Nikolaev Viacheslav O, Bünemann Moritz, Schmitteckert Eva, Lohse Martin J, Engelhardt Stefan
Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany.
Circ Res. 2006 Nov 10;99(10):1084-91. doi: 10.1161/01.RES.0000250046.69918.d5. Epub 2006 Oct 12.
Beta(1)- and beta(2)-adrenergic receptors (betaARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4>PDE2>PDE3. The beta(1)AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas beta(2)AR-mediated cAMP was under control of multiple PDE isoforms. beta(1)AR subtype-specific stimulation yielded approximately 2-fold greater cAMP responses compared with selective beta(2)-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (DeltaFRET, 17.3+/-1.3% [beta(1)AR] versus 8.8+/-0.4% [beta(2)AR]). Treatment with pertussis toxin to inactivate G(i) did not affect cAMP production. Localized beta(1)AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local beta(2)AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, beta(1)ARs induce far-reaching cAMP signals, whereas beta(2)AR-induced cAMP remains locally confined.
已知β1和β2肾上腺素能受体（βARs）对心肌细胞的收缩和生长具有不同的调节作用。我们验证了以下假说：这些差异归因于第二信使环磷酸腺苷（cAMP）的空间分隔。采用基于荧光共振能量转移（FRET）的方法，我们直接监测了成年心肌细胞中cAMP的时空分布。我们基于超极化激活的环核苷酸门控钾通道2（HCN2）的单个cAMP结合结构域开发了一种新型的cAMP-FRET传感器（称为HCN2-camps）。其胞质分布、高动态范围和灵敏度使HCN2-camps特别适合监测心肌细胞cAMP的亚细胞定位。我们构建了HCN2-camps转基因小鼠，并对新鲜分离的心肌细胞进行单细胞FRET成像。用异丙肾上腺素进行全细胞灌注显示cAMP适度升高。应用各种磷酸二酯酶（PDE）抑制剂揭示了通过PDE4>PDE2>PDE3对cAMP进行严格调控。β1AR介导的cAMP信号完全依赖于PDE4活性，而β2AR介导的cAMP受多种PDE同工型的控制。与选择性β2亚型刺激相比，β1AR亚型特异性刺激产生的cAMP反应大约高2倍，即使在用非选择性PDE抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）处理时也是如此（ΔFRET，β1AR为17.3±1.3%，β2AR为8.8±0.4%）。用百日咳毒素处理使G(i)失活并不影响cAMP的产生。局部β1AR刺激产生一个在整个细胞中传播的cAMP梯度，而局部β2AR刺激未引起明显的cAMP扩散。我们的数据表明，在成年心肌细胞中，β1ARs诱导产生影响深远的cAMP信号，而β2AR诱导的cAMP则局限于局部。
