C-S Lin1, X Liu, S Chow, T F Lue. 1. Knuppe Molecular Urology Laboratory, Department of Urology, School of Medicine, University of California, San Francisco, CA 94143-1695, USA. clin@urol.ucsf.edu
Abstract
OBJECTIVE: To investigate protein kinase G-I (PKG-I) expression and activation in cavernosal smooth muscle cells (CSMC) of young and old rats. MATERIALS AND METHODS: PKG-I expression in rat penis was examined by immunohistochemical staining, reverse transcription-polymerase chain reaction, and Western blot analysis. CSMC isolated from young (16-week-old) and old (28-month-old) rats were grown as monolayer cell cultures and treated with different dosages of cAMP or cGMP for different periods. Their proteins were then analysed for the expression of vasodilator-stimulated phosphoprotein (VASP), phosphorylated VASP (at serine 239), PKG-I, and protein kinase A (PKA). RESULTS: PKG-I expression was detected in the vascular and CSMC of the rat penis. There was little or no difference in the level of PKG-I expression between young and old rats. Treatment of CSMC with different dosages of cAMP or cGMP did not change the expression levels of VASP throughout the entire test period (up to 24 h). In contrast, the level of VASP phosphorylation at S239, i.e. the level of PKG-I activation, depended on the dosages of cAMP and cGMP and on the duration of treatment. Prolonged treatment (24 h) with either cAMP or cGMP resulted in down-regulation of both PKG-I and PKA. While cAMP and cGMP produced very similar results in nearly every aspect, there was a difference in one test, in which cGMP produced much less activated PKG-I than cAMP in the CSMC of 28-month-old-rats. CONCLUSIONS: For the first time we provide evidence for PKG-I activation in CSMC. Both cAMP and cGMP were capable of activating PKG-I in CSMC. Age seemed to compromise the ability of PKG-I in response to cGMP.
OBJECTIVE: To investigate protein kinase G-I (PKG-I) expression and activation in cavernosal smooth muscle cells (CSMC) of young and old rats. MATERIALS AND METHODS:PKG-I expression in rat penis was examined by immunohistochemical staining, reverse transcription-polymerase chain reaction, and Western blot analysis. CSMC isolated from young (16-week-old) and old (28-month-old) rats were grown as monolayer cell cultures and treated with different dosages of cAMP or cGMP for different periods. Their proteins were then analysed for the expression of vasodilator-stimulated phosphoprotein (VASP), phosphorylated VASP (at serine 239), PKG-I, and protein kinase A (PKA). RESULTS:PKG-I expression was detected in the vascular and CSMC of the rat penis. There was little or no difference in the level of PKG-I expression between young and old rats. Treatment of CSMC with different dosages of cAMP or cGMP did not change the expression levels of VASP throughout the entire test period (up to 24 h). In contrast, the level of VASP phosphorylation at S239, i.e. the level of PKG-I activation, depended on the dosages of cAMP and cGMP and on the duration of treatment. Prolonged treatment (24 h) with either cAMP or cGMP resulted in down-regulation of both PKG-I and PKA. While cAMP and cGMP produced very similar results in nearly every aspect, there was a difference in one test, in which cGMP produced much less activated PKG-I than cAMP in the CSMC of 28-month-old-rats. CONCLUSIONS: For the first time we provide evidence for PKG-I activation in CSMC. Both cAMP and cGMP were capable of activating PKG-I in CSMC. Age seemed to compromise the ability of PKG-I in response to cGMP.
Authors: Kenia P Nunes; Haroldo A Toque; Marcia H Borges; Michael Richardson; R Clinton Webb; Maria Elena de Lima Journal: J Sex Med Date: 2012-08-23 Impact factor: 3.802