| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MS 9101 Received 11 January 1999; accepted after revision 23 February 1999.
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
It has been known for a generation that gastrin (G) cells and other gut endocrine cells take up biogenic amine precursors, decarboxylate them and store the biogenic amine products in secretory vesicles (the APUD properties) (Pearse, 1969; Hakanson, 1970). These properties account for the capacity of enterochromaffin (EC) and enterochromaffin-like (ECL) cells to secrete amines (serotonin and histamine, respectively) on stimulation. But in the case of other cell types the significance of the APUD phenotype has remained uncertain. Recent studies suggest that the catecholamines histamine and serotonin (5-HT) are taken up by secretory vesicles via the activity of proton-amine exchangers (Liu et al. 1992; Peter et al. 1994). Two transporters that mediate this process have been cloned and characterized: vesicular monoamine transporter types 1 and 2 (VMAT1 and VMAT2) (Liu et al. 1992; Erickson et al. 1992; Peter et al. 1994). We recently found that in rat G cells, selected steps in the proteolytic conversion of precursor peptides to the secreted forms of gastrin were inhibited by raising intravesicular pH and we suggested that proton extrusion via VMAT activity might provide a physiological mechanism to modulate prohormone processing (Voronina et al. 1997).
The pyloric antral hormone gastrin occurs in multiple forms (Dockray et al. 1996). The results of pulse-chase labelling studies indicate that the precursor, progastrin, is cleaved first to a biosynthetic intermediate, 35-amino acid gastrin with a COOH-terminal glycine (G34Gly) (Varro et al. 1995). Two possible biosynthetic routes follow from G34Gly: either the peptidyl 
-amidating mono-oxygenase (PAM) converts the COOH-terminal Gly to a COOH-terminal amide, i.e. G34, which may in turn be cleaved to yield G17, or alternatively G34Gly is cleaved at a pair of lysines to yield G17Gly (Varro et al. 1995, 1997). These conversion steps determine biological activity. The two amidated gastrins G17 and G34 are gastric acid secretagogues and growth factors but they differ in their metabolic clearance rates, G34 being cleared approximately 5 times more slowly than G17 (Walsh et al. 1974, 1976; Walsh, 1994). The Gly-extended gastrins, and progastrin, are reported to be growth factors although they act at receptors distinct from the gastrin/CCK-B receptor at which amidated gastrins act (Seva et al. 1994; Wang et al. 1996; Singh et al. 1996; Hollande et al. 1997). The mechanisms controlling post-translational processing of progastrin therefore determine which of several alternative active products may be generated. In view of the recent evidence suggesting that VMAT activity might modulate progastrin processing (Voronina et al. 1997), we have sought to define the identity of the vesicular monoamine transporter in G cells. We report here evidence that VMAT1 can be expressed in rat antral G cells, that its activity is associated with modulation of G34 cleavage, and that dietary amines also modulate G34 cleavage but by a separate mechanism.
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Animals
Studies were made on male Wistar rats (250 g) fed ad libitum. Animals were killed by cervical dislocation followed by decapitation, and antral mucosa was dissected free of muscle for the studies described below.
Antibodies
Antibodies to the COOH-terminal sequences of rat VMAT1 (Pro-Leu-Gly-Glu-Asn-Ser-Asp-Asp-Pro-Ser-Ser-Gly-Glu) and rat VMAT2 (Ser-Tyr-Pro-Ile-Gly-Asp-Asp-Glu-Glu-Ser-Glu-Ser-Asp) were generated in rabbits immunized with the synthetic peptides conjugated to thyroglobulin as previously described (Varro et al. 1995). For immunocytochemical studies we also used mouse monoclonal antibodies to gastrin (Mab 28.2 directed at the C-terminal amide of G17; a gift from J. H. Walsh, University of California at Los Angeles), 5-HT (Dako, Denmark) or somatostatin (Mab 5607; a gift from J. H. Walsh).
Rat antral mucosa was fixed in 4 % paraformaldehyde in 0·1 M sodium cacodylate at pH 7·4 (4°C, 16 h) and washed in 20 % sucrose (4°C , overnight). Cryostat sections (5 µm) were prepared as previously described and processed for immunocytochemistry using horse anti-mouse IgG labelled with Texas Red and goat anti-rabbit IgG labelled with fluorescein isothiocyanate (FITC) (Macro et al. 1997). Rabbit antibodies to VMAT1 or VMAT2 were used in combination with mouse monoclonal antibodies to gastrin, somatostatin or 5-HT. In some experiments, samples of rat corpus mucosa were examined with antibodies to VMAT2. The patterns of colocalization of VMAT1 immunoreactivity together with gastrin or 5-HT immunoreactivity were examined using a Zeiss Axioplan 2 microscope and FITC/Texas Red dual filters. Estimates of the colocalization of VMAT1 and gastrin immunoreactivities were made by counting cells in at least six sections from three different animals. Absorption controls using antibodies preincubated with the appropriate antigen gave negative results in each case; immunostaining using VMAT1 antibodies was not, however, influenced by preincubation with a synthetic fragment of VMAT2 or vice versa.
Molecular identification of VMAT1 in antrum
A reverse transcriptase-polymerase chain reaction (RT-PCR) product corresponding to bases 575-846 of the published sequence of VMAT1 (Liu et al. 1992) was generated using a rat antral mucosal cDNA template with primers 5'-GCA CCA TCC CTC CTC CAG TCA C-3' and 5'-GAG TTC GGG CCA CAA ATA GCA G-3'. The product was inserted into the pGEM-Teasy vector (Promega, Southampton, UK) and the sequence of the cloned product confirmed by the dideoxy chain termination method. The vector was linearized using Nco1 and used as a template to produce a cRNA probe. Poly(A) enriched mRNA from antrum and corpus was generated using Trizol extraction (Gibco BRL, Paisley, UK) and the Poly A Tract system (Promega). Northern blots of 5 µg enriched mRNA were probed for VMAT1 using previously described protocols (Dimaline et al. 1993).
Western blot analysis
Antral or corpus mucosa was homogenized in 50 mM TrisCl (pH 7·0), centrifuged at 2000 g for 20 min and the supernatant stored at -40°C. Subsequently the supernatant was diluted two-fold in sample buffer and separated by electrophoresis on 8 % SDS polyacrylamide gels, transferred to nitrocellulose and processed for Western blot analysis using antibody to VMAT1 and detection of immune complexes by chemiluminescence (Pierce & Warriner, Chester, UK).
Biosynthetic labelling
The post-translational processing of progastrin and its derivatives was followed using a pulse-chase labelling protocol as previously described (Varro & Dockray, 1993; Varro et al. 1994). Antral mucosa was incubated with [35S]sulphate for 2 h at 22°C and chased at 37°C for up to 160 min. At the beginning of the chase period samples were incubated with the biogenic amine precursors L-DOPA and 5-hydroxytryptophan (5-HTP) (0·5 µM to 5 mM) or with dietary amines (ethylamine, methylamine, tyramine or tryptamine; 0·5-10 mM), and the VMAT inhibitors reserpine and tetrabenazine (0·5 nM to 25 µM). Labelled progastrin-derived peptides were extracted, immunoprecipitated with antibodies to the C-terminal amide of G17, and separated by HPLC with on-line scintillation counting as previously described (Varro et al. 1994, 1995).
Electron microscopy
Antral mucosa was incubated for 2 h in 10 mM tyramine. Tissue was fixed in 4 % formaldehyde and 2 % glutaraldehyde in 0·1 M sodium cacodylate buffer, pH 7·4, post-fixed in 1 % osmium tetraoxide, stained with uranyl acetate and embedded in Taab epoxy resin (Taab Laboratories, Reading, UK) as previously described (Voronina et al. 1997). Ultrathin sections (90 nm) were mounted on carbon-coated nickel grids. For immunogold studies, sections were treated with normal goat serum before incubating for 18 h with mouse monoclonal antibody to the C-terminus of amidated gastrin (Mab 28.2). Sites of antibody binding were labelled with 15 or 5 nm gold particles conjugated to goat anti-mouse IgG (BioCell Research Laboratories, Cardiff, UK). In control experiments, immunogold labelling was shown to be inhibited by preabsorption of antibodies with synthetic G17 (10 µM), indicating the specificity of the localization.
Statistics
Results are presented as means ± S.E.M.; comparisons were made by Student's t test and were considered significant when P < 0·05.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Immunocytochemistry
Antibodies to VMAT1 revealed cells that resembled endocrine cells in the antral mucosa (Fig. 1). In order to characterize the cell type, we performed double-labelling immunocytochemistry using antibodies to 5-HT, gastrin and somatostatin. A subpopulation of gastrin-immunoreactive cells (74·5 ± 6·1 % of total, n = 3 rats) also contained VMAT1 immunoreactivity (Fig. 1). In addition, a substantial subset of 5-HT-immunoreactive cells (67 ± 3 % of total) expressed VMAT1 immunoreactivity (Fig. 1). Somatostatin-immunoreactive cells (i.e. D cells) did not contain VMAT1 immunoreactivity. Occasional G cells contained 5-HT immunoreactivity, but most did not. Similarly, virtually all cells with 5-HT immunoreactivity did not exhibit either gastrin or somatostatin immunoreactivity; these cells were therefore tentatively identified as enterochromaffin (EC) cells. Antibodies to VMAT2 did not colocalize with gastrin, 5-HT or somatostatin in antral mucosa; they did, however, stain enterochromaffin-like (ECL) cells in the gastric corpus as previously reported (not shown) (Weihe et al. 1994; De Giorgio et al. 1996).
 |
View larger version [in this window] [in a new window] |
|
|
Double-immunofluorescence staining with mouse monoclonal antibodies to 5-HT (a), gastrin (b) or somatostatin (c) and rabbit antiserum to VMAT1 (d-f). Filled arrows indicate cells positive for VMAT1, open arrows indicate cells negative for VMAT1. Scale bar, 15 µm. | ||
Molecular identification of VMAT1 in antrum
In RT-PCR studies using primers to the published sequence of rat VMAT1 (Liu et al. 1992), we found a product of approximately 275 bp corresponding to the predicted size (Fig. 2). The sequence of the cloned PCR product was identical to that previously reported (Liu et al. 1992). The expression of VMAT1 in antral mucosa was also assessed by Northern and Western blot analysis (Fig. 2). In Northern blots, we found a single mRNA species in antral mucosa of approximately 3·2 kb. In Western blots, antral mucosal extracts contained a major band of 55 kDa and a minor band of 47 kDa. The VMAT1 signal in Western blots was abolished by prior incubation of antiserum with the synthetic peptide used for immunization; moreover, incubation of membranes with non-immune serum did not reveal the two bands. These data provide clear evidence for VMAT1 mRNA and protein in rat antrum, which are presumably derived from both G and EC cells.
 |
View larger version [in this window] [in a new window] |
|
|
A, identification by RT-PCR of a product of approximately 275 bp corresponding to VMAT1. B, identification of VMAT1 mRNA by Northern blot. The result of Northern blot analysis of poly(A) enriched mRNA from rat antrum examined with a probe for VMAT1 is shown. Note a band of 3·2 kb. C, identification of VMAT1 by Western blot. Protein (60 µg) in antral mucosal extracts was separated by SDS-PAGE and membranes were examined with antibodies to the C-terminus of rat VMAT1. Note a major band of 55 kDa and a minor one of 47 kDa. Preabsorption of antibodies with synthetic peptide abolished the localization and non-immune serum gave negative results (not shown). | ||
Biosynthesis
The data outlined above strongly suggest that many G cells express VMAT1. In order to define further the functional significance of VMAT1 in G cells we examined the consequences of incubation of antral mucosa with the biogenic amine precursors L-DOPA and 5-HTP and selective inhibitors of vesicular monoamine transporters. In pulse- chase labelling experiments, we confirmed that L-DOPA inhibited the cleavage of G34 to G17 and that this was reversed by reserpine (Fig. 3) (Voronina et al. 1997). We then compared the effects of reserpine, which has similarly high affinities for both VMAT1 and VMAT2, with tetrabenazine, which preferentially inhibits VMAT2 (Peter et al. 1994). At a concentration of 50 nM, reserpine produced approximately 70 % reversal of the inhibition of G34 cleavage by L-DOPA and 5-HTP. We found that the effect of L-DOPA on G34 cleavage was refractory to tetrabenazine. Thus, at concentrations 100-fold higher than those of reserpine there was no significant reversal of the effects of L-DOPA on G34 cleavage by tetrabenazine; in the case of 5-HTP, high concentrations of tetrabenazine (5 µM) did, however, produce a partial reversal of the inhibition of [35S]G34 cleavage (Fig. 4B), compatible with reports that at this concentration tetrabenazine inhibits VMAT1 by up to 50 % (Peter et al. 1994; Weihe et al. 1994).
 |
View larger version [in this window] [in a new window] |
|
|
Progastrin-derived peptides in rat antral mucosa were labelled in a pulse-chase protocol with [35S]sulphate and separated after immunoprecipitation by HPLC with on-line scintillation counting. In C, note that L-DOPA (0·5 mM) inhibited conversion of G34 to G17 compared with control (A), and that reserpine (50 nM, which acts on VMAT1 and VMAT2) blocks this effect (B), but tetrabenazine (5 µM, which exhibits specificity for VMAT2) does not (D). The total labelled amidated gastrin recovered was not influenced by incubation with amines or VMAT inhibitors. Here and elsewhere, c.p.m., counts per minute. | ||
 |
View larger version [in this window] [in a new window] |
|
|
In pulse-chase labelling experiments, rat antral mucosa was incubated with 0·5 mM L-DOPA (A) and 0·5 mM 5-HTP (B), with either 50 nM reserpine (Reserp) or 5 µM tetrabenazine (Tetraben). Samples were recovered at 160 min of chase. Control, no additions. Veh, vehicle; either L-DOPA or 5-HTP but no inhibitor. Data are expressed as the percentage of [35S]G17 in total labelled amidated gastrin (i.e. G34 + G17), and provide a measure of G34 cleavage. Data are means ± S.E.M. for n = 4-12 experiments, where each experimental sample corresponded to one animal. n.S.D., no significant difference. | ||
Dietary amines
Previous reports have suggested that certain dietary amines, including tyramine and tryptamine, may also be substrates for VMATs (Finn & Edwards, 1997; Romanenko et al. 1998). We therefore considered it interesting to examine whether these and other dietary amines might modulate G34 cleavage. At concentrations of 1-10 mM, tyramine produced a concentration-dependent inhibition of the cleavage of G34 (Figure 5 and Figure 6). At 10 mM, tryptamine had similar effects to tyramine (Fig. 7). There were, however, differences in the effects of dietary amines compared with L-DOPA and 5-HTP: (a) Reserpine did not inhibit the action of tryptamine (Fig. 7) or tyramine (not shown). (b) At 10 mM, tyramine and tryptamine also inhibited cleavage of progastrin at Arg94/95 (Figs 5 and 6), and there was a correlation between the hydrophobicity of dietary amines and their capacity to inhibit progastrin cleavage. Previous work has shown that L-DOPA and 5-HTP do not inhibit the latter cleavage although it can be suppressed by bafilomycin A1, which inhibits the vacuolar proton pump and blocks progression through the Golgi complex (Voronina et al. 1997). (c) The related amine, methylbenzylamine, inhibited G34 cleavage equally well in both D- and L-isomers, whereas the inhibition of G34 cleavage by L-DOPA was stereospecific (Fig. 6).
 |
View larger version [in this window] [in a new window] |
|
|
Antral mucosa was incubated in [35S]sulphate for 2 h at 22 °C and chased at 37 °C for 160 min in the presence or absence of 10 mM tyramine. Left panels, samples immunoprecipitated with antibody to progastrin. Right panels, samples precipitated with antibody to COOH-terminal amidated gastrins. A, results at the end of the pulse-labelling period (note the presence of immunoreactive progastrin, but not amidated gastrin). B, control samples taken after 160 min chase (note the appearance of amidated gastrin, mainly G17, and the loss of progastrin). C, samples taken after 160 min chase in the presence of tyramine (note that there is still some progastrin and that G34 rather than G17 is the predominant amidated gastrin). | ||
 |
View larger version [in this window] [in a new window] |
|
|
Representative experiments showing concentration-response relationships for the action of three dietary amines on G34 cleavage (A); the time course of the effect of 10 mM tyramine compared with control on cleavage of G34 (continuous lines) and progastrin (dashed lines) (B); and a correlation between hydrophobicity and the capacity of amines (all at 10 mM) to inhibit progastrin cleavage (C). D shows that the action of DOPA (0·5 mM) in inhibiting G34 is stereospecific, but that of methylbenzylamine (MBZ, 10 mM) is not; vertical bars correspond to +S.E.M. for n = 3-7 independent experiments, where each experimental sample corresponds to a single animal. | ||
 |
View larger version [in this window] [in a new window] |
|
|
Progastrin-derived peptides were labelled with [35S]sulphate for 2 h at 22 °C and chased at 37 °C for 160 min either in the presence of 10 mM tryptamine or 10 mM tryptamine plus 1 µM reserpine. Note tryptamine (B) inhibits G34 cleavage compared with control (A) and that reserpine does not influence this effect (C). | ||
The actions of tyramine and tryptamine are compatible with the notion that they act as weak bases that permeate the vesicle membrane, become protonated in the vesicle interior and raise the pH. We have previously noted that agents which raise intravesicular pH produce a loss of the electron-dense core of G cell secretory vesicles (Voronina et al. 1997). In the present study we found that G cells identified by the immunogold labelling technique showed enlarged secretory vesicles after incubation in tyramine, with evidence of a loss of the electron dense core (Fig. 8). In many cases, the remains of an electron dense core seemed to be eccentrically located within an enlarged vesicle. Interestingly, the immunogold labelling was stronger over the core than the remaining electron-lucent region of the vesicle.
 |
View larger version [in this window] [in a new window] |
|
|
Transmission EM showing immunogold localization of gastrin in rat antral mucosa. A and C, after incubation in 10 mM tyramine (120 min at 22 °C, 160 min at 37 °C), G cells exhibited enlarged secretory vesicles with evidence of loss of electron-dense cores, although not all vesicles within the same cell were affected. Immunogold labelling was associated with the remnants of the electron-dense core in enlarged vesicles and with the core of unaffected vesicles. B and D, control G cells exhibited the range of vesicle morphologies characteristic of G cells, with immunogold labelling of electron-dense cores. 15 nm gold particles were used A and B, 5 nm gold particles in C and D. Scale bar in A and B, 300 nm; in C and D, 100 nm. | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The main findings of the present study are that many G cells of the rat pyloric antral mucosa express VMAT1 and that the activity of VAMT1 is associated with inhibition of G34 cleavage. We propose, therefore, that VMAT1 is responsible for the long-recognized APUD property of G cells of being able to concentrate and store biogenic amines (Pearse, 1969). Moreover, the data suggest that this property provides a capacity for modulation of G34 cleavage, possibly secondary to raising intravesicular pH due to proton extrusion. Certain dietary amines that are potential VMAT substrates were also shown to modulate progastrin processing; however, in this case we found no evidence of VMAT-dependent activity. Instead we suggest that these dietary amines modulate progastrin processing secondary to an increased intravesicular pH, due to the properties of these compounds as weak bases that become protonated within secretory vesicles.
Previous reports of the distribution of the VMATs have noted their presence in monoamine-secreting neurons and in selected populations of endocrine cells, including those of the gut and adrenal medulla (Weihe et al. 1994; De Giorgio et al. 1996). Immunocytochemical studies suggest that the expression of VMAT1 and VMAT2 is mutually exclusive (Weihe et al. 1994; Peter et al. 1995; Erickson et al. 1996). In the gastric mucosa, it is agreed that VMAT2 is expressed in enterochromaffin-like (ECL) cells found in the corpus, and our own findings confirm this (Weihe et al. 1994; Dimaline & Struthers, 1996; De Giorgio et al. 1996; Erickson et al. 1996). The main function in ECL cells appears to be the sequestration of histamine in secretory vesicles. Previous studies have noted the presence of VMAT1 immunoreactive cells in the gut epithelium, including EC cells (Weihe et al. 1994; Erickson et al. 1996). In rat antral mucosa, we found examples of both EC and G cells expressing VMAT1. It is well recognized that EC cells release 5-HT on light mechanical stimulation (Bulbring & Lin, 1958), and so we suggest that in these cells, the function of VMAT1 is to load 5-HT into secretory vesicles. In the case of G cells, our data suggest that the presentation of substrates to VMAT1 regulates the conversion of G34 to G17. This step is known to be inhibited by raising intravesicular pH (Voronina et al. 1997), and since VMAT1 is a proton-amine exchanger we suggest that in G cells it acts to modulate post-translational processing via changes in intravesicular pH.
The immunocytochemical data revealed VMAT1 in only a subset of G cells. Given the localization of VMAT1 in vesicular membranes it would not be surprising if the protein was present in substantially lower abundance compared with gastrin. Since most if not all G cells have the capacity to store biogenic amines, it seems likely that we have underestimated the number of G cells expressing VMAT1 because its abundance is relatively low. Our studies of the processing of progastrin-derived peptides depend on the presentation of biogenic amine precursors to G cells. The mechanisms by which L-DOPA and 5-HTP enter the G cell have not been addressed directly. It is, however, known that there are plasma membrane transporter systems for L-DOPA, and presumably a similar transporter exists on the G cell plasma membrane (Soares-da-Silva et al. 1994). Conversion of the precursors to the corresponding amine is mediated by aromatic amino acid decarboxylase, which is known to be expressed in antral mucosa (Djali et al. 1998). We have previously presented evidence to suggest that sequestration of biogenic amines into secretory vesicles of G cells modulates prohormone cleavage by raising intravesicular pH (Voronina et al. 1997). The prohormone convertases that mediate the cleavage of progastrin-derived peptides have acidic pH optima and the activity of at least one of them (PC1) would be expected to be depressed by a shift in intravesicular pH towards neutrality (Davidson et al. 1988; Bailyes et al. 1992). The data are therefore compatible with the idea that amine loading in G cell secretory vesicles modulates processing secondary to an increase in intravesicular pH. It remains conceivable, however, that amines might also directly inhibit prohormone convertases, and this has been proposed to account for the action of reserpine in promoting cleavage of chromogranin A, chromogranin B, secretogranin II and proenkephalin in chromaffin cells (Lindberg, 1986; Watkinson & Robinson, 1992; Wolkersdorfer et al. 1996).
The present study has shown that dietary amines also modulate the cleavage of progastrin-derived peptides, although by a mechanism that appears to be distinct from the action of biogenic amines. These experiments were performed because at least two dietary amines, tyramine and tryptamine, are recognized substrates for the VMATs (Finn & Edwards, 1997; Romanenko et al. 1998), and we reasoned that either or both could mediate a physiological mechanism by which luminal nutrients influence progastrin processing. We found that in concentrations of 1-10 mM both amines inhibited cleavage of progastrin and G34. These effects were similar to those of weak bases such as chloroquine, proton ionophores such as monensin and inhibitors of the vacuolar proton pump such as bafilomycin. These are all thought to act on G cells partly by inhibiting progression of progastrin through the Golgi complex and partly by raising intravesicular pH (Voronina et al. 1997). The effects of the dietary and related amines were resistant to reserpine, were not stereospecific and appeared to be correlated with hydrophobicity. We propose, therefore, that these weak bases diffuse across the plasma and vesicular membranes, are sequestered in secretory vesicles and raise internal vesicular pH. Direct experimental evidence for an increase in G cell vesicular pH in response to dietary amines has previously been found in studies using isolated vesicles (Dial et al. 1991). The concentrations we used in the present experiments are comparable to those reported in some foods, for example tyramine is reported to occur in concentrations up to 5 mM in soy sauce and approximately 5 mmol kg-1 in certain cheeses (Halasz et al. 1994; Walker et al. 1996). Even so, it is likely that only a very small proportion of the amine is unprotonated at physiological pH and therefore available to penetrate the G cell. These effects would, however, be favoured in circumstances where acid secretion is inhibited and in support of this notion it has been shown that uptake of fluorescent amines was increased after inhibition of acid secretion (Lichtenberger et al. 1986).
Different progastrin products vary in their biological activity, and for this reason there is presently considerable interest in the mechanisms regulating the post-translational processing of progastrin (Dockray et al. 1996). The data suggest that progastrin and C-terminal Gly-extended gastrins are growth factors (but not secretagogues) and that amidated gastrins are both secretagogues and growth factors (Seva et al. 1994; Wang et al. 1996; Singh et al. 1996; Hollande et al. 1997). Moreover, the longer-chain gastrins such as G34 have a greater half-life than short-chain gastrins such as G17 (Walsh et al. 1974, 1976). Control of G34 cleavage would therefore be expected to determine the ratio of G34 : G17 in plasma and in turn to determine the absolute magnitude and duration of the changes in plasma gastrin following G cell stimulation.
The APUD properties of G cells and other peptide-producing endocrine cells in the gut have been recognized for many years (Pearse, 1969), but their molecular basis and physiological significance have been uncertain. The idea that these properties reflect a common developmental origin of the cells in question from the neural crest has not been supported by recent work (Langley, 1994). The present data establish that the molecular basis of the capacity to store biogenic amines in G cells is due to the expression of VMAT1, and indicate that this function has implications not just for the regulated exocytosis of biogenic amines but also for modulation of intravesicular prohormone processing. Modulation of VMAT expression would therefore indirectly regulate the precise patterns of progastrin-derived peptides produced in G cells exposed to biogenic amines. Some dietary amines are also substrates for the VMATs; however, it would seem that these compounds modulate progastrin processing by a VMAT-independent mechanism. The same compounds are gastrin secretagogues, so that dietary amines determine both acute secretory responses and the identity of the progastrin-derived peptides released (Lichtenberger et al. 1982; Delvalle & Yamada, 1990).
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Bailyes, E. M., Shennan, K. I. J., Seal, A. J., Smeekens, S. P., Steiner, D. F., Hutton, J. C. & Docherty, K. (1992). A member of the eukaryotic subtilisin family (PC3) has the enzymic properties of the type 1 proinsulin-converting endopeptidase. Biochemical Journal 285, 391-394 | [Medline] |
| Bulbring, E. & Lin, R. C. Y. (1958). The effect of intraluminal application of 5-hydroxytryptamine and 5-hydroxytryptophan on peristalsis; the local production of 5-HT and its release in relation to intraluminal pressure and propulsive activity. The Journal of Physiology 140, 381-407. | |
| Davidson, H. W., Rhodes, C. J. & Hutton, J. C. (1988). Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic J cell via two distinct site-specific endopeptidases. Nature 333, 93-96 | [Medline] |
| De Giorgio, R., Su, D., Doris, P, Edwards, R. H., Brecha, N. C. & Sternini, C. (1996). Vesicular monoamine transporter 2 expression in enteric neurons and enterochromaffin-like cells of the rat. Neuroscience Letters 217, 77-80 | [Medline] |
| Delvalle, J. & Yamada, T. (1990). Amino acids and amines stimulate gastrin release from canine antral G cells via different pathways. Journal of Clinical Investigation 85, 139-143 | [Medline] |
| Dial, E. J., Cooper, L. C. & Lichtenberger, L. M. (1991). Amino-acid and amine-induced release from isolated rat endocrine granules. American Journal of Physiology 260, G175-181 | [Medline] |
| Dimaline, R., Sandvik, A. K., Evans, D., Forster, E. R. & Dockray, G. J. (1993). Food stimulation of histidine decarboxylase messenger RNA abundance in rat gastric fundus. The Journal of Physiology 465, 449-458 | [Abstract] |
| Dimaline, R. & Struthers, J. (1996). Expression and regulation of a vesicular monoamine transporter (VMAT2) in rat stomach: a putative histamine transporter. The Journal of Physiology 490, 249-256 | [Abstract] |
| Djali, P. K., Dimaline, R. & Dockray, G. J. (1998). Novel form of L-aromatic amino acid decarboxylase mRNA in rat antral mucosa. Experimental Physiology 83, 617-627 | [Medline] |
| Dockray, G. J., Varro, A. & Dimaline, R. (1996). Gastric endocrine cells: gene expression, processing and targeting of active products. Physiological Reviews 76, 767-798 | [Medline] |
| Erickson, J. D., Eiden, L. E. & Hoffman, B. J. (1992). Expression cloning of a reserpine-sensitive vesicular monoamine transporter. Proceedings of the National Academy of Sciences of the USA 89, 10993-10997 | [Abstract] |
| Erickson, J. D., Schafer, M. K., Bonner, T. I., Eiden, L. E. & Weihe, E. (1996). Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter. Proceedings of the National Academy of Sciences of the USA 93, 5166-5171 | [Abstract] |
| Finn, J. P. III & Edwards, R. H. (1997). Individual residues contribute to multiple differences in ligand recognition between vesicular monoamine transporters 1 and 2. Journal of Biological Chemistry 272, 16301-16307 | [Abstract/Full Text] |
| Hakanson, R. (1970). New aspects of the formation and function of histamine, 5-hydroxytryptamine and dopamine in gastric mucosa. Acta Physiologica Scandinavica, suppl. 340, 1-134. | |
| Halasz, A., Barath, A., Simon-Sarkadi, L. & Holzapfel, W. (1994). Biogenic amines and their production by microorganisms in food. Trends in Food Science and Technology 5, 42-49. | |
| Hollande, F., Imdahl, A., Mantamadiotis, T., Ciccotosto, G. D., Shulkes, A. & Baldwin, G. S. (1997). Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line. Gastroenterology 113, 1576-1588 | [Abstract] |
| Langley, K. (1994). The neuroendocrine concept today. Annals of the New York Academy of Sciences 733, 1-17 | |
| Lichtenberger, L. M., Graziani, L. A. & Dubinsky, W. P. (1982). Importance of dietary amines in meal-induced gastrin release. American Journal of Physiology 243, G341-347 | [Medline] |
| Lichtenberger, L. M., Nelson, A. A. & Graziani, L. A. (1986). Amine trapping: physical explanation for the inhibitory effect of gastric acidity on the postprandial release of gastrin. Gastroenterology 90, 1223-1231 | [Abstract] |
| Lindberg, I. (1986). Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Journal of Biological Chemistry 261, 16317-16322 | [Abstract] |
| Liu, Y., Peter, D., Roghani, A., Schuldiner, S., Prive, G. G., Eisenberg, D., Brecha, N. & Edwards, R. H. (1992). A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter. Cell 70, 539-551 | [Medline] |
| Macro, J. A., Bate, G. W., Varro, A., Vaillant, C., Seidah, N. G., Dimaline, R. & Dockray, G. J. (1997). Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa. The Journal of Physiology 502, 409-419 | [Abstract] |
| Pearse, A. G. E. (1969). The cytochemistry and ultrastructure of polypeptide hormone-producing cells of the APUD series and the embryologic, physiologic and pathologic implications of the concept. Journal of Histochemistry and Cytochemistry 17, 303-313 | [Medline] |
| Peter, D., Jimenez, J., Liu, Y., Kim, J. & Edwards, R. H. (1994). The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors. Journal of Biological Chemistry 269, 7231-7237 | [Abstract] |
| Peter, D., Liu, Y., Sternini, C., De Giorgio, R., Brecha, N. & Edwards, R. H. (1995). Differential expression of two vesicular monoamine transporters. Journal of Neuroscience 15, 6179-6188 | [Abstract] |
| Romanenko, V. G., Gebara, R., Miller, K. M. & Njus, D. (1998). Determination of transport parameters of permeant substrates of the vesicular amine transporter. Analytical Biochemistry 257, 127-133 | [Medline] |
| Seva, C., Dickinson, C. J. & Yamada, T. (1994). Growth-promoting effects of glycine-extended progastrin. Science 265, 410-412 | [Medline] |
| Singh, P., Owlia, A., Varro, A., Dai, B., Rajaraman, S. & Wood, T. (1996). Gastrin gene expression is required for the proliferation and tumorigenicity of human colon cancer cells. Cancer Research 56, 4111-4115 | [Medline] |
| Soares-da-Silva, P., Fernandes, M. H. & Pinto-do-O, P. C. (1994). Cell inward transport of L-DOPA and 3-O-methyl-L-DOPA in rat renal tubules. British Journal of Pharmacology 112, 611-615 | [Medline] |
| Varro, A. & Dockray, G. J. (1993). Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A. Biochemical Journal 295, 813-819 | [Medline] |
| Varro, A., Dockray, G. J., Bate, G. W., Vaillant, C., Higham, A., Armitage, E. & Thompson, D. G. (1997). Gastrin biosynthesis in the antrum of patients with pernicious anemia. Gastroenterology 112, 733-741 | [Abstract] |
| Varro, A., Henry, J., Vaillant, C. & Dockray, G. J. (1994). Discrimination between temperature- and brefeldin A-sensitive steps in the sulfation, phosphorylation, and cleavage of progastrin and its derivatives. Journal of Biological Chemistry 269, 20764-20770 | [Abstract] |
| Varro, A., Voronina, S. & Dockray, G. J. (1995). Pathways of processing of the gastrin precursor in rat antral mucosa. Journal of Clinical Investigation 95, 1642-1649 | [Medline] |
| Voronina, S., Henry, J., Vaillant, C., Dockray, G. J. & Varro, A. (1997). Amine precursor uptake and decarboxylation: significance for processing of the rat gastrin precursor. The Journal of Physiology 501, 363-374 | [Abstract] |
| Walker, S. E., Shulman, K. I., Tailor, S. A. N. & Garner, D. (1996). Tyramine content of previously restricted foods in monoamine-oxidase inhibitor diets. Journal of Clinical Psychopharmacology 16, 383-388. | [Medline] |
| Walsh, J. H. (1994). Gastrin. In Gut Peptides, ed. Walsh, J. H. & Dockray, G. J., pp. 75-121. Raven Press, New York. | |
| Walsh, J. H., Debas, H. T. & Grossman, M. I. (1974). Pure human big gastrin: immunochemical properties, disappearance half time and acid stimulating action in dogs. Journal of Clinical Investigation 54, 477-485 | [Medline] |
| Walsh, J. H., Isenberg, J. I., Ansfield, J. & Maxwell, V. (1976). Clearance and acid-stimulating action of human big and little gastrins in duodenal ulcer subjects. Journal of Clinical Investigation 57, 1125-1131 | [Medline] |
| Wang, T. C., Koh, T. J., Varro, A., Cahill, R. J., Dangler, C. A., Fox, J. G. & Dockray, G. J. (1996). Processing and proliferative effects of human progastrin in transgenic mice. Journal of Clinical Investigation 98, 1918-1929 | [Abstract/Full Text] |
| Watkinson, A. & Robinson, I. (1992). Reserpine-induced processing of chromogranin A in cultured bovine adrenal chromaffin cells. Journal of Neurochemistry 58, 877-88 | [Abstract] |
| Weihe, E., Schafer, M. K. H., Erickson, J. D. & Eiden, L. E. (1994). Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat. Journal of Molecular Neuroscience 5, 149-164. | [Medline] |
| Wolkersdorfer, M., Laslop, A., Lazure, C., Fischer-Colbrie, R. & Winkler, H. (1996). Processing of chromogranins in chromaffin cell culture: effects of reserpine and -methyl-p-tyrosine. Biochemical Journal 316, 953-958 |
[Medline] |
We thank the MRC and The Wellcome Trust for financial support and Cathy McLean for skilled technical assistance. We are grateful to John Walsh for supplying the monoclonal antibodies to gastrin and somatostatin, and to Mark Cronin for useful discussion.
Corresponding author
A. Varro: Physiological Laboratory, University of Liverpool, Crown Street, PO Box 147, Liverpool L69 3BX, UK.
Email: avarro{at}liverpool.ac.uk
This article has been cited by other articles:
|
|
 |
|
  A. Pagliocca, P. Hegyi, V. Venglovecz, S. A. Rackstraw, Z. Khan, G. Burdyga, T. C. Wang, R. Dimaline, A. Varro, and G. J. Dockray Identification of ezrin as a target of gastrin in immature mouse gastric parietal cells Exp Physiol, November 1, 2008; 93(11): 1174 - 1189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kenny, C. Duval, S. J. Sammut, I. Steele, D. M. Pritchard, J. C. Atherton, R. H. Argent, R. Dimaline, G. J. Dockray, and A. Varro Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells Am J Physiol Gastrointest Liver Physiol, September 1, 2008; 295(3): G431 - G441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. A. Steele, R. Dimaline, D. M. Pritchard, R. M. Peek Jr., T. C. Wang, G. J. Dockray, and A. Varro Helicobacter and gastrin stimulate Reg1 expression in gastric epithelial cells through distinct promoter elements Am J Physiol Gastrointest Liver Physiol, July 1, 2007; 293(1): G347 - G354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Varro, S. Kenny, E. Hemers, C. McCaig, S. Przemeck, Timothy. C. Wang, K. Bodger, and D. M. Pritchard Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling Am J Physiol Gastrointest Liver Physiol, April 1, 2007; 292(4): G1133 - G1140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Varro, P-J. M. Noble, D. M. Pritchard, S. Kennedy, C. A. Hart, R. Dimaline, and G. J. Dockray Helicobacter pylori Induces Plasminogen Activator Inhibitor 2 in Gastric Epithelial Cells through Nuclear Factor-{kappa}B and RhoA: Implications for Invasion and Apoptosis Cancer Res., March 1, 2004; 64(5): 1695 - 1702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Wroblewski, P.-J. M. Noble, A. Pagliocca, D. M. Pritchard, C. A. Hart, F. Campbell, A. R. Dodson, G. J. Dockray, and A. Varro Stimulation of MMP-7 (matrilysin) by Helicobacter pylori in human gastric epithelial cells: role in epithelial cell migration J. Cell Sci., July 15, 2003; 116(14): 3017 - 3026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. WEIHE and L. E. EIDEN Chemical neuroanatomy of the vesicular amine transporters FASEB J, December 1, 2000; 14(15): 2435 - 2449. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
