Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug

Clonidine, clinically used in the treatment of hypertension, is a central a lpha(2)-adrenergic agonist chat reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was i nvestigated. This was verified by determination of NOS activity in vitro an d by analysis of inducible Ca2+-independent NOS (NOS-II) mRNA expression an d measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca2+-dcpendent NOS (NOS-I) competiti vely without affecting NOS-II and endothelial Ca2+-dependent NOS (NOS-III) activity. However, the value of K-i for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH4) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K-i for clonidine binding to NOS-I increased (from [7.9 +/- 0.4] x 10(-5) M to [8.0 +/- 0.4] x 10(-3) M) as BH4 concentration was increased (between 3.0 x 10(-7) M and 1.0 x 10(-3) M), at pH 7.5 and 37.0 degrees. In addition, clo nidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma ce lls, as induced by Escherichia coli lipopolysaccharide (LPS) plus interfero n-gamma (IFN-gamma). Finally, clonidine (1.0 x 10(-4) M to 1.0 x 10(-3) M) dose dependently increased the levels of LPS/IFN-gamma-induced nitrites, th e breakdown product of NO, in supernatants of rat C6 glioma cells. As repor ted for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely. BIOCHEM PHARMACOL 60;4:539-544, 2000. (C) 2000 Elsevier Science Inc.