Activation of the volume-sensitive chloride current in vascular endothelial cells requires a permissive intracellular Ca2+ concentration

Combined patch clamp and Ca²⁺-measurements (Fura-2) were used to study the dependence of volume-activated Cl^–-currents (I_Cl,vol) of endothelial cells from bovine pulmonary artery on the intracellular Ca²⁺-concentration [Ca²⁺]_i. Loading the cells with high concentrations of EGTA or BAPTA via ruptured membrane patches or by preincubating them with 50 M BAPTA-AM caused a substantial decrease of I_Cl,vol. This reduction was independent of the activation state of the current: the current amplitude was not only diminished if [Ca²⁺]_i was lowered at the peak of the volume-activated current, but this low Ca²⁺-concentration also prevented activation of the current by a second hypotonic challenge.I_Cl,vol is already maximally activated at intracellular Ca²⁺-concentrations between 50 and 100 nmol/l, a further increase of [Ca²⁺]_i does not affect the size of I_Cl,vol.These results indicate that a sustained full activation of I_Cl,vol in endothelial cells requires submicromolar concentrations of Ca²⁺, and that changes in [Ca²⁺]_i do not modulate the current.     

Cl− channel inhibition by glibenclamide is not specific for the CFTR-type Cl− channel

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl^– secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl^– currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl^– channels of HT₂₉ and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl^– channel which, potentially, may contribute to transepithelial Cl^– secretion.     

Small-conductance chloride channels induced by cAMP,Ca2+, and hypotonicity in HT29 cells: ion selectivity,additivity and stilbene sensitivity

Previous studies in HT₂₉ cells utilizing the cellattached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pflügers Arch 419:209–211] have revealed that the Cl^– channels induced by cAMP or by increasing cytosolic Ca²⁺, e.g. by addition of ATP, and by hypotonic cell swelling share in common their conductance, which was so small in our studies [Kunzelmann et al. (1992) Pflügers Arch (in press)] that we could not resolve it at the single-channel level. This prompted the question whether these Cl^– conductances can be distinguished in terms of their ion selectivity and sensitivity towards inhibitors. Whether these pathways are additive or not was also examined. The present study utilized the whole-cell patch-clamp and the CAN methods. A total of 160 patches were studied. In whole-cell patches 8-(4-chlorophenylthio)-cAMP (cAMP, 0.1±1 mmol/l) induced a significant depolarization by 5 mV and a twofold increase in conductance (G) from 6.2±1.5 nS to 11.7±3.2 nS (n=15). Total replacement of Cl^– by Br^– and I^– in cAMP-treated cells hyperpolarized the membrane voltage (V) significantly from –35±2.8 to –39±3.4 and –45± 3.3 mV respectively, but had no detectable effect on G, which was 11.9±3.3 nS in the case of Br^– and 11.8± 3.3 nS in the case of I^–. Hence, the permselectivity of the cAMP pathway was I^–>Br^–>Cl^–, but the conductances for these anions were all indistinguishable. For ATP at 10–100 mol/l the depolarization was least with I^–: from –41±1.1 to –36±2.4mV, intermediate for Br^– to –25±1.6 mV, and largest for Cl^– to –20±1.8 mV (n=18). ATP increased G from 3.4±0.3 nS to 12.9±2.8 nS (Cl^–), to 12.9±2.8 nS (Br^–) and to 12.9±2.7 (I^–) (n=18). These data indicate that the ATP-induced anion channel has a permeability sequence of I^–>Br^–>Cl^–. The conductance for all three anions was identical. Hypotonic cell swelling by 160 mosmol/l induced a depolarization that was smallest for I^–, from –42±4 to –32±2.1 mV, intermediate for Br^–: –29±1.8mV, and similar for Cl^–: –28±2 mV (n=20). G was increased from 2.8±0.8 nS to 15±2.5nS in the case of Cl^–, to 15±2.5 nS for Br^– and to 16±2.6 nS for I^– (n=20). Therefore, all three pathways are indistinguishable with respect to their anion selectivity. All three pathways are insensitive towards low concentrations of 4-nitro-2-(3-phenylpropylamino)benzoate, but are all blocked by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid, with a half-maximal inhibition around 0.6 mmol/l. Finally, the possible additivity was examined in three permutations. ATP (0.1 mmol/l) alone (n=14) had a slightly but not significantly larger effect on conductance than the combination of ATP and cAMP (1 mmol/l, n=14) and the combination of ATP and hypotonicity (193 mosmol/l, n=13). Similarly, the effects of hypotonicity and cAMP (n=11) were not additive. These data indicate that all three pathways share common properties. Hence, it is suggested that all three pathways converge on the same small Cl^– channel.Supported by DFG Gr 480/10 and BMFT 01 GA 8816     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

Thrombin potentiates volume-activated chloride currents in pulmonary artery endothelial cells

 In bovine pulmonary artery endothelial cells, ionic currents and the concentration of free intracellular Ca²⁺ ([Ca²⁺]_i) were measured with a combined patch clamp and Ca²⁺-fluorimetric method (Fura-2). Volume-activated Cl^––currents (I_Cl,vol) were activated by a 13 or 28% decrease in tonicity. Thrombin, 1 U/ml, strongly potentiated I_Cl,vol preactivated by low hypotonicity (13% HTS) but had no effect on I_Cl,vol preactivated by stronger hypotonic challenges (28% HTS). The thrombin-induced potentiation was not affected by buffering [Ca²⁺]_i at 50–100 nmol/l and omitting extracellular Ca²⁺. A peptide agonist of the thrombin receptor, SFLLRN, also potentiated I_Cl,vol, while an enzymatically inactive thrombin analogue, DIP-thrombin, was without effect. These results suggest that proteolytic activation of the thrombin receptor sensitises the activation of I_Cl,vol in endothelial cells in a Ca²⁺-independent mechanism. Received: 27 September 1996 / Received after revision: 16 January 1997 / Accepted: 30 January 1997     

Characterization of human sweat duct chloride conductance by chloride channel blockers

To characterize the chloride conductance of human sweat duct the effect of various analogues of diphenylamine-2-carboxylate was investigated on the transepithelial potential difference (PD_T) and resistance (R _T ) of isolated microperfused sweat ducts. Although the most powerful analogues which block Cl^– channels in various secretory and absorptive epithelia were ineffective, a number of analogues (in particular Cl substituted ones) were found which at high concentrations significantly and reversibly increased PD_T andR _T . The data suggest that the main chloride conductance pathway of sweat duct epithelium resides in the cell membranes rather than in the tight junctions. In addition the different blocking spectra of the chloride conductances of sweat duct and tracheal epithelium (Welsh MJ, Science 232:1648, 1986) suggest that the combined impairment of both conductances in cystic fibrosis does not result from a molecular defect in the Cl^– channels.     

The volume-activated chloride current in human endothelial cells depends on intracellular ATP

We have studied the effect of intracellular ATP on volume-activated Cl^–-currents in endothelial cells from human umbilical veins by means of the whole-cell patch clamp technique. The run-down of this current in ruptured patches during repetitive applications of hypotonic solutions (HTS) could be significantly reduced if the cells were internally perfused with a pipette solution that contained 4 mmol/l ATP. This run-down was much less pronounced if currents were recorded using nystatin-perforated patches. The amplitude of the current was drastically reduced and its activation became slower if the cells were superfused with a glucose-free medium with 1 mmol/l KCN. Adding 4 mmol/l ATPS, a poorly hydrolyzable ATP-analogue, to the patch pipette prevented run-down of the current during repetitive activations by HTS, even if the cells were superfused with glucose-free solution with 1 mmol/l KCN. It is concluded that activation of the mechanosensitive Cl^– conductance in human endothelial cells requires the presence of intracellular ATP, but not its hydrolysis.     

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]_o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl_o-induced retinal swelling were investigated in the present study, with emphasis on the Cl^– transport mechanisms. Increasing [KCl]_o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]_o was biphasic, with one component from 5 to 35 mM [KCl]_o and another from 35 to 100 mM. As Cl^– omission prevented swelling in all conditions, the effect of cotransporter or Cl^– channel blockers was examined to investigate the mechanisms of Cl^– influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]_o 25 mM (K25), and by 14–17% at [KCl]_o 54 mM (K54). The Cl^– channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90–95% at K54 (NPPB IC₅₀ 60.7 µM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC₅₀ 173.9 µM). Na⁺ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl^– influx at K25 and of Cl^– channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Müller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.     

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl^– transport and the effects of acetylcholine (ACh) and electrochemical Cl^– potential changes across the basolateral plasma membrane on intracellular Cl^– activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl^–-selective microelectrodes. In the resting state, the basolateral membrane potential (V _m) was about –40 mV and intracellular Cl^– activity was about 35 mmol/l. Addition of ACh (10^–910^–6 mol/l) hyperpolarizedV _m and decreased the Cl^– activity in a dose-dependent manner. ACh (10^–6 mol/l) hyperpolarizedV _m by 20 mV and decreased the cytosolic Cl^– activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl^– concentration to 1/9 control depolarizedV _m and decreased cytosolic Cl^– activity at a rate of 1.9 mmol/l · min. AV _m hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl^– activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedV _m by 8 mV with little change in Cl^– activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl^– activity induced by low-Cl^– Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl^– activity. Furosemide (1 mmol/l) slightly hyperpolarizedV _m and decreased Cl^– activity at a slow rate but affected Cl^– movements induced by ACh or low-Cl^– Ringer only slightly. Cl^– uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl^– permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl^– conductance pathway and a furosemide-sensitive Cl^– uptake mechanism.     

Volume-activated chloride channels in HeLa cells are blocked by verapamil and dideoxyforskolin

The possible role of Cl^– currents in regulatory volume decrease processes has been explored in HeLa cells using the whole-cell recording mode of the patch-clamp technique. Cells showed very small currents in voltage-clamp experiments performed with Cl^–-rich, permeant-cation-free (N-methyl-d-glucamine replacement) intracellular and bathing solutions. Exposure of the cells to hypotonic solutions visibly swelled the cells and activated, reversibly, an outward rectifying Cl^– current, which decayed at the most depolarised voltages used. Replacement of extracellular Cl^– by a series of halide anions, SCN^– and gluconate was consistent with an anion selectivity sequence: SCN^–>I^–>Br^–>Cl^– >F^–>gluconate. The volume-regulated Cl^– current was effectively inhibited by 100 M 5-nitro-2-(3-phenylpropylamino)-benzoic acid and by 100 M 4,4-diisothiocyanotostilbene-2,2-disulphonic acid, substances known to block Cl^– channels in a variety of cells. Chloride current activation by hypotonicity was dependent on the presence of ATP in the intracellular solution and this requirement could be replaced by the non-hydrolysable analogue ATP[S] and Mg²⁺-free ATP. The data suggest that the channels responsible for the current described are involved in the regulatory volume decrease in HeLa cells. The characteristics of this Cl^– current are similar to those of the current associated with expression of multidrug resistance P-glycoprotein. Furthermore, the currents in HeLa cells were inhibited rapidly and reversibly by verapamil and 1,9-dideoxyforskolin, which are known to inhibit P-glycoprotein function.     

Anion and cation channels in the basolateral membrane of rabbit parietal cells

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (E _m ) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl^–]₀), and of the pipette (intracellular) solution. The relationship betweenE _m and [Cl^–]₀ was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4-isothiocyanostilbene-2,2-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl^– conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl^– channel opening events could not be observed. The value ofE _m depended little on the basolateral K⁺ concentration, but inward-rectifier K⁺ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba²⁺. In cell-attached and cell-free patches, openings of single inward-rectifier K⁺ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca²⁺ activated these cation channels. The single K⁺ channel conductance was about 230 pS under the symmetrical high K⁺ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.     

Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: Studies with Cl−-sensitive microelectrode

The mechanism of uphill Cl^– accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl^–-selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential (V _m) was –39.2±0.4 mV and intracellular Cl^– activity (A _Cl ⁱ ) was 34.6±0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na⁺-free solution,A _Cl ⁱ decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level ofA _Cl ⁱ in 5 min. In the presence of furosemide (1 mmol/l), Cl^– uptake induced by Na⁺-readmission was inhibited by 44%. Superfusion with a K⁺-free solution gradually decreasedA _Cl ⁱ until it was close to the equilibrium level after 75 min; superfusion with a high-K⁺ (29.5 mmol/l) solution increasedA _Cl ⁱ significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K⁺-free to high-K⁺ and from high-K⁺ to K⁺-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (–7 mmol/l) inA _Cl ⁱ . These changes inA _Cl ⁱ were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl^– uptake after cessation of acetylcholine (ACh 1 mol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na⁺–K⁺–Cl^– cotransport. The additional inhibitory effect of DIDS to furosemide inhibition suggests an involvement of anion exchange transport, in parallel with the cotransport, in uphill Cl^– uptake into the cells.     

Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl– currents in macrovascular endothelial cells

 We have used the whole-cell patch-clamp technique to study the effects of inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P ₄], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P ₄] and inositol 1,3,4,5,6-pentacisphosphate [Ins(1,3,4,5,6)P ₅] on volume-activated Cl^– currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary artery (CPAE cells). Ins(1,4,5,6)P ₄ and Ins(3,4,5,6)P ₄ were applied intracellularly via the patch pipette at concentrations between 10 and 100 μM. Both tetrakisphosphates inhibited the Cl^– current I _Cl,Ca, which was activated by intracellular loading of the cells with 500 nM Ca²⁺ [for inhibition by Ins(1,4,5,6)P ₄: 58% at 10 μM, 75% at 100 μM; for Ins(3,4,5,6)P ₄: 44% at 10 μM, 65% at 100 μM]. Inhibition of I _Cl,Ca occurred without significant changes in its kinetic properties. The amplitude of I _Cl,vol activated by a 13.5 or 27% hypotonic solution at +100 mV was strongly reduced in cells loaded with either tetrakisphosphate, i.e. a 73% reduction for Ins(3,4,5,6)P ₄ and 89% for Ins(1,4,5,6)P ₄ at 100 μM. Both tetrakisphosphates also inhibited a current probably identical to I _Cl,vol which was activated by dialysing the cell with 100 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]). Ins(1,3,4,5,6)P ₅ at a concentration of 30 μM did not significantly reduce I _{Cl, vol}. The effects of Ins(3,4,5,6)P ₄ may represent an inhibitory pathway for the I _Cl,Ca and I _Cl,vol in macrovascular endothelium after sustained receptor-mediated activation of phospholipase C. Received: 8 September 1997 / Received after revision and accepted: 31 October 1997     

Small and intermediate conductance chloride channels in HT29 cells

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT₂₉ cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g _o) is caused by inhibition of small non-resolvable channels, which are impermeable to Na⁺ and SO₄ ^2–, but permeable to Cl^–. The regulation of these small Cl^–-conducting channels (g _o) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (P _o) of the ICOR or on the g_o) were observed. The P_o of the ICOR was reduced by 22% in a Ca²⁺-free solution. g _o was insensitive to changes in the Ca²⁺ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT₂₉ cells contain ICOR and small non-resolvable Cl^–-conducting channels which are similarly inhibited by C. I. Apart from a weak effect of Ca²⁺ on the ICOR, g _o and the ICOR do not seem to be directly controlled by Ca²⁺ or PKA. C. I. of normal and CF epithelia have a similar inhibitory potency on Cl^– channels.     

Evidence for a cytosolic inhibitor of epithelial chloride channels

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl^– conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl^– fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl^– channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl^– flux in membrane vesicles and single Cl^– channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl^– channel inhibition cannot be decided at present.     

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mol/l 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS). After prestimulation of inward I _sc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E₁ hypoosmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of I _sc. The stimulation of I _sc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive ⁸⁶Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T₈₄ cells is also associated with an increase in cytosolic Ca²⁺. It is concluded that the effects of hypo-osmotic exposure of T₈₄ cells on secretory I _sc are consistent with the activation of a DIDS-sensitive apical Cl^– conductance and a basal-lateral K⁺ conductance. With prior activation of inward I _sc by VIP via a cAMP-activated DIDS-insensitive apical Cl^– conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K⁺ current and loop-diuretic-sensitive Cl^– uptake.     

Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP,Ca2+ and hypotonic cell swelling

HT₂₉ cells were preincubated with forskolin (10^–5 mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10^–7 mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT₂₉ cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8±1.4 mV (n=77) and that of PMAHT was -43.6±2.4 mV (n=76). The conductance (G) of FORHT (9.4 ±0.9 nS, n=77) was significantly larger than that of PMAHT (3.7±0.4 nS, n=76). Acute application of forskolin (10^–5 mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6±1.9 nS for FORHT (n=22) and only 2.2±1 nS for PMAHT (n=13). ATP (10^–4 mol/l) depolarized V in both types of cells. It enhanced G by 16.7±4.1 nS in FORHT (n=14) and significantly less (by 5.5±1.2nS, n=14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10^–8 mol/l) also had a stronger and longer lasting effect in FORHT. Exposure to hypotonic bath solution (160 mosmol/l) depolarized V in both types of cells. The increase in G was by 15±2.2 nS in FORHT (n=18) and by 11±1.3 nS in PMAHT (n=23). After being returned to the normotonic media, the decline of G to the control value was delayed in FORHT when compared to PMAHT. Ionomycin (10^–7 mol/l) increased G significantly more (to 47±8.5 nS, n=13) in FORHT when compared to PMAHT (to 28±4 nS, n=13). The present data indicate that a 20-h exposure of HT₂₉ cells to FOR versus PMA alters markedly the CFTR concentration. The cells with high CFTR (FORHT) when compared to those with low CFTR (PMAHT) differ not only in their acute G response to cAMP, but also in that to ATP, NT, hypotonic cell swelling, and ionomycin. In contrast, the same pretreatment of CFPAC-1 cells did not alter the G changes induced by ionomycin or hypotonic cell swelling. These results indicate that changes in CFTR expression correlate with the Cl^– conductances induced by cAMP, Ca²⁺ and hypotonic cell swelling.     

Iodide efflux measurements with an iodide-selective electrode: A non-radioactive procedure for monitoring cellular chloride transport

A nonradioactive procedure using an I^–-selective electrode has been developed for assaying cellular Cl^– transport. NIH 3T3 fibroblasts stably transfected with the Cystic Fibrosis Trans-membrane Conductance Regulator (CFTR) Cl^– channel were grown in standard 35 mm culture dishes and used to test this assay system. For efflux measurements, the fibroblasts were first incubated in an I^–-loading buffer and then exposed to an I^–-free buffer. Efflux was monitored using the I^–-selective electrode. Application of either forskolin (5 µM) or 8-chlorphenylthio cAMP (500 µ M), to activate the CFTR Cl^– channels, resulted in over a 5-fold increase in I^– efflux as compared with control, untreated fibroblasts. No increase over basal efflux levels was observed in nontransfected NIH 3T3 fibroblasts treated with forskolin. The Cl^– channel blockers diphenylamine-2-carboxylate (DPC) (1 mM) and 5-nitro-2-(3- phenylpropylamino)-benzoic acid (NPPB) (25 µM) reduced forskolin- stimulated efflux by 50% and 23%, respectively. In addition to forskolin, both the tyrosine kinase inhibitor genistein and the protein kinase C activator phorbol 12,13-dibutyrate, were capable of stimulating I^– efflux. Thus, use of the I^–-selective electrode provides a fast and convenient method for studying Cl^– channels. The I^– efflux assay should be useful for monitoring drug and hormone-activated Cl^– transport pathways in a wide variety of cell types.     

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl− transport

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl^– movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2–9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l^–1 ADH was added to the bathing solutions. The basolateral membrane voltage (PD_bl) of principal cells was –77±1 mV and the luminal membrane voltage (PD₁) was –68±1 mV (mean ± SEM, n=124). Separate impalements with single-barrelled Cl^–-selective microelectrodes gave an apparent intracellular Cl^– activity of principal cells of 17±2 mmol.l^–1. Transepithelial PD and PD_bl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl^– channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO ₃ ^– by Cl^–. The intracellular Cl^– activity (a _cell ^Cl ) also remained unchanged with the addition of HCT, SITS or the Cl^– channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO ₃ ^– . With Cl^– replacement in both solutions, a _cell ^Cl decreased to 9 mmol.l^–1, but not until after 4–6 min, indicating a very low rate of Cl^– transport in these cells, even under conditions of maximal stimulation of NaCl reabsorption by mineralocorticoid plus ADH. The remaining a _cell ^Cl could be attributed to interference with the Cl^– selective electrodes by other cytosolic anions. We conclude that a _cell ^Cl of principal cells in the rat CCD is not far above passive equilibrium, and that these cells do not contribute significantly to transepithelial Cl^– reabsorption, which must occur by alternative routes such as the paracellular pathway, and/or through intercalated cells.Parts of this study were presented at the 65th meeting of the Deutsche Physiologische Gesellschaft at Würzburg, Federal Republic of Germany, 1988     

The benzazepine/benzothiazepine binding domain of the cardiac L-type Ca2+ channel is accessible only from the extracellular side

The whole-cell tight seal recording technique was used to investigate location of benzothiazepine binding site of the cardiac L-type Ca²⁺ channel. For this we utilized a permanently charged compound, SQ 32.428, out of a series of benzazepine drugs which have been characterized as competitive inhibitors of diltiazem binding. The non-permanently charged derivative SQ 32.910 was initially tested to electrophysiologically establish Ca²⁺ antagonistic properties of benzazepines. Upon extracellular application, either compound was able to completely block Ca²⁺ currents. At a stimulating frequency of 0.2 Hz IC₅₀ concentrations of SQ 32.910 and SQ 32.428 were determined as 35 nM and 15 M, respectively. Intracellular application of SQ 32.428 was then compared to control experiments in the absence of drug. Initially, adequate drug dialysis was confirmed with 100 M D890, which produced a progressive inhibition of Ca²⁺ currents within 10 min after whole-cell access. In contrast, internal application of 100 M SQ 32.428 did not change time-course of Ca²⁺ currents compared to control run-down. These results show that the benzazepine/benzothiazepine binding domain of the cardiac L-type Ca²⁺ channel is accessible only from the extracellular side and therefore suggest an extracellular location on the ₁-subunit of the Ca²⁺ channel protein.