Black E W, Strada S J, Thompson W J
Department of Pharmacology, University of South Alabama, College of Medicine, Mobile 36688.
J Pharmacol Methods. 1988 Aug;20(1):57-78. doi: 10.1016/0160-5402(88)90016-2.
Centrifugal elutriation was adapted and analyzed as a method to separate rat gastric parietal cells from other fundic mucosal cells. Elutriated parietal cell fractions provided sufficient purity by morphological criteria, and fluorescence activated cell sorting analyses, yield, reproducibility and maintenance of functional responses. These characteristics allowed the study and comparison of the kinetics of histamine, isoproterenol, and forskolin-induced cyclic AMP and 14C-amino-pyrine accumulations in the presence of 1-methyl-3-isobutyl xanthine (IBMX) in parietal-cell-rich and parietal-cell-poor fractions. All three acid secretagogues studied produced the same maximal rate of acid secretion as judged by 14C-aminopyrine accumulation. Each secretagogue action peaked at different times and had different accumulation kinetics. For each agonist, cyclic AMP accumulation preceded secretion. However, the rate, extent, and temporal changes of cyclic AMP accumulation were independent of aminopyrine accumulation. HPLC separation and drug inhibition studies of cyclic nucleotide phosphodiesterase activities indicated the presence of multiple, high affinity (Type IV), but not lower affinity (Types I and II), enzyme forms in gastric mucosal cells. IBMX did not distinguish between the two forms, but SQ 65442 and RO 20-1724 were selective inhibitors. Inhibition constants of IBMX for phosphodiesterase hydrolysis agreed closely with its EC50 for histamine-stimulated acid secretion (2-8 microM). Elutriated parietal cells maintained their responses to selective receptor antagonists in the micromolar concentration range.
采用离心淘析法并对其进行分析，以此作为从大鼠胃底其他黏膜细胞中分离胃壁细胞的一种方法。通过形态学标准、荧光激活细胞分选分析、产量、可重复性以及功能反应的维持情况，淘析得到的胃壁细胞组分具有足够的纯度。这些特性使得在富含胃壁细胞和缺乏胃壁细胞的组分中，能够研究和比较组胺、异丙肾上腺素和福斯高林在1-甲基-3-异丁基黄嘌呤（IBMX）存在的情况下诱导的环磷酸腺苷（cAMP）和14C-氨基吡啶积累的动力学。通过14C-氨基吡啶积累判断，所研究的所有三种胃酸分泌刺激剂产生的胃酸分泌最大速率相同。每种分泌刺激剂的作用在不同时间达到峰值，并且具有不同的积累动力学。对于每种激动剂，cAMP积累先于分泌。然而，cAMP积累的速率、程度和时间变化与氨基吡啶积累无关。环核苷酸磷酸二酯酶活性的高效液相色谱分离和药物抑制研究表明，胃黏膜细胞中存在多种高亲和力（IV型）而非低亲和力（I型和II型）的酶形式。IBMX无法区分这两种形式，但SQ 65442和RO 20-1724是选择性抑制剂。IBMX对磷酸二酯酶水解的抑制常数与其对组胺刺激的胃酸分泌的EC50（2-8 microM）密切一致。淘析得到的胃壁细胞在微摩尔浓度范围内维持其对选择性受体拮抗剂的反应。
