Elucidation of the cellular mechanisms of acid secretion

1. 建立了一种制备兔胃粘膜分离富集壁细胞的方法。通过对c -氨基吡啶的摄取来判断，这些细胞对各种分泌剂（包括组胺、氨基酚和胃泌素）表现出极好的反应。利用这些细胞，测定分泌诱导的细胞信使、cAMP和游离钙的变化。组胺增加细胞cAMP，而对其他促分泌剂没有反应。相反，组胺、氨基酚和胃泌素使细胞游离钙迅速升高。这种反应似乎是由独立的受体机制介导的。研究了^ 3h -组胺与家兔离体壁细胞的结合。然而，从使用各种组胺受体拮抗剂的实验来看，^ 3h -组胺的结合似乎并不代表实际的受体结合。分析了壁细胞膜糖脂的组成和结构。发现硫酸糖脂优先富集于分泌酸的粘膜和壁细胞。荧光标记的小管蛋白染色和肌动蛋白特异性抗体的间接免疫荧光染色显示，微丝与细胞内小管的结构密切相关。此外，微丝的抑制剂，如细胞松弛素D，无论分泌物是什么，都能强烈抑制酸的分泌。由于这些微丝破坏剂不影响第二信使的反应，因此这些药物的抑制作用应归因于远低于第二信使水平的机制。采用差速离心和身份梯度超离心制备了高富集H^+，K^+- atp酶的胃囊泡。兔制备了H^+、K^+- atp酶特异性多克隆抗体。

1. A method for preparing isolated enriched parietal cells from rabbit gastric mucosa has been developed. As judged by ^<14>C-aminopyrine uptake, these cells showed an excellent response to various secretagogues, including histamine, carbachol, and gastrin.2. Using these cells, secretagogue-induced changes in cellular messengers, cAMP and free calcium were determined. Histamine increased cellular cAMP which failed to respond to other secretagogues. In contrast, histamine, carbachol and gastrin brought about a rapid elevation of cellular free calcium. This response appeared to be mediated by independent receptor mechanisms.3. The binding of ^3H-histamine to isolated rabbit parietal cells was studied. However, from the experiments using various histamine receptor antagonists, the binding of ^3H-histamine did not appear to represent the actual receptor binding.4. The composition and ctructure of membrane glycolipids of parietal cells were analyzed. Sulfated glycolipids were found to be preferentially enriched in the acid-secreting mucosa and parietal cells.5. Staining with fluorescent-labelled phalloin and with indirect immunofluorescence using specific antibodies to actin, close structural association of microfilaments with intracellular canaliculi was revealed. Moreover, inhibotors of microfilament such as cytochalasin D strongly inhibited acid secretion irrespective of the secretagogues. Since these microfilaments-disrupting agents did not influence the response at the second messengers, the inhibition by these agents should be attributed to the mechanisms distal to the level of second messengers.6. The gastric vesicles containing highly enriched H^+,K^+-ATPase has been prepared by differential centrifugation and dentity gradient ultracentrifugation. Polyclonal antibodies specific to H^+,K^+-ATPase were raised in rabbits.