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To study vacuolar chloride (Cl(-)) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl(-) uptake into isolated tonoplast vesicles was measured using the Cl(-)-sensitive fluorescent dye lucigenin (N,N'-dimethyl-9,9'-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl(-), with a Stern-Volmer constant of 173 m(-1) in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl(-)-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K(m) of 17.2 mm and a V(max) of 4.8 mm min(-1). Vacuolar Cl(-) transport was not affected by sulfate, malate, or nitrate. In the presence of 250 microm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl(-) transport was actually significantly increased by 24%. To determine absolute fluxes of Cl(-) using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 x 10(7) m(-1). After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl(-) transport of 31 nmol m(-2) sec(-1) was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. [ABSTRACT FROM AUTHOR]

To study C1 conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was greater than 100 omega.cm2, and short circuit current (Isc = 2-20 microA/cm2), representing active secretion of Cl, increased greater than threefold with addition of 10 microM isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the C1-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5 mM, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360 +/- 5 nm, emission greater than 410 nm). SPQ leakage from the cells was less than 10% in 60 min at 37 degrees C. Intracellular calibration of SPQ fluorescence vs. [C1] (0-90 nM) was carried out using high-K buffers containing the ionophores nigericin (5 microM) and tributyltin (10 microM); SPQ fluorescence was quenched with a Stern-Volmer constant of 13 M-1. Intracellular Cl activity was 43 +/- 4 mM. Cl flux was measured in response to addition and removal of 114 mM Cl from the bathing solution. Addition of 10 microM isoproterenol increased Cl efflux from 0.10 to 0.27 mM/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1 mM). In the absence of isoproterenol, removal of external Na or addition of 0.5 mM furosemide, reduced Cl influx by greater than fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5 mM barium diminished Cl influx by greater than twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the real-time measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism. [ABSTRACT FROM AUTHOR]

The effect of the sulfhydryl reagent parachloromercuribenzoic acid (PCMB) on chloride transport was examined in rabbit renal brush-border membrane vesicles (BBMV). PCMB had no effect on the chloride conductive pathway. In the presence of an inside-alkaline pH gradient and a K/valinomycin voltage clamp, the addition of PCMB stimulatedCl uptake and induced a threefold overshoot above the equilibrium value, indicating Cl/OH exchange. The effect of PCMB was reversed by dithiothreitol. Cl/OH exchange was not observed in the absence of PCMB. PCMB-activated Cl/OH exchange persisted even when the membrane potential was made inside-negative relative to the controls, thus, demonstrating that PCMB's effect onCl uptake under pH-gradient conditions is not mediated by parallel Cl and H conductive pathways. PCMB-activated Cl/OH exchange was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) with IC values of 290 and 80 μ m, respectively. These results demonstrate that modification of sulfhydryl groups by PCMB activates Cl/OH exchange in BBMV. [ABSTRACT FROM AUTHOR]

The effects of bathing solution HCO/CO concentrations on baseline cell membrane voltages and resistances were measured in Necturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO/CO Ringer's solutions (2.4 mm HCO/air or 1 mm HEPES/air) or a high HCO/CO Ringer's (10 mm HCO/1% CO). The principal finding of these studies was that the apical membrane fractional resistance ( fR) was higher in tissues bathed in the 10 mm HCO/CO Ringer's, averaging 0.87±0.06, whereas fR averaged 0.63±0.07 and 0.48±0.08 in 2.4 mm HCO and 1 mm HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical ( R) and basolateral membrane ( R) resistances in tissues bathed in 10 mm HCO/1% CO Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO/CO Ringer's, the higher value of fR was found to be due to both an increase in R and a decrease in R. The higher values of fR and lower values of R confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K and Cl substitutions. The results of these studies suggest that an electrodiffusive Cl transport mechanism exists at the basolateral membrane of tissues bathed in a 10 mm HCO/1% CO Ringer's, which can explain in part the fall in R. The above observations are discussed in terms of a stimulatory effect of solution [HCO/PCO on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes. [ABSTRACT FROM AUTHOR]

The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent Cl conductance. Volume increase and conductance activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at -100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5 mM, but was independent of whether Ki was 2.4, 5, or 10 mM. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

Sodium (22Na) transport was studied in a basolateral membrane vesicle preparation from rabbit parotid. Sodium uptake was markedly dependent on the presence of both K+ and Cl- in the extravesicular medium, being reduced 5 times when K+ was replaced by a nonphysiologic cation and 10 times when Cl was replaced by a nonphysiologic anion. Sodium uptake was stimulated by gradients of either K+ or Cl (relative to nongradient conditions) and could be driven against a sodium concentration gradient by a KCl gradient. No effect of membrane potentials on KCl-dependent sodium flux could be detected, indicating that this is an electroneutral process. A KCl-dependent component of sodium flux could also be demonstrated under equilibrium exchange conditions, indicating a direct effect of K+ and Cl on the sodium transport pathway. KCl-dependent sodium uptake exhibited a hyperbolic dependence on sodium concentration consistent with the existence of a single-transport system with Km = 3.2 mM at 80 mM KCl and 23 degrees C. Furosemide inhibited this transporter with K0.5 = 2 X 10(4) M (23 degrees C). When sodium uptake was measured as a function of potassium and chloride concentrations a hyperbolic dependence on [K] (Hill coefficient = 0.87 +/- 0.09) and a sigmoidal dependence on [Cl] (Hill coefficient = 1.31 +/- 0.07) were observed, consistent with a Na/K/Cl stoichiometry of 1:1:2. Taken together these data provide strong evidence for the electroneutral coupling of sodium and KCl movements in this preparation and strongly support the hypothesis that a Na+/K+/Cl cotransport system thought to be associated with transepithelial chloride and water movements in many exocrine glands is present in the parotid acinar basolateral membrane. [ABSTRACT FROM AUTHOR]

The leukotriene, LTC4, exerts a stimulatory effect on chloride transport in the frog cornea. In the work described here, the mechanism of action of LTC4 to stimulate chloride transport was studied. In corneas pretreated with indomethacin, the effect of LTC4 was abolished, suggesting the involvement of cyclo-oxygenase products in the response. Incubation of corneas with LTC4 resulted in a significant stimulation in PGE2 synthesis, as determined by TLC-autoradiography and radioimmunoassay. In addition, LTC4 was found to stimulate cAMP synthesis in the cornea, and this stimulation was blocked with indomethacin. PGE2 was previously shown by us to be the dominant cyclo-oxygenase product formed in the frog cornea, and is capable of stimulating cAMP and chloride transport. We suggest that LTC4 stimulation of chloride transport is mediated via activation of the cyclo-oxygenase pathway, resulting in enhanced PGE2 synthesis. Elevated PGE2 levels induce cAMP synthesis, and ultimately, the stimulation of chloride transport. Further, the activation of cyclo-oxygenase was found to be dependent on phospholipase A2 activity. This was shown by the inhibition of the LTC4 effect in the presence of quinacrine. Similarly, inhibition of the LTC4 effect in the presence of trifluoperazine suggests that cyclo-oxygenase activation by LTC4 may be mediated via calmodulin. We have previously demonstrated that the frog cornea has the biosynthetic capacity to produce LTC4. Therefore LTC4 may function as an endogenous regulator of chloride transport in this tissue. [ABSTRACT FROM AUTHOR]

The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog, Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin. [ABSTRACT FROM AUTHOR]

Cell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium. [ABSTRACT FROM AUTHOR]

The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential difference Vsc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25 mM or exposure to 2 mM BaCl2 (K+ channel blocker). With Ba2+ the depolarization of the Vsc by 25 mM K+ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistance Ro remained unchanged, the relative basolateral membrane resistance Ri, which includes the lateral intercellular space, increased at the most by less than twofold after Ba2+. These effects in conjunction with the depolarization of the Vsc by 62 mV after increasing the stromal-side K+ from 2.5 to 100 mM in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a = Ro/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K+ conductance. This ratio decreased significantly after exposure to either 25 mM K+ or Ba2+. The decline of Ro/Ri with 25 mM K+ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25 mM K+, but rather perhaps a larger decrease of Ro than Ri. Also an increase of lateral space resistance may offset the effect of decreasing Ri with 25 mM K+. In contrast, Ro/Ri did transiently increase during voltage clamping of the apical membrane potential difference Vo and exposure to 25 mM K+ on the stromal side.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]

In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands of Squalus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170 mM KCl gradient was initially about five-fold higher than in the presence of a 170 mM KNO3 gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4 mM bumetanide and 40% by 0.8 mM N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170 mM NaCl gradient than in the presence of a 170 mM NaNO3 gradient. The effect of chloride was completely abolished by 0.4 mM bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound 3[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparent KD of 3.5 x 10(-6) M and a number of sites n of 104 pmol/mg protein. At 0.8 microM, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600 mM or when 600 1 mM bumetanide or furosemide was added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland. [ABSTRACT FROM AUTHOR]

Sodium transport mechanisms were investigated in plasma membrane vesicles prepared from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. The uptake of 22Na into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of chloride; it was reduced when chloride was replaced by nitrate, gluconate or sulfate. The stimulation of sodium uptake by chloride was seen in the presence of a chloride gradient directed into the vesicle and when the vesicles were equilibrated with NaCl, KCl plus valinomycin so that no chemical or electrical gradients existed across the vesicle (tracer exchange experiments). Furosemide decreased sodium uptake into the vesicles in a dose-dependent manner only in the presence of chloride, with a Ki of around 5 X 10(-6) M. Amiloride, at 2 mM, had no effect on the chloride-dependent sodium uptake. Similarly, potassium removal had no effect on the chloride-dependent sodium uptake and furosemide was an effective inhibitor of sodium uptake in a potassium-free medium. The results show the presence of a furosemide-sensitive sodium-chloride cotransport system in the plasma membranes of the medullary TALH. There is no evidence for a Na+/H+ exchange mechanism or a Na+ -K+ -Cl- cotransport system. The sodium-chloride cotransport system would effect the uphill transport of chloride against its electrochemical potential gradient at the luminal membrane of the cell. [ABSTRACT FROM AUTHOR]

We demonstrate that arachidonic acid (AA) stimulation of chloride transport across frog cornea is mediated via two independent pathways: (1) stimulation of prostaglandins and cAMP synthesis, and (2) a direct physical change in the membrane produced by substitution of different phospholipid acyl chains. AA is well known as a precursor in the synthesis of prostaglandins, which have been shown to stimulate cAMP synthesis and chloride transport in frog cornea. We show that frog cornea can convert exogenous AA to PGE2, but that in the presence of 10(-5) M indomethacin both the conversion to PGE2 and stimulation of cAMP are completely blocked. However, with indomethacin the action of AA to stimulate chloride transport (as measured by SCC) remains, but peak height of the response is reduced to 57% of that found when AA alone is given. Similarly, we show that propranolol completely blocks cAMP stimulation, but stimulation of SCC is reduced to 45% of the original response. Therefore, cAMP appears to be responsible for roughly half of the observed stimulation in SCC. By gas chromatographic analysis we show that significant quantities of AA can rapidly substitute into membrane phospholipids of corneal epithelium and L929 cells following the addition of AA to the medium. Modification of membrane phospholipid structure can affect membrane viscosity, membrane-bound enzyme activity, and the distribution and lateral mobility of integral proteins. It seems likely that such alterations in the properties of the membrane may modulate the rate of chloride transport, and this may constitute the second mechanism. Upon addition of AA, both mechanisms appear to stimulate chloride transport simultaneously, and are apparently additive. We show that prolonged exposure to AA results in a large incorporation of AA into phospholipid and consequently, a perturbation in the ratio of unsaturated to saturated fatty acids. We also find evidence of a compensatory cellular mechanism that alters the ratio of endogenously synthesized fatty acids and tends to reduce the membrane-perturbing effect of AA.U [ABSTRACT FROM AUTHOR]

The chloride conductance of the basolateral cell membrane of the Necturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl-. When extracellular Cl- activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl- conductance of the paracellular shunt but a low Cl- conductance of the cell membranes. Reduction of Cl- in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl- perfusion was abolished by exposure to HCO-3-free solutions and SITS treatment. In control conditions, intracellular Cl- activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl- in only the basolateral solution caused a decreased in intracellular Cl-. From an estimate of the net Cl- flux across the basolateral cell membrane and the electrochemical driving force, a Cl- conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl- conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl- by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl- transport across the basolateral cell membrane of the Necturus proximal tubule cell. [ABSTRACT FROM AUTHOR]

We examined the direct effects of isoproterenol (ISO) and L-norepinephrine (NE) on electrolyte transport in isolated rabbit cortical collecting tubules (CCT) perfused in vitro. The addition of either ISO (10(-6) M) or NE (10(-6) M) to the bath decreased transepithelial potential difference (PD), on average by 51 and 25%, respectively. These effects of ISO and NE were abolished by prior addition of the beta-adrenergic blocker, L-propranolol. ISO (10(-5) M) had no effect from lumen. Also, osmotic water permeability was not influenced by ISO. Ouabain and ISO had additive effects on PD. Elimination of chloride from both perfusate and bath, or addition of acetazolamide, abolished the effect of ISO on PD. Although isotopic sodium flux for lumen to bath was not influenced by ISO, chemical net chloride absorption increased from 1.1 +/- 0.4 to 2.7 +/- 0.6 peq . cm-1 . sec-1 (n = 8, p less than 0.005). In conclusion, both ISO and NE and capable of decreasing PD in rabbit CCT perfused in vitro. This effect is mediated by beta-adrenergic receptors and is accompanied by the increase in net chloride absorption. Although the mechanism responsible for this decrease in PD with ISO in unclear, active chloride absorption, active hydrogen secretion, or membrane chloride permeability changes may account for the effects of ISO. [ABSTRACT FROM AUTHOR]

Sodium and chloride influxes across the nonshort-circuited isolated skin of Rana esculenta were measured at widely varying external ionic concentrations. The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed. At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na/H; Cl/HCO) functioning at low external concentrations. At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components. The physiological significance of these results is discussed. [ABSTRACT FROM AUTHOR]

The ionic permeability of the basolateral membrane of Necturus gallbladder epithelium was studied with intracellular microelectrode techniques. After removal of most of the subepithelial tissue (to reduce unstirred layer thickness), impalements were performed from the serosal side, and ionic substitutions were made in the serosal solution while a microelectrode was kept in a cell. Thus, it was possible to obtain continuous (and reversible) records of transepithelial and cell membrane potentials and to measure intermittently the transepithelial resistance and the ratio of cell membrane resistances. From these data and the mean value of the equivalent resistance of the cell membranes in parallel (obtained from cable analysis in a different group of tissues), absolute cell membrane and shunt resistances and equivalent electromotive forces (emf's) were calculated. From the changes of basolateral membrane emf ( E) produced by the substitutions, the conductance ( G) and permeability ( P) of the membrane for K, Cl and Na were estimated. Potassium-for-sodium substitutions produced large reductions of both cell membrane potentials, of E, and of the resistance of the basolateral membrane ( R), indicating high G and P. Chloride substitution with isethionate or sulfate resulted in smaller changes of cell membrane potentials and E and in no significant change of R, indicating small but measurable values of G and P. Sodium substitutions with N-methyl- d-glucamine (NMDG) resulted in cell potential changes entirely attributable to the biionic potential produced in the shunt pathway ( P> P), and in no significant changes of P or E, indicating that G and P are undetectable. The question of the mechanism of Cl transport across the basolateral membrane was addressed by comparing the mean rate of transepithelial Cl transport ( J) and the predicted passive Cl flux across the basolateral membrane (from the membrane Cl conductance, potential, and Cl equilibrium potential). The conclusion is that only a very small fraction of the Cl flux across the basolateral membrane can be electrodiffusional. Since the paracellular Cl conductance is also too low to account for J, these results suggest the presence of a neutral mechanism of Cl extrusion from the cells. This could be a NaCl pump, a downhill KCl transport mechanism, or a Cl−HCO exchange mechanism. [ABSTRACT FROM AUTHOR]

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues. [ABSTRACT FROM AUTHOR]