Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (·O2 −) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented ·O2 − release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAMEtreated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca 2؉ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22␣ promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 ؎ 8% versus 79 ؎ 8% of maximum relaxation; n ‫؍‬ 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 ؎ 6% versus 68 ؎ 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor L-NAME (L-N(G)-nitroarginine methyl ester) (10؊5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without L-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with L-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCAoverexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 ؎ 5% versus 131 ؎ 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca 2؉ pump down-regulates activity of the vascular smooth muscle Ca 2؉ /calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.