Both PRA and SIM activate eNOS, but SIM is much less effective. Clinical benefits with PRA not explained by LDL reductions may be the result of an independent action of PRA on eNOS activation.

It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.

1 The goal of this work was to test the role of nitric oxide synthase (NOS) and its substrate Larginine in development of tolerance to nitroglycerin's (GTN) vasodilator actions. 2 GTN's eects on NOS activity and NO formation were tested in cultured bovine aortic endothelial cells (BAECs). The arginine to citrulline conversion assay showed that GTN stimulated NOS basal activity in BAECs by *40%, comparable with acetylcholine (ACh)-treated controls. Both eects were blocked by L-NMMA. Photometric assays showed that both GTN and AChstimulated NO formation. Both eects were potentiated by L-arginine and inhibited by L-NAME. L-NAME inhibited ACh responses *80% compared with *40% for GTN responses. 3 The aortic ring assay showed that 2 h pretreatment with GTN caused substantial tolerance to GTN's vasodilating eects as evidenced by a 38 fold rightward shift of the concentration-relaxation curve. In contrast to D-arginine, addition of L-arginine substantially inhibited this eect, reducing the rightward shift to 4.4 fold of control values. GTN tolerance was associated with a 40% reduction in L-arginine tissue levels. GTN had a biphasic eect on BAEC uptake of L-arginine, stimulating uptake at 5 and 15 min, and suppressing uptake after 1 and 4 h 4 In summary, acute GTN treatment stimulates endothelial NOS activity in producing NO and increases cellular uptake of L-arginine. Prolonged GTN exposure reduces GTN's vasodilator actions, decreases L-arginine tissue levels and depresses BAECs uptake of L-arginine. Supplementation of Larginine reduces development of GTN tolerance. These data indicate that GTN tolerance depends in part on activation of the NOS pathway.

The present study tested the hypothesis that altered vascular regulation of arachidonic acid enzymes in obese Zucker rats contributes to renal damage. Protein expression of CYP450 (cytochrome P450) and COX (cyclo-oxygenase) enzymes in renal microvessels was studied in obese and lean Zucker rats at 20-21 weeks of age. Body weight and blood glucose averaged 649+/-13 g and 142+/-10 mg/dl in obese Zucker rats compared with 437+/-10 g and 111+/-5 mg/dl in age-matched lean Zucker rats. Renal microvascular CYP4A and COX-2 protein levels were increased and CYP2C protein levels decreased in obese Zucker rats. TX (thromboxane) B2 excretion was 2-fold higher and PG (prostaglandin) E2 excretion significantly lower in obese Zucker rats. Additional studies investigated the ability of the COX-2 inhibitor, rofecoxib, to slow the progression of renal injury in obese Zucker rats. Rofecoxib treatment decreased urinary PGF2alpha and 8-isoprostane levels in obese Zucker rats. Renal microvessel mRNA expression of pro-inflammatory chemokines was decreased in COX-2-inhibitor-treated obese Zucker rats. Urinary albumin excretion, an index of kidney damage, averaged 95+/-11 mg/day in vehicle-treated and 9+/-1 mg/day in rofecoxib-treated obese Zucker rats. Glomerulosclerosis, characterized by mesangial expansion, tubulo-interstitial fibrosis and extracellular matrix accumulation, was prominent in obese Zucker rats compared with a lack of damage in age-matched lean Zucker rats and rofecoxib-treated obese Zucker rats. These results suggest that altered vascular arachidonic acid enzymes contribute to the renal damage, and that COX-2 inhibition decreases glomerular injury in obese Zucker rats.

Tolerance to glyceryl trinitrate (GTN) involves superoxide (O2.−) production by endothelial cells. Nitric oxide synthase (NOS) produces O2.− when L‐arginine (L‐arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O2.− synthesis in endothelial cells when L‐arg is limited. Production of O2.− by bovine aortic endothelial cells (BAEC, passages 3–5) was determined by spectrophotometrically measuring superoxide dismutase‐inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L‐arg. O2.− production was measured using BAEC either untreated or treated with L‐NAME or L‐arg alone or following treatment with GTN (10−9 to 10−6 M) for 30 min or DPTA NONOate (10−7 and 10−6 M) alone or with GTN or DPTA NONOate after pretreatment with nitro‐L‐arginine methyl ester (L‐NAME), L‐arg or their inactive enantiomers, D‐NAME or D‐arg (all 5×10−4 M) (n=6–7/group). L‐NAME alone produced a 69% reduction in O2.− levels. Treatment with L‐arg alone had no effect. Cells treated with GTN alone exhibited an increase in O2.−. This effect was prevented by pretreatment with either L‐NAME or L‐arg, and was unaffected by D‐NAME or D‐arg. We observed a dose‐response relationship in O2.− production to GTN over a range of 10−9 to 10−7 M. The NO donor, DPTA‐NONOate, unlike GTN, did not have a significant effect on O2.− production. In conclusion, endothelial NOS is a site of O2.− synthesis in endothelial cells activated by GTN. British Journal of Pharmacology (2000) 131, 1019–1023; doi:10.1038/sj.bjp.0703665