Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man. It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrate that 17 beta-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving K+ efflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates K+ channel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17 beta-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17 beta-estradiol on the gating of the large-conductance, Ca(2+)- and voltage-activated K+ (BKCa) channels, while 17 alpha-estradiol had no effect. Furthermore, blocking BKCa channels in intact arteries inhibited estrogen-induced relaxation. The effect of 17 beta-estradiol on BKCa channels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17 beta-estradiol relaxes coronary arteries by opening BKCa channels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease.

Cardiovascular diseases are often considered to be a predominantly male health problem, and it has been suggested that testosterone exerts deleterious effects on cardiovascular function; however, few experimental studies support this suggestion. Moreover, the cellular and molecular mechanism(s) underlying vascular responses to testosterone is unknown. The present study has investigated the acute effects of testosterone on porcine coronary artery smooth muscle at the tissue and cellular levels. Contractile studies demonstrated that testosterone or dihydrotestosterone (a nonaromatizable metabolite) relaxed these arteries by an endothelium-independent mechanism involving potassium efflux. Direct evidence from patch-clamp studies confirmed that testosterone opened K(+) channels in single coronary myocytes, and further analysis identified this protein as the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel. Moreover, inhibiting BK(Ca) channel activity significantly attenuated testosterone-induced coronary relaxation. These findings indicate that testosterone relaxes porcine coronary arteries predominantly by opening BK(Ca) channels in coronary myocytes, and this response may be associated with accumulation of cGMP. This novel mechanism may provide a better understanding of testosterone-induced vasorelaxation reported in recent experimental and early clinical studies.

It has been known for a number of years that neutrophils and macrophages secrete H2O2while fighting disease, and the levels obtained within the vasculature under these conditions can reach several hundred micromolar. Because the effect of H2O2on vascular smooth muscle is not fully understood, the present study examined the cellular effects of H2O2on coronary arteries. Under normal ionic conditions, H2O2relaxed arteries that were precontracted with prostaglandin F2α or histamine (EC50 = 252 ± 22 μM). The effect of H2O2was concentration dependent and endothelium independent. In contrast, H2O2did not relax arteries contracted with 80 mM KCl, suggesting involvement of K+ channels. Single-channel patch-clamp recordings revealed that H2O2increased the activity of the large-conductance (119 pS), Ca2+- and voltage-activated K+(BKCa) channel. This response was mimicked by arachidonic acid and inhibited by eicosatriynoic acid, a lipoxygenase blocker, suggesting involvement of leukotrienes. Further studies on intact arteries demonstrated that eicosatriynoic acid not only blocked the vasodilatory response to H2O2but unmasked a vasoconstrictor effect that was reversed by blocking cyclooxygenase activity with indomethacin. These findings identify a novel effector molecule, the BKCachannel, which appears to mediate the vasodilatory effect of H2O2, and suggest that a single signaling pathway, arachidonic acid metabolism, can mediate the vasodilatory and vasoconstrictor effects of H2O2and possibly other reactive oxygen species.

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.

Abstract-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 mol/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. Key Words: cAMP Ⅲ protein kinase G Ⅲ BK Ca channel Ⅲ coronary Ⅲ cross-activation E arly studies investigating the cellular effects of cyclic nucleotides demonstrated antagonism between cAMP and cGMP in most tissues. 1 An exception to this general rule was vascular smooth muscle (VSM), in which both nucleotides produced the same physiological response, ie, vasodilation; however, the signaling mechanisms by which cAMP or cGMP induced this response were unknown. Most investigations assumed that the vasodilatory response to either nucleotide was mediated via a distinct transduction cascade involving its corresponding nucleotide-activated protein kinase; however, research over the last 10 years has revealed a more complicated signaling process. Findings from the laboratories of Corbin and Lincoln demonstrated that "crossactivation" of the cGMP-dependent protein kinase (protein kinase G, PKG) by cAMP could be a key element in the signal transduction cascade of cAMP-induced vasodilation. 2,3 Subsequent studies have established that cGMP can also exert physiological effects by stimulating cAMP-dependent protein kinase (protein kinase A, PKA) activity. For example, PKA appears to mediate nitric oxide-dependent inhibition of aortic smooth muscle cell proliferation 4 and cGM...