PI3K Offsets b1-Adrenoceptor/PKA-Mediated Positive Inotr

磷脂酰肌醇3-激酶(PI3K)参与b2-肾上腺素能受体(b2-AR)/Gi介导的Gs-腺酰环化酶-cAMP-PKA信号的区隔，阻断b2-AR诱导的磷蛋白磷酸化以及心肌细胞正性变力和趋性反应。然而，目前还不清楚PI3K是否参与了b1-AR的信号转导，b1-AR是心肌细胞表达的主要b-AR亚型。在此，我们发现在A1-AR和b2-AR阻断剂存在的情况下，去甲肾上腺素选择性刺激b1-AR显著增加成年大鼠心肌细胞PI3K的活性。LY294002(5.10-6mol/L)抑制PI3K显著增强b1-AR引起的L型钙电流、细胞内钙瞬变和心肌细胞收缩的增加，但不改变受体介导的磷蛋白的磷酸化。LY294002的增强作用可被多肽抑制剂Bark-ct抑制GBG信号完全阻止，但不能被百日咳毒素干扰Gi功能，这表明从Gs解离的GBG异源二聚体在b1-AR激活的PI3K信号转导中起重要作用。此外，Forsklin直接刺激腺苷环化酶也可提高PI3K活性，抑制PI3K可增强对Forsklin引起的收缩反应的收缩反应或cAMP类似物CPT-cAMP激活PKA的收缩反应。相反，抑制PI3K既不影响基础收缩能力，也不影响高细胞外钙离子诱导的心肌细胞收缩增加。这些结果表明，GSA刺激的cAMP信号和Gs蛋白释放的游离GBG都可能参与b1-AR介导的PI3K激活，进而通过抑制依赖PKA的肌膜钙内流和随后增加的细胞内钙瞬变来抵消b1-AR诱导的正性变力作用，而不改变受体介导的磷蛋白磷酸化。

Phosphoinositide 3-kinase (PI3K) has been implicated in b2-adrenergic receptor (b2-AR)/Gi-mediated compartmentation of the concurrent Gs-adenylyl cyclase-cAMP-PKA signaling, negating b2-AR-induced phospholamban phosphorylation and the positive inotropic and lusitropic responses in cardiomyocytes. However, it is unclear whether PI3K is involved in the signal transduction of b1-AR, the predominant b-AR subtype expressed in cardiomyocytes. Here, we show that selective b1-AR stimulation by norepinephrine in the presence of a1-AR and b2-AR blockade markedly increases PI3K activity in adult rat ventricular myocytes. Inhibition of PI3K by LY294002 (5.10-6 mol/L) significantly enhances b1-AR-induced increases in L-type Ca2+ currents, intracellular Ca2+ transients, and myocyte contractility, without altering the receptor-mediated phosphorylation of phospholamban, a key cardiac PKA target protein. The potentiating effects of LY294002 are completely prevented by inhibiting Gbg signaling with a peptide inhibitor, bARK-ct, but not by disrupting Gi function with pertussis toxin, suggesting that the Gbg heterodimer dissociated from Gs plays an essential role in b1-AR-activated PI3K signaling. Moreover, direct stimulation of adenylyl cyclase by forskolin also elevates PI3K activity, and inhibition of PI3K enhances the contractile response to forskolin-induced contractile response or activation of PKA with a cAMP analogue, CPT-cAMP. In contrast, inhibition of PI3K affects neither the basal contractility nor high extracellular Ca2+-induced increase in myocyte contraction. These results suggest that both the Gsa-stimulated cAMP signaling and the free Gbg released from Gs proteins are likely involved in b1-AR-mediated PI3K activation, which, in turn, negates b1-AR-induced positive inotropic effect via inhibiting PKA-dependent sarcolemmal Ca2+ influx and the subsequent increase in intracellular Ca2+ transients, without altering the receptor-mediated phospholamban phosphorylation, in intact cardiomyocytes.