Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl⁻ channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na⁺ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl⁻ current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl⁻ current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn²⁺ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn²⁺ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl⁻ channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na⁺ transport in this surface epithelium under basal physiological conditions.     

The Effects of Mobile Phone Radiofrequency Radiation on Cochlear Stria Marginal Cells in Sprague–Dawley Rats

To investigate the possible mechanisms for biological effects of 1,800 MHz mobile radiofrequency radiation (RFR), the radiation-specific absorption rate was applied at 2 and 4 W/kg, and the exposure mode was 5 min on and 10 min off (conversation mode). Exposure time was 24 h short-term exposure. Following exposure, to detect cell DNA damage, cell apoptosis, and reactive oxygen species (ROS) generation, the Comet assay test, flow cytometry, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) staining, and a fluorescent probe were used, respectively. Our experiments revealed that mobile phone RFR did not cause DNA damage in marginal cells, and the rate of cell apoptosis did not increase (P > 0.05). However, the production of ROS in the 4 W/kg exposure group was greater than that in the control group (P < 0.05). In conclusion, these results suggest that mobile phone energy was insufficient to cause cell DNA damage and cell apoptosis following short-term exposure, but the cumulative effect of mobile phone radiation still requires further confirmation. Activation of the ROS system plays a significant role in the biological effects of RFR. Bioelectromagnetics. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996     

Current status of the role of Cl− ion in the oxygen-evolving complex

This minireview summarizes the current state of knowledge concerning the role of Cl⁻ in the oxygen-evolving complex (OEC) of photosystem II (PSII). The model that proposes that Cl⁻ is a Mn ligand is discussed in light of more recent work. Studies of Cl⁻ specificity, stoichiometry, kinetics, and retention by extrinsic polypeptides are discussed, as are the results that fail to detect Cl⁻ ligation to Mn and results that show a lack of a requirement for Cl⁻ in PSII-catalyzed H₂O oxidation. Mutagenesis experiments in cyanobacteria and higher plants that produce evidence for a correlation between Cl⁻ retention and stable interactions among intrinsic and extrinsic polypeptides are summarized, and spectroscopic data on the interaction between PSII and Cl⁻ are discussed. Lastly, the question of the site of Cl⁻ action in PSII is discussed in connection with the current crystal structures of the enzyme.     

Transport energetics of the Cl− pump in Aplysia gut

Basolateral membranes of Aplysia foregut epithelia contain an ATP-dependent Cl⁻ transporter (Cl⁻ pump). Increased activity of the Cl⁻ pump, coupled to apical and basolateral membrane depolarization, changed the Cl⁻ transport energetics across the apical membrane but did not change the vectorially-opposite Cl⁻ transport energetics across the basolateral membrane.     

Cl− Currents Activated by Extracellular Nucleotides in Human Bronchial Cells

The perforated-patch technique was used to study the response of human bronchial cells to extracellular nucleotides. ATP or UTP (100 μm) elicited a complex response consisting of a large transient membrane current increase followed by a relatively small sustained level. These two phases were characterized by different current kinetics. Throughout the transient phase (2–3 min) the membrane current (I _p ) displayed slow activation and deactivation kinetics at depolarizing and hyperpolarizing potentials respectively. At steady-state (I _s ) the relaxation at hyperpolarizing potential disappeared whereas at positive membrane potentials the current became slightly deactivating. The I _s amplitude was dependent on the extracellular Ca²⁺ concentration, being completely inhibited in Ca²⁺-free medium. Cell pre-incubation with the membrane-permeable chelating agent BAPTA/AM prevented completely the response to nucleotides, thus suggesting that both I _p and I _s were dependent on intracellular Ca²⁺. The presence of a hypertonic medium during nucleotide stimulation abolished I _s leaving I _p unchanged. On the contrary, niflumic acid, a blocker of Ca²⁺-activated Cl⁻ channels, prevented completely I _p without reducing significantly I _s . 1,9-dideoxyforskolin fully inhibited I _s but also reduced I _p . Replacement of extracellular Cl⁻ with aspartate demonstrated that the currents activated by nucleotides were Cl⁻ selective. I _p resulted five times more Cl⁻ selective than I _s with respect to aspartate. Taken together, our results indicate that ATP and UTP activate two types of Cl⁻ currents through a Ca²⁺-dependent mechanism. Received: 15 August 1996/Revised: 6 December 1996     

Regulation of Basolateral Cl− Channels in Airway Epithelial Cells: The Role of Nitric Oxide

The presence of basolateral Cl⁻ channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl⁻ conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl⁻ channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl⁻ channels via a cGMP-dependent pathway. Anion replacement and ³⁶Cl⁻ flux studies showed that NO affected both Cl⁻ and HCO ₃ ⁻ secretion. Two different types of Cl⁻ channels are known to be present in the basolateral membrane of epithelial cells: Zn²⁺-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl⁻ conductance in the presence of Zn²⁺ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl⁻ conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl⁻ channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.     

Cl− and F− anions regulate the architecture of protofibrils in fibrin gel

Ischemic heart disease is the leading cause of serious morbidity and mortality in Western society. One of the therapeutic approaches is based on the use of thrombolitic drugs that promote clot lysis. Even if the mechanisms leading to clot lysis are not completely understood, it is widely accepted that they depend on the complex biochemical reactions that occur among fibrin fibers and fibrinolitic agents, and by their ready diffusion into the fibers. Here we investigate the effects of specific anions on the architecture of protofibrils within fibrin fibers in fibrin gels prepared in a para-physiological solution. The results obtained through small-angle X-ray scattering (SAXS) demonstrate that the characteristic axial and longitudinal repeat distances among protofibrils are strongly affected by the action of Cl⁻ and F⁻ anions.     

Fish Gill Respiratory Cells in Culture: A New Model for Cl−-secreting Epithelia

Primary cultures of sea bass gill cells grown on permeable membranes form a confluent, polarized, functional tight epithelium as characterized by electron microscopy and electrophysiological and ion transport studies. Cultured with normal fetal bovine serum (FBS) and mounted in an Ussing chamber, the epithelium presents a small short-circuit current (I _sc : 1.4 ± 0.3 μA/cm²), a transepithelial voltage (V _t ) of 12.7 ± 2.7 mV (serosal positive) and a high transepithelial resistance (R _t : 12302 ± 2477 Ω× cm²). A higher degree of differentiation and increased ion transport capacities are observed with cells cultured with sea bass serum: numerous, organized microridges characteristic of respiratory cells are present on the apical cell surface and there are increased I _sc (11.9 ± 2.5 μA/cm²) and V _t (25.9 ± 1.7 mV) and reduced R _t (4271 ± 568 Ω× cm²) as compared with FBS-treated cells. Apical amiloride addition (up to 100 μm) had no effect on I _sc . The I _sc , correlated with an active Cl⁻ secretion measured as the difference between ³⁶Cl⁻ unidirectional fluxes, was partly blocked by serosal ouabain, bumetanide, DIDS or apical DPC or NPPB and stimulated by serosal dB-cAMP. It is concluded that the chloride secretion is mediated by a Na⁺/K⁺/2Cl⁻ cotransport and a Cl⁻/HCO₃ ⁻ exchanger both responsible for Cl⁻ entry through the basolateral membrane and by apical cAMP-sensitive Cl⁻ channels. This study gives evidence of a functional, highly differentiated epithelium in cultures composed of fish gill respiratorylike cells, which could provide a useful preparation for studies on ion transport and their regulation. Furthermore, the chloride secretion through these cultures of respiratorylike cells makes it necessary to reconsider the previously accepted sea water model in which the chloride cells are given the unique role of ion transport through fish gills. Received: 12 July 1996/Revised: 5 November 1996     

Effect of “Chemical” Hypoxia on the Potassium Conductance of the Membrane of Pheochromocytoma Cells

We studied the effect of “chemical” (induced by the action of sodium thiosulfate, STS) hypoxia on the potassium conductance of the membrane of pheochromocytoma cells. Application of 1 to 10 mM STS decreased in a dose-dependent manner the amplitude of integral potassium current without changes in the voltage dependence of its activation. The concentration dependence of the action of STS on the amplitude of potassium current was estimated using the Boltzmann equation. The value of concentration for 50% inhibition was 2.7 ± 0.2 mM, while the slope coefficient was 0.9 ± 0.2 mM⁻¹. In the presence of 10 mM STS, the decrease in the amplitude of potassium current reached, on average, 55%. Therefore, “chemical” hypoxia influences rather significantly the potassium conductance of the membrane of pheochromocytoma PC12 cells.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

Downregulation of Epithelial Sodium Channel (ENaC) by CFTR Co-expressed in Xenopus Oocytes is Independent of Cl− Conductance

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na⁺ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl⁻ into the cell and is negligible when Cl⁻ flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na⁺ current (I _amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I _amil by up to 80%, demonstrating that ENaC is inhibited when Cl⁻ is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I _amil downregulation and the CFTR-mediated plasma membrane Cl⁻ conductance, suggesting a direct correlation. However, I _amil downregulation was not affected when Cl⁻ flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl⁻ reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I _amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca²⁺-sensitive photoprotein aequorin showed that Ca²⁺ is not involved in I _amil downregulation by CFTR, although Ca²⁺ injection into the cytoplasm did inhibit I _amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca²⁺ and is independent of the direction and magnitude of Cl⁻ transport across the plasma membrane. Received: 15 December 1998/Revised: 5 March 1999     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Neurotrophic control of the transmembrane Cl− pump in mammalian muscle fibers

Resting membrane potential of both innervated and denervated rat diaphragm muscle fibers was investigated when the concentration of potential-producing ions was changed in the external fluid and following treatment with furosemide. It was found that equilibrium potential ( ) equalled resting potential level in innervated muscle for Cl^–, but shifts to more positive values compared with resting membrane potential following section of the nerve. Furosemide retards development of post-denervation depolarization of the muscle membrane. It is deduced that trophic influences originating from the motor nerve activates the furosemide-sensitive Cl^– influx system, leading to raised intracellular concentration of Cl^–, a shift in (E_Cl) and depolarization of the muscle membrane.S. V. Kurashov Medical Institute, Minsitry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 766–771, November–December, 1987.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

Summary The potential dependence of unidirectional³⁶Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl^– or Br^– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl^– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV _a , and intracellular Cl^– activity,a _Cl ^c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI _sc=27.0±2.0 A/cm², with NaCl-Ringer's bathing both surfaces,V _a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda _Cl ^c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV _a anda _Cl ^a were found to be positively correlated withI _sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV _a anda _Cl ^c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl^– conductance (G _Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG _Cl was deactivated,V _a was –70.1±5.0 mV (n=15, eight preparations) anda _Cl ^c was 40.0±3.8mm. The fractional apical membrane resistance (fR _a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV _a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera _Cl ^c norfR _a changed significantly within a 2 to 5-min period during whichG _Cl increased by 1.19±0.10 mS/cm². WhenV was stepped back to 40 mV,V _a instantaneously shifted to –67.8±3.9 mV whilea _Cl ^c andfR _a remained constant during deactivation ofG _Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D _MRC) cells was estimated and correlated with the Cl current (I _Cl though the fully activated (V=–100mV) Cl^– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR _a anda _Cl ^a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.     

Inhibition by Cl− channel blockers of the volume-activated,diffusional mechanism of inositol transport in primary astrocytes in culture

[³H]Inositol accumulated by rat brain cultured astrocytes is released when cells swell by exposure to solutions of decreased osmolarity. Activation of inositol efflux was proportional to reductions in osmolarity from 30%–70%. This volume-activated inositol efflux pathway was increased (27%) in Na⁺-free medium and decreased (22%) in Cl^–-free medium. It was independent of extracellular Ca²⁺ and was reduced (30%) in the presence of the intracellular chelator [1,2-bis(o-aminophenoxy) ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester] (BAPTA-AM). The inositol efflux pathway was markedly inhibited by Cl^– channel blockers, which at maximal inhibitory concentrations decreased inositol efflux by 70%–83%. The potency range of the drugs was: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)>1–9, dideoxyforskolin>4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)>niflumic acid. Inositol efflux was strongly inhibited by the SH blocker N-ethyl maleimide (NEM), which at 100 M abolished inositol release. Inositol efflux can be reversed by increasing its extracellular concentration, suggesting that the efflux is mediated by a diffusional pathway whose direction is given by the concentration gradient. The inhibition of volume-associated fluxes of inositol by Cl^– channel blockers supports the suggestion of an anion channel as the common pathway for inorganic and organic osmolytes in cultured astrocytes.