Endothelial-derived hyperpolarization factor (EDHF) contributes to PlGF-induced dilation of mesenteric resistance arteries from pregnant rats

The aim of this study was to investigate the cellular mechanism involved in the potent vasodilatory action of PlGF on mesenteric resistance arteries from pregnant rats. PlGF (3 nM) induced a vasodilation of 64 ± 3.8% that was completely abolished by endothelial denudation. Significant dilation (28 ± 4.0%) remained, however, in the presence of nitric oxide synthase and cyclooxygenase inhibition, and was associated with significant reductions in vascular smooth muscle cell calcium. Absence of dilation in potassium-depolarizing solution (30 mM) confirmed its dependence on endothelial-derived hyperpolarization factor. Subsequent studies established that vasodilation was abolished by pharmacologic inhibition of SK(Ca) (apamin) and BK(Ca) (iberiotoxin) but not IK(Ca) (tram-34) potassium channels. In summary, PlGF acts through the release of a combination of endothelium-derived relaxation factors. Based on the results of potassium channel blockade, we suggest that it induces endothelial hyperpolarization via SK(Ca) channel activation; this, in turn, leads to the release of a diffusible mediator that activates vascular smooth muscle BK(Ca) channels, hyperpolarization and vasodilation. This is the first study to identify the mechanism for PlGF/VEGFR-1 resistance artery dilation in the pregnant state, whose attenuation likely contributes to the systemic hypertension characteristic of pre- eclampsia.     

Role of potassium channels in the vascular response to endogenous and pharmacological vasodilators

Many endogenous and pharmacological vasodilators hyperpolarize vascular smooth muscle and this response appears to be due to an increased conductance to potassium ions. The hyperpolarization may contribute to the mechanism of dilation by causing voltage-dependent calcium channels to close. Recent evidence indicates that the response to hyperpolarizing vasodilators is mediated through activation of ATP-sensitive potassium (KATP) channels. Single KATP channels on isolated vascular smooth muscle cells are activated by cromakalim and calcitonin gene-related peptide (CGRP). This response is inhibited by glibenclamide. Cromakalim, CGRP and other vasodilators hyperpolarize and relax arteries in vitro and these responses are reversed by glibenclamide. The hypotensive effects of these agents in vivo are antagonized by glibenclamide. We propose that activation of KATP channels and the associated membrane hyperpolarization represents an important general mechanism of vasodilation.     

Vasoregulation at the molecular level: a role for the beta1 subunit of the calcium-activated potassium (BK) channel

Essential hypertension is among the most common and most costly medical conditions in the United States. Multiple defects in the kidneys, the vasculature, and the neuro-endocrine system may contribute to the development of this disorder. Within the past decade investigators have identified several molecular components of the vasculature that control tone and influence blood pressure. For example, the large conductance BK type calcium-activated potassium channel has recently been shown to play an important role in maintaining the dynamic equilibrium between vasoconstriction and vasodilation of vascular smooth muscle. Activation of vascular smooth muscle BK channels leads to hyperpolarization of the cell membrane, which causes deactivation of voltage-dependent calcium channels and vasodilation. In this review, we will summarize recently published data focusing on the role of the BK channel's accessory beta1 subunit as well as other modulators of BK channel activation that influence vascular tone and blood pressure.     

大电导钙激活钾通道在高血压病中的研究进展

大电导钙激活钾通道（BKCa）是血管平滑肌细胞（VSMCs）上表达最丰富的钾通道,对维持VSMCs的膜电位及血管收缩和舒张的动态平衡具有重要的调节作用。BKCa通道的激活可使细胞膜发生超极化,从而抑制电压依赖性钙通道的激活和钙离子内流,导致平滑肌舒张。对高血压患者的观察和高血压动物模型的研究发现,高血压血管张力升高时平滑肌细胞膜表面钾离子和钙离子通道表达和功能均发生异常,因此,有人推测高血压是离子通道重构导致平滑肌细胞去极化的结果。本文主要综述近年来BKCa通道在高血压病中的研究进展。     

大电导钙离子激活钾通道在心血管疾病中的研究进展

冠状动脉平滑肌细胞膜上存在许多大电导钙离子激活钾（BKCa）通道,在维持细胞正常生理活动中起重要作用。研究发现当细胞膜去极化或/（和）细胞内钙离子增加时,BKCa通道激活,开放增加,钾离子外流,细胞膜超极化,血管舒张。而在高血压、糖尿病、缺氧、心力衰竭和老化等许多病理情况下,BKCa通道功能发生改变,从而影响对血管功能的调节。本文主要综述近年来BK通道在心血管疾病中的研究进展。     

Endothelium-derived hyperpolarizing factor: where are we now?

The endothelium controls vascular tone not only by releasing nitric oxide (NO) and prostacyclin but also by other pathways causing hyperpolarization of the underlying smooth muscle cells. This characteristic was at the origin of the denomination endothelium-derived hyperpolarizing factor (EDHF). We know now that this acronym includes different mechanisms. In general, EDHF-mediated responses involve an increase in the intracellular calcium concentration, the opening of calcium-activated potassium channels of small and intermediate conductance and the hyperpolarization of the endothelial cells. This results in an endothelium-dependent hyperpolarization of the smooth muscle cells, which can be evoked by direct electrical coupling through myo-endothelial junctions and/or the accumulation of potassium ions in the intercellular space. Potassium ions hyperpolarize the smooth muscle cells by activating inward rectifying potassium channels and/or Na+/K(+)-ATPase. In some blood vessels, including large and small coronary arteries, the endothelium releases arachidonic acid metabolites derived from cytochrome P450 monooxygenases. The epoxyeicosatrienoic acids (EET) generated are not only intracellular messengers but also can diffuse and hyperpolarize the smooth muscle cells by activating large conductance calcium-activated potassium channels. Additionally, the endothelium can produce other factors such as lipoxygenases derivatives or hydrogen peroxide (H2O2). These different mechanisms are not necessarily exclusive and can occur simultaneously.     

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.     

cAMP-independent dilation of coronary arterioles to adenosine : role of nitric oxide, G proteins, and K(ATP) channels.

Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 microm in diameter) were isolated, cannulated, and pressurized to 60 cm H(2)O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K(ATP)) channels by glibenclamide, and inhibition of NO synthase by N(G)-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K(ATP) channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert its vasodilatory function. On one hand, adenosine opens endothelial K(ATP) channels through activation of pertussis toxin-sensitive G proteins. This signaling leads to the production and release of NO, which subsequently activates smooth muscle soluble guanylyl cyclase for vasodilation. On the other hand, adenosine activates smooth muscle K(ATP) channels and leads to vasodilation through hyperpolarization. It appears that the latter vasodilatory process is independent of G proteins and of cAMP/cGMP pathways.     

Blockade of the ATP-sensitive potassium channel modulates reactive hyperemia in the canine coronary circulation.

The mechanism of reactive hyperemia remains unknown. We hypothesized that reactive hyperemia was related to the opening of ATP-sensitive potassium channels during coronary occlusion. The resulting hyperpolarization of the smooth muscle cell plasma membrane might reduce calcium influx through voltage-dependent calcium channels and result in relaxation of smooth muscle tone and vasodilation. In eight open-chest, anesthetized dogs, 30-second coronary occlusions resulted in an average flow debt repayment of 200 +/- 41%. After low-dose (0.8 mumol/min) and high-dose (3.7 mumol/min) infusion of intracoronary glibenclamide, flow debt repayment fell to 76 +/- 14% and 50 +/- 8%, respectively (p less than 0.05 compared with control for both). The decline in flow debt repayment was due to a significant reduction both in maximum coronary conductance during reactive hyperemia and in its duration. In addition, there was a significant decline in the sensitivity of the coronary circulation to adenosine-induced vasodilation after glibenclamide. While more variable, there was no overall change in the sensitivity of the coronary vasculature to acetylcholine-induced vasodilation after glibenclamide. We conclude that reactive hyperemia is determined in a large part by the ATP-sensitive potassium channel, probably through its effect on membrane potential and voltage-sensitive calcium channels. Because reactive hyperemia was never fully abolished at the highest doses of glibenclamide tested, it is possible that additional mechanisms are involved in the genesis of this complex phenomenon.     

Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (-15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (-84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.     

The role of calcium in the regulation of normal vascular tone and in arterial hypertension

The vascular tone depends on periarterial neurogenic nerve stimulus and endothelial substances release. The most evident biochemical cause of the vascular smooth muscle contraction-relaxation process lies in the changing concentration of cytosolic Ca2+. Intracellular free calcium is the major determinant of vascular tone. The depolarization wave opens the slow calcium channels, which permit Ca2+ to enter in small quantities into the interior of the cell triggering the release of much larger quantities of calcium from the sarcoplasmic reticulum. The flux of Ca2+ to and from the cytosol is regulated by three principle mechanisms. The calcium voltage sensitive Ca2+ channel that are opened by the depolarization wave. The potassium channels (CK+) and the Na+/K(+)-ATPase pump. The CK+ opening permits the exit of potassium from the interior of the cell which tends to hyperpolarize the smooth muscle cell membrane and closes the calcium channel avoiding the entry of Ca2+. The activity of the sodium pump also produces membrane hyperpolarization. Thus, the activity of these two mechanisms counter-regulates the voltage dependent calcium channel. The massive release of Ca2+ from intracellular stores of the sarcoplasmic reticulum is done through two classes of channels. One is ryanodine sensitive, the other is the inositol 1,4,5-trisphosphate receptor. The endothelial cell dysfunction is accompanied by a decrease in the production and/or the release of nitric oxide and the increase of contracting factors. That induce a Ca2+ mobilization of extracellular and intracellular stores. Contraction of smooth muscle to hypoxia is mediated by an accumulation of intracellular Ca2+. The relaxant substances of vascular smooth muscle inhibit activation of the phospholipase C and open Ca2+ channels, or produce a stimulus to the exit of the Ca2+ through the plasmatic membrane, with a decrease of intracellular calcium. An endothelial dysfunction with decrease of nitric oxide release exists in different types of hypertension. Pregnancy-induced hypertension is associated with low calcium levels in the diet, improving with the treatment of calcium supplements.     

New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure

The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

Functional regulation of large conductance Ca2+-activated K+ channels in vascular diseases

The large conductance Ca²⁺-activated potassium channels, the BK channels, is widely expressed in various tissues and activated in a Ca²⁺- and voltage-dependent manner. The activation of BK channels hyperpolarizes vascular smooth muscle cell membrane potential, resulting in vasodilation. Under pathophysiological conditions, such as diabetes mellitus and hypertension, impaired BK channel function exacerbates vascular vasodilation and leads to organ ischemia. The vascular BK channel is composed of 4 pore-forming subunits, BK-α together with 4 auxiliary subunits: β₁ subunits (BK-β₁) or γ₁ subunits (BK-γ₁). Recent studies have shown that down-regulation of the BK β₁ subunit in diabetes mellitus induced vascular dysfunction; however, the molecular mechanism of these vascular diseases is not well understood. In this review, we summarize the potential mechanisms regarding BK channelopathy and the potential therapeutic targets of BK channels for vascular diseases.     

K+-induced dilation of hamster cremasteric arterioles involves both the Na+/K+-ATPase and inward-rectifier K+ channels

OBJECTIVE: The mechanism by which elevated extracellular potassium ion concentration ([K+]o) causes dilation of skeletal muscle arterioles was evaluated. METHODS: Arterioles (n = 111) were hand-dissected from hamster cremaster muscles, cannulated with glass micropipettes and pressurized to 80 cm H2O for in vitro study. The vessels were superfused with physiological salt solution containing 5 mM KCl, which could be rapidly switched to test solutions containing elevated [K+]o and/or inhibitors. The authors measured arteriolar diameter with a computer-based diameter tracking system, vascular smooth muscle cell membrane potential with sharp micropipettes filled with 200 mM KCl, and changes in intracellular Ca2+ concentration ([Ca2+]i) with Fura 2. Membrane currents and potentials also were measured in enzymatically isolated arteriolar muscle cells using patch clamp techniques. The role played by inward rectifier K+ (KIR) channels was tested using Ba2+ as an inhibitor. Ouabain and substitution of extracellular Na+ with Li+ were used to examine the function of the Na+/K+ ATPase. RESULTS: Elevation of [K+]o from 5 mM up to 20 mM caused transient dilation of isolated arterioles (27 +/- 1 microm peak dilation when [K+]o was elevated from 5 to 20 mM, n = 105, p <.05). This dilation was preceded by transient membrane hyperpolarization (10 +/-1 mV, n = 23, p <.05) and by a fall in [Ca2+]i as indexed by a decrease in the Fura 2 fluorescence ratio of 22 +/- 5% (n = 4, p <.05). Ba(2+) (50 or 100 microM) attenuated the peak dilation (40 +/- 8% inhibition, n = 22) and hyperpolarization (31 +/- 12% inhibition, n = 7, p <.05) and decreased the duration of responses by 37 +/-11% (n = 20, p < 0.05). Both ouabain (1 mM or 100 microM) and replacement of Na+ with Li+ essentially abolished both the hyperpolarization and vasodilation. CONCLUSIONS: Elevated [K+]o causes transient vasodilation of skeletal muscle arterioles that appears to be an intrinsic property of the arterioles. The results suggest that K+-induced dilation involves activation of both the Na+/K+ ATPase and KIR channels, leading to membrane hyperpolarization, a fall in [Ca2+]i, and culminating in vasodilation. The Na+/K+ ATPase appears to play the major role and is largely responsible for the transient nature of the response to elevated [K+]o, whereas KIR channels primarily affect the duration and kinetics of the response.     

Current views on mechanisms of vasodilation in response to ischemia and hypoxia

Myocardial function (and thus life) is entirely dependent on sufficient O₂ supply. Therefore, this supply is extremely well regulated via a refined system of interacting mechanisms. These have been subject to extensive research for more than 100 years. Surprisingly, remarkable dispute still arises among scientists concerning the factors and mechanisms involved in this regulatory system. During ischemia, myocardial cells have been shown to release vasoactive metabolites (eg, H⁺ and K⁺ ions, lactic acid, adenosine, and others), which cause spontaneous coronary dilation. On the other hand claims have been made that the endothelium itself could play a key role in hypoxic/ischemic vasodilation by releasing endothelium-derived relaxant factor (EDRF) (NO = nitric oxide) and other still partially unspecified vasoactive substances (eg, prostaglandins). Furthermore, it has been discussed that intravascular O₂ tension (pO₂) itself would exert a direct effect upon endothelial and/or vascular smooth muscle cells and thus produce per se a local reflectory vasodilation. In contrast, the intravascular CO₂ tension has also been shown to act upon coronary vascular resistance during myocardial ischemia. Recently, hints have been made about the membrane potential of arterial smooth muscle cells as a key factor in hypoxia/ischemia vasodilation. However, during hypoxia and metabolic inhibition, the membrane potential seems to be modulated primarily by the action of adenosinetriphosphate-dependent (ATP) potassium channels (K _ATP ⁺ channels). In conclusion, a number of factors contribute to ischemia/hypoxia-induced vasodilation. The present review contrasts recent findings on ATP-dependent K⁺ channels with other experimental evidence concerning other mechanisms involved in hypoxic/ischemic vasodilation.     

Opening of ATP-sensitive potassium channels causes generation of free radicals in vascular smooth muscle cells

Recent evidence suggests that opening of mitochondrial K_ATP channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K_ATP channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red™ for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 μM reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 μM) or pinacidil (100 μM), both potent mitochondrial K_ATP channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K_ATP channel inhibitor 5-hydroxydecanoate (5HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K_ATP channel blocker. Thus opening of K_ATP channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria. Received: 7 January 2002, Returned for revision: 31 January 2002, Revision received: 21 February 2002, Accepted: 14 March 2002     

Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.

OBJECTIVE: Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS: Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS: Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION: Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.