Cellular target of voltage and calcium-dependent K+ channel blockers involved in EDHF-mediated responses in rat superior mesenteric artery

1 We have investigated the cellular target of K+ channel blockers responsible for the inhibition of the EDHF-mediated relaxation in the rat mesenteric artery by studying their effects on tension, smooth muscle cell (SMC) membrane potential and endothelial cell Ca2+ signal ([Ca2+](endo)). 2 In arteries contracted with prostaglandin F-2 alpha (2.5 - 10 muM), relaxation evoked by ACh (0.01 - 3 muM) was abolished by a combination of charybdotoxin (ChTX, 0.1 muM) plus apamin (Apa, 0.1 muM) and was inhibited by 68 +/-6% (n=6) by 4-aminopyridine (4-AP, 5 mm). 3 ACh (0.001 - 3 muM) increased [Ca2+](endo) and hyperpolarized SMCs with the same potency, the pD(2) values were equal to 7.2 +/-0.08 (n=4) and 7.2 +/-0.07 (n=9), respectively. SMCs hyperpolarization to ACh (1 mum) was abolished by high K+ solution or by ChTX/Apa. It was decreased by 66 +/-5% (n=6) by 4-AP. 4 The increase in [Ca2+](endo) evoked by ACh (1 muM) was insensitive to ChTX/Apa but was depressed by 58 +/- 16% (n = 6) and 27 +/-4% (n=7) by raising external K+ concentration and by 4-AP, respectively. 5 The effect of 4-AP on [Ca2+](endo) was not affected by increasing external K+ concentration. In Ca-free/EGTA solution, the transient increase in [Ca2+](endo) evoked by ACh (1 muM) was abolished by thapsigargin (1 muM) and was decreased by 75 +/-7% (n=5) by 4-AP. 6 These results show that inhibition of EDHF-evoked responses by 4-AP may be attributed to a decrease in the Ca2+ release activated by ACh in endothelial cells. The abolition of SMCs hyperpolarization to ACh by ChTX/Apa is not related to an interaction with the [Ca2+](endo).