PI3K Offsets b1-Adrenoceptor/PKA-Mediated Positive Inotr

磷酸肌肽3-激酶（PI3K）参与了b2-肾上腺素能受体(b2-AR)/ gi介导的gs -腺苷基环化酶- camp - pka信号的区室化、b2-AR诱导的磷蛋白磷酸化以及心肌细胞的正性肌力和肌萎缩性反应。然而，目前尚不清楚PI3K是否参与b1-AR的信号转导，b1-AR是心肌细胞中表达的主要b-AR亚型。本研究表明，在a1-AR和b2-AR阻断的情况下，去甲肾上腺素选择性刺激b1-AR显著增加成年大鼠心室肌细胞PI3K活性。LY294002 （5.10-6 mol/L）对PI3K的抑制显著增强b1- ar诱导的L型Ca2+电流、细胞内Ca2+瞬态和心肌细胞收缩力的增加，而不改变受体介导的磷蛋白磷酸化，磷蛋白是心脏PKA的关键靶蛋白。LY294002的增强作用可以通过肽抑制剂bARK-ct抑制Gbg信号传导而完全阻断，但不能通过百日咳毒素破坏Gi功能来阻断，这表明与Gs分离的Gbg异源二聚体在b1- ar激活的PI3K信号传导中发挥重要作用。此外，福斯克林直接刺激腺苷酸环化酶也可提高PI3K活性，抑制PI3K可增强福斯克林诱导的收缩反应或cAMP类似物CPT-cAMP激活PKA的收缩反应。相反，抑制PI3K既不影响基础收缩力，也不影响高细胞外Ca2+诱导的肌细胞收缩增加。这些结果表明，gsa刺激的cAMP信号和Gs蛋白释放的游离Gbg可能都参与了b1- ar介导的PI3K激活，这反过来又通过抑制pka依赖性肌层Ca2+内流和随后细胞内Ca2+瞬态的增加，而不改变受体介导的磷酸蛋白磷酸化，在完整的心肌细胞中抑制b1- ar诱导的正性肌力效应。

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.