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MS 9451 Received 31 March 1999; accepted after revision 19 August 1999.
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ABSTRACT |
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INTRODUCTION |
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The ciliary body epithelium (CBE) forms the inner surface covering of the ciliary processes of the eye and is composed of two different epithelial cell layers: a non-pigmented ciliary epithelial (NPCE) cell layer and a pigmented ciliary epithelial (PCE) cell layer (Caprioli, 1992). Both PCE and NPCE cells are involved in the production of aqueous humour, an isotonic solution composed primarily of water, Na+, Cl- and HCO3-. The balance between the rate and quantity of aqueous humour produced and aqueous humour escape from the eye, via drainage pathways, is the primary determinant of intraocular pressure (IOP), and is subject to autonomic modulation (Caprioli, 1992).
Transport data from intact and dispersed ciliary epithelial tissue suggest that PCE cells have solute uptake properties and are functionally coupled to the NPCE cells which have solute efflux properties (Wiederholt et al. 1991; Edelman et al. 1994). In this cell coupled model, ions and water from the stroma are taken up by PCE cells and passed to the NPCE cells via apical gap junctions (Raviola & Raviola, 1978), where they are secreted at the basolateral membrane into the posterior chamber as aqueous humour.
Despite our understanding of this functional coupling between CE cells, the exact cellular transport mechanisms involved in fluid and ion secretion remain unresolved. However, it has now been shown that Na+, K+ and Cl- enter PCE cells via a furosemide- (frusemide-) and bumetamide-sensitive Na+-K+-2Cl- symport and diffuse from PCE to NPCE cells via the apical gap junctions. Na+, K+ and Cl- ions are then secreted from NPCE cells through Na+-K+ exchange pumps and via basolateral K+ and Cl- channels, and this is accompanied by paracellular Na+ movement. A HCO3--dependent transepithelial potential of approximately 1 mV, aqueous humour negative, provides a net electrochemical driving force (for review see Krupin & Civan, 1995; Jacob & Civan, 1996). In addition, the activity of volume-regulated K+ and Cl- channels in NPCE cells probably contributes to regulatory volume decrease (RVD) and transepithelial salt transport in the CBE following alterations in cellular osmotic gradients (Farahbakhsh & Fain, 1987; Yantorno et al. 1989, 1992; Civan et al. 1992, 1994; Adorante & Cala, 1995). In support of this, NPCE cells in the intact ciliary process have been shown to respond to hypotonic media with cell swelling accompanied by ion and water efflux (Farahbakhsh & Fain, 1987).
Chloride channels in the NPCE cells of the ciliary body epithelium have been suggested to be critical to the formation of aqueous humour, as well as in volume regulation of these cells (for review see Jacob & Civan, 1996). Several candidates for the volume-activated Cl- channel/channel regulator in NPCE cells have now been presented. These include the multidrug resistance gene product (MDR1) in native bovine ciliary epithelial cells (Wu et al. 1996; Wang et al. 1998), CIC-3 Cl- channel in a cultured transformed human NPCE cell line (Coca-Prados et al. 1996), and pICln in the transformed human NPCE cell line (Coca-Prados et al. 1995, 1996) and in acutely isolated NPCE cells from rabbit (Chen et al. 1998). To date, despite extensive investigation, none of these candidates have yet been unequivocably linked to the volume-activated Cl- channel(s) in NPCE cells. In addition, various mechanisms have also been suggested to be involved in linking cell swelling and activation of Cl- channels in NPCE cells. These signalling pathways include protein kinase C (PKC), Ca2+-calmodulin (CaM) and an epoxide (Civan et al. 1994; Coca-Prados et al. 1995, 1996).
The purpose of this study was to identify the electrophysiological and pharmacological properties of a hyposmotic (HOS)-activated Cl- channel in SV40-transformed rabbit NPCE cells, using whole-cell patch-clamp recordings and noise analysis. The rabbit CBE has been used extensively for studies of transepithelial ion transport and aqueous humour production, thus information from isolated cell studies can be correlated with existing data and transport models in this species (Farahbakhsh & Fain, 1987; Sears et al. 1991; for review see Jacob & Civan, 1996). Our results demonstrate that rabbit NPCE cells activate volume-sensitive Cl- channels in response to hyposmotic shock. These Cl- channels share some properties with other volume-activated Cl- channels, being sensitive to specific Cl- channel blockers and modulated by Ca2+ and phosphorylation. Noise analysis revealed that the HOS-activated Cl- channels in NPCE cells had a small unitary conductance. Since Cl- channel activation represents a rate-limiting step in fluid secretion, understanding the mechanisms of Cl- channel and cell volume regulation in NPCE cells can provide information about the physiology of aqueous humour secretion.
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METHODS |
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Cell culture
We used a cell line derived from simian virus-40 (SV40)-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells (Delamere et al. 1993). Although electrophysiological analysis of these cells has not previously been carried out, previous transport studies have indicated that SV40-transformed rabbit NPCE cells have many properties in common with native, acutely isolated rabbit ciliary epithelium and iris/ciliary body segments (Socci & Delamere, 1988; Chu et al. 1992). Positive viral transformation of a primary culture of rabbit NPCE cells was verified by expression of the large T-antigen (T-Ag) in the nuclei of SV40-transformed cells. Transformed rabbit NPCE cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM; Canadian Life Technologies) plus 10 % newborn calf serum (NCS) and 1 % gentamicin, in an atmosphere of 5 % CO2, 95 % O2. Prior to electrophysiological recording, cells were seeded onto 12 mm glass coverslips and incubated for a further 24 h at 37°C in a 5 % CO2, 95 % O2 atmosphere.
Solutions and chemicals
For whole-cell current recordings, cells attached to coverslips were placed in the recording chamber with a volume of 
1 ml and positioned on the stage of a Nikon inverted microscope. Cells were superfused at a rate of 1-2 ml min-1 with standard external recording solution containing (mM): Trizma HCl, 70; CaCl2, 1·5; MgCl2, 0·8; Hepes, 10; TEA-Cl, 5; BaCl2, 5; glucose, 10; sucrose, 105. External solution was adjusted to pH 7·4 with 1 n CsOH. Solution osmolarities were determined by freezing point depression (Osmette A, Fischer Scientific, Nepean, Ontario, Canada). The osmolarity of the standard external solution was 295 mosmol l-1. The external solution was made hyposmotic by omitting sucrose, which reduced the osmolarity to 191 mosmol l-1. For low Ca2+ experiments, extracellular CaCl2 was removed and 0·25 mM EGTA was added into standard external solution to make a nominally Ca2+-free external solution. Standard electrode-filling solution for whole-cell recordings was composed of (mM): Trizma HCl, 60; Trizma base, 60; aspartic acid, 60; Hepes, 15; CaCl2, 0·4; MgCl2, 1; EGTA, 1; ATP (Mg), 1; GTP (Na2), 0·1. The pH value was adjusted to 7·4 with 1 n CsOH. Free internal Ca2+ concentration was estimated to be 100 nM (Stockbridge, 1987). When [Cl-] was altered in the internal solutions, intracellular Trizma HCl was replaced with Trizma aspartate. In low Ca2+ experiments, intracellular [Ca2+] was buffered to < 10 nM by replacing 0·4 mM CaCl2 in the internal solution with 0·1 mM CaCl2 and 10 mM BAPTA.
Test solutions were applied by bath superfusion or by pneumatic pressure ejection (Picospritzer II, General Valve, Fairfield, NJ, USA) from micropipettes (tip diameter 1-2 µm) positioned 50-100 µm from the cell. For bath superfusion, test solutions were applied for a minimum of 10 complete (1 ml) bath exchanges. Cl- channel blockers DIDS, SITS, DNDS and niflumic acid were prepared as stocks, dissolved in external solution and bath superfused at the concentrations cited in Results. To investigate the potential involvement of protein phosphorylation, NPCE cells were pre-incubated (20 min) and superfused during electrophysiological recording with the membrane-permeant broad-action protein kinase inhibitor 1-(5-isoquinoline sulphonyl)-2-methylpiperazine (H-7) (Takahashi et al. 1990). DNDS was purchased from Molecular Probes Inc. H-7 was obtained from Calbiochem. All other chemicals and drugs were purchased from Sigma Chemical Co.
Electrophysiological recording techniques
Isolated epithelial cells were studied using tight-seal patch-clamp recording methods (Hamill et al. 1981). Membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments) and pCLAMP6 software (Axon Instruments) was used to generate voltage commands. Recording pipettes, with diameters of 1·5 mm outside and 1·1 mm inside, were fabricated from borosilicate glass (Sutter Instruments, Novato, CA, USA) using a two-stage vertical microelectrode puller (Narishige model PP83, Tokyo, Japan). Electrodes were coated with beeswax to reduce capacitance and had resistances of 2-4 M
. A sealed electrode-salt bridge combination was used as the reference electrode (Dri-ref-2; World Precision Instruments, Sarasota, FL, USA). Prior to seal formation, offset potentials were nulled using the amplifier circuitry. Liquid junction potentials (LJP), arising between the bath solution and the electrode were measured experimentally and were also calculated using a software program (JPCalc, version 2.00; P.H. Barry, Sydney, Australia). For whole-cell recordings the membrane potential (Vm) was defined as Vm = Vp - LJP, where Vp is the pipette potential. All the whole-cell current-voltage relationships shown have been corrected for LJPs which were 1-2 mV for standard solutions, 7·5 mV for low Cl- solutions, and 1·4 mV for low Ca2+ solutions. Cell capacitance ranged from 15 to 80 pF. Measures of series resistance (Raccess) were obtained directly from the amplifier and were generally less than 15 M. Eighty per cent series resistance compensation was used in most recordings. All experiments were conducted at room temperature (22-24°C).
For membrane current noise analysis, cells were voltage clamped at -62 mV during swelling and whole-cell currents were filtered with a 9-pole active filter having a corner frequency of 300 Hz. The filter reduced the output signal power by less than 1 % at frequencies below 180 Hz. Data above this frequency were not used in any measurement. The data were sampled at 1 ms intervals and digitized by a 12-bit analog-to-digital convertor. The data were processed in segments of 5000 points (5 s). Each segment was first fitted by a second-order polynomial function, using linear regression, to approximate the trend in increasing current. The fitted polynomial function was then subtracted from each point in the segment and the mean current of the segment was calculated. Finally, the total current variance in the block was measured as the mean of the squared residual current after subtracting the mean. Single channel conductance, 
, was obtained by fitting the noise versus membrane current relationship with the equation:
 2 = 02 + I (V - E) - I 2/N,
| (1) |
where 02 is the background variance, I is the total membrane current, V is the membrane potential, E is the reversal potential and N is the number of channels (DeFelice, 1981; Traynelis & Jaramillo, 1998). The second-order polynomial function was fitted by linear regression to obtain estimates of and N. The channel open probability, Po, was obtained from:
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Po = I/(N (V - E) ).
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Data are represented as the mean ± S.E.M. Student's unpaired t test was used to compare differences between two groups of data. Data were considered significant at P < 0·05.
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RESULTS |
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Hyposmotic shock (HOS) activates a Cl- current in NPCE cells
Figure 1A (left panel) shows typical whole-cell current recordings made from a representative cultured NPCE cell in standard external and internal solution in the absence of K+ and Na+. Cells were held at -62 mV and membrane potential was stepped from -102 to +118 mV in 20 mV increments. Under control conditions, whole-cell current was small, with a whole-cell conductance of 1·4 nS (2·1 ± 0·2 nS, n = 11) and 1·3 nS (2·0 ± 0·2 nS, n = 11) at -62 and +58 mV. Superfusion of the same NPCE cell with hyposmotic external solution (HOS) for 30 min (middle panel) activated a large current at potentials depolarized and hyperpolarized from the holding potential (VH = -62 mV) and increased whole-cell conductance to 20 nS (13·85 ± 1·7 nS, n = 11) and 11 nS (7·2 ± 0·7 nS, n = 11) at +58 and -62 mV, respectively. The HOS-activated current exhibited slightly time-dependent inactivation at more depolarized potentials (> 60 mV). Fifteen minutes after recovery in standard external solution the whole-cell current returned to control level (right panel). Figure 1B shows the time course of activation of the HOS current for the same cell shown in panel A. Current was measured at +58 mV at 2-15 min intervals from a VH of -62 mV over a 50 min period. In this cell, the HOS conductance activated after approximately 15 min of exposure to hyposmotic solution, continued to activate for the duration of the exposure to HOS solution, and recovered to control values on return to superfusion with standard extracellular solution. In eight other cells, in which HOS-activated current was measured at 5 min intervals following exposure to hyposmotic solution, the mean time to record a significant increase in current (activation time) was 15·4 ± 2·0 min.
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A, left panel shows patch-clamp recording from a representative NPCE cell taken 1 min after assuming the whole-cell configuration, in standard external solution and 90 mM [Cl-] internal solution (Control). The voltage protocol is shown at the top of the figure. Middle panel shows the same cell 30 min after superfusion with hyposmotic external solutions (HOS). The activation of a large current is apparent. The HOS-activated current recovered to the control levels (recovery, right panel) 15 min after return to standard external solution. B, time course for the activation of the HOS current shown in A. Current was measured at 2-15 min intervals at +58 mV from a holding potential of -62 mV in standard external solution or in external hyposmotic solution (HOS). C, current-voltage plot for the currents measured in the NPCE cell shown in A. Currents were measured 1 min after assuming whole-cell configuration in standard external solution ( | ||
Figure 1C shows the current-voltage relationship for the whole-cell currents recorded from the cell in panel A. In standard external solution, little whole-cell current was apparent at positive or negative potentials and current reversed at 0 mV. After 30 min superfusion with hyposmotic solution, a large current was reversibly activated. The HOS current exhibited mild outward rectification and reversed (Vrev) at -8 mV. The Vrev of the HOS current was close to that expected for a Cl--selective current (equilibrium potential for Cl-, ECl = -9·67 mV), when [Cl-]i = 60 mM and [Cl-]o = 91·6 mM.
The Cl- selectivity of the HOS-activated conductance was confirmed by examining the reversal potential for the induced current under experimental conditions where [Cl-]i was varied. Figure 2A shows the current-voltage relationships for currents recorded in three different cells, 20 min after superfusion with HOS external solution (91 mM [Cl-]), and with either 30, 60 or 90 mM [Cl-] in the pipette. Current-voltage (I-V ) relationships demonstrated that Vrev for the HOS current was shifted negative as [Cl-] was reduced in the pipette. Mean Vrev measured -29·67 ± 0·67 mV (n = 3, ECl = -26·31 mV), -13·46 ± 2·62 mV (n = 15, ECl = -9·67 mV) and 1·30 ± 1·93 mV (n = 5, ECl = 0·33 mV) for [Cl-]i of 30, 60 and 90 mM, respectively. Figure 2B shows the mean reversal potential of the HOS-activated current (dashed line) plotted against the theoretical ECl. Also plotted (continuous line) is the relationship for a Cl--selective current. The least-squares fit to the data for the HOS-activated current has a slope corresponding to 58 mV per 10-fold change in intracellular Cl- concentration, close to the Nernst predicted value of 59 mV per 10-fold change in intracellular Cl- concentration. This confirms that HOS-activated current in SV40-transformed NPCE cells is carried by Cl- ions.
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A, current-voltage plots for 3 representative NPCE cells are shown for [Cl-]i of 30 ( | ||
The stilbene-derived Cl- channel blockers, DIDS, SITS and DNDS, and niflumic acid have been demonstrated to block volume-activated anion conductances in a variety of cells (Botchkin & Matthews, 1995; Nilius et al. 1996; Strange et al. 1996; Mitchell et al. 1997; Okada, 1997). We tested the sensitivity of the HOS-activated Cl- conductance in NPCE cells to these drugs. Figure 3A shows whole-cell current traces recorded in a representative NPCE cell superfused sequentially with control solution, HOS solution and HOS solution containing 0·5 mM DIDS. The voltage protocol is shown above the control current traces. Cells were held at VH = -62 mV and the voltage was stepped from -102 to +118 mV in 20 mV increments. HOS solution activated a Cl- conductance (HOS) which was reversibly blocked by DIDS (HOS + DIDS) and recovered to pre-DIDS values on wash-out of the blocker (not shown). The DIDS block of the HOS-activated Cl- conductance exhibited voltage dependence. Figure 3B shows the mean amplitude of the HOS Cl- current measured at +58 and -62 mV in 11 cells before and after superfusion with DIDS. The HOS-activated current at +58 mV (33·9 ± 4·9 pA pF-1) was significantly reduced following a 5 min exposure to DIDS (16·2 ± 4·3 pA pF-1; n = 11; P < 0·05) but was unaffected by DIDS at the hyperpolarized potential of -62 mV. Figure 3C-E shows mean inhibition of the HOS-activated Cl- current following a 30 s pneumatic pressure application of 0·5 mM SITS, or a 3 min superfusion with either DNDS (0·5 mM) or niflumic acid (0·2 mM). Similar to DIDS, the two other stilbene-derived Cl- channel blockers, SITS and DNDS, significantly decreased the outward current at +58 mV from 33·41 ± 5·35 to 19·74 ± 3·85 pA pF-1 (P < 0·01, n = 7) and 32·9 ± 2·54 to 24·59 ± 6·35 pA pF-1 (P < 0·01, n = 10), respectively. Furthermore, like DIDS, DNDS and SITS had no significant effect on the amplitude of the HOS-activated Cl- current at -62 mV (P > 0·05). In contrast to the voltage-dependent block of Cl- current by the stilbene drugs, niflumic acid produced a voltage-independent block of the HOS-activated Cl- conductance. In 11 cells tested niflumic acid significantly reduced the amplitude of the Cl- current at both +58 mV (from 38·24 ± 3·69 to 17·0 ± 5·9 pA pF-1; P < 0·05) and -62 mV (from -18·95 ± 1·9 to -8·9 ± 2·08 pA pF-1; P < 0·05).
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A, whole-cell currents recorded from a representative NPCE cell. Currents were measured from a holding potential of -62 mV in response to voltage steps from -102 to +118 mV in standard external solution (Control) and hyposmotic external solution, in the absence (HOS) and in the presence of 0·5 mM DIDS (HOS + DIDS). B-E, mean (± S.E.M.) current amplitude of the HOS Cl- current. Current was measured at +58 and -62 mV before (Control; | ||
Modulation of HOS-activated Cl- current by Ca2+ and phosphorylation
In some cell types, an increase in cytosolic Ca2+ has been linked to activation of Cl- current (Basavappa et al. 1995; Szucs et al. 1996). Although an increase in [Ca2+]i has been reported to accompany cell swelling in freshly isolated NPCE cells from rabbit ciliary body, this was not linked to the regulation of swelling-activated Cl- conductance (Botchkin & Matthews, 1995). In our experiments, we examined Ca2+ dependence by activating the Cl- current in nominally Ca2+-free external solutions and with 10 mM BAPTA in the pipette internal recording solution ([Ca2+]i < 10 nM). Figure 4A shows mean current-voltage relationships recorded from eight cells under low Ca2+ conditions. Cells were held at VH = -62 mV and stepped to +58 mV at 5-15 min intervals. Superfusion with HOS solution was still able to elicit the Cl- current under Ca2+-free conditions. However, the onset of an increase in the current was significantly slower than that recorded in standard Ca2+-containing solution. The mean time to record a significant increase in current in nominally Ca2+-free conditions was 36·25 ± 6·0 min (n = 8) compared with 15·4 ± 2·0 min (n = 7; P < 0·05) in standard Ca2+-containing solution. Figure 4B shows the mean amplitude of the HOS-activated current measured at both +58 and -62 mV, 50 min after HOS superfusion. In Ca2+-free conditions, the HOS-activated current was reduced from 28·67 ± 3·01 pA pF-1 (n = 16) to 17·43 ± 4·08 pA pF-1 (n = 8; P < 0·05) at +58 mV and from -13·91 ± 1·97 to -6·34 ± 1·10 pA pF-1 at -62 mV. These results indicate that while Ca2+ is not required for current activation, Ca2+ participates in the regulation of the HOS-activated Cl- current.
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A, mean (± S.E.M.) time course for the HOS Cl- current measured from a VH of -62 mV in low Ca2+ external solution and 10 mM BAPTA internal solution in 8 NPCE cells. B, mean (± S.E.M.) current amplitude of the HOS Cl- current measured 50 min after superfusion with hyposmotic external solution at +58 and -62 mV. HOS Cl- current amplitude was measured in standard external and internal solutions ( | ||
In several cell types including rat brain neuronal cells (Kawasaki et al. 1994) and human ODM/SV40 NPCE cells (Civan et al. 1994; Coca-Prados et al. 1995, 1996), inhibition by kinase inhibitors such as the PKC inhibitor staurosporine, upregulates the volume-sensitive Cl- current. We examined whether phosphorylation by kinases is involved in the regulation of the HOS-activated Cl- current in rabbit NPCE cells. We used the kinase inhibitor H-7, which blocks PKA, PKC and PKG (Takahashi et al. 1990) and examined HOS activation of Cl- current in the presence and absence of H-7 included in the superfusate and the internal solution. The histogram in Fig. 5 shows the mean amplitude of the HOS-activated Cl- current in 16 cells tested in the absence of H-7 and another 5 cells after superfusion with 100 µM H-7. Currents were measured 5 min after HOS activation. In the absence of H-7, the mean amplitude of the HOS-activated current was -13·91 ± 1·97 and 28·67 ± 3·01 pA pF-1 at -62 and +58 mV, respectively. When cells were superfused for 20 min with H-7 prior to and during hyposmotic shock, the amplitude of the HOS-activated current was significantly increased over control to -26·57 ± 5·02 and 42·22 ± 7·27 pA pF-1 at -62 and +58 mV, respectively (P < 0·05).
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Bar graph showing mean (± S.E.M.) current amplitude of the HOS-activated Cl- current measured at +58 and -62 mV, 30 min after superfusion with hyposmotic external solution, in cells in the absence of H-7 ( | ||
Noise analysis of HOS-activated Cl- current
Noise analysis has been employed successfully to measure the single channel conductance of Cl- channels in a variety of cell types (for review see Traynelis & Jaramillo, 1998). We used noise analysis of whole-cell currents to estimate the mean single channel conductance and density of the HOS-activated Cl- channel in rabbit NPCE cells.
To ensure that whole-cell records contained only Cl- channel activity, currents were recorded as before in standard low K+ and Na+ solutions with external 70 mM Trizma HCl solution containing 5 mM TEA-Cl and 5 mM BaCl2 and with 60 mM Trizma HCl in the pipette solution. Whole-cell current was recorded at VH = -62 mV. After exposure of cells to standard external recording solution for 2 min, cells were then superfused with hyposmotic solution. Under these conditions, whole-cell current began to activate after 15-20 min of exposure to hyposmotic solution and remained elevated for another 15-20 min. Cells were maintained in the hyposmotic solution until maximum current activation occurred, as determined by the absence of any further increases in current during a 3 min period. Figure 6A shows noise variance versus membrane current from a typical recording fitted by eqn (1). For this representative cell, the estimated single channel conductance was 0·53 pS and the number of channels in the cell was 18 000. Figure 6B shows the time course of the current and Po for the cell shown in Fig. 6A. When the cell was held at -62 mV, the inward Cl- current and Po increased steadily during exposure to HOS solution and was almost saturated after 10 min. The mean single channel conductance from noise variance measurements on five NPCE cells during HOS stimulation was 0·65 ± 0·10 pS, and the average number of channels per cell was 18 000 ± 4550. Thus, in cultured rabbit NPCE cells under conditions that favour only Cl--permeable channels, cell swelling appears to primarily activate a high density of small conductance Cl- channels.
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A shows noise variance versus membrane current during the development of HOS-activated Cl- current in one cell, fitted by eqn (1) with | ||
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DISCUSSION |
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In this study, we have described the voltage dependence, kinetics and pharmacology of HOS-activated Cl- current in SV40-transformed rabbit NPCE cells. Our results demonstrate that hyposmotic shock activates an outwardly rectifying Cl- current in rabbit NPCE cells. The Cl- current showed only slight inactivation at positive potentials with ATP present in the pipette, and was blocked by the Cl- channel blockers niflumic acid, SITS, DIDS and DNDS. We also demonstrated that Ca2+ plays a regulatory role in activation of the HOS-stimulated Cl- current, as does kinase-dependent phosphorylation. Noise analysis of macroscopic current records indicated that, under conditions where Cl- current is isolated, cell swelling primarily activates a high density of small conductance (< 1 pS) Cl- channels in rabbit NPCE cells.
Properties of the macroscopic HOS-activated Cl- current in NPCE cells
The HOS-activated Cl- current in the rabbit NPCE cell line is similar in its voltage dependence and pharmacology to the swelling-activated Cl- current described in primary cultures of rabbit NPCE cells (Botchkin & Matthews, 1995), with the exception of Ca2+ dependence (see below), and shares some characteristics with volume-sensitive Cl- currents reported in a variety of other epithelial cell types, including that of NPCE cells from a SV40-transformed human NPCE cell line (Yantorno et al. 1992; Civan et al. 1992, 1994; Coca-Prados et al. 1996) and bovine pigmented epithelial cells (Wu et al. 1996).
In the SV40-transformed rabbit NPCE cells described here, as well as previously in non-transformed rabbit NPCE cells (Botchkin & Matthews, 1995), the swelling-activated Cl- current exhibited outward rectification and showed only slight and slow inactivation at larger positive potentials. A similar lack of time-dependent inactivation was also observed in retinal pigment epithelial cells (Botchkin & Matthews, 1993), in cardiac myocytes (Duan et al. 1995) and in smooth muscle cells (Dick et al. 1998). The voltage-dependent inactivation observed at positive potentials is thought to result from a progressive decrease in the number of active channels (Okada, 1997) and, in some cells, may involve extracellular cations such as Mg2+ (Anderson et al. 1995). Differences in voltage- and time-dependent inactivation in different cell types may also be explained by variations in the threshold potential for depolarization-induced inactivation which may be higher in some cell types and/or may reflect differing experimental conditions (Okada, 1997). In rabbit NPCE cells, absence of ATP in the pipette resulted in increased inactivation at more depolarized potentials and enhanced 'run-down' of current during hyposmotic stimulation (data not shown).
Volume-activated Cl- conductances display broad specificity to many types of Cl- channel blockers (for review see Nilius et al. 1996; Strange et al. 1996; Okada, 1997). Our study in transformed rabbit NPCE cells demonstrated that the stilbene-derivative Cl- channel blockers DIDS, SITS and DNDS inhibit this current in a reversible and voltage-dependent manner, whilst the DPC (N-phenylanthranilic acid) derivative niflumic acid produced a reversible voltage-independent block of the HOS-activated Cl- current. A similar block of volume-activated Cl- current by stilbene drugs has been reported for many cell types, including other ocular epithelia (Botchkin & Matthews, 1993; Mitchell et al. 1997). Furthermore, niflumic acid was also found to inhibit staurosporine-activated Cl- channels and cell shrinkage in a human NPCE cell line under isosmotic conditions (Coca-Prados et al. 1996), verifying that niflumic acid inhibits the volume-activated Cl- channels of NPCE cells.
The role of Ca2+ and second messengers in regulation of the HOS-activated Cl- current
Osmotic cell swelling is frequently accompanied by an increase in cytosolic Ca2+ (Botchkin & Matthews, 1995; Basavappa et al. 1995). However, the activation of a volume-activated Cl- channel was independent of Ca2+ in most cell types in which Ca2+ dependence was examined (for review see Nilius et al. 1996; Okada, 1997). A study in freshly isolated rabbit NPCE cells reported that, although swelling increases cytosolic Ca2+, the activation of this current is independent of changes in [Ca2+]i (Botchkin & Matthews, 1995). However, that study did not demonstrate whether the amplitude or time course of Cl- current activation was altered under Ca2+-deprived conditions. Recently, it has been reported that a permissive Ca2+ concentration is necessary for the full and sustained activation of the volume-sensitive Cl- current (Szucs et al. 1996). Our experiments demonstrated that, although Ca2+ was not required for activation of the HOS-activated current in rabbit NPCE cells, Ca2+ depletion suppressed current amplitude and slowed the onset of this current following HOS stimulation. This finding is supported by measurements in human NPCE cells, which reported that swelling-induced increases in [Ca2+]i may modulate net ion efflux during RVD (Adorante & Cala, 1995).
Studies in a human ODM/SV40 NPCE cell line have further suggested that volume-sensitive Cl- channels in these cells may be modulated by phosphorylation and this mechanism may involve Ca2+ and PKC (Civan et al. 1994; Coca-Prados et al. 1995, 1996). This conclusion was based on the demonstration that the PKC inhibitor staurosporine upregulated whole-cell Cl- currents in NPCE cells under both hypotonic and isosmotic conditions. Investigations into mechanisms regulating RVD and swelling-activated Cl- channel activation in NPCE cells also indicated that RVD is decreased by inhibiting Ca2+-CaM with trifluoperizine, and that volume-activated Cl- channels are upregulated by activation of Ca2+-CaM kinase and an epoxide (Civan et al. 1992, 1994). Our findings demonstrating enhancement of the HOS-activated Cl- current in rabbit NPCE cells following exposure to the non-specific kinase inhibitor H-7 support the involvement of a phosphorylation pathway(s) in modulating HOS-activated Cl- channel activation in NPCE cells, which may also include activation of Ca2+-dependent downstream mediators.
Single channel properties of HOS-activated Cl- channels
The noise analysis used here to estimate the mean single channel conductance and density of the HOS-activated Cl- channels assumes that current flows through a fixed number of channels with constant single channel conductance and varying open probability. These assumptions agree with single channel records from a wide range of ion channels, and the method has been employed successfully to measure the single channel conductance of Cl- channels in a variety of cell types (Duszyk et al. 1992; Lewis et al. 1993; Ho et al. 1994; Traynelis & Jaramillo, 1998).
An alternative measurement of glial swelling-activated Cl- channel conductance, based on combining voltage steps and swelling, gave larger values of single channel conductance than the conventional method (Jackson & Strange, 1995). However, this method requires the swelling-activated channels to also be voltage sensitive, and risks including other voltage-sensitive currents. Fitting data to eqn (1) requires measurements over a sufficiently wide current range to fit the parabolic component reliably. This is often difficult, and may explain discrepancies such as that reported by Jackson & Strange (1995). In the present case, we could fit most of the parabolic range (Fig. 6A) giving confidence in the model and the results.
In our study, noise analysis of whole-cell current indicated that hyposmotic shock activated a high density of channels with a single channel conductance of < 1 pS. Our estimate of 18 000 channels per cell is comparable to that reported for similar noise analysis measurements of volume-activated Cl- currents in T84 colonic epithelial cells (Ho et al. 1994) and other cell types such as T-lymphocytes (Lewis et al. 1993), HT29 colon carcinoma cells (Kunzelmann et al. 1992) and human endothelial cells (Nilius et al. 1996).
While our data do not eliminate the possibility that other Cl- channels in NPCE cells can be activated under hyposmotic conditions at more depolarized potentials, the Po of these channels would be expected to be very low at physiological membrane potentials compared with the small conductance HOS-activated Cl- channels described here. It is possible that conventional noise analysis, especially with a limited experimental bandwidth, may underestimate single channel unitary conductance. However, a comparison of single channel activity recorded from cell-attached or isolated membrane patches may not be representative of channel properties due to lack of information on membrane potential or alterations in channel behaviour as a result of loss of the supporting cellular cytoskeleton and milieu. Studies in human airway epithelia demonstrated that values for Cl- channel conductance measured using whole-cell noise analysis and single channel recording were closely comparable (Duszyk et al. 1992; Wilk-Blaszcak et al. 1992).
Although relatively few studies have examined the single channel characteristics of NPCE cells, a low conductance Cl- channel (7 pS) and an intermediate conductance (18 pS) channel have been described in bovine NPCE cells using single channel recording from cell-attached patches (Zhang & Jacob, 1997). A similar low conductance (6-7 pS) Cl- channel has also been identified in pigmented ciliary epithelial (PCE) cells (Zhang & Jacob, 1997). In NPCE cells both the 7 pS channel and the intermediate conductance Cl- channel, which had characteristics similar to the volume-activated organic osmolyte-anion channel (Jackson & Strange, 1995), were activated by hypotonic exposure.
Molecular identity of the volume-activated Cl- channel
Both ClC-2 and ClC-3 proteins belong to the ClC class of cloned Cl- channel proteins (Pusch & Jentsch, 1994; Lorenz et al. 1996), and represent viable candidates for the volume-activated Cl- channel (Clapham, 1998; Strange, 1998). Ciliary epithelial cells have been reported to express transcripts for ClC-3, and electrophysiological and volumetric evidence supports the association of ClC-3 with Cl- transport by human NPCE cells (Coca-Prados et al. 1996). The cDNA encoding the protein pICln, thought to be a swelling-induced chloride conductance regulatory protein, was also cloned from both rabbit ciliary epithelium (Chen et al. 1998; Wan et al. 1997) and human NPCE cells lines (Coca-Prados et al. 1996). Although recent data from other cell types as well as oocyte expression systems, have failed to support the concept that pICln is the volume-activated Cl- channel (Voets et al. 1996), overexpression of pICln protein from the ciliary body in oocytes resulted in outwardly rectifying current, and thus supports a role for pICln as a Cl- conductance regulatory protein related to Cl- channel activation in ciliary epithelial cells (Chen et al. 1998).
The HOS-activated Cl- current described here in SV40-transformed rabbit NPCE cells, has some characteristics in common with the Cl- currents recorded from cells transfected with ClC-3 (Kawasaki et al. 1994; Duan et al. 1997; Chen et al. 1998), as well as volume-activated Cl- currents of ODM/SV40 human NPCE cells (Coca-Prados et al. 1995, 1996). These include outward rectification, little or no inactivation at depolarized potentials, block by stilbenes, modulation by cytosolic Ca2+ and upregulation by kinase inhibitors. Our noise analysis data further suggest that a low conductance channel may give rise to swelling-activated Cl- conductance in rabbit NPCE cells. The data do not support a role for ClC-2 in rabbit NPCE cells, as the HOS-activated Cl- current was not inwardly rectifying, could not be activated by hyperpolarization under isotonic conditions and was not insensitive to SITS and DIDS blockage, all characteristics associated with the ClC-2 channel (Thieman et al. 1992; Okada, 1997). However, more studies providing evidence linking the molecular and electrophysiological results are still required before the identity of the volume-activated Cl- channel(s) in NPCE cells can be defined. For example, a volume-activated current regulated by P-glycoprotein, the product of the MDR1 multidrug resistance gene and putative swelling-activated Cl- channel regulatory protein, has also been identified in bovine NPCE cells (Wu et al. 1996; Wang et al. 1998), and may contribute to the regulation of swelling-activated channels in these cells.
Functional significance of Cl- channels in ciliary epithelial cells
Due to the rapid flow of ions and water through the ciliary epithelium, the activation of volume-sensitive Cl- channels is likely to be important in the secretion of aqueous humour (Caprioli, 1992). Optical measurements of intact rabbit ciliary epithelial processes (Farahbakhsh & Fain, 1987) have confirmed that anisosmotic swelling of CBE cells results in RVD. Experiments in human NPCE cells have further suggested that activation and upregulation of NPCE Cl- channels are essential in this process, by providing a transcellular pathway for Cl- movement (Civan et al. 1992; Coca-Prados et al. 1995, 1996). Thus, further exploration of the macroscopic and single channel properties of volume-activated Cl- channel(s) in ciliary epithelial cells is important not only in clarifying the identity of the Cl- channel(s) which contribute to RVD in NPCE cells, but also in understanding the mechanisms of aqueous humour secretion.
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REFERENCES |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Adorante, J. S. & Cala, P. M (1995). Mechanisms of regulatory decrease in nonpigmented human ciliary epithelial cells. American Journal of Physiology 268, C721-731 | [Medline] |
| Anderson, J. W., Jirsch, J. D. & Fedida, D. (1995). Cation regulation of anion current activated by cell swelling in two types of human epithelial cancer cells. The Journal of Physiology 483, 549-557 | [Abstract] |
| Basavappa, S., Chartouni, V., Kirk, K., Prpic, V., Ellory, J. C. & Mangel, A. W. (1995). Swelling-activated chloride currents in neuroblastoma cells are calcium dependent. Journal of Neuroscience 15, 3663-3666. | |
| Botchkin, L. M. & Matthews, G. (1993). Chloride current activated by swelling in retinal pigment epithelial cells. American Journal of Physiology 265, C1037-1045 | [Medline] |
| Botchkin, L. M. & Matthews, G. (1995). Swelling activates chloride current and increases internal calcium in nonpigmented epithelial cells from rabbit ciliary body. Journal of Cell Physiology 164, 286-294. | |
| Caprioli, J. (1992). The ciliary epithelia and aqueous humor. In Adler's Physiology of the Eye: Clinical Application, ed. Hart, W. M., pp. 228-241. Mosby Year Book Inc., St Louis, MO, USA. | |
| Chen, S., Wan, X. L. & Sears, M. (1998). pICln can regulate swelling-induced Cl- currents in either layer of rabbit ciliary epithelium. Biochemical and Biophysical Research Communications 246, 59-63 | [Medline] |
| Chu, T. C., Socci, R. R., Coca-Prados, M. & Green, K. (1992). Comparative studies of furosemide effects on membrane potential and intracellular chloride activity in human and rabbit ciliary epithelium. Ophthalmic Research 24, 83-91 | [Medline] |
| Civan, M. M., Coca-Prados, M. & Peterson-Yantorno, K. (1994). Pathways signalling the regulatory volume-decrease of cultured non-pigmented ciliary epithelial cells. Investigative Ophthalmology and Visual Science 35, 2876-2886 | [Abstract] |
| Civan, M. M., Peterson-Yantorno, K., Coca-Prados, M. & Yantorno, R. E. (1992). Regulatory volume decrease in cultured non-pigmented ciliary epithelial cells. Experimental Eye Research 54, 181-191 | [Medline] |
| Clapham, D. E. (1998). The list of volume-sensitive chloride currents continues to swell (and shrink). Journal of General Physiology 111, 623-624 | [Full Text] |
| Coca-Prados, M., Anguita, J., Chalfant, M. L. & Civan, M. M. (1995). PKC-sensitive Cl- channels associated with ciliary epithelial homologue of pICln. American Journal of Physiology 268, C572-579 | [Medline] |
| Coca-Prados, M., Sanchez-Torres, J., Peterson-Yantorno, K. & Civan, M. M. (1996). Association of CIC-3 channel with Cl- transport by human nonpigmented ciliary epithelium cells. Journal of Membrane Biology 150, 197-208 | [Medline] |
| DeFelice, L. J. (1981). Introduction to Membrane Noise, pp. 500. Plenum Press, New York. | |
| Delamere, N. A., Coca-Prados, M. & Aggarwal, S. (1993). Studies on the regulation of ascorbic acid transporter in a cell line derived from rabbit nonpigmented ciliary epithelium. Biochimica et Biophysica Acta 1149, 102-108 | [Medline] |
| Dick, G. M., Bradley, K. K., Horowitz, B., Hume, J. R. & Sanders, K. M. (1998). Functional and molecular identification of a novel chloride conductance in canine colonic smooth muscle. American Journal of Physiology 275, C940-950 | [Medline] |
| Duan, D., Fermini, B. & Nattel, S. (1995). Alpha-adrenergic control of volume-regulated currents in rabbit atrial myocytes: characterization of a novel regulatory mechanism. Circulation Research 76, 379-393. | |
| Duan, D., Winter, C., Cowley, S., Hume, J. R. & Horowitz, B. (1997). Molecular identification of a volume-regulated chloride channel. Nature 390, 417-421 | [Medline] |
| Duszyk, M., French, A. S. & Man, S. F. P. (1992). Noise analysis and single channel observation of 4 pS chloride channels in human airway epithelia. Biophysical Journal 61, 583-587. | [Abstract] |
| Edelman, J. L., Sachs, G. & Adorante, J. S. (1994). Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells. American Journal of Physiology 266, C1210-1221 | [Medline] |
| Farahbakhsh, N. A. & Fain, G. L. (1987). Volume regulation of nonpigmented cells from ciliary epithelium. Investigative Ophthalmology and Visual Science 28, 934-944 | [Abstract] |
| Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981). Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv 391, 85-100 | [Medline] |
| Ho, M. W. Y., Duszyk, M. & French, A. S. (1994). Evidence that channels below 1 pS cause the volume-activated chloride conductance in T84 cells. Biochimica et Biophysica Acta 1191, 151-156 | [Medline] |
| Jackson, P. S. & Strange, K. (1995). Single channel properties of a volume-sensitive anion conductance. Journal of General Physiology 105, 643-660 | [Abstract] |
| Jacob, T. J. C. & Civan, M. M. (1996). Role of ion channels in aqueous humor formation. American Journal of Physiology 271, C703-720 | [Medline] |
| Kawasaki, M., Uchida, S., Monkawa, T., Miyawaki, A., Mikoshiba, K., Marumo, F. & Sasaki, S. (1994). Cloning and expression of a protein kinase C-regulated chloride channel abundantly expressed in rat brain neuronal cells. Neuron 12, 597-604 | [Medline] |
| Krupin, T. & Civan, M. M. (1995). The physiologic basis of aqueous humor formation. In The Glaucomas, 2nd edn, ed. Ritch, R., Shields, M. B. & Krupin, T., pp. 250-281. Mosby, St Louis, MO, USA. | |
| Kunzelmann, K., Kubitz, R., Grolik, M., Warth, R. & Greger, R. (1992). Small conductance Cl- channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP. Pflügers Archiv 421, 238-246 | [Medline] |
| Lewis, R. S., Ross, P. E. & Cahalan, M. D. (1993). Chloride channels activated by osmotic stress in T lymphocytes. Journal of General Physiology 101, 801-826 | [Abstract] |
| Lorenz, L., Pusch, M. & Jentsch, T. J. (1996). Heteromultimeric ClC chloride channels with novel properties. Proceedings of the National Academy of Sciences of the USA 93, 13362-13366 | [Abstract/Full Text] |
| Mitchell, C., Zhang, J., Wang, L. & Jacob, T. J. C. (1997). Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. American Journal of Physiology 272, C212-222 | [Medline] |
| Nilius, B., Eggermont, J., Voets, T. & Droogmans, G. (1996). Volume-activated Cl- channels. General Pharmacology 27, 1131-1140 | [Medline] |
| Okada, Y. (1997). Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. American Journal of Physiology 273, C755-789 | [Medline] |
| Pusch, M. & Jentsch, T. J. (1994). Molecular physiology of voltage-gated chloride channels. Physiological Reviews 74, 813-827 | [Medline] |
| Raviola, G. & Raviola, E. (1978). Intercellular junctions in the ciliary epithelium. Investigative Ophthalmology and Visual Science 17, 958-981 | [Abstract] |
| Sears, M. L., Yamada, E., Cummins, D., Mori, N., Mead, A. & Murakami, M. (1991). The isolated ciliary bilayer is useful for studies of aqueous humor formation. Transactions of the American Ophthalmological Society 89, 131-152. | [Medline] |
| Socci, R. R. & Delamere, N. A. (1988). Characteristics of ascorbate transport in the rabbit iris-ciliary body. Experimental Eye Research 46, 853-861 | [Medline] |
| Stockbridge, N. (1997). EGTA. Computors in Biology and Medicine 17, 299-304. | |
| Strange, K. (1998). Molecular identity of the outwardly rectifying, swelling-activated anion channel: time to reevaluate pICln. Journal of General Physiology 111, 617-622 | [Full Text] |
| Strange, K., Emma, F. & Jackson, P. S. (1996). Cellular and molecular physiology of volume-sensitive anion channels. American Journal of Physiology 270, C711-730 | [Medline] |
| Szucs, G., Heinke, S., Droogmans, G. & Nilius, B. (1996). Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration. Pflügers Archiv 431, 467-469 | [Medline] |
| Takahashi, I., Kobayashi, E., Nakano, H., Murakata, C., Saitoh, H., Suzuki, K. & Tamaoki, T. (1990). Potent selective inhibition of 7-O-methyl UCN-01 against protein kinase C. Journal of Pharmacology and Experimental Therapeutics 255, 1218-1221 | [Abstract] |
| Thieman, A., Grunder, S., Pusch, M. & Jentsch, T. J. (1992). A chloride channel widely expressed in epithelial and non-epithelial cells. Nature 356, 57-60 | [Medline] |
| Traynelis, S. F. & Jaramillo, F. (1998). Getting the most out of noise in the central nervous system. Trends in Neurosciences 21, 137-145 | [Medline] |
| Voets, T., Buyse, G., Tytgat, J., Droogmans, G., Eggermont, J. & Nilius, B. (1996). The chloride current induced by expression of the protein pICln in Xenopus oocytes differs from the endogenous volume-sensitive chloride current. The Journal of Physiology 495, 441-447 | [Abstract] |
| Wan, X. L., Chen, S. & Sears, M. (1997). Cloning and functional expression of a swelling-induced chloride conductance regulatory protein, plCln, from rabbit ocular ciliary epithelium. Biochemical and Biophysical Research Communications 239, 692-696 | [Medline] |
| Wang, L., Chen, L., Walker, V. & Jacob, T. J. (1998). Antisense to MDR1 mRNA reduces P-glycoprotein expression, swelling-activated Cl- current and volume regulation in bovine ciliary epithelial cells. The Journal of Physiology 511, 33-44 | [Abstract/Full Text] |
| Wiederholt, M., Helbig, H. & Korbmacher, C. (1991). Ion transport across the ciliary epithelium: lessons from cultured cells and proposed role of carbonic anhydrase. In Carbonic Anhydrase, ed. Botie, F., Gross, G. & Storey, B. T., pp. 232-244. VHC, New York. | |
| Wilk-Blaszcak, M. A., French, A. S. & Man, S. F. P. (1992). 5 pS anion channels in airway epithelial cells. Biomedical Research 13, 143-148. | |
| Wu, J., Zhang, J. J., Koppel, H. & Jacob, T. J. C. (1996). P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells. The Journal of Physiology 491, 743-755 | [Abstract] |
| Yantorno, R. E., Carre, D. A., Coca-Prados, M., Krupin, T. & Civan, M. M. (1992). Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling. American Journal of Physiology 262, C501-509 | [Medline] |
| Yantorno, R. E., Coca-Prados, M., Krupin, T. & Civan, M. M. (1989). Volume regulation of cultured, transformed, nonpigmented epithelial cells from human ciliary body. Experimental Eye Research 49, 423-437 | [Medline] |
| Zhang, J. J. & Jacob, T. J. C. (1997). Three different Cl- channels in the bovine ciliary epithelium activated by hypotonic stress. The Journal of Physiology 499, 379-389 | [Abstract] |
Corresponding author
M. E. M. Kelly: Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Email: mkelly@is.dal.ca
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), 30 min after superfusion with HOS (
) and 15 min after return to standard external solution (
).
), 60 (
) or 90 mM (
) and after superfusion with, or application of, the Cl- channel blockers (
) DIDS (B), SITS (C), DNDS (D), or niflumic acid (NFA; E). Data have been normalized for cell capacitance (*P < 0·05; **P < 0·01).