The aim of this proposal is to identify the endogenous factors responsible for growth and differentiation of circular and longitudinal gastric and intestinal smooth muscle in the guinea pig and human, and characterize their cell surface receptors and the signal transduction pathways to which the receptors are coupled. The focus is on growth factors shown in preliminary studies to be effective in stimulating growth in cultures of intestinal circular and longitudinal muscle. Three classes of growth factors will be examined: (i) GRP/bombesin and its homologue, neuromedin B, which are released from enteric neurons; (ii) 5-HT which is released from mast cells in response to inflammation and which interacts with distinct 5-HT and S-HT4 receptors on smooth muscle; and (iii) peptide growth factors (TGF alpha, TGF beta, IGF-I and II) that are either normally expressed or induced in smooth muscle. In various cells, these factors interact with receptors coupled to signaling pathways via GTP-binding regulatory proteins or more directly via receptor/tyrosine kinases. In preliminary studies, we have shown that cultured intestinal muscle retains its characteristic phenotype and maintains its ability to express beta-tropomyosin and enteric smooth muscle-specific gamma-actin, possesses a complement of G protein-coupled and tyrosine kinase-coupled receptors capable of stimulating muscle growth, Ca2+ mobilization and contraction. The first specific aim is to identify the presence and secretion of growth factors (TGF alpha, IGF-I and IGF-II, TGF beta) in gastric and intestinal smooth muscle cultures by radioimmunoassay and immunocytochemistry, and determine their effects, and those of 5-HT, GRP and NMB on cell growth (thymidine uptake and protein synthesis), differentiation (expression of beta-tropomyosin and enteric smooth muscle-specific gamma-actin), and muscle function (Ca2+ mobilization, contraction and relaxation). The second specific aim is to characterize the expression and properties of cell surface receptors for growth factors in cultured gastric and intestinal smooth muscle cells by radioligand binding in conjunction with selective receptor protection. The third specific aim is to characterize the signal transduction pathways coupled to receptors mediating growth and differentiation by measuring the activity of effector enzymes (phosphoinositide-specific PLC beta-l and PLC~-l, and phosphatidylcholinespecific PLC, PLD and PLA2), and second messengers (IP3, diacylglycerol, arachidonic acid, and Ca2+, and the activities of protein kinase C, tyrosine kinase and MAP kinases- 2). The studies will provide a framework for understanding the intracellular mechanisms regulating growth and differentiation in enteric smooth muscle.
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