<p<0.01).
Thus the experimental data and statistical evaluation support the conclusion that
there is a net sodiiun ion influx in the
presence of the membrane potential and
that the net sodiiun ion current significantly
exceeds the estimated average SCC (by
two to three times).
Short-circuited membranes. By the shortcircuiting procedure the membrane potential is reduced to zero; therefore sodium
transport is not influenced by the membrane electromotive force. Under these experimental conditions all electrochemical
gradients between reservoirs have been
Number
It
2
3t
4
5t
6t
7
8
9t
lOf
11
12
13f
14
15
16
MeanJ
S.D.
Outflux/
Influx"
1.10
0.92
0.88
1.09
1.11
1.11
0.84
0.89
0.94
1.09
0.90
1.10
1.05
0.94
0.96
1.14
1.00
0.10
S./S,
1.06
0.93
0.94
1.12
1.13
1.14
0.83
0.87
0.93
1.07
0.89
1.09
1.05
0.92
0.93
1.06
1.00
0.10
"The outflux/influx ratio is derived from mass flow calculations and So/si is the outflux/influx slope ratio obtained
from a least squares approximation to a solution of the
diffusion equation. The theoretically expected ratio is
unity for equal flux. The means of both experimentally
measured
ratios are close to the expected value.
f:'Na used to measure outflux.
} Figures rounded to second decimal place.
to be larger than one. To circumvent any
difficulty which might occur by the consistent use of one isotope to measure flux
in a given direction, they were systematically alternated between influx and outflux measurements.
Nonshort-drcuited membranes. In this
condition the transmembrane potential is
maintained so that the sodium transport is
under the influence of the membrane's electromotive gradient. This potential gradient
in the isolated cat ciliary body is such that
it would assist sodium influx because the
epithelial surface of the membrane is negative with respect to the stromal surface.
During an experiment there are slight
variations in the membrane potential. It
usually shows an initial rise and a prolonged period of several hours of constant
or very gradually diminishing potential.
The average membrane potential for the
five hours has been calculated for each
experiment. They range from 1.20 to 2.27
.mv. Table II summarizes the results of 12
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406 Holland and Stockwell
Investigative Ophthalmology
August 1967
Table II. Summary of 12 experiments in which sodium ion flux was measured
simultaneously in the non-short-circuited, living ciliary body membrane.
I
No.
It
2t
3
4
5
et
7
8t
9t
iot
11
12
2
3
4
5*
Net Na flux
Na influx
(influx-outflux) Outflux/
Na outflux
(nM cm.-th-1) (nM cm.-^h-1) (fiM cmr2h-')
influx
14.62
12.73
10.30
8.02
12.39
8.09
12.61
8.01
9.05
9.46
7.09
7.78
11.38
11.50
6.47
5.91
10.31
6.97
9.83
6.59
8.96
9.34
5.52
6.13
3.24
1.23
3.82
2.10
2.09
1.12
2.78
1.42
0.09
0.12
1.57
1.65
0.78
0.90
0.63
0.74
0.83
0.86
0.78
0.82
0.99
0.99
0.78
0.79
6
s o /s t
0.85
0.95
0.65
0.68
0.81
0.90
0.80
0.79
0.98
1.02
0.75
0.78
7
8
Net Na ion
current
(coul. cm.-zh-1)
SCC (term.)
(coul.
cmrsh-})
0.31
0.12
0.37
0.20
0.20
0.11
0.27
0.14
0.01
0.01
0.15
0.16
0.06
0.04
0.06
0.07
0.04
0.04
0.07
0.06
0.05
0.06
0.07
0.06
1.77
0.83
0.17
0.06
8.24
Mean
10.01
0.82
0.12
1.13
0.11
0.01
2.21
0.10
2.48
S.D.
°The means of the flux ratios given in Columns 5 and 6 are significantly different from similar means of inert membranes when tested statistically. The isotopically measured sodium ion current (Column 7) is significantly larger (2
to 3 times) than the estimated average SCC (Column 8). These data show that there is a net influx of sodium ion
in21 the presence of the membrane potential.
f Na used to measure outflux.
eliminated, and any net transport which
remains would fulfill the conditions required for active transport. Table III summarizes the results of eleven experiments
under these conditions. The mean of the
flux ratios listed in Column 5 was evaluated with the pooled sample t test and null
hypothesis to determine whether there was
a statistically significant difference from
the mean flux ratio of the non-short-circuited membrane. The difference was
significant at the 0.05 level. The mean flux
ratio of the short-circuited membrane also
was tested with a one-tailed t statistic to
determine if it was significantly less than
the mean flux ratio of the inert cellulose
membrane. The difference also was found
to be significant (0.02 < p < 0.05).
In Columns 7 and 8 of Table III the
isotopically determined sodium ion current
is listed along with the SCC for each experiment. The mean value of these currents is approximately equal. Evaluating
the differences between these two currents
with a paired sample t test reveals that
there is no statistically significant difference.
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Thus the experimental evidence and
statistical evaluation support the conclusion
that the sodium ion is actively transported
by the ciliary body, and in the continuously
short-circuited membrane the net sodium
ion current and SCC are approximately
equal, within the limits of error and resolution of these methods.
Discussion
The present investigation shows that the
in vitro ciliary body transports the sodium
ion inwardly in the absence of electrochemical gradients, confirming the hypothesis
of an active transport. The net sodium influx is significantly larger in the presence
of the membrane potential, which in the
cat is oriented in a direction to assist cation
influx, i.e., the epithelial surface is negative relative to the stromal surface. It
should be pointed out that Cole12'13 has
observed an opposite orientation of potential in the rabbit and ox ciliary body membrane similarly isolated in vitro. Using the
frog skin model proposed by Ussing,14 he
has inferred that an inward active transport of the sodium ion generates a positive
Sodium, ion transport in ciliary body 407
Volume 6
Number 4
Table III. Summary of 11 experiments in which sodium ion flux was measured
simultaneously in the continuously short-circuited, living ciliary body membrane
1
No.
1
2t
3
4f
5
6
7t
8f
9
iot
11
2
3
4
5°
Net Na flux
Na influx
Na outflux
(influx-outflux) Outflux/
influx
(fiM cm.-zh-1) (jiM cvnr^hr1) (JLM cm.-2h-')
8.48
7.49
10.14
8.45
6.42
6.76
7.68
8.99
7.53
7.73
8.99
7.41
7.92
7.98
7.77
6.62
5.74
7.36
7.71
6.22
8.44
8.24
1.07
-0.42
2.17
0.68
-0.19
1.02
0.31
1.28
1.31
-0.71
0.75
0.87
1.06
0.79
0.92
1.03
0.85
0.96
0.86
0.83
,1.09
0.92
6
7
8
S0/Si
Net Na ion
current
(coul. cin.-sh-J)
SCC (avg.)
(coul.
cm.-zh-1)
0.89
1.07
0.80
0.93
0.98
0.87
0.98
0.91
0.82
1.06
0.92
0.10
-0.04
0.21
0.07
-0.02
0.10
0.03
0.12
0.13
-0.07
0.07
0.07
0.07
0.07
0.06
0.09
0.10
0.09
0.06
0.09
0.06
0.07
Mean
0.06
0.93
0.92
8.06
7.40
0.08
0.66
S.D.
0.08
0.09
0.01
0.10
0.85
1.08
0.86
"The mean of the flux ratios listed in Column 5 is significantly less than a similar mean in the inert membrane when
tested statistically; also, this mean is significantly larger than the non—short-circuited membrane. The mean of .the
isotopically determined sodium ion current (Column 7) is approximately equal to the mean SCC (Column 8) witriin
the limits of error of these methods. The data show that in the absence of electrochemical gradients there is a net sodium
influx. Eliminating the membrane potential by the short-circuiting procedure reduced this influx by approximately 60 per
cent.
t2lNa used to measure outflux.
potential on the epithelial surface of the
ciliary body; however, flux measurements
have not been made to confirm this hypothesis. The measured SCC was assumed
to be due to sodium ion transport. Hogben15 has shown that the SCC generated
in the isolated gastric mucosa is due to the
active transport of chloride and not sodium, and in many biologic membranes a
combined cation-anion pump functions.10
Thus, whether a measured SCC is due
totally or partially to sodium ion transport,
or is independent of it, must be verified
experimentally in individual membranes.
In an investigation partially completed,
we have compared the in vitro transciliary
membrane potential of the cat with that of
the rabbit. The results indicate that the
potential varies with the species and the
electrolyte-buffer solutions used. Rabbit
ciliary body membranes were mounted in
Tris-buffered Krebs' solution. Initially, approximately one half of the preparations
manifested a positive epithelial surface as
reported by Cole.12-13 The remainder, although initially showing a negative epithelial surface, became positive within 8 min-
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utes. The magnitude of our measured potentials ranged between 0.25 and 1.1 mv.,
compared with the average of 3.8 mv. in
Cole's experiments. When the electrolyte
solution was Krebs-phosphate, all rabbit
membranes showed a negative epithelial
surface within 2 minutes and usually remained negative. When the ciliary body
membrane of the cat was used, the epithelial surface was initially negative in all
solutions tried thus far, and remained negative throughout the experiment (3 to 5
hours). The magnitude of the cat ciliary
body potential was much higher than that
of the rabbit by the same techniques, but
tended to be lower in Tris-buffered than
in phosphate-buffered Krebs' solution. Although this investigation has not been
completed, the evidence supports the conclusion that the transciliary membrane potential manifested in vitro depends qualitatively and quantitatively on both the animal species and electrolyte solution used.
It is interesting to speculate on the possible relationship of these species differences in membrane potential to the wellknown differences in the chemical compo-
408 Holland and Stockwell
sition of the cat and rabbit aqueous humors.
Perhaps it is pertinent to consider the
possible relationship of our cat transciliary
membrane potential measurements, which
consistently show an electronegative epithelial surface in vitro, to measurements of
blood-aqueous humor potentials made in
living rabbits by a number of investigators.17"21 We have repeated this type of
measurement in the rabbit and have confirmed that the anterior chamber aqueous
is positive relative to the blood in the ear
vein in the reported range of magnitudes.
The posterior chamber is also positive relative to blood (as reported by Cole20) but
is less positive than the anterior chamber.
When measurements are made between
the two chambers, the posterior chamber
is 3 to 6 mv. electronegative relative to the
anterior chamber, as would be inferred
from measuring each chamber separately
relative to the blood in the ear vein. This
orientation of potential sign is compatible
with our observations of the cat and rabbit
in vitro transciliary membrane potentials
when using Krebs-phosphate medium.
Using the DuBois-Reymond moist chamber and nonpolarizing electrodes, Seidel"
investigated living and freshly enucleated
cat eyes to determine whether electrical
currents arising from secretory activity
could be demonstrated in the ciliary epithelium. These currents were known to have
a characteristic "inwardly directed" course,
i.e., "they are always directed from the cell
surface toward the cell base, therefore,
from outside to inside since the secretory
cell surface always assumes a negative potential. It is quite generally true that the
secretorily active (altered) cell parts are
negative relative to inactive (unaltered)
parts." He also states, "Upon the ground of
a large number of completely consistent results, I can report that the ciliary epithelium is the site of a powerful 'inwardly directed' electrical current, which runs from
the free cell surface toward its base, therefore scleralwards."22
Lehmann and Meesmann,17 when com-
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Investigative Ophthalmology
August 1967
menting on the relation of Seidel's observations to their own studies of the bloodaqueous humor potentials, state, "For our
measurements the currents found by him
(Seidel) do not come into consideration,
since they run in the opposite direction
from those of our measured potential."
Clarification of the meaning of these observed differences will require further investigation.
In the non-short-circuited membrane the
net sodium ion current, obtained from isotopic flux measurements, is two to three
times larger than the estimated average
SCC. It is probable that this difference is
produced by the simultaneous influx of an
anion, such as chloride, which would preclude electrical detection of the total sodium ion current. Such a combined cationanion pump has been demonstrated in several biologic membranes, including the skin
of a South American frog, the epinephrinestimulated frog skin, nasal gland of birds,
isolated rumen epithelium, and rabbit gall
bladder. The isolated intestinal mucosa of
a marine teleost actively transports sodium
with a zero membrane potential, indicating
a net anion transport in the same direction.10 A similar phenomenon has been observed in the cornea during the first 40
minutes in vitro.23
Ussing,24 in his Harvey Lecture, considers that in such cases the ion pump
transfers sodium together with some anion.
"In tissues where the inner epithelial border is permeable to the anion in question,
it will flow back into the cell during shortcircuiting and the pump will appear as a
pure sodium pump, whereas, if the anion
cannot; return or does so only to a small
extent, we have a mixed sodium and anion
pump." This mechanism may explain our
observation that, in the continuously shortcircuited ciliary body membrane, the net
sodium ion current appears approximately
to equal the SCC (within the statistical
resolution of these methods). Thus, the in
vitro cat ciliary body membrane appears
to function as a mixed sodiiun-anion pump
in the non-short-circuited state, but main-
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Number 4
ly as a sodium ion pump when short circuited. Chemical analytic determination of
aqueous/plasma ratios, dialysis of aqueous
against plasma,5 and in vivo kinetic turnover studies7 also support the hypothesis
that there is an active transport of chloride
ions by the ciliary epithelium. In vitro investigations are now under way to explore
this possibility.
The authors gratefully acknowledge the technical assistance of Mr. B. W. Baber.
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