BETA-ADRENOCEPTOR STIMULATION ACTIVATES LARGE-CONDUCTANCE CA2+-ACTIVATED K+ CHANNELS IN SMOOTH-MUSCLE CELLS FROM BASILAR ARTERY OF GUINEA-PIG

We studied the effect of isoproterenol on the Ca2+-activated K+ (BK) channel in smooth muscle cells isolated from the basilar artery of the guinea pig. Cells were studied in a whole-cell configuration to allow the clamping of intracellular Ca2+ concentration, [Ca2+](i). Macroscopic BK channel currents were recorded during depolarizing test pulses from a holding potential (V-H) of 0 mV, which was used to inactivate the outward rectifier. The outward macroscopic current available from a V-H of 0 mV was highly sensitive to block by external tetraethylammonium-Cl (TEA) and charybdotoxin, and was greatly augmented by increasing [Ca2+](i) from 0.01 to 1.0 mu M. With [Ca2+](i) between 0.1 and 1.0 mu M, 0.4 mu M isoproterenol increased this current by 58.6+/-17.1%, whereas with [Ca2+](i) at 0.01 mu M a sixfold smaller increase was observed. With [Ca2+](i) greater than or equal to 0.1 mu M, 100 mu M dibutyryl -adenosine 3':5: cyclic monophosphate (cAMP) and 1 mu M forskolin increased this current by 58.5+/-24.1% and 59.7+/-10.3%, respectively. The increase with isoproterenol was blocked by 4.0 mu M propranolol extracellularly, and by 10 U/ml protein kinase inhibitor intracellularly. Single-channel openings during depolarizing test pulses from a V-H of 0 mV recorded in the whole-cell configuration under the same conditions (outside-out-whole-cell recording) indicated a slope conductance of 260 pS. In conventional outside-out patches, this 260 pS channel was highly sensitive to block by external TEA, and in inside-out patches, its probability of opening was greatly augmented by increasing [Ca2+](i) from 0.01 to 1.0 mu M. Outside-out-whole-cell recordings with [Ca2+](i) greater than or equal to 0.1 mu M indicated that 100 mu M dibutyryl-cAMP increased the probability of opening of the 260-pS channel by 152+/-115%. In inside-out patches, the catalytic subunit of protein kinase A increased the probability of opening, and this effect also depended on [Ca2+](i), with a 35-fold larger effect observed with 0.1-0.5 mu M Ca2+ compared to 0.01 mu M Ca2+. We conclude that the BK channel in cerebrovascular smooth muscle cells can be activated by beta-adrenoceptor stimulation, that the effect depends strongly on [Ca2+](i), and that the effect is mediated by cAMP-dependent protein kinase A with no important contribution from a direct G-protein or phosphorylation-independent mechanism. Our data indicate that the BK channel may participate in beta-adrenoceptor-mediated relaxation of cerebral vessels, although the importance of this pathway in obtaining vasorelaxation remains to be determined.