Open BK Ca Channels Research Articles

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum.

Large-conductance Ca(2+)-activated K(+) (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca(2+), blocked by penitrem A, and had a conductance of 291 ± 20 pS (n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.

Original Research: Role of phosphodiesterases in modulation of BKCa channels in hypertensive pulmonary arterial smooth muscle

BKCa channels regulate pulmonary arterial pressure, and protein kinase C (PKC) inhibits BKCa channels, but little is known about PKC-mediated modulation of BKCa channel activity in pulmonary arterial smooth muscle. Studies were carried out to determine mechanisms of PKC modulation of BKCa channel activity in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), an animal model of pulmonary hypertension. Forskolin opened BKCa channels in FHR PASMC, which was blocked by PKC activation, and reversed by the phosphodiesterase (PDE) inhibitors IBMX, milrinone, and zaprinast. PDE inhibition also blocked the vasoconstrictor response to PKC activation in FHR pulmonary arteries. These results indicate that PKC inhibits cAMP-induced activation of BKCa channels and causes pulmonary vasoconstriction in hypertensive pulmonary arterial smooth muscle via PDE, which further suggests PDE inhibitors for treatment of pulmonary hypertension.

Effect of PKC Isozyme Inhibition on Forskolin-Induced Activation of BKCa Channels in Rat Pulmonary Arterial Smooth Muscle

Signaling mechanisms that elevate cyclic AMP (cAMP) activate large-conductance, calcium- and voltage-activated potassium (BKCa) channels in vascular smooth muscle and cause vasodilatation. In pulmonary vascular smooth muscle (PVSM), BKCa channel modulation is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel causes pulmonary vasoconstriction. Protein kinase C (PKC) modulates BKCa channels in systemic vascular smooth muscle, but little is known about the effect of PKC on BKCa channel activity in PVSM. A novel finding from our laboratory showed that PKC activates BKCa channels in rat pulmonary arterial smooth muscle and, having observed that cAMP-elevating agents also open BKCa channels, we hypothesized that PKC may open BKCa channels via a cAMP-dependent mechanism. Forskolin (10 microM), an activator of adenylyl cyclase, which increases cAMP concentration, opened BKCa channels in single pulmonary arterial smooth muscle cells (PASMC) of the Sprague-Dawley rat. The effect of forskolin was completely blocked by the PKC inhibitor Go 6983, which selectively blocks the alpha, beta, delta, gamma, and zeta PKC isozymes, and, by rottlerin, which selectively inhibits PKCdelta, and partially blocked by Go 6976, which selectively inhibits PKCalpha PKCbeta, and PKCmu. These results indicate that specific PKC isozymes mediate forskolin-induced activation of BKCa channels in PASMC, which suggests that a signaling pathway involving PKC activation and cAMP exists in pulmonary arterial smooth muscle to open BKCa channels.

Hypoxia Modulates Cyclic AMP Activation of BKCa Channels in Rat Pulmonary Arterial Smooth Muscle

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of potassium channel activity in pulmonary vascular smooth muscle are currently an area of great interest. Normally, signaling mechanisms that elevate cyclic AMP (cAMP) open potassium channels. Modulation of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCa channel has been implicated in the development of pulmonary vasoconstriction. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BKCa channel activity under normoxic and hypoxic conditions using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR). Forskolin (10 microM; n = 4), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and CPT-cAMP (100 microM; n = 3), a membrane-permeable derivative of cAMP, opened BKCa channels in single FHR PASMC under normoxic conditions. Exposure of FHRs to 4 weeks of 10% O2 (hypoxia) significantly attenuated the effect of both forskolin (n = 7) and CPT-cAMP (n = 14) on BKCa channel activity in PASMC. These results suggest that this phenomenon may serve as a physiological mechanism to cause hypoxic vasoconstriction in the pulmonary circulation via modulation of BKCa channels.

Diazepam, γ-aminobutyric acid, and progesterone open K + channels in myocytes from coronary arteries

Benzodiazepines enhance coronary blood flow and lower blood pressure, but the cellular basis of this action remains unclear. The present study now demonstrates a direct effect of diazepam, γ-aminobutyric acid (GABA), and progesterone on the large conductance, Ca 2+- and voltage-activated K + channel (BK Ca) in single myocytes isolated from porcine coronary arteries. These GABA receptor agonists significantly increased whole-cell (perforated patch) K + currents and stimulated the activity of single BK Ca channels in cell-attached patches dramatically. This effect is not mediated via cyclic AMP or cyclic GMP, but involves stimulation of Ca 2+ influx in response to activation of a bicuculline-sensitive GABA A-like receptor. We propose that localized, subsarcolemmal increases in Ca 2+ levels open BK Ca channels, thereby promoting K + efflux, membrane repolarization, and coronary relaxation. This transduction pathway can now account, at least in part, for the direct vasodilatory effects of diazepam, progesterone, and GABA.

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.