Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois;
Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Centre Marburg, Philipps-University of Marburg, Marburg, Germany;
Am J Physiol Heart Circ Physiol. 2014 Sep 1;307(5):H689-700. doi: 10.1152/ajpheart.00694.2013. Epub 2014 Jul 11.
Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.
尿皮质素 2（Ucn2）是一种具有心脏活性的肽，在正常和衰竭的心脏中都具有有益作用。在心肌细胞中，它引起 cAMP 和 Ca（2+）依赖性正性变力和变时作用。我们检验了这样一种假设，即 Ucn2 除了激活心脏一氧化氮（NO）信号外，还阐明了潜在的信号通路和机制。在分离的兔心室肌细胞中，Ucn2 引起 Akt（Ser473，Thr308）、内皮型一氧化氮合酶（eNOS）（Ser1177）和 ERK1/2（Thr202/Tyr204）的浓度和时间依赖性磷酸化增加。ERK1/2 磷酸化，但不是 Akt 和 eNOS 磷酸化，被 MEK1/2 抑制所抑制。增加的 Akt 磷酸化导致 Akt 激酶活性增加，并且是由促肾上腺皮质释放因子 2（CRF2）受体介导的（astressin-2B 敏感）。PI3K 的抑制减弱了 Ucn2 介导的 Akt 和 eNOS 磷酸化。蛋白激酶 A（PKA）的抑制减少了 Ucn2 诱导的 eNOS 磷酸化，但不影响 Akt 的磷酸化增加。相反，通过佛司可林直接受体非依赖性升高 cAMP 增加了 eNOS 的磷酸化，但不增加 Akt 的磷酸化。Ucn2 增加了细胞内 NO 浓度（[NO]i）、[cGMP]、[cAMP]和细胞缩短。eNOS 的抑制抑制了 [NO]i 和细胞缩短的增加。当 PI3K-Akt 和 cAMP-PKA 信号均被抑制时，Ucn2 诱导的 [NO]i 和细胞缩短的增加减弱。因此，在兔心室肌细胞中，Ucn2 引起 cAMP-PKA、PI3K-Akt 和 MEK1/2-ERK1/2 信号的激活。MEK1/2-ERK1/2 途径不是这些细胞中 NO 信号刺激所必需的。另外两个途径，cAMP-PKA 和 PI3K-Akt，在 eNOS 的 Ser1177 磷酸化上汇聚，并导致明显和持续的细胞 NO 产生，随后刺激 cGMP 信号。
