Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase.

The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.

Highly histamine-responsive clones from the human gastric adenocarcinoma cell line HGT-1.

The human gastric epithelial cell line HGT-1 possesses adenylate cyclase-coupled histamine H2 receptors. To test the cellular homogeneity or heterogeneity with respect to these receptors, we have isolated 7 clones from the HGT-1 line and studied their basal and histamine-stimulated adenylate cyclase activities. Basal adenylate cyclase activities of the clones did not differ significantly, nor did 10 mM NaF- nor 0.1 mM Gpp(NH)p-stimulated activities. However, histamine stimulation of adenylate cyclase varied among clones from 1.9 fold to 5.4 fold basal activity. The EC50 values, determined in 3 clones, were not significantly different. These findings support the heterogeneity of histamine responsiveness of the human gastric cell line HGT-1. In addition, they suggest that highly histamine-responsive clones may be useful models to study the gastric histamine H2-receptor and its specific antagonists in the human.

Inhibition of the histamine-stimulated adenylate cyclase activity of guinea pig gastric cells by the H2-receptor antagonists cimetidine, oxmetidine and SKF 93479.

The effect of cimetidine and two new histamine H2-receptor antagonists, oxmetidine and SKF 93479, on histamine-stimulated adenylate cyclase activity was studied in guinea pig gastric mucosal cells. Histamine stimulated the enzyme activity in concentration-dependent fashion. The concentration-response curve of histamine was progressively shifted to the right in the presence of increasing concentrations of each antagonist. The Schild plot gave a straight line for all three compounds, with a slope not significantly different from unity and this suggested a competitive antagonism. The calculated pA2 values were 8.45 +/- 0.20, 7.73 +/- 0.21 and 6.81 +/- 0.15 for SKF 93479, oxmetidine and cimetidine, respectively. These results are in accordance with the pharmacological potencies of the antagonists reported on isolated heart preparation and on gastric secretion in vivo. Therefore, the inhibition of histamine-sensitive adenylate cyclase of gastric cells may represent an additional tool for the in vitro evaluation of the H2-receptor antagonists.

Cytochrome b5-dependent delta 9 desaturation of fatty acids in gastric microsomes.

Stearyl-CoA was shown to stimulate the reoxidation rate of cytochrome b5 of gastric microsomes and to decrease the reduction rate of trypsin-purified hog liver cytochrome b5 by the NADH-cytochrome b5 reductase of these microsomes. This latter effect was (1) proportional to microsome concentration and to stearyl-CoA concentration with an apparent Km of 3.3 . 10(-6) M and a Vmax of 71 nmol per min and per mg microsomal protein, (2) insensitive to ATP and inhibited by 1.4 mM KCN, (3) mimicked by palmityl-CoA but not by stearic nor palmitic acid. Direct assays carried out using [14C]stearyl- and [14C]palmityl-CoA as substrates showed a production of 0.12 nmol of oleic and palmitoleic acid, respectively, per min per mg of microsomal protein. In the presence of Tb5 antibodies the reaction was inhibited by 40%. These results support the occurrence of cytochrome b5-dependent fatty acid delta 9 desaturation in gastric microsomes.

Characterization of a newly established human gastric cancer cell line HGT-1 bearing histamine H2-receptors.

A human gastric adenocarcinoma cell line, HGT-1, was established in vitro from the primary tumor of a 60-year-old patient. Histological examination of the tumor revealed a poorly differentiated adenocarcinoma. Primary tumor cells were cloned in soft agarose and gave rise to tumor colonies. The procedures enabling us to form a continuous cell line from the agarose colonies are described. The cultured cells grew as monolayers of closely apposed polygonal cells with a population-doubling time of 19.48 +/- 1.20 (S.E.) hr during exponential growth at passage 59. They had an epithelial morphology. Ultrastructural studies revealed the presence of microvilli and tight junctions. The HGT-1 cell line is tumorigenic in nude mice and has a hyperdiploid karyotype with a modal number of 57 chromosomes. It exhibits numerous marker chromosomes. These human gastric epithelial cells do not secrete mucus or carcinoembryonic antigen. They exhibit functional histamine H2-receptors mediating cellular cyclic adenosine 3':5'-monophosphate production and adenylate cyclase activation. In conclusion, the use of a soft-agarose clonogenic assay permitted us to develop a cancer cell line without the problems of fibroblastic cell contamination. The existence of histamine H2-receptors on gastric HGT-1 cells stresses the importance of this line as a model for studies of regulatory mechanisms involved in gastric secretion.

Specific activation of cyclic AMP-dependent protein kinase(s) by H2-histamine agonists in isolated gastric mucosal cells from guinea-pig.

Histamine stimulated cyclic AMP-dependent protein kinase activity in dispersed mucosal cells from guinea-pig gastric fundus (Ka = 5 microM). The H2-agonists dimaprit and impromidine produced similar effects, while the H1-agonist 2-(2-pyridyl) ethylamine had only a weak one. The H2-antagonist cimetidine competitively inhibited 0.1 mM histamine stimulation (Ki = 2 microM). In contrast, the H1-antagonist diphenhydramine had no effect up to 1 mM.