Phosphodiesterases Restrict Spontaneous Beating of Cardiac Pacemaker Cells

兔窦房结细胞(SANC)的自发性搏动由cAMP介导的依赖蛋白激酶A的局部肌膜下兰尼定受体(LCRs)控制。LCRs激活内向Na+/Ca~(2+)交换电流，使舒张末期除极速率增加，从而使自发搏动频率增加。SANC的基础cAMP升高，提示磷酸二酯酶(PDE)对cAMP的降解可能较低。令人惊讶的是，用广谱PDE抑制剂3-异丁基甲基黄嘌呤(IBMX)完全抑制PDE活性后，cAMP水平增加了9倍，cAMP介导的蛋白激酶依赖的磷蛋白磷酸化增加了一倍，SANC的放电频率增加了55%，表明SANC中PDE的基础活性很高。特异性PDE1to-5抑制剂的比较表明，PDE3的特异性抑制剂米力农使自发放电增加47%(其他作用很小)，使L型钙电流(ICa，L)的幅度增加46%，表明PDE3是基础状态下的主要成分活性的PDE。PDE抑制对自发放电的依赖依赖于肌膜下LCR，即抑制PDE可增加LCR的幅度和大小，缩短LCR周期，导致LCR钙释放提前和延长，Na+/Ca~(2+)交换电流和放电频率增加。当Ryanodine受体被Ryanodine阻断时，IBMX和米力农都不能放大LCRs，加快舒张期去极化速率，也不能增加SANC放电率，尽管保留PDE抑制了ICa的幅度增加，L。因此，基础成分PDE的激活为降低cAMP、限制cAMP介导的蛋白激酶A依赖的舒张期Ryanodine受体钙释放增加、限制自发搏动频率提供了一种新的、强大的机制。为了研究PDE抑制如何控制SR钙再充盈和LCR周期，我们比较了对照组和PDE抑制后SR钙再充盈的动力学。磷酸化蛋白(PLB)被用来作为SR泵送速率的指标，SR再充盈是通过AP启动的整体胞浆Ca~(2+)瞬变(T-90)衰减到90%的时间来估计的。广谱磷酸二酯酶抑制剂(IBMX)、特异性磷酸二酯酶-3抑制剂(米力农)或特异性PKA抑制肽(PKI)对PLB的分级磷酸化与T-90的比例变化平行。T-90和LCR周期的伴随变化与自发周期长度的变化高度相关。因此，抑制PDE活性增加cAMP介导的PKA依赖的磷酸化，加快肌浆网钙充盈速率，缩短LCR周期和钙时钟，增加SANC的自发搏动频率。

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase Adependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activated an inward Na+/ Ca2+ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase Adependent phospholamban phosphorylation, and increased SANC firing rate by ≈55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by ≈47% (effects of others were minor) and increased amplitude of L-type Ca2+ current (ICa,L) by ≈46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca2+ release, Na+/ Ca2+ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibitioninduced augmentation of ICa,L amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase Adependent increase of diastolic ryanodine receptor Ca2+ release, and restrict the spontaneous SANC beating rate. To study how PDE inhibition controls the SR Ca2+ refilling and LCR period we compared kinetics of SR Ca2+ refilling in control and after PDE inhibition. Phosphorylation of phospholamban (PLB) has been used as index of SR pumping rate and SR refilling was estimated by the time to 90% decay of the AP-initiated global cytosolic Ca2+ transient (T-90). Graded PLB phosphorylation by a broad-spectrum phosphodiesterase inhibitor (IBMX), specific phosphodiesterase-3 inhibitor (milrinone), or by specific PKA inhibitor peptide (PKI) were paralleled by proportional changes in T-90. Concomitant changes in T-90 and LCR period were highly correlated with changes in the spontaneous cycle length. Thus, suppression of PDE activity increases cAMP-mediated PKA-dependent phosphorylation, accelerates the SR Ca2+ refilling rate, decreases the LCR period, Ca2+ clock, and increases spontaneous beating rate of SANC.