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PubMed1996

Acidosis of rat dorsal vagal neurons in situ during spontaneous and evoked activity.

1. Rat brainstem slices were taken for simultaneous measurements of intracellular pH (pHi) and membrane currents or potentials in dorsal vagal neurons, dialysed with the pH-sensitive dye BCECF. 2. Intrinsic intracellular buffering power was 18 mM per pH unit, as determined by exposure to trimethylamine in CO2/HCO3(-)-free, Hepes-buffered saline. 3. Tonic spike activity led to a stable fall in pHi of 0.05-0.2 pH units from a baseline of 7.19 in current-clamp mode, whereas depolarization from -60 to 0 mV for 1 min in voltage-clamp mode produced an intracellular acidification of 0.3 pH units. The depolarization-evoked fall in pHi was suppressed by 1 mM Ni2+ or 0.2 mM Cd2+, but not by 0.5 microM TTX or CO2/HCO3(-)-free saline. 4. Kainate (100 microM) led to an an inward current of -620 pA and a threefold increase in membrane conductance, accompanied by a fall in pHi of 0.33 pH units. 5. GABA (1 mM) evoked a bicuculline-blockable conductance increase and fall in pHi of up to 0.5 pH units. The GABA-induced pHi decrease, but not the conductance increase, was suppressed in Hepes solution. 6. Neither tonic spike activity, nor resting current or conductance were markedly changed upon Hepes-induced intracellular alkalinizations of up to 0.35 pH units, or by an anoxia-induced fall in pHi of a maximum of 0.36 pH units. 7. The data show that neuronal activity produces profound changes in pHi. It appears that spontaneous spike discharge of dorsal vagal neurons is rather tolerant of major perturbations in pHi.

Acidosis↗
PubMed1987

Direct measurement of intracellular pH in identified glial cells and neurones of the leech central nervous system.

Neutral carrier pH-sensitive double-barrelled microelectrodes were used to investigate intracellular pH (pHi) in leech neuropile glial cells and in Retzius neurones. The mean pHi of the glial cells was 6.87 +/- 0.13 (+/- SD, n = 27) in HEPES-buffered saline (pHo 7.4) and 7.18 +/- 0.19 (n = 13) in solutions buffered with 2% CO2- 11 mM HCO3-. The distribution of H+ ions in both the glia and neurones was found not to be in electrochemical equilibrium. To investigate pHi regulation, the pHi was decreased by exposure to CO2 or by adding and then removing NH4Cl. Acidification by any method was followed by a recovery to normal pHi values within minutes. The pHi recovery from acidification in neuropile glial cells in HEPES-buffered saline and CO2-HCO3- buffered saline was, however, blocked by removing external Na. In HCO3(-)-free solutions the diuretic amiloride (2 mM) reduced the rate of pHi recovery. In the presence of HCO3-, the rate of acid efflux was stimulated; the stilbene 4-acetamido-4'-isothiocyanatostilbene-2,3'-disulfonic acid (SITS; 0.5 mM) slowed pHi recovery. In HEPES buffered and CO2-HCO3- buffered solutions pHi regulation in neurones was inhibited by removing external Na. In HCO3(-)-free solutions amiloride reduced the rate of pHi recovery considerably. In the presence of HCO3-, SITS or amiloride slowed but did not completely block pHi recovery. We conclude that leech glial cells and neurones have two mechanisms of pHi regulation, one being Na+-H+ exchange and the other Na+ and HCO3- dependent.

Animals↗
PubMed1988

Intracellular pH recovery from lactic acidosis of single skeletal muscle fibers.

Intracellular pH (pHi), measured with H+-selective microelectrodes, in quiescent frog sartorius muscle fibres was 7.29 +/- 0.09 (n = 13). Frog muscle fibres were superfused with a modified Ringer solution containing 30 mM HEPES buffer, at extracellular pH (pHo) 7.35. Intracellular pH decreased to 6.45 +/- 0.14 (n = 13) following replacement of 30 mM NaCl with sodium lactate (30 mM MES, pHo 6.20). Intracellular pH recovery, upon removal of external lactic acid, depended on the buffer concentration of the modified Ringer solution. The measured values of the pHi recovery rates was 0.06 +/- 0.01 delta pHi/min (n = 5) in 3 mM HEPES and was 0.18 +/- 0.06 delta pHi/min (n = 13) in 30 mM HEPES, pHo 7.35. The Na+-H+ exchange inhibitor amiloride (2 mM) slightly reduced pHi recovery rate. The results indicate that the net proton efflux from lactic acidotic frog skeletal muscle is mainly by lactic acid efflux and is limited by the transmembrane pH gradient which, in turn, depends on the extracellular buffer capacity in the diffusion limited space around the muscle fibres.

Acidosis, Lactic↗
PubMed1989

Stimulation of Cl- self exchange by intracellular HCO3- in rabbit cortical collecting duct.

In rabbit cortical collecting duct, Cl- self exchange accounts for most of the transepithelial Cl- tracer rate coefficient, KCl (nm/s); a small fraction is effected by Cl--HCO3- exchange and Cl- diffusion. We previously reported that changing from a CO2-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) bath to a 5% CO2-25 mM HCO3- bath stimulates Cl- self exchange. Here, we examine in further detail the individual components of the CO2-HCO3- system that stimulate KCl. Addition of 0.5% CO2 to a HEPES bath (final pH = 7.24) stimulated KCl by 70 +/- 19 nm/s, a delta KCl comparable to that induced by 1% CO2 (pH 7.12), 6% CO2 (pH 6.6), or 6% CO2-25 mM HCO3- (pH 7.4). The roles of intracellular pH (pHi) and HCO3- concentration were examined by clamping pHi using high K+ and nigericin. Increasing pHi from 6.9 to 7.6 in solutions without exogenous CO2 or HCO3- increased KCl by 71 +/- 17 nm/s. These results suggest that pHi might regulate anion exchange. However, during such a pHi-shift experiment, metabolically derived CO2 produces a concomitant change in intracellular HCO3- concentration [( HCO3-]i). To determine whether an increase in [HCO3-]i could stimulate Cl- self exchange, we replaced HEPES with 6% CO2-5 mM HCO3- isohydrically (pHi clamped at 6.9). With this increase in [HCO3-]i at constant pHi, KCl increased by 51 +/- 10 nm/s. These maneuvers had negligible effects on Cl- diffusion and Cl--HCO3- exchange. These experiments demonstrate that increases in cell [HCO3-] (or perhaps CO2) can stimulate transepithelial anion exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Mechanisms of pHi control and relationships between tension and pHi in human subcutaneous small arteries.

Intracellular pH (pHi) control and relationships between pHi and tension have been investigated in human subcutaneous small arteries. Isometric tension and pHi (using 2',7'-bis(carboxyethyl)- 5(6)-carboxyfluorescein) were estimated simultaneously. pHi recovery from an acute acid load was dependent on external Na+ and partially inhibited by the absence of HCO3(-) [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution] or by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In an HCO3(-)-buffered physiological salt solution (PSS), pHi recovery was partially blocked by hexamethylene amiloride (HMA), an inhibitor of Na+/H+ exchange, and completely blocked by DIDS and HMA together. Intracellular Cl- depletion of arteries did not affect the rate of pHi recovery in PSS from an acid load. pHi recovery from acute alkalosis was unaffected by external Na+ removal, reduced in HEPES buffer, and abolished by removal of external Cl-. These data suggest that human small arteries maintain pHi by Na+/H+ exchange and Na(+)-dependent HCO3(-) exchange in response to an acid load, and Na(+)-independent Cl-/HCO3(-) exchange to counteract intracellular alkalosis. Norepinephrine (NE)-, endothelin-1 (ET-1)-, arginine vasopressin (AVP)-, and K(+)-induced tension did not alter pHi in PSS, but there was a small fall with angiotensin II (ANG II). In HEPES, stimulation with K+, NE, ANG II, or AVP led to a fall in pHi, but this did not occur with ET-1. It is therefore unlikely in vivo that an increase in pHi in these arteries would be involved in either tension development or growth induced by these agonists.

Acetates↗
PubMed2001

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons.

To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O(2), we monitored pH(i) in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O(2) deprivation or anoxia (PO(2) approximately equal to 0 Torr), a large increase in pH(i) was seen in CA1 neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO(3)(-). Ionic substitution and pharmacological experiments showed that the large anoxia-induced pH(i) increase in HEPES buffer was totally Na(+) dependent and was blocked by HOE-694, strongly suggesting the activation of the Na(+)/H(+) exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na(+)/H(+) exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO. We conclude that 1) O(2) deprivation activates Na(+)/H(+) exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pH(i) during low-O(2) states in neurons.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2004

Reduced oxygen supply explains the negative force-frequency relation and the positive inotropic effect of adenosine in buffer-perfused hearts.

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 +/- 1.2 nM, maximal effect (Emax) = 104 +/- 2 and 84 +/- 2 mmHg at 0.1 microM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate (r = 0.67, P < 0.01; EC50 = 3.8 +/- 1.4 nM, baseline 228 +/- 21 beats/min to a minimum of 183 +/- 22 beats/min at 0.1 microM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.

Adenosine↗
PubMed1985

Influence of buffers on dV/dtmax recovery kinetics with lidocaine in myocardium.

Buffers are reported to modify electrical function of heart tissue. Since electrophysiological actions of antiarrhythmic drugs are examined in different buffer systems, we set out to examine the influence of buffers on lidocaine's electrophysiological actions by measuring recovery kinetics of maximum upstroke velocity (dV/dtmax) in lidocaine solutions buffered with HCO-3-CO2, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and tris(hydroxymethyl)aminomethane (Tris) at extracellular pH 7.4. Transmembrane potential and dV/dtmax were recorded from guinea pig papillary muscle. Recovery kinetics were determined by introducing progressively earlier test stimuli during diastole. During lidocaine (1.5 X 10(-5) M) exposure, the time constant (Tr) of dV/dtmax recovery significantly increased when 21 mM HCO-3-5% CO2 was replaced by either 5 mM HEPES (38 +/- 8%, mean, +/- SED) or 5 mM Tris (41 +/- 6%). This potentiation of Tr was 1) reversed by increasing Tris to 20 mM, and 2) also abolished by restoring HCO-3-CO2 to HEPES or Tris solutions. Decreasing HCO-3 (21-4 mM) and CO2 (5-1%) increased Tr by 27 +/- 1%. We propose that the mechanism for the potentiation of Tr is therefore related to buffer concentration rather than to the lack of HCO-3-CO2. We speculate that, by reducing surface pH, lowered buffer capacity can slow the rate of dV/dtmax recovery.

Action Potentials↗
PubMed2001

Vascular release of nonheme iron in perfused rabbit lungs.

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells.

We investigated changes in intracellular pH (pHi) of cultured rat glomerular mesangial cells (MCs) exposed to angiotensin II (ANG II) and arginine vasopressin (AVP). pHi of quiescent MCs, passage 2-5, and grown on glass cover slips, was assessed by spectrofluorometry using the pH-sensitive dye, 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The steady-state pHi of MCs in a pH 7.4, HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.10 +/- 0.02 (n = 68) and in a pH 7.4, HCO3-containing solution, was 7.23 +/- 0.03 (n = 47) (P less than 0.01). The pHi recovery following an NH+4-induced acid load was inhibited by removal of Na+ from the bath or by addition of the amiloride analogue, ethyl isopropyl amiloride (EIPA). These effects were observed in MCs bathed in HEPES- or in HCO3-buffered solutions, consistent with the action of a Na+-H+ exchanger. When cells were bathed in HEPES, a 10-min exposure to ANG II or AVP (10(-10) to 10(-6) M) caused early and transient acidification of MCs (maximal pH change was -0.10), followed by gradual alkalinization (maximal pHi change +0.15 above the initial value). The increase of pHi was dependent on the presence of Na+ in the bath and was inhibited by EIPA. In the presence of HCO3, ANG II or AVP induced merely a small gradual acidification of MCs (pHi change -0.05). These findings demonstrate that MCs utilize a Na+-H+ exchanger for acid extrusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1995

Stimulus-evoked changes of extra- and intracellular pH in the leech central nervous system. I. Bicarbonate dependence.

1. We have measured the effect of repetitive electrical nerve root stimulation on the extracellular potassium activity (aKe) and the extracellular pH (pHe) and intracellular pH (pHi) in segmental ganglia of the leech Hirudo medicinalis with double-barreled K(+)- and pH-sensitive microelectrodes. To investigate the influence of CO2/HCO3-, we compared the stimulus-evoked changes in aKe, pHe, and pHi in the presence and absence of 5% CO2-24 mM HCO3- in the saline. 2. An electrical nerve root stimulation at 20-30 Hz for 1 min caused a rapid increase of 1.11 +/- 0.79 (SD) mM in aKe, followed by an aKe undershoot of 0.17 +/- 0.15 mM when the stimulation was discontinued (n = 6). aKe transients were not significantly affected by CO2/HCO3-. 3. In 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered, nominally CO2/HCO3(-)-free saline, low stimulus intensities or stimulus durations up to a few seconds resulted in a fast alkaline pHe transient. This alkalinization was followed by a larger and longer-lasting extracellular acidification when the stimulation was intensified and prolonged. A stimulation at 20 Hz, 5 V for 1 min caused an average alkaline shift of 0.083 +/- 0.055 pH units, followed by an acidosis of 0.079 +/- 0.038 pH units (n = 63). A change from 5 mM HEPES-buffered saline to 20 mM HEPES-buffered saline attenuated the stimulus-evoked pHe transients by 50-60%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Effect of extracellular HCO(3)(-) on Na(+) channel characteristics in hippocampal CA1 neurons.

The effect of HCO(3)(-)/CO(2) on membrane properties of isolated hippocampal CA1 neurons was studied with the use of the whole cell configuration of the patch-clamp technique. Neurons were acutely dissociated from 21- to 30-day-old mice. In the current-clamp mode, HCO(3)(-)/CO(2) significantly hyperpolarized CA1 neurons by more than 10 mV and decreased their input resistance. In addition, the overall excitability of these neurons was lower in the presence of HCO(3)(-)/CO(2) than in HEPES. Spontaneous and evoked action potential firing frequency was lower in the presence of HCO(3)(-)/CO(2) than in its absence. In the voltage-clamp mode, both activation and steady-state inactivation of a fast Na(+) current were shifted in the hyperpolarized direction in such a way that the window currents were smaller in HCO(3)(-)/CO(2) than in HEPES. Recovery from inactivation and deactivation from the open state of the fast Na(+) current was slower in HCO(3)(-)/CO(2) than in HEPES. We conclude that HCO(3)(-)/CO(2) decreases the intrinsic excitability of CA1 neurons by altering not only the passive properties of the neuronal membranes but also by changing several characteristics of the fast Na(+) current, including activation and inactivation kinetics as well as the recovery from inactivation and deactivation.

Action Potentials↗
PubMed1995

Lymphocytic intracellular pH and Na+/H+ exchanger activity in hemodialysis patients.

We have evaluated intracellular pH (pHi) and Na+/H+ exchanger activity in peripheral lymphocytes from 16 patients on regular acetate hemodialysis. All the patients were taking oral NaHCO3 supplementation (30 mmol/day), to maintain predialysis arterial blood acid-base status within normal range (pH 7.36 +/- 0.02, PHCO3- 23.3 +/- 1.2 mM, pCO2 40.9 +/- 1.4 mm Hg). pHi was measured, using the fluorescent probe BCECF (2',7'-bis-carboxyethyl-5,6-carboxyfluorescein), both in nominal absence of bicarbonate (Hepes solution, pH 7.4; n = 10) and in the presence of HCO3-/CO2 buffer system (pH 7.4, [HCO3-] 25 mM, pCO2 40 mm Hg; n = 6). Predialysis pHi did not differ from controls when measured in the presence of HCO3-/CO2 (7.28 +/- 0.04 vs. 7.29 +/- 0.04, p = NS), but was lower in dialysis patients than in normal subjects (7.11 +/- 0.04 and 7.20 +/- 0.02, respectively; p < 0.05) when measured in Hepes solution. This suggested that bicarbonate-independent pHi regulation was abnormal in dialysis patients. To further characterize this abnormality of pHi regulation, lymphocytes were exposed to ethylisopropylamiloride, a specific Na+/H+ antiporter inhibitor, in Hepes solution; this maneuver induced a significantly lower decrement in pHi (0.04 +/- 0.04 vs. 0.15 +/- 0.03, p < 0.05) in dialysis patients than in controls, indicating reduced Na+/H+ exchanger activity in the patients. The rate of pHi recovery during the first 30 s after induction of various degrees of cell acidification (pHi range 6.2-7.0), which in the absence of HCO3-/CO2 is dependent on Na+/H+ exchanger activity, was also reduced in the patients as compared to controls (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Intracellular pH in human arterial smooth muscle. Regulation by Na+/H+ exchange and a novel 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)- and HCO3(-)-dependent mechanism.

We investigated in a physiological salt solution (PSS) containing HCO3- the intracellular pH (pHi) regulating mechanisms in smooth muscle cells cultured from human internal mammary arteries, using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and 22Na+ influx rates. The recovery of pHi from an equivalent intracellular acidosis was more rapid when the cells were incubated in CO2/HCO3(-)-buffered PSS than in HEPES-buffered PSS. Recovery of pHi was dependent on extracellular Na+ (Km, 13.1 mM); however, it was not attenuated by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), indicating the absence of SITS-sensitive HCO3(-)-dependent mechanisms. Recovery instead appeared mostly dependent on processes sensitive to 5-(N-ethyl-N-isopropyl)amiloride (EIPA), indicating the involvement of Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism. Differentiation between this HCO3(-)-dependent mechanism and Na+/H+ exchange was achieved after depletion of cellular ATP. Under these conditions, the NH4Cl-induced 22Na+ influx rate stimulated by intracellular acidosis was markedly attenuated in HEPES-buffered PSS but not in CO2/HCO3(-)-buffered PSS. EIPA also appeared to inhibit the two mechanisms differentially. In HEPES-buffered PSS containing 20 mM Na+, the EIPA inhibition curve for the intracellular acidosis-induced 22Na+ influx was monophasic (IC50, 39 nM), whereas in an identical CO2/HCO3(-)-buffered PSS, the inhibition curve exhibited biphasic characteristics (IC50, 37.3 nM and 312 microM). Taken together, the results indicate that Na+/H+ exchange and a previously undescribed EIPA-sensitive Na(+)- and HCO3(-)-dependent mechanism play an important role in regulating the pHi of human vascular smooth muscle. The involvement of the latter mechanism depends on the severity of the intracellular acidosis, varying from approximately 25% in severe intracellular acidosis up to 50% at lesser, more physiological, levels of induced acidosis.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Mechanisms of pHi recovery after global ischemia in the perfused heart.

A Na(+)-HCO3- coinflux carrier and the Na(+)-H+ antiport have both been shown to contribute to recovery from intracellular acidosis in cardiac tissue. We have investigated the participation of these mechanisms as well as metabolite (lactate and CO2) washout in the recovery of pHi after myocardial ischemia. Isovolumic ferret hearts were Langendorff-perfused with either HCO3(-)-buffered or nominally HCO3(-)-free (HEPES-buffered) medium at 30 degrees C. pHi was estimated from the chemical shift of the 31P-nuclear magnetic resonance signal of intracellular PO4-, and net H+ efflux rates were calculated at pHi 6.80. The H+ efflux rate during reperfusion, after 10 minutes of global ischemia, was 15.5 +/- 1.9 mmol.l-1 x min-1 (n = 10) in hearts perfused with HCO3(-)-buffered medium and 8.2 +/- 1.5 mmol.l-1 x min-1 (n = 9, p < 0.01) in hearts perfused with HEPES-buffered medium. HCO3- influx, assessed either by inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (20 microM) or by initially perfusing hearts with HEPES-buffered medium but reperfusing with HCO3(-)-buffered medium, accounted for 3.5-4.9 mmol.l-1 x min-1, and CO2 efflux accounted for 3.8-6.2 mmol.l-1 x min-1 of the additional H+ efflux in HCO3(-)-buffered medium. H(+)-coupled lactate efflux, measured by NAD(+)-linked spectrophotometric assay and inhibited by the sarcolemmal monocarboxylate transport inhibitor 4,4'-dibenzamidostilbene-2,2'-disulfonate (0.25 mM), contributed 3.7-6.2 mmol.l-1 x min-1. H+ efflux via the 5-(N-ethyl-N-isopropyl)amiloride-sensitive Na(+)-H+ antiport was 1.0-2.9 mmol.l-1 x min-1. pHi recovery after ischemia is therefore principally mediated by metabolite (lactate and CO2) washout. Na(+)-coupled acid extrusion contributed approximately 35% of total H+ efflux in this system. However, the associated Na+ entry (approximately 5 mmol.l-1 x min-1) may contribute to Ca2+ overload after reperfusion.

Animals↗
PubMed1994

Involvement of Na(+)-H+ antiporter in regulation of L-type Ca2+ channel current by angiotensin II in rabbit ventricular myocytes.

The present study investigated the possible involvement of a Na(+)-H+ antiporter in the regulation of L-type Ca2+ channels by angiotensin II (Ang II) in isolated rabbit ventricular cardiac myocytes by using both cell-attached and whole-cell patch-clamp current recording techniques. In cell-attached patch-clamp current recordings, an increase in the open-state probability of the Ca2+ channel (144.8 +/- 9.8% [mean +/- SEM], n = 11) was seen after exposure of the cells to Ang II (100 nmol/L). This effect was inhibited by pretreatment with losartan (10 mumol/L), a synthetic antagonist of the AT1 receptor. 5(N,N-Dimethyl)amiloride (100 mumol/L), an amiloride analogue, as well as Na(+)-deficient bath solution abolished Ang II-induced stimulation of the Ca2+ channel activities. In whole-cell patch-clamp current recordings, Ang II also increased the L-type Ca2+ current when a pipette solution of pH 7.1 containing 5 mmol/L HEPES (139 +/- 5%, n = 4) was used but did not significantly increase the current when a pipette solution of pH 7.5 containing 5 mmol/L HEPES or a pipette solution of pH 7.1 containing 30 mmol/L HEPES was used. These results suggest that Ang II-induced stimulation of the Ca2+ channels is mediated by a Na(+)-H+ antiporter and therefore provide a novel insight into signal transduction of Ang II receptor stimulation in cardiac myocytes.

Angiotensin II↗
PubMed1988

A human cell beryllium acute toxicity assay.

A rapid, inexpensive beryllium acute toxicity assay using human erythrocyte ATP levels has been developed. The assay uses a photometric measurement of the luciferin-luciferase reaction in erythrocytes incubated in HEPES buffer with the tested toxicant. Incubation in HEPES significantly increases the sensitivity of erythrocytes to beryllium when compared to incubation in either plasma or physiological saline. After a one-hour incubation period in HEPES buffer and beryllium there is a loss of 50% of the erythrocyte ATP at 3 micrograms/ml of beryllium, and an 80% loss of ATP at 5 micrograms/ml of beryllium. The source of human erythrocytes does not appear to influence the test. Erythrocytes from 10 individuals, one with chronic beryllium disease and another with an acute sensitivity to beryllium, all gave similar biphasic dose-response curves to beryllium.

Adenosine Triphosphate↗
PubMed1994

AMMONIA EXCRETION IN FRESHWATER RAINBOW TROUT (ONCORHYNCHUS MYKISS) AND THE IMPORTANCE OF GILL BOUNDARY LAYER ACIDIFICATION: LACK OF EVIDENCE FOR Na+/NH4+ EXCHANGE

Net ammonia fluxes (JAmm) were measured in adult freshwater rainbow trout in vivo under a variety of conditions designed to inhibit unidirectional sodium uptake (JinNa; low external [NaCl], 10(-4) mol l-1 amiloride), alter transbranchial PNH3 and NH4+ gradients [24 h continuous (NH4)2SO4 infusion, or exposure to 1 mmol l-1 external total ammonia at pH 8] and prevent gill boundary layer acidification (5 mmol l-1 Hepes buffer). Inhibition of JinNa with amiloride or low external [NaCl] under normal conditions reduced JAmm by about 20 %, but did not prevent the net excretion of ammonia during exposure to high concentrations of external ammonia. Increasing the buffer capacity of the ventilatory water with Hepes buffer (pH 8) reduced JAmm by 36 % and abolished the effect of amiloride on ammonia excretion. No evidence could be found to support a directly coupled apical Na+/NH4+ exchange. We suggest that any dependence of ammonia excretion on sodium uptake is caused by alteration of transbranchial PNH3 gradients within the gill microenvironment secondary to changes in net H+ excretion. Under normal conditions (pH 8, low external ammonia) gill boundary layer acidification facilitates over one-third of the total ammonia excretion. During exposure to high concentrations of external ammonia in poorly buffered water, estimates of transbranchial PNH3 gradients from measurements of bulk water pH and total ammonia concentration (TAmm) may be grossly in error because of boundary layer acidification. Prevention of boundary layer acidification with Hepes buffer during exposure to high cocncentrations of external ammonia revealed that the local transbranchial PNH3 gradient at the gill may in fact be positive (blood to water), negating the need for an active NH4+ transport mechanism. In freshwater trout, NH3 diffusion may account for all ammonia excretion under all experimental conditions used in the present study.

Journal Article↗