1. In addition to nitric oxide (NO) and prostacyclin (PGJ2) an as yet unidentified endothelium-derived hyperpolarizing factor (EDHF) contributes to the dilator effect of bradykinin in different vascular beds. We have investigated the nature and mechanism of action of this factor in freshly isolated bovine and porcine coronary artery segments which were preconstricted with the thromboxane mimetic U46619 (9,11-dideoxy-lla, 9ac-epoxymethano-prostaglandin F2a, 10-30 nM). 2. The concentration-response curve of bradykinin was significantly shifted to the right after inhibition of NO synthesis with NG-nitro-L-arginine (L-NNA, 30 FM), whereas cyclo-oxygenase blockade with diclofenac (1 AM) had no effect. Preconstriction of the segments with potassium chloride (40-60 mM) completely abrogated the NO/PGJ2-independent dilator response to bradykinin. In sandwich bioassay experiments, both the luminal and abluminal release of NO, but not that of EDHF, was readily detectable.3. Inhibitors of Ca2+-activated K+ channels (K+.), such as apamin (1 /M) and tetrabutylammonium (TBA, 3 mm), strongly attenuated the EDHF-mediated bradykinininduced relaxation, while glibenclamide (3/1M), an inhibitor of KATP channels, had no effect. 4. These relaxations were also significantly inhibited by the phospholipase A2 inhibitor, quinacrine (30 #M), and the cytochrome P450 inhibitors, SKF525a (30-100 SM) and clotrimazole (100 #M). Moreover, incubation of endothelium-denuded coronary artery rings with a cytochrome P450-derived arachidonic acid metabolite, 11,12-epoxyeicosatetraenoic acid, elicited a concentration-dependent (1-10 /IM) dilatation which was abolished both in the presence of TBA (3 mM) and following preconstriction of the segments with potassium chloride instead of U46619. 5. These findings suggest that EDHF released by bradykinin is a cytochrome P450-derived arachidonic acid metabolite, presumably an epoxide. This factor seems to hyperpolarize the underlying smooth muscle cell layers by opening Kca channels.

Abstract-Three 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), atorvastatin, pravastatin, and cerivastatin, inhibited phorbol ester-stimulated superoxide anion (O 2 Ϫ ) formation in endothelium-intact segments of the rat aorta in a time-and concentration-dependent manner (maximum inhibition of 70% after 18 hours at 1 to 10 mol/L). The HMG-CoA reductase product mevalonic acid (400 mol/L) reversed the inhibitory effect of the HCRIs, which, conversely, was mimicked by inactivation of p21 Rac with Clostridium sordellii lethal toxin but not by inactivation of p21 Rho with Clostridium botulinum exoenzyme (C3). A mevalonate-sensitive inhibition of phorbol ester-stimulated O 2 Ϫ formation by atorvastatin was also observed in porcine cultured endothelial cells and in a murine macrophage cell line. In the rat aorta, no effect of the HCRIs on protein kinase C, NADPH oxidase, or superoxide dismutase (SOD) activity and expression was detected, whereas that of endothelial nitric oxide (NO) synthase was enhanced Ϸ2-fold. Moreover, exposure of the segments to atorvastatin resulted in a significant improvement of endothelium-dependent NO-mediated relaxation, and this effect was abolished in the presence of SOD. Taken together, these findings suggest that in addition to augmenting endothelial NO synthesis, HCRIs inhibit endothelial O 2 Ϫ formation by preventing the isoprenylation of p21 Rac, which is critical for the assembly of NADPH oxidase after activation of protein kinase C. The resulting shift in the balance between NO and O 2 Ϫ in the endothelium improves endothelial function even in healthy blood vessels and therefore may provide a reasonable explanation for the beneficial effects of HCRIs in patients with coronary heart disease in addition to or as an alternative to the reduction in serum LDL cholesterol. Key Words: HMG-CoA reductase inhibitor(s) Ⅲ endothelial dysfunction Ⅲ coronary heart disease Ⅲ nitric oxide Ⅲ superoxide anion Ⅲ NADPH oxidase Ⅲ p21 Rac T he beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (HCRIs), collectively referred to as "statins," on coronary events have generally been attributed to the well-documented LDL cholesterol-lowering properties of these drugs. However, despite the strong association between drug concentration, blood lipid level, and patient benefit shown in the CARE, 4S, and WOSCOPS trials, HCRIs seem to exert at least part of their cardioprotective action by mechanisms other than simply lowering the lipid load of the vessel wall. Thus, HCRIs induce regression of atherosclerotic lesions in patients with coronary heart disease (CHD) with and without hypercholesterolemia 1 and improve endothelial dysfunction, a hallmark of atherosclerotic blood vessels, in patients with moderately elevated total serum cholesterol within 1 month. 2 At the cellular level, they modulate leukocyte activity and adhesiveness, 3,4 inhibit vascular smooth muscle cell proliferation both in vitro and in vivo, 5,6 and reduce the synthesis of che...

Signalling pathways determining the shear stress-induced production of NO from endothelial cells in situ were investigated using a bioassay system in which shear stress was increased by inducing vasoconstriction in an endothelium-intact donor segment (rabbit iliac artery) while maintaining a constant luminal perfusion rate. Shear stress-induced NO production, as assessed by changes in the tone of a preconstricted endothelium-denuded detector ring, was biphasic and consisted of an initial transient (20- to 25-minute) Ca(2+)-dependent phase followed by a Ca(2+)-independent plateau phase, which was maintained as long as the donor segment remained constricted. Stretching the donor segments to their in vivo length abolished the initial phase without affecting the plateau phase of NO release. Inhibition of the Na(+)-H+ exchanger using HOE 694 elicited an intracellular acidification which attenuated shear stress-induced NO production. The specific protein kinase C inhibitor, Ro 31-8220, was without effect, whereas the unspecific inhibitors, staurosporine and calphostin C, abolished the shear stress-induced production of NO. Erbstatin A, a tyrosine kinase inhibitor, attenuated the shear stress-induced tyrosine phosphorylation of specific cellular proteins and abrogated the associated NO production. In summary, these data indicate that shear stress activates the NO synthase at basal levels of [Ca2+]i via a mechanotransduction cascade that involves tyrosine phosphorylation and can be modulated by changes in pHi. The apparent fundamental alteration of the endothelial NO synthase under shear stress that renders its maintained activation independent of an increase in [Ca2+]i is probably the consequence of a change in the enzyme microenvironment.

These findings indicate that under physiological conditions, the production of EDHF is damped by NO. Therefore, it follows that when NO synthesis is impaired, alleviation of this intrinsic inhibition may, at least in part, maintain endothelial vasodilator function.