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PubMed1986

Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart.

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.

Animals↗
PubMed1991

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.

Aniline Compounds↗
PubMed1995

Inhibition of rat glomerular mesangial cell sodium/hydrogen exchange by hydrogen peroxide.

1. pHi regulation in glomerular mesangial cells (GMC) includes both Na+/H+ and Cl-/HCO3-exchange. As a fall in pHi may protect against H2O2-mediated GMC damage during ischaemia-reperfusion, the involvement of these mechanisms in the GMC pH1 response to H2O2 was assessed using confluent GMC grown in RPMI medium with 20% fetal calf serum (10-15 passages). 2. Cells were loaded with BCECF-AM and pH1 evaluated using standard fluorometric-ratio techniques. In HEPES buffer, GMC exposure to H2O2 dose-dependently (25 mumol/L-1 mmol/L) decreased pHi over 10 min from 7.3 +/- 0.1 to 6.7 +/- 0.1 (at 100 mumol/L) partly due to rapid non-competitive inhibition of amiloride-sensitive Na+/H+ exchange. 3. BCECF fluorescence in free solution was unchanged by H2O2 and averaged 100 +/- 9 nmol/2.6 x 10(6) cells/pH unit. Similarly, zero-Na+/high-K+ buffer, used to minimize passive H+ entry, did not prevent the fall in pHi while GMC H+-formation/extrusion, assessed by the rate of extracellular acidification in low-capacity buffer (0.05 mmol/L), was rapidly inhibited. 4. In contrast, following only a brief 3 min exposure to 1 mmol/L H2O2, HCO3-/CO2 buffer potentiated the inhibition of Na+/H+ exchange from 50 to 80% of control and reduced the acidification from pHi 6.6 +/- 0.1 to 7.15 +/- 0.05. This effect was reversed (to pHi 6.8 +/- 0.07) by pretreatment with 200 mumol/L DIDS, an inhibitor of Cl-/HCO3- exchange. 5. Thus, the decrease in GMC pHi in response to H2O2 in HEPES, partly mediated by inhibition of Na+/H+ exchange and a possible redistribution of intracellular H+, is antagonized in HCO3-/CO2 through a DIDS-sensitive Cl-/HCO3- exchange mechanism. This may act to negate potentially protective effects of low pHi and potentiate oxidative damage to membrane lipids, enzymes and intracellular organelles on reperfusion.

Animals↗
PubMed1999

Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide.

1. The effect of inhibition of brain carbonic anhydrase (CA) on the hyperpolarization-activated cation current (Ih) of thalamocortical (TC) neurones of the rat ventrobasal thalamic complex (VB) was investigated in an in vitro slice preparation using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF). 2. Recording of Ih before and after addition of 0.4-0.8 mM acetazolamide to the bathing fluid revealed a significant shift in the voltage dependence of activation (V ) of 5-7 mV to more positive potentials. 3. Simultaneous recording of Ih and BCECF fluorescence ratio (F420/F495) revealed an increase in Ih amplitude accompanied by an intracellular alkalinization upon application of acetazolamide. The CA inhibitor ethoxyzolamide (EZA, 50 microM) also led to an intracellular alkalinization and a subsequent 4-5 mV positive shift of V of Ih. 4. Acetazolamide and EZA both profoundly slowed the rapid fall of pHi upon switching from Hepes- to CO2/HCO3--buffered superfusate, indicating intracellular CA isoforms in TC neurones. 5. In slices bathed in Hepes-buffered saline, addition of acetazolamide had no effect on the amplitude and time course of activation of Ih, indicating that the action of acetazolamide on Ih was dependent on the presence of HCO3-. 6. Under current-clamp conditions, the neuronal response to hyperpolarizing current pulses in the presence of acetazolamide was decreased as compared to control. This resulted in a strongly reduced ability of TC neurones to produce rebound Ca2+-mediated spikes. 7. The present results implied that in TC neurones acetazolamide led to an intracellular alkalinization which causes, due to its pH sensitivity, an increase in Ih.

Acetazolamide↗
PubMed1992

Differential metabolism of hydroxyeicosatetraenoic acid isomers by mouse cerebromicrovascular endothelium.

Hydroxyeicosatetraenoic acid (HETE) derivatives of arachidonic acid are produced in the brain and have been implicated as pathologic mediators in various types of brain injury. To understand better their fate in the brain, particularly in cerebral microvessels, several HETEs were incubated with cultured mouse cerebromicrovascular endothelium for 1, 2, and 4 h, followed by HPLC analysis of medium and cellular lipids. 5(S)-, 8(RS)-, and 9(RS)-HETE were not metabolized by the cells, but were extensively incorporated, unmodified, into cell lipids. On the other hand, 11(RS)-, 12(S)-, and 15(S)-HETE were extensively metabolized and only minimally incorporated into cell lipids. Previously, the major 12-HETE metabolite was identified as 8-hydroxyhexadecatrienoic acid. In the present study, we identified the major 11-HETE metabolite as 7-hydroxyhexadecatrienoic acid and the major 15-HETE metabolite as 11-hydroxyhexadecatrienoic acid. omega-3 compounds, 15(S)- and 12(S)-hydroxyeicosapentaenoic acids (HEPE), were also metabolized to more polar compounds, but to a lesser extent than their tetraenoic acid, omega-6 counterparts. Comparison of 5-, 12-, and 15-HETE enantiomers revealed no differences in metabolism or incorporation between the R and S stereoisomers. These data suggest that many isomers of HETE and HEPE can be incorporated into cell lipids or metabolized by pathways that do not distinguish between enantiomers. These pathways, however, are sensitive to the position or number of double bonds and are selective based on the position of the hydroxyl group.

Animals↗
PubMed1993

Sodium-dependent antiporters in choroid plexus epithelial cultures from rabbit.

The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na(+)-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO3- plus 95% O2/5% CO2. Intracellular pH (pHi) was determined from the steady-state distribution of [14C]benzoate. Recovery after acidification with NH4Cl was rapid (t1/2 = 5 min) and was dependent on external Na+ (EC50 = 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na+/H+ antiport, blocked 80% of recovery when [Na+] was 5 mM with IC50 values of 100 nM. However, neither drug blocked recovery in normal [Na+]. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of Cl-/HCO3- antiports, blocked recovery of pHi in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na+ channel, nor with dimethylbenzamil, an inhibitor of Na+/Ca2+ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of 22Na+ uptake was very rapid (227 pmol/micrograms of DNA/min at pH 6.2), was dependent on external [Na+] with an EC50 value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of 22Na+ uptake by hexamethyleneamiloride was 60% with an IC50 value of 76 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Beta-adrenoceptor agonist stimulation of acid secretion by rat stomach in vitro is mediated by 'atypical' beta-adrenoceptors.

1. A previous study showed beta-adrenoceptor agonists stimulated acid secretion by rat stomach in vitro. The receptors could not be classed as either the beta 1- or beta 2-subtype. This study examines the effect of 2 'atypical' beta-agonists on acid secretion. 2. Basal and isoprenaline-stimulated acid secretion were compared in tissues bathed either in HEPES/O2- or HCO3-/CO2-buffer. Basal secretion was underestimated in HCO3- by an amount equal to the rate of base section. Tissues responded well in HEPES buffer and there was no base secretion following acid inhibition with SCH 28080. HEPES was used for the study. 3. SR 58611A stimulated acid in a concentration-related way (0.1-5 microM). Maximum response at 1 microM was equal to the response to a maximal concentration of isoprenaline. BRL 37344 (1 microM) also stimulated to the same extent. 4. Responses to isoprenaline (5 microM) and SR 58611A (1 microM) were reduced by propranolol (10 microM) but not by alprenolol (10 microM) or by practolol (12.5 microM) plus ICI 118551 (1 microM). 5. Exposure to SR 58611A (1 microM) led to desensitization to isoprenaline but not to bethanechol (1 microM) or histamine (50 microM). 6. We conclude that a HEPES/O2-buffer is advantageous when measuring gastric acid secretion in vitro and the stimulatory effect of beta-adrenoceptor agonists is mediated by 'atypical' receptors.

Adrenergic beta-Agonists↗
PubMed1996

Effects of trimetazidine on pHi regulation in the rat isolated ventricular myocyte.

1. We have examined the effects of trimetazidine (TMZ) on intracellular pH (pHi) regulation in rat isolated ventricular myocytes. pHi was recorded ratiometrically by use of the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphtorhodafluor). 2. Following an intracellular acid load (induced by 10 mM NH4Cl removal), pHi recovery in HEPES-buffered Tyrode solution was significantly slowed down upon application of 0.3 mM TMZ only when myocytes were pretreated for 5 h 30 min (slowing by approximately 50%; P < 0.01). This effect of TMZ on pHi recovery was shown to be not only time- but also dose-dependent with a large, quickly reversible, effect obtained with 1 mM TMZ applied for 2-3 h (slowing by approximately 64%; P < 0.001). This slowing of pHi recovery was also associated with a decrease of the NH4+ removal-induced acidification. 3. Relationship between intracellular intrinsic buffering power (beta i) and pHi was assessed in absence or presence of TMZ (0.3 mM or 1 mM). As expected, beta i increased roughly linearly with a decrease in pHi in all cases. However, both concentrations of TMZ significantly increased beta i (by approximately 55 and 65% at pHi 7.1, respectively). 4. When Na+/H+ exchange was inhibited by dimethyl amiloride (DMA; 40 microM), trimetazidine (1 mM) did not change the H+ flux estimated at pHi 7.1 (0.31 +/- 0.03 mequiv l-1 min-1, n = 5, control, versus 0.30 +/- 0.025 mequiv l-1 min-1, n = 5, TMZ), ruling out any effect of TMZ on background acid loading. 5. Acid efflux carried by Na+/H+ exchange was significantly decreased only when myocytes were pretreated with 1 mM TMZ, for 2-3 h (JeH = 2.86 +/- 0.38 mequiv l-1 min-1, n = 26, control, versus 1.66 +/- 0.26 mequiv l-1 min-1, n = 10, TMZ, estimated at pHi 7.1; P < 0.05). 6. In conclusion, the present work demonstrates that, following an intracellular acid load in HEPES-buffered medium, trimetazidine slows down pHi recovery in rat isolated ventricular myocytes, primarily through an increase of beta i. An effect on Na+/H+ exchange is also detected but only after long-term incubation of the myocytes with TMZ.

Animals↗
PubMed2005

Protein L-agarose for adsorption of autoantibodies: a potential tool for extracorporeal treatment.

This work investigated the adsorption of autoantibodies such as anti-SS-A/Ro, anti-SS-B/La, anti-Sm, and anti-dsDNA on protein L-agarose gel. In order to determine better conditions for IgG adsorption on this matrix, some buffer systems were tested. Adsorption data were analyzed using the Langmuir and Langmuir-Freundlich isotherm models. The experimental isotherms were best described by the Langmuir-Freundlich model, which indicated negative and positive cooperativities for binding in the presence of PBS and HEPES buffers, respectively. The K(d) values for phosphate buffered saline solution (PBS) and hydroxyethylpiperazine ethanesulfonic acid (HEPES) were 2.8 x 10(-7) M and 3.2 x 10(-7) M, respectively, which indicate a high affinity between IgG and the immobilized protein L. The amount of protein adsorbed per amount of protein loaded was high for anti-Sm (44%) and anti-dsDNA (46%), but low for anti-SS-B/La (9%). The amount of albumin adsorbed was lower than 0.06 mg/mL, which may remove the need for a plasma replacement solution in clinical apheresis.

Adsorption↗
PubMedDate not supplied

Cisapride stimulates contraction of idiopathic megacolonic smooth muscle in cats.

We have previously shown that cisapride, a substituted piperidinyl benzamide, stimulates contraction of healthy feline colonic smooth muscle. The purpose of the present investigation was to determine the effect of cisapride on feline idiopathic megacolonic smooth muscle function. Longitudinal smooth muscle strips from ascending and descending colon were obtained from cats with idiopathic megacolon, suspended in a 1.5 mM Ca(2+)-HEPES buffer solution (37 degrees C, 100% O2, pH 7.4), attached to isometric force transducers, and stretched to optimal muscle length (Lo). Control responses were obtained at each muscle site with acetylcholine (10(-8) to 10(-4) M), substance P (10(-11) to 10(-7) M), or potassium chloride (10 to 80 mM). Muscles were then stimulated with cumulative (10(-9) to 10(-6) M) doses of cisapride in the absence or presence of tetrodotoxin (10(-6) M) and atropine (10(-6) M), or in a 0 calcium HEPES buffer solution. In cats with idiopathic megacolon, cisapride stimulated contractions of longitudinal smooth muscle from both the ascending and the descending colon. Cisapride-induced contractions were similar in magnitude to those induced by substance P and acetylcholine in the ascending colon, but were less than those observed in the descending colon. Cisapride-induced contractions in megacolonic smooth muscle were only partially inhibited by tetrodotoxin and atropine, but were virtually abolished by removal of extracellular calcium. We concluded that cisapride-induced contractions of feline megacolonic smooth muscle are largely smooth muscle mediated and dependent on influx of extracellular calcium. Cisapride-induced contractions in megacolonic smooth muscle are only partially dependent on enteric cholinergic nerves. Thus, cisapride may be useful in the treatment of cats with idiopathic megacolon.

Acetylcholine↗
PubMed1994

Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.

Acid-Base Equilibrium↗
PubMed1976

Adverse effects of tris hydrochloride, a commonly used buffer in physiological media.

1. Tris 10mM, produced inhibition of motor responses to adrenergic motor nerve stimulation in all smooth muscles studied (rat anococcygeus and vas deferens perfused rabbit ear artery). Up to 30 mM Tris was without significant effect on those motor responses to cholinergic nerve stimulation examined (guinea-pig ileum). 2. a similar reduction in responses to endogenous motor agonists was seen only in the anococcygeus, while in the rabbit ear artery, responses to noradrenaline (NA) were potentiated. 3. tris consistently produced increased tone in the rat anococcygeus and rabbit aorta, but in no other tissue, Small reductions in tone were seen with Tris in spiral strips of rabbit ear artery. 4. inhibitory responses to adrenergic nerve stimulation and to NA in the rabbit jejunum and to inhibitory nerve stimulation in the rat anococcygeus were unaffected by Tris. 5. in the perfused rabbit heart Tris produced a negative inotropic effect and reduced responses to vagal stimulation. Responses to sympathetic nerve stimulation and to ACh and NA were not significantly altered. 6. up to 40 mM Tris had little effect on responses of the rat diaphragm to motor nerve stimulation. 7. the effects of Tris on the rat anococcygeus were not reduced by increased calcium levels but were reduced at lower incubation temperatures. The negative inotropic effect of Tris on the rabbit heart was also reduced at lower temperatures. 8. the buffer HEPES produced a reduction in responses to stimulation in the rat anococcygeus and vas deferens along, but at higher concentrations than those of Tris. Increases in tone in the anococcygeus and aorta were never seen with HEPES. 9. These results indicate that Tris in concentrations commonly used as a buffer in physiological salines can exert toxic effects on neuro-muscular transmission in smooth and cardiac muscle though not in skeletal muscle. The effects are variable, mainly presynaptic and appear to affect in particular motor and especially adrenergic transmission. They may be associated with intracellular metabolic actions of Tris.

Acetylcholine↗
PubMed1983

The intracellular pH of frog skeletal muscle: its regulation in isotonic solutions.

The behaviour of intracellular pH (pHi) was studied with micro-electrodes in frog semitendinosus muscle which was superfused with Ringer solution and with depolarizing solutions. The electrodes were introduced into the depolarized muscle about 40 min after contracture had subsided. All studies were done at external pH (pHo) of 7.35 and at 22 degrees C. The pHi in normal Ringer solution buffered with HEPES was 7.18 +/- 0.03 (S.E. of mean) (n = 10); the membrane potential, Vm, was -88 +/- 1.8 mV. When pHi was lowered to about 6.8 by replacing the HEPES by 5% CO2, 24 mM-HCO3 (constant pHo), it recovered at a very slow rate of 0.025 +/- 0.011 delta pHi h-1 (n = 6). When all the Na was replaced by N-methyl-D-glucamine (initial pHi 7.20 +/- 0.04, initial Vm -89 +/- 1.5 mV, n = 8), this slow alkalinization was converted into a slow acidification at a rate of 0.069 +/- 0.024 delta pHi h-1. In muscle depolarized in 15 mM-K (Vm approximately -50 mV), the rate of recovery from CO2 acidification was not increased above that in normal Ringer solution (2.5 mM-K). When, however, the muscle was depolarized in 50 mM-K to about -20 mV, the rate of recovery increased to 0.33 +/- 0.07 delta pHi h-1 (n = 6) when external Cl was kept constant, or to 0.21 +/- 0.03 (n = 9) when [K]. [Cl] product was kept constant. In the absence of Na, pHi recovery rate in 50 mM-K was reduced by at least 90%. Enhanced recovery from CO2-induced acidification was also observed in 2.5 mM-K when the fibres were depolarized to about -20 mV in one of two ways: (a) by previous exposure for 60 min to 50 mM-K at constant Cl, or (b) by reduction of external Cl to 5.9 mM in the presence of 0.5 mM-Ba. When pHi of depolarized fibres (50 mM-K) was lowered to about 6.8 by the weak acid dimethyl-2,4-oxazolidinedione (DMO), it recovered at a rate of 0.12 delta pHi h-1 in two experiments. In fibres depolarized in 50 mM-K and constant Cl, either 0.1 mM-SITS or 0.5 mM-amiloride slowed pHi recovery from CO2 exposure by about 50%. When the depolarization was achieved at constant [K]. [Cl] product, amiloride slowed pHi recovery by about 50%, while SITS had, at most, only a slight effect.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Chloride ion transport into pig jejunal brush-border membrane vesicles.

1. This study was carried out to determine the types and activities of carrier proteins which transport the chloride ion in pig jejunal brush-border membranes, with an emphasis on studying the properties of chloride conductance activity in vesicles prepared from these membranes. 2. Sodium-chloride co-transport activity was not detected in this tissue. A sodium-proton antiport with typical amiloride sensitivity was present. An anion exchanger linking chloride to hydroxyl or bicarbonate ions was also found in the pig jejunal brush-border membrane vesicles. 3. Chloride conductance activity in this system was specifically dependent on the buffering agents used for vesicle preparation. Conductance activity could not be demonstrated in vesicles prepared in imidazolium acetate or in HEPES-Tris buffers. HEPES-tetramethylammonium buffering of vesicles in the chloride uptake system produced a significant conductance response to a potassium gradient plus valinomycin. 4. Chloride conductance showed saturable kinetics with respect to substrate concentration, with a Michaelis-Menten constant (Km) of approximately 116 mM and a maximum velocity (Vmax) of 132 nmol (mg protein)-1 min-1. 5. Preliminary screening of potential inhibitors of chloride conductance showed only minimal inhibitor effects of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-sulphonic acid), anthracene-9-carboxylate, N-phenylanthranilate and piretanide. 6. The conductance activity in pig jejunal vesicles appears to have stringent buffer requirements, and to be relatively insensitive to the effects of reported conductance inhibitors.

Animals↗
PubMed1990

Consequences of CO2 acidosis for transmembrane Na+ transport and membrane current in rabbit cardiac Purkinje fibres.

1. The influence of sarcolemmal Na(+)-H+ exchange on intracellular Na+ activity (aiNa), intracellular pH (pHi) and membrane holding current (Ih) was investigated in rabbit cardiac Purkinje fibres. pHi and aiNa were measured with liquid sensor ion-selective microelectrodes. A two-microelectrode voltage clamp was used while recording pHi or aiNa.pHi was varied by alternating a nominally CO2-free HEPES buffer and a CO2-HCO3-buffer. 2. The intrinsic buffer capacity was calculated from the decrease in pHi after addition of CO2. The most accurate estimate was obtained when transmembrane pHi regulation was blocked and equalled 18.4 +/- 1.0 mequiv H+/pH unit (mean +/- S.E.M.). 3. aiNa started to rise when pHi fell below 7.0. A hyperpolarization paralleled the increase in aiNa. The magnitude of the rise in aiNa and the hyperpolarization were steeply dependent on pHi. 4. Inhibition of the Na(+)-K+ pump by K(+)-free superfusion increased aiNa. The rate of rise in aiNa was highly dependent on pHi. The rates of rise in 5% (pHi = 7.00 +/- 0.03), 7% (pHi = 6.89 +/- 0.04) and 15% CO2 (pHi = 6.74 +/- 0.02) at constant external pH (pHo) relative to the rate in HEPES solution (pHi = 7.24 +/- 0.02) were: 1.5 +/- 0.2, 2.4 +/- 0.1 and 3.1 +/- 0.2. The acid-induced rise in aiNa was abolished by 2 mM-amiloride. 5. Extracellular acidosis slowed down the recovery of pHi and depressed the rate of rise in aiNa upon intracellular acidification. When both pHi and pHo were decreased to 6.7 the acid-dependent rate increase fell to about 10% of the value found at pHo 7.4. 6. The tetrodotoxin (TTX)-sensitive Na+ current was not influenced by the change in pHi in the range 6.7-7.2. 7. Intracellular acidosis was associated with an early aiNa-independent depolarization and an inward shift in Ih. Current-voltage plots revealed that the initial inward current shift reversed at -81.5 mV on average, showed inward rectification and was largely depressed in the presence of 1 mM-Ba2+. These observations indicate a decrease in K+ conductance when pHi falls. 8. The increase in aiNa elicited a Na(+)-K+ pump-dependent outward current which could override the initial aiNa-independent current shift. At a pHo of 6.7 the initial fall in Ih remained, while the secondary outward current was largely depressed. 9. The rate of active Na+ extrusion and Na(+)-K+ pump current were suppressed by about 30% at pHi 6.7 compared to pHi 7.2.(ABSTRACT TRUNCATED AT 400 WORDS)

Acidosis↗
PubMed1991

Extracellular alkalinization evoked by GABA and its relationship to activity-dependent pH shifts in turtle cerebellum.

1. The effect of gamma-aminobutyric acid (GABA) on extracellular pH (pHo) was investigated in the turtle cerebellum, in vitro, using double-barrelled, H(+)-selective microelectrodes. Responses evoked by GABA were compared with pHo shifts evoked by repetitive stimulation of the parallel fibres. 2. In media buffered with 35 mM-HCO3- and 5% CO2, superfusion of GABA (1 mM) elicited an abrupt alkaline shift in the molecular layer, which averaged 0.05 +/- 0.02 pH units (+/- S.D., range 0.02-0.12 pH units). pHo often recovered in the continued presence of GABA, and displayed a rebound acidification upon wash-out. 3. The GABA-evoked alkaline shift was blocked by picrotoxin and was mimicked by the GABAA agonists isoguvacine and muscimol. The GABAB agonist baclofen did not elicit an alkaline shift. Alkaline shifts evoked by stimulation of the parallel fibres were unaffected by picrotoxin. 4. In nominally HCO3(-)-free solutions, buffered with 35 mM-HEPES, superfusion of GABA caused either no pHo change or a slow acid shift. In contrast, the alkaline shift evoked by stimulation of the parallel fibres became enhanced in HEPES-buffered media. 5. The alkaline shift evoked by GABA was accompanied by an increase in extracellular K+ ([K+]o) which averaged 1.7 mM above baseline. Experimental elevation of [K+]o to a comparable level always caused a pure acid shift in the extracellular space. 6. The GABA-evoked alkaline shift persisted when synaptic transmission was blocked using 4 mM-kynurenic acid or saline prepared with nominally zero Ca2+ and 10 mM-Mg2+. The alkaline shift evoked by repetitive stimulation of the parallel fibres was completely abolished in these media. 7. Although the GABA-evoked alkaline shift was blocked in nominally HCO3(-)-free media, substitution of 35 mM-formate for HCO3- restored the GABA response. Superfusion of 1 mM-GABA in formate saline produced an alkaline shift of 0.040 +/- 0.034 pH units. 8. These results indicate that gating of GABAA channels in the vertebrate CNS gives rise to an HCO3- efflux which can significantly increase the pH of the brain microenvironment. However, this mechanism cannot account for the extracellular alkalinization caused by parallel fibre stimulation. Extracellular alkaline shifts capable of modulating local synaptic operations may therefore be a consequence of either excitatory or inhibitory synaptic transmission.

Animals↗
PubMed1992

Intracellular pH regulation in isolated cochlear outer hair cells of the guinea-pig.

1. The intracellular pH (pHi) regulation mechanisms of the outer hair cell (OHC) isolated from the guinea-pig were studied using fluorescence ratio imaging microscopy. 2. The OHC pHi in the resting condition was 7.26 +/- 0.08 (mean +/- S.D., n = 49) when the standard solution buffered with HEPES-Tris was superfused. 3. Exposure to 25 mM-NH4+ in the absence of HCO3- caused biphasic changes in pHi; a transient increase (7.89 +/- 0.14, n = 22) followed by a slow decrease (7.57 +/- 0.12; mean +/- S.D.). Removal of external NH4+ by introducing the N-methyl-D-glucamine (NMDG+) solution in the absence of HCO3- markedly acidified the pHi to 6.38 +/- 0.12 with little pHi recovery. Subsequent application of the standard Na+ solution restored the pHi to the initial value. The recovery was inhibited by 0.5 mM-amiloride but not by 0.3 mM-DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). 4. In the presence of HCO3-, removal of both external NH4+ and Na+ promptly caused an intracellular acidification followed by a pHi recovery. The pHi recovery from an acid load was inhibited by 0.3 mM-DIDS or 10 microM-NPPB (5-nitro-2-(3-phenylpropyl-amino)-benzoate). However, the pHi in the steady state in the presence or absence of HCO3- was not altered by addition of 0.5 mM-amiloride or NMDG+ solution. 5. The intracellular buffering power obtained from the NH4+ exposure and withdrawal was -15.1 +/- 8.7 mM (pH unit)-1 (n = 6) and -14.3 +/- 5.8 mM (pH unit)-1, respectively. 6. Replacement of external Cl- with gluconate in the HCO3- solution increased the pHi from 7.22 +/- 0.12 to 7.51 +/- 0.20 (n = 6), which was inhibited by 0.3 mM-DIDS. Moreover, addition of DIDS to the HCO3- solution increased the pHi by 0.13 +/- 0.08 (n = 8). 7. When the external standard solution buffered with HEPES-Tris was replaced with the HCO3- solution, the basal pHi (7.27 +/- 0.10) was promptly acidified to 6.87 +/- 0.10 then relaxed slowly to 7.00 +/- 0.15 (n = 16). 8. The pHi showed an initial alkalinization and a subsequent slow acidification after the HCO3(-)-free standard solution replaced the HCO3- solution. The slow acidification was inhibited by low external Cl- concentration or by addition of 0.3 mM-DIDS.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetazolamide↗
PubMed1992

Na(+)-HCO3- symport in the sheep cardiac Purkinje fibre.

1. Intracellular pH (pHi) was recorded in isolated sheep cardiac Purkinje fibres using liquid sensor ion-selective microelectrodes in conjunction with conventional (3 M-KCl) microelectrodes (to record membrane potential). 2. In HEPES-buffered solution (pH0 7.4), pHi recovery from an intracellular acid load (20 mM-NH4Cl removal) was blocked by 1 mM-amiloride, consistent with the inhibition of Na(+)-H+ exchange. Replacement of the HEPES buffer with CO2-HCO3- caused a transient acidosis followed by an amiloride-resistant recovery of pHi to more alkaline levels (n = 43). This implies the presence of a HCO3(-)-dependent pHi regulatory mechanism. 3. Comparison of the membrane potential with the equilibrium potential for HCO3- ions (EHCO3) estimated during amiloride-resistant pHi recovery, showed that for polarized fibres (membrane potential Em approximately -80 mV), there was a net outward electrochemical driving force for HCO3- ions. Hence the amiloride-resistant pHi recovery cannot be explained in terms of passive HCO3- influx through membrane channels. 4. Removal of external Na+ (Na0+ replaced by N-methyl-D-glucamine) inhibited HCO3(-)-dependent pHi recovery, whereas removal of external Cl- (leading to depletion of internal Cl-; Cl0- replaced by glucuronate) or short-term removal of extracellular K+ had no inhibitory effect. We suggest that a Na(+)-HCO3- co-influx causes the recovery. Replacement of external Na+ with Li+ greatly reduced HCO3(-)-dependent pHi recovery indicating that Li0+ cannot readily substitute for Na0+ on the co-transport. 5. The stilbene drug DIDS (4,4-diisothiocyano-stilbene-disulphonic acid, 500 microM) slowed HCO3(-)-dependent pHi recovery. 6. Depolarization of the membrane potential in high K0+ (44.5 mM) solution or with 5 mM-BaCl2 had no effect upon the rate of HCO3(-)-sensitive pHi recovery. This observation, when coupled with the fact that activation of HCO3(-)-dependent pHi recovery was associated with no consistent change of membrane potential, suggests that the Na(+)-HCO3- co-influx is electroneutral and voltage insensitive. 7. HCO3(-)-dependent pHi recovery was unaffected by the Na(+)-K(+)-2Cl- co-transport inhibitor, bumetanide (150 microM). 8. The contribution of Na(+)-H+ exchange and Na(+)-HCO3- co-transport to net acid efflux was assessed. At a pHi of 6.6, we estimate that the co-transport should account for 20% of total acid equivalent efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

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