This study aimed to investigate the potential effect of in vivo administration of immunosuppressive agent FK-506 (tacrolimus) on the endothelial function of rat thoracic aortas with respect to nitric oxide (NO) synthesis. In vitro effect of the drug on NO synthesis in cultured rat coronary microvascular endothelial cells (CMEC) was also studied.In vivo administration of tacrolimus (1 mg/kg/d, intramuscular) to rats for 14 days resulted in decreased relaxant responses to the higher concentrations (1 to 30 muM) of acetylcholine in the aortas; however, responses to calcium ionophore A23187, sodium nitroprusside, L-arginine, and L-NAME did not change significantly. No changes were observed in phenylephrine-induced contractions in endothelium-denuded or -intact preparations. Administration of the vehicle for 14 days did not affect these parameters. In order to evaluate the in vitro effect of tacrolimus on NO release, CMEC isolated from rat hearts were incubated with either tacrolimus (0.01, 0.1 microM) or the vehicle. Basal, calcium ionophore-stimulated, or interleukin-1 beta-induced NO synthesis was determined by measuring total nitrite in the media. Neither tacrolimus nor the vehicle changed nitrite accumulation. It has been concluded that therapeutic concentrations of tacrolimus do not alter NO production in rat thoracic aorta or cultured CMEC; however, it impairs relaxant responses of rat aorta induced by higher concentrations of acetylcholine, possibly through changes in the downstream of receptor activation or through an imbalance between endothelium-dependent relaxant and contracting factors within the endothelium in favor of the contracting factor(s).
Hyperhomocysteinemia is widely recognized as an independent risk factor for coronary artery vascular disease, although the underlying mechanisms are not well understood. This study aims to investigate the effect of homocysteine on nitric oxide (NO) production in coronary microvascular endothelial cells (CMECs) and putative mechanisms mediating this effect. CMECs were isolated on Langendorff system by collagenase perfusion of hearts from male rats and cultured. The effect of homocysteine (0.01 to 1 mM) on basal and stimulated NO production was evaluated by measuring nitrite in the culture media after incubation with or without N(G)-nitro-L-arginine methyl ester (L-NAME) (1 mM), superoxide dismutase (100 U/mL), or catalase (1000 U/mL) for 24 h. Total nitrite was measured using Griess reaction after reduction of nitrate to nitrite with nitrate reductase. Homocysteine did not affect basal nitrite accumulation; however, it significantly increased the nitrite accumulation induced by the calcium ionophore A23187 or interleukin-1beta only at 1 mM. This effect of homocysteine was significantly inhibited by L-NAME, superoxide dismutase, and catalase. In conclusion, homocysteine increases NO release from stimulated CMECs without affecting basal NO production, which is probably accompanied by increased production of reactive oxygen species. It can be postulated that endothelial cells generate NO in order to minimize the damage caused by homocysteine.
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