August 2009
Volume 50, Issue 8
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Lens  |   August 2009
Whole-Cell Patch Clamping of Isolated Fiber Cells Confirms that Spatially Distinct Cl Influx and Efflux Pathways Exist in the Cortex of the Rat Lens
Author Affiliations
  • Kevin F. Webb
    From the Departments of Physiology and
  • Paul J. Donaldson
    From the Departments of Physiology and
    Optometry and Vision Science, School of Medical Sciences, University of Auckland, Auckland, New Zealand.
Investigative Ophthalmology & Visual Science August 2009, Vol.50, 3808-3818. doi:10.1167/iovs.08-2680
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      Kevin F. Webb, Paul J. Donaldson; Whole-Cell Patch Clamping of Isolated Fiber Cells Confirms that Spatially Distinct Cl Influx and Efflux Pathways Exist in the Cortex of the Rat Lens. Invest. Ophthalmol. Vis. Sci. 2009;50(8):3808-3818. doi: 10.1167/iovs.08-2680.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To test the hypothesis that lens fiber cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume.

methods. Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd3+, and short and long fiber cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA).

results. Cells from the influx zone were dominated by an outwardly rectifying Cl conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K+ conductance(s) with only a minimal contribution from the Cl conductance. Exposure of cells that exhibited a minimal baseline Cl conductance to hyposmotic challenge caused swelling and a transient increase in Cl current. In other cells that initially lacked a Cl conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential.

conclusions. In peripheral cells, Cl efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these cells also contain a Cl channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited.

The transparent properties of the lens are a direct result of its unique cellular structure. Disruption of the pseudocrystalline packing of the cortical fiber cells, by either cellular swelling or dilation of the extracellular space, increases intralenticular light scattering. 1 Thus, volume regulation at both the cellular and tissue levels is critically important for the maintenance of lens transparency. Earlier studies have shown that whole lenses placed in anisosmotic solutions are capable of regulating their volume. 2 3 4 When placed in hyposmotic medium, lenses initially swell before undergoing a regulatory volume decrease (RVD) via the loss of K+ and Cl ions and obligatory water loss. In other cell types, the efflux of KCl associated with RVD is mediated by the activation of K+ and Cl channels, 5 6 7 and/or potassium chloride cotransporters (KCCs). 8  
The relative contributions that Cl channels and transporters make to volume regulation in the lens has been examined by culturing rat lenses in the presence of reagents that modulate the activity of Cl transport proteins. 9 10 11 12 13 The application of the anion channel antagonists tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited volume regulation in lenses exposed to hyposmotic challenge. 11 13 In addition, lenses exposed to NPPB under isosmotic conditions increased their volume and exhibited light scattering. 11 Thus, under normal isosmotic conditions, the lens has a constitutively active Cl flux that regulates fiber cell volume. In response to hyposmotic challenge, this Cl flux can be upregulated to restore lens volume and maintain lens transparency. 
Histologic analysis of lenses treated with a variety of inhibitors 9 10 11 12 revealed that blocking Cl transport induces either one of two spatially distinct tissue damage phenotypes or, on occasion, a combination of the two phenotypes (Fig. 1) . In contrast to the regular cellular architecture observed in control lenses, lenses cultured in the presence of either the Cl channel inhibitor NPPB (Fig. 1A) , or the NKCC (Na+/2Cl/K+ cotransporter) blocker bumetanide (Chee et al., unpublished data, 2009), exhibited a localized band of tissue damage. This damage manifests itself as extracellular fluid accumulations between fiber cells located some 150 μm from the lens capsule. In contrast, the predominant effect of culturing lenses in the presence of [(dihydronindenyl)oxy] alkanoic acid (DIOA), a putative KCC inhibitor, was a swelling of fiber cells located at the lens periphery, although some deeper extracellular space dilations were evident (Fig. 1B)
The two distinct damage phenotypes generated by the inhibitors can be explained with reference to previous experimental measurements of lens membrane potential (Fig. 1C) . By measuring radial differences in the transmembrane potential and the concentration of Cl in the whole lens, the electrochemical gradient for Cl ion movement (ECl) can be calculated at different depths into the lens. 14 15 This analysis predicts that Cl will move from the extracellular space into fiber cells in the inner lens, but will be driven from the cytoplasm of fiber cells to the extracellular space in the lens periphery (Fig. 1C) . Therefore, one would expect that an inhibition of Cl fluxes would block the uptake of Cl from the extracellular space by fiber cells in the inner lens. This would cause an accumulation of Cl ions and water in the tortuous extracellular space and lead to the formation of extracellular space dilations (Fig. 1C) . In the lens periphery, the passive efflux of Cl ions from fiber cells would be blocked, thereby causing an intracellular accumulation of osmolytes and resultant fiber cell swelling. These two spatially segregated zones of cell swelling and extracellular space dilations have been deemed to be due to the inhibition of Cl influx and efflux in deeper and peripheral fiber cells, respectively. 12 Since these pathways are coupled together by gap junctions they generate a circulating flux of Cl ions, which contributes to the maintenance of steady state lens volume. The two spatially distinct damage phenotypes observed from pharmacologic experiments with Cl transport inhibitors, indicate that KCCs and Cl channels mediate ion uptake in the deeper cells, whereas KCCs mediate ion efflux in peripheral fiber cells (Fig. 1) . Since peripheral cells are coupled to deeper cells via gap junctions, these spatially distinct anion influx and efflux pathways must be balanced to maintain control of steady state cell volume in the lens. 16  
Although this model was initially developed with data obtained using inhibitors of Cl transport proteins that are notoriously nonselective, 17 the distinct morphologic effects obtained when using appropriate low doses of NPPB and DIOA (Fig. 1)suggest that these inhibitors are specific for their intended targets. This conclusion is supported by the use of molecular techniques to demonstrate the presence of specific anion transport proteins within appropriate cortical zones. Consistent with the damage caused to peripheral fiber cells by DIOA (Fig. 1B) , the differential expression of several KCC isoforms (KCC1, 3, and 4) was confirmed in the lens. 9 All three KCC isoforms were expressed in peripheral fiber cells, but in this region, their subcellular distribution was predominantly cytoplasmic. However, on exposure to hyposmotic challenge KCC1 and -4 underwent a translocation to the plasma membrane suggesting a physiological role for KCCs in mediating electroneutral ion efflux from peripheral fiber cells under both iso- and hyposmotic conditions. 9 Although the molecular identity of the Cl channels that mediate the circulating Cl fluxes remains unclear, patch clamp experiments on isolated fiber cells have allowed the functional properties of the channel(s) that mediate Cl fluxes to be characterized. 18 19 By relating fiber cell length to fiber cell position (cell layers) within lens sections, Webb et al. 18 showed that isolated fiber cells longer than 120 μm originate from the zone of ion influx. Fiber cells of this length exhibited a constitutively active outwardly rectifying Cl conductance that exhibited a lyotropic anion selectivity sequence (I > Cl), reminiscent of volume-sensitive Cl conductances seen in many cell types. 20 In contrast, the membrane properties of shorter fiber cells (<120 μm) isolated from the efflux zone were dominated by a K+ conductance and under isosmotic conditions appeared to lack a major contribution from the constitutively active Cl conductance seen in longer cells. 19  
Taken together, our data suggest that KCCs and Cl channels contribute to the maintenance of lens volume and that their activity can be modulated by changes in external osmolarity. To test this hypothesis, we exposed isolated fiber cells of various lengths, and therefore different stages of differentiation, to hyposmotic challenge, and used the patch clamp technique to monitor the activation of a characteristic outwardly rectifying Cl channel. By performing these experiments in the presence and absence of low effective concentrations of DIOA we were able to determine the relative contributions of the KCCs and Cl channels to Cl movement in fiber cells isolated from the efflux and influx zones. We show that in short fiber cells Cl efflux was primarily mediated by KCCs, and that the activity of the transporter could be upregulated by cell swelling. In addition, these cells also contain a Cl channel that exhibits a variable baseline activity level and that can be recruited to affect RVD if required. In contrast, longer fiber cells from the zone of influx contain a constitutively active Cl channel whose activity is increased by hyposmotic challenge. Our results confirm at the cellular level that there are spatially distinct Cl influx and efflux pathways in rat lenses and imply that their activity must be coordinated if overall lens volume and hence transparency are to be maintained. 
Methods
Isolation of Fiber Cells
Three- to four-week-old Wistar rats were killed by CO2 asphyxiation and cervical dislocation in accordance with protocols approved by the University of Auckland Animal Ethics Committee and in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Lenses were extracted from the eye and placed into artificial aqueous humor (AAH, in mM: NaCl 149, KCl 4.7, CaCl2 2.5, glucose 5, and HEPES 5 [pH 7.4] with the osmolarity adjusted to 300 mOsM · kg−1 with mannitol). With the aid of a dissecting microscope, sharpened forceps were used to gently remove the capsule and the fiber cells attached to it. The capsule with adherent cells was transferred to a tube (Eppendorf, Fullerton, CA) and incubated for 30 minutes in 1 mL of dissociation buffer (Na gluconate 170 mM, KCl 4.7 mM, HEPES 5 mM, glucose 5 mM, 0.125% wt/vol Sigma type 1A collagenase, at 37°C). Cells were gently vortexed before being centrifuged at 1000 rpm for 2 minutes. Pelleted cells were resuspended in 200 μL of AAH, which contained 1 mM GdCl3. The generic nonselective cation channel inhibitor Gd3+ was included to prevent the vesiculation of fiber cells that occurs on their isolation. 18 21 The cells were plated onto a poly-l-lysine coated glass coverslip that formed the bottom of a recording chamber, which in turn was mounted on the stage of an inverted microscope (Eclipse; Nikon, Melville, NY). Once adhered to the coverslip (∼5 minutes) the cells were overlaid with AAH+Gd3+ and continuously perfused using a peristaltic pump (Minipulse 3; Gilson, Middleton, WI) at a rate of 0.4 to 0.6 mL · min−1. Hyposmotic AAH was prepared by reducing the NaCl concentration to yield an osmolarity of 250 mOsM · kg−1. The KCC inhibitor DIOA was purchased from Sigma-Aldrich (St. Louis, MO) and made up to a concentration of 100 μM in either isosmotic or hyposmotic AAH that contained 1 mM Gd3+ and was introduced to the bath via the perfusion system. In the presence of Gd3+, DIOA precipitated to give a free concentration of around 6 μM as calculated by spectrophotometric analysis of precipitate formation. Cl-free AAH solutions were prepared by equimolar substitution of NaCl with Na gluconate, NaI, or NaNO3. All solutions were introduced to the bath via the perfusion system. All experiments were conducted at room temperature (∼20°C). 
Whole-Cell Patch Clamping
Patch electrodes were fabricated with a horizontal pipette puller (model P-77; Sutter Instruments, Novato, CA), fire polished, and when backfilled with filtered pipette solution (in mM: NaCl 10, K gluconate 130, HEPES 10, CaCl2 1.3, EGTA 10, MgCl2 4.1, [pH 7.4] 300 mOsM · kg−1), had resistances of between 3 and 5 MΩ. The pipettes were mounted directly to the head stage of a patch clamp amplifier (Multiclamp 700A; Axon Instruments, Union City, CA) and were positioned with a piezoelectric manipulator (Burleigh PCS-5000; Exfo Life Sciences, Mississauga, Ontario, Canada). Data were digitized with an analog-digital (A/D) converter (Digidata 1322A SCSI; Axon Instruments) and a 266-MHz computer (Pentium II; Mountain View, CA) computer (pClamp ver. 8.1 software package; Axon Instruments). Images were acquired via a cooled CCD camera (Photometrics Imagepoint; Roper Scientific, Tucson, AZ) attached to a framegrabber card under the control of an image analysis workstation (Imaging Workbench ver. 2.2; Axon Instruments). 
Whole Lens Dissection and Electrophysiological Recording
Four posterior incisions were made in the extracted rat eyes to create four scleral flaps that were pinned out into a custom-made recording chamber that was transferred to the stage of a dissecting microscope (Stemi SV 11; Carl Zeiss Meditec, Frankfurt, Germany). The pinned-out lens preparation was overlaid with warmed AAH (37°C) and continually perfused (∼2 mL/min) with AAH. The recording chamber (2 mL) was perfused by using three solution reservoirs independently controlled by three-way taps via a manifold with approximately 2 mL of downstream dead space. The lenses were impaled with glass microelectrodes (150F-10; Clark Electromedical Instruments, Edenbridge, UK) manufactured using a pipette puller (P-77 Micropipette puller; Sutter Instrument Co.) and had a resistance of between 3 and 5 MΩ when backfilled with 1 M KCl. The microelectrodes were attached to the head stage of a microelectrode amplifier (TEV-200; Dagan Corp., Minneapolis, MN). The bath was grounded by connection to an AgCl-coated silver wire 1 M KCl half cell via a 3% agarose 1 M KCl salt bridge. Lens potential recordings were sampled at 1 Hz with an A/D converter (DigiData 1200B; Axon Instruments) and recorded by software (Axoscope ver. 8.1; Axon Instruments) on a computer (Dell Computer, Round Rock, TX) running on a 500-MHz processor (Celeron; Intel, Santa Clara, CA) and a commercial operating system (Windows 2000; Microsoft, Redmond, WA). The head stage was mounted on a micromanipulator (World Precision Instruments, Sarasota FL) that was attached to the stage of the dissecting microscope. This electrophysiological setup was surrounded by a Faraday cage positioned on a vibration isolation table (Newport Corp., Irvine, CA). Mean lens potential was determined at baseline and both before and after exposure to the same solutions of varying ionic composition and osmolarity that isolated cells were exposed to. Summarized data were plotted on a spreadsheet (Excel; Microsoft) and statistical analysis was performed (Origin ver. 7.5; OriginLab Corp., Northampton, MA). 
Results
In a previous study 18 we used a calibration curve that related the axial length of fiber cells to the in situ position of fiber cells expressed as the number of cell layers in from the capsule (Fig. 1D) . The examination of pharmacologic damage phenotypes indicated that the sharp transition between zones of anion influx and efflux occurred in equatorial lens sections approximately 15 cell layers in from the capsule (Fig. 1D) . Thus, fiber cells in the zone of anion influx were in excess of ∼125 μm in length, whereas shorter, more peripheral fiber cells mediated anion efflux. The enzymatic dissociation of the lens yielded isolated fiber cells of a range of lengths from 23 to 605 μm that span the anion efflux and influx zones. Although these cells represent a continuum of fiber cell differentiation, it is possible to define two distinct “typical” membrane permeabilities in the short and long fiber cells isolated from the efflux and influx zones, respectively (Fig. 2) . Short fiber cells were usually dominated by K+ conductances with a minimal contribution from a Cl conductance (Figs. 2A 2B 2C 2D) . In contrast, in long fiber cells isolated from the influx zone, the dominant membrane conductance was an outwardly rectifying anion conductance that was reduced by the substitution of extracellular Cl with the impermeant anion gluconate (Figs. 2E 2F 2G 2H) . In the following sections we show that these typical membrane conductances were differentially regulated by changes in cell volume induced by either exposure to hyposmotic solutions or by the addition of DIOA, a putative inhibitor of KCC activity. 
Exposure of Isolated Fiber Cells of Various Lengths to Hyposmotic Challenge
In previous studies we have shown that anion channels in fiber cells exhibit a lyotropic anion selectivity sequence, 18 19 that is reminiscent of that observed for the volume-sensitive organic anion channel (VSOAC) activated in a variety of different cell types in response to exposure to hyposmotic solutions. 22 To determine whether the spontaneously active anion conductance that dominates the membrane properties of long fiber cells is also volume-sensitive, fiber cells isolated from the zone of ion influx were exposed to a solution rendered hyposmotic by omission of NaCl (Fig. 3) . Because fiber cells isolated from this region have a very long and thin morphology fiber cells isolated from this region, it proved difficult to resolve whether hyposmotic challenge was inducing cell swelling in these long fiber cells, although it appears that the tips of the fibers were somewhat swollen (Fig. 3B) . Despite the absence of a distinct morphologic effect, the reduction of extracellular osmolarity resulted in an increase in the characteristic outwardly activating conductance component of the whole-cell current (Figs. 3C 3D) . This increase in conductance occurred over a time course of minutes, and principally involved an increase in current at depolarizing potentials (Fig. 3E) . When the current–voltage relationships were examined, it was apparent that the increase in outward current triggered by lowering the osmolarity in AAH in which Cl is the major anion was not associated with a shift in reversal potential, and inward current was largely unaltered (Fig. 3F) . The subsequent replacement of extracellular Cl with hyposmotic solutions containing either I (Table 1)or NO3 (Fig. 3F , Table 1 ) produced a hyperpolarization of the reversal potential and an increase in current magnitude, whereas replacement with gluconate depolarized the reversal potential and greatly reduced the outward current (Fig. 3F , Table 1 ). Taken together, these observations suggest that the activity of the lyotropic anion channel that dominates the membrane behavior of isolated fiber cells can be increased by a decrease in extracellular osmolarity. 
In contrast to the longer fiber cells, exposure of the short fiber cells isolated from the zone of ion efflux to hyposmotic challenge produced a detectible cellular swelling of all shorter cells (Figs. 4 5) . However, the swelling of shorter fiber cells was not always accompanied by changes in membrane current and the responses of these shorter cells could be divided into two distinct classes. Although most short fiber cells are usually dominated by K+ conductances, some also exhibit an additional variable contribution from a constitutively active anion conductance. 19 In peripheral fiber cells that contained an active anion conductance, hyposmotic challenge resulted in an initial increase in outward current (Fig. 4E)that was similar in character to that observed in longer fiber cells. However, unlike in longer fiber cells, the observed increase in outward current in response to hyposmotic challenge in these shorter cells was not maintained and was followed by a decline in outward current toward baseline levels (Figs. 4E 4F) , a result reminiscent of RVD. Once again anion-substitution experiments verified that this increase in outward current was mediated by a lyotropic anion channel (Table 1)
The other class of response observed in short fiber cells was most evident in those cells whose membrane properties were dominated by an inwardly rectifying K+-conductance (Fig. 5) . In these cells, although exposure to hyposmotic solutions caused cell swelling (Fig. 5B) , no increase in outwardly directed current was observed (Fig. 5E) . Of interest, in this example, a transient outward current was observed during the voltage step to +100 mV (Fig. 5E)that is suggestive of the activation of a normally quiescent membrane current. In this subgroup of cells, it appears that either RVD is not occurring, or that it is mediated by an electroneutral transport process. To test the possibility that this electroneutral process is mediated by the KCC, DIOA was applied to the hyposmotic bathing solution. Although difficult to quantify precisely, because these cells swell at their ends but shorten at the same time, it appears that in response to the addition of DIOA, the cell underwent further swelling (Fig. 5C)and exhibited a marked increase in outward current, suggesting the activation of a previously quiescent outwardly rectifying lyotropic anion conductance (Figs. 5F 5G 5H ; Table 1 ). Thus, it appears in this subset of short fiber cells that an electroneutral transport process, rather than a Cl channel, mediates ion efflux to effect RVD in response to an applied hyposmotic challenge. However, on blockage of this ion efflux by DIOA cell swelling was exacerbated and a volume-sensitive Cl channel was then activated. 
The time dependence of the effects of hyposmotic challenge on the current voltage relationships recorded from fiber cells isolated from both the influx and efflux zones are summarized and compared in Figure 6 . In longer fiber cells isolated form the zone of influx the initial hyposmotically induced increase in outward current was maintained throughout the experimental recording (Fig. 6A) . This illustrates that not only are the Cl channels in these cell constitutively active and sensitive to changes in volume sensitive, but when exposed to hyposmotic cell swelling, they exhibit a sustained activation. In contrast, short fiber cells isolated from the efflux zone exhibited only a minor Cl channel activity or no baseline Cl channel activity. In the former, hyposmotic challenge produced a transient increase in outward current that was followed by a decline in current that is indicative of an RVD (Fig. 6B) . In the latter cells with no baseline Cl activity, hyposmotic challenge produced no change in outward current, but the subsequent addition of DIOA resulted in a transient increase in outward current that is again indicative of an RVD (Fig. 6C)
Steady State Volume Regulatory Mechanisms in Isolated Fiber Cells
Under isosmotic conditions, morphologic damage is induced in cultured lenses by pharmacologic inhibition of anion transport pathways. It thus appears that interfering with either Cl channels or transporters in the resting lens disrupts volume homeostasis in cortical fiber cells. To test whether an electroneutral ion efflux, presumably mediated by KCCs also have a role in steady state volume regulation in isolated fiber cells, DIOA was added to isolated fiber cells under isosmotic conditions, causing the swelling of peripheral fiber cells isolated from the zone of ion efflux (Fig. 7) . Although cell swelling was obvious by microscope and in time-lapse video microscopy, it was difficult to quantify cell volume from these two-dimensional differential interference contrast (DIC) images. However, it can be seen from a qualitative examination that while short isolated cells from the zone of ion efflux display obvious cellular swelling in response to DIOA treatment (Figs. 7A 7B 7C 7D) , the longer cells isolated from the zone of ion influx are minimally if at all affected by the addition of DIOA (Fig. 7E 7F) . This observation is in agreement with the pharmacologic damage observed in the peripheral zone of ion efflux when lenses were cultured in the presence of DIOA. These observations imply that KCCs play a major role in mediating ion efflux, which contributes to steady state volume homeostasis in cells from the zone of ion efflux. 
Since the addition of DIOA leads to cell swelling under isosmotic conditions (Fig. 7) , it is expected that this volume change would potentiate the activity of volume-sensitive Cl conductances, as demonstrated previously under hyposmotic conditions (Fig. 5) . In the short fiber cell shown in Figure 8the application of DIOA under isosmotic conditions caused marked cell swelling (Fig. 8B)and a transient increase in whole-cell current (Figs. 8E 8F)that is consistent with upregulation of a Cl conductance. From this conductance increase, it can be implied that the KCCs are in fact the major anion efflux pathway in peripheral cells under isosmotic conditions. However, when this transporter is inhibited with DIOA, ion efflux is blocked, the cells swell, and a volume-sensitive Cl channel is activated to create an alternative parallel pathway to mediate ion efflux. 
Exposure of Whole Lenses to Osmotic Stress
To directly test whether the observed modulation in anion conductance in isolated fiber cells in response to hyposmotic challenge in either the presence or absence of DIOA is a physiological feature of the lens and not some artifact of the fiber cell isolation procedure, we impaled whole lenses with a microelectrode and monitored the response of the intracellular lens membrane potential (V m) to changes in the extracellular anion composition (Fig. 9 , Table 1 ). As in previous studies, 19 23 24 25 V m was found to be modulated by extracellular anion replacement in a fashion substantially similar to that in isolated cells (V m, Table 1 ). Under isotonic conditions, both NO3 and I addition caused a hyperpolarization of whole lens V m, whereas replacement with gluconate caused depolarization (Fig. 9A) . These relative alterations in V m were preserved under hypotonic conditions (Fig. 9B)and when lenses were further challenged by exposure to DIOA (Table 1) . Although it is theoretically possible to use the Goldman-Hodgkin-Katz (GHK) equation to calculate the relative permeability changes induced by osmotic challenge, it requires that the cation-to-anion permeability ratio be low and remain constant throughout the experiment. 26 Since under osmotic challenge this condition is unlikely to be met, no calculation of relative permeability ratios was attempted. However, the shifts in V m recorded from intact lenses show that the constituent fiber cells possess a constitutively active anion channel, the activity of which can be upregulated under conditions of hyposmotic stress. 
Discussion
The results presented in this study represent a validation of our hypothesis that there are spatially distinct ion influx and efflux pathways in the cortex of the rat lens. 27 This model was initially advanced to explain the spatially distinct damage phenotypes of peripheral cell swelling and deeper extracellular space dilations that were observed when rat lenses were cultured in the presence of a variety of Cl channel and transporter inhibitors. 9 10 12 In the lens cortex, fiber cells continuously undergo a process of differentiation that creates an inherent gradient of cells at different stages of elongation that are in turn influenced by electrochemical gradients that alter with distance into the lens. Previously, we have hypothesized that the measured change in the electrochemical gradient for Cl 28 is the underlying cause of the spatially distinct ion influx and efflux pathways revealed by culturing lenses in the presence of specific Cl transport inhibitors. 12 In this study, we showed that differences in the functional expression of Cl channels and KCCs also contribute to the formation of the spatially distinct ion efflux and influx zones. 
By using fiber cell length as a criterion to assign isolated fiber cells to these putative influx or efflux zones, we have used whole-cell patch clamping to confirm that differences in Cl transport mechanisms exist at the single cell level and that the cells isolated from these two zones respond differently to hyposmotic challenge and to the addition of DIOA. Our results show that the membrane properties of longer cells from the influx zone are dominated by a constitutively active Cl channel, whose activity is upregulated by exposure to hyposmotic challenge (Fig. 3 , Table 1 ). In contrast, the membrane properties of shorter more peripheral fiber cells from the efflux zone are dominated by K+ conductance(s), and in these cells the anion channel was either not active or exhibited a relatively small contribution to overall membrane properties (Figs. 4 5) . In these cells hyposmotic challenge caused cell swelling, but had either no effect on (Fig. 5)or evoked a transient increase in (Fig. 4)outward current. In these cells the addition of DIOA, under either isosmotic (Fig. 8)or hyposmotic conditions (Fig. 5) , induced cell swelling and the activation of an outwardly rectifying Cl channel (Table 1) . If we assume that DIOA blocks KCCs then our results suggest that in short fiber cells ion efflux is primarily mediated by the KCCs. Furthermore, if the ion efflux mediated by the KCCs is blocked, peripheral cells swell, and a Cl channel that is normally quiescent or that exhibits only a low level of activity becomes activated and mediates RVD. As fiber cells elongate, it appears that this Cl channel becomes constitutively active and eventually dominates the membrane properties of longer fiber cells located deeper into the lens. In these cells hyposmotic challenge produces a further sustained augmentation of the outward current, indicating that the channel retains its volume sensitivity. 
This interpretation, however, relies on the fact that DIOA is a specific inhibitor of KCC activity. Unfortunately, at higher concentrations DIOA has also been shown to inhibit Cl channels, NKCCs, and more recently IR K channels. 5 In our experiments, we used a free concentration of DIOA of ∼6 μM, which is considered in the appropriate range to specifically block KCCs. 29 At this concentration, the effect of addition of DIOA to short fiber cells under isotonic and hypotonic conditions is the activation of an outwardly rectifying Cl conductance. This observation effectively rules out the possibility that DIOA inhibits either a Cl or K+ channel. In addition to the activation of a Cl conductance, DIOA also causes cell swelling in whole lenses (Fig. 1)and isolated short fiber cells (Fig. 7) . In contrast, the addition of the NKCC inhibitor bumetanide to organ-cultured lenses caused peripheral cell shrinkage in the same area where DIOA caused cell swelling, indicating that NKCC mediates ion influx in these peripheral fiber cells (Chee et al., unpublished observation, 2009). Considering the concentration of DIOA used and the consistency of the morphologic effect induced by its addition to isolated and whole lenses, we believe that it is reasonable to conclude that DIOA acts via the inhibition of a KCC in peripheral fiber cells. The inhibition of KCCs in these cells in turn causes an isosmotic cell swelling that activates a normally quiescent, volume-sensitive, lyotropic Cl channel that in the absence of KCC activity mediates a volume regulation. 
Redundancy in volume regulatory pathways is a common feature of volume regulation in other cells (Jentsch et al. 22 ) and roles for both KCCs and Cl channels in the maintenance of cell volume in peripheral fiber cells isolated from the efflux zone is supported by the both the current and previous studies. Chee et al. 9 showed that the lens expresses, in a differentiation-dependent manner, three of the four known KCC isoforms (1, 3, and 4); that application of DIOA to whole lenses culture under isosmotic conditions causes peripheral cell swelling; and that hyposmotic challenge promotes the insertion of KCC1 and -4 into the plasma membrane of peripheral fiber cells. Consistent with these observations in whole lenses, isolated short fiber cells exposed to DIOA under both hyposmotic (Figs. 4 5)and isosmotic (Fig. 8)conditions exhibited cell swelling, and this swelling was associated with an increase in outward current. Since KCCs 30 and VSOAC 22 are both volume sensitive, cell swelling should potentiate the activity of each mechanism in parallel. If a given flux of anions across the cell membrane is necessary to control cell volume, this flux will thus be shared between both the channel and transporter-mediated pathways according to their current level of activity. Since the transporter is electrically silent, only the anion conductance is visible under whole-cell patch clamp recording. The variability in response to hyposmotic challenge seen in short isolated fiber cells (Figs. 4 5)could thus indicate a variable contribution of these two pathways to volume regulation under osmotic stress. The existence of two interacting anion efflux pathways could also explain the heterogeneity in baseline anion conductance seen previously in short fiber cells. 19  
Since in a variety of cell types RVD is mediated by a transient activation of a Cl conductance, we assumed that the initial increase and subsequent decrease in outward current observed in short fiber cells exposed to hyposmotic challenge in the presence and absence of DIOA also represents RVD in these cells. Although this is a reasonable assumption, it of course should be confirmed by correlating the time course of cell swelling with the observed changes in outward current. Although changes in the morphology of short fiber cells are apparent, changes in longer fiber cells in response to hyposmotic challenge were less obvious (Fig. 7) , and in both cases the changes in morphology were difficult to quantify. Quantification of cell volume changes from DIC images of isolated fiber cells were constrained in long fiber cells by limited imaging resolution and in short cells by the observed changes in fiber cell geometry. In short fiber cells, osmotic stress induced swelling at the tips and a shortening of the cell that made it difficult to calculate fiber cell geometry and therefore volume from the two-dimensional images (Figs. 7C 7D) . Accurately quantifying cell volume changes and therefore the progression of RVD requires an alternative approach such as the use of a fluorescent indicator of cell volume. Despite this caveat about the need to quantify changes in cell volume in future experiments, it is apparent that once this volume-sensitive outward current is activated, the time course of current activation is quite different between fiber cells isolated from the efflux and influx zones (Fig. 6) . In short cells from the efflux zone, the observed reduction in outward current with time implies that the activation of the channel has initiated a process of RVD that restores cell volume, and this restoration of cell volume reduces the activity of volume-sensitive Cl conductance. By analogy, the sustained activation of the outward current observed in long fiber cells indicates that RVD is not occurring in these cells isolated from the zone of ion influx. This is consistent with the view that with distance into the lens there is a decline in the transmembrane potential that promotes a reversal of ECl, which in turn drives a Cl influx, not the efflux of Cl necessary to effect RVD. Thus, in long fiber cells isolated from the influx zone hyposmotic challenge activates the volume-sensitive Cl conductance, increases Cl influx, and exacerbates cell swelling. 
The demonstrated volume sensitivity of the KCCs and Cl channels in fiber cells isolated from the influx zone has implications for the maintenance of the ordered lens tissue architecture required for lens transparency. Diabetic lens cataract is characterized by a distinct band of tissue liquefaction in the outer cortex that is flanked by regions of normal fiber cell morphology. 31 This band of advanced tissue damage appears to be initiated by the swelling of fiber cells located in the influx zone. 31 In the diabetic rat lens, hyperglycemia results in the depletion of antioxidant GSH, increased oxidative stress and the accumulation the impermeable osmolyte sorbitol. 32 We have hypothesized 16 33 that these combined stresses induce fiber cell swelling that in turn activates the volume-sensitive KCCs and Cl channels that our work has shown to be expressed in cortical fiber cells. However, because of the existence of distinct ion efflux and influx zones in the outer cortex (Fig. 1)the volume-sensitive activation of KCCs and Cl channels will have spatially different morphologic effects. In peripheral fiber cells, the activation of KCCs and Cl channels results in RVD that acts to preserve fiber cell volume and therefore tissue architecture in the periphery of the diabetic lens. In deeper fiber cells located in the influx zone the hyperglycemia-induced accumulation of osmolytes activates the volume-sensitive KCCs and Cl channels, promoting an increased ion influx and an augmentation of cell swelling, specifically in this zone of the lens. 
Because our results indicate that volume-dependent activation of the Cl channels in these long fiber cells is maintained and is not transient as observed for peripheral fiber cells, we propose that this prolonged activation of ion influx pathways in this zone initiates a destructive cascade that mimics the observed globulization that occurs when fiber cells are isolated in presence of Ca2+ ions. 19 21 34 35 The initial cell swelling activates ion influx pathways that in turn lead to membrane potential depolarization, Ca2+ influx, gap junction closure, the activation of Ca2+-dependent proteases, cytoskeletal degradation, and ultimately the localized band of tissue liquefaction typically observed in diabetic cataract. Thus, it seems that the ultimate cause of diabetic cataract may not be the osmotic load imposed by the membrane-impermeable glucose metabolites trapped in fiber cells, but the destructive calcium influx mediated by cation channels gated open by membrane stretch and depolarization. 
 
Figure 1.
 
A working model for volume regulation in the outer cortex of the rat lens. (A) Confocal images of equatorial sections of organ-cultured rat lenses under isosmotic conditions for 18 hours in the presence of (A) the Cl channel inhibitor NPPB (10 μM) and (B) the KCC inhibitor DIOA (10 μM) exhibited extracellular dilations (inset) and peripheral cell swelling, respectively. (C) Molecular model of transporters and channels that contribute to volume regulation in different regions of the rat lens. In this model, radial differences in transmembrane voltage 14 contribute to the establishment of spatially distinct ion efflux and influx zones. Since these two zones are connected by gap junction channels, a circulating flux is established that helps to maintain cell volume by balancing ion influx and efflux. (D) Plots of fiber cell length versus numbers of cell layers in from the lens capsule obtained from an earlier study 18 overlaid with the flux reversal point that separates the ion efflux and influx zones. Using this plot, it is evident that isolated fiber cells that are greater than 120 μm in length originate from the zone of ion influx.
Figure 1.
 
A working model for volume regulation in the outer cortex of the rat lens. (A) Confocal images of equatorial sections of organ-cultured rat lenses under isosmotic conditions for 18 hours in the presence of (A) the Cl channel inhibitor NPPB (10 μM) and (B) the KCC inhibitor DIOA (10 μM) exhibited extracellular dilations (inset) and peripheral cell swelling, respectively. (C) Molecular model of transporters and channels that contribute to volume regulation in different regions of the rat lens. In this model, radial differences in transmembrane voltage 14 contribute to the establishment of spatially distinct ion efflux and influx zones. Since these two zones are connected by gap junction channels, a circulating flux is established that helps to maintain cell volume by balancing ion influx and efflux. (D) Plots of fiber cell length versus numbers of cell layers in from the lens capsule obtained from an earlier study 18 overlaid with the flux reversal point that separates the ion efflux and influx zones. Using this plot, it is evident that isolated fiber cells that are greater than 120 μm in length originate from the zone of ion influx.
Figure 2.
 
Differentiation dependent changes in fiber cell membrane properties. (A) Image of a short (56 μm) fiber cell isolated from the zone of ion efflux. Whole-cell currents recorded in AAH from the cell shown in (A) exhibit a characteristic inwardly activating current component (B) that is eliminated by exposure to 5 mM of the K+ channel blocker Ba2+ (C). (D) Current voltage curves recorded from the cell in (A) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The Ba2+ inhibition of the inward current component, the positive shift in reversal potential, and the lack of effect of gluconate-substitution indicated that the membrane properties of short fiber cells are dominated by a potassium conductance. (E) Image of a longer fiber cell (275 μm) isolated from the zone of ion influx. Whole-cell currents recorded in AAH from the cell shown in (E) exhibited a larger characteristically outwardly rectifying current (F) that was significantly reduced in magnitude by the substitution of extracellular Cl with the impermeant anion gluconate (G). (H) Current voltage curves recorded from cell shown in (E) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The lack of effect of Ba2+ and the large reduction in outward current and a positive shift in reversal potential on replacement of extracellular Cl with gluconate indicates that the membrane properties fiber cells isolated from the zone of ion influx are dominated by an anion conductance.
Figure 2.
 
Differentiation dependent changes in fiber cell membrane properties. (A) Image of a short (56 μm) fiber cell isolated from the zone of ion efflux. Whole-cell currents recorded in AAH from the cell shown in (A) exhibit a characteristic inwardly activating current component (B) that is eliminated by exposure to 5 mM of the K+ channel blocker Ba2+ (C). (D) Current voltage curves recorded from the cell in (A) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The Ba2+ inhibition of the inward current component, the positive shift in reversal potential, and the lack of effect of gluconate-substitution indicated that the membrane properties of short fiber cells are dominated by a potassium conductance. (E) Image of a longer fiber cell (275 μm) isolated from the zone of ion influx. Whole-cell currents recorded in AAH from the cell shown in (E) exhibited a larger characteristically outwardly rectifying current (F) that was significantly reduced in magnitude by the substitution of extracellular Cl with the impermeant anion gluconate (G). (H) Current voltage curves recorded from cell shown in (E) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The lack of effect of Ba2+ and the large reduction in outward current and a positive shift in reversal potential on replacement of extracellular Cl with gluconate indicates that the membrane properties fiber cells isolated from the zone of ion influx are dominated by an anion conductance.
Figure 3.
 
The effect of hypotonic challenge on fiber cells isolated from the zone of ion influx. Images of and whole-cell currents recorded from a fiber cell (475 μm) isolated from the influx zone bathed in isotonic AAH (A, C) and after exposure to hypotonic AAH (B, D). Note the lack of any obvious change in cell morphology in response to hypotonic challenge. (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion influx respond to hypotonic challenge with a sustained activation of outward current. (F) Whole-cell current–voltage relationships for the cell in isotonic AAH (A, • 300 mOsM · kg−1), hypotonic Cl-based AAH (B, ▵ 250 mOsM · kg−1), hypotonic NO3 based AAH (▴), and gluconate-based AAH (▪). Scale bar, 50 μm.
Figure 3.
 
The effect of hypotonic challenge on fiber cells isolated from the zone of ion influx. Images of and whole-cell currents recorded from a fiber cell (475 μm) isolated from the influx zone bathed in isotonic AAH (A, C) and after exposure to hypotonic AAH (B, D). Note the lack of any obvious change in cell morphology in response to hypotonic challenge. (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion influx respond to hypotonic challenge with a sustained activation of outward current. (F) Whole-cell current–voltage relationships for the cell in isotonic AAH (A, • 300 mOsM · kg−1), hypotonic Cl-based AAH (B, ▵ 250 mOsM · kg−1), hypotonic NO3 based AAH (▴), and gluconate-based AAH (▪). Scale bar, 50 μm.
Table 1.
 
Comparison of the Effects of Anion Replacement on E rev in Isolated Fiber Cells and V m in Whole Lenses
Table 1.
 
Comparison of the Effects of Anion Replacement on E rev in Isolated Fiber Cells and V m in Whole Lenses
Isotonic (300 mOsmkg−1) Hypotonic (250 mOsmkg−1) Hypotonic + DIOA (250 mOsmkg−1)+10μM)
CI I NO3 Gluc CI I NO3 Gluc CI I NO3 Gluc
Isolated fiber cells E rev (mV) −20.28 ± 3.30 −34.76 ± 4.94 −40.00 ± 6.50 −9.54 ± 4.70 −13.27 ± 5.02 −20.16 ± 6.74 −31.03 ± 2.68 4.17 ± 0.50 −36.28 ± 12.08 −43.65 ± 11.33 −43.10 ± 12.35 −7.70 ± 4.30
(33) (25) (13) (9) (7) (4) (3) (3) (3) (3) (2) (2)
Whole lens in vitro V m (mV) −60.36 ± 3.26 −67.13 ± 2.96 −77.04 ± 3.89 −57.12 ± 4.42 −60.99 ± 4.03 −65.68 ± 3.33 −72.19 ± 5.03 −61.79 ± 10.51 −42.28 ± 0.29 −55.82 ± 1.55 −53.71 ± 5.94 −51.36 ± 8.69
(11) (7) (5) (10) (3) (3) (3) (3) (3) (3) (2) (2)
Figure 4.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a baseline anion conductance. Images of and whole-cell currents recorded from a short fiber cell (55 μm) isolated from the efflux zone bathed in isosmotic AAH (A, C), and some 8 minutes after exposure to hyposmotic AAH (B, D). (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion efflux respond to hyposmotic challenge with a transient activation of outward current. (F) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge.
Figure 4.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a baseline anion conductance. Images of and whole-cell currents recorded from a short fiber cell (55 μm) isolated from the efflux zone bathed in isosmotic AAH (A, C), and some 8 minutes after exposure to hyposmotic AAH (B, D). (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion efflux respond to hyposmotic challenge with a transient activation of outward current. (F) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge.
Figure 5.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a dominant K+ conductance. Images of, and whole-cell currents recorded from, a short fiber cell (58 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to hyposmotic AAH (B, E), and after exposure to hyposmotic AAH+10 μM DIOA (C, F). A flicker-like current (E, ▴) that is indicative of an apparent activation of an outward current at very depolarized potentials was evident. (G) Time course of normalized whole-cell current recorded at +100 mV showing that fiber cells isolated from the zone of ion efflux that exhibited a dominant K+ conductance responded to hyposmotic challenge with no significant increase in outward current. However, the subsequent addition of DIOA induced the activation of an outward current. (H) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and after hyposmotic challenge in the absence (▴) and presence of DIOA (○).
Figure 5.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a dominant K+ conductance. Images of, and whole-cell currents recorded from, a short fiber cell (58 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to hyposmotic AAH (B, E), and after exposure to hyposmotic AAH+10 μM DIOA (C, F). A flicker-like current (E, ▴) that is indicative of an apparent activation of an outward current at very depolarized potentials was evident. (G) Time course of normalized whole-cell current recorded at +100 mV showing that fiber cells isolated from the zone of ion efflux that exhibited a dominant K+ conductance responded to hyposmotic challenge with no significant increase in outward current. However, the subsequent addition of DIOA induced the activation of an outward current. (H) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and after hyposmotic challenge in the absence (▴) and presence of DIOA (○).
Figure 6.
 
A comparison of responses of isolated fiber cells from the influx and efflux zones to hyposmotic challenge. (A) Pooled whole-cell current-voltage relationships (n = 4) of cells from the zone of ion influx recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. (B) Pooled whole-cell current–voltage relationships (n = 19) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. Note the acute current increase caused by hyposmotic swelling (▵) and the return toward control levels observed at steady state (○), indicating that RVD has occurred. (C) Pooled whole-cell current-voltage relationships (n = 11) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•), 6 minutes after hyposmotic challenge (▪), and hyposmotic challenge in the presence of DIOA after 2 (▵) and 6 (▾) minutes. In this subgroup of cells from the efflux zone, hyposmotic challenge alone did not activate an outward current. Outward current activation occurred only in the presence of hyposmotic plus DIOA (▵), before returning toward control levels observed at steady state (▾), indicating that an RVD had occurred. Note data points for hyposmotic challenge in the presence and absence of DIOA overlay each other in (C).
Figure 6.
 
A comparison of responses of isolated fiber cells from the influx and efflux zones to hyposmotic challenge. (A) Pooled whole-cell current-voltage relationships (n = 4) of cells from the zone of ion influx recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. (B) Pooled whole-cell current–voltage relationships (n = 19) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. Note the acute current increase caused by hyposmotic swelling (▵) and the return toward control levels observed at steady state (○), indicating that RVD has occurred. (C) Pooled whole-cell current-voltage relationships (n = 11) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•), 6 minutes after hyposmotic challenge (▪), and hyposmotic challenge in the presence of DIOA after 2 (▵) and 6 (▾) minutes. In this subgroup of cells from the efflux zone, hyposmotic challenge alone did not activate an outward current. Outward current activation occurred only in the presence of hyposmotic plus DIOA (▵), before returning toward control levels observed at steady state (▾), indicating that an RVD had occurred. Note data points for hyposmotic challenge in the presence and absence of DIOA overlay each other in (C).
Figure 7.
 
The effects of DIOA on the morphology of isolated fiber cells. Images of isolated fiber cells of various lengths incubated in isosmotic AAH in the absence (A, C, E) and presence (B, D, F) of 100 μM DIOA. A short (29 μm) fiber cell (A, B) and slightly longer (63 μm) fiber cell (C, D) isolated from the efflux zone both underwent cell swelling on application of DIOA. A long (383 μm) fiber cell (E, F) isolated from the zone of ion influx remained largely unaffected by exposure to DIOA
Figure 7.
 
The effects of DIOA on the morphology of isolated fiber cells. Images of isolated fiber cells of various lengths incubated in isosmotic AAH in the absence (A, C, E) and presence (B, D, F) of 100 μM DIOA. A short (29 μm) fiber cell (A, B) and slightly longer (63 μm) fiber cell (C, D) isolated from the efflux zone both underwent cell swelling on application of DIOA. A long (383 μm) fiber cell (E, F) isolated from the zone of ion influx remained largely unaffected by exposure to DIOA
Figure 8.
 
Electrophysiological effects of DIOA exposure on fiber cells isolated from the zone of ion efflux. Images of and whole-cell currents recorded from a short fiber cell (23 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to AAH+10 μM DIOA (B, E), and after washout of DIOA with AAH (C, F). Addition of DIOA caused an increase in cell volume. (G) Time course of normalized whole-cell current recorded at +100 mV showing that the addition of DIOA to isosmotic AAH induced the activation of an outward current. (H) Whole-cell current-voltage relationships of the cell under study recorded in isosmotic AAH (•), in the presence of DIOA (▵), and after washout of DIOA (○).
Figure 8.
 
Electrophysiological effects of DIOA exposure on fiber cells isolated from the zone of ion efflux. Images of and whole-cell currents recorded from a short fiber cell (23 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to AAH+10 μM DIOA (B, E), and after washout of DIOA with AAH (C, F). Addition of DIOA caused an increase in cell volume. (G) Time course of normalized whole-cell current recorded at +100 mV showing that the addition of DIOA to isosmotic AAH induced the activation of an outward current. (H) Whole-cell current-voltage relationships of the cell under study recorded in isosmotic AAH (•), in the presence of DIOA (▵), and after washout of DIOA (○).
Figure 9.
 
Modulation of an anion conductance by hyposmotic stress in whole lenses. Representative traces of intracellular microelectrode recordings of membrane potential (V m) obtained from intact rat lenses exposed to different extracellular anions (Cl, NO3 , I, and gluconate) under isosmotic (top) or hypotonic conditions (bottom).
Figure 9.
 
Modulation of an anion conductance by hyposmotic stress in whole lenses. Representative traces of intracellular microelectrode recordings of membrane potential (V m) obtained from intact rat lenses exposed to different extracellular anions (Cl, NO3 , I, and gluconate) under isosmotic (top) or hypotonic conditions (bottom).
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Figure 1.
 
A working model for volume regulation in the outer cortex of the rat lens. (A) Confocal images of equatorial sections of organ-cultured rat lenses under isosmotic conditions for 18 hours in the presence of (A) the Cl channel inhibitor NPPB (10 μM) and (B) the KCC inhibitor DIOA (10 μM) exhibited extracellular dilations (inset) and peripheral cell swelling, respectively. (C) Molecular model of transporters and channels that contribute to volume regulation in different regions of the rat lens. In this model, radial differences in transmembrane voltage 14 contribute to the establishment of spatially distinct ion efflux and influx zones. Since these two zones are connected by gap junction channels, a circulating flux is established that helps to maintain cell volume by balancing ion influx and efflux. (D) Plots of fiber cell length versus numbers of cell layers in from the lens capsule obtained from an earlier study 18 overlaid with the flux reversal point that separates the ion efflux and influx zones. Using this plot, it is evident that isolated fiber cells that are greater than 120 μm in length originate from the zone of ion influx.
Figure 1.
 
A working model for volume regulation in the outer cortex of the rat lens. (A) Confocal images of equatorial sections of organ-cultured rat lenses under isosmotic conditions for 18 hours in the presence of (A) the Cl channel inhibitor NPPB (10 μM) and (B) the KCC inhibitor DIOA (10 μM) exhibited extracellular dilations (inset) and peripheral cell swelling, respectively. (C) Molecular model of transporters and channels that contribute to volume regulation in different regions of the rat lens. In this model, radial differences in transmembrane voltage 14 contribute to the establishment of spatially distinct ion efflux and influx zones. Since these two zones are connected by gap junction channels, a circulating flux is established that helps to maintain cell volume by balancing ion influx and efflux. (D) Plots of fiber cell length versus numbers of cell layers in from the lens capsule obtained from an earlier study 18 overlaid with the flux reversal point that separates the ion efflux and influx zones. Using this plot, it is evident that isolated fiber cells that are greater than 120 μm in length originate from the zone of ion influx.
Figure 2.
 
Differentiation dependent changes in fiber cell membrane properties. (A) Image of a short (56 μm) fiber cell isolated from the zone of ion efflux. Whole-cell currents recorded in AAH from the cell shown in (A) exhibit a characteristic inwardly activating current component (B) that is eliminated by exposure to 5 mM of the K+ channel blocker Ba2+ (C). (D) Current voltage curves recorded from the cell in (A) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The Ba2+ inhibition of the inward current component, the positive shift in reversal potential, and the lack of effect of gluconate-substitution indicated that the membrane properties of short fiber cells are dominated by a potassium conductance. (E) Image of a longer fiber cell (275 μm) isolated from the zone of ion influx. Whole-cell currents recorded in AAH from the cell shown in (E) exhibited a larger characteristically outwardly rectifying current (F) that was significantly reduced in magnitude by the substitution of extracellular Cl with the impermeant anion gluconate (G). (H) Current voltage curves recorded from cell shown in (E) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The lack of effect of Ba2+ and the large reduction in outward current and a positive shift in reversal potential on replacement of extracellular Cl with gluconate indicates that the membrane properties fiber cells isolated from the zone of ion influx are dominated by an anion conductance.
Figure 2.
 
Differentiation dependent changes in fiber cell membrane properties. (A) Image of a short (56 μm) fiber cell isolated from the zone of ion efflux. Whole-cell currents recorded in AAH from the cell shown in (A) exhibit a characteristic inwardly activating current component (B) that is eliminated by exposure to 5 mM of the K+ channel blocker Ba2+ (C). (D) Current voltage curves recorded from the cell in (A) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The Ba2+ inhibition of the inward current component, the positive shift in reversal potential, and the lack of effect of gluconate-substitution indicated that the membrane properties of short fiber cells are dominated by a potassium conductance. (E) Image of a longer fiber cell (275 μm) isolated from the zone of ion influx. Whole-cell currents recorded in AAH from the cell shown in (E) exhibited a larger characteristically outwardly rectifying current (F) that was significantly reduced in magnitude by the substitution of extracellular Cl with the impermeant anion gluconate (G). (H) Current voltage curves recorded from cell shown in (E) bathed in AAH (•), on exposure to Ba2+ (▴), and after the substitution of extracellular Cl with the impermeant anion gluconate (○). The lack of effect of Ba2+ and the large reduction in outward current and a positive shift in reversal potential on replacement of extracellular Cl with gluconate indicates that the membrane properties fiber cells isolated from the zone of ion influx are dominated by an anion conductance.
Figure 3.
 
The effect of hypotonic challenge on fiber cells isolated from the zone of ion influx. Images of and whole-cell currents recorded from a fiber cell (475 μm) isolated from the influx zone bathed in isotonic AAH (A, C) and after exposure to hypotonic AAH (B, D). Note the lack of any obvious change in cell morphology in response to hypotonic challenge. (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion influx respond to hypotonic challenge with a sustained activation of outward current. (F) Whole-cell current–voltage relationships for the cell in isotonic AAH (A, • 300 mOsM · kg−1), hypotonic Cl-based AAH (B, ▵ 250 mOsM · kg−1), hypotonic NO3 based AAH (▴), and gluconate-based AAH (▪). Scale bar, 50 μm.
Figure 3.
 
The effect of hypotonic challenge on fiber cells isolated from the zone of ion influx. Images of and whole-cell currents recorded from a fiber cell (475 μm) isolated from the influx zone bathed in isotonic AAH (A, C) and after exposure to hypotonic AAH (B, D). Note the lack of any obvious change in cell morphology in response to hypotonic challenge. (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion influx respond to hypotonic challenge with a sustained activation of outward current. (F) Whole-cell current–voltage relationships for the cell in isotonic AAH (A, • 300 mOsM · kg−1), hypotonic Cl-based AAH (B, ▵ 250 mOsM · kg−1), hypotonic NO3 based AAH (▴), and gluconate-based AAH (▪). Scale bar, 50 μm.
Figure 4.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a baseline anion conductance. Images of and whole-cell currents recorded from a short fiber cell (55 μm) isolated from the efflux zone bathed in isosmotic AAH (A, C), and some 8 minutes after exposure to hyposmotic AAH (B, D). (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion efflux respond to hyposmotic challenge with a transient activation of outward current. (F) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge.
Figure 4.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a baseline anion conductance. Images of and whole-cell currents recorded from a short fiber cell (55 μm) isolated from the efflux zone bathed in isosmotic AAH (A, C), and some 8 minutes after exposure to hyposmotic AAH (B, D). (E) Time course of normalized whole-cell current recorded at +100 mV showing that exposure of fiber cells isolated from the zone of ion efflux respond to hyposmotic challenge with a transient activation of outward current. (F) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge.
Figure 5.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a dominant K+ conductance. Images of, and whole-cell currents recorded from, a short fiber cell (58 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to hyposmotic AAH (B, E), and after exposure to hyposmotic AAH+10 μM DIOA (C, F). A flicker-like current (E, ▴) that is indicative of an apparent activation of an outward current at very depolarized potentials was evident. (G) Time course of normalized whole-cell current recorded at +100 mV showing that fiber cells isolated from the zone of ion efflux that exhibited a dominant K+ conductance responded to hyposmotic challenge with no significant increase in outward current. However, the subsequent addition of DIOA induced the activation of an outward current. (H) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and after hyposmotic challenge in the absence (▴) and presence of DIOA (○).
Figure 5.
 
The effect of hyposmotic challenge on fiber cells isolated from the zone of ion efflux that exhibit a dominant K+ conductance. Images of, and whole-cell currents recorded from, a short fiber cell (58 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to hyposmotic AAH (B, E), and after exposure to hyposmotic AAH+10 μM DIOA (C, F). A flicker-like current (E, ▴) that is indicative of an apparent activation of an outward current at very depolarized potentials was evident. (G) Time course of normalized whole-cell current recorded at +100 mV showing that fiber cells isolated from the zone of ion efflux that exhibited a dominant K+ conductance responded to hyposmotic challenge with no significant increase in outward current. However, the subsequent addition of DIOA induced the activation of an outward current. (H) Whole-cell current–voltage relationships of the cell under study recorded in isosmotic AAH (•) and after hyposmotic challenge in the absence (▴) and presence of DIOA (○).
Figure 6.
 
A comparison of responses of isolated fiber cells from the influx and efflux zones to hyposmotic challenge. (A) Pooled whole-cell current-voltage relationships (n = 4) of cells from the zone of ion influx recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. (B) Pooled whole-cell current–voltage relationships (n = 19) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. Note the acute current increase caused by hyposmotic swelling (▵) and the return toward control levels observed at steady state (○), indicating that RVD has occurred. (C) Pooled whole-cell current-voltage relationships (n = 11) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•), 6 minutes after hyposmotic challenge (▪), and hyposmotic challenge in the presence of DIOA after 2 (▵) and 6 (▾) minutes. In this subgroup of cells from the efflux zone, hyposmotic challenge alone did not activate an outward current. Outward current activation occurred only in the presence of hyposmotic plus DIOA (▵), before returning toward control levels observed at steady state (▾), indicating that an RVD had occurred. Note data points for hyposmotic challenge in the presence and absence of DIOA overlay each other in (C).
Figure 6.
 
A comparison of responses of isolated fiber cells from the influx and efflux zones to hyposmotic challenge. (A) Pooled whole-cell current-voltage relationships (n = 4) of cells from the zone of ion influx recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. (B) Pooled whole-cell current–voltage relationships (n = 19) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•) and 2 (▵) and 8 (○) minutes after hyposmotic challenge. Note the acute current increase caused by hyposmotic swelling (▵) and the return toward control levels observed at steady state (○), indicating that RVD has occurred. (C) Pooled whole-cell current-voltage relationships (n = 11) of cells from the zone of ion efflux, recorded in isosmotic conditions in AAH (•), 6 minutes after hyposmotic challenge (▪), and hyposmotic challenge in the presence of DIOA after 2 (▵) and 6 (▾) minutes. In this subgroup of cells from the efflux zone, hyposmotic challenge alone did not activate an outward current. Outward current activation occurred only in the presence of hyposmotic plus DIOA (▵), before returning toward control levels observed at steady state (▾), indicating that an RVD had occurred. Note data points for hyposmotic challenge in the presence and absence of DIOA overlay each other in (C).
Figure 7.
 
The effects of DIOA on the morphology of isolated fiber cells. Images of isolated fiber cells of various lengths incubated in isosmotic AAH in the absence (A, C, E) and presence (B, D, F) of 100 μM DIOA. A short (29 μm) fiber cell (A, B) and slightly longer (63 μm) fiber cell (C, D) isolated from the efflux zone both underwent cell swelling on application of DIOA. A long (383 μm) fiber cell (E, F) isolated from the zone of ion influx remained largely unaffected by exposure to DIOA
Figure 7.
 
The effects of DIOA on the morphology of isolated fiber cells. Images of isolated fiber cells of various lengths incubated in isosmotic AAH in the absence (A, C, E) and presence (B, D, F) of 100 μM DIOA. A short (29 μm) fiber cell (A, B) and slightly longer (63 μm) fiber cell (C, D) isolated from the efflux zone both underwent cell swelling on application of DIOA. A long (383 μm) fiber cell (E, F) isolated from the zone of ion influx remained largely unaffected by exposure to DIOA
Figure 8.
 
Electrophysiological effects of DIOA exposure on fiber cells isolated from the zone of ion efflux. Images of and whole-cell currents recorded from a short fiber cell (23 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to AAH+10 μM DIOA (B, E), and after washout of DIOA with AAH (C, F). Addition of DIOA caused an increase in cell volume. (G) Time course of normalized whole-cell current recorded at +100 mV showing that the addition of DIOA to isosmotic AAH induced the activation of an outward current. (H) Whole-cell current-voltage relationships of the cell under study recorded in isosmotic AAH (•), in the presence of DIOA (▵), and after washout of DIOA (○).
Figure 8.
 
Electrophysiological effects of DIOA exposure on fiber cells isolated from the zone of ion efflux. Images of and whole-cell currents recorded from a short fiber cell (23 μm) isolated from the efflux zone bathed in isosmotic AAH (A, D), after exposure to AAH+10 μM DIOA (B, E), and after washout of DIOA with AAH (C, F). Addition of DIOA caused an increase in cell volume. (G) Time course of normalized whole-cell current recorded at +100 mV showing that the addition of DIOA to isosmotic AAH induced the activation of an outward current. (H) Whole-cell current-voltage relationships of the cell under study recorded in isosmotic AAH (•), in the presence of DIOA (▵), and after washout of DIOA (○).
Figure 9.
 
Modulation of an anion conductance by hyposmotic stress in whole lenses. Representative traces of intracellular microelectrode recordings of membrane potential (V m) obtained from intact rat lenses exposed to different extracellular anions (Cl, NO3 , I, and gluconate) under isosmotic (top) or hypotonic conditions (bottom).
Figure 9.
 
Modulation of an anion conductance by hyposmotic stress in whole lenses. Representative traces of intracellular microelectrode recordings of membrane potential (V m) obtained from intact rat lenses exposed to different extracellular anions (Cl, NO3 , I, and gluconate) under isosmotic (top) or hypotonic conditions (bottom).
Table 1.
 
Comparison of the Effects of Anion Replacement on E rev in Isolated Fiber Cells and V m in Whole Lenses
Table 1.
 
Comparison of the Effects of Anion Replacement on E rev in Isolated Fiber Cells and V m in Whole Lenses
Isotonic (300 mOsmkg−1) Hypotonic (250 mOsmkg−1) Hypotonic + DIOA (250 mOsmkg−1)+10μM)
CI I NO3 Gluc CI I NO3 Gluc CI I NO3 Gluc
Isolated fiber cells E rev (mV) −20.28 ± 3.30 −34.76 ± 4.94 −40.00 ± 6.50 −9.54 ± 4.70 −13.27 ± 5.02 −20.16 ± 6.74 −31.03 ± 2.68 4.17 ± 0.50 −36.28 ± 12.08 −43.65 ± 11.33 −43.10 ± 12.35 −7.70 ± 4.30
(33) (25) (13) (9) (7) (4) (3) (3) (3) (3) (2) (2)
Whole lens in vitro V m (mV) −60.36 ± 3.26 −67.13 ± 2.96 −77.04 ± 3.89 −57.12 ± 4.42 −60.99 ± 4.03 −65.68 ± 3.33 −72.19 ± 5.03 −61.79 ± 10.51 −42.28 ± 0.29 −55.82 ± 1.55 −53.71 ± 5.94 −51.36 ± 8.69
(11) (7) (5) (10) (3) (3) (3) (3) (3) (3) (2) (2)
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