Vascular tone is controlled by a variety of signals including potent vasoconstrictors and thrombotic agents like 5-hydroxytriptamine (5-HT) and Thromboxane A2 (TXA2). The critical role of 5-HT and TXA2 receptors in vascular physiology and pathophysiology is supported by anti-thrombotic models that use both 5-HT- and TXA2-receptor antagonists. Thus, the long-term goal of this research program is to identify the mechanisms and proteins underlying the signaling cascades of TXA2 and 5-HT2A receptors in smooth muscle cells from blood vessels subject to thrombotic episodes, like aorta, coronary, and cerebral arteries. Src tyrosine kinases (classical regulators of cell migration and differentiation) are emerging as new signals in vascular contraction, as their inhibition diminishes Angiotensin (Angll)- and 5-HT-induced vasoconstriction. However, mechanisms of the Src-constricting cascade remained elusive. In this regard, we recently discovered that one mechanism in 5-HTAngll- and Phenylephrine (Phe)-Src tyrosine kinase constricting pathway is K+ channel inhibition in human coronary arteries and rat aorta. We will now test the hypothesis that Src tyrosine kinases may be obligatory steps in 5-HT- and TXA2-induced contraction, may differentially stimulate distinct signaling cascades depending on the membrane receptor being activated, and thus, may be located in specialized membrane compartments forming macromolecular complexes with their signaling partners. Our preliminary data using rat aorta indicate that: 1) Src seems to be an absolute requirement for both 5-HT- and TXA2-induced contractions but not for Phe-induced contraction; 2) 5-HT-induced contraction may result from activation of c-Src; and 3) c-Src and 5-HT receptors may be organized in caveolae forming a macromolecular signaling complex. In this proposal, we will use native tissue mainly from rat aortas and a multidisciplinary approach to perform the following Specific Aims to determine: 1) if Src activation is an obligatory step in 5-HT-, TXA2 (U46619)-, and Phe-induced vasoconstriction, and investigate downstream pathways; 2) the identity of vascular Src tyrosine kinases, their localization in single cells, and their activity in response to 5-HT, TXA2, and Phe; 3) the molecular mechanisms of 5-HT-, TXA2, and Phe-Src-constricting pathways, and their potential organization in macromolecular complexes; 4) the localization of receptor-Src complexes in lipid rafts and possible reorganization upon agonist stimulation; and 5) the c-Src subproteome in resting conditions, and after vasoconstriction with 5-HT. The elucidation of primary steps involved in 5-HT and TXA2 smooth muscle stimulation should help in the design of new therapeutic ways to control cardiovascular disease. ? ?
