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

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, [ 3 H]‐P1075, and by electrophysiological techniques. 2 Saturation binding experiments gave a K D value of 9.2±5.2 n M and a binding capacity (B Max ) of 140±40 fmol mg −1 protein for [ 3 H]‐P1075 binding to A10 cells; from the B Max value a density of binding sites of 5–10 per μm 2 plasmalemma was estimated. 3 K ATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [ 3 H]‐P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [ 3 H]‐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 EC 50 values of 170±40 n M and 870±190 n M , respectively. The hyperpolarization induced by levcromakalim (3 μ M ) was completely reversed by glibenclamide with an IC 50 value of 86±17 n M . 5 Voltage clamp experiments were performed in the whole cell configuration under a physiological K + gradient. Levcromakalim (10 μ M ) induced a current which reversed around −80 mV; the current‐voltage relationship showed considerable outward rectification. Glibenclamide (3 μ M ) abolished the effect of levcromakalim. 6 Analysis of the noise of the levcromakalim (10 μ M )‐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 [ 3 H]‐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.British Journal of Pharmacology (1997) 122 , 1119–1126; doi: 10.1038/sj.bjp.0701514