Nitric oxide synthase induction by ouabain in vascular smooth muscle cells from normotensive and hypertensive rats.
OBJECTIVE
To investigate the effect of ouabain on inducible nitric oxide synthase (iNOS) activity and expression in cytokine-stimulated vascular smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).


METHODS
VSMC were treated for 24 h and afterwards, nitric oxide (NO) release was determined by the production of nitrite, a stable metabolite of NO. Activity of iNOS was measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline and iNOS protein expression by Western blotting.


RESULTS
Ouabain (0.01-1 mmol/l) further enhanced interleukin-1beta (II-1beta)-induced nitrite production by WKY and SHR VSMC, although a more pronounced effect was observed in SHR cells (maximum response 52.1 +/- 5.2 and 71.2 +/- 6.4% of 11-1beta effect in WKY and SHR cells, respectively). Such response on NO release was mimicked by the calcium ionophore A 23187 (0.01-1 micromol/l) and abolished by the voltage-operated calcium channels (VOCC) nifedipine (0.1 micromol/l). Expression of iNOS showed that ouabain increased the synthesis of the enzyme in WKY and SHR VSMC stimulated with II-1beta, and this effect was higher in SHR cells. The increased iNOS expression was significantly reduced by nifedipine.


CONCLUSIONS
Ouabain stimulation of iNOS expression and activity in II-1beta-stimulated VSMCs from WKY rats and SHR seems to be related to increased intracellular calcium influx through VOCC. The more pronounced effect observed in SHR VSMC could be explained by an altered calcium entry in the hypertensive strain.
Pacheco
M E
ME
Departamento de Farmacologia y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Marín
J
J
Manso
A M
AM
Rodríguez-Martínez
M A
MA
Briones
A
A
Salaices
M
M
Redondo
J
J
D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006973:Hypertension / Q000201:enzymology* / Q000473:pathology; D007375:Interleukin-1 / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009543:Nifedipine / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000187:drug effects; D052247:Nitric Oxide Synthase Type II; D010042:Ouabain / Q000494:pharmacology*; D051381:Rats; D011918:Rats, Inbred SHR; D017208:Rats, Wistar
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D007476:Ionophores; D000001:Calcimycin; D010042:Ouabain; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D009543:Nifedipine; D002118:Calcium