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，所述信号通路包括改变离子电导（Na+、Ca 2+、K+）以引起肌浆Ca 2+的振荡。除了离子信号机制，乙酰胆碱和神经激肽A激活ERK和p38 MAP激酶在犬结肠平滑肌。在哺乳动物平滑肌中，将神经递质受体偶联到这些下游效应蛋白激酶的途径定义不明确。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，神经激肽A是信号转导的早期环节。具体目标是：1。明确Src家族酪氨酸激酶的哪些成员在结肠平滑肌中表达.同种型选择性抗体将用于鉴定结肠平滑肌提取物中的Src、Fyn、Yes、Lck和林恩。逆转录酶PCR将用于Src家族酪氨酸激酶的cDNA克隆和测序。SRC家族酪氨酸激酶将通过MonoQ和免疫亲和色谱法分离和表征。2.确定Src家族的哪些成员被乙酰胆碱和神经激肽A激活.在免疫沉淀后测定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，以及使用百日咳毒素的抑制性G蛋白的作用。3.明确Src家族酪氨酸激酶与PI-3激酶的关系。在抑制PI-3激酶后测量Src家族激酶活性，并且在抑制Src家族激酶后测量PI-3激酶活性。4.确定Src家族受体酪氨酸激酶的功能。将测试同种型选择性酪氨酸激酶抑制剂对完整和培养的结肠平滑肌中的肌肉收缩、离子电导和肌浆Ca 2+浓度的影响。腺病毒载体将用于在培养细胞中表达显性阴性和组成型活性Src突变体。将抑制性抗体电穿孔到培养的细胞中将用于直接确定Src家族酪氨酸激酶的功能。这些结果将明确结肠平滑肌中表达的Src家族酪氨酸激酶，并确定它们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.