Warrier Sunita, Belevych Andriy E, Ruse Monica, Eckert Richard L, Zaccolo Manuela, Pozzan Tullio, Harvey Robert D
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106-4970, USA.
Am J Physiol Cell Physiol. 2005 Aug;289(2):C455-61. doi: 10.1152/ajpcell.00058.2005. Epub 2005 Mar 23.
beta-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by beta-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of beta-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types.
β-肾上腺素能受体激活通过刺激环磷酸腺苷（cAMP）生成及随后激活蛋白激酶A（PKA）来调节心肌细胞功能。此外，毒蕈碱受体激活既抑制又促进这些依赖cAMP的效应。然而，一直以来并非总能将毒蕈碱反应与细胞cAMP活性变化的直接测量结果相关联。最近开发出了基因编码生物传感器，使得在单细胞水平监测cAMP和PKA活性的实时变化成为可能。一种这样的生物传感器由分别用青色和黄色荧光蛋白标记的PKA调节亚基和催化亚基组成。影响这些标记的PKA亚基缔合的cAMP活性变化可作为荧光共振能量转移的变化来检测。在本研究中，开发了一种基于腺病毒的方法，用于在成年心肌细胞中表达这种重组蛋白复合物，并利用它来监测由β-肾上腺素能和毒蕈碱受体激活产生的cAMP活性变化。使用该系统表达的生物传感器能够检测由生理相关水平的β-肾上腺素能受体激活产生的cAMP活性变化，而不会破坏正常的功能反应。还能够直接证明这些细胞中毒蕈碱受体激活产生的cAMP活性抑制和刺激变化的复杂时间模式。我们开发的基于腺病毒的方法应有助于在多种细胞类型中使用这种生物传感器来研究cAMP和PKA依赖性信号传导机制。
