Original Articles

Regulation of intracellular Ca2+ and calcineurin by NO/PKG in proliferation of vascular smooth muscle cells

Authors: Shi-jun LI, Ning-ling SUN

Abstract

Aim: To determine whether Ca2+/calcineurin mediated the inhibitory effects of
nitric oxide /cGMP-dependent protein kinase (NO/PKG) on the proliferation of
vascular smooth muscle cells (VSMC). Methods: Proliferation and viability of
primary VSMC from rat aorta were measured using [3-(4,5-dimethyl thiazol-2-yl)-2,
5-diphenyl tetrazolium bromide] (MTT) assay and acridine orange and ethidium
bromide staining, respectively. Cytosolic Ca2+ was determined by Fluo-3/AM.
Calcineurin protein and its activity were assayed using immunoblotting and free
inorganic phosphate analysis, respectively. Results: (±)-S-nitroso-,N-acetylpenicillamine
(SNAP) and Sp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic
monophosphorothioate (Sp-8-pCPT-cGMPS) decreased phenylephrine (PE)-induced
proliferation of VSMC by 27.3% and 36.6%, respectively, but Rp-8-[(4-
chlorophenyl)thio]-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPTcGMPS)
increased PE-induced proliferation of VSMC. SNAP, Sp-8-pCPT-cGMPS,
and Rp-8-pCPT-cGMPS did not affect the viability of VSMC. Calcineurin protein
was decreased by 63.1% and its activity was decreased by 59.7% in smooth muscle
cells (SMC) pretreated with verapamil (Ver) and then stimulated by PE. In SMC
pretreated with Ver, the absorbance of cells stimulated by PE decreased by 22.0%
and was further inhibited by the additional treatment of SNAP and Sp-8-pCPTcGMPS.
In SMC pretreated with cyclosporin A (CsA), the absorbance of cells
stimulated by PE decreased by 36.7%, but could not be further altered by the
additional treatment of SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGMPS. In
addition, Ver inhibited PE-induced intracellular Ca2+ variations, which could be
further inhibited by SNAP and Sp-8-pCPT-cGMPS, but not by Rp-8-pCPT-cGMPS.
Moreover, the increase in calcineurin activity induced by PE was inhibited by
SNAP and Sp-8-pCPT-cGMPS, but was promoted by Rp-8-pCPT-cGMPS.
Conclusion: NO/PKG regulates calcineurin activity via the modulation of intracellular
Ca2+ concentration, and thus partially inhibits the proliferation of VSMC
without affecting their viability.
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