Laboratorio de Investigación Cardiovascular, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla 41013, Spain.
Cardiovasc Res. 2010 Aug 1;87(3):459-66. doi: 10.1093/cvr/cvq063. Epub 2010 Feb 26.
The aim of this study was to elucidate the signalling pathways implicated in the modulation of cardiac L-type Ca(2+) channels by urocortin (Ucn) in ventricular myocytes.
Adult rat ventricular myocytes were stimulated in vitro with Ucn for 20-40 min. L-type calcium currents (I(CaL)) were measured with the patch-clamp technique, whereas quantification of activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was assessed by sandwich-ELISA. Ucn induced a significant increase in I(CaL) density that was not prevented by the protein kinase A (PKA) inhibitor KT-5720 or the non-selective antagonist of guanine nucleotide exchange factor brefeldin A. The Ucn effect was antagonized by astressin, a corticotropin-releasing factor receptor-2 (CRF-R2) antagonist, and significantly reduced by protein kinase C (PKC) and ERK1/2 inhibitors. The cyclic AMP (cAMP) analogue 8-pCPT-2'OMe-cAMP, which selectively activates the exchange protein activated by cAMP (Epac), was ineffective in modifying I(CaL). Analysis of phospho-ERK1/2 showed that Ucn induced a significant activation of the ERK1/2 pathway in ventricular myocytes and this effect was prevented by pre-incubation with PKC inhibitors.
The present study provides evidence of new mechanisms involved in the modulation of L-type Ca(2+) channels by Ucn in adult ventricular myocytes. We propose that the marked increase in I(CaL) density induced by Ucn is mediated through CRF-R2 and involves PKC-dependent activation of the ERK1/2 pathway, whereas PKA and Epac signalling are not implicated.
本研究旨在阐明促肾上腺皮质素释放激素（Ucn）对心室肌细胞 L 型钙通道的调节所涉及的信号通路。
在体外，用 Ucn 刺激成年大鼠心室肌细胞 20-40 分钟。采用膜片钳技术测量 L 型钙电流（I(CaL)），通过夹心 ELISA 法评估细胞外信号调节激酶 1/2（ERK1/2）的激活情况。Ucn 诱导 I(CaL)密度显著增加，而蛋白激酶 A（PKA）抑制剂 KT-5720 或鸟嘌呤核苷酸交换因子布雷菲德菌素 A 的非选择性拮抗剂不能阻止这种增加。Ucn 的作用被阿斯特林（一种促肾上腺皮质素释放因子受体-2（CRF-R2）拮抗剂）拮抗，并且被蛋白激酶 C（PKC）和 ERK1/2 抑制剂显著降低。环磷酸腺苷（cAMP）类似物 8-pCPT-2'OMe-cAMP，可选择性激活 cAMP 激活的交换蛋白（Epac），对 I(CaL)无作用。磷酸化 ERK1/2 的分析表明，Ucn 可显著激活心室肌细胞中的 ERK1/2 通路，而 PKC 抑制剂的预孵育可阻止这种作用。
本研究提供了新的证据，证明 Ucn 在成年心室肌细胞中调节 L 型钙通道的新机制。我们提出，Ucn 诱导的 I(CaL)密度显著增加是通过 CRF-R2 介导的，涉及 PKC 依赖性 ERK1/2 通路的激活，而 PKA 和 Epac 信号不参与。
