Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

Ca2+-activated Cl- channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion.

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC-1) and CFTR-deficient (CFPAC-1; DeltaF508 mutation) cell lines and measured HCO3- extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC-1 cells, cAMP causes parallel activation of Cl- channels and of HCO3- extrusion by DIDS-sensitive and Na+-independent Cl-/HCO3- exchange, both effects being inhibited by Cl- channel blockers NPPB and glibenclamide. 2) In CFPAC-1 cells, cAMP fails to stimulate Cl-/HCO3- exchange and Cl- channels, except after promoting surface expression of DeltaF508-CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 micromol/l or stimulating purinergic receptors with ATP (10 and 100 micromol/l) leads to parallel activation of Cl- channels and HCO3- extrusion. 3) K+ channel function is required for coupling cAMP- and Ca2+-dependent Cl- channel activation to effective stimulation of Cl-/HCO3- exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl-/HCO3- exchange is directly correlated to activation of apical membrane Cl- channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP-activated Cl- channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+-dependent Cl- channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.     

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and physiological evidence for functional gamma-aminobutyric acid (GABA)-C receptors in growth hormone-secreting cells

The neurotransmitter gamma-aminobutyric acid (GABA), released by hypothalamic neurons as well as by growth hormone- (GH) and adrenocorticotropin-producing cells, is a regulator of pituitary endocrine functions. Different classes of GABA receptors may be involved. In this study, we report that GH cells, isolated by laser microdissection from rat pituitary slices, possess the GABA-C receptor subunit rho2. We also demonstrate that in the GH adenoma cell line, GH3, GABA-C receptor subunits are not only expressed but also form functional channels. GABA-induced Cl- currents were recorded using the whole cell patch clamp technique; these currents were insensitive to bicuculline (a GABA-A antagonist) but could be induced by the GABA-C agonist cis-4-aminocrotonic acid. In contrast to typical GABA-C mediated currents in neurons, they quickly desensitized. Ca2+i recordings were also performed on GH3 cells. The application of either GABA or cis-4-aminocrotonic acid led to Ca2+ transients of similar amplitude, indicating that the activation of GABA-C receptors in GH3 cells may cause membrane depolarization, opening of voltage-gated Ca2+ channels, and a subsequent Ca2+ influx. Our results point at a role for GABA in pituitary GH cells and disclose an additional pathway to the one known via GABA-B receptors.     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

Membrane currents and pharmacology of retinal bipolar cells: a comparative study on goldfish and mouse.

We obtained solitary bipolar cells using enzymatic (papain) dissociation of the goldfish and mouse (C57BL/6J, adult) retinae and measured the membrane currents of these cells by whole-cell patch clamp. Bipolar cells of these two species showed two main differences. A. Ca current 1. In the mouse, depolarization evoked a transient Ca current that had maximal amplitude at about -30 mV. 2. The Ca conductance was activated by voltage steps to potentials greater than -60 mV and inactivated fully at potentials greater than -20 mV. 3. The mouse Ca current was insensitive to Cd2+ or dihydropyridine. 4. Contrary to mouse, goldfish bipolar cells had a sustained Ca current, which was activated over a more positive potential range (greater than -30 mV), blocked by either 50 microM Cd2+ or 10 microM nifedipine, and markedly augmented by 10 microM Bay K8644. 5. The transient character of the Ca current in mouse bipolar cells may help to shape phasic responses of ganglion cells, while in goldfish the sustained nature of Ca current may contribute to shape tonic responses of ganglion cells. B. Pharmacology 1. We examined the effects of the inhibitory transmitters, glycine and GABA, on bipolar cells. 2. GABA produced strong inhibitory effects on bipolar cells of both goldfish and mouse. 3. The highest GABA sensitivity was found at the bipolar cell axon terminal, the site of reciprocal connection with amacrine cells. 4. GABA increased the Cl conductance. 5. Unlike GABA, glycine was effective only on the mouse bipolar cells. Axon terminals showed the highest glycine sensitivity. 6. Glycine-induced currents were also carried by Cl ions. 7. Since ECl in intact cells is assumed to be -55 mV, both GABA and glycine are thought to generate hyperpolarizing responses in cells maintained at their resting potential (ca. -45 mV). 8. The present study suggests that inhibition from amacrine cells to bipolar cells, found in both species, is mediated by different transmitters.     

Direct presynaptic regulation of GABA/glycine release by kainate receptors in the dorsal horn: an ionotropic mechanism.

In the spinal cord dorsal horn, excitatory sensory fibers terminate adjacent to interneuron terminals. Here, we show that kainate (KA) receptor activation triggered action potential-independent release of GABA and glycine from dorsal horn interneurons. This release was transient, because KA receptors desensitized, and it required Na+ entry and Ca2+ channel activation. KA modulated evoked inhibitory transmission in a dose-dependent, biphasic manner, with suppression being more prominent. In recordings from isolated neuron pairs, this suppression required GABA(B) receptor activation, suggesting that KA-triggered GABA release activated presynaptic GABA(B) autoreceptors. Finally, glutamate released from sensory fibers caused a KA and GABA(B) receptor-dependent suppression of inhibitory transmission in spinal slices. Thus, we show how presynaptic KA receptors are linked to changes in GABA/glycine release and highlight a novel role for these receptors in regulating sensory transmission.     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.     

Taurine-evoked chloride current and its potentiation by intracellular Ca2+ in immature rat hippocampal CA1 neurons

Taurine is one of the most abundant free amino acids in the immature mammalian central nervous system. In the present study, whole-cell patch-clamp recordings were made to examine taurine-evoked currents ( I(Tau)) in acutely dissociated immature rat hippocampal CA1 neurons. Taurine at low concentrations (/=3 mM) activated both glycine and GABA(A) receptors. Moreover, elevation of intracellular Ca(2+) via non-NMDA receptor activation enhanced I(Tau) reversibly. The results indicate that taurine may act as a native ligand of glycine receptors and modulate neurotransmissions in the immature hippocampus, and under certain conditions it can also activate GABA(A) receptors. The potentiation of I(Tau) by intracellular Ca(2+) may contribute to the protection effect of taurine under some cell-damaging conditions.     

Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus

Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.     

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9- anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.     

Changes in smooth muscle cell pH during hypoxic pulmonary vasoconstriction: a possible role for ion transporters

Hypoxic pulmonary vasoconstriction (HPV) occurs in smooth muscle cells (SMC) from small pulmonary arteries (SPA) and is accompanied by increases in free cytoplasmic calcium ([Ca2+]i) and cytoplasmic pH (pHi). SMC from large pulmonary arteries (LPA) relax during hypoxia, and [Ca2+]i and pHi decrease. Increases in pHi and [Ca2+]i in cat SPA SMC during hypoxia and the augmentation of hypoxic pulmonary vasoconstriction by alkalosis seen in isolated arteries and lungs suggest that cellular mechanisms, which regulate inward and outward movement of Ca2+ and H+, may participate in the generation of HPV. SMC transport systems that regulate pHi include the Na+ - H+ transporter which regulates intracellular Na+ and H+ and aids in recovery from acid loads, and the Na+ -dependent and Na+ -independent Cl-/HCO3- transporters which regulate intracellular chloride. The Na+ -dependent Cl-/HCO3- transporter also aids in recovery from acidosis in the presence of CO2 and HCO3-. The Na+ -independent Cl-/HCO3- transporter aids in recovery from cellular alkalosis. The Na+ - H+ transporter was present in SMC from SPA and LPA of the cat, but it seemed to have little if any role in regulating pHi in the presence of CO2 and HCO3-. Inhibiting the Cl-/HCO3- transporters reversed the normal direction of pHi change during hypoxia, suggesting a role for these transporters in the hypoxic response. Future studies to determine the interaction between pHi, [Ca2+]i and HPV should ascertain whether pHi and [Ca2+]i changes are linked and how they may interact to promote or inhibit SMC contraction.     

Cadmium ions modulate GABA induced currents in molluscan neurons

The effect of Cd2+, as one of the most widespread toxic environmental pollutants, was studied on gamma-aminobutyric acid (GABA) evoked responses of identified neurons in the central nervous system of the pond snail, LYmnaea stagnalis L. (Gastropoda). In the experiments, the modulation of the action of GABA both on neuronal activity (current clamp recording) and on the a GABA activated membrane Cl- current (voltage clamp studies) has been shown. It was found that: 1. GABA could evoked three different various types of response in GABA sensitive neurons: i) hyperpolarization with strong inhibition of ongoing spike activity, ii) short depolarization with an increase of spike the activity, iii) biphasic respone with a short excitation followed by a more prolonged long inhibition. 2. In low-Cl- solution the inhibitory action of GABA was reduced or eliminated, but the excitatory one was not or only moderately affected. 3. CdCl2 inhibited the GABA evoked hyperpolarization, but left intact or only slightly reduced the excitation evoked by GABA. 4. The inward Cl- current evoked by GABA at a -75 mV holding potential was slightly augmented in the presence of I micromol/l Cd2+, but was reduced or blocked at higher cadmium concentrations. The effect of Cd2+ was concentration and time dependent. 5. Parallel with reducing the GABA evoked current, cadmium increased both the time to peak and the half inactivation time of the current. 6. CdCl2 alone, in 50 micromol/l concentration, induced a 1-2 nA inward current. The blocking effect of cadmium on GABA activated inhibitory processes can be an important component of the neuro-toxic effects of this heavy metal ion.     

Intracellular pH regulation by HCO3-/Cl- exchange is activated during early mouse zygote development

We report here that at least one major pHi-regulatory mechanism, the HCO3-/Cl- exchanger, is quiescent in unfertilized mouse eggs but becomes fully activated during early development following fertilization. Zygotes (8-12 h postfertilization) exhibited a marked intracellular alkalinization upon external Cl- removal, which is indicative of active HCO3-/Cl- exchangers, in contrast to the very small response observed in eggs. In addition, efflux of Cl- from eggs upon external Cl- removal was much slower than that from zygotes, indicating additional pathways for Cl- to cross the plasma membrane in zygotes. Furthermore, while zygotes quickly recovered from an induced alkalosis, eggs exhibited only a slow, incomplete recovery. Following in vitro fertilization (IVF), increased HCO3-/Cl- exchanger activity was first detectable about 4 h postfertilization and reached the maximal level after about 8 h. The upregulation of HCO3-/Cl- exchanger activity after fertilization appeared to occur by activation of existing, inactive exchangers rather than by synthesis or transport of new exchangers, as the increase in activity following IVF was unaffected by inhibition of protein synthesis or by disruption of the Golgi apparatus or the cytoskeleton. This activation may depend on the Ca2+ transients which follow fertilization, as suppression of these transients, using the Ca2+ chelator BAPTA, reduced subsequent upregulation of HCO3-/Cl- exchanger activity by about 50%. Activation of pHi-regulatory systems may be a widespread feature of the earliest period of embryonic development, not restricted to species such as marine invertebrates as previously believed.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].