Patients with Helicobacter pylori-associated gastritis have an increased release of gastrin. The mechanisms by which H. pylori affects the endocrine cells are unclear. We have used primary cultures containing canine antral G cells to examine the effects of human blood mononuclear cells, purified monocytes and lymphocytes, recombinant cytokines, and NH4Cl on gastrin release. Mononuclear cells and purified monocytes in direct contact with G cells stimulated gastrin release dose dependently. Separating mononuclear cells from G cells by Transwell filters with 0.4-micron pore size still produced a significant increase of gastrin release. Three human recombinant cytokines, interferon-gamma, tumor necrosis factor-alpha, and interleukin-2, but not interleukin-6 and interleukin-1 beta, each produced dose-dependent increases of gastrin stimulation. NH4Cl did not stimulate gastrin release. We conclude that mononuclear cells and purified monocytes prepared from human blood, as well as several cytokines, stimulate gastrin release from antral G cells. These factors may play an important role in the pathogenesis of H. pylori-associated hypergastrinemia.
To study the control of histamine release, we developed techniques for culturing fundic mucosal mast cells. After enzyme dispersion, enrichment by elutriation, and overnight suspension culture, mast cells accounted for 30% of the cells present. Histamine release into the medium, measured by radioenzymatic assay, was stimulated by the lectin concanavalin A (Con A). Ragweed antigen released histamine in antisera-sensitized cultures. Con A-induced histamine release was enhanced by adenosine, but adenosine alone was inactive. The relative potency of adenosine analogues was consistent with interaction at an adenosine A1-receptor site. The calcium ionophore A23187 (0.1-1 microM) also induced histamine release. Phorbol esters that activate protein kinase C, such as phorbol 12-myristate 13-acetate, did not release histamine but enhanced release when added to low concentrations of A23187. In contrast, inactive phorbols, such as 4 alpha-phorbol 12,13-didecanoate, failed to enhance A23187-induced release. Parallel studies with canine hepatic mast cells yielded comparable results. We conclude that canine fundic mast cells possess receptors for immunoglobulin E and adenosine. Our data are consistent with increases in cytosolic calcium and protein kinase C activation working synergistically to stimulate fundic mast cells.
Using enzyme-dispersed canine oxyntic mucosal cells, we studied regulation of histamine release from fractions in which mast cells were largely removed by density gradient. Histamine-like immunoreactivity was demonstrated using peroxidase-anti-peroxidase immunohistochemistry. Histamine-containing cells in the small cell elutriator fractions (SCEF) were further separated by albumin step density gradients. Approximately 2.5% of cells in the low density fraction (LDF) contained histamine-like immunoreactivity; this fraction was largely depleted of the more dense mast cells (0.5%). These two fractions were cultured for 48-64 h on a Matrigel substrate. The cell content of histamine and release into the medium were measured by radioenzymatic assay. Gastrin, carbachol, and forskolin increased histamine release from the LDF. The induction of histamine release by gastrin was evident within 5 min and was sustained for at least 60 min. The response to gastrin was dose dependent between concentrations of 10(-11) and 10(-8) M. In contrast, in the mast cell-enriched SCEF, basal release was higher and gastrin was without effect; however, concanavalin A stimulated and epinephrine inhibited histamine release indicating that histamine-release mechanisms were intact in this fraction. Our methods provide a preparation of low density oxyntic mucosal histamine cells that demonstrate gastrin-responsive histamine release; we speculate that enterochromaffin-like cells account for this gastrin response.
To investigate the involvement of guanosine 3',5'-cyclic monophosphate (cGMP) in the cholinergic activation of gastric acid and pepsinogen secretion, we studied the subcellular and cellular relation between particulate guanylate cyclase and muscarinic cholinergic receptor sites. Subcellular fractionation of homogenates from rabbit gastric glands showed that particulate guanylate cyclase and muscarinic receptors were distributed in similar patterns, which differed from the pattern found for Na+-K+-ATPase, a marker for basal-lateral plasma membranes. Assuming a basal-lateral membrane localization for particulate guanylate cyclase and cholinergic receptors, these results suggested a heterogeneity of glandular basal-lateral membranes. The distributions of these markers among fractions enriched in isolated canine parietal or chief cells were also followed. Na+-K+-ATPase correlated with parietal cell distribution (r = 0.86) and guanylate cyclase with chief cell distribution (r = 0.76). The distribution of quinuclidinyl benzilate (QNB) binding sites indicated association of muscarinic receptors with both cell types. The similar subcellular and cellular distributions of guanylate cyclase and QNB binding sites may reflect a functional relationship of these markers in muscarinic-activated pepsinogen secretion. As seen in most other tissues, gastric glandular guanylate cyclase was not stimulated by various gastric secretagogues. We found that small changes in Ca2+ concentration, within the micromolar range, can regulate glandular guanylate cyclase activity. These results are discussed in terms of the cholinergic activation of parietal and chief cell function.
Evidence in vivo indicates that endogenous and exogenous prostaglandins can alter gastrin secretion. We have used primary cultures containing canine antral G-cells to study the cellular actions of prostaglandins on gastrin secretion, comparing the effects of prostaglandin E2 (PGE2) and its synthetic analogue enprostil. Enprostil (10(-10)-10(-6) M) inhibited gastrin secretion in response to bombesin, carbachol, and forskolin, the latter a receptor-independent activator of adenylate cyclase. This inhibition by enprostil was reversed by treatment with pertussis toxin (200 ng/ml, 8 h). However, enprostil did not inhibit the postreceptor stimuli 8-bromoadenosine 3',5'-cyclic monophosphate (10(-3) M), calcium ionophore A-23187 (10(-7) M), or 4 beta-phorbol 12-myristate 13-acetate (10(-8) M). In contrast, whereas PGE2 inhibited forskolin-stimulated gastrin release, PGE2 did not inhibit the response to carbachol or bombesin in control cultures. However, in pertussis toxin-treated cultures, PGE2 inhibition was reversed and, in contrast, the responses to bombesin, carbachol, and possibly forskolin were augmented. Indomethacin at a dose of 10(-5) M did not alter basal or bombesin-stimulated gastrin secretion. However, the somatostatin antibody CURE-S6 enhanced the response to forskolin and enhanced inhibition by PGE2, suggesting that endogenous somatostatin produced an inhibitory tone in these cultures and excluding the possibility that PGE2 acted via release of endogenous somatostatin. Our data suggest that in cultured antral cells gastrin release is regulated by inhibitory and stimulatory prostaglandin mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)
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