Shi-jun Li1, Ning-ling Sun. 1. Department of Cardiology, People's Hospital, Peking University, Beijing 100044, China. lsj_020626@163.com
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-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. 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.
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-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. 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.
Authors: Shanta M Messerli; William Morgan; Shanda R Birkeland; Jeremiah Bernier; Michael J Cipriano; Andrew G McArthur; Robert M Greenberg Journal: Mol Biochem Parasitol Date: 2006-08-28 Impact factor: 1.759
Authors: Andrew W Holt; Danielle N Martin; Patti R Shaver; Shaquria P Adderley; Joshua D Stone; Chintamani N Joshi; Jake T Francisco; Robert M Lust; Douglas A Weidner; Brian M Shewchuk; David A Tulis Journal: Cell Signal Date: 2016-06-11 Impact factor: 4.315