SIGNAL CROSS-TALK IN VASCULAR SMOOTH MUSCLE CELLS

血管平滑肌细胞中的信号串扰

基本信息

批准号：
2224644
负责人：
JERRY G WEBB
金额：
$ 17.61万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-04-01 至 1997-03-31
项目状态：
已结题

项目摘要

The goal of these studies is to understand mechanisms of cross- communication between the major transmembrane signal pathways (i.e., inositol phosphates, cAMP) in vascular smooth muscle cells that are key to the regulation of vascular tone. We have observed a pronounced effect of the pressor peptide, angiotensin II, to synergistically enhance the action of dilator agonists (isoproterenol, PGI-2, adenosine) to stimulate cAMP formation in cultured vascular smooth muscle cells. The objectives of this proposal are to determine the molecular basis for this observed synergism and to evaluate the significance of such signal cross-talk to the modulation of vascular tone. A working hypothesis is that angiotensin II enhancement of hormone-induced cAMP formation results from the action of angiotensin II to elevate intracellular free Ca2+ which acts synergistically with the activated stimulatory guanine nucleotide regulatory protein, G2, to stimulate a calmodulin-sensitive form of adenylyl cyclase in the membrane of vascular smooth muscle cells. It is also proposed that calcium-mobilizing constrictor agonists which enhance cAMP stimulation in vascular smooth muscle cells will sensitize blood vessels to the relaxant effects of dilator agents that act through adenylyl cyclase activation. To test these hypotheses, studies will be conducted with isolated aortic rings and with particulate membranes and partially purified adenylyl cyclases isolated from a homogeneous population of cultured vascular smooth muscle cells. Experiments are proposed: i) to determine the effects of calcium-mobilizing constrictor agonists on the relaxation of aortic rings by dilator agents (isoproterenol, prostacyclin) that act through cAMP in comparison to dilator agents (nitroprusside, ANF) that act through alternative mechanisms; 2) to characterize Ca2+-calmodulin regulation of basal and stimulated adenylyl cyclase in particulate membranes and in a partially- purified enzyme preparation where G2 remains effectively coupled to the catalytic unit of adenylyl cyclase; and 3) to examine the influence of activated G proteins (G5, Gi,Go) and the beta-gamma subunit complex of these proteins on the regulation of different adenylyl cyclase isoforms solubilized from vascular smooth muscle cells and separated using calmodulin-sepharose affinity chromatography (preliminary studies support the feasibility of this approach). These studies should provide new information on the cellular processes that participate in hormonal control of vascular tone and have the potential to substantially broaden our perspective on the possible mechanisms that may underlie hypertension and other forms of vascular disease.

这些研究的目的是了解交叉机制主要跨膜信号通路之间的通讯（即血管平滑肌细胞中的磷酸肌醇 (cAMP) 是维持血管平滑肌细胞功能的关键血管张力的调节。我们观察到了明显的效果升压肽，血管紧张素 II，协同增强作用扩张剂激动剂（异丙肾上腺素、PGI-2、腺苷）刺激 cAMP 在培养的血管平滑肌细胞中形成。本次活动的目标建议确定这种观察到的协同作用的分子基础并评估这种信号串扰对血管张力的调节。一个可行的假设是血管紧张素 II 激素诱导的 cAMP 形成的增强是由于血管紧张素 II 升高细胞内游离 Ca2+，其作用与激活的刺激性鸟嘌呤核苷酸协同作用调节蛋白 G2，刺激钙调蛋白敏感形式血管平滑肌细胞膜上的腺苷酸环化酶。这是还提出钙动员收缩剂激动剂可以增强血管平滑肌细胞中的 cAMP 刺激将使血液变得敏感血管扩张剂的松弛作用通过腺苷酸环化酶激活。为了检验这些假设，研究将用孤立的主动脉环和颗粒膜进行从均质中分离的部分纯化的腺苷酸环化酶培养的血管平滑肌细胞群。实验是建议：i) 确定钙动员收缩剂的作用激动剂对扩张剂舒张主动脉环的影响（异丙肾上腺素、前列环素）通过 cAMP 发挥作用，与通过替代作用的扩张剂（硝普钠，ANF）机制； 2) 表征Ca2+-钙调蛋白对基础和刺激颗粒膜和部分腺苷酸环化酶纯化的酶制剂，其中 G2 保持有效偶联腺苷酸环化酶的催化单元； 3) 检验影响激活的 G 蛋白（G5、Gi、Go）和 β-γ 亚基复合物这些蛋白质对不同腺苷酸环化酶亚型的调节从血管平滑肌细胞中溶解并使用分离钙调蛋白-琼脂糖亲和层析（初步研究支持该方法的可行性）。这些研究应该提供新的有关参与激素控制的细胞过程的信息血管张力并有可能大大拓宽我们的对高血压的可能机制的看法其他形式的血管疾病。