guanosine-5--o-(3-thiotriphosphate) and Cardiomegaly

We have recently reported enhanced levels of G(i)alpha proteins in genetic and other experimentally induced models of hypertension, whereas the levels of G(s)alpha were decreased in hypertensive rats expressing cardiac hypertrophy. The present studies were undertaken to investigate whether the decreased levels of G(s)alpha are associated with cardiac hypertrophy per se and used an aortocaval fistula (AV shunt; volume overload) rat model that exclusively expresses cardiac hypertrophy. Cardiac hypertrophy in Sprague-Dawley rats (200-250 g) was induced under anesthesia, and, after a period of 10 days, the hearts were used for adenylyl cyclase activity determination, protein quantification, and mRNA level determination. A temporal relationship between the expression of G(s)alpha proteins and cardiac hypertrophy was also examined on days 2, 3, 7, and 10 after induction of AV shunt in the rat. The heart-to-body-weight ratio (mg/g) was significantly increased in AV shunt rats after 3, 7, and 10 days of induction of AV shunt compared with sham-operated controls, whereas arterial blood pressure was not different between the two groups. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) stimulated adenylyl cyclase activity in a concentration-dependent manner in heart membranes from both groups; however, the degree of stimulation was significantly decreased in AV shunt rats. In addition, the stimulatory effects of isoproterenol were also diminished in AV shunt rats compared with control rats, whereas glucagon-stimulated adenylyl cyclase activity was not different in the two groups. The inhibitory effects of oxotremorine (receptor-dependent G(i) functions) and low concentrations of GTPgammaS on forskolin-stimulated adenylyl cyclase activity (receptor-independent G(i) functions) were not different in the two groups. In addition forskolin and NaF also stimulated adenylyl cyclase activity to a lesser degree in AV shunt rats compared with control rats. The levels of G(i)alpha-2 and G(i)alpha-3 proteins and mRNA, as determined by immunoblotting and Northern blotting, respectively, were not different in both groups; however, the levels of G(s)alpha(45) and G(s)alpha(47), and not of G(s)alpha(52), proteins were significantly decreased in AV shunt rats by days 7 and 10 compared with control rats, whereas no change was observed on days 2 and 3 after induction of AV shunt. These results suggest that the decreased expression of G(s)alpha proteins may not be the cause but the effec

Topics: Adenylyl Cyclases; Animals; Aorta, Thoracic; Arteriovenous Shunt, Surgical; Blood Pressure; Blotting, Northern; Body Weight; Cardiomegaly; Colforsin; Glucagon; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Guanosine 5'-O-(3-Thiotriphosphate); Isoproterenol; Male; Myocardium; Organ Size; Protein Isoforms; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Fluoride

alpha 1-Adrenergic agonists activate a hypertrophic response in cultured neonatal ventricular myocytes, which include an increase in cell size, organization of contractile proteins into sarcomeric units, and the induction of the atrial natriuretic factor (ANF) gene. Previous findings have supported a role for ras in this signaling pathway. Utilizing microinjection techniques to delivery affinity-purified neutralizing antibodies to G alpha q,11 into cultured ventricular myocytes, the current studies demonstrate a functional requirement for the heterotrimeric G protein, Gq, in the alpha 1-adrenergic induction of the ANF gene, changes in cell size, organization of myofilaments, and phosphoinositide hydrolysis. Expression of a constitutively active mutant of G alpha q leads to the expression of ANF protein in these cells. Taken together, these data suggest that G q-dependent pathways are necessary and sufficient to activate defined features of the hypertrophic response. In attempts to further delineate the relative roles of ras and Gq in this pathway, we found that G alpha q is required for alpha 1-adrenergic phosphoinositide hydrolysis, though ras does not appear to be necessary for this response. In addition, we coexpressed an inhibitory ras mutant, along with the constitutively active G alpha q. Expression of ANF protein stimulated by the G alpha q mutant was not inhibited. Thus, both ras- and Gq-dependent pathways are necessary to fully transduce defined features of alpha 1-adrenergic-stimulated hypertrophy of neonatal cardiac ventricular myocytes, but activated Gq may be able to induce ANF expression independent of inhibitory ras.

Topics: Animals; Animals, Newborn; Atrial Natriuretic Factor; Cardiomegaly; Cells, Cultured; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Heart Ventricles; Hydrolysis; Luciferases; Microinjections; Myocardium; Oncogene Protein p21(ras); Phenylephrine; Phosphatidylinositols; Rats; Receptors, Adrenergic, alpha-1