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.     

Acetylcholine-induced change in intracellular Cl− activity of the mouse lacrimal acinar cells

Using double-barreled Cl^–-sensitive microelectrodes, intracellular Cl^– activity (A _Cl ⁱ ) in the mouse lacrimal acinar cells in vitro was determined in both resting and secretory phases. In the resting stateA _Cl ⁱ was 31 mmol/l which was 1.4 times higher than that predicted for the passive distribution according to the membrane potential (V _m) of –41 mV. Addition of acetylcholine (ACh, 1M) hyperpolarizedV _m to –63 mV and decreasedA _Cl ⁱ to 20 mmol/l which was still twice the equilibrium activity. A-23178 produced similar changes inV _m andA _Cl ⁱ to those induced by ACh. It was concluded that Cl^– was actively accumulated in the acinar cells and, in the secretory phase, Cl^– efflux was enhanced by the increased driving force and Ca²⁺-mediated increase in the Cl^– permeability across the cell membrane.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

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.     

Dual effect of N-ethylmaleimide on Cl− transport across the thin ascending limb of Henle's loop

Effects of SH reagents on Cl^– transport were studied in the isolated hamster thin ascending limb of Henle's loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10^–4 M in the bath decreased the relative permeability for Cl^–/Na⁺ (P _Cl/P _Na), as determined by the transmural diffusion voltage (V _T) generated under a NaCl concentration gradient, from 2.71±0.16 to 1.11±0.09 (P<0.001). The effect of PCMBS was prevented by the pretreatment with 10^–3 M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10^–3 M in the bath exhibited a dual action on Cl^– permeability of the TAL: It inhibited the Cl^– permeability in fresh preparations, whereas it stimulated the Cl^– permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextranmaleimide did not show any inhibitory effect onP _Cl/P _Na, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM onP _Cl/P _Na was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca²⁺ free, the stimulatory effect of NEM was unaffected, although the basal level ofP _Cl/P _Na was reduced. These observations suggest that the conductive Cl^– pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca²⁺ may be independent of the SH regulatory sites.     

Electrical properties and electrolyte transport in bovine tracheal epithelium: Effects of ion substitutions,transport inhibitors and histamine

The active transport of Na⁺ and Cl^– across the tracheal epithelium of the cow was investigated in vitro, using the short-circuit technique, by means of ion substitutions, transport inhibitors and by measuring²²Na and³⁶Cl fluxes. Under short-circuit conditions, short-circuit current (i _o) was 168±5A cm^–2 (mean±SEM,n=30), i.e. 6.2±0.2 Eq h^–1cm^–2 and resistance (R) was 248±10cm². Net Na⁺ flux toward the submucosa (J _{Na net} ^L-S ) and net Cl^– flux toward the lumen (J _{Cl net} ^S-L ) were of the same magnitude, i.e. 2.7±0.2 and 2.9±0.2 Eq h^–1 cm^–2, respectively. The permeability coefficients were 3.6·10^–6 forP _Na and 7.8·10^–6 cm s^–1 forP _Cl. Under open-circuit conditions, the transepithelial electrical potential difference () was 43±2 mV (lumen negative,n=20).J _{Na net} ^L-S andJ _{Cl net} ^S-L were close to zero.Bilateral substitution of Cl^– with SO ₄ ^2– or isethionate, or administration of furosemide 5·10^–3M or bumetanide 10^–4M in the submucosal bathing medium produced a 40 to 50% decrease ini ₀; furosemide abolishedJ _{Cl net} ^S-L . Bilateral substitution of Na⁺ with choline or Mg²⁺, or addition of ouabain 10^–4M to the submucosal bath abolishedi ₀;J _{Na net} ^L-S andJ _{Cl net} ^S-L were suppressed by ouabain. Amiloride 10^–4M in the luminal bath reducedi ₀ by 23% and diminishedJ _{Na net} ^L-S by 80%. Histamine 10^–4M, added to the submucosal bathing medium, reducedJ _{Na net} ^L-S and increasedJ _{Cl net} ^S-L , under short-circuit conditions. In open-circuit conditions, histamine had little effect on ion fluxes. This substance had no effect on the electrical properties, as shown previously.These results are consistent with the model proposed by Silva et al. [20] for a Cl^–-secreting, Na⁺-reabsorbing epithelium.Supported by the Swiss National Foundation (SNF), grant no. 3.588-0.79     

Cl−-channels in the apical cell membrane of the rectal gland “induced” by cAMP

Isolated rectal gland tubules (n1000) of dogfish (Squalus Acanthias) were perfused in vitro. Individual channels in the apical and basolateral cell membrane were recorded with the patch clamp method. K⁺-channels were present in excised membrane patches of the basolateral membrane in stimulated (dbcAMP + forskolin + adenosine) and in nonstimulated state. Cl^–-channels were found only in patches of the apical cell membrane when the tubule was stimulated. Cell attached recordings and simultaneous transepithelial PD measurements were obtained while the segment was stimulated. It is shown that concomitant with the increase in lumen negative PD silent membrane patches of the apical cell membrane suddenly develop Cl^–-channel activity. It is concluded that stimulation of rectal gland tubules activates Cl^–-channels in the apical cell membrane.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-2 Correspondence to first author at the above address     

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.     

Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons

K⁺ and Cl^– homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K⁺ and Cl^– efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl^– channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl^– current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C₂-ceramide, or serum deprivation. Cl^– channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl^– channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K⁺ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl^– and K⁺ homeostasis in regulating different apoptotic events.     

Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by31P NMR spectroscopy

Intracellular pH (pH_i) was measured in the isolated, perfused rabbit mandibular salivary gland by³¹P NMR spectroscopy. In the unstimulated gland perfused with HCO ₃ ^– /CO₂-buffered Ringer's solution, pH_i was 7.27±0.01. Continuous stimulation with acetylcholine elicited dose- and time-dependent changes in pH_i. 10^–6 mol/l acetylcholine caused a brief intracellular acidosis (–0.19±0.06 pH units) followed by an increase in pH_i to a more alkaline steady-state value (7.33±0.02). In the absence of perfusate HCO ₃ ^– or in the presence of 10^–4 mol/l DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid), the transient acidosis was abolished and pH_i increased rapidly to give a sustained alkalosis (7.49±0.03 and 7.44±0.03 respectively). In the presence of 10^–3 mol/l amiloride, the response to acetylcholine was a rapid decrease in pH_i to 7.02±0.02. The data suggest that, during perfusion with HCO ₃ ^– /CO₂-buffered solutions, stimulation with acetylcholine results in a transient loss of HCO ₃ ^– from the acinar cells (causing a transient acidosis), and, independently, the activation of Na⁺–H⁺ exchange (causing a sustained alkalosis). In the unstimulated gland, DIDS and the HCO ₃ ^– -free perfusate caused decreases in pH_i to 7.12±0.02 and 7.04±0.01 respectively. In contrast, amiloride had little effect. The relatively high value of pH_i maintained by the unstimulated gland is therefore probably not due to Na⁺–H⁺ exchange.     

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     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

The effect of hyperosmotic challenge upon ion transport in cultured renal epithelial layers (MDCK)

Exposure of the basal-lateral surfaces of MDCK epithelia, mounted in Ussing chambers, to medium made hyperosmotic by the non-electrolyte mannitol, resulted in a marked inhibition of the adrenalinestimulated inward short-circuit current (Cl^– secretion). This inhibition was unaccompanied by a reversal of the adrenaline-stimulated increment in tissue conductance, indicating that the inhibition was due to modulation of ion transport at the basal-lateral membranes. Loop-diuretic-sensitive ⁸⁶Rb(K⁺) efflux mediated by the Na⁺-K⁺ — 2 Cl^– cotransporter at the basal-lateral membranes was markedly stimulated by hypertonic exposure. A diuretic-sensitive K⁺ (Cl^–) loss was observed in shrunken cells upon prolonged exposure (20 min), showing that the net direction of cotransport flux was outward. ⁸⁶Rb(K⁺) efflux stimulated by adrenaline (100 M), exogenous ATP (100 M) and A23187 (10 M) was attenuated in shrunken cells, suggesting that basal-lateral K⁺ conductance is reduced in hyperosmotic media. Cotransport stimulation by hyperosmotic medium was asymmetric, apical bathing hypertonicity being ineffective. These data are consistent with a low hydraulic permeability of the apical membranes.     

Mechanism of Cl− transport in eel intestinal brush-border membrane vesicles

Cl^– transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. ³⁶Cl^– uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K⁺ diffusion potential obtained by imposing a K⁺ concentration gradient (C _out>C_in) in the presence of valinomycin, (c) an inwardly directed H⁺ ion concentration gradient. The membrane-potential-driven Cl^– transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropylamino)-benzoic acid. Arachidonic acid also inhibited Cl^– uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na⁺ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl^– influx was the same in the presence of inwardly directed gradients of Li⁺, Na⁺ or K⁺, arguing against the presence of Na⁺-Cl^–, as well as K⁺-Cl^– cotransport. The absence of a significant contribution of the Na⁺-K⁺-2Cl^– cotransport mechanism to the Cl^– uptake in seawater eel intestinal BBMV was indicated from the observations that Cl^– uptake was not stimulated by the simultaneous presence of inwardly directed Na⁺ and K⁺ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na⁺ and K⁺. Furthermore, no evidence for the dependence of Cl^– uptake on the Na⁺ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K⁺ and valinomycin. The finding that the Cl^– uptake in the presence of a H⁺ gradient was not inhibited by 1 mM SITS and was significantly reduced in the presence of [K⁺]_in=[K⁺]_out and valinomycin, suggests that no anion exchanger is present in our experimental system. We conclude that Cl^– uptake in eel intestinal BBMV does not occur via an electroneutral Na⁺-dependent Cl^– transport mechanism (either cotransport or double exchange) and is realized by a Cl^– conductance.     

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 effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK)

Regulatory volume decrease after exposure to hypo-osmotic media in MDCK epithelial cells results from activation of both K⁺ and Cl^– conductances. Swelling-stimulated ⁸⁶Rb(K) losses were observed only across the basal-lateral membrane and were relatively insensitive to 10 mM Ba²⁺. The effect of hypo-osmotic media upon MDCK epithelia mounted in Ussing chambers has been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc) followed by inhibition of inward I _sc in both control layers and in layers where inward current (due to transepithelial Cl^– secretion) was first stimulated by 5 M prostaglandin E₁ (PGE₁). The transient stimulation of inward current by hypo-osmotic media was not markedly attenuated by 10 mM Ba²⁺ in PGE₁-stimulated layers. After stimulation of inward (Cl^–-secretory) current to high levels by 10M adrenaline, the predominant effect of basal-lateral exposure to hypo-osmotic media was an inhibition of the inward current. This inhibition was partially reversed by 40M 4,4-diisothiocyanatostilbene-2,2-disulphonate (DIDS). The stimulation, then inhibition, of inward I _sc is likely to be the result of separate swelling-induced K⁺ and Cl^– conductances (respectively) at the basal-lateral membrane. The swelling-stimulated Cl^– conductance is distinct from the apical Cl^– conductance regulated by PGE₁ or adrenaline.     

Role of protein kinase C in the regulation of inositol phosphate production and Ca2+ mobilization evoked by ATP and acetylcholine in rat lacrimal acini

Stimulation of rat lacrimal acinar cells with ATP and acetylcholine (ACh) induced a rapid accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] and its degradation products, resulting in an initial release of Ca²⁺ from intracellular stores. However, after pretreating the acini with U73122 no increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) or Ins(1,4,5)P ₃ production was observed. A short pre-treatment with the phorbol ester 4--phorbol-12--myristate-13--acetate (PMA) significantly attenuated the ATP- and ACh-induced increase in [Ca²⁺]_i and over- all inositol phosphate production. In contrast, staurosporine enhanced Ins(1,4,5)P ₃ and inositol 1,3,4-trisphosphate [Ins(1,3,4)P ₃] production and [Ca²⁺]_i above control values in ATP- and ACh-stimulated cells. Stimulation of phospholipase C by iono-mycin-evoked changes in [Ca²⁺]_i were unaltered by pretreatment with staurosporine and PMA. The data show that a change in protein kinase C activity during cell stimulation affects the inositol phosphate metabolism and thereby the cellular Ca²⁺ signalling processes in lacrimal acinar cells.     

Characterization of an outwardly rectifying chloride channel in a human submandibular gland duct cell line (HSG)

We have used the single-channel patch-clamp technique to study ion channels in the plasma membrane of the HSG human submandibular gland duct cell line. In cell-attached and excised inside-out patches, at least six channel types were observed. When the pipette contained an isotonic KCl-rich solution and the bath an isotonic NaCl-rich solution, the predominant channel type seen in excised inside-out patches was a Cl⁻ channel with an outwardly rectifying current/voltage (I/V) relation that had a conductance of 12 pS at positive pipette potentials and 43 pS at negative pipette potentials. The channel was only seen in excised patches and its open probability was not significantly increased by membrane depolarization. The channel selectivity sequence (relative to Cl⁻) was estimated from reversal potential measurements to be: SCN⁻ (1.8)>NO ₃ ⁻ (1.4)> I⁻ (1.1) ∼ Cl⁻ (1) ∼ Br⁻ (0.8) > acetate (0.35). In inside-out patches the channel was blocked by addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (100 μmol/l) to the bath but not by 9-anthracene carboxylic acid (9-AC) (100 μmol/l). The channel was not activated by increases in the free Ca²⁺ concentration on the cytosolic surface. This is the first report of an outwardly rectifying Cl⁻ channel in a salivary epithelium. The properties of this channel are not in accordance with the properties of the Cl⁻ conductances in the acinar or duct tissues which have been studied so far and its physiological role is unclear.     

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.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.