Regulation Microvascular Smooth Muscle Ca2+ & BK+ ECM-Integrin-Cytoskeletal Axis

血管整合素和血管壁细胞外基质成分之间的相互作用是重要的 小动脉张力和血流控制的决定因素。在项目2中，我们集中在两个血管功能方面，其中细胞外基质-整合素相互作用敏锐地调节血管张力：1)通过电压门控的L型钙离子通道和大电导钙激活的K*通道进行血管内压的机械转导；2)生物活性的基质密码素(即具有血管活性的细胞外基质蛋白的蛋白分解片段)的作用。项目2的中心假设是，A5B1内质蛋白在介导生理性伸展到mVSM离子通道以增强肌张力的过程中起关键作用，而avB3内质蛋白作为一种配基受体发挥作用，抑制肌张力并启动血管扩张，以响应血管壁神经。这一假说将通过膜片钳方法记录单个大鼠或小鼠mVSM细胞的Cal和BK(大电导钙激活的K*通道)电流以及分离的小动脉的直径反应，并结合腺病毒方法来操纵小动脉平滑肌中特定蛋白的表达。有两个具体的目标Aim A将专注于纵向细胞伸展如何通过整合素转导来增强CBL和BK通道以及肌源性张力。我们预测：ASPI整合素而不是avp3整合素转导机械力来调节CBL和BK通道；talin-1、paxlin、a-actinin和p130Cas是通过整合素向VSM通道传递压力所需的关键焦点黏附蛋白；拉伸诱导整合素链连接的激酶和磷酸酶增强VSM通道。目的B将重点研究AVP整合素如何作为一种Matricritin受体来抑制肌源性张力。我们预测：纤维粘连蛋白、骨桥蛋白和胶原蛋白含有诱导小动脉扩张的基质结合位点；Matriritins通过抑制mVSM Cal通道和/或激活BK通道发挥作用；Matricritin通过Avps整合素诱导的信号转导是由Src对p3整合素尾部的磷酸化介导的。这些研究将扩大我们对生理拉伸如何调节关键的VSM离子通道以及ECM蛋白的蛋白分解片段如何发挥其血管活性作用的了解。

Interactions between vascular integrins and ECM components of the blood vessel wall are important determinants of arteriolar tone and blood flow control. In Project 2, we focus on two aspects of vascular function in which ECM-integrin interactions acutely regulate vascular tone: 1) mechanotransduction of intravascular pressure through voltage-gated, L-type Ca^* channels (CBL) and BK (large-conductance calcium-activated K*) channels, and 2) the effects of biologically active matricryptins (i.e., proteolytic fragments of ECM proteins with vasoactive properties). The central hvpothesis of Project 2 is that a5B1 inteqrin plavs a critical role in mediatinq the transduction of physiological stretch to mVSM ion channels to enhance mvoqenic tone, while avB3 inteqrin functions as a matricryptin receptor to inhibit myoqenic tone and initiate vasodilation in response to vessel wall iniurv. The hypothesis will be tested using patch-clamp methods to record CaL and BK (large-conductance calcium-activated K* channel) currents in single rat or mouse mVSM cells and diameter responses of isolated arterioles in conjunction with adenoviral methods to manipulate the expression of selected proteins in arteriolar smooth muscle. There are two specific aims Aim A will focus on how longitudinal cell stretch is transduced through integrins to potentiate CBL and BK channels and myogenic tone. We predict that: aSpi integrin but not avp3 integrin transduces mechanical force to regulate CBL and BK channels; talin-1, paxillin, a-actinin and p130Cas are critical focal adhesion proteins required for force transmission through integrins to VSM channels; stretch-induced potentiation of by integrin-linked kinases and phosphatases. Aim B will focus on how avpS integrin functions as a matricryptin receptor to inhibit myogenic tone. We predict that: fibronectin, osteopontin and collagen contain matricryptic sites that induce arteriolar dilatation; matricryptins act by inhibition of mVSM CaL channels and/or activation of BK channels; matricryptin-induced signaling through avpS integrin is mediated by phosphorylation of the p3 integrin tail by Src. The studies will expand our knowledge of how physiologic stretch regulates key VSM ion channels and how proteolytic fragments of ECM proteins exert their vasoactive effects.