Elucidation of the cellular mechanisms of acid secretion

1.本文报道了一种从家兔胃粘膜分离富集壁细胞的方法。通过对β-<14>C-氨基比林的摄取判断，这些细胞对各种促分泌素表现出极好的反应，包括组胺、卡巴胆碱和胃泌素。使用这些细胞，促分泌素诱导的细胞信使，cAMP和游离钙的变化进行了测定。组胺使细胞内cAMP增加，而对其他促分泌素无反应。相反，组胺、氨甲酰胆碱和胃泌素引起细胞游离钙的快速升高。这种反应似乎是由独立的受体机制介导的。研究了^3H-组胺与离体兔壁细胞的结合。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能代表实际的受体结合。分析壁细胞膜糖脂的组成和结构。发现硫酸化糖脂优先富集在泌酸粘膜和壁细胞中。染色与荧光标记的鬼笔蛋白和间接免疫荧光使用特异性抗体肌动蛋白，密切的结构关联的微丝与细胞内小管被揭示。此外，微丝的抑制剂如细胞松弛素D强烈抑制酸分泌，而不管促分泌素。由于这些微生物活性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第二信使水平的远端机制。采用差速离心和密度梯度离心法制备了高浓度H^+，K^+-ATP酶的胃囊泡。制备了H^+，K^+-ATP酶的多克隆抗体。

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.