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.
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