Activation of BKCa channels via cyclic AMP- and cyclic GMP-dependent protein kinases by eugenosedin-A in rat basilar artery myocytes

Background and purpose: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K-DR)- or large-conductance Ca2+ -activated potassium (BKCa)-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and - independent protein kinases. Experimental approach: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+- and Ca2+ channel activities. Key results: Eugenosedin-A (1 mu M) did not affect the K-DR current but dramatically augmented BKCa channel activity in a concentration-dependent manner. Increased BKCa current was abolished by charybdotoxin (ChTX, 0.1 mu M) or iberiotoxin (IbTX, 0.1 mu M), but not affected by a small-conductance K-Ca blocker (apamin, 100 mu M). BKCa current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 mu M), a soluble guanylate cyclase inhibitor (ODQ, 10 mM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 mu M and Rp-cGMP, 100 mu M), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 mu M and KT5823, 0.3 mu M). Eugenosedin-A reversed the inhibition of BKCa current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 mu M). Eugenosedin-A also prevented BKCa current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca+2 current (I-Ca,(L)), but without modifying the voltage-dependence of the current. Conclusions and implications: Eugenosedin-A enhanced BKCa currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.