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 受体偶联的途径。重点放在增长上 初步研究显示的有效刺激因素 肠道环行肌和纵行肌培养中的生长。三 将审查生长因子的类别：(I)GRP/蛙皮素及其 肠神经元释放的同系物--神经梅毒素B；(Ii) 5-羟色胺是肥大细胞在炎症和 与血管上不同的5-羟色胺和S-羟色胺受体相互作用； 和(Iii)多肽生长因子(转化生长因子α、转化生长因子β、胰岛素样生长因子-I和胰岛素样生长因子II) 在平滑肌中正常表达或诱导表达。在各种情况下 细胞，这些因子与受体相互作用，耦合到信号 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，我们已经证明了培养的肠道肌肉 保留其特有的表型，并保持其表达能力 β-原肌球蛋白和肠平滑肌特异的γ-肌动蛋白， 具有G蛋白偶联和酪氨酸激酶偶联的补体 受体能够刺激肌肉生长，钙离子动员和 收缩。 第一个特定的目标是确定存在和分泌 生长因子(转化生长因子α、胰岛素样生长因子-I和胰岛素样生长因子-II、转化生长因子β)在胃和胃组织中的表达 用放射免疫法和放射免疫法培养肠平滑肌 免疫细胞化学，并测定其作用及5-羟色胺、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 肌肉特异的γ-肌动蛋白)和肌肉功能(钙离子动员， 收缩和放松)。第二个具体目标是描述 细胞表面生长受体的表达和特性 体外培养的胃肠平滑肌细胞因子 放射配基结合结合选择性受体保护。 第三个具体目标是描述信号转导的特征 与受体偶联的途径介导生长和分化 效应酶活性的测定(磷脂酰肌醇特异性PLC L和L，以及磷脂酰胆碱特异的PLC、PLD和PLA2)， 和第二信使(IP3、二酰甘油、花生四烯酸和钙离子， 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，以了解 肠道中调节生长和分化的细胞内机制 平滑的肌肉。

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.