G PROTEINS INHIBITION RESCUES CONTRACTILE RESPONSE--BETA2 ADRENERGIC STIMULATION

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SUMMARY OF WORK Previous studies showing that beta-adrenergic receptor (betaAR) stimulation cannot increase cardiac contractility in transgenic mice overexpressing the human beta2ARs (TG4) were interpreted to indicate that the baseline cardiac contractility of TG4 mice was already enhanced to the maximal level, due to a greater number of spontaneous active beta2ARs (R*) in the absence of agonist. To test this hypothesis and the underlying mechanisms, we investigated effects of beta2AR stimulation on contraction and intracellular Ca2+ transient in TG4 ventricular myocytes and adenylyl cyclase activity in TG4 cardiac membranes. We also examined possible involvement of inhibitory G proteins and betaAR kinase1 (betaARK1) in the loss of TG4 cardiac betaAR response. Baseline contractility of TG4 ventricular myocytes was increased by three-fold relative to wild type controls. Beta2AR stimulation by zinterol had no effect on either the contractility or adenylyl cyclase activity in TG4 mice, whereas an adenylyl cyclase activator, forskolin, markedly increased the contraction amplitude, suggesting that TG4 cardiac beta2ARs might be desensitized or uncoupled from down-stream signaling cascades. BetaARK1 activity was not significantly altered in TG4 hearts. However, pertussis toxin (PTX) treatment fully rescued the intracellular Ca2+ transient, contractile and adenylyl cyclase responses to beta2AR stimulation, indicating PTX-sensitive G proteins are responsible for the unresponsiveness of cardiac beta2ARs in TG4 mice. This was further confirmed by the increased incorporation of a photoreactive GTP analog, [alpha-32P]GTP azidoanilide, into alpha- subunits of Gi2 and Gi3 following beta2AR stimulation by zinterol or isoproterenol plus a1betaAR blocker, CGP 20712A. The beta2AR stimulatory effect on Gi protein was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. In contrast, a beta1AR agonist, norepinephrine (10-6 M), can not enhance the incorporation of Gi proteins. Thus, we conclude that the loss of cardiac contractile response to beta2AR stimulation during chronic spontaneous beta2AR activation is not due to the enhanced baseline contractility or to betaARK1-mediated receptor desensitization, but due to an activation of the beta2AR-coupled PTX-sensitive G proteins, Gi2 and Gi3.