Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells.
Homocysteine found in the plasma of patients with coronary heart disease, induces vascular smooth muscle cell (VSMC) proliferation and increases deposition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is largely unknown. We hypothesized that homocysteine induces ECM production via intracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeling for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with fura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca(2+)](i) transients with an EC(50) of 60 +/- 5 nM. The EC(50) for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular calcium; however, thapsigargin pretreatment, which depletes intracellular Ca(2+) stores, abolished the homocysteine effect, demonstrating its dependence on intracellular Ca(2+) stores. Extracellular sodium depletion significantly (P < 0.05) increased [Ca(2+)](i) also suggesting a possible role of sodium-calcium exchange in the process. To begin to elucidate the intracellular pathways by which homocysteine might act, VSMC were pretreated with specific inhibitors and stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented the release of Ca(2+) by homocysteine. Interestingly, pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) greatly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretreatment with either the phospholipase A(2) activator neomycin, the antioxidant and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium channel 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 Ca(2+). Effects of homocysteine on collagen production, as ascertained by immunoblot analysis, correlated with its effect in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM deposition, 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 range of angiotensin II concentrations which normally have no effect on intracellular Ca(2+). After homocysteine pretreatment, VSMC were extremely responsive to angiotensin II at concentrations well below the physiologic range. These data taken together suggested that an initial effect of homocysteine is to induce release of intracellular Ca(2+) in VSMC and may induce vascular reactivity. The transient in Ca(2+) correlates with the effect on ECM associated with homocysteine.
Mujumdar
V S
VS
Department of Physiology and Biophysics, Center of Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA.
Hayden
M R
MR
Tyagi
S C
SC
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D015736:Felodipine / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D005978:Glutathione / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology*; D006711:Homocystine / Q000494:pharmacology*; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D017035:Pravastatin / Q000494:pharmacology; D051381:Rats; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D005456:Fluorescent Dyes; D006710:Homocysteine; C049925:fura-2-am; D000804:Angiotensin II; D006711:Homocystine; D019284:Thapsigargin; D003094:Collagen; D005978:Glutathione; D019311:Staurosporine; D009355:Neomycin; D017035:Pravastatin; D013755:Tetradecanoylphorbol Acetate; D015736:Felodipine; D002118:Calcium; D016257:Fura-2; D019331:NG-Nitroarginine Methyl Ester