PDE3, PDE4 and PKC regulate local Ca2+ releases and cardiac pacemaker firing

为了验证（PDE 3 + PDE 4）的组合活性通过调节cAMP/PKA信号传导影响基础自发SANC放电的假设，我们使用受磷蛋白（PLB）在Ser 16位点的磷酸化作为SANC中cAMP/PKA依赖性蛋白磷酸化的标志物。特异性PDE 3抑制剂西洛酰胺（Cil，0.3 mkmol/L）或PDE 4抑制剂咯利普兰（Rol，2 mkmol/L）使PLB磷酸化增加20%，但Cil+Rol的组合使PLB磷酸化增加110%，与广谱PDE抑制剂IBMX产生的效果（140%）相似。L-型Ca ~（2+）电流（伊卡，L）确保LCR的存在，提供可用于SR中泵送的Ca ~（2+）。单独的Cil或Rol分别使伊卡，L的幅度增加60%和4%，而（Cil+Rol）或IBMX使伊卡，L增加100%。Cilostamide使SANC自发放电率（穿孔膜片钳）增加20%（从14813到17513次/min，n=8），而咯利普兰在2、20或100 mkmol/L时不产生自发放电加速。与IBMX相似，（Cil+Rol）使SANC的自发搏动频率增加50%（从1348次/min增加到19711次/min，n=9），这是由于LCR数量、大小的显著增加和LCR周期的减少，这预示着伴随着自发周期长度的减少。当RyR被ryanodine阻断，LCR被取消时，IBMX和（Cil+Rol）均不能加速DD率或增加SANC放电率，提示Ca ~（2+）循环在PDE依赖性控制自发搏动中起关键作用。我们的结论是，PDE 3和PDE 4调节自发SANC放电，和PDE 4的关键作用，揭示了只有当PDE 3和PDE 4同时抑制。因此，（PDE 3 + PDE 4）的协同作用抑制了基础cAMP/PKA依赖的PLB磷酸化，降低了伊卡，L幅度，从而减少了RyR Ca 2+释放，延长了LCR周期，并限制了自发SANC放电率。

To test the hypothesis that combined activity of (PDE3+PDE4) impacts on basal spontaneous SANC firing via regulation of cAMP/PKA signaling, we used phosphorylation of phospholamban (PLB) at Ser16 site as a marker of cAMP/PKA-dependent protein phosphorylation in SANC. Specific PDE3 inhibitor, cilostamide (Cil, 0.3 mkmol/L), or a PDE4 inhibitor, rolipram (Rol, 2 mkmol/L), increased PLB phosphorylation by 20%, but the combination of Cil+Rol increased PLB phosphorylation by 110%, an effect similar to that (140%) produced by broad spectrum PDE inhibitor IBMX. L-type Ca2+ current (ICa,L) ensures LCR existence, providing Ca2+ available for pumping in SR. Cil or Rol alone increased the amplitude of ICa,L by 60% and 4%, respectively, while (Cil+Rol) or IBMX increased ICa,L by 100%. Cilostamide increased the spontaneous SANC firing rate (perforated patch-clamp) by 20% (from 14813 to 17513 beat/min, n=8), while rolipram produced no acceleration of spontaneous firing at 2, 20 or 100 mkmol/L. Similar to IBMX, (Cil+Rol) increased the spontaneous SANC beating rate by 50% (from 1348 to 19711 beat/min, n=9), the effect was due to a marked increase in the LCR number, size and decrease in the LCR period that predicted the concomitant decrease in the spontaneous cycle length. When RyR were disabled by ryanodine and LCRs were abolished, both IBMX and (Cil+Rol) failed to accelerate DD rate or increase SANC firing rate indicating key role of Ca2+ cycling for PDE-dependent control of spontaneous beating. We conclude that both PDE3 and PDE4 regulate spontaneous SANC firing, and a crucial role of PDE4 is revealed only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of (PDE3+PDE4) suppresses basal cAMP/PKA-dependent PLB phosphorylation and reduces ICa,L amplitude to decrease RyR Ca2+ release, prolong the LCR period and limit the spontaneous SANC firing rate.