DIRECT COUPLING OF B2 ADRENERGIC RECEPTOR TO INHIBITORY G PROTEINS IN MYOCYTES

β-肾上腺素能受体（β AR）与刺激性G 激活腺苷酸环化酶的蛋白（Gs）被认为是一种 β AR信号的“教条”。然而，我们最近的研究表明， 证明百日咳毒素（PTX）治疗特异性增强 β 2 AR而非β 1 AR刺激的正性肌力作用， 大鼠心室肌细胞，这表明β 2 AR是耦合到一个 除了已建立的Gs之外，PTX敏感性G蛋白（Xiao，et al 摩尔Pharmacl. 1995; 45：322-9）。最近产生的a2 AR过表达 转基因小鼠（TG 4）提供了一个独特的机会，进一步探索 PTX敏感性G蛋白在α 2 AR刺激中的作用。以前的研究 表明基线心肌收缩力是 与野生型（WT）小鼠相比， （β 1和β 2）aAR激动剂异丙肾上腺素（ISO）未能进一步增加 心肌收缩力（Milano.等人Science 1994;264：582-6）。这些结果 已被解释为表明基线心肌 TG 4小鼠的收缩力处于最大水平，这是由于 在不存在下， 激动剂（即R* 状态）。在这项研究中，我们研究了 β 2 AR刺激对单个心肌细胞内钙和收缩力的影响 从TG 4和野生型（WT）小鼠心脏分离的心脏细胞。 在控制条件下，基线收缩是3.2倍， TG 4细胞的细胞毒活性高于WT细胞。锌特罗对β 2 AR的刺激没有 对WT和TG 4心室肌细胞收缩幅度的影响。 然而，PTX揭示了一种有效的正性肌力作用和增加 在两种WT中，锌特罗刺激β 2 AR后细胞内钙 和TG 4心脏细胞，并进一步增加TG 4细胞基线 收缩性此外，我们提供的证据表明，β 2AR，而不是 β 1 AR激动剂刺激光反应性GTP类似物的掺入 [α-32 P]GTP叠氮酰苯胺转化为Gi的α亚基（具体地， Gialpha 3）蛋白;这通过PTX预处理消除。这些结果 证明了自发激活的（R*）和 激动剂激活的β 2 AR在功能上与Gialpha 3偶联， 反过来，抑制心脏对β 2 AR刺激的变力反应。

A sole coupling of beta-adrenergic receptor (betaAR) to stimulatory G protein (Gs) to activate adenylyl cyclase has been considered as a "dogma" of betaAR signaling. However, our recent studies have demonstrated that pertussis toxin (PTX) treatment specifically potentiates the positive inotropic effect of beta2AR but not of beta1AR stimulation in rat ventricular myocytes, suggesting that beta2AR is coupled to a PTX-sensitive G protein(s) in addition to the established Gs (Xiao, et al Mol. Pharmacl. 1995; 45:322-9). A recently generated a2AR overexpression transgenic mouse (TG4) provides a unique opportunity to further explore the role of PTX-sensitive G proteins in a2AR stimulation. Previous studies in TG4 mice in vivo indicate that the baseline myocardial contractility is markedly increased over that in the wild type (WT) mice, and that a mixed (beta1&beta2) aAR agonist, isoproterenol (ISO) failed to further increase cardiac contractility (Milano. et al. Science 1994;264: 582-6). These resul have been interpreted to indicate that the baseline myocardial contractility in the TG4 mouse is at the maximal level, and that this is du to a greater number of a2ARs in the active form in the absence of agonists (i.e. the R* state). In this study, we have examined effects of beta2AR stimulation on intracellular calcium and contractility in single heart cells isolated from both TG4 and wild type (WT) mouse hearts. Under control conditions, the baseline contraction is 3.2-fold greater in TG4 cells over that of WT cells. beta2AR stimulation by zinterol has no effect on contraction amplitude in both WT and TG4 ventricular myocytes. However, PTX unmasks a potent positive inotropic effect and an increase in intracellular calcium after beta2AR stimulation by zinterol in both WT and TG4 heart cells and further increases the TG4 cell baseline contractility. Additionally, we provide evidence that beta2AR but not beta1AR agonists stimulate incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into the alpha subunits of Gi (specifically Gialpha3) proteins; this is abolished by PTX pretreatment. These results demonstrate that both the spontaneously activated (R*) and agonist-activated beta2ARs are functionally coupled to Gialpha3, which, in turn, inhibit the cardiac inotropic response to beta2AR stimulation.