SIGNAL TRANSDUCTION MECHANISMS IN COLONIC SMOOTH MUSCLE

兴奋性神经递质通过多种信号通路产生结肠平滑肌收缩，包括改变离子电导（Na+, Ca2+, K+）引起肌浆Ca2+振荡。除了离子信号机制外，乙酰胆碱和神经激肽A还能激活犬结肠平滑肌中的ERK和p38 MAP激酶。在哺乳动物平滑肌中，将神经递质受体与这些下游效应蛋白激酶偶联的途径定义不清。我们的工作假设是Src家族的非受体蛋白酪氨酸激酶和磷脂激酶，磷脂酰肌醇-3激酶（PI-3激酶）被乙酰胆碱激活，而神经激肽A是信号转导的早期sep。具体目标是：1.；确定哪些Src家族酪氨酸激酶的成员在结肠平滑肌中表达。同种异构体选择性抗体将用于鉴定结肠平滑肌提取物中的Src、Fyn、Yes、Lck和Lyn。逆转录酶PCR将用于克隆和测序src家族酪氨酸激酶的cdna。SRC-家族酪氨酸激酶将被分离并通过MonoQ和免疫亲和层析进行表征。2. 确定Src家族的哪些成员被乙酰胆碱和神经激肽a激活。免疫沉淀后检测神经递质刺激的激酶活性。将定义激活的时间和浓度依赖性。将研究M3和M2毒蕈碱受体的相对贡献，以及百日咳毒素抑制G蛋白的作用。3. 明确src家族酪氨酸激酶与PI-3激酶之间的关系。抑制PI-3激酶后检测Src家族激酶活性，抑制Src家族激酶后检测PI-3激酶活性。4. 确立src家族受体酪氨酸激酶的功能。同种异构体选择性酪氨酸激酶抑制剂将对完整和培养结肠平滑肌的肌肉收缩、离子电导和肌浆Ca2+浓度的影响进行测试。腺病毒载体将用于在培养细胞中表达显性阴性和本构活性Src突变体。电穿孔抑制抗体进入培养细胞将用于直接确定Src-家族酪氨酸激酶的功能。研究结果将明确结肠平滑肌中表达的src家族酪氨酸激酶，并确定其在神经递质信号转导和结肠平滑肌收缩中的作用。

Excitatory neurotransmitters produce colonic smooth muscle contraction by multiple signaling pathways which include altering ionic conductances (Na+, Ca2+, K+) to cause oscillations in myoplasmic Ca2+. In addition to ionic signaling mechanisms, acetylcholine and neurokinin A activate ERK and p38 MAP kinase in canine colonic smooth muscle. Pathways coupling neurotransmitter receptors to these downstream effector protein kinases are poorly defined in mammalian smooth muscles. Our working hypothesis is that non-receptor protein tyrosine kinases of the Src family and the phospholipid kinase, phosphatidylinositol-3 kinase (PI-3 kinase), are activated by acetylcholine and neurokinin A is an early sep in signal transduction. Specific aims are: 1. Define which members of the Src- family tyrosine kinases are expressed in colonic smooth muscle. Isoform- selective antibodies will be used to identify Src, Fyn, Yes, Lck and Lyn in colonic smooth muscle extracts. Reverse-transcriptase PCR will be used to clone and sequence cDNAs for Src-family tyrosine kinases. SRC- family tyrosine kinases will be isolated and characterized by MonoQ and immuno-affinity chromatography. 2. Determine which members of the Src- family are activated by acetylcholine and neurokinin A. Kinase activity stimulated by neurotransmitters will be assayed after immunoprecipitation. Time and concentration dependence of activation will be defined. The relative contribution of M3 and M2 muscarinic receptors will be investigated as will the role of inhibitory G proteins using pertussis toxin. 3. Define the relationship between Src-family tyrosine kinases and PI-3 kinase. Src-family kinase activities will be measured after inhibition of PI-3 kinase, and PI-3 kinase activity will be measured after inhibition of Src family kinases. 4. Establish the function of Src-family receptor tyrosine kinases. Effects of isoform- selective tyrosine kinase inhibitors will be tested on muscle contraction, ionic conductance and myoplasmic Ca2+ concentration in intact and cultured colonic smooth muscle. Adenovirus vectors will be used to express dominant negative and constitutively active Src mutants in cultured cells. Electroporation of inhibitory antibodies into cultured cells will be used to directly determine the function of Src- family tyrosine kinases. The results will define the Src-family tyrosine kinases expressed in colonic smooth muscle, and define their role in neurotransmitter signal transduction and colonic smooth muscle contraction.