Background-NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (eNOS). Endothelial dysfunction occurs in atherosclerosis. Methods and Results-With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. eNOS protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 mol/L) was 0.42Ϯ0.05 (mol/L)/s (nϭ10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to 0.08Ϯ0.04 (mol/L)/s (nϭ10, PϽ0.0001). NO peak concentration in normal arteries was 0.9Ϯ0.09 mol/L (nϭ10) and in atherosclerotic segments, 0.1Ϯ0.03 mol/L (nϭ10, PϽ0.0001). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive eNOS in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (nϭ13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for eNOS, as was the endothelium of normal arteries. Conclusions-In clinically relevant human atherosclerosis, eNOS protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis. (Circulation. 1998;97:2494-2498.)

Aging is an important determinant of vascular disease. Endothelium-derived nitric oxide (NO) is protective as a vasodilator and inhibitor of platelet function. This study was designed to directly measure effects of prolonged aging on endothelial NO release in isolated blood vessels and to delineate differences between the systemic and pulmonary circulation.Aortas and pulmonary arteries from 5-6-mo-old (young), 18-19-mo-old (middle-aged), and 32-33-mo-old (old) normotensive female rats were used. Blood pressure and plasma estradiol-17 ␤ (E 2 ) remained unchanged. In isolated blood vessels, NO release was induced by the receptor-independent agonist calcium ionophore A23187 (10 mol/liter) and measured in situ on the endothelial surface of vessels using a porphyrinic microsensor. In vessels suspended in organ chambers isometric tension was recorded. In the aorta, the initial rate of NO release and peak NO concentration were reduced in middle-aged and old rats ( P Ͻ 0.0006 vs. young rats, n ϭ 6). Furthermore, endothelium-dependent relaxations to calcium ionophore and acetylcholine (both 10 Ϫ 10 -10 Ϫ 5 mol/liter) were also reduced in aortas from old as compared with young rats ( n ϭ 6, P Ͻ 0.05). The initial rate of NO release and peak NO concentration significantly correlated with maximal relaxation to calcium ionophore A23187 (correlation coefficients r ϭ 0.916, P Ͻ 0.0018 and r ϭ 0.961, P Ͻ 0.0001, respectively, n ϭ 7). In pulmonary arteries, however, the initial rate of NO release as well as peak NO concentration did not decrease with age ( n ϭ 6 for each age group, NS). In both blood vessels, the NO release was unaffected by superoxide dismutase in all age groups ( n ϭ 6, NS).Thus, aging specifically reduces initial rate and peak concentrations of endothelial NO release from aorta but not pulmonary artery indicating reduced NO production. As arterial pressure did not change with aging, the chronic exposure of the aorta to higher pressure and/or pulsatility than in the pulmonary artery may be the cause. This appears important as NO plays a protective role by preventing vasoconstriction, thrombosis and atherosclerosis.

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide is a vasodilator that can be rapidly inactivated by superoxide (reaction rate constant, K = 3.6 x 10(9) L/mol per second). The measurement of nitric oxide concentration in biological systems is a challenging analytic problem because nitric oxide is also rapidly inactivated by Fe(II), Fe(III), and O2, all of which are found in great abundance in biological systems. To date, no currently used instrumental technique has been suitable for direct in situ measurement of NO in isolated resistance arteries. We designed the present study to perform for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and to delineate the effects of hypertension on the release of NO and/or its interaction with superoxide. We describe here an adaptation of the recently published design of a porphyrinic sensor for direct in vitro measurement of NO in a single cell. The most significant advantage of this modified porphyrinic microsensor is that its small size makes it ideal for NO measurement in resistance arteries with an internal diameter of 200 microns or less. Small segments of the third-order branch of the mesenteric artery were isolated from normotensive Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats and placed in an organ chamber filled with Hanks' balanced salt solution buffer (2 mL, 37 degrees C). The tip of the porphyrinic microsensor was inserted into the lumen of an isolated vascular ring, and NO release was monitored in situ after maximal stimulation of NO synthase with the receptor-independent agonist calcium ionophore A23187 (10 mumol/L). Maximal surface concentration of NO measured after A23187 administration was significantly smaller in 15-week-old hypertensive rats (0.28 +/- 0.03 mumol/L, n = 10) than in age-matched normotensive rats (0.38 +/- 0.03 mumol/L, n = 10, P < .03). However, in the presence of the superoxide scavenger superoxide dismutase (100 U/mL), the peak NO level from the hypertensive rats was 0.37 +/- 0.04 mumol/L (n = 10), which was comparable to that observed for the normotensive rats in the absence and presence of superoxide dismutase. In summary, our results demonstrate that in rat mesenteric resistance arteries hypertension is associated with increased NO decomposition by superoxide, whereas NO release remains unaffected. This may be important in the pathogenesis of hypertension and its cardiovascular complications.

Endothelin-1 is a 21-amino acid endothelial vasoconstrictor peptide that may be the physiological antagonist of endothelium-derived relaxing factor (EDRF). Endothelin-1 (10(-11)-3 x 10(-7) M) evoked potent contractions of isolated internal mammary arteries, internal mammary veins, and saphenous veins, which were enhanced in internal mammary veins as compared with internal mammary arteries (concentration shift, 6.3-fold; p less than 0.05) but not in the saphenous veins. Endothelial removal augmented the response to the peptide (at 3 x 10(-7) M) in internal mammary arteries (p less than 0.05) but not in veins. In the artery, EDRF released by acetylcholine or bradykinin reversed endothelin-1-induced contractions; in saphenous veins, both agonists were much less effective compared with the artery and veins contracted with norepinephrine (p less than 0.005-0.01). This inhibition of endothelium-dependent relaxations in veins occurred at half-maximal contractions but was most prominent at maximal contractions to the peptide. Nitric oxide similarly inhibited contractions to endothelin-1 and norepinephrine in internal mammary arteries, whereas in veins that were contracted with endothelin-1 but not with norepinephrine, the relaxations were blunted (p less than 0.005). The nitric oxide donor SIN-1 and sodium nitroprusside induced complete relaxations of internal mammary arteries but were less effective in veins contracted with endothelin-1 (p less than 0.005). Thus, in normal human arteries, EDRF inhibits endothelin-1-induced contractions, whereas the peptide specifically attenuates the effects of EDRF and nitrovasodilators in veins. This may be important in pathological conditions associated with increased levels of endothelin-1 and in veins used as coronary bypass grafts.

The endothelium is a physical barrier between the blood and vascular smooth muscle, a source of enzymes activating and deactivating cardiovascular hormones and a site of production of relaxing and contracting factors. In addition, the endothelium is a source of growth inhibitors and promoters of vascular smooth muscle cells. Monoaminooxidase deactivates catecholamines and serotonin. Angiotensin converting enzyme transforms angiotensin I into angiotensin II and breaks down bradykinin into inactive products. Nitric oxide is a potent vasodilator and inhibitor of platelet function that under most circumstances is released together with prostacyclin, which exerts similar effects. Both substances play an important protective role in the coronary circulation in that they cause continuous vasodilation and inhibition of platelet function. In addition, the endothelium is a source of contracting factors such as endothelin-1, thromboxane A2, and endoperoxides. Endothelium-derived growth inhibitors include heparin (sulfates) and transforming growth factor beta 1, while basic fibroblast growth factors and platelet-derived growth factor and possibly endothelin promote proliferation. Because of its strategic anatomic position, the endothelium is a primary target for injuries and cardiovascular risk factors. In particular, aging, low density lipoproteins, hypertension, diabetes, and ischemia alter endothelium function. In arterial coronary bypass grafts, the release of nitric oxide is more pronounced than in vein grafts. Alterations of endothelial function may contribute to vasospasm, thrombus formation, and vascular proliferation and in turn myocardial ischemia, all common events in patients with coronary artery disease.

Endothelial cells regulate vascular tone through the release of nitric oxide and other relaxing factors. The role of these substances was studied in isolated intramyocardial porcine coronary resistance arteries suspended in myographs for isometric tension recording. The inhibitor of nitric oxide formation NG-monomethyl-L-arginine (L-NMMA; 10(-7)-10(-4)M), but not D-NMMA, caused endothelium-dependent contractions that could be reversed by L-arginine but not by D-arginine. In preparations with endothelium, L-NMMA potentiated the contractions induced by acetylcholine and the relaxations to 3-morpholino-sydnonimine. Under both conditions, the effect of endothelial removal was slightly more pronounced than that of L-NMMA. Bradykinin, serotonin, and the alpha 2-adrenergic agonist clonidine evoked endothelium-dependent relaxations. L-NMMA as well as the inhibitor of guanylate cyclase methylene blue (10(-5) M) prevented the relaxations induced by clonidine, reduced those to serotonin, but hardly affected those to bradykinin. Thus, in porcine coronary resistance arteries, endothelium-derived nitric oxide is continuously produced from L-arginine. Endothelium-dependent relaxations to clonidine are fully mediated and those to serotonin partially mediated by nitric oxide; its release does not involve a Gi protein. An endothelium-derived relaxing factor different from nitric oxide must mediate the relaxations to bradykinin and contribute to those evoked by serotonin.

In rat coronary arteries, NO is synthesized via the endothelial L-arginine pathway and released after stimulation with acetylcholine. In SHR, chronic antihypertensive therapy with either angiotensin receptor antagonists, an angiotensin-converting enzyme inhibitor, or a calcium antagonist specifically increased the normal endothelium-dependent relaxations to acetylcholine, probably because of their blood pressure-lowering effects, whereas the responsiveness of vascular smooth muscle to NO remained unaffected.

Endothelial cells release nitric oxide from L-arginine, and this pathway can be inhibited by the analogue of L-arginine, NG-monomethyl-L-arginine (L-NMMA). The effect of L-NMMA on endothelium-dependent relaxation of epicardial porcine coronary arteries was studied in isolated blood vessels suspended in organ chambers for isometric tension recording. Endothelium-dependent relaxations to bradykinin, serotonin, and the alpha 2-adrenergic agonist clonidine were evaluated in the presence and absence of L-NMMA (10(-5)-10(-3) M). L-NMMA, as well as the inhibitor of guanylate cyclase methylene blue (10(-5) M) and hemoglobin (10(-5) M), inhibited endothelium-dependent relaxation to serotonin and clonidine. The effect of L-NMMA could be reversed by L-arginine but not by D-arginine. In contrast, L-NMMA, methylene blue, and hemoglobin caused a weak inhibition of the endothelium-dependent relaxation evoked by bradykinin; indomethacin and tranylcypromine had no effect. The inhibitor of Gi proteins pertussis toxin (100 ng/ml) abolished the relaxations evoked by clonidine and markedly reduced those evoked by serotonin but did not affect those caused by bradykinin. In the presence of pertussis toxin, L-NMMA induced a further reduction of the relaxations to serotonin, suggesting that inhibition of Gi proteins does not completely prevent the activation of the L-arginine pathway. Thus endothelium-dependent relaxations to serotonin and to the alpha 2-adrenergic agonist clonidine are mediated through the release of nitric oxide formed from L-arginine in endothelial cells, whereas bradykinin evokes endothelium-dependent relaxations via a different pathway.