The intermediate conductance calcium-activated potassium channel KCa3.1 regulates vascular smooth muscle cell proliferation via controlling calcium-dependent signaling.

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF ß-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Ecto-5'-nucleotidase, CD73, is an endothelium-derived hyperpolarizing factor synthase.

OBJECTIVE: Adenosine dilates human coronary arteries by activating potassium channels in an endothelial cell-independent manner. Cell surface ecto-5'-nucleotidase (CD73) rapidly dephosphorylates extracellular adenosine 5'-monophosphate to adenosine. We tested the hypothesis that coronary vasodilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that acts as an endothelium-derived hyperpolarizing factor. METHODS AND RESULTS: Videomicroscopy showed that adenine nucleotides, but not inosine, potently dilated and hyperpolarized human coronary arteries independent of nitric oxide, prostacyclin, and classical endothelium-derived hyperpolarizing factors, whereas endothelial denudation, adenosine receptor antagonism, adenosine deaminase, or CD73 blockers reduced vasodilations. Liquid chromatography-electrospray ionization-mass spectrometry revealed adenosine accumulation in perfusates from arteries in the presence of adenosine 5'-diphosphate. CD73 was localized on the cell surface of endothelial cells, but not of vascular smooth muscle cells, and its deficiency suppressed vasodilation of mouse coronary arteries to adenine nucleotides and augmented vasodilation to adenosine. Adenosine dose-dependently dilated and hyperpolarized human coronary arteries to a similar extent as adenosine 5'-diphosphate. CONCLUSIONS: Coronary vasodilation to adenine nucleotides is associated with endothelial CD73-dependent production of extracellular adenosine that acts as an endothelium-derived hyperpolarizing factor by relaxing and hyperpolarizing underlying vascular smooth muscle cells via activating adenosine receptors. Thus, CD73 is a novel endothelium-derived hyperpolarizing factor synthase in human and mouse coronary arteries.

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation.

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Mechanism of vasodilation to adenosine in coronary arterioles from patients with heart disease.

Adenosine is a key myocardial metabolite that elicits coronary vasodilation in a variety of pathophysiological conditions. We examined the mechanism of adenosine-induced vasodilation in coronary arterioles from patients with heart disease. Human coronary arterioles (HCAs) were dissected from pieces of the atrial appendage obtained at the time of cardiac surgery and cannulated for the measurement of internal diameter with videomicroscopy. Adenosine-induced vasodilation was not inhibited by endothelial denudation, but A(2) receptor antagonism with 3,7-dimethyl-1-propargylxanthine and adenylate cyclase (AC) inhibition with SQ22536 significantly attenuated the dilation. In contrast, A(1) receptor antagonism with 8-cyclopentyl-1,3-dipropylxanthine significantly augmented the sensitivity to adenosine. Moreover, dilation to A(2a) receptor activation with 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine hydrochloride was reduced by the A(1) receptor agonist (2S)-N(6)-(2-endo-norbornyl)adenosine. The nonspecific calcium-activated potassium (K(Ca)) channel blocker tetrabutylammonium attenuated adenosine-induced dilation, as did the intermediate-conductance K(Ca) blocker clotrimazole. Neither the large-conductance K(Ca) blocker iberiotoxin nor small-conductance K(Ca) blocker apamin altered the dilation. In conclusion, adenosine endothelium independently dilates HCAs from patients with heart disease through a receptor-mediated mechanism that involves the activation of intermediate-conductance K(Ca) channels via an AC signaling pathway. The roles of A(1) and A(2) receptor subtypes are opposing, with the former being inhibitory to AC-mediated dilator actions of the latter. These observations identify unique fundamental physiological characteristics of the human coronary circulation and may help to target the use of novel adenosine analogs for vasodilation in perfusion imaging or suggest new strategies for myocardial preconditioning.