Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
J Cell Physiol. 2012 Sep;227(9):3201-7. doi: 10.1002/jcp.24008.
Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.
腺苷 A(1) 受体 (A(1)R) 诱导蛋白激酶 Cε (PKCε) 向分离的大鼠心肌细胞中的横向 (t) 管状膜易位与 β(1)-肾上腺素能刺激的收缩功能降低有关。内皮素-1 通过蛋白激酶 D (PKD) 的 PKCε 介导的激活被 β(1)-肾上腺素能刺激的蛋白激酶 A (PKA) 抑制，这表明心脏中可能存在 A(1)R 信号转导调节的类似机制。我们研究了 β(1)-肾上腺素能刺激对 A(1)R 诱导的 PKCε 易位的影响。使用 PKCε 和 β-COP 抗体的免疫荧光成像和 Western blot 用于定量分离的大鼠心肌细胞中 PKCε 和 t-管状结构的共定位。A(1)R 激动剂 CCPA 增加了成像检测到的 PKCε 和 t-小管的共定位。β(1)-肾上腺素能受体激动剂异丙肾上腺素 (ISO) 抑制了 CCPA 的这种作用。强效 PKA 激活剂 Forskolin 模拟了 PKA 药理学抑制剂 H89 和膜可渗透的 PKA 肽 PKA 抑制剂 PKI，减弱了 ISO 对 A(1)R 介导的 PKCε 易位的负性作用。用分离的完整心脏进行 Western blot 显示，A(1)R 诱导的 PKCε/β-COP 共定位增加。这种增加被 A(1)R 拮抗剂 DPCPX 和 ISO 减弱。ISO 诱导的衰减被 H89 逆转。结论是，肾上腺素能刺激抑制 A(1)R 诱导的 PKCε 易位至 PKCε 锚定位点 RACK2，RACK2 是包含 β-COP 的衣被小体的组成部分，并与心脏的 t-管状结构相关。由于这种易位先前与 A(1)R 的抗肾上腺素能特性相关，因此显然，腺苷和 β(1)-肾上腺素能激动剂对功能的相互作用在心脏中是复杂的。
