Binding and effects of K(ATP) channel openers in the vascular smooth muscle cell line, A10

Abstract

1. The ATP-sensitive K+ channel (K(ATP) channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated K(ATP) channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a K(D) value of 9.2 ± 5.2 nM and a binding capacity (B(Max)) of 140 ± 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the B(Max) value a density of binding sites of 5-10 per μm2 plasmalemma was estimated. 3. K(ATP) channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 ± 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 ± 40 nM and 870 ± 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 μM) was completely reversed by glibenclamide with an IC50 value of 86 ± 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 μM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 μM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 μM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 μm-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional K(ATP) channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional K(ATP) channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.