Modulation of Ca2+ channel activity by ATP metabolism and internal Mg2+ in guinea-pig basilar artery smooth muscle cells.
1. Single smooth muscle cells were isolated from the basilar artery of the guinea-pig and, within 10 h, inward currents through voltage-gated Ca2+ channels were recorded using the amphotericin or conventional whole-cell voltage-clamp techniques. 2. In amphotericin whole-cell recordings, bath application of 2,4-dinitrophenol (DNP, an uncoupler of mitochondrial ATP production) induced an initial stimulation (14% increase in 5 of 11 cells) and then pronounced inhibition (50% decrease in 9 of 11 cells within 9.5 min) of voltage-dependent Ca2+ current (I(Ca)) elicited by depolarizing to +10 mV in 1.5 mM extracellular Ca2+ solution. By contrast, inhibition of glycolysis by replacing glucose in the bath with 2-deoxy-D-glucose had no effect. 3. Na+ current through Ca2+ channels (I[(Ca)(Na)]) recorded in the absence of extracellular divalent cations also responded to DNP, again with stimulation followed by inhibition of current. The stimulation of I[(Ca)(Na)] was associated with a leftward shift of the Ca2+ channel activation curve which averaged -9 mV. A combination of 2-deoxy-D-glucose, mannoheptulose and 3-0-methyl-glucose had only minor effects on I[(Ca)(Na)], whereas rotenone had an effect similar to that of DNP in six of eight cells. 4. The amplitude of I[(Ca)(Na)] in conventional whole-cell recordings was not different from that in amphotericin whole-cell recordings, even without ATP in the recording pipette and with metabolic poisons in the bath solution. Furthermore, attempts to dephosphorylate the Ca2+ channels in ATP-free conditions did not prevent I[(Ca)(Na)], and a high concentration of Mg-ATP with or without a phosphorylation-supporting medium in the recording pipette did not increase its amplitude. 5. In the absence of ATP, Mg2+ inhibited whole-cell I[Ca)(Na)] with a K(d) of about 100 mu M at -10 mV and induced a leftward shift of the Ca2+ channel activation curve. When ATP and a phosphorylation-supporting medium were in the recording pipette the blocking effect of free Mg2+ was reduced but the shift in the Ca2+ channel activation curve was unaffected. 6. From these data it is suggested that inhibition of mitochondrial, but not glycolytic, ATP production has stimulatory and inhibitory effects on voltage-gated Ca2+ channels of basilar artery smooth muscle cells. Effects of intracellular Mg2+ on the Ca2+ channels were modulated by ATP and mimicked the effects of metabolic poisoning by DNP. A hypothesis is discussed in which the intracellular free Mg2+ concentration may be a key factor coupling ATP production to Ca2+ channels.
McHugh
D
D
Department of Pharmacology, University of Leeds, Leeds, UK.
Beech
D J
DJ
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001488:Basilar Artery / Q000166:cytology / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D006168:Guinea Pigs; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*
D015220:Calcium Channels; D000666:Amphotericin B; D000255:Adenosine Triphosphate; D008274:Magnesium; D019297:2,4-Dinitrophenol