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.
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