nitroarginine and 1-(2-trifluoromethylphenyl)imidazole

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Biological Factors; Catalase; Dose-Response Relationship, Drug; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Enzyme Inhibitors; Hydrazines; Hydrogen Peroxide; Imidazoles; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Nitroarginine; Nitroprusside; Oligonucleotides, Antisense; RNA, Messenger; Vasodilation; Vasodilator Agents

This study examined the effects of trifluoromethylphenylimidazole (TRIM) on tone, and calcium entry, in mouse anococcygeus stimulated by either thapsigargin (Tg; 100 nM) which activates capacitative calcium entry (CCE), or high K (60 mM) which activates voltage-operated calcium channels. TRIM (1 - 333 microM) produced concentration-related relaxation of Tg-induced tone (EC(50), 42 microM) but was much less effective against high K. In single smooth muscle cells loaded with FURA-2, TRIM reduced the increase in fluorescence ratio produced by Tg but had no effect on that produced by high K. The relaxations of Tg-induced tone, and reduction in fluorescence ratio, were obtained in the presence of L-N(G)-nitroarginine and were thus independent of nitric oxide synthase inhibition; further, TRIM had no discernible effect on nitrergic responses. TRIM provides a novel drug for the selective inhibition of CCE and a template for the development of more potent inhibitors.

Topics: Animals; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Imidazoles; In Vitro Techniques; Male; Mice; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Synthase; Nitroarginine; Potassium; Thapsigargin

We have previously reported that 1-(2-trifluoromethylphenyl) imidazole (TRIM) is a potent inhibitor of mouse cerebellar neuronal NOS (nNOS) in vitro with very much reduced activity against bovine aortic endothelial NOS (eNOS). Using purified rat brain nNOS as enzyme source we have now probed the mechanism of action of TRIM. nNOS activity was linear over the first 5 min incubation. Optimal enzyme activity occurred in the presence of NADPH (0.5 mM), calcium chloride (75 microM), tetrahydrobiopterin (12 microM) and calmodulin (10 microg/ml) as cofactors. TRIM was a poor inhibitor of nNOS (IC50, 462.0 microM) compared with L-N(G) nitro arginine (L-NOARG, IC50, 0.32 microM). Removal of tetrahydrobiopterin (but not calmodulin) from the incubation medium greatly enhanced the nNOS inhibitory activity of TRIM (IC50, 32.0 microM) but not L-NOARG (IC50, 0.34 microM). In the absence of added tetrahydrobiopterin, TRIM competed with L-arginine for the substrate binding site on the nNOS enzyme with a Ki value of 47.3 microM. The present experiments suggest that TRIM interferes with the binding of both L-arginine and tetrahydrobiopterin to their respective sites on the nNOS enzyme.

Topics: Animals; Brain; Cattle; Citrulline; Enzyme Inhibitors; Imidazoles; Kinetics; Neurons; Nitric Oxide Synthase; Nitroarginine; Rats; Tritium