Activation of protein kinase A by endothelin-1

血管活性肽内皮素-1 （ET1）通过多种信号通路刺激血管平滑肌收缩，促进血管平滑肌肥大。以前，有不同的报道称ET1可以升高血管平滑肌细胞（VSMC）中的cAMP，但由于影响不大，这种升高的功能后果尚未得到探讨。我们发现，尽管ET1对cAMP的影响很小，但它可以显著激活蛋白激酶A （PKA）——一种被认为与抑制VSMC收缩和增殖有关的效应酶。然而，我们的初步数据表明，在VSMC中，ET1与β激动剂异丙肾上腺素（ISO）诱导的PKA激活在几个关键方面有所不同：(i) ET1诱导的PKA激活持续时间远短于ISO诱导的PKA激活持续时间；（ii） et1诱导的PKA激活促进了肥厚性生长，而iso诱导的PKA激活则没有这种作用；（iii）当被ET1激活时，PKA磷酸化一组蛋白质，其中一些蛋白质与ISO刺激时PKA磷酸化的蛋白质不同。这些初步数据表明，PKA激活模式（ET1 vs. ISO）深刻影响VSMC中PKA激活的一些功能后果。本提案的主要目的是：a)确定ET1激活PKA的信号机制，以及它们与ISO诱导的信号机制有何不同；b)开始理解PKA如何促进ET1诱导的VSMC肥大，以及为什么当ISO激活时PKA没有这样做。为了实现我们的主要目标，我们提出了三个具体目标：1)确定VSMC中ET1瞬时激活PKA的机制；2)检测蛋白激酶B/Akt是否以pka依赖的方式被ET1磷酸化和激活；3)鉴定以pka依赖方式被ET1或ISO差异磷酸化的蛋白。这项研究具有基础和实际意义，因为它可能揭示PKA激活的新的激动剂特异性后果，并可能有助于更好地了解高血压、动脉粥样硬化、再狭窄和血管痉挛的发病机制。

DESCRIPTION (provided by applicant: The vasoactive peptide endothelin-1 (ET1) stimulates contraction and promotes hypertrophy of vascular smooth muscle, through diverse signaling pathways. Previously, it has been variably reported that ET1 can elevate cAMP in vascular smooth muscle cells (VSMC), but the functional consequences of this elevation have not be explored because the effects were modest. We have found that despite its minimal effect on cAMP, ET1 can dramatically activate protein kinase A (PKA) - an effector enzyme thought to be implicated in inhibition of VSMC contraction and proliferation. However, our preliminary data indicates that in VSMC, PKA activation by ET1 differs from that induced by beta-agonist, isoproterenol (ISO), in several key ways: (i) the duration of PKA activation by ET1 is much shorter than that induced by ISO; (ii) ET1-induced PKA activation promotes hypertrophic growth, whereas ISO-induced PKA activation has no such effect; and (iii) when activated by ET1, PKA phosphorylates a complement of proteins, some of which are different from those phosphorylated by PKA during ISO stimulation. These preliminary data indicate that the mode of PKA activation (ET1 vs. ISO) profoundly influences some of the functional consequences of PKA activation in VSMC. The major objective of this proposal is a) to identify the signaling mechanisms of PKA activation by ET1 and how they are different from those induced by ISO, and b) to begin understanding how PKA promotes ET1-induced hypertrophy of VSMC and why it does not do this when activated by ISO. To achieve our major objective, we propose three specific aims: 1) identify the mechanisms of transient PKA activation by ET1 in VSMC; 2) examine whether protein kinase B/Akt is phosphorylated and activated by ET1 in a PKA-dependent manner; and 3) identify the proteins that are differentially phosphorylated by ET1 or ISO in a PKA-dependent manner. This study is of fundamental and practical importance, as it may uncover the new agonist-specific consequences of PKA activation and may lead to a better understanding the pathogenesis of hypertension, atherosclerosis, restenosis and vasospasm.