十二指肠腔膜侧多巴胺受体介导胃源性多巴胺引起十二指肠碳酸氢根分泌的在体与离体研究

Duodenal bicarbonate secretion plays a vital role in maintaining the alkaline microenvironment of brush border and protecting mucosa from H+ in gastric juice. Clinical research suggests that patients with dopaminergic dysfuction (such as Parkinson's disease) suffers from a higer incidence of gastroduodenal ulcer, while a lower incidence has been reported in the patients with the dopaminergic hyperfunction, which indicates that dopamine might contribute to the preservation of gastrointestinal mucosa. Previous studies have shown that gastric parietal cells can synthesis dopamine, and dopamine receptors are widespreadly distributed in the duodenal mucosa. However, it remains unknown whether gastric dopamine can enter duodenum via pylorus and stimulate duodenal bicarbonate secretion by combining with the dopamine receptors on the luminal membrane. It is also worthwhile to find out the subtypes of DA receptors, key protiens in signaling pathways and the channels or transporters of cell membrane involved in this process. To discover the mechanism underlying the dopamine regulating duodenal bicarbonate secretion, the following methods will be employied. In vivo stomach perfusion, radioimmunoassay (RIA) and high pressure liquid (HPLC) etc.will be used to measure the content of DA in gastric juice. The location and quantitative detecting techniques including immunohistochemistry, molecularbiology, enzyme linked immunosorbent assay and laser cutting will be used to determine the expression and alterations of dopamine receptors, key protiens in signaling pathways, and relative membrane channels or transporters in duodenal epithelia. In addition, the real-time pH titration, intracellular pH measurement, calcium-sensitive fluorescent probe, dopamine receptor RNA inteference and relevant animal models with altered endogenous DA will be applied. The present study will provide some theoretical and experimental information for the mechanism underlying the regulation of gastric DA on the duodenal HCO3- secretion and the cause of the high ulcer incidence in PD condition.

十二指肠粘膜HCO3-的分泌具有维持刷状缘碱性微环境、保护粘膜免受酸损伤的作用。临床发现胃十二指肠溃疡在多巴胺（DA）系统功能减退者（如帕金森病）发病率高，亢进者（如精神分裂症）发病率明显降低。提示DA具有胃肠粘膜保护作用。胃粘膜可以合成DA，DA受体广泛分布于十二指肠粘膜，但胃DA能否通过幽门刺激十二指肠腔面膜DA受体引起HCO3-分泌、其作用的受体亚型、信号通路和相关膜通道/转运体等并不清楚。本研究采用在体胃灌流、高压液相和放免等方法测定胃液DA含量；采用免疫组化、分子生物学、酶联免疫和激光切割等技术对十二指肠粘膜DA受体、信号通路中关键蛋白、膜转运体和通道等进行定位和定量检测；采用实时pH滴定、胞内pH和钙敏感荧光探针、基因干扰结合相关动物模型等研究DA对HCO3-分泌的影响。为胃源性DA调节十二指肠HCO3-分泌的机制、帕金森病胃十二指肠溃疡高发病率的病因提供理论与实验依据。

十二指肠粘膜碳酸氢根分泌（Duodenal bicarbonate secretion, DBS）可维持刷状缘碱性微环境、保护粘膜免受酸的损伤。临床研究发现DA具有胃肠粘膜保护作用。胃粘膜可以合成DA，DA受体广泛分布于十二指肠粘膜，但胃DA能否通过幽门刺激十二指肠腔面膜DA受体引起HCO3-分泌、其作用的受体亚型、信号通路等并不清楚。本研究采用在体胃灌流、高压液相和放免等方法测定胃液DA含量，发现胃酸分泌增多时，胃液中DA的含量显著增多；应用免疫荧光结合激光共聚焦扫描技术观察发现DA的五种受体亚型在十二指肠粘膜上均有表达。D1受体仅在粘膜的固有层表达，D5受体在十二指肠腺和隐窝的基底膜侧和腔面膜侧均有表达，并通过cAMP通路降低紧密连接蛋白表达进而增加粘膜的通透性；而D2受体家族的三种亚型（D2、D3、D4）在十二指肠绒毛、十二指肠腺和隐窝的腔面膜侧均有大量表达。进一步发现人十二指肠粘膜DA受体的定位和表达与大鼠基本一致。采用实时pH滴定发现在腔面侧而非基底侧加入DA有效地促进DBS并呈剂量依赖，其可被D2受体家族拮抗剂Sulpride、D2受体亚型特异性拮抗剂L741,626以及在体D2-siRNA敲低所抑制，而D1受体家族拮抗剂SCH23390无影响；进一步发现腔面侧加入D2受体家族激动剂Quinpirole可剂量依赖性模拟DA促进DBS的作用。此外，DA明显增强十二指肠绒毛上皮细胞内pH敏感的荧光探针（BCECF-AM）的强度，并可被D2受体家族激动剂Quinpirole剂量依赖性模拟。提示：腔面侧D2受体介导DA引起的DBS。进一步应用cAMP测定、钙通道阻断剂和钙敏感荧光探针检测发现钙通路而非cAMP参与DA引起的DBS，具体机制尚需进一步研究。本研究发现胃源性DA可与腔膜侧D2受体相结合通过钙通路引起DBS,为帕金森病胃肠功能紊乱的病因提供理论与实验依据。