4-Aminopyridine affects rat arterial smooth muscle BK(Ca) currents by changing intracellular pH

1. The hypothesis whether or not 4-AP can affect vascular smooth muscle BK(Ca) currents was tested using the patch-clamp technique, pH- and calcium-fluorimetry, and freshly isolated rat arterial smooth muscle cells. 2. Application of 4-AP reversibly inhibited BK(Ca) currents at an intracellular calcium ([Ca](i)) of 250 nM with a half-block of 2.5 mM at +50 mV. 3. The presence of 2 μM thapsigargin, 10 μM heparin, and 10 μM ryanodine did not alter the effect of 4-AP on BK(Ca) currents at [Ca](i) 250 nM. 4. At [Ca](i)<100 nM 4-AP did not inhibit BK(Ca) currents. 5. Application of 4-AP to the intracellular or extracellular side of excised BK(Ca) channels did not alter channel activity or channel amplitude. 6. Replacement of the pH-sensitive calcium buffer EGTA by the pH-insensitive calcium buffer BAPTA in the intracellular solution turned the 4-AP-induced inhibition of BK(Ca) currents into a stimulation at [Ca](i) 250 nM. 7. Application of 4-AP to single cells increased intracellular pH, which was accompanied by a reduction of [Ca](i) in EGTA-loaded cells and a stable [Ca](i) in BAPTA-loaded cells. 8. Thus, these results suggest that in isolated vascular smooth muscle cells at [Ca](i)>100 nM 4-AP affects BK(Ca) currents via an alteration of intracellular pH

Effects of some sterically hindered phenols on whole-cell Ca(2+) current of guinea-pig gastric fundus smooth muscle cells

1. The aim of the present study was to investigate the effects of extracellular application of some sterically-hindered phenols, namely 3-t-butyl-4-hydroxyanisole (BHA), 3,5-di-t-butyl-4-hydroxyanisole (DTBHA) and the dimer of BHA, 2,2′-dihydroxy-3,3′-di-t-butyl-5,5′-dimethoxydiphenyl (DIBHA), on the whole-cell Ca(2+) current (I(Ca)) of freshly isolated smooth muscle cells from the guinea-pig gastric fundus, in the presence of a range of Ca(2+) concentrations (1 – 5 mM) using the patch-clamp technique. The influx of Ca(2+) had characteristics of L-type I(Ca) (I(Ca(L))). 2. BHA as well as DTBHA inhibited I(Ca(L)) in a concentration-dependent manner, during depolarization to 10 mV from a holding potential of −50 mV. Bath application of BHA (50 μM) and DTBHA (30 μM) decreased I(Ca(L)) by 48.9% and 45.2%, respectively. This inhibition was only partially reversible. In contrast, DIBHA (up to 50 μM) was devoided of effects on I(Ca(L)). 3. BHA inhibition of I(Ca(L)) was voltage-dependent and inversely related to the external concentration of Ca(2+). On the other hand, DTBHA inhibition was only voltage-dependent. 4. BHA and DTBHA shifted the voltage range of the steady-state inactivation curve to more negative potentials by 8 mV at the mid-potential of the curve, without affecting the activation curve. Furthermore, BHA and DTBHA did not modify the time-course of the current decay. 5. We conclude that the inhibition of I(Ca(L)) by BHA and DTBHA is qualitatively similar to that of a Ca(2+) channel blocker and is characterized by the stabilizing effect of the inactivated state of the channel