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PubMed1989

pH regulation in hepatoma cells: roles for Na-H exchange, Cl-HCO3 exchange, and Na-HCO3 cotransport.

Regulation of intracellular pH (pHi) was studied in Fu5, a rat hepatoma cell line that maintains a variety of differentiated functions. Microspectrofluorimetry of the pH-sensitive dye 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) was used to measure pHi in 10-15 cells growing on cover glasses that were mounted in a flow-through chamber on the stage of a microscope. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solutions, pHi was 7.14, and intrinsic buffer capacity was inversely related to pHi. Amiloride (0.1 mM) caused pHi to decrease by 0.33 pH units in 4 min. Recovery from an acid load (using either NH4 prepulse technique or Na-free solutions) was completely blocked by amiloride. In HCO3-CO2-buffered solutions, pHi was 7.15, and buffer capacity was relatively insensitive to pHi between pHi of 6.6 and 7.2. Amiloride caused pHi to decrease by only 0.09 units. Recovery from an acid load was Na dependent, occurred in Cl-free solutions, and was totally blocked by the combination of amiloride plus 0.5 mM dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2DIDS); recovery occurred when either amiloride or H2DIDS was removed. Removal of external Cl caused a rapid, H2DIDS-blockable alkalinization that was faster in HCO3-CO2 than in HEPES. The apparent Km for Clout for relaxation of Cl-free alkalinization was 4.5 mM. Rate of HCO3 transport during Cl-free treatment increased at alkaline resting pHi. It is concluded that Fu5 cells have two Na-dependent base-loading mechanisms and an acid-loading Cl-HCO3 exchanger. In solutions containing HCO3-CO2, the Na-H exchanger accounts for approximately 40% of recovery from an acid load, and a Na-HCO3 cotransporter accounts for the remainder. Recovery from an alkaline load appears to occur through the activity of the Cl-HCO3 exchanger.

Amiloride↗
PubMed1990

Zinc blocks apical membrane anion exchange in gallbladder epithelium.

The effect of Zn2+ on Cl- transport across the apical membrane of Necturus gallbladder epithelium was studied with intracellular conventional and Cl(-)-selective microelectrodes and measurements of apparent base secretion. Most studies were done on tissues incubated in HEPES-buffered solutions; intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels were elevated by adding to the serosal bathing medium either theophylline or dibutyryl cAMP. Under these conditions, Zn2+ (added to mucosal solution) had no effect on membrane voltages, apparent cell membrane resistance ratio, or rapid depolarization induced by reducing mucosal solution [Cl-]. However, Zn2+ reduced the rate of cell membrane repolarization during exposure to the low-Cl- solution and decreased significantly the rate of fall of intracellular Cl- activity (alpha Cli) elicited by lowering mucosal solution [Cl-]. Both effects were time dependent, became significant after 10 min, and were slowly reversible. In tissues not stimulated by cAMP and incubated in a HCO3-CO2-buffered solution, Zn2+ also reduced the rate of fall of alpha Cli on lowering mucosal solution [Cl-]. Base secretion from cells to mucosal solution was assessed from changes in mucosal pH on stopping superfusion with a poorly buffered (1 mM HEPES) medium in the presence of 1 mM amiloride or a Na(+)-free medium, without cAMP stimulation. Exposure to Zn2+ reduced the alkalinization observed with both protocols. We conclude that Zn2+ has no effect on apical membrane Cl- conductance stimulated by cAMP and inhibits Cl(-)-HCO3- exchange. The slow onset and reversal of the effects suggests slow binding of Zn2+, a covalent modification of the exchanger, or an effect requiring Zn2+ transport to the cell interior.

Amiloride↗
PubMed1993

Cell survival in rabbit gastric glands: effect of extracellular pH, osmolarity, and anoxia.

Although backdiffusion of luminal acid is regarded as a common mechanism of gastric injury, the extracellular pH (pHo) at which cells are irreversibly injured is not well defined. Exclusion of the fluorescent dye propidium iodide was used to estimate cell survival in rabbit gastric glands incubated in buffers of pHo 8.0-2.0. Mean survival (+/- SE) for n = 6 experiments at 2 h in a HEPES buffer of 300 mosM at pHo 8.0, 7.0, 6.0, 4.0, and 2.0 was 80 +/- 3, 91 +/- 2, 90 +/- 2, 71 +/- 2, and 17 +/- 4%, respectively. Survival at acidic pHo was improved in a high KCl buffer: 78 +/- 3 and 38 +/- 7% at pHo 4.0 and 2.0, respectively. Survival in HCO3- buffers was 73 +/- 3, 88 +/- 2, and 92 +/- 3% at pHo 8.0, 7.4, and 6.0. Brief (5 min) exposure to pHo 4.0 followed by reexposure to pHo 7.4 had no effect on acid secretion as estimated by [14C]aminopyrine uptake or cellular viability over 4 h. The influence on cell survival of changes in pHo under conditions of chemical anoxia and in buffers of different osmolarity was investigated. Chemical anoxia was induced using 2.5 mM KCN and 2.0 mM iodoacetic acid (IAA) to inhibit oxidative phosphorylation and anaerobic glycolysis. Survival in glands exposed to KCN and IAA in HEPES buffer was 54 +/- 5, 82 +/- 3, and 87 +/- 2% at pHo 8.0, 7.0, and 6.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopyrine↗
PubMed1993

Effect of ursodeoxycholic acid on intracellular pH regulation in isolated rat bile duct epithelial cells.

To determine if ursodeoxycholic acid (UDCA) induces a HCO3(-)-rich hypercholeresis by stimulating HCO3- secretion from bile duct epithelial (BDE) cells, we studied the effect of UDCA, sodium tauroursodeoxycholate (TUDCA), and cholic acid on intracellular pH (pHi) regulation and HCO3- excretion in BDE cells isolated from normal rat liver. Exposure of BDE cells to UDCA (0.5-1.5 mM) produced a dose-dependent initial acidification [from -0.05 to -0.16 pH units (pHu)], which was lower in Krebs-Ringer bicarbonate than in N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid (HEPES), because of the higher cell-buffering power in the presence of HCO3-. In contrast, TUDCA (1 mM) had no effect on pHi in either media. BDE acidification induced by UDCA (1.5 mM) in KRB was not inhibited by Cl- depletion excluding activation of Cl(-)-HCO3- exchange. Most BDE cells spontaneously recovered their basal pHi during the UDCA infusion (0.5-1 mM) by a secondary activation of the Na(+)-H+ exchanger (amiloride inhibition of pHi recovery; n = 4), and pHi overshot basal levels by 0.1-0.2 pHu after UDCA withdrawal. The activity of Cl(-)-HCO3- exchange (Cl- removal/readmission maneuver) as well as the activities of Na(+)-H+ exchange and Na(+)-HCO3- symport (NH4Cl acid load in HEPES and KRB, respectively) were unaffected by UDCA (0.5 mM) compared with controls. Cholic acid (1.5 mM), which does not produce a hypercholeresis, also acidified BDE cells in KRB media. These studies indicate that UDCA does not stimulate HCO3- excretion from isolated rat BDE cells but modifies pHi in BDE cells as a weak acid.

Ammonium Chloride↗
PubMed1995

Interstitial buffer capacity influences Na+/H+ exchange kinetics and oxyntic cell pHi in intact frog gastric mucosa.

We studied the influence of interstitial buffer capacity and CO2-HCO3- on oxyntic cell intracellular pH (pHi) in intact frog gastric mucosa. Oxyntic cells in stripped gastric mucosa of Rana esculenta were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and pHi was assessed fluorometrically. In the presence of a constant serosal and luminal pH, oxyntic cell pHi was dependent on the serosal but not the luminal concentration of cell-impermeable buffer ions such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid. The stepwise increase in oxyntic cell pHi from 6.74 +/- 0.05 with 1 mM HEPES to 7.23 +/- 0.08 was almost completely inhibited by removal of serosal Na+ and by amiloride and dimethyl amiloride, suggesting that it was largely due to Na+/H+ exchange. Increasing the serosal concentration of a CO2-HCO3- buffer from 1% CO2-4 mM HCO3- to 10% CO2-40 mM HCO3- increased oxyntic cell pHi from 7.03 +/- 0.06 to 7.39 +/- 0.07. This CO2-HCO(3-)-dependent pHi increase was also Na+ and amiloride sensitive, but high HCO3- concentrations increased pHi even in the absence of Na+, K+, or Cl-, and in the presence of omeprazole, bafilomycin A1, or acetazolamide. We suggest that in intact frog gastric mucosa, cellular proton extrusion acidifies the interstitial pH immediately adjacent to the basolateral membrane of the oxyntic cells in the absence of a high interstitial buffer capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

GTP requirement for isoproterenol activation of calcium channels in vascular myocytes.

The effects of activating the beta-adrenoceptor pathway on calcium current (ICa) in rabbit portal vein (PV) were studied in myocytes freshly isolated by collagenase and elastase treatment. ICa was measured at room temperature (20 degrees C) using whole cell, voltage-clamp methods from a holding potential of -60 mV in cells dialyzed with a pipette solution containing (mM) 100 CsCl, 20 tetraethylammonium chloride, 5 NaCl, 5 MgATP, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The cells were superfused with a solution containing (mM) 140 NaCl, 5 KCl, 1 MgCl2, 5 CaCl2, 10 HEPES, and 10 glucose. Only L-type ICa was present in these myocytes, averaging 3.5 +/- 0.3 pA/pF at +10 mV under control conditions. With 0.1 mM guanosine 5'-triphosphate (GTP) added to the pipette solution, 1 microM isoproterenol (Iso) or forskolin (Fsk) uniformly increased ICa: Iso by 45 +/- 5% and Fsk by 88 +/- 11%. This augmentation of ICa was not associated with significant changes in the voltage dependence of activation or inactivation but was associated with a small increase in the rate of inactivation of ICa. Fsk was also associated with an increased rate of ICa activation. The Iso effect was blocked by pretreatment with 1 microM propranolol and reversed by propranolol after Iso exposure. The ICa response to 10 microM Iso or Fsk was smaller than the response to 1 microM, with some cells showing a steady-state reduction in ICa. When the latter occurred, the voltage dependence of availability was shifted to the left by 5 +/- 0.4 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Dissociation between positive inotropic and alkalinizing effects of angiotensin II in feline myocardium.

The present study examines the intracellular pH (pHi) dependence of angiotensin (ANG) II-induced positive inotropic effect in cat papillary muscles contracting isometrically (0.2 Hz, 30 degrees C). Muscles were loaded with the fluorescent dye 2'-7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester for simultaneous measurement of pHi and contractility. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (n = 4), there was a temporal dissociation between the positive inotropic and the alkalinizing effects of ANG II (0.5 microM). The positive inotropic effect of ANG II peaked at 9.7 +/- 0.8 min (240 +/- 57% above control) without significant changes in pHi. The increase in pHi became significant (0.05 +/- 0.01 pH units) only after 16 min of exposure to the drug, when the positive inotropic effect of ANG II was already fading. In HCO3- buffer (n = 7), the ANG II-induced positive inotropic effect occurred without significant pHi changes. In the presence of 5 microM ethyl isopropyl amiloride (EIPA, to specifically inhibit the Na+/H+ exchanger), the alkalinizing effect of ANG II was changed to a significant decrease in pHi, despite which ANG II still increased contractility by 87 +/- 16% (n = 6). The results indicate that in HEPES buffer only a fraction of the ANG II-induced positive inotropic effect can be attributed to a pHi change, whereas in a physiological CO2-HCO3- medium the positive inotropic effect of ANG II is independent of pHi changes. Furthermore, an ANG II-induced increase in myocardial contractility was observed even when ANG II administration elicited a decrease in pHi, as occurred after Na+/H+ exchanger blockade. The results show that in feline myocardium, the increase in contractility evoked by ANG II in a physiological CO2-HCO3- medium is not due to an increase in Ca2+ myofilament sensitivity secondary to an increase in myocardial pHi.

Ammonium Chloride↗
PubMed1998

Effect of ANG II on pHi, [Ca2+]i, and contraction in rabbit ventricular myocytes from infarcted hearts.

In this study we examined Na+/H+ exchange activity, Ca2+ transients, and contractility in rabbit ventricular myocytes isolated from normal and chronically (8-12 wk) infarcted left ventricles. Myocytes from infarcted hearts (post-MI myocytes) were isolated from the peri-infarcted region of the left ventricle. Intracellular pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured with the fluorescent pH indicators seminaphthorhodafluor 1 and fluo 3, respectively, and contractility was assessed from changes in cell shortening during field stimulation. Experiments were performed at extracellular pH 7. 4 in the presence and absence (HEPES buffer) of CO2 and HCO-3. Our findings demonstrate that 1) myocytes after myocardial infarction (post-MI) were significantly larger than normal, 2) post-MI hypertrophy was not accompanied by changes in non-CO2 intracellular buffering power, 3) post-MI hypertrophy did not significantly affect the ability of Na+/H+ exchange to mediate pHi recovery from intracellular acidosis, 4) the stimulatory effect of ANG II (100 nM) on Na+/H+ exchange was significantly reduced in post-MI myocytes, 5) in HCO-3-buffered solutions, ANG II did not significantly stimulate pHi recovery from acidosis in post-MI myocytes, 6) the angiotensin AT1 receptor mediates the stimulatory action of ANG II on Na+/H+ exchange in normal and post-MI myocytes, and 7) the stimulatory effect of ANG II on the Ca2+ transient and contraction was blunted in post-MI myocytes bathed in HEPES-buffered solution. A suppressed ventricular responsiveness to ANG II may be beneficial in the intact myocardium by attenuating ATP consumption and by reducing intracellular Na+ accumulation during ischemia-reperfusion.

Angiotensin II↗
PubMed1997

Furosemide-induced airway relaxation in guinea pigs: relation to Na-K-2Cl cotransporter function.

This study tested the hypothesis that airway relaxation to furosemide is mediated via the Na-K-2Cl cotransporter. If this mechanism exists in airway smooth muscle like in vascular smooth muscle, changes in airway relaxation should be associated with changes in Na-K-2Cl cotransporter function, and both should be substrate dependent. Tracheal rings from newborn guinea pigs were bathed in standard (STD) or varying low Cl- concentration ([Cl-]) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Isometric relaxation to 300 microM furosemide or 10(-8) to 10(-5) M salbutamol was measured. Airway segments were incubated with rubidium-86 (86Rb) in STD or varying low [Cl-] HEPES, with and without 300 microM furosemide or 25 microM salbutamol. Furosemide was unable to reduce 86Rb uptake at 10 mM [Cl-], although relaxation was still observed in 10 mM [Cl-]. Salbutamol did not affect 86Rb uptake. This study demonstrated that there is a furosemide-sensitive Na-K-2Cl cotransporter in newborn guinea pig trachea. However, the effect of furosemide on cotransporter function did not always directly correspond to differences in relaxation, suggesting that the Na-K-2Cl cotransporter may play a major, but not exclusive, role in furosemide-induced airway relaxation.

Animals↗
PubMed1997

Protection of perfused lung from oxidant injury by inhibitors of anion exchange.

Hyperoxic lung injury is enhanced in isolated perfused lungs (IPL) in the presence of L-arginine. Reactive O2 species such as superoxide anion (O2-.) produced during hyperoxia are known to react with nitric oxide to form the strong oxidant species peroxynitrite. The appearance of O2-. in red blood cell membranes in vitro and in buffer-perfused lung preparations can be inhibited by the stilbene compound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) DIDS also inhibits anion exchange across the cell membrane regulated by a family of anion exchange proteins (AE). In this study, we hypothesized that anion exchange inhibitors would prevent lung injury from hyperoxia and L-arginine (O2 + L-Arg) by decreasing O2-. flux into the vascular space of the IPL. We found that both DIDS and a structurally distinct anion transport blocker, dipyridamole, protected the rabbit IPL from pulmonary hypertension and edema produced by O2 + L-Arg. The protective effect was associated with increased nitrite concentrations in the perfusate. Protection also was conferred when sodium bicarbonate in the perfusion buffer was replaced with either sodium thiosulfate or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In lungs perfused with thiosulfate or HEPES-containing buffer, protection from O2 and L-arginine was also associated with diminished detection of reducing activity consistent with O2-. in the vascular space. Western blot analysis of lung protein and immunocytochemical staining of lung sections using antibodies against rabbit red blood cell AE1 and mouse gastric AE2 peptide showed that lung contains membrane protein antigenically similar to gastric AE2. These data suggest the possibility that inhibition of AE or other anion transporters may play an important role in mediating oxidative lung injury.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid.

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.

Animals↗
PubMed2002

Troglitazone induces a cellular acidosis by inhibiting acid extrusion in cultured rat mesangial cells.

We studied the effect of troglitazone on cellular acid-base balance and alanine formation in isolated rat mesangial cells. Mesangial cells were grown to confluency in RPMI 1640 media on 30-mm chambers used to monitor both cellular pH using the pH-sensitive dye 2'7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein and metabolic acid production as well as glutamine metabolism. Troglitazone (10 microM) induced a spontaneous cellular acidosis (6.95 +/- 0.02 vs. 7.47 +/- 0.04, respectively; P < 0.0001) but without an increase in lactic acid production. Alanine production was reduced 64% (P < 0.01) consistent with inhibition of the glutamate transamination. These findings pointed to a decrease in acid extrusion rather than an increase in acid production as the underlying mechanism leading to the cellular acidosis. To test their acid extrusion capabilities, mesangial cells were acid loaded with NH and then allowed to recover in Krebs-Henseleit media or in Krebs-Henseleit media minus bicarbonate (HEPES substituted), and the recovery response (Delta pH(i)/min) was monitored. In the presence of 10 microM troglitazone, the recovery response to the NH acid load was virtually eliminated in the bicarbonate-buffered media (0.00 +/- 0.001 vs. 0.06 +/- 0.02 pH(i)/min, P < 0.0001 vs. control) and reduced 75% in HEPES-buffered media (0.01 +/- 0.01 vs. 0.04 +/- 0.02 pH(i)/min, P < 0.002 vs. control). These results show that troglitazone induces a spontaneous cellular acidosis resulting from a reduction in cellular acid extrusion.

Acid-Base Equilibrium↗
PubMed1995

Amiloride-sensitive Na+/H+ antiporter in basolateral membrane of hamster ascending thin limb of Henle's loop.

The mechanisms of intracellular pH (pHi) regulation were investigated in the in vitro microperfused hamster ascending thin limb (ATL) of Henle's loop with the fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. pHi of ATL cells was 7.05 +/- 0.02 (n = 30) when microperfused with a CO2/HCO(3-)-buffered solution. In HEPES-buffered solution, pHi was 7.10 +/- 0.02 (n = 16), which was significantly higher than the value in CO2/HCO(3-)-buffered solution (P < 0.05, n = 16). In HEPES-buffered solution, elimination of Na+ and addition of 1 mM amiloride to basolateral solution decreased the pHi by 0.12 +/- 0.03 (n = 6) and 0.11 +/- 0.02 (n = 5) at 1 min, respectively. The same manipulations in the luminal solution had no effect on pHi. One millimolar of N-ethylmaleimide (NEM) added to either side of ATL caused no significant change in pHi. Elimination of K+ on either side of ATL did not alter pHi. After adding 20 mM NH4Cl to basolateral solution, pHi instantaneously increased from 7.17 +/- 0.01 to 7.51 +/- 0.03 (n = 3), and then returned to steady-state level of 7.21 +/- 0.05 (n = 15) in 3 min. Removal of NH4Cl from basolateral solution then caused a rapid fall in pHi to 6.31 +/- 0.05 (n = 15), followed by spontaneous recovery at a rate of 0.43 +/- 0.06 unit/min (n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

APB suppresses synaptic input to retinal horizontal cells in fish: a direct action on horizontal cells modulated by intracellular pH.

1. Membrane potentials and cone-driven light responses were recorded from the H1-type horizontal cells in isolated retinas. Membrane potentials and intracellular pH were recorded also in enzymatically dissociated solitary horizontal cells. 2. In isolated retinas the glutamate analogue 2-amino-4-phosphonobutyrate (APB) hyperpolarized horizontal cells and reduced their light responses in a dose-dependent manner (5-200 microM). 3. The action of APB depended on the formulation of the saline; APB was effective in L-15 saline buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) but not in a commonly used, nominally CO2-free bicarbonate/Tris-buffered saline. 4. The major factor controlling the potency of APB was intracellular pH. APB was ineffective during retinal perfusion with NH4Cl-containing or CO2-free bicarbonate saline, both of which are known to alkalinize cells. In contrast, APB was effective in salines formulated to acidify the retinal neurons. These included both HEPES and Tris-buffered salines containing a weak acid and bicarbonate/Tris-buffered saline gassed with CO2. 5. APB reduced the size of glutamate-evoked depolarizations in solitary horizontal cells but had no independent action in the absence of glutamate. This reduction of glutamate-induced depolarization was observed in salines formulated to block voltage-dependent calcium and potassium currents. 6. The magnitude of APB's antagonistic action on solitary horizontal cells increased in a dose-dependent manner from 10 to 200 microM. The antagonism was increased by intracellular acidification and was reduced or eliminated by alkalinization. 7. We conclude that APB can reduce glutamate-evoked and, by inference, the photoreceptor neurotransmitter-evoked depolarization of horizontal cells by acting directly on the horizontal cells. This effect of APB is modulated by intracellular pH.

Aminobutyrates↗
PubMed1994

Ligand-gated currents of alpha and beta ganglion cells in the cat retinal slice.

1. We studied the receptor pharmacology of the ligand-gated currents of ON- and OFF- alpha and beta ganglion cells in a cat retinal slice preparation using the whole cell recording variation of the patch-clamp technique. Cat retinal slices were cut in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer and incubated in a bicarbonate-buffered solution. Ganglion cells were voltage clamped at -70 mV in HEPES-buffered Ringer solution. The pipette solution contained a low concentration of Cl- to distinguish mixed cationic from Cl(-)-mediated conductances, and Lucifer yellow (0.5%) was included for identification of the cell type. 2. In Ringer solution containing 1.2 mM Mg2+, current-voltage (I-V) curves of responses to the excitatory amino acid agonist (EAA) N-methyl-D-aspartate (NMDA) (200 microM) revealed a J-shaped function. In Mg(2+)-free Ringer solution containing 200 microM Cd2+ to block synaptic transmission, NMDA (200 microM) elicited an inward current 5-8 times larger at -70 mV. In both conditions I-V curves of the NMDA-induced currents reversed near 0 mV. These results suggest that there are NMDA EAA receptors present directly on the dendrites of alpha and beta ganglion cells. Responses to NMDA were blocked by +/- 2-amino-7-phosphonoheptanoic acid (AP7) (200 microM). 3. In Ringer solution containing 200-1,000 microM Cd2+ to block synaptic transmission, both ON- and OFF- alpha and beta cells responded to kainic acid (10-50 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) (20-70 microM), and quisqualic acid (0.1-30 microM) with inward currents that reversed near 0 mV. These responses were blocked by the quinoxaline EAA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM). The metabotropic agonists 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (25 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) in the presence of Cd2+ evoked little or no response for all cells tested. 4. In the presence of Cd2+, alpha and beta cells responded to gamma-amino-butyric acid (GABA) (200 microM) and glycine (200 microM) with inward currents that reversed near -35 mV, the calculated chloride equilibrium potential Ecl. Responses to GABA and glycine were both strongly desensitizing. (+)Bicuculline methyl chloride (20 microM) blocked an average of 90% of the inward current evoked by 200 microM GABA on all ganglion cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Benzolamide inhibits low-threshold calcium currents in hippocampal pyramidal neurons.

1. Benzolamide is a poorly permeant sulfonamide inhibitor of the enzyme carbonic anhydrase. We studied the effect of benzolamide on low-threshold (LT) Ca currents in neonatal hippocampal CAl neurons. 2. In hippocampal slices, benzolamide (2-10 microM) inhibited the LT current 30-75% in voltage-clamped CAl pyramidal cells (n = 6). In slices bathed in N-2-hydroxypiperazine-N'-2-ethane-sulfonic acid (HEPES)-buffered Ringer, benzolamide also reduced the LT current, indicating that the action of the drug was not bicarbonate dependent. 3. Benzolamide inhibited LT Ca currents 20-75% in acutely dissociated CAl neurons in HEPES (n = 18): inhibition was 36 +/- 8% (mean +/- SE; n = 7) and 50 +/- 8% (n = 7) at 10 and 50 microM benzolamide, respectively. By contrast, high-threshold calcium currents recorded in CAl pyramidal cells (n = 18) and dorsal root ganglion neurons (n = 4) were virtually unaffected by benzolamide. 4. These results indicate that benzolamide inhibits LT Ca channels in central neurons and suggest caution in the use of this agent to inhibit extracellular carbonic anhydrase in excitable tissues.

Animals↗
PubMed1997

Differential sensitivity to intracellular pH among high- and low-threshold Ca2+ currents in isolated rat CA1 neurons.

The effects of intracellular pH (pHi) on high-threshold (HVA) and low-threshold (LVA) calcium currents were examined in acutely dissociated rat hippocampal Ca1 neurons with the use of the whole cell patch-clamp technique (21-23 degrees C). Internal pH was manipulated by external exposure to the weak base NH4Cl or in some cases to the weak acid Na-acetate (20 mM) at constant extracellular pH (7.4). Confocal fluorescence measurements using the pH-sensitive dye SNARF-1 in both dialyzed and intact cells confirmed that NH4Cl caused a reversible alkaline shift. However, the external TEA-Cl concentration used during ICa recording was sufficient to abolish cellular acidification upon NH4Cl wash out. With 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the pipette, NH4Cl exposure reversibly enhanced HVA currents by 29%, whereas exposure to Na-acetate markedly and reversibly depressed HVA Ca currents by 62%. The degree to which NH4Cl enhanced HVA currents was inversely related to the internal HEPES concentration but was unaffected when internal ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was replaced by equimolar bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). When depolarizing test pulses were applied shortly after break-in (Vh = -100 mV), NH4Cl caused a proportionally greater increase in the sustained current relative to the peak. The dihydropyridine Ca channel antagonist nifedipine (5 microM) blocked nearly all of this sustained current. A slowly inactivating nifedipine-sensitive (L-type) HVA current could be evoked from a depolarized holding potential of -50 mV; NH4Cl enhanced this current by 40 +/- 3% (mean +/- SE) and reversibly shifted the tail-current activation curve by +6-8 mV. L-type currents exhibited more rapid rundown than N-type currents; HVA currents remaining after prolonged cell dialysis, or in the presence of nifedipine, inactivated rapidly and were depressed by omega-conotoxin (GVIA). NH4Cl enhanced these N-type currents by 76 +/- 9%. LVA Ca currents were observed in 32% of the cells and exhibited little if any rundown. These amiloride-sensitive currents activated at voltages negative to -50 mV, were enhanced by extracellular alkalosis and depressed by extracellular acidosis, but were unaffected by exposure to either NH4Cl or NaAC. These results demonstrate that HVA Ca currents in hippocampal CA1 neurons are bidirectionally modulated by internal pH shifts, and that N-type currents are more sensitive to alkaline shifts than are L- or T-type (N > L > T). Our findings strengthen the idea that distinct cellular processes governed by different Ca channels may be subject to selective modulation by uniform shifts in cytosolic pH.

Animals↗
PubMed1999

Effect of divalent cations on AMPA-evoked extracellular alkaline shifts in rat hippocampal slices.

The generation of activity-evoked extracellular alkaline shifts has been linked to the presence of external Ca(2+) or Ba(2+). We further investigated this dependence using pH- and Ca(2+)-selective microelectrodes in the CA1 area of juvenile, rat hippocampal slices. In HEPES-buffered media, alkaline transients evoked by pressure ejection of RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) averaged approximately 0.07 unit pH and were calculated to arise from the equivalent net addition of approximately 1 mM strong base to the interstitial space. These alkaline responses were correlated with a mean decrease in [Ca(2+)](o) of approximately 300 microM. The alkalinizations were abolished reversibly in zero-Ca(2+) media, becoming indiscernible at a [Ca(2+)](o) of 117+/-29 microM. Addition of as little as 30-50 microM Ba(2+) caused the reappearance of an alkaline response. In approximately one-fourth of slices, a persistent alkaline shift of approximately 0.03 unit pH was observed in zero-Ca(2+) saline containing EGTA. In HEPES media, addition of 300 microM Cd(2+), 100 microM Ni(2+), or 100 microM nimodipine inhibited the alkaline shifts by roughly one-half, one-third, and one-third, respectively, whereas Cd(+) and Ni(2+) in combination fully blocked the response. In bicarbonate media, by contrast, Cd(+) and Ni(2+) blocked only two-thirds of the response. In the presence of bicarbonate, Ni(2+) caused an unexpected enhancement of the alkalinization by approximately 150%. However, when the extracellular carbonic anhydrase was blocked by benzolamide, addition of Ni(2+) reduced the alkaline shift. These results suggested that Ni(2+) partially inhibited the interstitial carbonic anhydrase and thereby increased the alkaline responses. These data indicate that an activity-dependent alkaline shift is largely dependent on the entry of Ca(2+) or Ba(2+) via voltage-gated calcium channels. However, sizable alkaline transients still can be generated with little or no external presence of these ions. Implications for the mechanism of the activity-dependent alkaline shift are discussed.

Animals↗