Open AccessRegulation of intracellular Ca 2+ and calcineurin by NO/PKG in proliferation of vascular smooth muscle cells
Author(s) -
LI Shijun,
SUN Ningling
Publication year - 2005
Publication title -
acta pharmacologica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.514
H-Index - 90
eISSN - 1745-7254
pISSN - 1671-4083
DOI - 10.1111/j.1745-7254.2005.00049.x
Subject(s) - vascular smooth muscle , calcineurin , chemistry , endocrinology , medicine , cyclic guanosine monophosphate , viability assay , mtt assay , cgmp dependent protein kinase , nitric oxide , cell growth , biochemistry , microbiology and biotechnology , biology , protein kinase c , kinase , cell , transplantation , smooth muscle , mitogen activated protein kinase kinase
Aim: To determine whether Ca 2+ /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 Ca 2+ 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 ‐acetyl‐penicillamine (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 Ca 2+ 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 Ca 2+ concentration, and thus partially inhibits the proliferation of VSMC without affecting their viability.