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Aim:To determine whether Ca~(2+)/calcineurin mediated the inhibitory effects ofnitric oxide/cGMP-dependent protein kinase (NO/PKG) on the proliferation ofvascular smooth muscle cells (VSMC).Methods:Proliferation and viability ofprimary 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 ethidiumbromide staining,respectively.Cytosolic Ca~(2+) was determined by Fluo-3/AM.Calcineurin protein and its activity were assayed using immunoblotting and freeinorganic phosphate analysis,respectively.Results:(e)-S-nitroso-N-acetyl-penicillamine (SNAP) and Sp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclicmonophosphorothioate (Sp-8-pCPT-cGMPS) decreased phenylephrine (PE)-in-duced 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 proteinwas decreased by 63.1% and its activity was decreased by 59.7% in smooth musclecells (SMC) pretreated with verapamil (Ver) and then stimulated by PE.In SMCpretreated with Vet,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 cellsstimulated by PE decreased by 36.7%,but could not be further altered by theadditional treatment of SNAP,Sp-8-pCPT-cGMPS,and Rp-8-pCPT-cGMPS.Inaddition,Ver inhibited PE-induced intracellular Ca~(2+) variations,which could befurther 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 bySNAP and Sp-8-pCPT-cGMPS,but was promoted by Rp-8-pCPT-cGMPS.Conclusion:NO/PKG regulates calcineurin activity via the modulation of intracel-lular Ca~(2+) concentration,and thus partially inhibits the proliferation of VSMCwithout affecting their viability.
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 ethidiumbromide staining, respectively.Cytosolic Ca 2+ by Fluo-3 / AM.Calcineurin protein and its activity were assayed using immunoblotting and free inorganic phosphate analysis, respectively. Results: (e) -S-nitroso-N-acetyl- penicillamine (SNAP) and Sp- 8- (4-chlorophenylthio ) -guanosine-3 ’, 5’-cyclic monophosphorothioate (Sp-8-pCPT-cGMPS) decreased phenylephrine (PE) -in-duced proliferation of VSMC by 27.3% and 36.6% 5-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) increased PE-induced proliferation of VSMC.SNAP, Sp-8-pCPT-cGMPS, and Rp-8-pCPT-cGM PS 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. SMC pretreated with Vet, 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. SMC pretreated with cyclosporin A (CsA), the absorbance of cellsstimulated 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.Inaddition, Veristed 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 intracel-lular Ca 2+ concentration, and thus partially inhibits the proliferation of VSMCwithout affecting their viability.