Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery.
ABSTRACT Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH(i)) changers such as sodium acetate (SA) and NH(4)Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH(4)Cl. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R (340/380)). SA (20 mM) increased and NH(4)Cl (20 mM) decreased R (340/380) by 0.2 +/- 0.03 and 0.1 +/- 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R (340/380) by 0.9 +/- 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH(4)Cl, implying that SA and NH(4)Cl may affect [Ca(2+)](i) (p < 0.05). Accordingly, we studied the effects of SA and NH(4)Cl on Ca(2+)-activated K(+) current (IK(Ca)) under patch clamp technique. Caffeine produced transient outward current at holding potential (V (h)) of 0 mV, caffeine induced transient outward K(+) current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH(4)Cl. In addition, IK(Ca) was significantly increased by acidotic condition when pH(i) was lowered by altering the NH(4)Cl gradient across the cell membrane. Finally, the effects of SA and NH(4)Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was -28 +/- 2.3 mV in a single cell level and was depolarized by 13 +/- 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH(4)Cl depolarized RMP by 10 +/- 1.9 and 16 +/- 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH(4)Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca(2+)](i) and Ca(2+)-activated K channels in rabbit basilar artery.
- SourceAvailable from: physoc.org[show abstract] [hide abstract]
ABSTRACT: In smooth muscle cells freshly dispersed from the rabbit portal vein, effects of intracellular pH (pHi) on Ca2+ channel currents were studied with the whole-cell clamp method using nystatin in the pipette. pHi was modified with ammonium chloride (NH4Cl) and propionate. Changes in intracellular Ca2+ concentration ([Ca2+]i) and pHi were also measured with the fluorescent indicator fura-2 and a pH-sensitive dye, respectively, together with the mechanical response in intact tissues. Intracellular alkalinization caused by an application of NH4Cl (20 mM) markedly potentiated and acidification caused by propionate (20 mM) inhibited inward Ca2+ channel currents, without much change in the kinetics. Tension development induced by 60 mM K- was inhibited by NH4Cl (20 mM) and potentiated by propionate (20 mM), whereas the peak [Ca2+]i level reached during K+ contracture was reduced in the presence of NH4Cl and increased in the presence of propionate. It was concluded that the modification of Ca2+ channel currents caused by pHi is not directly related to the effects of pHi on the mechanical response to excess K+. The direct effects of pHi on [Ca2+]i and on contractile machinery are considered to be mainly responsible for the mechanical effect of pHi.Experimental Physiology 10/1994; 79(5):669-80. · 2.79 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca(2+)-uptake which increased linearly with time for > 1 h. The Ca(2+)-uptake occurred with Km values of 0.20 +/- 0.03 microM for Ca2+ and 400 +/- 34 microM for MgATP2-. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13 +/- 0.02 and 0.56 +/- 0.04 microM, respectively. These properties of SR Ca(2+)-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4-6.8).Molecular and Cellular Biochemistry 11/1995; 151(2):149-55. · 2.33 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: 1. Intracellular pH (pHi) and intracellular [Ca2+] ([Ca2+]i) were measured during changes to superfusate PCO2 and/or [NaHCO3]. Changes to superfusate PCO2 produced sustained changes to pHi and [Ca2+]i, while changes to [NaHCO3] altered only extracellular pH (pHo). 2. Carbachol or caffeine induced a transient rise of [Ca2+]i due to Ca2+ release from an intracellular store. This Ca2+ transient was reduced by extracellular acidosis, but increased by intracellular acidosis. Alkalosis in either compartment produced opposite effects to acidosis. Changes to the Ca2+ transient mirrored those to phasic tension previously reported in this preparation. 3. A raised superfusate [K+] also induced a Ca2+ transient, due to transmembrane influx of Ca2+. This transient was depressed by extracellular acidosis, but unaffected by changes to pHi. The L-type Ca2+ current was similarly affected by changes to pHo, but not by alteration of pHi. 4. The results suggest that extracellular acidosis depresses the Ca2+ transient by reducing transmembrane influx through the L-type Ca2+ channel. The increase in the carbachol- and caffeine-induced Ca2+ transients by intracellular acidosis is due to enhancement of Ca2+ uptake into intracellular stores as a result of a raised resting [Ca2+]i.The Journal of Physiology 05/1998; 508 ( Pt 1):131-43. · 4.38 Impact Factor