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1 Departments of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California
2 Department of Physiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California
3 Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California
4 Membrane Biology Laboratory, West Los Angeles Department of Veterans Affairs Medical Center, Los Angeles, California
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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1-subunit protein content. These results indicate that rat gastric ECL cells regulate their histamine content during 24-h fasting not by a change in HDC gene or protein expression but by regulation of substrate concentration for HDC and a decreased histamine secretory pool. enterochromaffin-like cell; oligonucleotide expression microarray; transcriptome; histamine; secretion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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In the absence of the buffering effect of food, acid secreted by the parietal cell produces a highly acidic fluid that is potentially damaging to the upper gastrointestinal tract. Also, acid secretion is an energy-demanding process that is unnecessary without food. Therefore, it is expected that the quantity of histamine produced and released from ECL cells will decrease with fasting. Furthermore, this reduction in histamine production likely results in decreased storage of histamine in the ECL cell. This will reduce but not abolish acid secretion in the absence of food, since a basal rate of exocytosis of histamine occurs even in the absence of exogenous stimulation of the ECL cell (11).
Given the contrasting needs of the organism for gastric acid secretion with fasting and feeding, it is likely that the physiological state of the ECL cell and the enzymatic pathways involved in histamine production and release are changed to reduce histamine availability during fasting but maintain the ability of this cell to rapidly increase histamine production and secretion with feeding. A major factor in the ability of the ECL cell to regulate acid secretion should therefore be the histamine content of the secretory vesicles of the ECL cell. In addition to histamine regulation, many other ECL cell functions are likely to change with feeding or fasting for more precise regulation of acid production and regulation of the status of the ECL cell.
Potential mechanisms whereby steady-state histamine levels in fasting ECL cells could be altered include a decrease in histamine synthesis by downregulation of the rate-limiting enzyme histidine decarboxylase (HDC), by changes in translational or posttranslational processing of HDC affecting its activity, or by depletion of the substrate for this enzyme, L-histidine. The latter effect could be achieved by upregulation of a L-histidine-degrading pathway catalyzed by histidine ammonia lyase (histidase) and urocanate hydratase (urocanase) forming the 4-imidazolone 5-propionate intermediate that is then converted to L-glutamate or by downregulation of the L-histidine transporter in the ECL cell. Histamine stores could also be depleted by downregulation of vesicular monoamine transporter 2 (VMAT-2), the vesicular histamine transporter, resulting in decreased histamine accumulation in the secretory vesicles.
Gastrin is a major trophic factor for the ECL cell. Fasting has been shown to decrease plasma concentrations of gastrin in rodents (10, 20, 25) and in humans (14). Several groups have postulated that reduced gastrin levels lead to reduced HDC mRNA levels in ECL cells and hence reduction of histamine biosynthesis (6, 9, 10, 25). The possibility that histamine biosynthesis is attenuated by a decrease in the L-histidine content of ECL cells, thus depleting them of the substrate for HDC, has not yet been investigated. Moreover, ECL-specific levels of mRNA of HDC and other proteins involved in histamine synthesis and secretion or of other potential factors affecting ECL cell function during fasting have not been examined.
To determine the impact of fasting on factors modulating histamine production, transport, and secretion by the ECL cell and on the functional state of the cell, we obtained a highly enriched (>95%) population of ECL cells from stomachs of fasted and fed rats and measured ECL cell gene expression with whole rat genome expression microarrays and methods described previously (17). We identified changes in gene expression by this cell by comparing the transcriptome of purified ECL cell populations from normally fed and 24-h fasted rats to that of a mixed cell population of the whole fed or fasting gastric mucosa (subtractive hybridization). Expression of several key genes of histamine synthesis and secretion was then compared with that found in fed or fasting gastric mucosal homogenates, as measured by reverse transcriptase (RT)-quantitative polymerase chain reaction (qPCR), confocal microscopy, and Western blotting. Microscopy of cell populations isolated from fasting and fed stomachs also allowed analysis of any change in the morphology of the ECL cell population.
The results of these studies show that there are significant changes in the expression of genes encoding proteins modulating the ability of the ECL cell to produce, accumulate, or secrete histamine in the absence of changes in mRNA and protein content of HDC. There are also significant changes in expression of other genes that result in changes in ECL cell neuroendocrine properties, inducing an apparently relatively quiescent state. These findings result in further insight into the mechanisms underlying peripheral regulation of gastric acid secretion during different physiological states and could provide targets for drugs that alter gastric acid secretion via their impact on the ECL cell.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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ECL cell purification from rat stomach to 
95% purity.
ECL cell suspensions purified to 95% purity were prepared as described previously (17). Briefly, 10 stomachs were used to yield 120 ml of primary mucosal digested cell suspension in buffer A [in mM: 140 NaCl, 1.2 MgSO4, 1 CaCl2, 10 HEPES, and 11 glucose, with 0.5 g/l bovine serum albumin (BSA) and 300 mg/l DTT, pH 7.4]. The primary digest was subjected to Nycodenz gradient centrifugation before counterflow elutriation. Eight tubes of Nycodenz (Accudenz, Accurate Chemical & Scientific) gradients containing two layers of 14% and 7% Nycodenz (wt/vol) in buffer A were prepared and spun at 1,100 rpm for 8 min in a Sorvall centrifuge. Cells on top of the 14% layer were collected, washed twice, and resuspended in 8 ml of buffer A. This suspension was injected into a zonal rotor of an elutriator (Beckman) spinning at 1,500 rpm with a counterflow rate of 13 ml/min. Cell debris and small cells were washed out during an initial wash period of 5 min. The speed of the centrifuge was then reduced to 1,200 rpm with a concurrent gradual increase of flow rate to 21 ml/min. Approximately 100–150 ml was collected containing small- to medium-size cells. Cells were washed twice and concentrated to a total volume of 2 ml in buffer A. Acridine orange (100 nM) was added to the ECL suspensions, and the cells were sorted with the UCLA Flow Cytometry Core and the FACStar PLUS service. The excitation wavelength was 488 nM; sorting was achieved by gating cells showing emission of both 532 and 585 nM.
Cells were sorted for 4 h, pooled in buffer A without DTT containing 10 U of RNase inhibitor (Promega), and pelleted by centrifugation in a Sorvall centrifuge for 5 min at 1,100 rpm. The majority of cells were resuspended in 100–200 µl of RNAlater (Qiagen) to inhibit RNase activity. A small portion of cells was used for immunohistochemical assessment using an polyclonal antibody against HDC (ALPCO) and a polyclonal antibody against VMAT-2 (ABR) and for typical morphological ECL cell features such as vesicles loaded with acridine orange in stacked (585-nm emission) and nonstacked (532-nm emission) form (8). Purity was at least 95% (17).
cRNA labeling and rat expression oligonucleotide microarray hybridization.
Each transcriptome analysis was performed with "subtractive hybridization" on 44K whole rat genome expression oligonucleotide microarrays (Agilent Technologies) as described previously (17).
To obtain total RNA from mixed gastric mucosal samples, rat stomachs of each group (fasted or fed ad libitum; see above) were freshly resected and opened, and the oxyntic mucosa was scraped off the stomach wall with the sharp edge of a slide. The fragments were filtered through a nylon sieve in 1.5 ml of RNALater (Qiagen) and homogenized on ice. One hundred microliters of this homogenate was used directly to isolate total gastric mucosal RNA with a NucleoSpin RNA II Kit (BD Biosciences). The same kit was also used to isolate total RNA from 106 cells from the purified cell suspension (see above). The typical RNA concentration was 200–300 ng/µl. The RNA was assessed regarding purity and stability with a Bioanalyser 2100 (Agilent Technologies).
Fluorescently labeled cRNA was generated with a RT reaction with a poly d(T)-T7 promoter primer followed by T7 polymerase-based linear amplification in the presence of fluorophore-labeled nucleotides Cy3- or Cy5-CTP according to the manufacturer's protocol (Low RNA Input Fluor Linear Amp Kit, Agilent Technologies). The final cRNA concentration of typically 300–500 ng/µl and the Cy3- or Cy5-cytidine incorporation of 5–10 pmol/µg cRNA were determined with a Nanodrop spectrophotometer ND-1000 (Nanodrop Technologies). Labeled cRNA (3.5 µg) from whole gastric mucosal epithelium was combined with labeled cRNA from each purified cell suspension and hybridized to the 44K rat oligonucleotide expression array (Agilent Technologies) according to the manufacturer's protocol. Each set of three microarray experiments contained one dye swap experiment. All microarrays were scanned, and the intensities were normalized over background with a microarray scanner from Agilent Technologies including proprietary software. All microarray data sets (single channel) were imported into the microarray data analysis software Genespring 7.3 (Agilent Technologies), and normalized intensities were compared. The software is able to calculate the significance of difference between different sets of data [i.e., 3 sets of normal ECL cell preparations (ECL), 3 sets of ECL cells from fasted rats (ECLFas) compared with 5 sets of normal gastric mucosal homogenates (ST) and 6 sets of gastric mucosal homogenates from fasted animals (STFas)]. The microarray data were deposited for public access in the Gene Expression Omnibus (GEO) NCBI database with the series number GSE6204 with 17 sample data submissions (GSM143172, GSM143311-15, GSM143319-23 GSM160703-04, GSM160729-33, and GSM160737).
RT-real-time qPCR.
Total RNA from gastric mucosal fundic homogenates from fed and 24-h fasted rats were generated as described above for the generation of control cRNA probes for the microarray experiments. One microgram of total RNA was added to the RT reaction using Omniscript (Qiagen) RT and an oligo(dT)12–18 primer (Invitrogen) according to the manufacturer's protocol. Two microliters of RT product was then added to a real-time qPCR reaction together with the Dynamo SYBR Green qPCR Kit (Finnzymes) according to the manufacturer's protocol. Primers used were rat HDC: S-CTGTGGTTTGTGATTCGGTCCTTC, AS-GGATAGCCCATTGGTCAGGTCTTT; rat VMAT-2: S-CCAGCTCCTCACTAACCCATTCAT, AS-TCAGCAAGGTCGTTAGAGGTGTCC; rat Slc38a5 (SN2, SNAT5): S-ACCCTCACTGTGCCTGTTGTCCT, AS-CCAGGCAGAAGGGACTCAGGAA; rat urocanase: S-AACAGGTTGGCCCAGGAGAAGT, AS-GCAGAAGGCAGAACCGTCATAAAT; rat histidase: S-GTCTTAGAAGCCCACGGATTGAAA, AS-AGGGCTTTGGCTGGGTATTCAC; rat ZXDC zinc finger: S-GGTCGGATGAAGCACTGAACTCTG, AS-TGGGTGGTGTCTGAAGAAGTTGGT; rat Na-K-ATPase 1: S-TGGCCTATGGACAGATCGGTATGA, AS-AAGGGAGTAGGGGAAGGCACAGAA.
Real-time qPCR was performed in six well strips with a DNA Engine Opticon 2 unit (MJ Research). The cycle of threshold (CT) is determined as the fluorescent signal (binding of SYBR Green to double-stranded cDNA) of 1 SD over background. The efficiency of each primer pair was also measured by cloning of each PCR product into pCR-4-TOPO cloning vector (Invitrogen) and amplification with known amounts of cDNA starting material (10 pg–10 ng). The slope of the linear curve of CT vs. cDNA starting material is used to calculate the efficiency of the primer pair (efficiency = 10[–1/slope]).
Target primer pair amplifications were compared with reference primer pair amplifications (Na-K-ATPase 1-subunit) in the same experiment for each RT product tested. All reactions were carried out in duplicate, and eight separate amplifications for each primer pair were performed. The relative expression ratio of the target gene compared with the reference gene Na-K-ATPase 1-subunit was calculated according to Pfaffl (21), using the formula:
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SDS-polyacrylamide gel electrophoresis and Western blot analysis.
Total cellular protein for Western blot analysis was prepared from ECL cell-enriched suspensions after Nycodenz gradient separation and elutriation. The cells were resuspended in buffer containing (in mM) 10 PIPES, 2 EGTA, and 2 EDTA, pH 7.0, supplemented with one tablet of protease inhibitor cocktail tablets ("complete, EDTA-free," Roche Applied Science) and homogenized with a tight Dounce homogenizer (Wheaton, Millville, NY). The homogenate was centrifuged at 1,000 g for 5 min at 4°C. Gel samples were prepared by mixing 10 µg of total protein with 30 µl of gel sample buffer [4% SDS, 0.05% bromphenol blue (wt/vol), 20% glycerol, 1% ß-mercaptoethanol (vol/vol) in 0.1 M Tris buffer pH 6.8]. The samples (10 µg protein/lane) and unstained recombinant protein standards (Sigma) were loaded on a 4–12% SDS-polyacrylamide gel (NuPage 4–12% Bis-Tris, Invitrogen), and the gel was run in 50 mM MES buffer according to the manufacturer's instructions. After SDS-polyacrylamide gel electrophoresis, proteins were transferred by electrophoresis to nitrocellulose membranes (BioPlot-NC, Costar, Cambridge, MA) for 6 h at 4°C. These membranes were washed in distilled water, and the immobilized protein was stained in 0.2% Ponceau protein stain in 3% TCA solution for 10 min. After an image was taken, membranes were washed twice with Tris-buffered saline [TBS; 10 mM Tris, 150 mM NaCl, 0.05% Tween (vol/vol)] and incubated in TBS containing 5% milk powder (wt/vol). After 30 min, the membranes were incubated in the primary antibody solution [polyclonal antibody against VMAT-2 (1:1,000, ABR), polyclonal antibody against HDC (1:1,000, ALPCO), and monoclonal antibody against Na-K-ATPase 1 (1:1,000, Upstate)]. After 1 h, membranes were washed twice with TBS and incubated with the secondary antibody solution [anti-mouse or anti-rabbit IgG conjugated to alkaline phosphatase (Promega, Madison, WI), diluted 1:4,000 in TBS]. After 1 h, the membranes were washed twice and incubated for 15 min in TBS. After a final wash, the membranes were incubated for 10 min in AP buffer (in mM: 100 Tris, 100 NaCl, 5 MgCl2, pH 9.5) containing 0.3% nitro blue tetrazolium solution (vol/vol) and 0.15% 5-bromo-4-chloro-3-indolyl-1-phosphate solution (vol/vol) according to the manufacturer's instructions (Promega).
Confocal microscopy for ECL cell identification.
Cells from ECL cell-enriched cell suspensions isolated from fasting and fed stomachs were fixed on slides with 100% methanol for 10 min at –20°C. The slides were washed twice in TBS containing 0.2% Tween 20 (TBS-Tween) and incubated for 30 min at room temperature in TBS-Tween containing 10 mg/ml BSA (TBS-Tween-BSA) with 0.3% Triton X-100 added for permeabilization of the cell membranes. Slides were then washed twice in TBS-Tween and incubated for 1 h at room temperature with primary antibody solution [polyclonal antibody against VMAT-2 (1:100, ABR) and polyclonal antibody against HDC (1:100, ALPCO)], 100 times diluted in TBS-Tween-BSA. Then the slides were washed twice in TBS-Tween and incubated in TBS-Tween-BSA containing a 100-fold dilution of the secondary antibody against rabbit IgG Fc region conjugated with rhodamine. The slides were washed for 15 min in TBS-Tween and mounted in 70% glycerol in PBS. Slides were placed in a confocal microscope (Zeiss LSM510) with a x20 objective, an excitation of 543 nm, and an LP560 long-pass filter to detect rhodamine-specific emission. Total cells in the same field were visualized by maximal opening of the pinhole and maximal photomultiplier tube (PMT) amplification. The VMAT- and HDC-containing cells were counted.
Total histamine determination on whole gastric mucosal homogenates of 24-h fasted and ad libitum-fed rats.
Gastric mucosal homogenates were centrifuged at 10,000 rpm in an Eppendorf centrifuge. This separates membranes, nuclei, and mitochondria from the soluble fraction of the homogenate. The total protein concentration of this supernatant was used to normalize the samples used for total histamine determination with an LDN Histamine Food EIA (Labor Diagnostika Nord). Briefly, 100 µl of serial diluted supernatant was acetylated together with controls and standards provided by the kit according to the manufacturer's instructions. Acetylated histamine was quantified with a specific monoclonal antibody against acetylated histamine in an ELISA format. The final total protein concentration of each acetylated sample of supernatant from fed or fasted mucosal homogenate in the assay was 15, 1.5, or 150 pg. The ELISA is provided as microtiter assay strips, and the microplate is read after addition of substrate in a plate reader at 450 nm according to the manufacturer's instructions. A standard curve was generated with the extinction values at 450 nm and GraphPad Prism 4 software (GraphPad Software) of standards with known histamine concentrations according to the manufacturer, and sample histamine concentrations were determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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20%, Fig. 1, A and C). However, after FACS sorting, there was an 50% reduction in the number of ECL cells from the fasting animals (7.24 ± 2.85% ECL of total cells) compared with the fed animals (15.1 ± 2.39% ECL of total cells). This decrease is due to inefficient sorting of ECL cells during FACS using the acridine orange signal in the acidic secretory vesicles. The signal is reduced and shifted to a longer wavelength because of a significant reduction in size and increased acidity of ECL cell vesicles. The latter is presumably due to a decrease in proton efflux via VMAT-2 resulting also in a decreased accumulation of histamine (Fig. 2). However, the resulting cell suspension showed >95% ECL cell purity as shown by the typical morphology of ECL cells by acridine orange uptake (Fig. 2) and by staining of the cell suspensions with an anti-HDC antibody (data not shown).
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ECL transcriptome under fasting conditions.
We compared ECL cell-specific transcriptomes of fasted rats to those of rats with access to food ad libitum by subtractive hybridization of labeled cRNA-derived probes to the whole rat genome microarray covering 98% of the rat genome (44K spotted oligonucleotides) (17). Gene expression values of the purified ECL cells from fed and fasted animals were compared with each other, using normalization of the data with the mean of gene expression of ß-actin and GAPDH (change in gene expression).
The data were also subtracted from the expression in gastric fundic mucosal cell suspensions from fed and fasted animals, respectively (ratio). Thus we reconfirmed significant gene expression of most of the ECL cell-specific genes, which had been identified previously (17). This experimental design enables measurement of the quantity of message for each gene independently of the total number of ECL cells isolated after FACS since the same number of purified ECL cells is used to generate equal amounts of total RNA and cRNA probes.
That the FACS-sorted cells from 24-h fasted rats are indeed highly purified ECL cells, albeit with a reduction in total number, is shown by the finding that HDC gene expression was not significantly changed (normalized intensity of 237,369 ± 23,347 in purified fasting ECL cells and 26,007 ± 6,734 in fasting gastric fundic mucosa) compared with fed rats (normalized intensity of 231,994 ± 9,857 in purified ECL cells and 26,912 ± 11,770 in gastric fundic mucosa). Furthermore, expression of one of the transcriptional regulators of the HDC promoter, gut-enriched Krüppel-like factor (Klf4) (1), was unchanged in the fasting sample as shown by the normalized intensity of 134,393 ± 18,454 in purified ECL cells from fed rats vs. 120,171 ± 33,123 after 24-h fasting. The unchanged steady-state expression of the HDC gene and one of its major transcriptional regulators in purified ECL cells after fasting compared with fed animals indicates regulation of histamine content in the ECL cells themselves by a pathway other than downregulation of HDC transcription.
Comparison of gene expression from purified ECL cells from 24-h fasted to normally fed rats showed 18 genes that were significantly induced (Table 1). Of these, seven genes appear to be uniquely expressed in the fasted ECL cell (ratio >7), while the remainder, including urocanase and histidase, appear to be expressed also in cells other than the ECL cell, anticipating a purification factor of >7 in the purified ECL cell sample. These genes are involved in reduction of L-histidine content, transcriptional regulation inducing cell quiescence, change in cell shape and inhibition of migration, induction of lipolysis, and inhibition of the mitogen-activated kinase pathway in the ECL cells from fasted animals.
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Gene expression of proteins regulating ECL cell histamine content during fasting: microarray analysis.
Histamine content in ECL cells is regulated by histamine synthesis from L-histidine and by transport from cytosolic histamine into the secretory vesicle of the cell. Since HDC gene expression in the purified ECL cells in 24-h fasted rats was unchanged, we examined the possibility that cytosolic L-histidine availability was decreased during fasting. The microarray data clearly showed significant upregulation of two rate-limiting enzymes of L-histidine degradation, histidase that converts L-histidine to urocanic acid (24) and urocanase that converts urocanic acid to 4-imidazolone-5-propionic acid (22), during fasting. Moreover, gene expression of the sodium-coupled neutral amino acid transporter SN2 (SNAT5), facilitating L-histidine uptake into the cell (19), was significantly downregulated during fasting.
Gene expression for VMAT-2, the transport protein responsible for histamine accumulation into the secretory vesicle (7), was also significantly decreased in fasting rats. This corresponds to the observed decrease in size and numbers of secretory vesicles and increased acidity of the remaining vesicular spaces in purified ECL cells from fasting animals with high-magnification confocal microscopy and acridine orange as a pH-sensitive probe (Fig. 2).
It is of interest that the urocanase gene on the rat chromosome 4 (q34) is immediately followed by a transcription factor, ZXDC, whose gene expression is repressed during fed conditions but significantly induced during fasting. This factor contains a transcriptional repressor domain (SFP1) regulating G2/M transition in addition to a DNA/RNA binding zinc finger domain, indicating control of cell cycle in addition to transcriptional activity (2). Its exact function remains to be elucidated, but it is presumably involved in regulation of gene expression in ECL cells during fasting.
Gene expression of proteins regulating ECL cell histamine content during fasting: RT-qPCR analysis of gastric fundic mucosal samples.
As identified in the microarray experiment, we confirmed downregulation of gene expression of VMAT-2, responsible for histamine uptake in secretory vesicles, and Slc38a5, responsible for L-histidine uptake into ECL cells. In addition, histidase and urocanase gene expression were significantly upregulated (Table 3 and Fig. 3). We also confirmed the significant expression of the zinc finger transcription factor ZXDC, whose gene locus is placed immediately following the urocanase gene, suggesting cotranscriptional activity in this area of the genome.
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This comparison emphasizes the importance of the measurement of gene expression in the purified cell samples to enable conclusions as to the selective changes in ECL cell gene expression resulting from the different states of the cell during fasting and feeding.
Confirmation of decreased VMAT-2 protein levels compared with steady-state HDC protein levels in ECL cell-enriched suspensions before FACS of fasted vs. normally fed animals.
We observed a significant decrease in size and numbers of secretory vesicles and increased acidity of the remaining vesicular spaces in purified ECL cells from fasting animals (Fig. 2), which could be due to the decreased expression of VMAT-2 as observed in the analysis of the transcriptome. We tested the content of both HDC- and VMAT-2-positive cells in ECL cell-enriched suspensions before FACS sorting (Fig. 1). The analysis showed a significantly decreased ratio of VMAT-2-positive cells to all cells of the suspension in the fasting sample compared with the normally fed sample, with, however, equal ratios of HDC-positive cells in both samples. This result was confirmed by Western blot analysis with antibodies against VMAT-2, HDC, and Na-K-ATPase 1-subunit (normalization), showing a significant decrease of VMAT-2 protein (P < 0.001, Fig. 4) in protein homogenates of ECL cell-enriched suspensions before FACS from fasted compared with fed animals. In contrast, HDC protein expression was slightly, but not significantly, decreased. However, the well-known proteolytic processing of full-length HDC was significantly increased during 24-h fasting, resulting in a significant decrease of the intensity of the 74-kDa band. This is in good agreement with previously published results showing stabilization of constructs containing the NH2- and COOH-terminal PEST sequences, whose proteolytic processing is significantly inhibited by gastrin, leading to stabilization of these proteins (12).
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DISCUSSION |
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In contrast, the data presented here clearly show that ECL cell-specific expression of HDC and one of its major transcriptional activators, Klf4 (1), was not changed during the physiological condition of 24-h fasting. Instead, the microarray analysis identified significant changes in the expression of several other genes contributing to the significantly decreased histamine content in the cells noted with fasting. Thus genes involved in accumulation of histamine in secretory vesicles and transport of the HDC substrate L-histidine into the cell are downregulated, while those involved in degradation of L-histidine are upregulated. The net result of these effects is less total histamine synthesis in the fasting ECL cell and decreased release of the hormone. These findings were confirmed by RT-qPCR of a gastric fundic mucosal sample.
Analysis of VMAT-2 and HDC protein content on Western blots of ECL cell-enriched cell suspensions showed significantly decreased protein levels of VMAT-2 but not HDC after 24-h fasting. However, the proteolytic processing of the full-length 74-kDa form of HDC was significantly decreased in this condition. Expression studies of HDC constructs containing the NH2- or COOH-terminal PEST domains show stabilization of these proteins in the presence of gastrin and degradation in the absence of the ligand (12). Also, a recently published study identified a gastrin response element in the VMAT-2 promoter region (4). Whether HDC is tightly associated with VMAT-2 in a complex at the exocytotic vesicular membrane in gastric ECL cells resulting in HDC full-length stabilization by blocking the proteolytic PEST sequences in this enzyme remains to be elucidated.
Hence fasting for only 24 h alters ECL cell gene and protein expression to decrease its releasable histamine content, so the cell is less able to stimulate gastric acid secretion. This change in the ECL cell could be an important protective mechanism, because continual release of large quantities of histamine could lead to high gastric acid output under fasting conditions and potentially exacerbate gastric or esophageal mucosal injury (16). Figure 5 illustrates a hypothetical model showing how net histamine synthesis and transport into secretory vesicles is decreased with fasting.
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In summary, we report a detailed analysis of gene and protein expression of key proteins involved in regulating ECL cell histamine content in response to 24 h of fasting. This study provides an explanation for previously observed discrepancies in the effect of short-term gastrin on HDC gene transcription compared with HDC translation and/or activity (6, 25) as well as insight into the regulation of ECL cell histamine content during short-time gastrin reduction during fasting. It appears that during this physiological manipulation the reduced demand for ECL cell release of histamine is achieved by increased L-histidine substrate depletion resulting in decreased HDC activity, despite constant HDC gene and protein expression. In addition, there is decreased histamine accumulation in exocytotic vesicles due to decreased VMAT-2 in the cells.
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
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