FUNCTION OF GUT PEPTIDES IN ISOLATED GUT MUSCLE CELLS

肠肽在分离的肠肌细胞中的功能

• 批准号: 6634872
• 负责人: S Murthy Karnam
• 金额: $ 21.39万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6634872
• 关键词: adenylate cyclase; atrial natriuretic peptide; enzyme activity; enzyme inhibitors; gastrointestinal function; guinea pigs; laboratory mouse; laboratory rabbit; muscle cells; muscle relaxation; myenteric plexus; neuroregulation; neurotransmitter receptor; neurotransmitters; phosphodiesterases; protein kinase; receptor coupling; smooth muscle; tissue /cell culture; vasoactive intestinal peptide

The objective of this proposal is to characterize the receptors, signaling pathways and cellular mechanisms involved in relaxation of gastric smooth muscle by peptide (VIP, PACAP) and non-peptide (NO) neurotransmitters of the myenteric plexus. Several advances made during the previous funding period form the basis of the proposal. We have identified two receptor types for which VIP and PACAP have high affinity: a cognate, seven-transmembrane VIP2/PACAP3 receptor coupled via GS to adenylyl cyclase, and a distinct, single-transmembrane natriuretic peptide clearance receptor (NPR-C) coupled via Gi1 and Gi2 to activation of a constitutive NO synthase (NOS) in smooth muscle cells. We have identified the NOS isoform as eNOS by RT-PCR in muscle cultures and by in situ RT-PCR in single muscle cells. The concurrent stimulation of cAMP and cGMP has important implications with respect to their regulation by specific phosphodiesterases (PDE), activation and cross-activation of cA-kinase and cG-kinase, and feedback regulation of adenylyl cyclase. Preliminary studies have identified the isoforms of PDE (PDE3, PDE4, PDE5) and adenylyl cyclase (types V and VI) expressed in gastric muscle. Accordingly, the first aim is to characterize NPR-C and VIP2/PACAP3 receptors: the studies involve experiments in eNOS minus/minus and NPR-C minus/minus mice, reconstitution studies in COS-1 cells co-transfected with NPR-C and eNOS, site-directed mutagenesis to identify binding and G protein coupling domains, and chimeric constructs and site-directed mutagenesis to identify VIP2/PACAP3 receptor subtypes. The second aim is to characterize the regulation of adenylyl cyclases V/VI by cA-kinase, G proteins and Ca2+ influx; and the regulation of cAMP levels by cAMP-specific PDE4 and PDE3 and cGMP levels by cGMP- specific PDE5. The third aim is to characterize the concurrent activation and cross-activation of cA- and cG-kinase, and their role in phosphorylation of major targets in smooth muscle, including PLC-beta and PLA2, IP3 and ryanodine receptors, and MLC phosphatase. The combined approach embodied by these aims, all of which are supported by preliminary studies, should advance our knowledge of the cellular mechanisms mediating visceral smooth muscle relaxation.

该建议的目的是表征受体， 信号通路和蜂窝机制与放松 通过肽（VIP，PACAP）和非肽（NO）的胃平滑肌 肌丛的神经递质。 在 先前的资金期是该提案的基础。 我们有 鉴定出VIP和PACAP高的两种受体类型 亲和力：耦合的同源，七跨膜VIP2/PACAP3受体 通过GS到Adenylyl环化酶和​​独特的单跨膜 NATRIARITE肽清除受体（NPR-C）通过GI1和GI2耦合 激活平滑肌中构型NO合酶（NOS） 细胞。 我们已经通过RT-PCR在肌肉中确定了NOS同工型为eNOS 培养物和单个肌肉细胞中的原位RT-PCR。 并发 CAMP和CGMP的刺激对 它们对特定磷酸二酯酶（PDE），激活和 CA-激酶和CG激酶的交叉激活，以及反馈调节 腺苷酸环化酶。 初步研究已经确定了 PDE（PDE3，PDE4，PDE5）和Adenylyl Cyclase（V型和VI）表示 在胃肌中。因此，第一个目的是表征NPR-C 和VIP2/PACAP3受体：研究涉及eNOS的实验 减去/减和NPR-C减去小鼠，COS-1的重建研究 与NPR-C和ENOS共转染的细胞，定向诱变为 识别结合和G蛋白耦合结构域以及嵌合构建体 和定向的诱变，以鉴定VIP2/PACAP3受体亚型。 第二个目的是表征腺苷循环酶的调节 v/vi通过CA-激酶，G蛋白和Ca2+流入；和监管 CAMP特异性PDE4和PDE3以及CGMP水平的营地水平由CGMP- 特定PDE5。第三个目的是表征并发 CA和CG激酶的激活和交叉激活及其在 平滑肌中主要靶标的磷酸化，包括PLC-beta 和PLA2，IP3和Ryanodine受体和MLC磷酸酶。组合 这些目标体现的方法，所有这些都得到了支持 初步研究应提高我们对细胞的了解 介导内脏平滑肌松弛的机制。