GROWTH AND FUNCTION OF CULTURED GASTROINTESTINAL MUSCLE

这一建议的目的是确定内生因素 负责圆形和纵向的生长和分化 豚鼠和人的胃和肠平滑肌，以及 表征它们的细胞表面受体和信号转导 与受体结合的途径。重点是增长 初步研究表明， 在肠环肌和肠纵肌培养物中生长。三 将检查生长因子的类别：（i）GRP/蛙皮素及其 同源物，神经介肽B，其从肠神经元释放;（ii） 5-HT，其响应于炎症而从肥大细胞释放， 其与平滑肌上不同的5-HT和S-HT 4受体相互作用; 和（iii）肽生长因子（TGF α、TGF β、IGF-I和II）， 在平滑肌中正常表达或诱导。以各种 在细胞中，这些因子与受体相互作用， 通过GTP结合调节蛋白或更直接地通过 受体/酪氨酸激酶。 在初步研究中，我们已经表明，培养的肠肌肉， 保留其特征表型并保持其表达 β-原肌球蛋白和肠平滑肌特异性γ-肌动蛋白， 具有互补的G蛋白偶联和酪氨酸激酶偶联 能够刺激肌肉生长、Ca 2+动员和 收缩。 第一个具体的目的是确定的存在和分泌 胃和胃粘膜中的生长因子（TGF α、IGF-I和IGF-II、TGF β） 通过放射免疫测定法进行肠平滑肌培养， 免疫细胞化学法检测其作用，并测定5-HT、GRP的作用 和NMB对细胞生长（胸苷摄取和蛋白质合成）的影响， 分化（β-原肌球蛋白和肠平滑肌的表达） 肌肉特异性γ-肌动蛋白），和肌肉功能（Ca 2+动员， 收缩和松弛）。第二个具体目标是描述 细胞表面生长受体的表达和特性 培养的胃和肠平滑肌细胞中的因子， 放射性配体结合结合选择性受体保护。 第三个具体目标是表征信号转导 与介导生长和分化的受体偶联的途径， 测量效应酶（磷酸肌醇特异性PLC）的活性 β-l和PLC~-l，以及磷脂酰胆碱特异性PLC、PLD和PLA_2）， 和第二信使（IP 3，甘油二酯，花生四烯酸，和Ca 2+， 以及蛋白激酶C、酪氨酸激酶和MAP激酶的活性- 2）。这些研究将提供一个框架， 调节肠上皮细胞生长和分化的细胞内机制 平滑肌

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.