Studies on the regulation of gastrin secretion

The regulation of gastrin secretion in vivo is complex as a result of the multiplicity of nervous, luminal, endocrine and paracrine signals that are received by the G cell. The objective of these studies was to investigate the mechanisms that control G-cell function in normal and pathophysiological situations. Gastrin secretion was examined in lean and obese Zucker rats, in the dog and in the human using in vivo and in vitro methods. Hypergastrinemia in the obese Zucker rat was found to be a progressive condition, which appears between 3 and 8 weeks after birth and increases in severity with age. Elevated gastrin levels persisted from the isolated, vascularly perfused stomach preparation with adult obese animals displaying secretory rates 3-fold higher than their lean littermates. Basal gastrin secretion was unaltered by atropine treatment in both groups indicating that post-ganglionic cholinergic inputs to the G cell of the obese rat were normal. This was supported by perfusion experiments in which electrical activation of the anterior vagus nerve augmented gastrin release equally (2-fold) from lean and obese stomachs, an effect that was abolished by the addition of atropine. Basal somatostatin secretion from the perfused stomachs of lean and obese rats was not different nor was the density of the antral D-cell population. This suggested that somatostatin was probably not involved in the hypergastrinemia of the obese animal. G-cell numbers in the antra of both groups of animals were similar at 3 weeks of age, but were doubled in 6 month-old obese rats compared to lean controls. It was hypothesized that G-cell hyperplasia secondary to increased food consumption in obese rats was responsible for hypergastrinemia. This possibility was investigated in pair-feeding experiments wherein the food intake of obese animals was restricted to match that of lean littermates from 5 to 8 weeks of age. Dietary restriction reduced the antral G-cell population of the obese rat to a similar level as that seen in lean animals, supporting the view that hyperphagia is the trigger for G-cell hyperplasia. However, basal gastrin levels in vivo and from the in vitro stomach preparation of pair-fed obese animals were not significantly lower than those of obese animals fed ad libitum. Thus, abnormal feeding behaviour in the obese phenotype cannot account for gastrin hypersecretion and G-cell hyperplasia is not the primary cause of hypergastrinemia. The role of bombesin/gastrin releasing peptide (BN/GRP) in the hypergastrinemic condition of the obese rat was subsequently examined. GRP content in antral extracts of obese animals was markedly greater than their lean counterparts. Furthermore, the gastrin responses of perfused stomachs of the obese animal to exogenous BN were attenuated compared to the lean group. These results provided circumstantial support for the involvement of a BN/GRP drive in the hypergastrinemia of the obese Zucker rat. However, this conclusion could not be validated by perfusion experiments employing two putative BN/GRP antagonists, [D-Arg1, D-Pro2, D-Trp7-9, Leu11] substance P and [Leu 1 3 -y-CH 2 NHLeu14] bombesin. Basal and BN-stimulated gastrin release from the perfused stomachs of both groups of animals were unaffected by treatment with either antagonist. Thus, a causal role for BN/GRP neurons in the hypergastrinemia of obese rats is suggested, but additional support awaits improvements in the synthesis of BN/GRP antagonists. The control of gastrin secretion at the cellular level in the dog and human was investigated using isolated antral G cells in primary culture. Preferential enrichment for G cells by centrifugal elutriation and short-term culture resulted in preparations in which gastrin-containing cells accounted for 8.5% of the total viable adherent cell population. BN, from 0.01 to 100 pM, stimulated gastrin release from cultured canine G cells in a concentration-dependent manner. The gastrin response achieved with such low concentrations of BN suggested a direct stimulatory role for this peptide via high-affinity receptors. The BN analog, [Leu13-\j/-CH2NH-Leu14] bombesin, completely blocked BNinduced gastrin secretion from 0.01 to 1 pM and produced > 50% inhibition at the higher concentrations of BN tested. In comparison, [D-Arg1, D-Pro2, D-Trp 79, Leu11] substance P augmented basal gastrin levels when given alone. Consequently, this anatgonist elicited only modest suppression of BN-evoked gastrin secretion. Differential inhibition of BN-mediated gastrin release from cultured canine cells and from the isolated perfused stomachs of Zucker rats suggests the existence of a heterogeneous population of BN/GRP receptors among species. In the final series of experiments, receptor-dependent and -independent regulation of gastrin secretion from cultured human antral G cells was investigated. With minor modifications, methods used in the canine preparation were successfully applied to produce cultures of human antral mucosal cells that were enriched for G cells. Gastrin-containing cells accounted for 15% of the total adhered cell population. Forskolin, A23187 and 6PMA stimulated gastrin secretion from cultured human G cells in a concentration-dependent fashion. These results indicated that gastrin release in the human could be mediated by elevations in cytosolic cAMP levels, calcium influx or activation of protein kinase C. A direct stimulatory role for BN/GRP was supported by experiments demonstrating concentration-dependent enhancement of gastrin release by BN from 0.01 fM to 10 nM. In contrast to findings from the canine preparation, [Leu13-\|/-CH2NH-Leu14] bombesin augmented basal gastrin levels by itself and produced weak inhibition of BN-induced gastrin secretion from human antral G cells. This further supports the hypothesis that species differences exist with regard to gastric BN/GRP receptors of the Zucker rat, dog and human. Somatostatin potently suppressed forskolinand BN-mediated gastrin release, but did not significantly alter basal gastrin levels. These results suggest that BN and somatostatin directly activate and inhibit G-cell function via specific and sensitive receptors. Furthermore, the adenylate cyclase and phosphatidyl inositide second messenger systems appear to be intracellular mediators of gastrin secretion from human antral G cells. These investigations clearly demonstrated the complexity of the control of gastrin secretion in the Zucker rat, dog and human. Further clarification of the mechanisms that regulate G-cell function in the intact animal relies on improved methodology and the integration of information obtained from various experimental models.