Blocker Of Cl Research Articles

Excitatory Action of GABA in the Terminal Nerve Gonadotropin-Releasing Hormone Neurons

The terminal nerve (TN)-gonadotropin-releasing hormone (GnRH) neurons have been suggested to function as a neuromodulatory system that regulates the motivational and arousal state of the animal and have served as a model system for the study of GnRH neuron physiology. To investigate the synaptic control of the TN-GnRH neurons, we analyzed electrophysiologically the effect of GABA on the TN-GnRH neurons. GABA generally hyperpolarizes most of the neurons in the adult brain by activating GABA(A) receptors while the activation of GABA(A) receptors depolarizes some specific neurons in the mature brain. Here we examined the GABA(A) receptor-mediated responses in the TN-GnRH neurons of adult teleost fish, the dwarf gourami, by means of gramicidin-perforated patch-clamp and cell-attached patch-clamp recordings. The reversal potential for the currents through GABA(A) receptors under the voltage clamp was depolarized relative to the resting membrane potential. GABA(A) receptor activation depolarized TN-GnRH neurons under the current clamp and had excitatory effect on their electrical activity, whereas the stronger GABA(A) receptor activation had bidirectional effect (excitatory-inhibitory). This excitatory effect is suggested to arise from high [Cl(-)](i) and was shown to be suppressed by bumetanide, the blocker of Cl(-)-accumulating sodium-potassium-2-chloride co-transporter (NKCC). The present results demonstrate that GABA(A) receptor activation induces excitation in TN-GnRH neurons, which may facilitate their neuromodulatory functions by increasing their spontaneous firing frequencies. The excitatory actions of GABA in the adult brain have recently been attracting much attention, and the easily accessible large TN-GnRH neurons should be a nice model system to analyze their physiological functions.

Blockers of K+/Cl- transporter/channels diminish proliferation of osteoblastic cells

In the preset report, we studied if blockers of the K+/Cl- cotransporter, and Cl- and K+ channels modify the proliferation of MC3T3-E1 osteoblastic cells. A blocker of the K+/Cl- cotransporter, DIOA (100 and 500 microM), diminished the cell proliferation. NPPB (100 and 300 microM, a blocker of Cl- channel) and quinine (100 and 500 microM, a blocker of K+ channel) also suppressed the proliferation of MC3T3-E1 cells. These blockers of the K+/Cl- cotransporter/channels (DIOA, NPPB and quinine) would increase the intracellular Cl- concentration ([Cl-]i). On the other hand, our previous study (Biochem. Biophys. Res. Commun. 361, 1038-1043, 2007) reported that a decrease in [Cl-]i diminished the proliferation of MC3T3-E1 osteoblastic cells. Taken together, the observations indicate that both increases and decreases in [Cl-]i diminish the proliferation of MC3T3-E1 osteoblastic cells.

E-Ring Isoprostanes Stimulate a Cl− Conductance in Airway Epithelium via Prostaglandin E2-Selective Prostanoid Receptors

Isoprostanes comprise a class of membrane lipid metabolites produced during oxidative stress, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. They are widely recognized to evoke a variety of biological responses in airway and pulmonary vascular smooth muscle, lymphatics, and innervation. However, their effects on airway epithelium are largely unstudied. We examined the electrophysiological responses evoked by several different isoprostane species in bovine airway epithelium using the Ussing chamber technique. The E-ring isoprostanes 15-E(1t)-IsoP and 15-E(2t)-IsoP evoked a substantial increase in short-circuit current (I(SC)), whereas four different F-ring isomers were ineffective. 15-E(2t)-IsoP-evoked I(SC) was mimicked by the prostaglandin E(2)-selective prostanoid receptor (EP)-agonist prostaglandin E(2) but not by agonists of EP(1)/EP(3)-, FP-, or TP receptors (sulprostone, fluprostenol, and U46619, respectively). This response was significantly reduced by the EP(4)-receptor blocker GW627386 but not by blockers of other prostanoid receptors (ICI 192,605 [TP-selective], SC19220 [EP(1)-selective], AH6809 [DP/EP(1)/EP(2)-selective], and AL8810 [FP-selective]). 15-E(2t)-IsoP-evoked I(SC) was reduced by blockers of Cl(-) channels (niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid), of Na(+)/K(+)/2Cl(-) co-transport (furosemide and bumetanide), of adenylate cyclase (MDL 12,330A), or of guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by blockers of Na(+) conductances (amiloride). We conclude that 15-E(2t)-IsoP activates a transepithelial Cl(-) conductance in bovine airway epithelium through an EP(4) receptor coupled to adenylate cyclase and soluble guanylate cyclase.

Swelling-Activated Cl−Channels Support Cl−Secretion by Bovine Ciliary Epithelium

To determine whether swelling-activated Cl- currents (ICl,swell) observed in isolated nonpigmented ciliary epithelial (NPE) cells contribute to Cl- secretion across the ciliary epithelium. Ion transport across intact bovine ciliary epithelium was monitored electrically. Native isolated bovine NPE cells were harvested enzymatically. Cell volume changes were measured by calcein-fluorescence quenching. Bilateral reduction in osmolality transiently increased short-circuit current (Isc), averaging 60% to 70%. Bilateral pretreatment with 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker, reduced Isc stimulation by approximately 60%, suggesting that transcellular ICl,swell largely mediates the increased current. The hypotonically-triggered Isc stimulation was also inhibited by phloretin, a blocker of swelling-activated Cl- channels and by flufenamic acid, a blocker of Cl- and nonselective cation channels. Cyclamate substitution for bath Cl- reduced the baseline Isc and the increase in hypotonically-triggered Isc. In that case, addition of either NPPB or flufenamic acid did not produce further inhibition. The transepithelial responses were correlated with regulatory volume responses of freshly harvested NPE cells. Hypotonicity elicited a regulatory volume decrease (RVD) over a period comparable to that of the hypotonicity-triggered increase in Isc. The RVD was also inhibited by Cl--channel blockers and by Cl- substitution. ICl,swell of NPE cells is functionally expressed in intact ciliary epithelium and is oriented to subserve aqueous humor formation. NPE cell volume can be measured with calcein-fluorescence quenching. ICl,swell may be stimulated by increased stromal fluid uptake and delivery to the NPE cells, facilitating Cl- secretion and increasing fluid release into the posterior chamber.

Quercetin and NPPB‐induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transpithelial Cl− secretion due to activation of the Na+/K+/2Cl− cotransporter (NKCC), and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC) and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity and alpha-ENaC mRNA expression was diminished by 24 h-pretreatment with quercetin (an activator of NKCC) or NPPB (a blocker of Cl− channel), while 24 h-pretreatment with bumetanide (a blocker of NKCC) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effects on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of NKCC, and that modification of activity of Cl− transporter/channel participating in the transepithelial Cl− secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport). Supported by Grants-in-Aid from JSPS (17390057, 17590191).

Spontaneous oscillation and mechanically induced calcium waves in chondrocytes

The characteristics of spontaneous calcium (Ca(2+)) oscillation and mechanically induced Ca(2+) waves in articular chondrocytes were studied. In some, but not all, chondrocytes in sliced cartilage and primary cultures, we observed spontaneous oscillation of intracellular Ca(2+) that never spread to adjacent cells. In contrast, a mechanical stimulus to a single cell by touching with a glass rod induced an increase of intracellular Ca(2+) that spread to neighboring cells in a wave-like manner, even though there was no physical contact between the cells. This indicated the release of some paracrine factor from the mechanically stimulated cells. Application of ultrasonic vibration also induced an oscillation of intracellular Ca(2+). The application of a uridine 5'-triphosphate (UTP), UTP, induced a transient increase in intracellular Ca(2+) and the release of adenosine 5'-triphosphate (ATP) in cultured chondrocytes. A P2 receptor antagonist (suramin) and blockers of Cl(-) channels, niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), reduced the UTP-induced ATP release. The results indicated that Cl(-) channels were involved in the extracellular release of ATP following mechanical or P2Y receptor stimulation. Thus, ATP stimulation of P2Y receptors elicits an increase in intracellular Ca(2+), triggering further release of ATP from adjacent cells, thereby expanding the Ca(2+) wave in chondrocytes.

Two-Photon Imaging Reveals Somatodendritic Chloride Gradient in Retinal ON-Type Bipolar Cells Expressing the Biosensor Clomeleon

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.

Cyclic nucleotides induce long-term augmentation of glutamate-activated chloride current in molluscan neurons.

1. Literature data indicate that serotonin induces the long-term potentiation of glutamate (Glu) response in molluscan neurons. The aim of present work was to elucidate whether cyclic nucleotides can cause the same effect. 2. Experiments were carried out on isolated neurons of the edible snail (Helix pomatia) using a two-microelectrode voltage-clamp method. 3. In the majority of the cells examined, the application of Glu elicited a Cl- -current. The reversal potential (Er) of this current lied between -35 and -55 mV in different cells. 4. Picrotoxin, a blocker of Cl- -channels, suppressed this current equally on both sides of Er. Furosemide, an antagonist of both Cl- -channels and the Na+/K+/Cl- -cotransporter, had a dual effect on Glu-response: decrease in conductance, and shift of Er to negative potentials. 5. A short-term (2 min) cell treatment with 8-Br-cAMP or 8-Br-cGMP caused long-term (up to 30 min) change in Glu-response. At a holding potential of -60 mV, which was close to the resting level, an increase in Glu-activated inward current was observed. This potentiation seems to be related to the right shift of Er of Glu-activated Cl- -current rather than to the increase in conductance of Cl- -channels. The blocking effect of picrotoxin rested after 8-Br-cAMP treatment. 6. The change in the Cl- -homeostasis as a possible mechanism for the observed effect of cyclic nucleotides is discussed.

Quercetin and NPPB-induced diminution of aldosterone action on Na + absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl − secretion over 30-fold due to activation of the Na +/K +/2Cl − cotransporter and stimulated the transepithelial Na + absorption, activity of epithelial Na + channel (ENaC), and α-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na + absorption, ENaC activity, and α-ENaC mRNA expression was diminished by 24 h-pretreatment with quercetin (an activator of Na +/K +/2Cl − cotransporter participating in Cl − entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl − channel participating in Cl − release from the cytosolic space), while 24 h-pretreatment with bumetanide (a blocker of Na +/K +/2Cl − cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na + absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na + absorption, activity of ENaC or α-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na + transport), aldosterone has an inhibitory action on ENaC (transepithelial Na + transport) via activation of the Na +/K +/2Cl − cotransporter, and that modification of activity of Cl − transporter/channel participating in the transepithelial Cl − secretion influences the aldosterone-stimulated ENaC (transepithelial Na + transport).

Properties of Cl--Stimulated Mg2+-ATPase Sensitive to Ligands of Inhibitory Receptors in Brain Plasma Membranes of the Bream Abramis brama

Properties of Cl−-stimulated Mg2+-ATPase in the brain plasma membranes of the bream Abramis brama L. were studied; this enzyme is composed of “basal” Mg2+-ATPase activity that can be stimulated by 40–80% by Cl− ions (Cl−-ATPase). These anions stimulate the basal Mg2+-ATPase starting with 8 mM concentration, their maximal effect being observed at a concentration of 30–100 mM. The Cl−-ATPase activity was found at a low molarity of HEPES-Tris buffer ( 30 mM). The “basal” Mg2+-ATPase activity was detected in the whole studied pH range (5.5–9.0), with maximum at pH 7.2–7.8 values, whereas optimum to reveal Cl−-ATPase was at high and low H+ concentrations (pH 6.0 and 8.5, respectively). At physiological pH values (7.2–7.5) the Cl−-ATPase activity was not revealed, but was detected after preincubation of the enzyme with 10 µM GABA. The “basal” Mg2+-ATPase, like Cl−-ATPase, hydrolyzed ATP with a maximal rate, while CTP, ITP, and ADP only slightly, and did not hydrolyze GTP and AMP. The Cl−-ATPase activity decreased in the presence of divalent cations in the following order: Mg2+ > Co2+ > Mn2+ = Cd2+ > Al3+ = Cu2+, and it was not found in the presence of Ca2+ and Zn2+. Anions of halogen series activated the “basal” Mg2+-ATPase in the descending order: Cl− > Br− > J− > F−. Among other monovalent anions, HCO3− activated the enzyme, NO3− practically had no effect, and SCN− inhibited its activity. Blockers of Cl− transport (ethacrinic acid, furosemide, and SITS) and GABA-receptor ligands (pentobarbital, diazepam, and picrotoxin) suppressed the enzyme activity. Out of SH-reagents, PCMB inhibited the enzyme, while NEM did not affect it. The H+-ATPase blocker oligomycin inhibited the enzyme, while the blocker of Na+,K+-ATPase ouabain and the blocker of Ca2+,Mg2+-ATPase ruthenium red had no effect. The properties of the Cl−-stimulated Mg2+-ATPase of fish brain are discussed in comparison with those of the rat brain Cl−-ATPase. The conclusion is made that the bream brain enzyme differs markedly from Cl−-ATPase (the ATP-dependent Cl−-pump) of mammalian brain.

Modulation of intracellular Cl − homeostasis by lectin-stimulation in Jurkat T lymphocytes

We investigated changes in intracellular Cl − concentration ([Cl −] i) during lectin-induced activation and proliferation in human Jurkat T lymphocytes. [Cl −] i was measured using Cl − fluorescence dye ( N-(6-methoyquinolyl) acetoxy-acetyl-ester, MQAE) methods. Lectins, phytohemagglutinin and concanavalin A, dose-dependently increased [Cl −] i and triggered intracellular Cl − oscillation in human Jurkat T lymphocytes. However, some mitochondria metabolism inhibitors, such as m-chlorocarbonylcyanide phenylhydrazone (CCP) and 2,4-dinitrophenol, increased [Cl −] i without triggering any Cl − oscillation. Furthermore, both lectins and metabolism inhibitors-induced elevation in [Cl −] i were blocked by removal of extracellular Cl − from perfusion solution or by application of anthracene-9-carboxylate, a blocker of Cl − channels. Since an extracellular Cl −-free condition and application of 9-AC also inhibited PHA-induced proliferation, we suggested that elevation of [Cl −] i via activation of Cl − channels and increase in incidence of Cl − oscillation would play an important role in modulation of Jurkat T cell activation and proliferation.

Gossypol induces chloride secretion in rat proximal colon

The effect of gossypol on electrolyte transport was investigated in rat colon mounted in Ussing chambers. The addition of gossypol to the mucosal or serosal side led to an increase in mucus secretion, which we did not quantify. Mucosally or serosally added gossypol also induced a rise in short circuit current ( I sc) and tissue conductance ( G t). Part of the mucosally added gossypol seemed to be bound to the mucus because the effects on I sc and G t were smaller when gossypol was added to the mucosal side. Serosally added gossypol had an effect on I sc at a concentration of 10 μmol l −1. Mucus secretion was reduced in low Ca 2+ buffer. The increase in I sc was diminished by blockers of Cl − channels, K + channels, of the Na +/K + ATPase and of the Na +/K +/2 Cl − cotransporter. Measurements of unidirectional ion fluxes showed that gossypol added to the mucosal side had no effect on net Na + transport, but increased Cl − secretion. The effect of mucosally added gossypol was significantly reduced by the use of low Cl − buffers and abolished when the buffer was additionally depleted of HCO 3 −. Calmodulin antagonists inhibited the effect on secretion. These findings indicate that gossypol induces chloride secretion via a calmodulin-dependent mechanism. High concentrations of gossypol induced a strong increase in G t that could be blocked by W7, a blocker of calmodulin-dependent myosin light chain kinase. This indicates that the rise in G t is not due to an unspecific toxic effect, but instead, to specific opening of tight junctions.

Do seminiferous tubules secrete a fluid rich in KHCO3?

Do seminiferous tubules secrete a fluid rich in KHCO3?

Selective inhibition of Cl(-) conductance in toad skin by blockers of Cl(-) channels and transporters.

We compared the effects exerted by two classes of Cl(-) transport inhibitors on a Cl(-)-selective, passive anion transport route across the skin of Bufo viridis, the conductance (G(Cl)) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G(Cl) with IC(50) of 6 +/- 1, 54 +/- 12, and 607 +/- 125 microM, respectively; they had no effect on the cAMP-induced G(Cl). The voltage for half-maximal activation of G(Cl) (V(50)) increased compared with controls, but effects on the maximal G(Cl) at more positive clamp potentials were small. Cl(-) channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G(Cl) with IC(50) of 8.3 +/- 1.2, 10.5 +/- 0.6, 16.5 +/- 3.4, and 36.5 +/- 11.4 microM, respectively, and also inhibited the cAMP-induced G(Cl), albeit with slightly larger IC(50). V(50) was not significantly changed compared with controls; the maximal G(Cl) was strongly reduced. We conclude that the pathway for Cl(-) is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.

CFTR and lysozyme secretion in human airway epithelial cells

Lysozyme is secreted in large quantities in human airways (10-20 mg/day), where it helps to defend against bacterial and fungal infection. Lysozyme expression is restricted to the serous cells of the submucosal glands, which also express high levels of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. It is often assumed that mucus secretion in human airways is coupled to anion secretion through CFTR Cl(-) channels located in the apical membrane. Therefore, a defect in CFTR function could cause abnormal mucus secretion leading to persistent bacterial infection and inflammation of the airways. In this study we measured simultaneous secretion of lysozyme and Cl(-) from human airway epithelial serous cells. Secretion of lysozyme was measured by a turbidimetric assay that relies on the ability of lysozyme to disrupt the wall of the bacterium Micrococcus lysodeikticus, thus causing a fall in the optical density of the sample. Secretion of Cl(-) was measured as short-circuit current in a modified Ussing chamber. Activation of Cl(-) secretion by stimulation of cAMP- or Ca(2+)-dependent pathways caused comparable increases in lysozyme secretion. Similarly, blockers of Cl(-) secretion, such as diphenylamine-2-carboxylate (DPC), also reduced lysozyme secretion. However, while treatment of airway submucosal gland cells with antisense oligonucleotides directed against CFTR reduced Cl(-) secretion, it had no significant effect on the total amount of lysozyme secretion. These results suggest a role for functional CFTR in regulation of lysozyme secretion in human airways.

Optical imaging reveals cation--Cl(-) cotransporter-mediated transient rapid decrease in intracellular Cl(-) concentration induced by oxygen--glucose deprivation in rat neocortical slices.

In brain slices from young (postnatal day (P) 10--15) rat somatosensory cortex, real-time neuronal intracellular Cl(-) concentration ([Cl(-)](i)) recordings were made by an optical technique measuring 6-methoxy-N-ethlquinolinium iodide (MEQ) fluorescence. Oxygen--glucose deprivation (in vitro model of ischemia) induced a long-lasting [Cl(-)](i) increase preceded by a rapid, transient [Cl(-)](i) decrease that could not be inhibited by blockers of Cl(-) pumps, Cl(-) channels, or Cl(-) antiporters, but was sensitive to cation-Cl(-) cotransporter inhibitors (bumetanide and furosemide). Use of low external Na(+) or high external K(+) revealed that the Na(+),K(+)-2Cl(-) cotransporter was inhibited by bumetanide and furosemide, whereas the K(+)-Cl(-) cotransporter was preferentially inhibited by furosemide under our experimental conditions. With a reduced inward driving force for Na(+) (reducing Na(+),K(+)-2Cl(-) cotransport), the transient [Cl(-)](i) decrease was only rarely induced by oxygen-glucose deprivation. In contrast, with a reduced outward driving force for K(+) (reducing K(+)-Cl(-) cotransport), the transient [Cl(-)](i) decrease still occurred. These results suggest that the transient [Cl(-)](i) decrease was primarily mediated by a rapid inhibition of the inwardly directed Na(+),K(+)-2Cl(-) cotransporter. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that the isoform involved is NKCC1. We hypothesize that the initial rapid Cl(-) efflux might effectively delay the irreversible Cl(-) influx that mediates neuronal injury.

Molecular characterisation of pancreatic zymogen granule ion channel and regulator proteins involved in exocytosis.

In pancreatic acinar cells Ca(2+)-dependent secretagogues promote the fusion of zymogen granules (ZG) with the apical plasma membrane (PM) and exocytosis of digestive enzymes. In addition to exocytotic fusion complexes between SNARE proteins in the ZG membrane (ZGM) and the apical PM, enzyme secretion elicited by Ca(2+)-dependent secretagogues requires cytosolic Cl and K+ and is inhibited by blockers of Cl- and K+-channels. We have identified a Cl-conductance activated by ATP, and a K+-conductance (with properties similar to ATP-sensitive K+-channels), regulated by the granule matrix protein Zg-16p in the ZGM. Both conductances are inversely regulated by a 65-kD mdr1 gene product. We have also identified a novel Ca(2+)-activated anion conductance in ZGM, the Ca(2+)-sensitivity of which increases 50-fold when Cl is replaced by 1. This conductance is blocked by micromolar H2-DIDS or DTT, reminiscent of a family of epithelial Ca(2+)-activated Cl -channels (CaCC). Expression of a CaCC in exocrine pancreas has been confirmed by RT-PCR analysis, and by immunoblotting and immunogold labeling of ZG membranes. These data suggest that ion channels in the ZGM are essential elements in pancreatic exocytosis.

Effect of inflammatory mediators on the electrophysiology of the human oviduct.

The effects of histamine and other inflammatory mediators on the electrophysiology and intracellular free calcium ([Ca(2+)](i)) of human oviductal epithelial cells, grown as a polarized layer in primary culture, were studied. Transepithelial potential difference (PD) and short-circuit current (I(scc)) were recorded using a modified Ussing chamber. Resistance (R) was calculated from the measurements of PD and I(scc). Basally applied histamine produced transient increases in PD and I(scc) with a small decrease in R. The histamine effect was reduced by triprolidine (H(1) receptor antagonist) but was unaffected by H(2) (ranitidine) or H(3) (thioperamide) receptor antagonists. Blockers of Na(+), K(+), or Na(+)/K(+)/2Cl(-) channels did not affect histamine action. Blockers of Cl(-)/HCO(3)(-) channels or Ca(2+) channels reduced the histamine effect. Platelet activating factor (PAF), applied apically, increased PD and I(scc). Histamine produced a transient increase in fluorescence of Fura 2-AM dye, indicating an increase in [Ca(2+)](i). Triprolidine pretreatment inhibited histamine-stimulated [Ca(2+)](i) increase. Cimetidine, (H(2) receptor antagonist), ranitidine, or thioperamide reduced the histamine effect. Histamine increased contractions of both circular and longitudinal smooth muscles in oviduct segments, an effect that was antagonized by triprolidine or thioperamide but not by ranitidine. Histamine's action on Ca(2+) and Cl(-) movements may adversely affect oviductal fluid production and decrease fertility. PAF's effects on Cl(-) movements may be important for normal embryo transport.

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.

H+ ATPase and Cl- interaction in regulation of MDCK cell pH.

MDCK cells display several acid-base transport systems found in intercalated cells, such as Na+-H+ exchange, H+-K+ ATPase and Cl-/HCO-3 exchange. In this work we studied the functional activity of a vacuolar H+-ATPase in MDCK cells and its chloride dependence. We measured intracellular pH (pHi) in monolayers grown on glass cover slips utilizing the pH sensitive probe BCECF. To analyze the functional activity of the H+ transporters we observed the intracellular alkalinization in response to an acute acid load due to a 20 mm NH+4 pulse, and calculated the initial rate of pHi recovery (dpHi/dt). The cells have a basal pHi of 7.17 +/- 0.01 (n = 23) and control dpHi/dt of 0.121 +/- 0.006 (n = 23) pHi units/min. This pHi recovery rate is markedly decreased when Na+ was removed, to 0.069 +/- 0.004 (n = 16). It was further reduced to 0.042 +/- 0. 005 (n = 12) when concanamycin 4.6 x 10(-8) m (a specific inhibitor of the vacuolar H+-ATPase) was added to the zero Na+ solution. When using a solution with zero Na+, low K+ (0.5 mm) plus concanamycin, pHi recovery fell again, significantly, to 0.023 +/- 0.006 (n = 14) as expected in the presence of a H+-K+-ATPase. This result was confirmed by the use of 5 x 10(-5) m Schering 28080. The Na+ independent pHi recovery was significantly reduced from 0.069 +/- 0.004 to 0.042 +/- 0.004 (n = 12) when NPPB 10(-5) m (a specific blocker of Cl- channels in renal tubules) was utilized. When the cells were preincubated in 0 Cl-/normal Na+ solution for 8 min. before the ammonium pulse, the pHi recovery fell from 0.069 +/- 0.004 to 0.041 +/- 0.007 (n = 12) in a Na+ and Cl- free solution. From these results we conclude that: (i) MDCK cells have two Na+-independent mechanisms of pHi recovery, a concanamycin sensitive H+-ATPase and a K+ dependent, Schering 28080 sensitive H+-K+ ATPase; and, (ii) pHi recovery in Na+-free medium depends on the presence of a chloride current which can be blocked by NPPB and impaired by preincubation in Cl--free medium. This finding supports a role for chloride in the function of the H+ ATPase, which might be electrical shunting or a biochemical interaction.