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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Effect of Omeprazole on Gastric Adenosine A₁ and A_2A Receptor Gene Expression and Function

Department of Physiology, University of British Columbia, Vancouver, British Columbia, Canada

Received for publication April 7, 2004
Accepted May 20, 2004.

	Abstract

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Adenosine has been shown to inhibit immunoreactive gastrin (IRG) release and to stimulate somatostatin-like immunoreactivity (SLI) release by activating adenosine A₁ and A_2A receptors, respectively. Since the synthesis and release of gastrin and somatostatin are regulated by the acid secretory state of the stomach, the effect of achlorhydria on A₁ and A_2A receptor gene expression and function was examined. Omeprazole-induced achlorhydria was shown to suppress A₁ and A_2A receptor gene expression in the antrum and corporeal mucosa, but not in the corporeal muscle. Omeprazole treatment produced reciprocal changes in A₁ receptor and gastrin gene expression, and parallel changes in A_2A receptor and somatostatin gene expression. The localization of A₁ and A_2A receptors on gastrinsecreting G-cells and somatostatin-secreting D-cells, respectively, suggests that changes in adenosine receptor expression may modulate the synthesis and release of gastrin and somatostatin. Thus, the effect of omeprazole on adenosine receptor-mediated changes in IRG and SLI release was also examined in the vascularly perfused rat stomach. After omeprazole treatment, the A₁ receptor-mediated inhibition of IRG and SLI release induced by N⁶-cyclopentyladenosine (A₁ receptor-selective agonist) was not altered, but the A_2A receptor-mediated augmentation of SLI release induced by 2-p-(2-carboxyethyl-)phenethylamino-5'-N-ethylcarboxamidoadenosine (A_2A-selective agonist) was significantly attenuated. These findings agree well with the corresponding omeprazole-induced decrease in antral A_2A receptor mRNA expression. Overall, the present study suggests that adenosine receptor gene expression and function may be altered by omeprazole treatment. Acid-dependent changes in adenosine receptor expression may represent a novel purinergic regulatory feedback mechanism in controlling gastric acid secretion.

Adenosine elicits its actions by activating specific G-protein-coupled receptors that are classified into the A₁, A_2A, A_2B, and A₃ receptor subtypes (Fredholm et al., 2001). The inhibitory and stimulatory effects of adenosine on IRG and SLI release are likely mediated by the activation of A₁ and A_2A receptors, respectively (Schepp et al., 1990; Yip and Kwok, 2004; Yip et al., 2004b). Furthermore, the basal release of SLI is likely to be under the control of the A_2A receptors since ZM 241385 (A_2A receptor-selective antagonist) was shown to suppress basal SLI release (Yip and Kwok, 2004). The A₁ and A_2A receptors were shown to be expressed throughout the antral and corporeal mucosa, and in the enteric plexi (Yip and Kwok, 2004; Yip et al., 2004b). The cellular localization of A₁ receptors on gastrin-containing G-cells and A_2A receptors on somatostatin-containing D-cells further suggests that adenosine may act directly to alter the release of IRG and SLI. Although adenosine A₁ and A_2A receptors may regulate gastric acid secretion by modulating gastrin and somatostatin release, it has not been determined whether the gene expression of these receptors could be altered by changes in the acid-secretory state of the stomach. Our laboratory has demonstrated that fasting, a condition that increases gastric acidity (Matsumoto et al., 1989), up-regulated gastric A₁ and A_2A receptor gene expression and altered gastrin and somatostatin gene expression (Yip and Kwok, 2002). The proton pump inhibitor, omeprazole, has also been demonstrated to alter gastrin and somatostatin gene expression (Brand and Stone, 1988; Wu et al., 1990; Sandvik et al., 1995). However, it is unclear whether omeprazole-induced achlorhydria also alters adenosine receptor gene expression. The objective of the present study was to examine whether short-term omeprazole treatment alters adenosine A₁ and A_2A receptor gene expression and function. The gene expression of gastrin and somatostatin was also measured since changes in adenosine receptor expression may be associated with changes in gastrin and somatostatin gene expression and release. To examine whether omeprazole-induced changes in adenosine receptor gene expression also result in corresponding changes in adenosine receptor-mediated SLI and IRG release, the effect of selective adenosine agonists on SLI and IRG release were also studied following omeprazole treatment.

	Materials and Methods

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Animals were treated in accordance with the guidelines of the University of British Columbia Committee on Animal Care.

Omeprazole Treatment
Male Wistar rats (250–270 g) housed in light- and temperature-controlled rooms with free access to food and water were used. As suggested by the manufacturer, omeprazole (50 µmol/ml; AstraZeneca, Mölndal, Sweden) was thoroughly dispersed in 0.25% Methocel (Dow Corning, Midland, MI) containing 2 mg/ml NaHCO₃ (pH 9.0) using the Tissumizer tissue homogenizer (Tekmar-Dohrmann, Mason, OH). The drug was aliquoted, frozen, and stored at –20°C. To prevent drug degradation due to repeated freezing and thawing, a new aliquot of drug was thawed overnight at 4°C and brought to room temperature before administration. Test groups were treated by gavage with a dose of 400 µmol/kg omeprazole once daily between 9:00 and 10:00 AM for either 1 or 3 days. These regimens were selected since gastric acid secretion is decreased by up to 80% after 1 day of treatment with 400 µmol/kg omeprazole (Larsson et al., 1988; Lee et al., 1992), and steady-state inhibition is achieved after 3 days of treatment at this dose (Carlsson et al., 1986). Rats were not treated for more than 3 days since G-cell and/or D-cell densities are altered after 4 days (Bolkent and Yilmazer, 1997) and 5 days (Pawlikowski et al., 1992) of treatment. Control groups were treated similarly with the vehicle. Animals were anesthetized with an i.p. injection of 60 mg/kg sodium pentobarbital (Somnotol; MTC Pharmaceuticals, Cambridge, ON, Canada), 24 h after the last treatment, and tissue extraction occurred between 10:00 and 11:00 AM.

Quantification of Adenosine A₁ and A_2A Receptor and Gastric Peptide Gene Expression
The quantitative real-time RT-PCR developed to measure A₁ and A_2A receptor gene expression has previously been described (Yip and Kwok, 2004; Yip et al., 2004b). Two-step real-time RT-PCR assays were used to quantify the gene expression of adenosine A₁ and A_2A receptors, gastrin, and somatostatin in various gastric regions following omeprazole treatment. Primers and probes were designed using the Primer Express Sequence Design software program (v. 1.0; Applied Biosystems, Foster City, CA). The reporter dye, 6-carboxyfluorescein, and the quencher dye, 6-carboxytetramethylrhodamine, were linked to the 5' and 3' ends of the probes, respectively. The sequences of the forward primers, reverse primers, and probes used are listed in Table 1. The primers and probes were synthesized by the Nucleic Acid Protein Services Unit (University of British Columbia) and Synthegen, LLC (Houston, TX), respectively.

A₁ and A_2A Receptor Gene Expression. RNA transcripts expressing the entire coding region of the A₁ and A_2A receptors were used as the standards for real-time RT-PCR. The standards were synthesized from plasmids containing the A₁ or A_2A receptor transcript by in vitro transcription using the Riboprobe in vitro transcription kit and T7 RNA polymerase (Promega, Madison, WI), as previously described (Yip and Kwok, 2004; Yip et al., 2004b). Adenosine receptor RNA standards were treated with DNase I (Amersham Biosciences Inc., Piscataway, NJ) and purified. Standard concentrations were determined using the RiboGreen Reagent Quantitation kit (Molecular Probes, Eugene, OR), the FL600 Microplate Fluorescence reader (Bio-Tek Instruments, Winooski, VT), and the KC4 Kineticalc Software (version 2.6; Bio-Tek Instruments), according to the manufacturer's instructions. RNA standards were serially diluted to 1 x 10³ to 1 x 10¹² copies/µl in RNase-free water, aliquoted, stored at –80°C, and thawed only once before use.

Gastrin and Somatostatin Gene Expression. Relative gastrin and somatostatin gene expression levels were measured using a sample of rat antrum total RNA to produce the standards for the standard curve. Antral total RNA (1 µg) was reverse transcribed into 10 µl of cDNA using SuperScript II RNase H-Reverse Transcriptase. This sample was designated as the 100 ng/µl sample since 100 ng of total RNA was reverse transcribed to produce 1 µl of cDNA. This sample was then serially diluted to various concentrations (0.1 ng/µl to 100 ng/µl) to produce a relative standard curve.

Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) and 18S rRNA Gene Expression. The expression of GAPDH mRNA and 18S rRNA was measured using the Rodent GAPDH Control Reagents Kit (Applied Biosystems) and the TaqMan ribosomal RNA Control Reagents Kit (Applied Biosystems), respectively, according to the protocol described in the PE Applied Biosystems User Bulletin 2. A relative standard curve was constructed using the Rodent Control RNA Standard (50 ng/µl) provided with the kit. Rodent Control RNA (100 ng) was reverse transcribed into 10 µl of cDNA using SuperScript II RNase H-Reverse Transcriptase. This sample was designated as the 10 ng/µl sample since 10 ng of Rodent Control RNA was used to produce 1 µl of cDNA. The 10 ng/µl sample was diluted to 7.5, 5, 2.5, 1, 0.5, and 0.1 ng/µl, and these samples were used to construct the relative standard curve for the real-time RT-PCR assay. The 18S rRNA and GAPDH real-time RT-PCR assays were performed according to the manufacturer's instruction.

Tissue and Total RNA Extraction
The corporeal mucosa, corporeal muscle, and antrum were dissected out. These regions were examined since the corporeal mucosa contains somatostatin-secreting D-cells, the corporeal muscle contains somatostatin-releasing nerve fibers, and the antrum contains D-cells, gastrin-secreting G-cells, and somatostatin-releasing nerve fibers. Care was taken to avoid inclusion of tissue at the antro-corpus border. Since the antrum is a relatively small tissue, total RNA was extracted from the whole antrum to ensure consistency among samples. The corpus was separated into the corporeal mucosa and muscle. The whole corpus tissue was rinsed in ice-cold saline, and the mucosa was removed from the corpus with gentle scraping using a sterile glass slide. The mucosa was then rinsed off the slide using TRIzol reagent (Invitrogen, Carlsbad, CA), and total RNA was extracted immediately using the same reagent. The corporeal muscle and antrum were flash frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNA was extracted from all tissues using TRIzol reagent, according to the manufacturer's instructions, and concentrations were determined using the RiboGreen Quantitation Kit (Molecular Probes). Total RNA samples were treated with DNase I to remove any residual DNA contamination that may remain in samples after total RNA extraction. DNase I treatment was performed at room temperature in 1x first strand buffer [50 mM Tris-HCl (pH 8.3 at 25°C), 75 mM KCl, 3 mM MgCl₂] containing 1 U DNase I/µg total RNA. The reaction was allowed to proceed for 15 min, before 1 µl of 25 mM EDTA was added to stop the reaction. Samples were then incubated for an additional 10 min at 65°C.

Two Step Real-Time RT-PCR
Reverse Transcription. DNase I-treated tissue RNA (1 µg) was reverse transcribed in a total volume of 10 µl containing 200 ng of random hexamers, 20 U of RNAguard RNase inhibitor (Amersham Biosciences Inc.), 1x first strand buffer, 10 mM dithiothreitol, 0.5 mM deoxynucleoside-5'-triphosphate mix, and 100 U of SuperScript II RNase H-Reverse Transcriptase. At least six concentrations of each standard and a sample containing DNase I-treated RNase-free water in place of the template (negative control) were reverse transcribed simultaneously. The reverse transcribed RNA standards were used to construct the standard curve for the real-time RT-PCR assay.

PCR. Each assay consisted of at least six standard curve samples, a negative control sample, and unknown samples. All reactions were performed in triplicate. The PCR reaction mixture (25 µl) consisted of 1x TaqMan buffer A; 200 µM concentrations, each, of dATP, dCTP, and dGTP; 400 µM dUTP; 0.01 U/µl AmpErase uracil-N-glycosylase; and 0.025 U/µl AmpliTaq Gold DNA polymerase from the TaqMan PCR Core Kit (Applied Biosystems). Reaction mixtures also contained 0.5 µl of tissue cDNA, standard cDNA, or negative control; 100 nM probe; 100 nM (A_2A receptor, gastrin and somatostatin assay) or 300 nM (A₁ assay) concentrations, each, of the forward and reverse primers; and 7.5 mM MgCl₂ (A₁ receptor, gastrin, and somatostatin assay) or 4.5 mM MgCl₂ (A_2A receptor assay). The reaction was performed using the ABI Prism 7700 Sequence Detector (Applied Biosystems) with the following cycling parameters: 2-min hold at 50°C for uracil-N-glycosylase incubation, 10-min hold at 95°C for AmpliTaq Gold activation, followed by 40 cycles of amplification consisting of a 15-s denaturation step at 95°C and 1-min anneal/extend period at 60°C.

Data Collection and Analysis. Data were collected during each PCR cycle and analyzed using the Sequence Detection Software (v. 1.6.3; Applied Biosystems). An amplification plot showing normalized reporter emissions versus cycle number was generated. The threshold cycle (C_T), the cycle during which an increase in fluorescence is associated with exponential growth, was determined by the software using the fluorescence emitted during the first 15 cycles. A standard curve of C_T versus log (initial standard concentrations) was then generated. The initial concentration of each unknown sample was determined by interpolation using the C_T value determined by the assay. The correlation coefficient of each standard curve was >0.95, and the C_T of the negative control exceeded 40 cycles in every assay, indicating the absence of DNA contamination. To compare gene expression levels between the test and control animals, measurements were first normalized with the gene expression of the endogenous control (18S rRNA). This was performed according to the procedures described in the PE Applied Biosystems User Bulletin 2. The adenosine A₁ receptor, A_2A receptor, gastrin, or somatostatin gene expression level of each individual sample was divided by the 18S rRNA level to obtain a normalized value of measurement. The mean ± S.E.M. of this normalized value was calculated for control and omeprazole-treated groups. The mean of each control group was expressed as 100%, and gene expression levels of the omeprazole-treated groups were expressed as a percentage of the control. Statistical significance was determined using the two-tailed unpaired Student's t test and was performed using GraphPad Prism (v. 3.0; GraphPad Inc., San Diego, CA); P 0.05 was considered significant.

Stomach Perfusion
Rats were housed in light- and temperature-controlled rooms with free access to food and water. Omeprazole-treated animals were deprived of food for 12 to 14 h but had free access to water before stomach perfusion. Rats were anesthetized with Somnotol (60 mg/kg). The procedures used to prepare the stomach for perfusion have previously been described (Kwok et al., 1990). Following the exposure of the stomach through an abdominal midline incision, the superior mesenteric artery and vasculatures supplying the left and right adrenal glands and kidneys were occluded or cut between double ligatures. The pancreas and spleen were then dissected along the greater curvature of the stomach, while preserving the right gastroepiploic artery. A cannula was secured into the gastroduodenal junction for drainage of any gastric contents. The spleen, pancreas, and small and large intestines were then dissected out. Arterial perfusion was achieved through a cannula inserted into the aorta with the tip lying adjacent to the celiac artery. An injection of 2 ml of saline containing 600 U of heparin (Sigma-Aldrich, St. Louis, MO) was introduced into the gastric circulation through this arterial cannula, followed by perfusate. Venous effluent was collected via the portal vein cannula. The preparation was equilibrated for 30 min before 5-min samples were collected into ice-cold scintillation vials containing 0.3 ml of Trasylol (aprotinin; 10,000 KIU/ml; Miles Laboratories, Etobicoke, ON, Canada). Aliquots (0.5 ml) of samples were immediately transferred into ice-cold test-tubes containing 0.05 ml of aprotinin and stored at –20°C until assayed. The stomach was perfused at a rate of 3 ml/min using a peristaltic pump (Cole-Parmer Instrument Co., Vernon Hills, IL). The perfusate was composed of Krebs' solution (120 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄·7H₂O, 1.5 mM KH₂PO₄, 25 mM NaHCO₃, and 5.1 mM dextrose) containing 0.2% bovine serum albumin (RIA grade; Sigma-Aldrich) and 3% dextran (Clinical grade; Sigma-Aldrich). The perfusate was continuously gassed with a mixture of 95% O₂ and 5% CO₂ to maintain a pH of 7.4. Both the perfusate and the preparation were kept at 37°C by thermostatically controlled heating units throughout the experiment. Drugs were introduced into the perfusate via side-arm infusion at a rate calculated to give the final perfusion concentrations. N⁶-Cyclopentyladenosine (CPA) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine HCl (CGS 21680) were purchased from Sigma-Aldrich. Drugs were first dissolved in a small volume of DMSO (BDH, Toronto, ON, Canada) and subsequently diluted with saline or perfusate to 0.03 or 0.5% DMSO before perfusing into the stomach. At these concentrations, DMSO did not alter basal IRG or SLI release. The perfusion concentrations of the A₁ receptor agonist, CPA, and A_2A receptor agonist, CGS 21680, were 0.1 or 1 µM. The lower concentration (0.1 µM) was selected to approximate the EC₅₀ of CPA (0.067 µM with a 95% confidence interval between 0.014 and 0.325 µM) (Yip et al., 2004b) and CGS 21680 (0.06 µM with a 95% confidence interval between 0.02 and 0.17 µM) (Yip and Kwok, 2004) in inhibiting and stimulating IRG and SLI release, respectively. The higher concentration (1 µM) was also examined since CPA and CGS 21680 elicited their maximal effects at this concentration.

Measurement of Gastric Peptides and Data Analysis
The specific RIA used for the measurement of IRG (Jaffe and Walsh, 1978; Fujimiya and Kwok, 1997) and SLI (McIntosh et al., 1987; Kwok et al., 1990) have previously been described. The gastrin antibody (PM1) used to measure IRG was kindly provided by Dr. R. Pederson (Department of Physiology, University of British Columbia). The inter- and intra-assay variation was less than 6 and 4%, respectively. The monoclonal antibody, SOMA-3 (Medical Research Council-Regulatory Peptide Group, University of British Columbia), was used to measure SLI. The inter- and intra-assay variation of the RIA was less than 12 and 8%, respectively. The drugs used in the present study did not cross-react with these antibodies.

Although the basal release rate of IRG and SLI release varied among animals, previous experiments have demonstrated that basal IRG (Pederson et al., 1984; Kwok et al., 1990) and SLI release (Kwok et al., 1990) were maintained in the perfused stomach. Therefore, results were expressed as mean ± S.E.M. of IRG or SLI release (percentage), which was calculated as follows: [release (pg/min) during a 5-min period ÷ release (pg/min) during period 1] x 100. Results were also expressed as percentage inhibition of IRG release, which was calculated as follows: [mean basal IRG release (periods 1–3) – mean IRG release in the presence of drug (periods 4–7)] pg/min ÷ [mean basal IRG release (periods 1–3)] pg/min x 100. The percentage change of SLI release was calculated as follows: (mean SLI release in the presence of drug – mean basal SLI release) pg/min ÷ (mean basal SLI release) pg/min x 100. Statistical significance (P < 0.05) was determined using one-way ANOVA followed by Dunnett's multiple comparison test, and the paired or unpaired Student's t test when appropriate.

	Results

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Effect of Omeprazole Treatment on 18S rRNA and GAPDH Gene Expression. Omeprazole treatment was found to alter GAPDH mRNA levels in various gastric regions. The gene expression of GAPDH in the corporeal mucosa was significantly decreased to 65 ± 5% of control levels after 3 days of omeprazole treatment. In the corporeal muscle, the GAPDH mRNA level was significantly increased to 130 ± 9% after 1 day of treatment (Fig. 1A). In all gastric regions examined, 18S rRNA levels were not altered by 1 or 3 days of omeprazole treatment (Fig. 1B). Since omeprazole treatment did not alter 18S rRNA levels, this gene was used as the endogenous control for the subsequent quantification of gastric A₁ receptor, A_2A receptor, somatostatin, and gastrin gene expression using real-time RT-PCR.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Effect of omeprazole treatment on gastric housekeeping gene expression. GAPDH mRNA (A) and 18S rRNA (B) levels were measured in rats treated with vehicle or omeprazole (OM) for 1 or 3 days; n 7. Results are expressed as a percentage of the control as described under Materials and Methods; , P < 0.05.

Effect of Omeprazole Treatment on Adenosine A₁ and A_2A Receptor Gene Expression. Figure 2A shows that antral adenosine A₁ receptor gene expression was significantly reduced to 66 ± 12% and 47 ± 8% of control levels after 1 and 3 days of omeprazole treatment, respectively. In the corporeal mucosa, A₁ receptor gene expression was significantly decreased to 62 ± 6% of control levels after 1 day of treatment, but changes were not apparent after 3 days of treatment. The gene expression of the A_2A receptor was significantly reduced in the antrum to 57 ± 7% of control levels after 3 days of treatment (Fig. 2B). A_2A receptor gene expression was also decreased in the corporeal mucosa, to 67 ± 14% of control levels, after 1 day of treatment. However, changes were not detected in this tissue after 3 days of treatment. In the corporeal muscle, neither A₁ receptor nor A_2A receptor gene expression was altered by omeprazole treatment.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of omeprazole treatment on adenosine A1 and A2A receptor gene expression. A1 receptor (A) and A2A receptor (B) mRNA levels were measured in rats treated with omeprazole (OM) for 1 or 3 days; n 7. Results are expressed as a percentage of the control as described under Materials and Methods; , P < 0.05; , P < 0.01; , P < 0.001.

Effect of Omeprazole Treatment on Gastrin and Somatostatin Gene Expression. Antral gastrin gene expression was significantly increased to 201 ± 18% and 268 ± 42% of controls after 1 and 3 days of omeprazole treatment, respectively (Fig. 3A). In contrast, after 1 and 3 days of omeprazole treatment, antral somatostatin gene expression was significantly reduced to 71 ± 6% and 58 ± 4% of controls, respectively. The somatostatin gene expression in the corporeal mucosa was also reduced to 54 ± 13% and 54 ± 10% of control levels after 1 and 3 days of treatment, respectively (Fig. 3B). In the corporeal muscle, a significant decrease (45 ± 6%) in somatostatin gene expression was observed following 3 days of omeprazole treatment.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of omeprazole treatment on gastric peptide gene expression. Gastrin (A) and somatostatin (B) mRNA levels were measured in rats treated with omeprazole (OM) for 1 or 3 days; n 7. Results are expressed as a percentage of the control, as described under Materials and Methods; , P < 0.05; , P < 0.01; , P < 0.001.

Effect of Omeprazole on CPA-Induced Changes in IRG Release. Previous studies have shown that adenosine A₁ receptors are involved in the inhibition of IRG release. Thus, the effect of omeprazole treatment on CPA (A₁-selective agonist)-induced changes in IRG release was examined. Figure 4A shows the effect of 0.1 µM CPA on IRG release in animals after 1-day treatment with omeprazole. Basal IRG release during periods 1 to 3 in control (222 ± 46 to 238 ± 52 pg/min) and treated (175 ± 19 to 198 ± 23 pg/min) animals was comparable. The administration of CPA (0.1 µM) resulted in a significant inhibition of basal IRG release in both control and treated animals. However, no significant difference in CPA-induced IRG release was observed between these two groups. Figure 4B shows the effect of 1 µM CPA on IRG release in controls and animals treated with omeprazole for 3 days. The basal release of IRG during periods 1 to 3 in control (203 ± 24 to 212 ± 29 pg/min) and treated (231 ± 36 to 251 ± 46 pg/min) animals was also comparable. The perfusion of CPA (1 µM) significantly inhibited IRG release. However, this response also did not differ between 3-day omeprazole-treated and control animals. For comparison, results are also expressed as percentage inhibitions and summarized in Fig. 5. No significant changes in the inhibitory effect of CPA (0.1 µM and 1 µM) on IRG release occurred between 1-day and 3-day omeprazole-treated and vehicle-treated control animals.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Effect of CPA on gastric IRG release in 1-day (A) and 3-day (B) omeprazole (OM)-treated animals. Results are expressed as IRG release (percentage) as described under Materials and Methods. Each column represents the mean ± S.E.M. , P < 0.05, and , P < 0.001 when compared with period 3 using repeated measures ANOVA followed by Dunnett's multiple comparison test (n 5). The IRG release during each 5-min period was not significantly different between omeprazole-treated and control animals when compared using the Student's unpaired t test.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effect of omeprazole (OM) treatment on CPA-induced changes in IRG release. The percentage inhibition of IRG release between omeprazole-treated animals and their respective controls was compared using the Student's unpaired t test and was found not to differ. Each column represents the mean ± S.E.M. (n 5).

Effect of Omeprazole on CPA- and CGS 21680-Induced Changes of SLI Release. Previous studies have demonstrated that adenosine A₁ and A_2A receptors are involved in the inhibition and augmentation of SLI release, respectively. Thus, the effect of omeprazole treatment on CPA and CGS 21680 (A_2A selective agonist)-induced changes in SLI release were examined. Figure 6 shows the effect of 1 µM CGS 21680 on SLI release in control and 3 day omeprazole-treated animals. The basal release of SLI during periods 1 to 3 in controls (152 ± 48 to 157 ± 51 pg/min) was not significantly different from that of treated animals (194 ± 45 to 197 ± 47 pg/min). In these experiments, SLI release was enhanced by CGS 21680 in both groups of animals. However, CGS 21680-induced SLI release was significantly attenuated by 3 days of omeprazole treatment. The percentage changes in SLI release induced by CGS 21680 (0.1 and 1 µM) in control and 1- and 3-day omeprazole-treated animals are summarized in Fig. 7. After 3 days of omeprazole treatment, CGS 21680-induced augmentation of SLI release was shown to be significantly reduced.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effect of CGS 21680 (CGS; 1 µM) on gastric SLI release in control (A) and 3-day omeprazole (OM)-treated (B) animals. Results are expressed as SLI release (percentage) as described under Materials and Methods. Each column represents the mean ± S.E.M. (n 5); , P < 0.001 when compared with period 3 using repeated measures ANOVA followed by Dunnett's multiple comparison test. The SLI release during each 5-min period was compared between omeprazole-treated and control rats using the Student's unpaired t test and found to differ significantly during periods 6, 7, and 8; , P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of omeprazole (OM) treatment on CGS 21680 (CGS)-induced changes in SLI release. The percentage change in SLI release between omeprazole-treated animals and their respective controls was compared using the Student's unpaired t test. Each column represents the mean ± S.E.M. (n 5); , P < 0.05.

Previous studies have demonstrated that the administration of 0.1 µM CPA significantly inhibited gastric SLI release, whereas the administration of 1 µM CPA significantly stimulated SLI release (Yip and Kwok, 2004). These findings were also observed in the current study (Figs. 8 and 9). Figure 8A shows the effect of 0.1 µM CPA on SLI release after 1 day of omeprazole treatment. Basal SLI release did not differ significantly between control (227 ± 33 to 234 ± 30 pg/min) and treated (194 ± 27 to 197 ± 21 pg/min) animals. The administration of 0.1 µM CPA significantly reduced SLI release. However, this inhibitory response did not differ between control and 1-day omeprazole-treated animals. Figure 8B shows the effect of 1 µM CPA on SLI release after 3 days of omeprazole treatment. Basal SLI release was comparable between control (158 ± 33 to 165 ± 37 pg/min) and treated (134 ± 26 and 138 ± 30 pg/min) animals. The administration of CPA (1 µM) significantly increased SLI release. This stimulatory response was shown to be attenuated after 3 days of omeprazole treatment. The effect of omeprazole treatment on CPA-induced changes in SLI release is summarized in Fig. 9. The inhibition of SLI release induced by 0.1 µM CPA was not altered by either 1 or 3 days of omeprazole treatment. However, 3 days of treatment did significantly attenuate the stimulatory effect of 1 µM CPA on SLI release (Fig. 9).

View larger version (22K): [in this window] [in a new window]   Fig. 8. Effect of CPA on gastric SLI release in 1-day (A) and 3-day (B) omeprazole (OM)-treated animals. Results are expressed as SLI release (percentage) as described under Materials and Methods. Each column represents the mean ± S.E.M.; , P < 0.05, and , P < 0.001 when compared with period 3 using repeated measures ANOVA followed by Dunnett's multiple comparison test (n 5). The SLI release during each 5-min period was compared between omeprazole-treated and control rats using the Student's unpaired t test. In 1-day treated rats perfused with 0.1 µM CPA (A), SLI release did not differ from controls. In 3-day treated rats perfused with 1 µM CPA (B), SLI release was found to differ significantly during periods 4 and 5; , P < 0.05, and , P < 0.01.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Effect of omeprazole (OM) treatment on CPA-induced changes in SLI release. The percentage change in SLI release between omeprazole-treated animals and their respective controls was compared using the Student's unpaired t test. Each column represents the mean ± S.E.M. (n 5); , P < 0.05.

	Discussion

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In the present study, the gene expression of GAPDH, a commonly used housekeeping gene, was shown to be significantly increased in the corporeal muscle and decreased in the corporeal mucosa after 1 and 3 days of omeprazole treatment, respectively. These findings are consistent with another study demonstrating increased GAPDH mRNA levels in the rat corpus after omeprazole treatment (Sandvik et al., 1995). In contrast, 18S rRNA gene expression was not altered by omeprazole treatment. Therefore, 18S rRNA was used as the endogenous control for the measurement of A₁ receptor, A_2A receptor, somatostatin, and gastrin gene expression. Results demonstrate that both gastrin and somatostatin mRNA levels were altered by omeprazole treatment. The observation that 1 and 3 days of omeprazole treatment led to increased gastrin mRNA levels agrees well with other studies showing increased expression after 4 and 14 days of omeprazole treatment (Wu et al., 1990; Dockray et al., 1991). The present study also demonstrates decreased antral somatostatin gene expression after 1 and 3 days of treatment, which also agrees well with the findings of other investigators (Brand and Stone, 1988; Wu et al., 1990; Sandvik et al., 1995). Similar to previous studies (Tari et al., 1991; Sandvik et al., 1995), the present study also demonstrates reduced somatostatin mRNA expression in the corporeal mucosa and muscle following omeprazole treatment.

We have previously demonstrated that adenosine may regulate gastric acid secretion by suppressing IRG and stimulating SLI release via activation of A₁ and A_2A receptors, respectively (Yip and Kwok, 2004; Yip et al., 2004b). The present study suggests that the gene expression and function of these receptors may be regulated by changes in the acid-secretory state of the stomach. Omeprazole treatment inhibited A₁ receptor gene expression in the antrum (1- and 3-day treatment) and corporeal mucosa (1-day treatment), and A_2A receptor gene expression in the antrum (3-day treatment) and corporeal mucosa (1-day treatment). The precise cellular localization of both receptors has been examined in these tissues (Yip and Kwok, 2004; Yip et al., 2004b). A₁ receptors were shown to be expressed on D-cells and G-cells, whereas A_2A receptors are expressed on D-cells. Thus, it is plausible that omeprazole-induced changes in adenosine receptor gene expression may occur in the mucosal G-cells and/or D-cells of the corpus and antrum. Both of these adenosine receptors have also been localized in the enteric plexi. However, changes in A₁ and A_2A receptor gene expression were not observed in the corporeal muscle after omeprazole treatment, suggesting that adenosine receptor gene expression in the corporeal enteric plexi was unlikely altered by omeprazole treatment.

Conditions that increase intracellular acidity, such as hypoxia or ischemia, have previously been shown to up-regulate A₁ and A_2A receptor gene expression in DDT1-MF2 cells (Nie et al., 1998) and PC12 cells (Kobayashi and Millhorn, 1999), respectively. In addition, fasting, which increases gastric acidity (Matsumoto et al., 1989), has been shown to up-regulate both gastric A₁ and A_2A receptor mRNA levels (Yip and Kwok, 2002). In the present study, adenosine receptor gene expression was reduced following omeprazole-induced achlorhydria. This observation fits the proposal that adenosine receptor gene expression may respond to changes in acidity.

When omeprazole-induced changes in adenosine receptor and gastric regulatory peptide gene expression were compared, a reciprocal relationship was observed between changes in A₁ receptor and gastrin gene expression. Although a clear relationship between A_2A receptor and somatostatin gene expression was not apparent, the expression of both genes was reduced in gastric tissues after various lengths of omeprazole treatment. Omeprazole-induced achlorhydria may decrease adenosine receptor expression and subsequently alter the synthesis and release of gastrin and somatostatin. Expression of both gastrin and somatostatin genes is enhanced by cAMP formation (Shiotani and Merchant, 1995; Montminy et al., 1996). Activation of A₁ and A_2A receptors inhibits and stimulates cAMP formation, respectively (Ralevic and Burnstock, 1998). Thus, activation of adenosine receptors may alter cAMP levels to regulate gastrin and somatostatin gene expression. Studies have shown that A_2A receptor stimulation can induce the expression of genes regulated by cAMP (Chae and Kim, 1997; Ravid et al., 1999). Therefore, a reduction in A_2A receptor gene expression may lead to a subsequent decrease in somatostatin gene expression.

Although the possible regulatory mechanism(s) involved in the modulation of adenosine receptor gene expression by omeprazole was not examined in the present study, the expression of the A₁ and A_2A receptor gene may be regulated by changes in gastric acidity, as suggested for gastrin and somatostatin gene expression. Omeprazole, however, has also been shown to induce the expression of various genes, through the activation of specific intracellular signaling pathways (Backlund et al., 1997; Kikuchi and Hossain, 1999). Therefore, the direct regulation of A₁ and A_2A receptor gene expression by omeprazole cannot be ruled out.

Omeprazole-induced changes in adenosine receptor mRNA expression may result in similar changes in receptor density and, thus, receptor function. The present study demonstrates that changes in gastric adenosine receptor gene expression can occur with corresponding changes in adenosine receptor function. We have previously suggested that high concentrations of CPA (1 µM) may stimulate SLI release through the nonspecific activation of A_2A receptors (Yip and Kwok, 2004b). After 3 days of omeprazole treatment, the stimulatory effect of 1 µM CPA on SLI release was decreased, suggesting a reduction in A_2A receptor expression. This proposal is supported by the observation that the stimulatory effect of the A_2A receptor agonist, CGS 21680, on SLI release was also significantly attenuated after 3 days of omeprazole treatment. The reduced A_2A receptor-mediated function is consistent with the reduced level of A_2A receptor gene expression observed in the antrum after 3 days of omeprazole treatment. Together, these results suggest that omeprazole may decrease the number of A_2A receptors in the antrum by reducing A_2A receptor gene expression. Our previous studies have implicated the involvement of the A_2A receptor in the tonic control of basal SLI release by endogenously released adenosine (Yip and Kwok, 2004). Furthermore, in comparison to the A₁ receptors, the relative abundance of the gastric mucosal A_2A receptors was found to be relatively low (Yip and Kwok, 2004; Yip et al., 2004b), suggesting the existence of a fairly small A_2A receptor reserve in this region. Thus, changes in A_2A receptor expression may readily alter somatostatin secretion.

Contrary to the A_2A receptor, the A₁ receptor is highly expressed in the gastric mucosa (Yip et al., 2004b). A₁ receptors are expressed on all G-cells, all corporeal D-cells, and some antral D-cells (Yip et al., 2004b). Thus, a large A₁ receptor population may be present in the rat gastric mucosa. In the present study, omeprazole treatment reduced A₁ receptor gene expression in the antrum and corporeal mucosa but did not alter A₁ receptor-mediated inhibition of IRG and SLI release. The large reserve of A₁ receptors may prevent decreased A₁ receptor expression from altering gastrin and somatostatin release, thus resulting in a discrepancy between changes in A₁ receptor mRNA levels and A₁ receptor function. However, this discrepancy may also be due to other factors. The present study examined the effect of short-term (1- and 3-day) omeprazole treatment. Although changes in actual receptor expression may require more time to occur, the effect of a longer treatment regimen was not examined. In rats treated with omeprazole for more than 3 days, both G-cell and D-cell density have been shown to be altered (Pawlikowski et al., 1992; Bolkent and Yilmazer, 1997). Any changes in adenosine receptor mRNA expression may be complicated by the alterations in G-cell and D-cell numbers, since adenosine receptors are present on both these endocrine cell types. The adenosine receptors are also expressed in different regions of the stomach, including the enteric plexi and vasculature. Thus, it is also possible that changes in adenosine receptor mRNA expression may occur in anatomical structures that are not involved in the control of IRG and SLI release. The regulation of adenosine receptor gene expression by post-transcriptional mechanisms, such as changes in the translational rate (Ren and Stiles, 1994; Chu et al., 1996; Lee et al., 1999), also cannot be ruled out. Thus, the disparity between adenosine receptor mRNA levels and receptor function may result from various factors.

The results of this study suggest that adenosine A₁ and A_2A receptor gene expression may respond to changes in the acid-secretory state of the stomach. Omeprazole treatment inhibited both A₁ and A_2A receptor gene expression in gastric tissues. Although A₁ receptor-mediated inhibition of IRG and SLI release was not altered by omeprazole treatment, A_2A receptor-mediated augmentation of SLI release was attenuated. The omeprazole-induced inhibition of A_2A receptor function corresponds with the reduced level of antral A_2A receptor mRNA. These findings suggest that gastric acidity may modulate the purinergic control of IRG and SLI release through the alteration of adenosine receptor expression. The possible modification of adenosine receptor expression by changes in intraluminal acidity may represent a novel purinergic regulatory feedback mechanism to control gastric acid secretion.

	Footnotes

 
This work was supported by the Canadian Apoptosis Research Foundation Society, the Canada Foundation for Innovation, the Wah Sheung Fund, and the former British Columbia Health Research Foundation. L.Y. was supported by the Cordula and Gunter Paetzold Fellowship and the University of British Columbia Graduate Fellowship.

A portion of this work was included in Linda Yip's Ph.D. thesis (Yip, 2004). Part of this work was presented at the Purines 2004 meeting (Yip et al., 2004a), and a portion of this work has previously been published in abstract form (Yip and Kwok, 2002).

doi:10.1124/jpet.104.069708.

ABBREVIATIONS: IRG, immunoreactive gastrin; CPA, N⁶-cyclopentyladenosine; CGS 21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine; SLI, somatostatin-like immunoreactivity; RT-PCR, reverse transcription-polymerase chain reaction; RIA, radioimmunoassay; C_T, threshold cycle; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ZM 241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance.

Address correspondence to: Dr. Yin Nam Kwok, Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. E-mail: kynkwok{at}interchange.ubc.ca

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