cAMP-dependent vasodilators cross-activate the cGMP-dependent protein kinase to stimulate BK(Ca) channel activity in coronary artery smooth muscle cells.

cAMP-dependent vasodilators are used to treat a variety of cardiovascular disorders; however, the signal transduction pathways and effector mechanisms stimulated by these agents are not fully understood. In the present study we demonstrate that cAMP-stimulating agents enhance the activity of the large-conductance, calcium-activated potassium (BK(Ca)) channel in single myocytes from coronary arteries by "cross-activation" of the cGMP-dependent protein kinase (protein kinase G, PKG). Single-channel patch-clamp data revealed that 10 micromol/L isoproterenol, forskolin, or dopamine opens BK(Ca) channels in coronary myocytes and that this effect is attenuated by inhibitors of PKG (KT5823; Rp-8-pCPT-cGMPS), but not by inhibiting the cAMP-dependent protein kinase (protein kinase A, PKA). In addition, a membrane-permeable analog, CPT-cAMP, also opened BK(Ca) channels in these myocytes, and this effect was reversed by KT5823. Direct biochemical measurement confirmed that dopamine or forskolin stimulates PKG activity in coronary arteries but does not elevate cGMP. Finally, the stimulatory effect of cAMP on BK(Ca) channels was reconstituted in a cell-free, inside-out patch by addition of purified PKG activated by either cGMP or cAMP. In contrast, channel gating was unaffected by exposure to the purified catalytic subunit of PKA. In summary, findings from on-cell and cell-free patch-clamp experiments provide direct evidence that cAMP-dependent vasodilators open BK(Ca) channels in coronary myocytes by cross-activation of PKG (but not via PKA). Biochemical assay confirmed this cross-activation mechanism of cAMP action in these arteries. This signaling pathway is a novel mechanism for regulation of potassium channel activity in vascular smooth muscle and other cells.

A novel transduction mechanism mediating dopamine-induced vascular relaxation: opening of BKCa channels by cyclic AMP-induced stimulation of the cyclic GMP-dependent protein kinase.

Dopamine dilates the coronary, renal and other vascular beds; however, the signaling pathway underlying this effect is unclear. In this study the signal-transduction process mediating dopamine-induced relaxation of porcine coronary arteries was investigated in isolated vessels and single arterial myocytes. Dopamine-induced relaxation of arteries was mediated through the DA- receptor and involved K+ efflux, and subsequent patch-clamp studies demonstrated that either dopamine or fenoldopam, a selective DA-1 agonist, increased the opening probability of the large-conductance, calcium- and voltage-activated K+ (BKCa) channel in coronary myocytes. Moreover, blockade of this channel by iberiotoxin prevented dopamine-induced coronary relaxation. Dopamine stimulation of BKCa channels was completely prevented by a DA-1-receptor antagonist, but was unaffected by propranolol. Furthermore, inhibiting adenylyl cyclase activity prevented stimulation of BKCa channel activity, whereas chlorophenylthio (CPT)-cyclic adenosine monophosphate (AMP), a membrane-permeable analog of cyclic AMP, mimicked the effects of dopamine. Interestingly, inhibiting the cyclic AMP-dependent protein kinase (PKA) did not affect the response to dopamine, whereas dopamine-induced channel activity was completely blocked by inhibiting the activity of the cyclic guanosine monophosphate (GMP)-dependent protein kinase (PKG). These findings demonstrate that activation of DA-1 receptors causes stimulation of BKCa channel activity by a mechanism involving cyclic AMP-dependent stimulation of PKG, but not PKA, and further suggest that this cross-reactivity mediates dopamine-induced coronary vasodilation.