Myogenic Control: Circular Muscle of Rectosigmoid Colon

生肌控制：直肠乙状结肠环状肌

• 批准号: 7425762
• 负责人: KHALIL N BITAR
• 金额: $ 9.08万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7425762
• 关键词: Acetylcholine; Actins; Actomyosin Adenosinetriphosphatase; Adult; Affect; Aging; Aging-Related Process; Agonist; Animals; Aspartate; Blood Vessels; Brain; Calcium; Cell membrane; Cell physiology; Cells; Ceramides; Colon; Complementary DNA; Complex; Consumption; Coupling; Cytoskeletal Modeling; Data; Defecation; Dehydration; Failure; Figs - dietary; Funding; Genus Cola; Glycine; HSPB1 gene; Heat shock proteins; Human; Hydrolysis; Inflammation; Inflammatory; Laboratories; Large Intestine; Lead; Lecithin; Light; Localized; Location; Maintenance; Mediating; Membrane; Mitogen-Activated Protein Kinases; Molecular Weight; Motor; Movement; Muscle; Muscle Contraction; Muscle function; Mutate; Myosin ATPase; Myosin Light Chain Kinase; Myosin Regulatory Light Chains; Numbers; Oryctolagus cuniculus; Particulate; Pathway interactions; Pattern; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Production; Protein Dephosphorylation; Protein Kinase; Protein Overexpression; Rattus; Recombinant Proteins; Regulatory Pathway; Reporting; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Smooth Muscle; Smooth Muscle Myocytes; Source; Sphingolipids; Sphingomyelins; Tissues; Transgenic Mice; Western Blotting; aged; caldesmon; cell motility; extracellular; functional disability; gastrointestinal; genetic regulatory protein; mutant; programs; response; rhoA GTP-Binding Protein; trafficking

The recruitment of signal transduction molecules to the membrane is crucial for the efficient coupling of extracellular signals and contractile response. Signaling molecules, including protein kinases and phosphatases, need to traffic to specific cellular locations for effective action on their downstream targets. The trafficking is dynamic and is difficult to follow. Preliminary data indicate i:hat acetylcholine- and ceramide-induced contraction of isolated ci'cular smooth muscle cells from the rabbit colon, is associated with: 1) PKCa and RhoA translocation to the membrane 2) an immuno-complexing of PKCa with RhoA in the particulate fraction, and 3) an increase in the association of translocated PKCa and of translocated RhoA with HSP27 in the particulate fraction. Preliminary results also indicate a role for phosphorylated HSP27 in modulating the association of PKCa with RhoA in the particulate fraction. We have generated mutants in Human HSP27 cDNA where in, Ser-15, Ser-78, and Ser-82 were replaced with aspartate or glycine to mimic constitutively phosphorylated (3D constructs) or non- phosphorylated (3G constructs) HSP27. Preliminary observations suggest that PKCa and RhoA failed to translocate to the cell membrane during agonist-induced contraction in rabbit colon smooth muscle cells that were transfected with 3G constructs. Further, failure of translocation was correlated with lack of association of PKCa with RhoA on the membrane, a significant decrease (48.4 ¿ 4% P<0.005) in the association of actin with myosin and an inhibition of acetylcholine-induced contraction. Furthermore, there was an increase in the association of PKCa with RhoA in cells transfected with 3D constructs. Similar results were observed in smooth muscle cells obtained from the colons of transgenic mice overexpressing the phospho-mimic form (3D) of HSP27. We therefore propose to: 1) Examine the membrane-cytoskeletal reorganization and activation of PKCa, RhoA and HSP27 that are activated during agonist-induced contraction. 2) Examine the interaction of RhoA, PKCa and HSP27 using recombinant proteins. 3) Examine the effect of expression of HSP27 mutants on the association of translocated PKCa and RhoA with HSP27 and the formation of a dynamic complex between HSP27-Actin-PKCa-RhoA in transfected cells and in transgenic mice. These pathways, not clearly defined in gastrointestinal smooth muscle, are of functional physiological significance in the normal adult and are affected due to inflammation or due to the aging process.

将信号转导分子募集到膜上对于有效偶联 细胞外信号和收缩反应。信号分子，包括蛋白激酶和 磷酸酶，需要运输到特定的细胞位置，以有效地作用于其下游靶标。 贩运活动是动态的，很难跟踪。 初步数据表明，乙酰胆碱和神经酰胺诱导的离体环平滑肌收缩 来自兔结肠的肌细胞，与：1）PKCa和RhoA易位到膜2） PKCa与颗粒部分中RhoA的免疫复合，以及3） 易位的PKCa和易位的RhoA与颗粒部分中的HSP 27。初步结果还 表明磷酸化HSP 27在调节颗粒中PKCa与RhoA的结合中的作用 分数我们已经在人HSP 27 cDNA中产生了突变体，其中Ser-15，Ser-78和Ser-82被 用天冬氨酸或甘氨酸替代以模拟组成型磷酸化（3D构建体）或非磷酸化（3D构建体）。 磷酸化（3G构建体）HSP 27。初步观察表明，PKCa和RhoA未能 在激动剂诱导的兔结肠平滑肌细胞收缩过程中， 用3G构建体转染。此外，易位失败与缺乏相关性有关。 在细胞膜上，PKC α与RhoA的结合显著减少（48.4 <$4% P<0.005）， 与肌球蛋白和抑制乙酰胆碱诱导的收缩。此外， 在用3D构建体转染的细胞中PKCa与RhoA的关联。中观察到类似的结果 从过表达磷酸模拟形式的转基因小鼠的结肠获得的平滑肌细胞 (3D)HSP 27的 因此，我们建议：1）检查膜细胞骨架重组和激活的PKC α， RhoA和HSP 27在激动剂诱导的收缩期间被激活。2)检查RhoA的相互作用， 使用重组蛋白的PKC α和HSP 27。3)检测HSP 27突变体的表达对细胞凋亡的影响。 易位的PKCa和RhoA与HSP 27的关联以及它们之间动态复合物的形成 转染细胞和转基因小鼠中的HSP 27-肌动蛋白-PKCa-RhoA。这些途径，没有明确定义， 胃肠平滑肌，在正常成人中具有功能生理学意义， 由于炎症或由于衰老过程而受到影响。