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 alpha(2)-adrenergic agonist that 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 investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca(2+)-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca(2+)-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca(2+)-dependent NOS (NOS-III) activity. However, the value of K(i) for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH(4)) 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 BH(4) 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, clonidine (1.0 x 10(-4) M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-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, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.
Venturini
G
G
Department of Biology, University of Roma Tre, I-00146, Rome, Italy.
Colasanti
M
M
Persichini
T
T
Fioravanti
E
E
Federico
R
R
Ascenzi
P
P
D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology*; D003000:Clonidine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005910:Glioma / Q000201:enzymology / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D000959:Antihypertensive Agents; D004791:Enzyme Inhibitors; D009573:Nitrites; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; D003000:Clonidine