Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells

Homocysteine found in the plasma of patients with coronary heart disease, i nduces vascular smooth muscle cell (VSMC) proliferation and increases depos ition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is larg ely unknown. We hypothesized that homocysteine induces ECM production vis i ntracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeli ng for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with f ura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca2 +](i) transients with an EC50 of 60 +/- 5 nM. The EC50 for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular Calcium; how ever, thapsigargin pretreatment, which depletes intracellular Ca2+ stores, abolished the homocysteine effect, demonstrating its dependence on intracel lular Ca2+ stores. Extracellular sodium depletion significantly (P < 0.05] increased [Ca2+](i) also suggesting a possible role of sodium-calcium excha nge in the process. To begin to elucidate the intracellular pathways by whi ch homocysteine might act, VSMC were pretreated with specific inhibitors an d stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented th e release of Ca2+ by homocysteine. Interestingly, pretreatment with the nit ric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) great ly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretre atment with either the phospholipase A(2) activator neomycin, the antioxida nt and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibi tor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium c hannel blocker, felodipine completely inhibited the homocysteine-induced Ca 2+ signal in VSMC. This suggests the role of multiple signaling pathways in the homocysteine effect on VSMC Ca2+ Effects of homocysteine on collagen p roduction, as ascertained by immunoblot analysis, correlated with its effec t in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM depositio n, has the potential to affect vascular reactivity. To determine the effect of homocysteine on the ability of VSMC to react to potent agonist such as angiotensin II, VSMC were pretreated with homocysteine and exposed to a ran ge of angiotensin II concentrations which normally have no effect on intrac ellular Ca2+ After homocysteine pretreatment, VSMC were extremely responsiv e to angiotensin II at concentrations well below the physiologic range. The se data taken together suggested that an initial effect of homocysteine is to induce release of intracellular Ca2+ in VSMC and may induce vascular rea ctivity. The transient in Ca2+ correlates with the effect on ECM associated with homocysteine. (C) 2000 Wiley-Liss, Inc.