John DelValle

Post-translational processing of gastrin in neoplastic human colonic tissues

Gastrin has been postulated to stimulate proliferation in colorectal neoplasms. Although gastrin mRNA has been demonstrated to be present in colon cancer cell lines, the intact peptide had not been recovered from human colorectal neoplasms. We demonstrate that gastrin and its precursors are present in both colorectal neoplasia and adjacent normal-appearing colonic mucosa. In colonic tissue, the glycine-extended precursor form of the peptide is over 10-fold more abundant than the amidated gastrin, and progastrin is more than 700-fold more abundant. In contrast, amidated gastrin in the human antrum is the predominant form of gastrin by a factor of 10. Furthermore, the ratio of gastrin precursors to gastrin is significantly increased in neoplastic colonic mucosa when compared with normal colonic tissue. These data suggest that the processing of gastrin is unique in the human colon and that further differences in processing occur in neoplastic colonic tissue.

Progastrin and its glycine-extended posttranslational processing intermediates in human gastrointestinal tissues

We examined the posttranslational modification of gastrin in human gastrointestinal and Zollinger-Ellison tumor tissues using antibodies specific for progastrin and its glycine-extended processing intermediates. Gel filtration of antral and duodenal extracts on Sephadex G-50 revealed multiple molecular forms of immunoreactive glycine-extended processing intermediates corresponding to known molecular forms of gastrin and cholecystokinin. Immunoaffinity chromatography studies of antral mucosal and gastrinoma extracts identified a molecular form of glycine-extended processing intermediates that was characterized by an amino terminus identical to that of gastrin heptadecapeptide. Large differences in the relative contents of precursors and products of gastrin synthesis were found in gastrinoma tissue as compared to gastrointestinal mucosal extracts. These studies suggest the potential importance of glycine-extended peptides as intermediates in the posttranslational processing of gastrin and cholecystokinin in humans and indicate that gastrin processing mechanisms may be altered in Zollinger-Ellison tumors.

Effect of transforming growth factor alpha and interleukin 8 on somatostatin release from canine fundic D cells

BACKGROUND & AIMS Helicobacter pylori infection in patients who have peptic ulcer disease is associated with altered regulation of gastric secretion, hypergastrinemia, and diminished somatostatin expression in gastric mucosa. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 are the predominant cytokines produced in the gastric mucosa of patients with H. pylori infection. The aim of this study was to examine whether IL-8 and TNF-alpha could regulate somatostatin release from isolated canine gastric D cells. METHODS Canine gastric D cells were isolated from fundic mucosa and enriched by centrifugal elutriation. Secretagogue-stimulated somatostatin release was measured by radioimmunoassay. RESULTS TNF-alpha dose dependently increased somatostatin release after 2 hours of treatment. The stimulatory effect of TNF-alpha was additive to that of epinephrine but was unaffected by a maximal concentration of cholecystokinin. IL-8 did not alter basal or secretagogue (cholecystokinin, epinephrine)-mediated somatostatin release. The stimulatory effect of TNF-alpha (10 ng/mL) was potentiated by the addition of IL-8 (1 nmol/L), inhibited by octreotide and staurosporine, but unaffected by indomethacin. Pretreatment of D cells with TNF-alpha (10 ng/mL) for 24 hours abolished the subsequent stimulatory effect of this cytokine and secretagogues on somatostatin release. CONCLUSIONS TNF-alpha was shown to regulate somatostatin release from cultured D cells in a divergent manner.

Tumour necrosis factor alpha stimulates gastrin release from canine and human antral G cells: possible mechanism of the Helicobacter pylori–gastrin link

There is evidence that gastric Helicobacter pylori (Hp) infection promotes duodenal ulceration by releasing gastrin. We therefore asked how Hp releases gastrin. Tumour necrosis factor alpha (TNF‐α) is up‐regulated in Hp gastritis and stimulates hormone release from pituitary cells, so we tested its effect on primary cultures of canine antral G cells and human antral fragments. TNF‐α pretreatment (100ngmL–1) of canine G cells significantly increased both basal (by 89%: P<0.01) and bombesin‐stimulated (by 39% P<0.05) gastrin release. A similar pattern of increase was seen following TNF‐α (20ngmL−1) pretreatment of human antral fragments: basal gastrin release was increased by 38% (P < 0.05) and bombesin‐stimulated by 26% (P < 0.05). This effect persisted during immunoblockade with anti‐somatostatin antibody S6. We propose that TNF‐α provides the link between Hp infection and gastrin release and thus contributes to duodenal ulceration.

Glycine-extended gastrin regulates HEK cell growth

Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH2) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH2, glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH2 and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH2 can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH2 and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for 125I-labeled Leu15-G17-NH2and125I-Leu15-G2-17-Gly. Both G-NH2 and G-Gly induced a dose-dependent increase in [3H]thymidine incorporation, and both peptides together significantly increased [3H]thymidine incorporation above the level of either peptide alone. G-NH2 and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH2 and G-Gly lead to a significant reduction in [3H]thymidine incorporation. G-NH2 but not G-Gly increased intracellular Ca2+concentration. We conclude that G-NH2 and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH(2)) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH(2), glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH(2) and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH(2) can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH(2) and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for (125)I-labeled Leu(15)-G17-NH(2) and (125)I-Leu(15)-G(2-17)-Gly. Both G-NH(2) and G-Gly induced a dose-dependent increase in [(3)H]thymidine incorporation, and both peptides together significantly increased [(3)H]thymidine incorporation above the level of either peptide alone. G-NH(2) and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH(2) and G-Gly lead to a significant reduction in [(3)H]thymidine incorporation. G-NH(2) but not G-Gly increased intracellular Ca(2+) concentration. We conclude that G-NH(2) and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.

Glycine-extended processing intermediates of gastrin and cholecystokinin in human plasma.

The biosynthesis of biologically active gastrin and cholecystokinin (CCK) requires the formation of carboxyl-terminally amidated peptides from glycine-extended precursors of gastrin and CCK. In previous studies we and others have identified and characterized glycine-extended forms of gastrin (Ggly) and CCK (CCK-gly) in the human gastrointestinal tract. To explore the potential biologic importance of these peptides in humans, we examined their release into the circulation. Ingestion of a standard meal induced a biphasic rise in plasma G/CCK-gly concentration, but only the initial increase correlated with gastrin release. Intraduodenal lipid infusion caused a selective rise in CCK-gly immunoreactivity with no increase in gastrin or G-gly. Gel filtration chromatography revealed that the predominant molecular form of G/CCK-gly in basal plasma coeluted with CCK8-gly, but in response to meal stimulation, increases in other molecular forms were noted. Measurement of glycine-extended intermediates of progastrin and procholecystokinin posttranslational processing in plasma may aid in determining their physiologic importance in health and disease.

Secretagogue-induced Ca2+ oscillations in isolated canine gastric chief cells

Agonist-induced changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) of isolated canine gastric chief cells were evaluated by microspectrofluorometry of superfused fura-2 loaded cells. Application of high concentrations of carbachol (CCh, 10(-5) M) or cholecystokinin octapeptide (10(-8) M) resulted in biphasic Ca2+ mobilization comprising an initial large transient followed by a small sustained elevation above the prestimulation level. Submaximal concentrations of CCh (10(-6) M) or cholecystokinin (10(-9) M) led to either a transient series of large amplitude Ca2+ spike(s) or a higher frequency of sustained Ca2+ oscillations of smaller amplitude. Cholecystokinin at 10(-10) M induced only sustained Ca2+ oscillations. Elimination of Ca2+ from the medium had no immediate effect on oscillations indicating an intracellular source of Ca2+. Thus the Ca2+ signalling mode in chief cells is dependent on agonist concentrations.

Cellular mechanisms of somatostatin action in the gut

We have used isolated canine parietal cells to examine the receptor and postreceptor events mediating the inhibitory effects of somatostatin on acid secretion. Somatostatin-14 (S14) and somatostatin-28 (S28) dose dependently inhibited parietal cells stimulated by secretagogues that activate both the adenylate cyclase/cyclic adenosine monophosphate and the inositol phospholipid/protein kinase C cascades. The inhibitory action was mediated via a specific cell surface receptor that consists of a single subunit protein (molecular weight 99,000 d). This receptor recognized S14 and S28 equally well. Somatostatin inhibited parietal cell activity via mechanisms that are both dependent on and independent of a pertussis toxin-sensitive inhibitory guanine nucleotide binding protein.

The liver plays an important role in the regulation of somatostatin-14 in the rat☆

Since little is known about the in vivo disposition of circulating somatostatin-14 (SRIF-14), we examined hepatic processing of SRIF-14 in the rat. Three minutes after the intraportal injection of iodine 125 (125I)-labeled SRIF-14, 16.0 +/- 2.0% of the injected dose is localized to the liver. In the presence of unlabeled SRIF-14, hepatic uptake can be decreased by 68%. Five minutes after the intraportal injection of 125I-SRIF-14, 9.5 +/- 1.4% of the tracer is localized to the liver, more than any other organ tested. Serial collections of bile reveal peak radioactivity at between 10 and 20 minutes. Simultaneous administration of unlabeled SRIF-14 decreases biliary radioactivity by 40%. HPLC analysis of radioactive bile reveals a chromatographic profile similar to that of intact SRIF and is 73% immunoprecipitable by an anti-SRIF antibody. Pretreatment with chloroquine, a lysosomal enzyme inhibitor, does not significantly decrease biliary radioactivity. We conclude that the data are consistent with saturable hepatic uptake and predominantly nonlysosomal transcellular transport.

Canine prosomatostatin: Isolation of a cDNA, regulation of gene expression, and characterization of post-translational processing intermediates

Somatostatin is a tetradecapeptide (SS-14) initially isolated from the hypothalamus that is also found in D cells of the stomach and pancreas where it exerts an inhibitory action on a variety of gastrointestinal functions. Since many of concepts important to an understanding of gastrointestinal physiology are derived from experiments in the dog we examined somatostatin gene expression and post-translational processing in the canine fundus, antrum and pancreas. The canine somatostatin cDNA which is highly homologous to other known mammalian somatostatins was used to examine somatostatin expression in isolated canine fundic D-cells. Somatostatin expression induced by cholecystokinin (10(-8) M) was inhibited by the somatostatin analog, octreotide (10(-7) M). To examine somatostatin processing in the canine gut we noted that synthesis of SS-14 and somatostatin octacosapeptide (SS-28) involves endoproteolytic cleavage of prosomatostatin (proSS) at both paired and single basic amino-acid residues, respectively. Antisera capable of recognizing the amino-terminal residues of SS-28, SS-28(1-14) and SS-28(1-12) were characterized and identified concentrations of SS-28(1-12) but not SS-28(1-14) in the fundus, antrum and pancreas equivalent to those of SS-14. Since previous biosynthetic studies in canine fundic D-cells showed that SS-14 was synthesized without the appearance of a SS-28 intermediate, we hypothesize that proSS is sequentially cleaved at a dibasic site to produce SS-14 followed by monobasic cleavage that results in the formation of SS-28(1-12). Furthermore, equivalent amounts of SS-14 and SS-28(1-12) were co-released from canine fundic D-cells by CCK (10(-8) M) suggesting that the generation of these products occurs within the same regulated pathway of secretion.