Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells

PURPOSE: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport. METHODS: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2). RESULTS: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. CONCLUSIONS: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.     

Medullary thick limb basolateral Cl- channels

Chloride transport by the medullary thick ascending limb is a major determinant of urinary concentrating and diluting power and also of tubuloglomerular feedback. In this review, we discuss the electrophysiologic, regulatory, and molecular features of a Cl- channel present in basolateral membranes from the medullary thick ascending limb. Functional and immunohistochemical data support the view that rbCIC-Ka, a CIC Cl- channel cloned from rabbit kidney medulla, may be the Cl- channel which mediates basolateral Cl- efflux.     

Transcellular chloride pathways in ambystoma proximal tubule

The transport mechanisms of Ambystoma proximal tubule that mediate transcellular Cl- absorption linked to Na+ were investigated in isolated perfused tubules using Cl--selective and voltage-recording microelectrodes. In control solutions intracellular activity of Cl- (aiCl) is 11.3 +/- 0.5 mm, the basolateral (V1), apical (V2), and transepithelial (V3) potential differences are -68 +/- 1.2 mV, +62 +/- 1.2 mV and -6.4 +/- 0.3 mV, respectively. When Na+ absorption is decreased by removal of organic substrates from the lumen, aiCl falls by 1.3 +/- 0.3 mm and V2 hyperpolarizes by +11.4 +/- 1.7 mV. Subsequent removal of Na+ from the lumen causes aiCl to fall further by 2.3 +/- 0.4 mm and V2 to hyperpolarize further by +15.3 +/- 2.4 mV. The contribution of transporters and channels to the observed changes of aiCl was examined using ion substitutions and inhibitors. Apical Na/Cl or Na/K/2Cl symport is excluded because bumetanide, furosemide or hydrochlorothiazide have no effect on aiCl. The effects of luminal HCO-3 removal and/or of disulfonic stilbenes argue against the presence of apical Cl-base exchange such as Cl-HCO3 or Cl-OH. The effects of basolateral HCO-3 removal, of basolateral Na+ removal and/or of disulfonic stilbenes are compatible with presence of basolateral Na-independent Cl-base exchange and Na-driven Cl-HCO3 exchange. Several lines of evidence favor conductive Cl- transport across both the apical and basolateral membrane. Addition of the chloride-channel blocker diphenylamine-2-carboxylate to the lumen or bath, increases the aiCl by 2.4 +/- 0.6 mm or 2.9 +/- 1.0 mm respectively. Moreover, following inhibition by DIDS of all anion exchangers in HCO-3-free Ringer, the equilibrium potential for Cl- does not differ from the membrane potential V2. Finally, the logarithmic changes in aiCl in various experimental conditions correlate well with the simultaneous changes in either basolateral or apical membrane potential. These findings strongly support the presence of Cl- channels at the apical and basolateral cell membranes of the proximal tubule.     

A novel, volume-correlated Cl- conductance in marginal cells dissociated from the stria vascularis of gerbils

Using the whole-cell patch-clamp technique, we examined Cl-selective currents manifested by strial marginal cells isolated from the inner ear of gerbils. A large Cl-selective conductance of approximately 18 nS/pF was found from nonswollen cells in isotonic buffer containing 150 mM Cl-. Under a quasi-symmetrical Cl- condition, the "instantaneous' current-voltage relation was close to linear, while the current-voltage relation obtained at the end of command pulses of duration 400 msec showed weak outward rectification. The permeability sequence for anionic currents was as SCN- > Br- approximately = Cl- > F- > NO3- approximately = I- > gluconate-, corresponding to Eisenmann's sequence V. When whole-cell voltage clamped in isotonic bathing solutions, the cells exhibited volume changes that were accounted for by the Cl- currents driven by the imposed electrochemical potential gradients. The volume change was elicited by lowered extracellular Cl- concentration, anion substitution and altered holding potentials. The Cl- conductance varied in parallel with cell volume when challenged by bath anisotonicity. The whole-cell Cl- current was only partially blocked by both 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 0.5 mM) and diphenylamine-2-carboxylic acid (DPC, 1.0 mM), but 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS, 0.5 mM) was without effect. The properties of the present whole-cell Cl- current resembled those of the single Cl- channel previously found in the basolateral membrane of the marginal cell (Takeuchi et al., Hearing Res. 83:89-100, 1995), suggesting that the volume-correlated Cl- conductance could be ascribed predominantly to the basolateral membrane. This Cl- conductance may function not only in cell volume regulation but also for the transport of Cl- and the setting of membrane potential in marginal cells under physiological conditions.     

Membrane function and vascular reactivity

The resting potential and monovalent ions in the marginal cells and scala media were measured before and 20 min after the onset of anoxia using ion-sensitive microelectrodes. The resting potential of the marginal cells decreased from 62.7 to -2.4 mV. The K+ activity decreased from 77.7 to 53.2 mEq/l, while the Na+ activity increased from 2.6 to 24.7 mEq/l. The Cl- activity did not change significantly. In the scala media, the endocochlear potential decreased rapidly from 80.9 to -28.0 mV after the onset of anoxia. The K+ activity decreased from 119.0 to 96.5 mEq/l, the Na+ activity increased from 1.3 to 9.5 mEq/l and that of Cl- decreased from 127.0 to 115.1 mEq/l. The electrochemical gradients determined for each ion based on the ionic changes in the scala media and marginal cells, suggested the existence of an Na/K pump and Na-K-2Cl cotransport at the basolateral membrane of the marginal cells, and a rheogenic K pump and Na-K-2Cl transport at the luminal membrane of the marginal cells. The Na+ and K+ must be recycled at the basolateral membrane and luminal membrane of marginal cells, respectively.     

Ion exchangers mediating NaCl transport in the proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. We identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanism's of uphill Cl- entry across the apical membrane of proximal tubule cells. For steady state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recycling of formate from lumen to cell may occur by H(+)-coupled formate transport and nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. Oxalate recycling from lumen to cell may take place by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. To identify the Na(+)-H+ exchanger (NHE) isoform(s) expressed on the brush border membrane of proximal tubule cells, we developed isoform-specific monoclonal and polyclonal antibodies. We found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the thin and thick limbs of the loop of Henle. NHE3 is also present in a population of subapical intracellular vesicles, suggesting possible regulation by membrane trafficking. The inhibitor sensitivity of Na(+)-H+ exchange in renal brush border vesicles indicates that it is mediated by NHE3 under baseline conditions and during up-regulation by metabolic acidosis. Increased apical membrane Na(+)-H+ exchange activity in response to metabolic acidosis and during renal maturation is associated with increased NHE3 protein expression. Finally, we found that the organic anion-dependent absorption of Cl- is markedly down-regulated in metabolic acidosis in parallel with the up-regulation of brush border membrane Na(+)-H+ exchange. Thus, differential regulation of apical membrane ion exchangers may provide a mechanism to regulate the relative rates of NaHCO3 and NaCl reabsorption.     

Calcium uptake and binding by membrane fractions of human placenta: ATP-dependent calcium accumulation

An ATP-dependent calcium (Ca2+) sequestration activity was demonstrated in membrane vesicles prepared from the human term placenta. Microsomal and brush border membrane fractions accumulated Ca2+ within a vesicular space by a saturable process requiring Mg2+ and ATP. The "uptake" activity was enriched six-fold in a microsomal membrane fraction and was only 1.5-fold enriched in purified brush border membranes compared to the activity present in the filtered homogenate. Mitochondrial inhibitors such as azide and oligomycin did not inhibit Ca2+ uptake in these preparations. The process was temperature dependent and displayed Michaelis-Menten-like kinetics with respect to free Ca2+ concentrations. At 30 degrees C, the Vmax was 1.05 nmole/mg/min; Km = 74 nM for free Ca2+ in the microsomal fraction. Oxalate and phosphate enhanced uptake in both fractions. Ca2+ uptake activity was not associated with Ca2+-stimulated ATPase, alkaline phosphatase, or other brush border markers during cell fractionation. The characteristics of the Ca2+ uptake process contrasted sharply with those of Ca2+-stimulated ATPase, and a Ca2+-stimulated, Mg2+-dependent ATPase activity could not be identified in these membrane vesicle preparations.     

Pheromonal influences

The mechanism of uptake of sparfloxacin, a new quinolone, by intestinal brush-border membrane vesicles was investigated to clarify whether there is a common transport process for new quinolones mediated by the diffusion potential across the intestinal membrane bilayer. Sparfloxacin was taken up pH-dependently by rat intestinal brush-border membrane vesicles, behaviour analogous to that of organic cations including enoxacin and ciprofloxacin. Transient overshooting uptake of this quinolone was observed in the presence of an outward H+ gradient. Momentary dissipation of the H+ gradient by addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone did not affect the uptake of sparfloxacin, and a marked but incomplete reduction in the H+-sensitive overshooting uptake of sparfloxacin was apparent in the voltage-clamped brush-border membrane vesicles. Furthermore, a valinomycin-induced K+-diffusion potential (interior negative) and an inward C1--diffusion potential stimulated the initial uptake of sparfloxacin at pH 5.5. Sparfloxacin uptake was inhibited by tetracaine and imipramine. The inhibitory effect of these cations correlated well with changes in membrane surface charges induced by the presence of tetracaine or imipramine. These results indicate that sparfloxacin transport across the brush-border membrane depends upon the inside-negative ionic diffusion potential, that the H+- or K+-diffusion-potential-dependent uptake of sparfloxacin by intestinal brush-border membrane vesicles is affected by the membrane surface potential and that inhibition of sparfloxacin uptake originates from changes in the membrane surface potential caused by the organic cations.     

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.     

Hepatic uptake of choline in rat liver basolateral and canalicular membrane vesicle preparations

Choline, an endogenous quaternary ammonium ion, is transported into the liver by both saturable and nonsaturable processes. The objective of the present investigation was to determine the driving force(s) for uptake of choline in rat liver basolateral membrane (blLPM) and canalicular membrane (cLPM) vesicles. Choline is transported into an osmotically sensitive intravesicular space in both blLPM and cLPM. Uptake of [3H]choline into both blLPM and cLPM exhibited temperature dependence (0 degree C vs. 37 degrees C). A valinomycin-induced inside-negative K+ diffusion potential significantly stimulated initial uptake of [3H]choline in both vesicles. Choline uptake in blLPM and cLPM was not stimulated in the presence of an inwardly directed sodium gradient or an outwardly directed H+ gradient, and ATP did not enhance choline uptake in cLPM. Choline itself and structurally similar derivatives, such as hemicholinium-3 and succinylcholine, inhibited [3H]choline uptake 11 to 92% (at 10-fold higher concentrations) in blLPM and cLPM. Other cations, including N1-methylnicotinamide, thiamine and d-tubocurarine, and cardioglycosides did not inhibit choline transport in either vesicle preparation. In addition, [3H]choline uptake into both blLPM and cLPM was enhanced when vesicles were preloaded with nonradiolabeled choline (trans-stimulation). Kinetic studies indicated that choline was transported into blLPM by both saturable and passive processes and into cLPM predominantly by a saturable process. These results suggest that the transport of choline is likely mediated by a potential-sensitive conductive pathway in both blLPM and cLPM. The electrogenic pathway in cLPM may play a role in the reabsorption of choline from bile.     

Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.     

Role of chloride transport in the development of the rat blastocyst

Preimplantation mammalian development culminates in the generation of a fluid-filled cavity, the blastocoel, which requires the vectorial transport of ions across the trophectoderm, followed by the movement of water. Experiments were carried out to establish the role of Cl- transport in blastocoel formation in the rat. These included investigations of the effect of Cl- substitution and the Cl- transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, furosemide, and anthracene-9-carboxylic acid, on the development of morulae into blastocysts in culture, and on the rate at which blastocoel fluid is accumulated. In addition, a novel technique was developed in which the Cl(-)-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl) quinolium (SPQ) was loaded into the blastocoel cavity, in order to characterize the pathways of transtrophectodermal Cl- transport. We established that 1) blastocoel formation in the rat requires the external presence of Cl- ions; 2) transport across the trophectoderm appears to be via a transcellular pathway, since rates of blastocyst development, fluid accumulation, and SPQ-monitored Cl- efflux are all reduced in the presence of Cl- transport inhibitors; and 3) Cl- transport occurs via Cl-/HCO3- exchange.     

Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver

We investigated the effects of 17 alpha-ethinylestradiol treatment of rats on various transport functions in isolated basolateral and canalicular liver plasma membrane vesicles. Both membrane subfractions were purified to a similar degree from control and cholestatic livers. Although moderate membrane lipid alterations were predominantly observed in basolateral vesicles, no change in basolateral Na+/K(+)-ATPase activity was found. Furthermore, while Na(+)-dependent taurocholate uptake was decreased by approximately 40% in basolateral vesicles, the maximal velocity of ATP-dependent taurocholate transport was decreased by 63% in canalicular membranes. In contrast, only minimal changes or no changes at all were observed for electrogenic taurocholate transport in "cholestatic" canalicular membranes and total microsomes, respectively. However, canalicular vesicles from cholestatic livers also exhibited marked reductions in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione and in Na(+)-dependent uptake of adenosine, while in the same vesicles HCO3-/SO4- exchange and Na+/glycine cotransport activities were markedly stimulated. These data show that in addition to the previously demonstrated sinusoidal transport abnormalities ethinylestradiol-induced cholestasis is also associated with multiple canalicular membrane transport alterations in rat liver. Hence, functional transport alterations at both polar surface domains might ultimately be responsible for the inhibitory effects of estrogens on the organic anion excretory capacity and on bile formation in rat liver.     

Cyclic AMP-dependent anion secretion in human small and large intestine

Cyclic AMP-dependent Cl- secretion is the major secretion pathway in human intestine. The aim of the present study was to examine mechanisms involved in cAMP-dependent anion secretion in human small and large intestine. Surgical resection specimens from both jejunum and distal colon were studied under short circuited conditions. Addition of the phosphodiesterase inhibitor IBMX induced an increase in the short-circuit current (Isc) equivalent to the net increase in Cl- secretion. The Isc was inhibited by diphenylamine decarboxylate (DPC; Cl- channel blocker), bumetanide (basolateral Na+/K+/2Cl- cotransporter), BaCl2 (basolateral K+ channel) and Cl- free buffer in both segments and indomethacin (cyclo-oxygenase inhibitor) in colon alone. Diphenylamine decarboxylate appears to directly inhibit secretion in jejunum, although its inhibitory effect is possibly mediated by inhibition of cyclo-oxygenase in the colon. A small component of IBMX-stimulated Isc was inhibited by acetazolamide. Cyclic AMP-dependent secretion is largely apical Cl- secretion, although a small component appears to be HCO3. Secretion is dependent on basolateral K+ channels and Na+/K+/2Cl- cotransporters and, in the colon, is inhibited by indomethacin, implying a role for cyclo-oxygenase metabolites. The chloride channel blocker DPC inhibits secretion in both areas. This class of compounds may have potential for treatment of secretory diarrhoea.     

Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro

It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K+. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K+ secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage (Vt) and resistance (Rt) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = Vt/Rt). Further, K+ secretion (JK+,probe) was measured with a K(+)-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, Vt was 8 mV (apical side positive), Rt was 10 ohm-cm2 and Isc was 850 microA/cm2 (N = 27). JK+,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the Na+/Cl-/K+ cotransporter. On the basolateral but not apical side, oubain and bumetanide each caused a decline of Vt and an increase of Rt suggesting the presence of the Na,K-ATPase and the Na+/Cl-/K+ cotransporter in the basolateral membrane. The responses to [Cl-] steps demonstrated a significant Cl- conductance in the basolateral membrane and a small Cl- conductance in the paracellular pathway or the apical membrane. The responses to [Na+] steps demonstrated no significant Na+ conductance in the basolateral membrane and a small Na+ or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K+] steps demonstrated a large K+ conductance in the apical membrane. Apical application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and basolateral elevation of K+ caused an increase in Vt and a decrease in Rt consistent with stimulation of the apical K+ conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117-124].(ABSTRACT TRUNCATED AT 400 WORDS)     

Selected pyrethroid insecticides stimulate glutamate uptake in brain synaptic vesicles

We aimed to ascertain whether pyrethroid insecticides could influence the vesicular transport of the excitatory amino acid glutamate. The incubation of rat cortical synaptic vesicles with resmethrin and permethrin, consistently stimulated both ATP-dependent and -independent uptake of [3H]glutamate, while not evoking depletion of its vesicular content. Both processes were counteracted by valinomycin, a dissipator of the transmembrane potential gradient (deltapsi(sv)). Meanwhile, the vesicular influx of 36Cl- anions was impaired by pyrethroid concentrations which did not affect the ATP-dependent uptake of [14C]methylamine, as a marker for the proton gradient (deltapH). Thus, the stimulation of glutamate transport appeared to involve mainly the deltapsi(sv). A self-attenuating effect of selected pyrethroids on putatively enhanced excitatory transmission in severe intoxication is suggested.     

Effect of frusemide on Cl- channel in rat peritoneal mast cells

Frusemide can be used as an antiasthma drug and appears to inhibit the release (conditioned by activation of Cl- channels) of mast cell proinflammatory mediators. We studied the cause of the effects of frusemide, checking its action on Cl- channels. The patch-clamp technique was used to study single-channel currents, and differences in electrical potential of the cellular membrane of rat peritoneal mast cells were measured. In inside-out configuration, outwardly-rectifying Cl- channels were identified whose conductance was 2.4/1.7 pS at positive and negative voltages. In cell-attached configuration, the open probability (Po) of the channel increased with depolarization or with the presence of cyclic adenosine monophosphate (cAMP) in the incubation medium. Po increased with a rise of cytoplasmic free calcium concentration [Ca2+] and was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and by 4-4'-diisothiocyanatoostilbene-2-2'-disulphonic acid (DIDS). These channels seem to be the main cause of mast cell Cl- conductance. Frusemide (10(-5) and 10(-3) M) did not affect Cl- channel activity when using excised patches. In cell-attached configuration experiments, the presence of frusemide (from 10(-5) to 10(-3) M) in the cell incubation medium, increasingly reduced Po (median inhibitory concentration (IC50) = 4.3 x 10(-7) M). In similar conditions, bumetanide also inhibited Po (IC50 = 5.7 x 10(-3) M). The results of this study suggest that frusemide can inhibit mast cell Cl- channels only via an indirect mechanisms, which probably involves an inhibition of a Na(+)-K(+)-2Cl- symport.     

Cl- absorption across the thick ascending limb is not altered in cystic fibrosis mice. A role for a pseudo-CFTR Cl- channel

The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.     

Hyperpolarization-activated Cl- current elicited by pituitary adenylate cyclase activating polypeptide in Xenopus oocytes

We examined the electrophysiological effect of pituitary adenylate cyclase activating polypeptide (PACAP) in isolated Xenopus laevis oocytes in vitro. In conventional two-electrode voltage clamp experiments, PACAP (1-10 microM) activated an inward rectifier current at membrane potentials more negative than -60 mV without causing any significant change in currents at potentials more positive than -60 mV both in the follicle-enclosed oocyte and in the defolliculated oocyte. This current reversed at -22.5 mV, close to the theoretical value of Cl- equilibrium potential and the reversal potential of this current was shifted positively by reducing [Cl-]o. This current was blocked by Cl- channel blocker SITS and Ba2+. Furthermore, VIP and adenylate cyclase activator forskolin did not elicit the currents. In conclusion, PACAP elicited the hyperpolarization-activated Cl- current in Xenopus laevis oocytes. This current may modulate the membrane potential of the oocyte, thereby affecting the oocyte physiology.     

Volume-activated Cl- channels

1. An increase in cell volume activates, in most mammalian cells, a Cl- current, ICl,vol. This current is involved in a variety of cellular functions, such as the maintenance of a constant cell volume, pH regulation, and control of membrane potential. It might also play a role in the regulation of cell proliferation and in the processes that control transition from proliferation to differentiation. This review focuses on various aspects of this current, including its biophysical characterisation and its functional role for various cell processes. 2. Volume-activated Cl- channels show all outward rectification. Iodide is more permeable than chloride. In some cell types, ICl,vol inactivates at positive potentials. Single channel conductance can be divided mainly into two groups: small (< 5 pS) and medium conductance channels (around 50 pS). 3. The pharmacology and modulation of these channels are reviewed in detail, and suggest the existence of an heterogeneous family of multiple volume-activated Cl- channels. 4. Molecular candidates for this channel (i.e. ClC-2, a member of the ClC-family of voltage-dependent Cl- channels, the mdr-1 encoded P-glycoprotein, the nucleotide-sensitive pICln protein and phospholemman) will be discussed.