Control of Colonic Motility in Health and Disease

描述（由申请方提供）：结肠粘膜的病原性炎症伴有环行肌收缩力抑制，这对腹泻症状有重要作用。先前的研究已经确定，收缩性的抑制是由介导兴奋-收缩偶联的细胞信号分子表达的特定变化引起的。特别是，L-型钙通道的成孔α 1c亚基表达的减少，这减少了钙内流，是这种效应的关键因素。细胞信号分子表达的改变由炎症反应介质介导，例如从肌层外的免疫细胞释放的TNF α。我们的假设是TNF α通过激活转录因子NF-κ B下调L型钙通道α 1c亚基的基因表达和调节α 1c mRNA的稳定性来抑制收缩性。将在对人结肠环形平滑肌细胞（HCCSMC）原代培养物进行的研究中检验这一假设。该提案的具体目的是调查：1）HCCSMC肌条中NF-κ B活化是否下调L型钙通道α 1c亚基的表达，2）NF-κ B活化下调L型钙通道α 1c亚基表达的分子机制，（α 1c通过NF-κ B的p50和p65亚基应答TNF α，3）所述信号传导分子调节HCC SMC中NF-κ B亚基应答TNF α的活化，4）TNF α是否改变a1 c mRNA的稳定性，如果是，这种作用是否由丝裂原活化蛋白激酶（MAPK）介导，和5）TNF α处理环形肌条是否抑制它们的收缩性，如果是，这种作用是否由p50、p65、PKCzeta和MAPK介导。这些实验将利用互补的药理学和分子方法。这些发现将为治疗干预提供新的细胞靶点，以调节结肠平滑肌的生理和病理生理重要收缩。抑制NF-κ B活化是治疗炎症性疾病的新兴疗法。

DESCRIPTION (provided by applicant): Pathogenic inflammation of the colonic mucosa is accompanied by suppression of circular muscle contractility, which makes important contributions to the symptom of diarrhea. Previous studies have established that the suppression of contractility results from specific changes in the expression of cell signaling molecules that mediate excitation-contraction coupling. In particular, a decrease in the expression of the pore-forming alpha1c subunit of L-type calcium channels, which reduces calcium influx, is a key factor in this effect. The alterations in the expression of cell signaling molecules are mediated by inflammatory response mediators, such as TNFalpha released from the immunocytes in the muscularis externa. Our hypothesis is that TNFalpha suppresses contractility by down-regulating the gene expression of the alpha1c subunit of L-type calcium channels through the activation of transcription factor NF-kappaB and by modulating alpha1c mRNA stability. This hypothesis will be tested in studies performed on primary cultures of human colonic circular smooth muscle cells (HCCSMC). The specific aims of the proposal are to investigate: 1) whether NF-kappaB activation in HCCSMC muscle strips down-regulates the expression of alpha1c subunit of L-type calcium channels, 2) the molecular mechanisms of down-regulation of (alpha1c by p50 and p65 subunits of NF-KappaB in response to TNFalpha, 3) which signaling molecules regulate the activation of NF-kappaB subunits in HCCSMC in response to TNFalpha, 4) whether TNFalpha alters the stability of a1c mRNA and, if so, is this effect mediated by mitogen-activated protein kinases (MAPKs), and 5) whether TNFalpha treatment of circular muscle strips suppresses their contractility and, if so, is this effect mediated by p50, p65, PKCzeta and MAPKs. The experiments will utilize complimentary pharmacologic and molecular approaches. The findings will suggest new cellular targets for therapeutic interventions to modulate physiologically and pathophysiologically important contractions of colonic smooth muscle. The inhibition of NF-kappaB activation is an emerging therapy in treating inflammatory disorders.