CHANGES IN TYPE-VI ADENYLYL-CYCLASE ISOFORM EXPRESSION CORRELATE WITHA DECREASED CAPACITY FOR CAMP GENERATION IN THE AGING VENTRICLE

We investigated the developmental regulation of the beta-adrenergic re ceptor-G(s)-adenylyl cyclase pathway in myocardial membranes from feta l, neonatal, adult, and mature adult rats by measuring the density of the beta-adrenergic receptor and the activities of the stimulatory gua nine nucleotide-binding protein G(s) and the adenylyl cyclase enzyme. Total beta-adrenergic receptor content (in femtomoles per milligram pr otein) was greatest in the fetal (124.4+/-20.5 fmol/mg) and neonatal ( 122.3+/-16.1 fmol/mg) stages and gradually decreased in the adult (90. 9+/-8.0 fmol/mg) and mature adult (70.0+/-9.6 fmol/mg) stages. An equi valent pattern was seen for adenylyl cyclase activity: the basal activ ity of the effector enzyme or that measured in the presence of 0.1 mmo l/L isoproterenol with 0.1 mmol/L Gpp(NH)p, 10 mmol/L NaF, or 0.05 mmo l/L forskolin was greater in the fetus and the neonate than in the adu lt and the mature adult. These data suggested that decreased stimulati on of the catalytic unit by G(s) could be the underlying cause of dimi nished adenylyl cyclase activity with aging. However, quantification o f G(s) by reconstitution into S49 cyc- membranes (in picomoles cAMP pe r microgram for 10 minutes) demonstrated no significant decrease durin g development from fetus (1.55+/-0.1 pmol/mug) to neonate (1.9+/-0.5 p mol/mug) and subsequent aging to adult (2.6+/-0.2 pmol/mug) and mature adult (1.9+/-0.2 pmol/mug). When Northern blot analysis was used to c haracterize the relative amounts of mRNA coding for G(salpha), no sign ificant differences were seen among the developmental stages studied. Unlike G(salpha), the inhibitory protein G(ialsph2) was subject to dev elopmental regulation; however, Concomitant evaluation of G(ialpha2) l evels both by ADP-ribosylation with pertussis toxin and by immunoblott ing showed the greatest amount in the fetus (0.82+/-0.07 pmol/mg) and the neonate (0.51+/-0.1 pmol/mg). The adult and mature adult both cont ained 0.09+/-0.02 pmol/mg. Also, the steady-state G(ialpha2), mRNA lev el paralleled the amount of protein. Similarly, the level of type V mR NA coding for adenylyl cyclase was greater in the mature adult than in the fetal and neonatal stages. In contrast, the level of type VI aden ylyl cyclase mRNA decreased with age, paralleling the decline in the f unctional activity of adenylyl cyclase with age. Taken together, these data suggest that the age-related changes in the activity of the myoc ardial beta-adrenergic receptor-G(s)-adenylyl cyclase pathway is not p rimarily regulated by alteration in the level of G(s) or G(i) but by t he density of beta-adrenergic receptors and by the activity of the cat alyst adenylyl cyclase. In particular, steady-state mRNA measurements show that a decrease in the content of the type VI isoform of adenylyl cyclase correlates with a decrease in catalytic activity with age.