Department of Pharmacology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
J Cardiovasc Pharmacol Ther. 2012 Jun;17(2):190-8. doi: 10.1177/1074248411416815. Epub 2011 Aug 9.
G protein-coupled receptors for adenosine (A(1), A(3), A(2A), and A(2B)), bradykinin (B(1)) and opioids (δ) are all involved in the mechanism of ischemic preconditioning. Although the heart is comprised of many tissue types, it has been assumed that preconditioning's protective signaling occurs in the cardiomyocyte. We critically tested that hypothesis by testing for the presence of each of these receptors in isolated adult rabbit ventricular myocytes that had been transfected with cyclic nucleotide-gated (CNG) ion channels. Because subsarcolemmal cyclic adenosine monophosphate (cAMP) opens the CNG channels, we could monitor cAMP levels within a single cardiomyocyte by measuring channel current with a patch pipette. The presence of a receptor would be confirmed if we could alter cAMP in the cell with a selective agonist to the receptor being studied. Superfusion with the β-adrenergic G(s)-coupled receptor agonist isoproterenol (50 nmol/L) transiently increased cAMP levels and, therefore, channel current. Pretreatment with selective agonists to A(1) or A(3) adenosine receptors (ARs) that are G(i)-coupled markedly attenuated the response to isoproterenol, indicating inhibition of adenylyl cyclase by increased G(i) activity. Agonists to bradykinin or δ-opioid receptors also attenuated isoproterenol's response. A(2A)AR and A(2B)AR are G(s)-coupled. The A(2A)AR-selective agonist CGS21680 increased current through CNG channels but only in the presence of phosphodiesterase (PDE) inhibitors, indicating low surface receptor activity and high intracellular PDE activity. As we previously reported, BAY 60-6583, an A(2B)AR-selective agonist which mimics preconditioning's protection in rabbit heart, neither increased nor decreased membrane current in transfected cardiomyocytes, suggesting the absence or a markedly limited number of A(2B)AR in the sarcolemma. However, reverse transcription polymerase chain reaction (RT-PCR) of purified cardiomyocytes yielded an A(2B)AR band, implying that rabbit cardiomyocytes do indeed express A(2B)AR. These data reveal that all receptors reported to be involved in ischemic preconditioning do exist on or within the cardiomyocyte.
G 蛋白偶联受体家族中的腺苷（A1、A3、A2A 和 A2B）、缓激肽（B1）和阿片（δ）受体均参与了缺血预处理的机制。尽管心脏由多种组织类型组成，但人们一直认为预处理的保护信号发生在心肌细胞中。我们通过测试转染了环核苷酸门控（CNG）离子通道的成年兔心室肌细胞中这些受体的存在来严格检验这一假说。因为细胞溶质层下的环腺苷酸单磷酸（cAMP）可以打开 CNG 通道，所以我们可以通过用膜片钳测量通道电流来监测单个心肌细胞内的 cAMP 水平。如果我们可以用研究受体的选择性激动剂改变细胞内的 cAMP，那么就可以确认受体的存在。用 β-肾上腺素能 G(s)-偶联受体激动剂异丙肾上腺素（50 nmol/L）灌流可以短暂增加 cAMP 水平，从而增加通道电流。用选择性激动剂预处理 A1 或 A3 腺苷受体（AR），这些受体与 G(i)偶联，明显减弱了异丙肾上腺素的反应，表明 cAMP 合成酶的活性增加导致腺苷酸环化酶抑制。缓激肽或 δ-阿片受体激动剂也减弱了异丙肾上腺素的反应。A2AAR 和 A2BAR 与 G(s)偶联。A2AAR 选择性激动剂 CGS21680 增加了 CNG 通道的电流，但仅在磷酸二酯酶（PDE）抑制剂存在的情况下，表明表面受体活性低而细胞内 PDE 活性高。正如我们之前报道的，BAY 60-6583，一种模拟兔心预处理保护作用的 A2BAR 选择性激动剂，在转染的心肌细胞中既没有增加也没有减少膜电流，这表明在质膜中缺乏或存在明显数量有限的 A2BAR。然而，用纯化的心肌细胞进行逆转录聚合酶链反应（RT-PCR）得到了 A2BAR 带，这意味着兔心肌细胞确实表达了 A2BAR。这些数据表明，所有与缺血预处理有关的受体都存在于心肌细胞内或表面。
