G PROTEINS INHIBITION RESCUES CONTRACTILE RESPONSE--BETA2 ADRENERGIC STIMULATION

工作概要 先前的研究表明，β-肾上腺素能受体（β AR） 刺激不能增加转基因小鼠的心肌收缩力 过表达人β 2 AR（TG 4）被解释为表明， TG 4小鼠的基线心肌收缩力已经增强到 最大水平，由于自发活性β 2 AR数量较多（R*） 在没有激动剂的情况下。为了验证这一假设和潜在的 机制，我们研究了β 2 AR刺激对收缩的影响， TG 4心室肌细胞内Ca 2+瞬变和腺苷酸 TG 4心脏膜中的环化酶活性。 我们还研究了 抑制性G蛋白和β AR激酶1（β ARK 1）参与了 TG 4心脏β AR反应丧失。TG 4的基线收缩力 心室肌细胞相对于野生型增加了三倍 对照锌特罗刺激β 2 AR对两种细胞均无影响。 收缩力或腺苷酸环化酶活性，而 腺苷酸环化酶激活剂，毛喉素，显着增加收缩 这表明TG 4心脏β 2 AR可能是脱敏的， 与下游信号级联分离。BetaARK 1活性不是 在TG 4心脏中显著改变。 百日咳毒素（PTX） 治疗完全挽救了细胞内Ca 2+瞬变，收缩和 腺苷酸环化酶对β 2 AR刺激的反应，表明 PTX敏感的G蛋白是导致 TG 4小鼠的心脏β 2 AR。这一点得到了进一步证实， 掺入光反应性GTP类似物，[α-32 P]GTP叠氮酰苯胺， 在β 2 AR刺激后， 锌特罗或异丙肾上腺素加α 1 β AR阻滞剂，CGP 20712 A。 beta2AR 对Gi蛋白的刺激作用被选择性β 2 AR所消除 阻断剂ICI 118，551或PTX治疗。相反，β 1 AR激动剂， 去甲肾上腺素（10-6 M）不能增强Gi蛋白的掺入。 因此，我们的结论是，心脏收缩反应的损失，β 2 AR 慢性自发β 2 AR激活期间的刺激不是由于 增强的基线收缩力或β ARK 1介导的受体 脱敏，但由于激活β 2AR-偶联 PTX敏感性G蛋白，Gi 2和Gi 3。

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.