External Cl Research Articles

Dual mode of N-methyl-D-aspartate-induced neuronal death in hippocampal slice cultures in relation to N-methyl-D-aspartate receptor properties.

Hippocampal slices prepared from neonatal rats were cultured for several weeks, and excitotoxicity induced in CA1 pyramidal neurons by N-methyl-D-aspartate was evaluated at different culture stages. CA1 neurons cultured for one week exhibited cell death predominantly within 1-3 h after a 15-min N-methyl-D-aspartate insult (early death), whereas those cultured for three weeks showed cell death mainly a few hours to 24 h after the insult (delayed death). CA1 neurons cultured for two weeks were in a transitional state, expressing only weak early and delayed forms of cell death in response to N-methyl-D-aspartate. The N-methyl-D-aspartate-induced early cell death observed in the one-week group depended on external Cl- but did not require external Ca2+; rather, early cell death was enhanced in Ca2+-free solution. This early cell death was accompanied by cell swelling, but cell swelling alone produced by osmotic changes failed to induce cell death. There was no evidence that CA1 neurons in the one-week group were more responsive to N-methyl-D-aspartate than those in the two other groups. Delayed cell death examined in the three-week group depended on external Ca2+ but was independent of Cl-. The density of N-methyl-D-aspartate-induced whole-cell currents recorded from CA1 neurons in Mg2+-free solution remained unchanged during three weeks of culture. However, the N-methyl-D-aspartate receptor channel in the three-week group was more resistant to Mg2+ block than that in the one- or two-week group. The incidence of N-methyl-D-aspartate-induced delayed cell death was higher in the three-week group than in the two-week group in normal solution but not in Mg2+-free solution. Thus, Mg2+ block-resistant properties of the N-methyl-D-aspartate receptor channel acquired during prolonged culture may account for the high incidence of N-methyl-D-aspartate-induced delayed cell death in the three-week group. However, the N-methyl-D-aspartate receptor subunits expressed in the CA1 subfield did not show any feature specific to the three-week group. These results show that two mechanistically distinct modes of N-methyl-D-aspartate-induced neuronal death are manifested differentially at different culture stages, depending on the intrinsic neuron properties (i.e. early cell death) and on the properties or the responsiveness of the N-methyl-D-aspartate receptor (i.e. delayed cell death).

The Ca2+-inactivated Cl- channel at work: selectivity, blocker kinetics and transport visualization.

Removal of extracellular divalent cations activated a Cl- channel in the plasma membrane of Xenopus laevis oocytes. This so-called Ca2+-inactivated Cl- channel (CaIC) was present in every oocyte and was investigated using two-electrode whole-cell voltage clamp and single-channel patch-clamp techniques. Beside other Cl- channel inhibitors, anthracene-9-carboxylic acid (9-AC) and 3' azido-3'deoxythymidine (AZT), a nucleoside analog we used as an antiviral drug, blocked at least partly the CalC-mediated currents. Using the Cl--sensitive dye 6-methoxy-N-(sulfopropyl)quinolinium (SPQ) we could visualize the transport of Cl- from the oocyte cytoplasm to the surrounding medium after activation of the CaIC by Ca2+ removal. In the absence of external Cl- and Ca2+, the emission intensity of SPQ declined continuously, indicating a quenching of fluorescence by the efflux of Cl- in the millimolar range. In the presence of external Ca2+, no emission changes could be observed during the same time period. Chelating external Ca2+ in absence of Cl- immediately activated Ca2+-inactivated Cl- channels leading to subsequent emission decrease of SPQ. Investigations on the selectivity of the CaIC revealed only poor discrimination between different anions. With single-channel measurements, we found an anion selectivity sequence I- > Br- > Cl- >> gluconate as it is also typical for maxi Cl- channels. Contrary to the majority of all other transport systems of the Xenopus oocyte, which show reduced activity due to membrane depolarization or endocytotic removal of the transport protein from the plasma membrane during oocyte maturation, the CaIC remained active in maturated oocytes. Single-channel measurements on maturated oocytes, also known as eggs, showed the presence of Ca2+-inactivated Cl- channels. However, this egg CaIC revealed an altered sensitivity to external Ca2+ concentrations. All these data confirm and extend our previous observations on the CaIC and give clear evidence that this channel is peculiar among all Cl- channels described up to now.

The asymmetry of chloride transport at 38 degrees C in human red blood cell membranes.

Band 3-mediated Cl- exchange in human red blood cells and resealed ghosts was measured at 38 degrees C by the continuous flow tube method. When external Cl- concentration, C(o), is varied with constant internal Cl- concentration, C(i), the flux fits a simple Michaelis-Menten saturation curve (MM fit), with K1/2o = 3.8 +/- 0.4 mM. When the Cl- concentration is varied simultaneously at both sides of the membrane in resealed ghosts (C(i) = C(o) = C(i = o)), the flux rises toward a flat maximum between 200 and 450 mM Cl-, and then decreases at very high C(i = o). An MM fit to the data with C(i = o) < 500 mM gives K1/2s of 106 +/- 13 mM; fits including modifier site inhibition (MS fit) give an over threefold higher K1/2s. Despite this uncertainty, the intrinsic asymmetry of unloaded transport sites, A (defined as E(o)/E(i) with C(i) = C(o), where E(i) is the fraction of unloaded inward-facing sites and E(o) is the fraction of unloaded outward-facing sites), calculated from K1/2s and K1/2o, ranges only from 0.046 to 0.107. A new method, which uses the initial slope of a plot of Cl- flux versus C(i = o), gives A values of 0.023 to 0.038. Flufenamic acid (FA) inhibits Cl- exchange by binding to an external site different from the transport site. At 38 degrees C, FA binds 24-36 times more tightly to E(o) than to E(i). Estimates of A from FA inhibitory potency range from 0.01 to 0.05. All methods, including bicarbonate data from the preceding paper, indicate that at 38 degrees C, like 0 degree C, far more band 3 molecules are in the E(i) than in the E(o) form. The agreement of various methods supports the ping-pong model for anion exchange, and demonstrates that the intrinsic asymmetry is very slightly, if at all, affected by temperature.

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Halothane acts as a partial agonist of the alpha6 beta2 gamma2S GABA(A)receptor.

Whole-cell patch clamp recording was performed on human embryonic kidney 293 cells stably transfected with rat cDNAs for the alpha6, beta2, and gamma2S subunits of the GABA(A) receptor. The volatile anesthetic halothane directly activated a current in the absence of the ligand gamma-aminobutyric acid (GABA). Both the current amplitude and the rate of desensitization increased in a dose-dependent manner with an EC50 of 1.0+/-0.2 mM and a Hill coefficient (nh) of 1.5+/-0.1. The EC50 and nh for GABA to activate the receptor were 1.0+/-0.3 microM and 1.4+/-0.2, respectively. The peak amplitude of the halothane-activated current was about 4% of the maximal GABA response, which was not changed when the concentration of Ca2+ in the external solution was decreased from 2 mM to 0.2 mM. The reversal potential of both halothane- and GABA-activated currents changed with the external Cl- concentration as predicted by the Nernst equation for chloride ions. The halothane- and GABA-activated currents were blocked by both the noncompetitive GABA(A) receptor antagonist picrotoxin and the competitive GABA(A) receptor antagonist bicuculline. Schild plots revealed that the K(i)s for bicuculline to competitively antagonize the currents activated by halothane and GABA are similar (0.69 and 0.72 microM, respectively). These results indicate that halothane activates the alpha6 beta2 gamma2S GABA(A) receptor to induce a current similar to the GABA-induced current.

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel.

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)-liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium.

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K-Cl cotransport. The anion exchange inhibitor 4,4'diisothiocyanato-2,2'disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.

Chloride and fluid secretion by cultured human polycystic kidney cells

Epithelial cells cultured from the renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) secrete fluid via a process stimulated by adenosine 3',5'-cyclic monophosphate (cAMP). We have investigated the hypothesis that fluid secretion by these cells is dependent on cAMP-mediated chloride secretion. Individual cultured ADPKD cells were suspended within a polymerized collagen matrix and stimulated to form cysts. Individual cultured cysts were placed in a chamber on the stage of an inverted microscope equipped with epifluorescent and video analysis attachments. The rate of fluid secretion, cell volume and changes in intracellular Cl- were measured. In the absence of secretagogues, fluid was absorbed from the cyst cavity (-2.36 +/- 0.64 nl/min/cm2 inner surface area). 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) plus 3-isobutyl-1-methlyxanthine (IBMX) induced a rapid reversal in the net movement of fluid to secretion (6.79 +/- 1.28 nl/min/cm2). Bumetanide reversibly reduced fluid secretion to 0.95 +/- 0.60 nl/min/cm2. Cell volume rapidly decreased by 7.5 +/- 0.9% with the initiation of secretion and bumetanide caused an additional loss (4.2 +/- 1.0%). Furosemide had a similar effect on forskolin-induced fluid secretion. Cellular chloride concentration was monitored with the use of the indicator, 6-methoxy-N-ethylquinolinium chloride (MEQ). Removal of Cl- from the bath reduced intracellular [Cl-] (MEQ fluorescence increased by 11.4 +/- 2.3%). In cysts pretreated with furosemide to prevent Cl- entry, the application of forskolin caused a decrease in Cl- concentration (MEQ fluorescence increased by 9.3 +/- 2.6%). Using monolayers of cultured ADPKD cells, grown on permeant supports, we compared the changes in short circuit current (ISC) induced by forskolin in the presence and absence of external Cl-. Forskolin increased ISC (from 8.9 +/- 2.7 to 10.6 +/- 2.7 microA/cm2) in the presence of Cl-, but did not significantly affect ISC in its absence. These data indicate that cultured ADPKD cells can direct fluid transport in either the absorptive or the secretory direction, and that cAMP stimulates secretion and this secretion is accompanied by a net loss of cell solute. Inhibition of secretion by bumetanide or furosemide caused an additional loss of cell solute, including Cl-. The ionic transepithelial current induced by forskolin is dependent on the presence of Cl-. These data support the thesis that chloride secretion drives fluid secretion by cultured ADPKD cells.

PH Changes in Frog Rods upon Manipulation of Putative pH-regulating Transport Mechanisms

Rod intracellular pH (pH i) in the intact frog retina was measured fluorometrically with the dye 2′,7′-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pH i changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na + by K + or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na +/H + exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pH i. (2) Subsequent application of the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl − by gluconate (95 mM) caused a rapid pH i rise both in normal Na + and low-Na + perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na +/H + exchange and bicarbonate transport control rod pH i, modulating the light-sensitive current. Part of the bicarbonate transport is by Na +-independent HCO 3 −/Cl − exchange, but a further Na +-coupled bicarbonate import mechanism is implicated. Copyright © 1996 Published by Elsevier Science Ltd.

Lowering extracellular Na+ concentration causes NMDA receptor-mediated neuronal death in cultured rat hippocampal slices.

Reduction of the transmembrane Na+ gradient is expected to induce neurotoxic glutamate release via reversed uptake. We describe neuronal death induced by lowering extracellular Na+ concentration in cultured rat hippocampal slices. When slices were exposed to 3.6 mM Na+ for 30 min, almost all the neurons in the CA1 region were degenerated within 20-24 h. The marginal concentration of external Na+ for induction of neurotoxicity was 6.6 mM. N-methyl-D-aspartate (NMDA) receptor antagonists, MK-801 at 1-3 microM and (+/-)-CPP at 30 microM, significantly decreased these neurotoxic effects. A non-NMDA receptor antagonist, DNQX at 30-100 microM, had no protective effect against neurotoxicity. Removal of external Ca2+ completely eliminated neuronal death, but replacing external Cl- with SO4(2-) had no protective effect against neurotoxicity. A drastic 40-fold increase in glutamate release was produced by 30 min exposure to 3.6 mM Na+, and this release was partially independent of external Ca2+. These findings suggest that the low-Na(+)-induced neurotoxicity we observed is mediated by excessive release of glutamate via reversed uptake and subsequent Ca(2+)-influx through NMDA receptors. The model reported here may be useful for investigation of the mechanism of neuronal injury mediated by endogenous glutamate.

Lowering extracellular Na + concentration causes NMDA receptor-mediated neuronal death in cultured rat hippocampal slices

Reduction of the transmembrane Na + gradient is expected to induce neurotoxic glutamate release via reversed uptake. We describe neuronal death induced by lowering extracellular Na + concentration in cultured rat hippocampal slices. When slices were exposed to 3.6 mM Na + for 30 min, almost all the neurons in the CA1 region were degenerated within 20–24 h. The marginal concentration of external Na + for induction of neurotoxicity was 6.6 mM. N-methyl- d-aspartate (NMDA) receptor antagonists, MK-801 at 1–3 μM and (±)-CPP at 30 μM, significantly decreased these neurotoxic effects. A non-NMDA receptor antagonist, DNQX at 30–100 μM, had no protective effect against neurotoxicity. Removal of external Ca 2+ completely eliminated neuronal death, but replacing external Cl − with SO 4 2− had no protective effect against neurotoxicity. A drastic 40-fold increase in glutamate release was produced by 30 min exposure to 3.6 mM Na + and this release was partially independent of external Ca 2+. These findings suggest that the low-Na +-induced neurotoxicity we observed is mediated by excessive release of glutamate via reversed uptake and subsequent Ca 2+-influx through NMDA receptors. The model reported here may be useful for investigation of the mechanism of neuronal injury mediated by endogenous glutamate.

The electrical response ofAvena coleoptile cortex to auxins

The electrical response to auxin perfusion by cortex tissue of peeledAvena saliva L. coleoptiles was examined using microelectrode measurements of the membrane potential. Applied at 10 μM, the known auxins indole-3-acetic acid (IAA), α-naphthalene acetic acid (αNAA), 2,4-dichlorophenoxyacetic acid, and indole-3-butyric acid, the two analogues β-naphthalene acetic acid and 2,3-dichlorophenoxyacetic acid, and acetic acid transiently (1–30 min) depolarized the membrane potential; but a subsequent hyperpolarization, possibly resulting from activation of the plasma-membrane H+-ATPase, occurred only with active auxins. The magnitude and duration of the depolarizations induced by the different compounds varied. The αNAA-induced depolarization consisted of two depolarization events. The first reached a maximum within 2–3 min and the second between 6 and 10 min. The magnitude of the first αNAA-induced depolarization varied with external concentration of Cl− suggesting that it represents an efflux of Cl−. A known anion-channel blocker, anthracene-9-carboxylic acid, at 1 μM prevented the Cl−-sensitive depolarization induced by αNAA. The same concentration failed to block αNAA-induced elongation of peeled coleoptile segments, though higher concentrations (20 μM and 100 μM) increasingly inhibited growth. Applied at 10 μM, the naturally occurring auxin, IAA, induced a single depolarization (maximal between 6 and 8 min) which was insensitive to external Cl− concentration. The depolarization induced by 10 μM IAA, as well as that induced by acetic acid and the second depolarization induced by 10 μM αNAA, increased in magnitude when the pH of the perfusate was lowered from 6.0 to 4.5. When IAA was applied at a higher concentration (100 μM) the magnitude of the depolarization increased, lengthened, and also contained an early, Cl−-sensitive, depolarization (maximal between 2 and 4 min). The Cl−-sensitive depolarization induced by αNAA also increased when αNAA was increased from 10 to 100 μM, but, while 10 and 100 μM IAA and 10 μM αNAA induced elongation of peeledAvena coleoptile segments, 100 μM αNAA did not. Our results suggest that: (i) the putative Cl− currents induced by 10 μM αNAA and 100 μM IAA are the result of the opening of a population of Cl−-permeable anion channels, (ii) activation of these channels is not required for the auxin-induced growth ofAvena coleoptiles, (iii) the pH-sensitive depolarization induced by αNAA and IAA is not an auxin-specific response and might result from a weak acid protonophore effect, and (iv) the delayed hyperpolarization is a specific response to auxins inAvena coleoptiles.

External-anion-dependent anionic current in blastoderm cells of early medaka fish embryos.

1. Anionic current was studied by a whole-cell variation of the patch-clamp technique in blastoderm cells dissociated from medaka (Oryzias latipes) embryos at the early blastula stage. The blastoderm cells were mechanically dissociated without using proteolytic enzymes. 2. The anionic current was deactivated by hyperpolarizing steps. The steady-state current-voltage (I-V) relationship of the anion current was accurately represented by the Boltzmann relation with z = 1.01 +/- 0.02 (+/- S.E.M., n = 4) for voltage-dependent activation when internal calcium ions were buffered at 100 nM by BAPTA-Ca2+ buffer. 3. When the internal calcium concentration was reduced to 10 nM, this anionic current became an external-calcium-dependent current and was remarkably decreased by removal of external calcium ions. Furthermore, this anionic current was almost abolished when the internal calcium concentration was greatly reduced by chelating Ca2+ ions with 10 mM BAPTA. 4. This current was not affected by replacing external Na+ with TEA+ or N-methyl-D-glucamine. The reversal potentials shifted by +58.6 mV for a 10-fold decrease in the external Cl-concentration. External Cl-ions were substituted with various anions and respective current-reversal potentials were measured. The order of permeability was I- > Br- > Cl- > F-. Both the outward and inward currents almost disappeared in external Cl(-)-free solutions. 5. The voltage dependency of the anionic current shifted in a positive direction with the reduction in the external Cl- concentration, the potentials at which half the channels were activated (V 1/2) being -59.8 +/- 1.2 mV (n = 4), -40.7 +/- 0.8 mV (n = 4), -14.9 +/- 0.7 mV (n = 4), and -0.7 +/- 0.6 mV (n = 4) for 135, 90, 67, and 45 mM Cl-, respectively. This implies that the inward current, brought by efflux of internal anions, decreases with reductions in the external Cl- concentration. In the presence of external 135 mM Br-, the voltage dependency of the anionic current shifted in the negative direction (V 1/2 = -84.5 +/- 1.0 mV and z = 0.91 +/- 0.02 (n = 3)) in comparison with that in the external Cl- medium. It further shifted in the negative direction in the presence of 135 mM I- (V 1/2 = -93.8 +/- 1.5 mV and z = 0.85 +/- 0.03 (n = 3)). The inward current of the anion channel is considered to be affected by both the type and the concentration of external halide ions. 6. At the beginning of the blastula stage, the blastocoel cavity is formed for the first time. A mechanism is necessary to transport ions to the cavity without losing them to pond water outside the embryos. Since this anion channel is external-anion dependent, the current cannot be activated in the membrane facing the pond water, where concentrations of anions are much lower than those of the intracavernous solution. Only after blastocoel formation are blastoderm cells first exposed to the extracellular fluid with high ionic concentrations. Then, the anion channel would become effective for maintaining the Cl- equilibrium potential.

Prostaglandin E2 inhibits Na-K-2Cl cotransport in medullary thick ascending limb cells

Prostaglandin E2 (PGE2) is known to inhibit transepithelial Cl transport in medullary thick ascending limb (mTAL), but the mechanism of inhibition or the transport pathway affected has not been identified. We undertook this study to examine the effect of PGE2 on Na-K-2Cl cotransport in mouse mTAL cells in culture. In nanomolar concentrations, PGE2 inhibited the Na- and Cl-dependent, bumetanide-sensitive K influx by 45%, and this inhibition was also observed in the presence of 3 mM ouabain. Although PGE2 also inhibited ouabain-sensitive K flux, that inhibition was abolished in the presence of apical nystatin, suggesting that the pump inhibition was secondary to diminished Na entry into the cells. The effect of PGE2 was concentration dependent. Inhibition was observed at a concentration of < 1 nM, and half-maximal effect was observed at 2.5 nM. The effect of PGE2 was not mediated by an action on cytosolic Ca because cytosolic Ca was unchanged after the addition of PGE2. PGE2 reduced the maximal velocity for the cotransporter but had no effect on the affinity of the cotransporter for external Na, K, or Cl. Specific [3H]bumetanide binding was reduced in the presence of PGE2, suggesting that PGE2 affected bumetanide-sensitive K influx by downregulating the number of functioning Na-K-2Cl cotransporters. These results suggest that Na-K-2Cl cotransport in the mTAL cells may be under tonic inhibitory control of PGE2.

Ionic mechanisms of Ca(2+)-dependent electrolyte transport across equine sweat gland epithelium.

1. The ionic mechanism involved in Ca(2+)-stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short-circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/- 40.4 omega cm2. 3. Ca(2+)-mobilizing agents, A23187 (1 microM) or thapsigargin (0.01-1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl- by gluconate significantly reduced the ISC by 63% when stimulated by 0.1 microM thapsigargin. Residual ISC could be abolished (> 99%) by elimination of HCO3- from the bathing solution. 5. Basolateral addition of bumetanide (0.1 mM), ouabain (0.01 mM) and acetazolamide (45 microM) and apical addition of methyl isobutyl amiloride (MIA, 1-100 microM) all had inhibitory effects on the thapsigargin-stimulated ISC to various extents. 6. Substantial current inhibition could be obtained using 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and diphenylamine-2-carboxylate (DPC) in a concentration-dependent manner. 7. The K+ channel blocker barium (5 mM) was effective on both sides of the epithelium with a much larger effect on the basolateral side. 8. The inhibitory effects of acetazolamide, amiloride, MIA, DIDS and DPC on the thapsigargin-stimulated ISC were also observed when a Cl(-)-free solution was used. 9. The results provide evidence for Ca(2+)-stimulated HCO3- as well as Cl- secretion by equine sweat gland epithelium.

CFTR Channels Expressed in CHO Cells Do Not Have Detectable ATP Conductance

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mM Mg middle dot ATP or Tris middle dot ATP solution in the pipette and 140 mM NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (Vm), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as Vm was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mM MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near Vm = -55 mV under these conditions, however the whole cell resistance measured at -100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 Gomega). We conclude that CFTR does not mediate detectable electrodiffusion of ATP.

P-glycoprotein regulates a volume-activated chloride current in bovine non-pigmented ciliary epithelial cells.

1. The whole-cell patch clamp technique was used to investigate the swelling-activated currents in bovine non-pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). The I-V relationship was shifted in the direction expected for a Cl- current when the external Cl- was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), also inhibited this current. 5. The volume-activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time-dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl- current. 8. An antibody (C219) to the MDR1 gene product, P-glycoprotein, caused a functional block of the swelling-activated Cl- current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB-1 demonstrated the presence of P-glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non-pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)-independent, ATP-dependent Cl- current and that the activity of this current is associated with P-glycoprotein. 11. It is suggested that this Cl- current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.

Intracellular dialysis of cyclic nucleotides induces inward currents in turtle vomeronasal receptor neurons

Turtle vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the whole-cell and cell-attached configurations. The mean resting potential was -48, and the response to an injected current step consisted of either a single spike or a train of spikes. An injected current of 3-30 pA was required to depolarize the neuron to spike threshold near -50 mV. Voltage-clamped vomeronasal receptor neurons displayed transient inward currents followed by sustained outward currents in response to depolarizing voltage steps. In cell-attached recordings, 10 microM forskolin added to the bath caused a transient increase of spike rate. Intracellular application of cAMP evoked ann inward current in a dose-dependent manner from the neurons voltage clamped at -70 mV; 0.1 mM cAMP was sufficient to elicit an inward current in the neurons. The magnitude of the response to cAMP reached a plateau at 1 mM with an average peak amplitude of 176 +/- 34 pA. Intracellular application of 1 mM cGMP also evoked an inward current with an average peak amplitude of 227 +/- 61 pA. The reversal potentials of the induced components were estimated to be 10 +/- 7 mV for cAMP and -4 +/- 16 mV for cGMP. The reversal potential of the cAMP-induced current in external Cl(-)-free solution was similar to that in normal Ringer's solution, suggesting that Cl- current is not significantly involved in the current. The present results represent the first evidence of cyclic nucleotide-activated conductance in the vomeronasal receptor membranes.

Electron Probe X-Ray Microanalysis of Rabbit Ciliary Epithelium

Rabbit iris–ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl -Ringer's solution, the ciliary epithelial intracellular Na +, K +and Cl -concentrations were estimated to be 15±3, 162±14 and 46±5mmolkg -1intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl -channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl -transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl -by NO 3 -was twice as effective as replacement by gluconate in leaching Cl -out of the intracellular compartment. An unexpected finding was that NO 3 -replacement of internal Cl -substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E 1P form and inhibiting enzyme activity.

Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein.

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.