Postnatal Changes in the G-Proteins, Cyclic Nucleotides and Adenylyl Cyclase Activity in Rabbit Heart Cells

Abstract

We have studied the postnatal changes in the levels of isoforms of stimulatory (Gs) and inhibitory (Gi) G-proteins, cAMP and cGMP in washed particulate membranes (WPM) from whole ventricles as well as from isolated ventricular myocytes and have also measured adenylyl cyclase (AC) activity in WPM prepared from isolated myocytes of adult (AD) and newborn (NB) rabbit heart. Immunoblot analysis for the levels of Giα1, Gα2 Giα3 and Gsα subunits showed that Giα2 and Giα3 were higher in WPM from whole ventricles of NB compared to AD. This ratio was much higher in WPM from isolated ventricular myocytes since Giα2 and Giα3 were either absent or present in extremely low immunodetectable levels in WPM from AD ventricular myocytes. Giα1 levels were not different for AD compared to NB WPM, whether prepared from whole ventricle or from isolated myocytes. Two forms of Gsα, a small form (Gsα-S) and a large form (Gsα-L), were immunodetected at 43 and 48 kDa, respectively. The Gsα-S form was higher in AD WPM and the Gsα-L form was higher in NB WPM while the total Gsα(L+S) was not different. The Gsα results for WPM from isolated myocytes were not different from the results for WPM from whole ventricles. Basal levels of cAMP were 80% higher in NB compared to AD whole ventricles and were 200% higher in NB compared to AD isolated myocytes. Levels of cGMP were 4-5 fold higher in NB than in AD myocytes and ventricular tissue. Basal AC activity was higher in NB than in AD WPM from isolated myocytes and was enhanced by Gpp(NH)p pretreatment in AD but not in NB WPM. The isoproterenol-induced increase in AC activity was higher in AD compared to NB WPM and was completely abolished by Gpp(NH)p pretreatment in NB but not in AD WPM. Forskolin caused a greater increase in AC activity in NB than in AD WPM. The post-natal decrease in the levels of Giα2 and Giα3, particularly in isolated ventricular myocytes, may help to explain the smaller effects of isoproterenol and greater muscarinic influence on ICa+, as we previously showed, and the smaller effect of isoproterenol on AC activity in NB compared to AD WPM. It is not clear if the post-natal differences in the distribution of types of Gsα may also play a role in modulating the effect of isoproterenol. The postnatal decrease in the levels of cAMP and cGMP may be significant in regulating phosphodiesterases, protein phosphatases and kinases