Experiments were designed to examine the effect of oxidized low density lipoproteins (Ox-LDLs) on the expression and the release of endothelin from cultured endothelial cells and intact blood vessels. Ox-LDLs (30-300 jtg/ml), but not native low density lipoproteins (200 ,ug/ml), stimulated the expression of preproendothelin mRNA in porcine and human endothelial cells, leading to a time-and concentrationdependent release of the peptide into the culture medium. The Ox-LDL-stimulated release of endothelin was mimicked by acetylated low density lipoprotein and abolished by downregulation of protein kinase C by phorbol ester. In the intact porcine aorta, Ox-LDLs, but not native low density lipoproteins, also increased the release of peptide in an endothelium-and concentration-dependent manner. The maximal effect was observed at a concentration of 100 ,g/ml. Incubation of the intact porcine aorta with the scavenger receptor antagonist dextran sulfate decreased the formation of endothelin evoked by Ox-LDLs. The Ox-LDL-stimulated production of the peptide was further augmented in the presence of thrombin (4 units/ml) and was unaffected by nitric oxide-generating compound 3-morpholinosydnonimine (10`M).These results suggest that Ox-LDL may be an endogenous mediator of the augmented release of endothelin observed in hyperlipidemia and atherosclerosis. The increased production of the peptide could contribute to vasospastic events and may promote vascular smooth muscle proliferation and progression of atherosclerotic vascular disease.

Sustained inhibition of NO synthesis (N omega-nitro-L-arginine [L-NNA], 20 mg.kg-1.d-1, 7 days) was investigated at rest and during exercise in conscious dogs. At rest, L-NNA did not alter mean arterial blood pressure but markedly increased total peripheral resistance (+73 +/- 14%, P < .01). Exaggerated hypertension was observed during exercise (+132 +/- 5 mm Hg after L-NNA versus +113 +/- 5 mm Hg before L-NNA, P < .01). L-NNA decreased the resting coronary artery diameter by 6 +/- 1% and suppressed its exercise-induced dilation but had no effect on coronary blood flow and resistance. L-NNA decreased flow repayment volumes during reactive hyperemia, but corresponding flow debt volumes remained unchanged. The cyclooxygenase inhibitor diclofenac (10 mg/kg) had no effect on reactive hyperemia parameters before L-NNA but reduced flow repayment volumes, durations, and corresponding debt-to-repayment ratios in L-NNA-treated dogs (all P< .05). In vitro, indomethacin blunted the residual relaxation to bradykinin of large coronary arteries taken from L-NNA-treated, but not from control, dogs. Bradykinin-induced increase in 6-ketoprostaglandin F1 alpha production was greater in coronary arteries taken from L-NNA-treated dogs (+ 179 +/- 41 pg/mm2) than from control dogs (+ 66 +/- 18 pg/mm2) (P < .05). These results indicate that (1) NO is of major importance in the control of systemic but not coronary resistance vessels at rest and during exercise, and (2) after L-NNA, the cyclooxygenase pathway is involved in myocardial reactive hyperemia and in the residual relaxation to bradykinin of isolated coronary arteries. Thus, in conscious dogs, the cyclooxygenase pathway might act as a protective mechanism of the coronary circulation when endothelial nitric oxide synthesis is altered.

In addition to biological and clearance receptors for atrial natriuretic factor (ANF), cultured vascular smooth muscle cells (VSMC) from the rabbit renal cortex possess ectoenzymes degrading this hormone. We examined whether neutral endopeptidase (NEP) was implicated in this process. The presence of NEP in VSMC was demonstrated as follows. 1) NEP activity measured from the hydrolysis of a synthetic substrate by intact cells cultured in a medium containing 10% fetal calf serum was 1,609 +/- 65 pmol.min-1 x mg-1 [surface localization of the enzyme was confirmed by low activity (4% of total) in the cytosol; release of NEP activity in the medium was negligible]; 2) a monoclonal antibody directed against rabbit NEP specifically stained VSMC membranes; and 3) mRNA from VSMC hybridized a NEP cDNA probe with a single band as shown by Northern blot analysis. The role of NEP in ANF catabolism was demonstrated by incubating 125I-ANF or unlabeled ANF for increasing periods of time with VSMC in the presence of thiorphan (1-100 microM). Intact hormone estimated by trichloroacetic acid precipitation or radio-immunoassay, respectively, increased markedly compared with control in the presence of this specific inhibitor of NEP. NEP activity was stimulated (x1.6) in quiescent VSMC deprived from serum during 3 days. This effect was dose dependent and was not observed with creatine kinase activity measured as control. NEP expression at the cell surface estimated by sorting of immunostained cells was also increased in the absence of serum. Northern blot analysis showed increases in the mRNA band of NEP with increasing periods of serum deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the effects of chronic in vivo inhibition of NO synthase on endothelium-dependent hyperpolarization, cell-membrane potential (in individual vascular smooth-muscle cells) and changes in tension (in isolated rings) were recorded from isolated canine coronary arteries and guinea-pig carotid arteries and aortas. In coronary arteries taken from control dogs and contracted with U46619, acetylcholine- and bradykinin-induced endothelium-dependent relaxations, which were unaffected by short-term in vitro exposure to indomethacin but were inhibited partially by L-nitro-arginine (LNA). In coronary arteries taken from dogs treated over the long term in vivo with LNA (30 mg/kg on the first day and 20 mg/kg the 7 following days, i.v.), the response to acetylcholine and bradykinin was inhibited when compared with arteries from control dogs. Short-term in vitro exposure to LNA or indomethacin or both did not influence the effects of either agonist. In these arteries, the hyperpolarizing response to acetylcholine, observed in the presence of LNA and indomethacin, was enhanced, whereas that to bradykinin was partially inhibited. In the guinea pig isolated aorta, the relaxation to bradykinin was abolished by long-term in vivo treatment with L-nitro-arginine-methyl-ester (L-NAME; 1.5 mg/ml, in the drinking water for > or =4 days). In the isolated guinea pig carotid artery studied in the presence of LNA and indomethacin, acetylcholine induced a hyperpolarization that was not significantly affected by long-term in vivo treatment with L-NAME. These findings indicate that endothelium-dependent hyperpolarizations are maintained during long-term inhibition of NO synthase and probably act as a back-up mechanism to elicit endothelium-dependent relaxations.