Regulation of intracellular Ca2+ and calcineurin by NO/PKG in proliferation of vascular smooth muscle cells.
OBJECTIVE
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-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-pCPT-cGMPS) 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-pCPT-cGMPS. 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.


CONCLUSIONS
NO/PKG regulates calcineurin activity via the modulation of intracellular Ca2+ concentration, and thus partially inhibits the proliferation of VSMC without affecting their viability.
Li
Shi-jun
SJ
Department of Cardiology, People's Hospital, Peking University, Beijing 100044, China. lsj_020626@163.com
Sun
Ning-ling
NL
D000818:Animals; D000831:Animals, Newborn; D001013:Aorta, Thoracic; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013873:Thionucleotides / Q000494:pharmacology*; D014700:Verapamil / Q000494:pharmacology
C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D013873:Thionucleotides; D010656:Phenylephrine; D014700:Verapamil; D017869:Cyclic GMP-Dependent Protein Kinases; D019703:Calcineurin; D010396:Penicillamine; D006152:Cyclic GMP; D002118:Calcium