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REGULATION OF CALCIUM CHANNELS IN VASCULAR SMOOTH MUSCLE

血管平滑肌钙通道的调节

基本信息

批准号：
2445167
负责人：
NICHOLAS SPERELAKIS
金额：
$ 23.99万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-07-01 至 2000-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2445167
关键词：
G protein adenosine triphosphate calcium channel calmodulin dependent protein kinase electrophysiology enzyme inhibitors laboratory rat muscle contraction neural transmission neurotransmitters phosphorylation protein kinase C vascular smooth muscle vasoactive agent vasomotion voltage /patch clamp voltage gated channel

项目摘要

DESCRIPTION: Calcium ion channels in the membrane of vascular smooth muscle (VSM) cells play an important role in excitation-contraction coupling and in setting vasomotor tone. Ca2+ channels may be regulated either directly or indirectly by neurotransmitters, vasoactive hormones and therapeutic agents; regulatory processes may include the formation or release of intracellular messengers such as Ca2+, cAMP, cGMP, IP3/IP4, and diacylglycerol. The objective of the proposed studies is to investigate in detail the specific characteristics and regulatory mechanisms of calcium channels in freshly-isolated VSM cells from several different types of vessels in the rat (small mesenteric artery, aorta, portal vein). Since blood vessels from different vascular beds have different degrees of excitability, the proportion of various ion channels and/or their regulatory processes may differ. Macroscopic and microscopic (single-channel) Ca2+ current (ICa) will be recorded using whole-cell voltage clamp and patch clamp techniques. Intracellular agents will be delivered through the use of the intracellular perfusion technique. A possible role of phosphorylation, mediated by various protein kinases (PK), in regulating ICa will be explored. Specific aims include: (1) Assessing the role of cAMP- and cGMP-dependent PK systems (PK-A and PK- G) and the effect of phosphorylation and dephosphorylation on ICa. Techniques will include the application of cyclic nucleotide analogs, PK-A (cat subunit) and PK-G, and phosphatases (PPases), as well as the use of PK and PPase inhibitors. (2) The role of PK-C in the modulation of ICa, will be explored. Responses to exogenous PK-C activators (i.e. DAG, OAG), to purified PK-C, and to PK inhibitors will be determined. (3) The role of Ca2+-calmodulin-dependent PK (Ca/CaM- PKII), will be investigated using specific activators and inhibitors of this PK. The influence of intracellular levels of calcium on Ca2+ channel activity will also be studied. (4) The role of metabolism and intracellular levels of ATP on ICa will be further studied through the use of stable ATP analogs, phosphorylation blockers and metabolic poisons. (Preliminary data showed that lowering intracellular ATP levels inhibited Ca2+ channel activity and ICa.) (5) The modulation of ICa by G-proteins and G-protein gating will be examined. The results of these studies should provide comprehensive information on the complex mechanisms involved in calcium channel regulation in VSM cells, and should help to define how calcium influx, cellular excitability, and contraction are altered by important vasoactive substances.

描述：血管平滑肌膜上的钙离子通道肌肉（VSM）细胞在兴奋收缩中发挥重要作用耦合和设定血管舒缩张力。 Ca2+ 通道可能是直接或间接受神经递质调节，血管活性激素和治疗剂；监管程序可能包括细胞内信使的形成或释放，例如 Ca2+、cAMP、cGMP、IP3/IP4 和二酰甘油。拟议研究的目的是详细调查钙的具体特征和调节机制来自几种不同类型的新鲜分离的 VSM 细胞中的通道大鼠血管（肠系膜小动脉、主动脉、门脉静脉）。由于来自不同血管床的血管具有不同程度的兴奋性，各种离子的比例渠道和/或其监管流程可能有所不同。宏观微观（单通道）Ca2+电流（ICa）将是使用全细胞电压钳和膜片钳记录技术。细胞内制剂将通过使用来递送细胞内灌注技术。一个可能的角色是磷酸化，由多种蛋白激酶（PK）介导，将探索调节 ICa。具体目标包括：（1）评估 cAMP 和 cGMP 依赖性 PK 系统（PK-A 和 PK- G) 以及磷酸化和去磷酸化对ICa的影响。技术将包括环核苷酸类似物的应用， PK-A（猫亚基）和 PK-G，以及磷酸酶 (PPase)，以及使用 PK 和 PPase 抑制剂。 (2) PK-C的调节作用的ICa，将被探索。对外源 PK-C 激活剂的反应（即 DAG、OAG）、纯化的 PK-C 和 PK 抑制剂将决定。（3）Ca2+-钙调蛋白依赖性PK（Ca/CaM- PKII），将使用特定的激活剂进行研究，该 PK 的抑制剂。细胞内钙水平的影响还将研究 Ca2+ 通道活性。（四）作用 ICa 上的代谢和细胞内 ATP 水平将进一步通过使用稳定的 ATP 类似物、磷酸化阻断剂进行研究和代谢毒物。（初步数据显示，降低细胞内 ATP 水平抑制 Ca2+ 通道活性和 ICa。） (5) G蛋白和G蛋白门控对ICa的调节被检查。这些研究的结果应提供关于所涉及的复杂机制的全面信息 VSM 细胞中的钙通道调节，应该有助于定义如何调节钙流入、细胞兴奋性和收缩发生改变由重要的血管活性物质引起。