Our recent work demonstrates that several vascular smooth muscle (VSM) integrins are capable of acutely regulating the tone of resistance blood vessels. The activation of two VSM integrins, alpha v beta 3 and alpha 5 beta 1,produces transient constriction, followed by sustained dilation, of arterioles. Patch clamp methods show that L-type Ca 2+ current in arteriolar myocytes is regulated by both integrins. The signaling pathway through alpha 5 beta 1 integrin involves phosphorylation of the pore-forming alpha channel subunit by pp60c-Src. Potentiation of Ca 2+ current is consistent with alpha 5 beta 1-mediated vasoconstriction but does not explain the sustained vasodilation. However, the vasodilation is blocked by IC channel antagonists, and large-conductance, Ca+ - activated K+ (BK) current in arteriolar myocytes is potentiated by alpha 5 beta 1 ligands, suggesting a specific role for the BK channel. We also find that cloned BK channels expressed in HEK cells are potentiated by integrin activation in the absence of intracellular Ca 2+ changes. The central hypothesis of this proposal is that BK current in arteriolar myocytes is potentiated by alpha 5 beta 1 integrin ligands through a signaling pathway that involves phosphorylation of the channel alpha subunit by an integrin-linked tyrosine kinase. To test this hypothesis, we propose three aims, utilizing a combination of methods involving isolated arterioles, electrophysiology and molecular biology: A) to determine how arteriolar diameter and native arteriolar smooth muscle BK channels are acutely regulated by alpha 5 beta 1 integrin; B) to define the mechanism by which VSM BK current is potentiated by alpha 5 beta 1 integrin; C) to elucidate the tyrosine kinase(s) involved in potentiation of BK current by alpha 5 beta 1 integrin. The results will provide a more complete picture of a previously unappreciated signaling mechanism whereby the extracellular matrix (ECM) can regulate microvascular tone and organ blood flow. These studies will have wide-ranging implications for vascular ion channel regulation in both normal and disease states. For example, it is known that vascular function is compromised subsequent to changes in ECM composition and integrin expression in pathological conditions where vascular remodeling occurs, such as hypertension, atherosclerosis and restenosis. In addition, our results will establish a paradigm for understanding how other ion channels in other tissues may be regulated by integrins.
