Regional distribution of the Na+ and K+
currents around the crystalline lens of rabbit
Oscar A.
Candia1,2 and
Aldo C.
Zamudio1
Departments of 1 Ophthalmology and 2 Physiology and
Biophysics, Mount Sinai School of Medicine, New York, New York
10029-6574
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ABSTRACT |
Early studies described asymmetrical
electrical properties across the ocular lens in the
anterior-to-posterior direction. More recent results obtained with a
vibrating probe indicated that currents around the lens surface are not
uniform by showing an outwardly directed K+ efflux at the
lens equator and Na+ influx at the poles. The latter
studies have been used to support theoretical models for fluid
recirculation within the avascular lens. However, the existence of a
nonuniform current distribution in the lens epithelium from the
anterior pole to the equator has never been confirmed. The present work
developed a modified short-circuiting technique to examine the net
flows of Na+ and K+ across arbitrarily defined
lens surface regions. Results indicate that passive inflows of
Na+ occur at both the anterior polar region and posterior
lens surface, consistent with suggestions derived from the vibrating
probe data, whereas K+ efflux plus the
Na+-K+ pump-generated current comprise the
currents at the equatorial surface and an area anterior to it.
Furthermore, Na+-K+ pump activity was absent at
the posterior surface and its polar region in all lenses examined, as
well as from the anterior polar region in most lenses. The latter
unexpected observation suggests that the monolayered epithelium, which
is confined to the anterior surface of the lens, does not express an
active Na+-K+ pump at its anterior-most aspect.
Nevertheless, this report represents the first independent confirmation
that positive currents leave the lens around the equator and reenter
across the polar and posterior surfaces.
Ussing-type chamber; short-circuit current; ion transport
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INTRODUCTION |
BECAUSE OF ITS
HIGH [K+], low [Na+], and general
gross properties, early observations compared the crystalline lens to a
giant spherical cell (17, 18). From this paradigm,
numerous physiological studies described the overall functional
characteristics of the lens, e.g., intralenticular potentials, input
resistances, and impedance analyses (11, 12, 15, 22,
24-26). In contrast, other early investigations emphasized
the influence of the lens epithelium, which comprises a single layer of
cuboidal cells on the anterior side only, in conferring to the lens
asymmetrical electrophysiological properties (7-9, 14,
19). When the lens is isolated in a double chamber that
separates the anterior from the posterior lens surfaces, an anteriorly
directed positive electrical potential develops, in all studied
species, as a result of an electrogenic Na+-K+
pump located in the surface, basolateral membrane of the epithelium (13). Logically, it was assumed that the activity of this
monolayer was relatively uniform and that a positive current must
emanate from the anterior pole to the equator. However, Patterson and coworkers (23, 27, 29, 30), using the vibrating probe, showed in frog and rat lenses a nonhomogeneous distribution of currents
around the lens surface.
The findings of Patterson's group were not widely received, and no
other laboratory attempted to reproduce their results. Nevertheless, on
the basis of Patterson's initial model, in which ionic currents exited
around the equator and reentered the lens at the poles, Mathias et al.
(21) developed a theoretical model for electrolyte and
fluid circulation inside the lens. That model also included information
obtained from impedance analysis (5, 20).
Our experience with the vibrating probe in the rabbit lens suggested
that this device was unreliable. We present here an alternative, novel
approach to empirically examine the ionic currents about the lens
surface. It entails the use of three O-rings of different sizes to
separate the anterior surface from the posterior surface of the lens at
three different parallels between either the anterior or the posterior
pole and the equator, thereby enabling the study of the currents at
seven predefined zones.
In this article we report that in the isolated rabbit lens, a positive
current leaves from around the equator and an area anterior to it and
reenters the lens across its posterior face as well as a small area
around the anterior pole. The current across this anterior polar area
is insensitive to ouabain despite the presence of the lens epithelium.
Our results represent the first independent confirmation of initial
observations of Patterson and collaborators and identify additional
asymmetrical aspects in this remarkable organ.
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METHODS |
Theoretical considerations.
To simplify the analysis, we performed all experiments in
Cl
-free solutions where the predominant movement of
charges between lens and solutions is due to Na+ and
K+ fluxes. Furthermore, when the isolated lens is placed in
a bath, the net interchange of these charges between solution and lens must be zero; i.e., the total charges leaving the lens must be equal to
the total charges entering. However, this balance can be accomplished
not necessarily because there is no charge movement across the lens
surface but because a net charge movement across a particular area of
the lens surface is compensated by an equal and opposite charge
movement across another lens area.
We will adopt the convention for the lens immersed in a bath that
positive currents from the bath to the lens are positive and that
positive currents from the lens to the bath are negative. Thus a net
K+ flow from the lens to the bath will have a negative sign.
When the lens is immersed in a saline solution, it is essentially
short-circuited by the low-resistance solution around its surface, and
no potential difference (PD) can be detected between electrodes placed
on any two points in the proximity of the lens surface. However, if the
lens is isolated in an Ussing-type chamber (7-9) and
two separate areas in the anterior-posterior direction are electrically
isolated, there are two possible outcomes: 1) the total
current at each one of the two areas is independently balanced, in
which case the translens PD (PDt) will be zero; or 2) such individual balance does not occur, in which case one
side will develop a PD with respect to the other as a consequence of the asymmetry between the two delimited areas.
Despite the separation of the two areas, short-circuiting restores the
distribution of currents to the open-bath condition. The short-circuit
current (Isc) represents the difference between inward and outward currents at each surface. However, the
Isc is a current that circulates through the
lens and an external circuit, thereby completing a closed loop. Because
of this, the Isc enters the lens across one
surface and leaves across the other. In this situation, the sign of the
current must be based on the direction of circulation (e.g., clockwise
or counterclockwise within the loop) and not on whether the current
enters or leaves the lens. Thus the sign of the
Isc should be based on its direction in the
external circuit. We have chosen to define the
Isc as positive when a positive current flows
from the anterior to the posterior pole in the external circuit and,
consequently, from the posterior to the anterior pole through the lens.
Of course, the polarity of the Isc will depend
on the natural polarity of the lens and how it is mounted in the
chamber. As shown later, this precise definition of the
Isc is necessary to determine the direction in
which current and ion fluxes traverse the surface of the lens. When a
"flat" epithelium is mounted in a bicameral chamber, the only
detectable asymmetry is that between the apical and basolateral sides.
Because of its semispherical shape, the lens offers the opportunity for
a comparison between multiple pairs of its total surface and in doing
so allows for a determination of the transport and permeability
properties of various arbitrarily defined zones. To exploit the
advantages inherent in lens geometry, we designed a special chamber
that enabled separation of six pairs of areas.
Chambers.
Two chambers were used in the experiments to be described. One, a
three-compartment chamber, shown diagrammatically (Fig. 1), was exclusively utilized to determine
the resistance ratio between near equal areas of the posterior and
anterior surfaces (RP/RA). The lens was initially supported between two
centrally perforated Lucite disks with O-rings as surfaces of contact.
Each O-ring touched the lens with just enough pressure to prevent
leaks, without distorting the shape of the lens. This was accomplished by advancing four screws on the disk that brought them closer together
against the resistance of a large O-ring that sealed a center
compartment. The two-disk lens assembly was mounted between the halves
of an Ussing-type chamber with conventional PD-measuring and
current-sending electrodes. A third PD electrode was placed in the
center compartment through an opening in the large O-ring. This opening
also served to fill the center compartment with Ringer's solution. The
surface area in contact with the outer solution given by the diameter
of the small O-rings (7.9 mm) was 0.65 cm2 on each side.
The equatorial surface bathed by the solution in the center compartment
was about 2.04 cm2 for a typical ~10.5-mm equatorial
diameter lens. With this arrangement the PD drop between the center and
external compartments could be determined. The ratio of PDs is equal to
the resistance ratio between the respective surfaces.

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Fig. 1.
Schematic of the 3-compartment chamber used to determine
the resistance ratio of nearly equal areas of the posterior and
anterior surfaces (RP/RA) of the rabbit lens. PD, potential difference;
PDt, translens potential difference; PDp,
potential difference between the posterior and center compartment.
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The second chamber, used in all the other experiments, is shown in Fig.
2. It consists of a lower Lucite
hemichamber with a glass-made recirculation attachment, a Lucite upper
hemichamber with its glass-made recirculation device, and a center
spacer that fits between both hemichambers. The spacer served as an
atrium around the lens to prevent the upper chamber from touching the organ. Lenses were placed on one of three O-ring assemblies (Fig. 2 and
inset) with the anterior epithelial side either up or down. This O-ring lens assembly was then positioned on the lower hemichamber. The spacer was placed on the top of the O-ring assembly, and the upper
chamber was in turn situated over the spacer. The whole system was
secured by two nylon thumbnuts screwed onto threaded rods also used as
guides. Once the hemichambers were filled with the bathing solutions,
the upper hemichamber was at atmospheric pressure. The fluid in the
lower chamber, because its level was about 10 cm below that of the
fluid in the upper chamber, exerted a negative pressure on the lower
face of the lens that kept it in place and provided a seal that
electrically isolated the two surfaces of the lens bathed by the upper
and lower solutions. The O-ring assembly had a center aperture of
either 4.8, 6.4, or 7.9 mm in diameter. The lens, with an equatorial
diameter between 10 and 12 mm, was not subjected to an external
pressure, since the internal diameter of the spacer was 15 mm. From the
placement of the lens on either of the three O-ring assemblies, with
its anterior or posterior pole facing the O-ring, six positions
(P1-P6) were defined that allowed for the separation of seven lens
zones that are described below.

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Fig. 2.
Photograph of the vertically arranged Ussing-type chamber
used to mount rabbit lenses. It consists of 2 hemichambers connected to
glass bubblers. Between the hemichambers there is a Lucite disk with a
central hole. An O-ring is placed in the periphery of the central hole.
The lens, shown separated from the O-ring, is positioned on it. A
cylindrical spacer prevents the upper hemichamber from touching the
lens. The lens can be positioned with either its posterior or anterior
side down on any of 3 different diameter O-rings (inset).
Heating coils were placed within the glass attachments, which also were
connected to a source of humidified 5% CO2 that provided
bubbling and circulation. PD-sensing and current-sending electrodes
were inserted through ports present in the upper and lower Lucite
hemichambers.
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A common protocol was to start with the posterior lens pole on the
smallest ring (P1) to study zone 1 (Z1; the posterior polar region);
after recording in this position, open the chamber and change to the
larger rings (P2 and P3, respectively), revert the lens (P4), and then
sequentially change to the smaller rings with the anterior pole now
facing downward. Many other sequences were tested. In most experiments,
each individual lens was examined in at least three positions. As shown
in Fig. 2, the chamber has inlets in which to insert agar bridges for
the measurements of PD, Isc, and translens
resistance (Rt).
Translenticular electrical measurements.
Lenses from adult albino rabbits that had been killed by
CO2 asphyxiation were mounted in either of the chambers
described. Agar-NaCl-filled polyethylene tubing served as a salt bridge
connecting each bathing compartment to Ag-AgCl electrodes for
PDt measurements. A second pair of bridges, connected to an
automatic voltage-clamp system (28), was used to
short-circuit PDt, with the current needed to keep
PDt at 0 mV (i.e., the Isc)
continuously recorded. For Rt measurements, the
current needed to offset the short-circuited condition by ±10 mV was
measured every 2-5 min for a few seconds.
Description of lens zones.
The three O-ring separations resulted in the seven zones Z1-Z7
(Fig. 3), in which Z1 with Z7, Z2 with
Z6, and Z3 with Z5 constituted symmetrical pairs. Z4 was without a
pair. The general dimensions of these zones for an idealized spherical
lens of 11 mm in diameter also are shown in Fig. 3. Regarding the
mounting positions, P1, P2, and P3 were symmetrical to P6, P5, and P4,
respectively. Regardless of the O-ring used and the zones included, the
anterior side is defined as the one that includes the anterior pole,
and by default, the area on the other side of the O-ring is the
posterior side. Figure 3 also shows the equivalent resistance circuit
when the O-ring is in P2, separating Z1 and Z2 from Z3, Z4, Z5, Z6, and Z7.

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Fig. 3.
Diagram showing the 7 zones (Z1-Z7) defined by the 6 O-ring positions and the relative surface areas of each zone on the
approximately spherical rabbit lens. Left: an equivalent
resistance diagram applicable when measuring resistance between points
a and p with the O-ring in position 2 (P2).
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Solutions.
The bathing medium used during the dissection and bathing of lenses in
the chambers lacked Cl
and had the following composition
(in mM): 1.8 calcium gluconate, 1.2 MgSO4, 2 K2HPO4, 119 sodium gluconate, 25 NaHCO3, and 10 glucose. The pH of this solution when
bubbled with 5% CO2-95% air was ~7.45. Its osmolality
measured 285 mosmol/kgH2O. Lenses were incubated in
the Cl
-free solution for at least 1 h before being
mounted in the chamber.
In some experiments described in RESULTS, a
Cl
-free, elevated-K+ solution with reduced
Na+ was used. For this, potassium gluconate replaced an
equimolar amount of the Na+ salt.
Determination of resistances of individual zones.
When resistance across the lens isolated in an Ussing-type chamber with
the anterior and posterior surfaces separated by a circular O-ring
(Fig. 2) is measured, the obtained value is the series combination of
the resistance of both surfaces with the associated lens fibers. With
the use of smaller O-rings, the resistance of one side becomes larger,
whereas the other becomes smaller (see Fig. 3). In general, the total
measured resistance is larger as the size of the O-ring decreases and
the separation of the anterior zones moves away from the equator toward
one of the poles.
In our experimental approach, the lens surface is divided into seven
zones, but there are only six O-ring positions from which to measure
the combined resistances. The seventh value necessary to solve the
equation is provided by the resistance value obtained with the
three-compartment chamber. The two 7.9-mm O-rings used in the
three-compartment chamber separate the lens into three zones: A
(anterior), E (equatorial), and P (posterior). This O-ring placement
corresponds to P3 and P4 in the two-compartment chamber. Thus the
resistances measured at these positions in the two-compartment chamber
are
and
where RP3 and RP4
are the resistances measured at positions P3 and P4, respectively, and
RE, RA, and RP are the resistances of the equatorial, anterior, and
posterior zones defined for the three-compartment chamber.
Also, in this chamber the resistance ratio is r = RP/RA. It can be shown that
which can be simplified to
which can be converted into a quadratic equation
and solving for RA
and then
and
RE is identical to the resistance of zone 4, R4.
Once this value is known, the resistance of the other six zones,
R1, R2,
R3, R5,
R6, and R7, can be
obtained. The combined resistances of Z4-Z7
(R4-7) is the parallel combination of RA
and RE. To obtain R3, the resistance of Z3, the
O-ring is placed in P2 in the two-compartment chamber. This position is shown in Fig. 3. The measured resistance between points a
and p, RP2, is equal to
where R1-2 is the combined
resistance (in parallel) of Z1 and Z2. Then
and
which can also be converted into a quadratic equation
and solving for R3
By changing the position of the O-ring and following the same
procedure, the resistances of the remaining zones can be determined.
 |
RESULTS |
Determination of the posterior-to-anterior surface resistance
ratio.
Six experiments were performed with the three-compartment chamber with
the lens bathed in Cl
-free Ringer's. The average
PDt was 19 ± 4 mV, with the anterior side positive.
The electrical potential difference between the posterior and center
compartment (Fig. 1, PDp) was 17 ± 5 mV. This
observation confirms the presence of the Na+-K+
pump in the epithelium covering the anterior face and equatorial region. However, the main reason for the use of this chamber was the
determination of RP/RA, a value needed for the processing of data
obtained with the bicameral chamber. When a pulse of current was sent
across the lens between the outer compartments (no current flow in to
or out of the center compartment), a potential deflection of 3.00 mV
across the lens was simultaneously recorded as a 1.79 ± 0.16-mV
deflection across the posterior-to-center electrodes. These
values correspond to an RP/RA of 1.48 ± 0.12. The
Rt was 1.87 ± 0.30 k
· cm2. This value represents the series
resistance of the 0.65-cm2 surface in contact with the
external solutions plus that of the lens fibers between them. Because
the O-rings used in this chamber were the larger size used for P3 and
P4 of the two-compartment chamber, the area of the lens surface in
contact with the bathing solutions was the addition of Z1, Z2, and Z3
or Z5, Z6, and Z7 for the posterior and anterior side, respectively.
Determination of Isc at each position.
Although it was not always possible to obtain information from every
position for practical reasons of time or because the lens deteriorated
during the manipulation necessary to change O-rings, this was
accomplished in 11 experiments. Two such experiments are shown in Figs.
4 and 5;
resistances were examined in the six possible positions.

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Fig. 4.
Representative short-circuit current (Isc)
recordings obtained upon the sequential remounting of the rabbit lens
at 6 different separations between the anterior and posterior surfaces
as defined by the O-ring position. Upward deflections are the points at
which the translens resistance was recorded. The length of the
deflection is proportional to the electrical conductance. Toward the
end of the experiment shown, the lens was returned to P5. In this
situation, the additions of BaCl2 and ouabain to the lower
chamber bathing the anterior side of the lens were without effect. Such
additions to the upper hemichamber exposed the posterior-most and
equatorial lens surfaces to the drugs, resulting in elimination of the
Isc.
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Fig. 5.
Experiment in which the rabbit lens was sequentially remounted in
symmetrical positions, i.e., P1 with P6, P5 with P2, and P3 with P4 to
illustrate the reversals in the current flow across the lens. At the
end of the experiment, in P4, BaCl2 plus quinidine were
added first to the posterior bath and then to the anterior bath.
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Experiment 19, shown in Fig. 4, was started in P1 where the
lens is sitting with its posterior polar region on the smallest O-ring.
As expected, the Isc indicated that a positive
current was entering the posterior pole and leaving across the much
larger surface isolated above the O-ring. Changing from P1 to P2
increased the Isc, indicating that a positive
current that had been entering the lens anterior to the O-ring across
Z2 was now part of the posterior inward current. The
Isc increased further at P3. Also, the
electrical conductance progressively increased from P1 to P3. This also
was expected, because the largest O-ring more evenly divides the
anterior and posterior surfaces. The change to P4 reduced the
Isc to ~6 µA from 13.4 µA, indicating that
across the equatorial Z4, a net positive Isc was
leaving the lens. In P5, the Isc became
negative. This indicates that a net positive current was entering the
lens across its anterior pole (Z7) and its surrounding Z6, and leaving
throughout Z1-Z5. Because we already know (from determination at
P1, P2, and P3) that a positive current enters Z1-Z3, a much
larger positive current must be leaving Z4 and Z5. This is actually
expected, because the Na+-K+ pumps that produce
an outward current are concentrated in the equatorial zone (Z4) and
anterior epithelium (6, 10). Finally, with the use of the
smallest O-ring with the anterior pole down (P6), the
Isc remained negative. This result, which is a
novel finding, indicates that a positive current entered across the anterior polar region delimited by the 4.8-mm-diameter O-ring. One
could expect that the Na+-K+ pumps known to be
present at the epithelium covering the anterior pole would produce an
outwardly directed positive current. Although an unlikely possibility,
K+ entering the anterior pole may produce the observed
Isc. However, after the lens was returned to P5,
BaCl2 had absolutely no effect on the
Isc when added to the anterior bath. Even more
perplexing was the fact that ouabain did not affect this
Isc either. BaCl2 and ouabain had
their usual effects when added to the posterior-side solution bathing
Z1-Z5 in this position.
Although anterior-side ouabain had a minor effect in some experiments
done with P5, which includes Z6 and Z7, in 10 experiments in which the
glycoside was added to an isolated Z7, there was no effect in the
Isc. Clearly, the Na+-K+
pump was physiologically inactive in this region of the epithelium. One
must conclude that the Isc is carried by a
Na+ current that was recirculating across the anterior pole.
Experiment 112, shown in Fig. 5, also was started in P1, but
the lens was then inverted so that the anterior polar region sat on the
smallest O-ring (P6). The Isc continued to flow
in the same direction in the external circuit, but across the lens it
now went from the anterior to the posterior side, thus requiring the
reversal of the scale according to the definition in
METHODS. Compared with P6, P5 extends the area of the
anterior surface limited by the O-ring. The orientation of the lens was
then again inverted while the same O-ring was used, resulting in P2. In
comparison with P1, one can see that the Isc was
larger with P2, indicating that additional current was entering the
lens across Z2. By using the largest O-ring, the
Isc at P3 was obtained, which was usually the
largest compared with that of the other positions. As observed here, in
30% of lenses the Isc oscillates because of the
concerted opening and closing of the K+ channels, which we
have examined in previous publications (2, 3). Although it
was necessary to interrupt the Isc, open the chamber, and turn the lens over to reposition it in P4, the
oscillations continued. Additions of 5 mM BaCl2 plus
10
4 M quinidine to the posterior side in this position,
which includes Z1-Z4, reduced the Isc and
stopped the oscillation. This can be interpreted as a reduction of
K+ current leaving via the K+ channels in Z4
that were closed by the blockers. Adding the blockers to the anterior
side (Z5-Z7) produced an increase in Isc.
In this position the Isc is given by the
Na+ current entering across Z5-Z7 minus the
K+ current leaving the lens across these zones. Clearly,
from the response of the Isc to the blockers,
the K+ current was larger across the posterior side (which
includes in this position equatorial Z4) than across the anterior side.
Determination of the Isc at each lens zone.
With the posterior polar region on the smaller O-ring (P1), one
measures directly the Isc of Z1. In P2, one
measures the Isc of Z1 plus Z2 and can calculate
the Isc of Z2 by subtraction. In general, the
following matrix can determine the Isc across each zone
Based on the sign definition for Isc (see
METHODS), this matrix is consistent with the convention
that positive currents entering the lens are positive and positive
currents leaving the lens are negative.
Table 1 shows currents measured in 47 lenses from at least 2 positions. In some experiments, the
Isc for a given position was determined for a
second time, and an average value was entered in Table 1. The average
Isc for every position (shown at the bottom of
Table 1) was used in the matrix above to calculate the
Isc at every zone. These values are summarized
in Table 2 and Fig.
6. As can be seen, current gets into the
lens not only across the three posterior regions, as always thought
(4, 7), but also across the anterior pole (Z7). This is a
new finding for the rabbit lens, confirmatory of the prediction in rat
and frog lenses (29). It should be noted that the
equatorial zone is by far the largest, and it is possible that the low
density of the current is simply the result of a transition from an
inward current at its most posterior part to outward current at the
equator proper and its anterior part. A similar situation may exist in Z6, where in some experiments a positive current entering the lens was
found. Its density may be an indication of a transition from outward to
inward current as the anterior pole is approached. In any case, the
matrix dictates that the total current leaving the lens (negative) must
be equal to the current entering the lens.
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Table 1.
Short-circuit currents recorded at 6 different O-ring positions
separating the anterior from the posterior surfaces of isolated rabbit
lenses
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Fig. 6.
Summary of the average current flows across the 7 predefined zones of the rabbit lens. Net Na+ and/or
K+ currents (in µA/zone) entering (positive) or leaving
(negative) are shown.
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A close examination of the experiments in Table 1 shows that in some
cases (e.g., experiments 47, 62, and
79), the Isc in P6 was positive. This
is an indication that, contrary to the average, a positive current was
leaving across Z7. As shown in Fig. 7, in
these cases the addition of BaCl2 to the anterior side
reversed the polarity of the Isc. This is an
indication that there was a K+ current leaving the lens
across K+ channels (presumably driven by an electrochemical
gradient) that was larger than the inward Na+ current.

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Fig. 7.
Example of the Isc reversal evoked
upon addition of BaCl2 to the anterior side of the rabbit
lens mounted in P6. The Isc was initially
positive because the K+ efflux across the anterior Z7 was
larger than the Na+ influx. After K+ channel
blockade, the negative Isc represents
exclusively the Na+ influx. Inset: explanation
of the Isc reversal. The control
Isc is equal to JK JNa, where J is flux. After
addition of BaCl2, the Isc is equal
to JNa.
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To ascertain the presence of K+ channels in Z7 and to
compare their permeability with that of Na+ channels, the
following protocol was implemented in P6. The solution bathing the
anterior polar region was replaced by one in which the K+
concentration was increased by 30.7 mM (to 34.7 mM), and the Na+ concentration was reduced by the same amount to 109.3 mM. If the permeability of Na+ and K+ of the
zone bathed by the solution in which the change was made were the same,
and assuming a minimal effect on the pump, there would be no immediate
change in the Isc because the same number of
Na+ ions are replaced by K+, and the reduction
in the unidirectional Na+ influx would be compensated with
an equal increase in K+ influx. If the permeability of
K+ were larger than that of Na+, the
Isc would become more negative or less positive
because the additional K+ current is larger than the
reduced Na+ current entering the lens. This is what
happened in six experiments shown in Table
3. Making the same ionic change in the
posterior solution produced the opposite change in the
Isc, indicating that the overall K+
permeability of the other six zones was larger than that of the Na+. Also shown in Table 3 is the effect of the same ionic
change when the O-ring is in P1, separating Z1 from the other zones. Increasing the K+ concentration (with an equal reduction in
Na+) in the posterior solution increased the
Isc in four experiments, indicating that the
K+ permeability of Z1 was larger than that of
Na+. In the other two experiments in which the
Isc decreased, one must conclude that the
Na+ permeability of Z1 was larger. This is consistent with
the increase in resistance in these experiments compared with the
decrease in the other four. When the change is done on the anterior
side that includes Z2 through Z7, the Isc
decreased, indicating an overall larger K+ permeability
around this area of the lens surface.
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Table 3.
Effects of equal but opposite changes in bathing solution
[K+] and
[Na+] on Isc and electrical
resistance across rabbit lenses isolated in P6 and P1
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Determination of the electrical resistance of each zone.
Table 4 shows the mean resistance values
of lenses determined at the six previously defined positions. An
initial comparison can be made between values registered at symmetrical
positions P1 and P6. P1 had a larger resistance than P6. Because Z2
through Z6 are common in these positions, the resistance ratio P1/P6
therefore indicates the resistance ratio of Z1/Z7, although their
values are not revealed. A similar comparison can be made between P2 and P5. The ratio now is larger (1.88 vs. 1.42), indicating that the
resistance of the now included Z2 is even larger than that of Z6,
making the ratio larger. This type of analysis gave a general idea of
the resistances of various zones but cannot determine their exact
values. Using the analysis described in METHODS, we have
calculated the resistances of each zone, which also are shown in Table
4. The lowest resistance was found in Z4, followed by that of Z6 and
Z7; the highest was that of Z2. When these values were normalized for
an area of 1 cm2 (k
· cm2), the
resistances of Z4, Z6, and Z7 were still the lowest, but the difference
between the other zones was greatly reduced. Of course, these
resistances are mainly given by Na+ and K+
currents driven by the prevailing gradients. Analysis of these currents
is outside of the scope of this presentation.
 |
DISCUSSION |
The crystalline lens, with its high-protein-containing fibers
representing almost its total mass and the single-layered epithelium on
its anterior face, has been the subject of innumerable biochemical, cell biology, and molecular biology studies (18, 21). Only a handful of investigators have studied the electrophysiology of the
lens. Although certain elements of asymmetry in ionic movement had been
described (6, 19), our laboratory was the first to isolate
the toad lens in an Ussing-type chamber and record an
Isc that was originated by the activity of the
Na+-K+ pump in the lens epithelium
(7-9). We, as well as others, assumed that the
activity of the Na+-K+ pump was relatively
uniform on the anterior epithelium and that the asymmetry was strictly
in the anterior-to-posterior direction.
The results from Patterson's group (23, 27, 29, 30)
indicated a totally new concept for lens electrophysiology: ionic currents leaving the lens at the equatorial region and reentering at
each pole. However, the reports by Patterson and coworkers did not
account for all the currents that should be moving across the lens
surface. We designed the present experiments to empirically reexamine
Patterson's model with a more direct method in which the
Isc was recorded at various arbitrarily defined
zones. Our initial results were in accord with Patterson's
conclusions, suggesting that his model was, in general, correct. This
is based on the belief that the methodology that we have used is sound
and the theoretical analysis valid. Also in support of this, Gao et al. (16) estimated that the pump current density at the frog
lens equator was about 20 times larger than that at the anterior pole.
The O-ring provided an excellent seal that separated two areas of the
lens surface without damage. Our results were reproducible after the
lens was removed and repositioned on various O-rings. The
short-circuiting technique, as applied in this work, simulates the
condition of a freely submerged lens, with the
Isc being a measure of the net current entering
on one side of the O-ring and leaving on the other. These currents are
originated by the activity of the Na+-K+ pumps
located mainly in the epithelial cells by two mechanisms: the current
produced directly by the pump and indirectly by the passive flow of
K+ and Na+ due to gradients created by the
pump. We chose Cl
-free solutions to simplify the
identification of currents that could produce the
Isc.
Table 2 and Fig. 6 show the average currents and their direction
recorded across the seven zones. It should be emphasized that these
zones were chosen on the basis of sizes of O-rings that could support
the lens with a certain degree of reproducibility. Although we tried to
use lenses of uniform size (~10.5-mm diameter), the separation of
zones was clearly arbitrary. This was particularly evident in P4, P5,
and P6, where inward and outward currents were registered (see Table
1). Our previous work with a simple separation of anterior from
posterior surfaces determined that across the posterior surface of the
lens, the inward current was essentially a Na+ current
(4). The division of this surface into Z1, Z2, and Z3 did
not change this conclusion. The current was inward and rather uniform
among these three zones (see Table 2). Ouabain and BaCl2
had only minor or no effect when added to the posterior side in P1, P2,
and P3. Thus, as previously reported, the main and largest component of
the inward current was a Na+ influx. When Z4 was examined,
the current was outward. The components of this current were the pump
current plus the diffusion of K+, as ouabain and
BaCl2 brought the Isc to near zero
(e.g., Fig. 4). This effect was observed regardless of the position of
the O-ring as long as the inhibitor and blocker were added to the side
that included Z4.
Examination of the Isc in P5 and P6 revealed a
finding unique to this article. The currents across Z5 and Z6 remained
negative (outward) but in some experiments reversed to a positive
(inward) current. The effects of ouabain and BaCl2 were
variable, suggesting that in these zones, three currents may coexist:
the Na+-K+ pump current, the K+
diffusion current, and the Na+ diffusion current, with the
net dictated by their relative contributions. In the majority of
experiments, the Isc in P6 was negative. This indicates an inward current across Z7 that exits across all the other
zones to complete the circuit around the external loop. Notice that
according to the adopted convention, a negative
Isc in P6 denotes a positive inward current
across Z7 (Isc, Z7 = 0
Isc, P6). Although this inward current was the
norm, in some experiments the current was outwardly directed. This was
due to the fact that the outward K+ current was larger than
the opposite Na+ current. Blocking the K+
current with BaCl2 reversed the outward current or
increased the inward current. In the majority of cases, ouabain had no
effect on the current across Z7. This novel result indicates that the lens epithelium, in an area of about 3-4 mm in diameter concentric to the anterior pole does not translocate Na+ for
K+. The reason for this deficiency is not clear. Possibly,
this region contains an inactive pump or the enzyme is simply absent.
The density of the Na+ current in Z7 is at least 4.7 µA/cm2 (Table 2) and could be larger because an outward
K+ current may be subtracting from the Na+
current in some cases. As we have observed repeatedly before, amiloride
had no effect on the Na+ currents of the lens (data not
shown). The activity of the Na+-K+ pumps is
concentrated in Z4, Z5, and Z6. The total outward current of these
zones is 14.1 µA. Although the largest contributor is Z4, this is due
to its largest area, because its density is lower than that of Z5 and
Z6. The lower density may be due to the fact that Z4 included a
subequatorial posterior area without pumps.
Previous studies (2, 4) and our present experiments show
that the Isc can be totally inhibited by
BaCl2 and ouabain when applied to Z4, Z5, and Z6. The
ouabain component is ~40%, so the total pump current is ~5.64 µA
(14.1 × 0.40) and the outward Na+ current of the pump
is 16.9 µA (5.64 × 3, for a 3:2 Na+-K+
ratio). The inward current across Z1, Z2, Z3, and Z7 totals, on
average, 14.1 µA. Because 16.9 µA of Na+ must get back
into the lens across these zones, 2.8 µA must be carried by
K+ leaving across these zones. Because the K+
influx translocated by the Na+-K+ pump should
be 11.25 µA (16.9 × 2/3), 8.46 µA should be diffusing out
across Z4, Z5, and Z6. Notice that 8.46 µA is 60% of the 14.1 Isc. Figure 8
shows schematically vectors representing the currents originated by the
diffusion of Na+ and K+ and the
Na+-K+ pump across three main areas of the lens
surface. This diagram accounts for all currents entering and leaving
the lens and indicates that small fractions of the K+
outflow maybe across Z1 through Z3 and Z7.

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|
Fig. 8.
Summary of the flows of Na+ and
K+ across the anterior, equatorial, and posterior surfaces.
Although not indicated, there is a circulation of ions around the lens
between the points of exit and entry.
|
|
The electrical resistance of the different lens zones is not uniform
when normalized by unit area. Z2 and Z3 had the largest resistances,
and Z4, Z6, and Z7 had the lowest. The low resistance of those zones is
probably a reflection of a high density of K+ channels. The
equimolar replacement of Na+ by K+ demonstrated
that the K+ permeability of Z7 was larger than that of
Na+. The immediate change in Isc is
dictated by the change in the unidirectional influxes of
Na+ and K+. If the permeability of the two
cations were the same, their combined influx would be proportional to
their combined concentration, which was kept constant in this protocol.
The overall permeability of the other zones also was larger for
K+, but some experiments showed that in Z1, the
permeability to Na+ could be larger than that of
K+. Despite the observed K+ permeability,
BaCl2 had minor effects on certain zones, possibly because
K+ is at near equilibrium across the surfaces of those zones.
A general interpretation of our results suggests that the
Na+-K+ pumps present at the equatorial region
and the anterior face (except for a small anterior polar region)
produce an outward Na+ current that circulates around the
lens surface and gets back into the lens across its posterior face and
its anterior pole. Simultaneously, the Na+-K+
pump is translocating K+ into the lens. This K+
flux leaves the lens predominantly across the equatorial area and Z5.
The other zones contribute less to the K+ outflow. Thus the
internal circulation of Na+ involves currents from the
anterior pole and posterior region to the pumps located at the equator
and anterior region. The internal circulation of K+ may be
more localized because diffusional currents and
Na+-K+ pumps overlap in their distribution.
The pathways for the Na+ and K+ circulation
inside the lens cannot be determined from these experiments, but
Na+ will have to flow from the anterior polar and posterior
polar regions to the equatorial and anterior zones where the
Na+-K+ pumps are located. K+ will
have to travel a shorter distance because the pumps and K+
channels are predominately in the same zones (i.e., Z4 through Z6).
These ions also must pass in opposite directions, which is not
different from their antipodal movement in a flat epithelium.
Although we have measured parameters indicative of the activity of the
Na+-K+-2Cl
cotransporter in the
rabbit lens, its contribution to lens currents will be minor because
its contribution to the Isc is virtually undetectable (1). Recent publications based on structural
observations have indirectly suggested that there is an internal
recirculation of Cl
between fibers and interfibrillar
space (31). Thus the movements of not only Na+
and K+ but also Cl
inside the lens may play
an important role in the maintenance of the orderly structure,
transparency, and malleability of the lens.
In short, our results confirm the original findings of Patterson's
group and provide experimental support to the model of Mathias et al.
(21) for internal fluid circulation in the lens.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Eye Institute Grants EY-00160
and EY-01867 and by an unrestricted grant from Research to Prevent
Blindness, Inc., New York, NY.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: O. A. Candia, Depts. of Ophthalmology and Physiology & Biophysics, Mount
Sinai School of Medicine, 100th St. and 5th Ave., New York, NY
10029-6574 (E-mail: oscar.candia{at}mssm.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00360.2001
Received 30 July 2001; accepted in final form 5 October 2001.
 |
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Am J Physiol Cell Physiol 282(2):C252-C262
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