Abstract-Flow-induced dilation (FID) is dependent largely on hyperpolarization of vascular smooth muscle cells (VSMCs) in human coronary arterioles (HCA) from patients with coronary disease. Animal studies show that shear stress induces endothelial generation of hydrogen peroxide (H 2 O 2 ), which is proposed as an endothelium-derived hyperpolarizing factor (EDHF). We tested the hypothesis that H 2 O 2 contributes to FID in HCA. Arterioles (135Ϯ7 m, nϭ71) were dissected from human right atrial appendages at the time of cardiac surgery and cannulated with glass micropipettes. Changes in internal diameter and membrane potential of VSMCs to shear stress, H 2 O 2 , or to papaverine were recorded with videomicroscopy. In some vessels, endothelial H 2 O 2 generation to shear stress was monitored directly using confocal microscopy with 2Ј,7Ј-dichlorofluorescin diacetate (DCFH) or using electron microscopy with cerium chloride. Catalase inhibited FID (%max dilation; 66Ϯ8 versus 25Ϯ7%; PϽ0.05, nϭ6), whereas dilation to papaverine was unchanged. Shear stress immediately increased DCFH fluorescence in the endothelial cell layer, whereas treatment with catalase abolished the increase in fluorescence. Electron microscopy with cerium chloride revealed shear stress-induced increase in cerium deposition in intimal area surrounding endothelial cells. Exogenous H 2 O 2 dilated (%max dilation; 97Ϯ1%, ED 50 ; 3.0Ϯ0.7ϫ10 Ϫ5 mol/L) and hyperpolarized HCA. Dilation to H 2 O 2 was reduced by catalase, 40 mmol/L KCl, or charybdotoxin plus apamin, whereas endothelial denudation, deferoxamine, 1H-1,2,4 -oxadiazole-[4,3-a]quinoxalin-1-one, or glibenclamide had no effect. These data provide evidence that shear stress induces endothelial release of H 2 O 2 and are consistent with the idea that H 2 O 2 is an EDHF that contributes to FID in HCA from patients with heart disease. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2003;92:e31-e40.)Key Words: human Ⅲ coronary microcirculation Ⅲ flow-induced dilation Ⅲ hydrogen peroxides P hysiologically, shear stress plays a critical role in the regulation of vascular tonus and vascular homeostasis, contributing to the maintenance of tissue perfusion and vascular integrity. Shear stress-induced release of nitric oxide (NO) from endothelial cells is widely recognized as one of the most important and common mechanisms for shear-induced vasomotion. For example, flow-induced release of NO is responsible for the mediation of flow-induced vasodilation (FID). [1][2][3] Animal studies have reported that the contribution of NO to FID is reduced as oxidative stress increases in the presence of risk factors for cardiovascular disease such as hypercholesterolemia 4 and hypertension. 5 In humans, in vivo and in vitro studies have demonstrated that relaxant factor(s) other than NO compensate to maintain FID when NO availability is reduced. 6,7 We recently reported that FID is mediated largely by endothelium-derived hyperpolarizing factor (EDHF) in human coronary arterioles (HCAs...

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.

Background-Kϩ channel activation in vascular smooth muscle cells (VSMCs) plays a key role in regulating vascular tone. It has been proposed that endothelium-derived hyperpolarizing factor (EDHF) contributes to microvascular dilation more than nitric oxide (NO) does. Whether hyperpolarization is important for coronary arteriolar dilation in humans is not known. Bradykinin (BK), an endogenous vasoactive substance, is released from ischemic myocardium and regulates coronary resistance. Therefore, we tested the effects of inhibiting NO synthase, cyclooxygenase, and K ϩ channels on the changes in diameter and membrane potential (Em) in response to BK in isolated human coronary microvessels. Methods and Results-Arterioles (97Ϯ4 m; nϭ120) dissected from human right atrial appendages (nϭ78) were cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in vessel diameter (video microscopy) and VSMC Em (glass microelectrodes) were measured simultaneously. In vessels constricted and depolarized (Em; Ϫ50Ϯ3 to Ϫ28Ϯ2 mV) with endothelin-1 (ET), dilation to BK was associated with greater membrane hyperpolarization (Ϫ48Ϯ3 mV at 10 Ϫ6 mol/L) than dilation to sodium nitroprusside (SNP) (Ϫ34Ϯ2 mV at 10 Ϫ4 mol/L) for similar degrees of dilation. Treatment with N

Abstract-ATP-sensitive K ϩ channels (K ATP ) contribute to vasomotor regulation in some species. It is not fully understood the extent to which K ATP participate in regulating vasomotor tone under physiological and pathophysiological conditions in the human heart. Arterioles dissected from right atrial appendage were studied with video microscopy, membrane potential recordings, reverse transcription-polymerase chain reaction, and immunohistochemistry. Hypoxia produced endothelium-independent vasodilation and membrane hyperpolarization of vascular smooth muscle cells, both of which were attenuated by glibenclamide. Aprikalim, a selective K ATP opener, also induced a potent endothelium-independent and glibenclamide-sensitive vasodilation with membrane hyperpolarization. Reverse transcription-polymerase chain reaction detected mRNA expression for K ATP subunits, and immunohistochemistry confirmed the localization of the inwardly rectifying Kir6.1 protein in the vasculature. In patients with type 1 or type 2 diabetes mellitus (DM), vasodilation was reduced to both aprikalim (maximum dilation, DM(ϩ) 90Ϯ2% versus DM(Ϫ) 96Ϯ1%, PϽ0.05) and hypoxia (maximum dilation, DM(ϩ) 56Ϯ8% versus DM(Ϫ) 85Ϯ5%, PϽ0.01) but was not altered to sodium nitroprusside or bradykinin. Baseline myogenic tone and resting membrane potential were not affected by DM. We conclude that DM impairs human coronary arteriolar dilation to K ATP opening, leading to reduced dilation to hypoxia. This reduction in K ATP function could contribute to the greater cardiovascular mortality and morbidity in DM.

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K+ (BK(Ca)) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BK(Ca) channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11,12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BK(Ca) channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously.

Endothelium-dependent hyperpolarization of vascular smooth muscle cells (VSMCs) plays a crucial role in regulating vascular tone, especially in resistance vessels. It has been proposed that metabolites of arachidonic acid (AA), formed by cytochrome P-450 monooxygenase (P450), are endothelium-derived hyperpolarizing factors (EDHFs). These metabolites have been reported to mediate dilation to endogenous vasoactive compounds, such as bradykinin and acetylcholine. However, it is not known whether these metabolites of AA contribute to dilation of human resistance vessels. This is important since it has been proposed that EDHF serves as a compensatory mechanism to maintain dilation in disease states. Therefore, we studied the effect of AA on vessel diameter and VSMC membrane potential in isolated human coronary microvessels. Arterioles (81+/-5 microm, n=70) were dissected from right atrial appendages at the time of cardiac surgery and cannulated at a distending pressure of 60 mm Hg and zero flow. Changes in internal diameter were recorded with videomicroscopy. Some vessels were impaled with glass microelectrodes to measure membrane potential of VSMCs while internal diameters were simultaneously recorded. After constriction (47+/-2%) with endothelin-1, AA (10(-10)to 10(-5)mol/L) induced substantial dilation of human coronary microvessels, which was abolished by removal of the endothelium. Treatment with 17-octadecynoic acid (17-ODYA, 10(-5) mol/L; a P450 inhibitor) attenuated maximal dilation to AA (49+/-9% versus 91+/-4% [control]; P<0.05 versus control), whereas indomethacin (INDO, 10(-5) mol/L; a cyclooxygenase inhibitor) and N omega-nitro-L-arginine methyl ester (L-NAME, 10(-4) mol/L; a NO synthase inhibitor) were without effect. Both 17-ODYA and miconazole (10(-5) mol/L, a chemically distinct P450 inhibitor) further reduced the dilation to AA in the presence of INDO. The presence of 40 mmol/L KCl or charybdotoxin (10(-8) mol/L, a blocker of large-conductance Ca2+-activated K+ channels) impaired dilation to AA (19+/-9% [KCI] versus 76+/-5% [control] and 47+/-6% [charybdotoxin] versus 91+/-3% [control]; P<0.05 for both). After depolarization with endothelin-1 (-26+/-1 mV from -48+/-3 mV [before endothelin]), AA (10(-5)mol/L) in the presence of INDO and L-NAME induced hyperpolarization of VSMCs (-57+/-5 mV). In the presence of 17-ODYA together with INDO and L-NAME, endothelin produced similar depolarization (-26+/-2 mV from - 48+/- 3 mV), but hyperpolarization to AA was reduced (-33+/-2 mV; P<0.05 versus absence of 17-ODYA). AA metabolites formed primarily by P450 produce potent endothelium-dependent dilation of human coronary arterioles via opening of Ca2+-activated K+ channels and hyperpolarization of VSMCs. These findings support an important role for P450 metabolites in the regulation of human coronary arteriolar tone.

Arterioles from atria or ventricles were cannulated for videomicroscopy. Membrane potential of vascular smooth muscle cells (VSMCs) was measured simultaneously. After constriction with endothelin-1, increases in flow induced an endothelium-dependent vasodilation. Nomega-Nitro-L-arginine methyl ester 10(-4) mol/L modestly impaired FID of arterioles from patients without coronary artery disease (CAD), whereas no inhibition was seen in arterioles from patients with CAD. Indomethacin 10(-5) mol/L was without effect, but 40 mmol/L KCl attenuated maximal FID. Tetraethylammonium 10(-3) mol/L but not glibenclamide 10(-6) mol/L reduced FID. Charybdotoxin 10(-8) mol/L impaired both FID (15+/-3% versus 75+/-12%, P<0.05) and hyperpolarization (-32+/-2 mV [from -28+/-2 mV after endothelin-1] versus -42+/-2 mV [-27+/-2 mV], P<0.05). Miconazole 10(-6) mol/L or 17-octadecynoic acid 10(-5) mol/L reduced FID. By multivariate analysis, age was an independent predictor for the reduced FID. Conclusions-We conclude that shear stress induces endothelium-dependent vasodilation, hyperpolarizing VSMCs through opening Ca(2+)-activated K(+) channels in human coronary arterioles. In subjects without CAD, NO contributes to FID. NO and prostaglandins play no role in patients with CAD; rather, cytochrome P450 metabolites are involved. This is consistent with a role for endothelium-derived hyperpolarizing factor in FID of the human coronary microcirculation.

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.