REGULATION OF CALCIUM CHANNELS IN VASCULAR SMOOTH MUSCLE

血管平滑肌钙通道的调节

• 批准号: 2219656
• 负责人: NICHOLAS SPERELAKIS
• 金额: $ 22.39万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2219656
• 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

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：血管细胞膜上的钙离子通道 肌肉(VSM)细胞在兴奋收缩中起重要作用 偶联和设定血管舒缩音调。CA2+通道可能是 由神经递质直接或间接调节， 血管活性激素和治疗剂；调节过程可能 包括细胞内信使的形成或释放，如 Ca~(2+)、cAMP、cGMP、IP3/IP4和二酰甘油。 拟议研究的目的是详细调查 钙离子的特性及其调控机制 几种不同类型新鲜分离的VSM细胞中的通道 大鼠血管(肠系膜小动脉、主动脉、门静脉) 脉)。由于来自不同血管床的血管 不同程度的兴奋性，各种离子的比例 渠道和/或它们的监管过程可能不同。宏观 而微观(单通道)钙电流(ICA)将 采用全细胞电压钳和膜片钳记录 技巧。细胞内的药物将通过使用 细胞内灌流技术。一个可能的角色是 由多种蛋白激酶(PK)介导的磷酸化，在 将探索对ICA进行监管。具体目标包括：(1) 评估cAMP和cGMP依赖的PK系统的作用(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(CAT亚基)和PK-G，以及磷酸酶(PPase)，以及 使用PK和PPase抑制剂。(2)PK-C在调制中的作用 将对ICA进行探索。对外源性PK-C激动剂的反应 (即DAG、OAG)、纯化的PK-C和PK抑制剂将是 下定决心。(3)钙调素依赖的蛋白激酶(Ca/CaM-PK)的作用。 PKII)，将使用特定的激活剂和 这种PK的抑制剂。细胞内钙离子水平的影响 对Ca~(2+)通道活性也将进行研究。(四)角色定位 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、磷酸化阻滞剂进行研究 和新陈代谢毒药。(初步数据显示，降低 细胞内ATP水平抑制Ca~(2+)通道活性和ICa。 (5)G蛋白和G蛋白门控对ICA的调节作用 接受检查。这些研究的结果应该提供 关于涉及的复杂机制的全面信息 VSM细胞中钙通道的调节，并应有助于定义如何 钙内流、细胞兴奋性和收缩发生改变 通过重要的血管活性物质。