Pivotal role of Cu,Zn-superoxide dismutase in endothelium-dependent hyperpolarization.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived H2O2 is an EDHF in mesenteric arteries of mice and humans and in porcine coronary microvessels. However, the mechanism for the endothelial production of H2O2 as an EDHF remains to be elucidated. In this study, we tested our hypothesis that Cu,Zn-superoxide dismutase (Cu,Zn-SOD) plays a pivotal role in endothelium-dependent hyperpolarization, using control and Cu,Zn-SOD-/- mice. In mesenteric arteries, EDHF-mediated relaxations and hyperpolarizations were significantly reduced in Cu,Zn-SOD-/- mice with no inhibitory effect of catalase, while endothelium-independent relaxations and hyperpolarizations were preserved. Endothelial H2O2 production also was significantly reduced in Cu,Zn-SOD-/- mice. In Langendorff isolated heart, bradykinin-induced increase in coronary flow was significantly reduced in Cu,Zn-SOD-/- mice, again with no inhibitory effect of catalase. The exogenous SOD mimetic tempol significantly improved EDHF-mediated relaxations and hyperpolarizations and coronary flow response in Cu,Zn-SOD-/- mice. These results prove the novel concept that endothelial Cu,Zn-SOD plays an important role as an "EDHF synthase" in mice, in addition to its classical role to scavenge superoxide anions.

Endothelium-derived hydrogen peroxide accounts for the enhancing effect of an angiotensin-converting enzyme inhibitor on endothelium-derived hyperpolarizing factor-mediated responses in mice.

UNLABELLED: Background- We have recently identified that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans, for which endothelial nitric oxide synthase (eNOS) is an important source. Angiotensin-converting enzyme (ACE) inhibitors are known to enhance EDHF-mediated responses. In this study, we examined whether endothelium-derived H2O2 accounts for the enhancing effect of an ACE inhibitor on EDHF-mediated responses and, if so, what mechanism is involved. METHODS AND RESULTS: Control and eNOS-/- mice were maintained with or without temocapril (10 mg/kg per day orally) for 4 weeks, and isometric tensions and membrane potentials of mesenteric arteries were recorded. In control mice, temocapril treatment significantly enhanced EDHF-mediated relaxations and hyperpolarizations to acetylcholine (n=8 each). Catalase, a specific scavenger of H2O2, abolished the beneficial effects of temocapril, although it did not affect endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of K(Ca) channels (n=6 each). Western blot analysis demonstrated that the temocapril treatment significantly upregulated the expression of eNOS. By contrast, this enhancing effect of temocapril was absent in eNOS-/- mice (n=6). CONCLUSIONS: These results indicate that endothelium-derived H2O2 accounts for the enhancing effect of temocapril on EDHF-mediated responses caused in part by eNOS upregulation, further supporting our H2O2 theory.

Influence of diabetes mellitus, hypercholesterolemia, and their combination on EDHF-mediated responses in mice.

The endothelium synthesizes and releases several vasodilator substances, including vasodilator prostaglandins, NO, and EDHF. NO-mediated relaxations are reduced by various risk factors, such as diabetes mellitus and hypercholesterolemia. However, it remains to be elucidated whether EDHF-mediated relaxations also are reduced by those factors and their combination. In this study, we addressed this point in mice. We used small mesenteric arteries from control, diabetic (streptozotocin-induced), apolipoprotein-E-deficient (ApoE-/-), and diabetic ApoE-/- mice. In control mice, endothelium-dependent relaxations to acetylcholine were largely mediated by EDHF. This EDHF-mediated component was slightly reduced in diabetic mice, preserved in ApoE-/- mice, and markedly reduced in diabetic ApoE-/- mice with an increase in NO-mediated component and a negative contribution of indomethacin-sensitive endothelium-derived contracting factor (EDCF). Endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of calcium-activated K channels, were attenuated in ApoE-/- and diabetic ApoE-/- mice. Endothelium-dependent hyperpolarizations were significantly reduced in diabetic mice, preserved in ApoE-/- mice, and again markedly reduced in diabetic ApoE-/- mice. These results indicate that hypercholesterolemia alone minimally affects the EDHF-mediated relaxations, and diabetes mellitus significantly attenuated the responses, whereas their combination markedly attenuates the responses with a compensatory involvement of NO and a negative contribution of EDCF.

Important role of superoxide dismutase in EDHF-mediated responses of human mesenteric arteries.

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have identified hydrogen peroxide (H2O2) as an EDHF in mouse and human mesenteric arteries and porcine coronary microvessels. We also have recently demonstrated that Cu,Zn-SOD plays an important role in EDHF synthesis in mouse mesenteric arteries. However, it remains to be determined whether SOD also plays an important role in EDHF-mediated responses of human arteries. In this study, we addressed this point in human mesenteric arteries. We used small mesenteric arteries of patients who underwent gastrectomy operations. Isometric tensions and membrane potentials were recorded in the presence of indomethacin and N-nitro-L-arginine to inhibit the synthesis of prostacyclin and NO, respectively. Pretreatment with Tiron, a cell-permeable SOD-mimetic, significantly enhanced the EDHF-mediated relaxations and hyperpolarizations to bradykinin, and this effect was abolished by catalase, indicating that this enhancing effect was achieved by H2O2. By contrast, Tiron did not affect endothelium-independent relaxations, indicating that the enhancing effect of Tiron is not caused by the enhancement of vascular smooth muscle responses. These results indicate that SOD plays an important role in EDHF-mediated relaxations and hyperpolarizations of human mesenteric arteries.

Acute vasodilator effects of HMG-CoA reductase inhibitors: involvement of PI3-kinase/Akt pathway and Kv channels.

3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase inhibitors (statins) have several non-lipid-lowering actions; however, characteristics of their acute vasodilator effects remain to be elucidated. In this study, acute vasodilator effects of statins were examined in isolated rat blood vessels. After incubation with cerivastatin (1 microM) for 2 hours, acetylcholine-induced endothelium-dependent relaxations were enhanced in the rat aorta. This effect was abolished by a nitric oxide synthase (NOS) inhibitor, L-NNA, and by a PI3 kinase inhibitor, LY294002. Western blot analysis showed that the extent of phosphorylation of Akt, an active form of Akt, was increased by cerivastatin while it was reduced by LY294002, suggesting an involvement of PI3 kinase/Akt-dependent activation of endothelial NOS. At higher concentrations (1-300 microM), both cerivastatin and fluvastatin, but not pravastatin, directly relaxed the blood vessels, regardless of the presence or absence of the endothelium. These relaxations were abolished by KCl and were significantly inhibited by an inhibitor of Kv channel, 4-aminopyridine. These results indicate that multiple mechanisms are involved in the acute vasodilator effects of statins, including augmentation of nitric oxide-mediated endothelium-dependent relaxations through the PI3 kinase/Akt pathway and endothelium-independent relaxations via Kv channel-mediated smooth muscle hyperpolarizations. These acute vasodilator effects of statins may account, at least in part, for their beneficial effects on cardiovascular diseases associated with impaired organ blood flow.

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in human mesenteric arteries.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived hydrogen peroxide (H(2)O(2)) is an EDHF in mice. The present study was designed to examine whether this is also the case in humans. Bradykinin elicited endothelium-dependent relaxations and hyperpolarizations in the presence of indomethacin and N(omega)-nitro-l-arginine, which thus were attributed to EDHF, in human mesenteric arteries. The EDHF-mediated relaxations were significantly inhibited by catalase, an enzyme that specifically decomposes H(2)O(2), whereas catalase did not affect endothelium-independent hyperpolarizations to levcromakalim. Exogenous H(2)O(2) elicited relaxations and hyperpolarizations in endothelium-stripped arteries. Gap junction inhibitor 18alpha-glycyrrhetinic acid partially inhibited, whereas inhibitors of cytochrome P450 did not affect the EDHF-mediated relaxations. These results indicate that H(2)O(2) is also a primary EDHF in human mesenteric arteries with some contribution of gap junctions.

Endothelial nitric oxide synthase-independent effects of an ACE inhibitor on coronary flow response to bradykinin in aged mice.

ACE inhibitors are known to ameliorate cardiovascular complications in aging; however, their effects on the coronary circulation in relation to aging and eNOS dependence remain to be examined. Coronary flow responses to bradykinin with or without ACE inhibitors were examined in Langendorff-perfused hearts from young (16-20 weeks) and aged (16-20 months) control and eNOS mice. Western blot analysis was performed for cardiac eNOS, nNOS, and ACE. Baseline coronary flow was comparable between young and aged mice of both strains. Aging did not affect bradykinin-induced coronary flow in either strain. Interestingly, both acute and chronic treatment with an ACE inhibitor markedly augmented the flow response in aged control and eNOS mice. Aged eNOS mice were markedly hypertensive and had larger ventricular mass than control mice. The antihypertensive effect of temocapril was greater in aged eNOS mice, associated with reduction in the ventricular weight in both strains. Western blot analysis demonstrated an increased expression of eNOS in aged control mice, and ACE expression was increased in eNOS mice. These results indicate that coronary flow response to bradykinin is preserved in aged mice even in the absence of eNOS, and an ACE inhibitor augments this response by both eNOS-dependent and -independent mechanisms.

Up-regulated neuronal nitric oxide synthase compensates coronary flow response to bradykinin in endothelial nitric oxide synthase-deficient mice.

It has been reported that endothelium-dependent relaxations are preserved in isolated coronary arteries of endothelial nitric oxide synthase-deficient (eNOS-/-) mice with a possible involvement of nNOS. However, it remains to be examined whether nNOS compensates coronary flow response in a beating heart of eNOS-/- mice and if so, whether and where nNOS is up-regulated. Coronary flow response to bradykinin was examined in Langendorff-perfused hearts from WT and eNOS-/- mice. Bradykinin-induced coronary flow was greater in eNOS-/- mice than in WT mice, and indomethacin had no inhibitory effect on it. Bradykinin receptor antagonist HOE-140 abolished the bradykinin response in both strains. Non-selective NOSs inhibitor L-NNA inhibited the bradykinin-induced coronary flow in both strains, whereas specific inhibitors of nNOS, SMTC, and 7-NI, significantly attenuated the coronary flow response only in eNOS-/- mice. A guanylate cyclase inhibitor ODQ also attenuated the bradykinin response in eNOS-/- mice. Immunohistochemistry revealed the presence of nNOS mainly in coronary vascular smooth muscle cells (VSMCs) in both strains and Western blot analysis demonstrated a marked increase in cardiac nNOS expression in eNOS-/- mice. These results indicate that nNOS compensates coronary flow response to bradykinin in eNOS-/- mice, for which up-regulation of nNOS in VSMCs may be involved.