Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.

中文翻译：

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Ca（2 +）/钙调蛋白激活的磷酸二酯酶1A在兔心脏窦房结细胞中高表达并调节起搏器功能。

窦房结细胞（SANC）中1型和8型腺苷酸环化酶（ACs）的组成型Ca（2 +）/钙调蛋白（CaM）激活在富含脂质筏的微域内产生cAMP，以启动cAMP-蛋白激酶A（PKA）信号传导，调节这些细胞的基础状态节律性动作电位。在其他细胞类型中越来越多的证据表明这些细胞内Ca（2+）激活的抵消酶，AC和磷酸二酯酶（PDE）之间的平衡。我们假设在SANC中Ca（2 +）/ CaM激活的PDE 1A型的表达和活性高于其他心脏细胞类型。我们发现，窦房结组织中PDE1A蛋白的表达比左心室高5倍，而其mRNA表达在相应的分离细胞中高12倍。PDE1活性（对尼莫地平敏感）占SANC裂解物中总PDE活性的39％，而左心室心肌细胞（LVC）仅占4％。此外，SANC裂解液中的总PDE活性在富含脂质筏的部分中最低（10％），而缺乏脂质筏的部分中最高（76％）（在蔗糖密度梯度上达到平衡沉降）。在完整细胞中，PDE1A免疫标记未定位在细胞表面膜上（结构化照明显微镜成像），而是位于超极化激活的环状核苷酸门控钾通道（HCN4）的免疫标记内部约150nm内，该通道位于脂质筏中。丰富的微环境。在透化SANC，其中表面膜离子通道不起作用，尼莫地平增加自发SR Ca（2+）循环。响应于广谱PDE抑制剂IBMX（通过荧光共振能量转移显微镜检测），HL-1细胞中的PDE1A mRNA沉默可提高自发搏动率，降低cAMP并提高cGMP水平。我们得出结论，SANC脂质筏域中通过由Ca（2 +）/ CaM激活的AC生成的cAMP进行信号传递受到非脂质筏域中Ca（2 +）/ CaM激活的PDE1A的cAMP降解的限制。这表明[Ca（2 +）]-CaM或不同的AC和PDE1A亲和力的局部梯度调节cAMP的产生及其降解，而这种平衡决定了驱动Ca（2 +）-AC-cAMP-PKA信号的强度SANC起搏器功能。广谱PDE抑制剂（通过荧光共振能量转移显微镜检测）。我们得出结论，SANC脂质筏域中通过由Ca（2 +）/ CaM激活的AC生成的cAMP进行信号传递受到非脂质筏域中Ca（2 +）/ CaM激活的PDE1A的cAMP降解的限制。这表明[Ca（2 +）]-CaM或不同的AC和PDE1A亲和力的局部梯度调节cAMP的产生及其降解，而这种平衡决定了驱动Ca（2 +）-AC-cAMP-PKA信号的强度SANC起搏器功能。广谱PDE抑制剂（通过荧光共振能量转移显微镜检测）。我们得出结论，SANC脂质筏域中通过由Ca（2 +）/ CaM激活的AC生成的cAMP进行信号传递受到非脂质筏域中Ca（2 +）/ CaM激活的PDE1A的cAMP降解的限制。这表明[Ca（2 +）]-CaM或不同的AC和PDE1A亲和力的局部梯度调节cAMP的产生及其降解，而这种平衡决定了驱动Ca（2 +）-AC-cAMP-PKA信号的强度SANC起搏器功能。