Somekawa Satoshi, Fukuhara Shigetomo, Nakaoka Yoshikazu, Fujita Hisakazu, Saito Yoshihiko, Mochizuki Naoki
Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, Japan.
Circ Res. 2005 Sep 30;97(7):655-62. doi: 10.1161/01.RES.0000183880.49270.f9. Epub 2005 Aug 25.
Gap junctions (GJs) constituted by neighboring cardiac myocytes are essential for gating ions and small molecules to coordinate cardiac contractions. cAMP is suggested to be a potent stimulus for enhancement of GJ function. However, it remains elusive how cAMP potentiates the GJ of cardiomyocytes. Here we demonstrated that the gating function of GJ is enhanced by the protein kinase A (PKA)-dependent signal, and that the accumulation of connexin43 (Cx43), the most abundant Cx in myocytes, is enhanced by an exchange protein directly activated by cAMP (Epac) (Rap1 activator)-dependent signal. The gating function of GJs was analyzed by microinjected dye transfer method. The accumulation of Cx43 was analyzed by quantitative immunostaining. Using the PKA-specific activator N6-benzoyladenosine-3',5'-cyclic monophosphate (6Bnz) and Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT), we could delineate the two important downstream signals of cAMP for enhanced GJ neoformation. Whereas 6Bnz potentiated gating function of GJs with slight accumulation of Cx43 at cell-cell contacts, 8CPT remarkably enhanced the accumulation of Cx43 with a slight effect on gating. We further noticed that adherens junctions (AJs) were maturated by 8CPT, as marked by increased neural-cadherin immunostaining. Because AJ formation precedes the GJ formation, AJ formation accelerated by Epac-Rap1 signal may result in enhanced GJ formation. The involvement of Epac-Rap1 signal in GJ neoformation was further confirmed by evidence that inactivation of Rap1 by overexpression of Rap1GAP1b perturbed the accumulation of Cx43 at cell-cell contacts. Collectively, PKA and Epac cooperatively enhance functional GJ neoformation in cardiomyocytes.
由相邻心肌细胞构成的缝隙连接（GJs）对于离子和小分子的通道形成至关重要，从而协调心脏收缩。有研究表明，环磷酸腺苷（cAMP）是增强缝隙连接功能的有效刺激因素。然而，cAMP如何增强心肌细胞的缝隙连接仍不清楚。在这里，我们证明了缝隙连接的通道形成功能通过蛋白激酶A（PKA）依赖的信号增强，并且心肌细胞中最丰富的连接蛋白43（Cx43）的积累通过环磷酸腺苷直接激活的交换蛋白（Epac）（Rap1激活剂）依赖的信号增强。通过显微注射染料转移法分析缝隙连接的通道形成功能。通过定量免疫染色分析Cx43的积累。使用PKA特异性激活剂N6-苯甲酰腺苷-3',5'-环一磷酸（6Bnz）和Epac特异性激活剂8-（4-氯苯硫基）-2'-O-甲基腺苷-3',5'-环一磷酸（8CPT），我们可以描绘出cAMP增强缝隙连接新生的两个重要下游信号。虽然6Bnz增强了缝隙连接的通道形成功能，在细胞间接触处Cx43有轻微积累，但8CPT显著增强了Cx43的积累，对通道形成功能影响较小。我们进一步注意到，8CPT使黏附连接（AJs）成熟，神经钙黏蛋白免疫染色增加即为标志。由于AJ形成先于缝隙连接形成，由Epac-Rap1信号加速AJ形成可能导致缝隙连接形成增强。通过Rap1GAP1b过表达使Rap1失活扰乱细胞间接触处Cx43的积累这一证据，进一步证实了Epac-Rap1信号参与缝隙连接新生。总之，PKA和Epac协同增强心肌细胞中功能性缝隙连接的新生。
