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.

Novel form of L-aromatic amino acid decarboxylase mRNA in rat antral mucosa.

Gut endocrine cells possess the capacity to take up and decarboxylate biogenic amine precursors. Decarboxylation is mediated by aromatic L-amino acid decarboxylase (AADC) which is encoded by mRNAs differing in their 5' untranslated regions (UTR) depending on the usage of alternative first exons, 1a and 1b, each with its own acceptor site (-13 and -8 bases relative to the translation start site, respectively). We describe here a novel splice variant of exon 1a-AADC mRNA in rat antral mucosa. Both exon 1a and 1b mRNAs were expressed in rat antral mucosa, but the 1a form was spliced into the acceptor site usually associated with exon 1b (-8). An enteroendocrine cell line (STC-1) expressed exon 1a or exon 1b mRNAs spliced into the -8 acceptor site of exon 2. Transient transfection of a range of cell lines with reporter constructs revealed that all three 5' UTRs efficiently supported expression of the Luciferase reporter. There is therefore a novel, functional 5' UTR of AADC mRNA in rat antral mucosa; alternative AADC splice variants could provide the capacity for control at the level of mRNA translation.

Immunohistochemical study of leucine-enkephalin and its secretory effects in the isolated pig lacrimal gland.

BACKGROUND: In our previous studies immunohistochemical studies have demonstrated the presence of leucine-enkephalin (Leu-Enk) in the intrinsic nerves of the pig lacrimal gland, which are discernible in the interlobular and interacinar areas from where branches that innervate the acinar tissues are sent. Since the intrinsic nerves have been shown to contain Leu-Enk, this study aimed to investigate the secretory effects of this neuropeptide in isolated segments of the pig lacrimal gland and to reconfirm its presence in the neuronal tissue of the lacrimal gland. METHODS: Leu-Enk was identified using immunohistochemical techniques, while total protein output was measured in the effluent samples by an automated on-line colorimetric method. The Ca2+ and Mg2+ concentrations in the effluent samples were determined using an atomic absorbance spectrophotometer. RESULTS: Leu-Enk (10(-12)-10(-7) M) evoked marked increases in total protein output from superfused lacrimal gland segments. The secretory effect of Leu-Enk was not blocked by pretreatment of the tissue with atropine but was substantially reduced by a combination of phentolamine and propranolol. The competitive antagonist, naloxone, has no effect on basal protein output, but when combined with Leu-Enk it caused a significant reduction in total protein output. Combining theophylline (10(-3) M) with Leu-Enk resulted in a marked potentiation of total protein output. In superfused lacrimal gland segments Leu-Enk (10(-8) M) evoked a net efflux of magnesium (Mg2+ release) and a net influx of calcium (Ca2+ intake). CONCLUSION: The results indicated a physiological role for Leu-Enk in the regulation of protein secretion in the pig lacrimal gland.