Regulation of Arteriolar Tone & K Channels by Integrins

描述(申请人提供)：我们最近的工作表明，几种血管平滑肌(VSM)整合素能够敏锐地调节阻力血管的张力。两种VSM整合素--αvβ3和α5β1的激活可导致小动脉短暂收缩，随后持续扩张。膜片钳方法显示，小动脉肌细胞上的L钙电流受两种整合素的调节。通过α5β1整合素的信号通路涉及pp60c-Src对形成孔道的α通道亚单位的磷酸化。钙电流的增强与α5β1介导的血管收缩是一致的，但不能解释持续的血管扩张。然而，IC通道拮抗剂阻断血管扩张，小动脉细胞的大电导、钙激活的K+(BK)电流被α5β1配体增强，提示BK通道具有特殊的作用。我们还发现，在HEK细胞中表达的克隆的BK通道在没有细胞内钙变化的情况下被整合素激活而增强。这一建议的中心假设是，小动脉肌细胞的BK电流是由整合素α5β1整合素配体通过一种信号通路增强的，该信号通路涉及整合素连接的酪氨酸激酶对通道α亚基的磷酸化。为了验证这一假说，我们提出了三个目标，利用分离的小动脉、电生理学和分子生物学的方法：A)确定α5β1整合素如何强烈地调节小动脉直径和天然小动脉平滑肌BK通道；B)确定α5β1整合素增强VSM BK电流的机制；C)阐明参与α5β1整合素增强BK电流的酪氨酸激酶(S)。这一结果将提供一个更完整的图景，说明以前未被认识的信号机制，即细胞外基质(ECM)可以调节微血管张力和器官血流。这些研究将对正常和疾病状态下的血管离子通道调节产生广泛的影响。例如，众所周知，在发生血管重塑的病理条件下，如高血压、动脉粥样硬化和再狭窄，ECM成分和整合素的表达发生变化，导致血管功能受损。此外，我们的结果将为理解其他组织中的其他离子通道如何受到整合素的调节建立一个范例。

DESCRIPTION (provided by applicant): 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.