Ontogeny of G-protein expression: control by beta-adrenoceptors.

Cardiac cell homeostasis is maintained in the face of excessive beta-adrenoceptor stimulation through the process of desensitization. Desensitization is not an inherent property of these cells but rather is acquired during development; neonates given beta-agonists actually show heterologous sensitization, involving changes in the expression and catalytic activity of adenylyl cyclase (AC) as well as an increased receptor/G-protein coupling. The current study examines the role of specific G-protein components, G(s)alpha and G(i)alpha, in the ontogeny of beta-adrenoceptor responses and in the transition from agonist-induced sensitization to desensitization. Between postnatal days (PN) 6 and 15 there was a significant decrease in the 52 kDa isoform of G(s)alpha with no accompanying change of the 45 kDa form; over the same period, G(i)alpha3 also declined substantially. In contrast, the 45 kDa isoform of G(s)alpha and G(i)alpha1,2 remained fairly constant over the same period and fluoride-stimulated AC activity increased. Treatment with isoproterenol on PN2-5 did not result in any significant changes in G(s)alpha expression but robustly decreased G(i)alpha1,2. These changes were accompanied by heterologous sensitization of AC activity at the level of AC itself, evidenced by equivalent increases in the enzymatic response to fluoride and forskolin-Mn2+. Isoproterenol given to older animals (PN11-14) also caused specific loss of G(i) protein, in this case targeting G(i)alpha3, whereas G(s)alpha again was unchanged; in contrast to the younger group, the older animals displayed heterologous desensitization of AC at the level of G-protein function (specific loss of the fluoride response). These results indicate that the normal ontogenetic increase of cardiac beta-adrenoceptor coupling to AC is not dependent on the absolute amount of G-proteins, nor on the relative balance of stimulatory (G(s)) and inhibitory (G(i)) subunits. However, the ability of receptor stimulation to downregulate G(i)alpha1,2, an event which is specific to immature cardiac cells, is likely to be an important component of the resistance of the fetal/neonatal heart to agonist-induced desensitization and hypertrophy. The maintenance of cardiac beta-adrenoceptor signaling in the face of intense stimulation is likely to play an important role in the physiologic adaptations necessary to the perinatal transition.

Agonist-induced sensitization of beta-adrenoceptor signaling in neonatal rat heart: expression and catalytic activity of adenylyl cyclase.

Agonist stimulation of neonatal cardiac beta-adrenoceptors produces heterologous sensitization of adenylyl cyclase (AC) signaling, rather than desensitization, as seen in adults. We examined the ontogenetic patterns of AC expression and activity, and evaluated isoproterenol effects on this pattern. [(3)H]Forskolin binding showed an increase in AC concentration across the period (birth to 25 days of age) in which agonist-induced sensitization is replaced by desensitization; binding affinity also increased, suggesting a shift in conformation and/or isoform. Indeed, catalytic properties of AC changed substantially with development, as evaluated by AC responses to forskolin versus Mn(2+). In contrast, there were only minor changes in the levels of mRNAs encoding the two major isoforms. Neonates given repeated isoproterenol treatment showed an enhancement of [(3)H]forskolin binding B(max) and a precocious shift to the mature affinity state and corresponding catalytic properties. Although isoproterenol caused significant increases in AC mRNAs, the effects were small and showed no isoform preference. Thus, a primary mode for ontogenetic increases in cardiac cellular responsiveness to adrenergic stimulation is the increase in AC activity attendant upon an absolute increase in the membrane concentration of AC molecules, along with changes in the catalytic properties of AC. The lack of correlation between mRNA and AC protein suggests that the primary regulatory events are post-transcriptional. The induction of AC by beta-adrenoceptor stimulation in the fetus and neonate accounts for heterologous, agonist-induced sensitization, a phenomenon that preserves cellular responses during the period of the perinatal transition.

Ontogeny of cardiac beta-adrenoceptor desensitization mechanisms: agonist treatment enhances receptor/G-protein transduction rather than eliciting uncoupling.

In the fetus and neonate, beta-adrenoceptor stimulation fails to produce physiological desensitization. The current study explores the mechanisms underlying the response pattern in neonatal rats. Homologous cardiac beta-adrenergic desensitization caused by isoproterenol treatment in vivo was demonstrable in adult rats by the immediate (2h) and specific loss of the ability of isoproterenol, but not glucagon, to stimulate adenylyl cyclase in vitro. Homologous desensitization was absent when the same treatment was given to neonates. By 12 h post-treatment, the adults showed heterologous desensitization (loss of the response to glucagon), an effect which was once again absent in the immature rats. The absence of desensitization in neonates did not reflect a deficiency in the activity or subcellular distribution of beta ARK1, the enzyme that initiates the phosphorylation and consequent desensitization of beta-adrenoceptors. On the other hand, neonates showed relatively poor receptor-Gs transduction as assessed by the GTP-induced shift in receptor ligand binding. Repeated isoproterenol treatment of adult rats led to uncoupling of receptor-G-protein transduction but the same treatment in neonates enhanced transduction. Furthermore, neonatal sympathectomy with 6-OHDA interfered with the ontogenetic rise in beta-adrenoceptor-Gs interactions. These results indicate that the maintenance of agonist responses in the face of neonatal adrenergic stimulation does not reflect simply an absence of the ability to elicit homologous or heterologous desensitization but rather represents an active regulatory mechanism in which neural input exerts a positive trophic role at the level of G-protein function.

Ontogeny of regulatory mechanisms for beta-adrenoceptor control of rat cardiac adenylyl cyclase: targeting of G-proteins and the cyclase catalytic subunit.

Fetal and neonatal tissues are resistant to catecholamine-induced desensitization of essential physiological responses. We examined the mechanisms underlying the ontogeny of desensitization in neonatal rat heart for the beta-adrenergic receptor/adenylyl cyclase signaling cascade. Animals of different ages received isoproterenol daily or 4 days and cardiac membrane preparations were evaluated on the 5th day (6, 15, 25 days old and adults). Measurements were made of basal activity, activity stimulated by two agonists (isoproterenol or glucagon) that operate at different receptors but that share Gs as the transduction intermediate, or by forskolin-Mn' to assess total catalytic capacity of the cyclase subunit; we also assessed inhibition of activity by carbachol which acts via muscarinic cholinergic receptors and G. Adult rats exhibited robust desensitization of the adenylyl cyclase response but the effect was heterologous in that equivalent loss of activity was seen for basal, isoproterenol- and glucagon-stimulated activity forskolin-Mn(2+)-stimulated activity was also decreased. Two factors contributed to desensitization; generalized reduction in membrane protein concentrations caused by cell enlargement (reduced surface-to-volume ratio), and specific interference with the G-protein component that couples receptors to the cyclase. Thus, after adjustment for changes in membrane protein, the desensitization of the forskolin-Mn2, response was no longer evident, but the effects on the other measures were still present. In addition, isoproterenol treatment produced crosstalk with the carbachol/Gi signaling pathway, with significant reductions in the ability of carbachol to inhibit adenylyl cyclase activity. Heterologous desensitization by isoproterenol was also present in 15 and 25 day old rats, but involved only selective components of the effects seen in adults. At 25 days, uncoupling of signals operating through Gs and Gi was obtained without a reduction in forskolin-Mn(2+)-stimulated activity. At 15 days, only the effect on Gs coupling was seen. At 6 days, agonist-induced desensitization was not detectable and instead, heterologous sensitization was found. In these youngest animals, isoproterenol treatment produced a parallel increase in basal, isoproterenol-, glucagon- and forskolin-Mn(2+)-stimulated activity, unaccompanied by changes in membrane protein concentrations, indicating an increase in adenylyl cyclase catalytic activity. These results indicate that the ability to elicit desensitization is not an inherent property of cardiac cells but rather is acquired in distinct stages during development. Sensitization by agonists early in development may be important in preserving physiological responsiveness during ontogenetic surges of adrenergic activity.