Modulation of gastrin processing by vesicular monoamine transporter type 1 (VMAT1) in rat gastrin cells.

1. Gastrointestinal endocrine cells produce biogenic amines which are transported into secretory vesicles by one of two proton-amine exchangers, vesicular monoamine transporters type 1 and 2 (VMAT1 and 2). We report here the presence of VMAT1 in rat gastrin (G) cells and the relevance of VMAT1 function for the modulation of progastrin processing by biogenic and dietary amines. 2. In immunocytochemical studies VMAT1, but not VMAT2, was localized to subpopulations of G cells and enterochromaffin (EC) cells; neither was found in antral D cells. The expression of VMAT1 in antral mucosa was confirmed by Northern blot analysis, which revealed an mRNA band of approximately 3.2 kb, and by Western blot analysis, which revealed a major protein of 55 kDa. 3. In pulse-chase labelling experiments, the conversion of the amidated gastrin G34 to G17 was inhibited by biogenic amine precursors (L-DOPA and 5-hydroxytryptophan). This inhibition was stereospecific and sensitive to reserpine (50 nM), which blocks VMAT1 and VMAT2, but resistant to tetrabenazine, which is a selective inhibitor of VMAT2. 4. Dietary amines such as tyramine and tryptamine also inhibited G34 cleavage. This effect was associated with a loss of the electron-dense core of G cell secretory vesicles. It was not stereospecific or reserpine sensitive, but was correlated with hydrophobicity. 5. Thus rat antral G cells can express VMAT1; transport of biogenic amines into secretory vesicles by VMAT1 is associated with inhibition of G34 cleavage, perhaps by raising intravesicular pH. Dietary amines also modulate cleavage of progastrin-derived peptides, but do so by a VMAT1-independent mechanism; they may act as weak bases that passively permeate secretory vesicle membranes and raise intravesicular pH.

Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa.

1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.

Gastrin biosynthesis in the antrum of patients with pernicious anemia.

BACKGROUND & AIMS: The gastrin precursor progastrin produces multiple alternative active products, but the pathways of posttranslational processing in human antral mucosa have not yet been studied directly. The aim of this study was to investigate the biosynthetic relationships and release kinetics of newly synthesized progastrin-derived peptides in the antrum of patients with pernicious anemia. METHODS: Antral mucosal explants were incubated with [35S]sulfate and [3H]tyrosine to label progastrin and its derivatives, which were detected by online scintillation counting after immunoprecipitation and high-performance liquid chromatography. RESULTS: [35S]- and [3H]progastrin were detected within 2.5 hours, and labeled G34Gly and G34 were readily detected after 5-hour incubation. Pulse-chase studies indicated conversion of progastrin to G17 via G34Gly and G34. Secretion of newly synthesized G34, but not G34Gly, was routinely detected; G17Gly was present only in trace quantities in cell extracts and media. In control samples, progastrin synthesis was about 10 times lower than in pernicious anemia samples, although the proportions of different labeled amidated gastrins after 5-hour incubation were similar. CONCLUSIONS: In the antrum of patients with pernicious anemia, Gly-gastrins, particularly G34Gly, are biosynthetic intermediates and not major secretory products. Some G34 is secreted preferentially under basal conditions.

Control of preprogastrin messenger RNA translation by gastric acid in the rat.

BACKGROUND & AIMS: Plasma gastrin and tissue preprogastrin messenger RNA (mRNA) increase in rats treated with the proton pump inhibitor omeprazole, but changes in mRNA alone cannot account for calculated changes in gastrin synthesis. The possibility that there is control of preprogastrin mRNA translation rates was investigated. METHODS: Preprogastrin mRNA translation was assessed by incorporation of [3H]tyrosine into progastrin in rat antral mucosa in vitro at 22 degrees C; preprogastrin mRNA was determined by Northern blot analysis. RESULTS: During incubation, incorporation of [3H]tyrosine into progastrin was linear up to 4 hours, and preprogastrin mRNA was unchanged. Fasting (24 hours) decreased plasma gastrin levels by 75% and progastrin translation by 40%, but preprogastrin mRNA was unchanged. Conversely, omeprazole increased plasma gastrin levels 8-fold, preprogastrin mRNA 2-3-fold, and progastrin translation 6-fold. In a cell-free translation system, preprogastrin was increased in samples from omeprazole-treated rats in direct proportion to the increase in preprogastrin mRNA abundance. CONCLUSIONS: Stimulation of gastrin cells by achlorhydria or inhibition by fasting lead, respectively, to increased and decreased preprogastrin translation rates that are more pronounced than changes in mRNA abundance. Therefore, luminal acid controls preprogastrin mRNA translation independently of changes in mRNA abundance or gastrin release.