Characterization of the effects of two new arginine/citrulline analogues on constitutive and inducible nitric oxide synthases in rat aorta

1 New potent inhibitors of nitric oxide synthase (NOS) may be useful in the treatment of septic shock, a disorder characterized by a vascular hyporeactivity to catecholamines caused by an overproduction of nitric oxide (NO − ). We examined the effects of L ‐thiocitrulline (L‐TC) and S‐methyl‐ L ‐thiocitrulline (L‐SMTC), novel NOS inhibitors, on the constitutive and inducible NOS in rat aorta and compared those effects with inhibition due to N G ‐methyl‐ L ‐arginine (L‐NMA). 2 Phenylephrine evoked similar concentration‐contraction curves in the control rings and in the rings treated with these different NOS inhibitors (10 μ M ), whereas 100 μ M of L ‐NMA, L ‐TC or L ‐SMTC increased significantly, and to a similar extent, contractions evoked by phenylephrine in aortic rings with endothelium without significantly affecting the maximal responses. 3 Relaxations evoked by acetylcholine, adenosine triphosphate, or calcium ionophore were significantly inhibited in a dose‐dependent manner by L ‐NMA, L ‐SMTC, or L ‐TC (10–100 μ M ). The potencies of these inhibitors in reducing the relaxations of these vasodilators were not significantly different. 4 In endotoxin‐treated preparations with endothelium, the three L ‐arginine analogues (10 μ M ) significantly potentiated contractile responses to phenylephrine (pEC 50 : 6.73±0.12 and 7.3±0.12, 7.34±0.13, or 7.22±0.14; in the absence and the presence of L ‐NMA, L ‐TC, or L ‐SMTC respectively) and increased maximal contractions from 1.53±0.15 g to 1.95±0.13 g, 2.08±0.12 g, and 2.03±0.13 g with L ‐NMA, L ‐TC, and L ‐SMTC respectively. A higher concentration of these NOS inhibitors (100 μ M ) further increased contractions evoked by this α 1 ‐agonist without further enhancing the maximal contractions; however, contractions evoked by 10 nM phenylephrine were significantly greater in the presence of L ‐SMTC or L ‐TC than in the presence of L ‐NMA (100 μ M ) (L‐NMA: 0.4±0.11 g; L ‐TC: 0.78±0.14 g and L ‐SMTC: 0.82±0.17 g). The effects of these inhibitors on NO − synthesis induced by endotoxin were significantly reversed by addition of L ‐arginine (1 mM) but not by L ‐citrulline (1 mM). In LPS‐treated rings with endothelium, all three NOS inhibitors (100 μ M ) shifted the concentration‐contraction curves evoked by phenylephrine significantly to the left (pEC 50 : 7.19±0.03 and 7.79±0.08, 8.01 +0.07, or 8.02 +0.07, in the absence and the presence of L ‐NMA, L ‐TC, or L ‐SMTC, respectively) and increased significantly maximal contractions from 2.05±0.05 g to 2.38±0.14 g, 2.5±0.12 g, and 2.4 +0.21 g with L ‐NMA, L ‐TC, and L ‐SMTC, respectively. L ‐TC and L ‐SMTC were significantly more potent than L ‐NMA in potentiating contractions evoked by 10 nM and 30 nM phenylephrine. 5 L ‐TC and L ‐SMTC produced dose‐dependent increases in tone in LPS‐treated aortic rings with and without endothelium. In LPS‐treated rings with endothelium, L ‐NMA induced contractions but in preparations without endothelium low concentrations of L ‐NMA induced small contractions while high concentrations of this inhibitor evoked relaxations. In both preparations L ‐TC and L ‐SMTC were significantly more potent than L ‐NMA in increasing vascular tone. 6 These results suggest that L ‐SMTC, L ‐TC and L ‐NMA were equipotent on basal and agonist‐stimulated NO − synthesis produced by the constitutive isoform of NOS, whereas the two new L ‐arginine analogues were more potent than L ‐NMA in inhibiting the production of NO − induced by endotoxin in rat aorta.