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Correspondence to: Surinder K. Aggarwal, Dept. of Zoology, Michigan State University, East Lansing, MI 488241115.
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Summary |
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Cisplatin treatment (9 mg/kg) causes bloating of the stomach, an increase in gastric acid, and ulceration in rats. Gastrin, a gut peptide, plays an important role in regulating gastric acid production. To study the role of gastrin in this increased gastric acid production after cisplatin treatment, male Wistar rats (100150 g) were treated with cisplatin (9 mg/kg) in five divided doses over 5 consecutive days. The rats were sacrificed 1, 6, 10, or 15 days after the last treatment. As measured by immunocytochemistry, in situ hybridization, Northern blot, and dot-blot techniques, gastrin was found to be below detectable limits just 1 day after cisplatin treatment. However, 1015 days after the last injection, the levels for both gastrin and its mRNA gradually recovered to normal. Northern blot studies showed that decreased somatostatin mRNA parallels the changes of gastrin and its mRNA. These results suggest that after cisplatin treatment the increased gastric acid production in rat stomach is independent of gastrin. This decrease of gastrin production is not under the influence of somatostatin, which also decreased after cisplatin treatment. (J Histochem Cytochem 47:10571062, 1999)
Key Words: gastrin, cisplatin, ulcer, immunocytochemistry, in situ hybridization, stomach, Northern blot, dot-blot, rat
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Introduction |
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CISPLATIN (cis-dichlorodiammineplatinum II; CDDP), a broad-spectrum chemotherapeutic drug, has been proved effective in the treatment of bladder, lung, ovarian (
Under low chloride ion concentrations inside the cell, cisplatin hydrolyzes into monoaqua (monovalent) and diaqua (divalent) species. The diaqua form of cisplatin with its divalent charge has been demonstrated to bind to calmodulin and inhibit its binding to calcium. Without the calciumcalmodulin complex, acetylcholine release is inhibited, resulting in bloating of the stomach and an increase in its acid content (
Gastrin, one of the gut peptides, which is primarily produced and secreted in the stomach and proximal duodenum, is a potent stimulant of gastric acid secretion and proliferation of the acid-secreting oxyntic cells of the gastric mucosa. It acts both directly on gastrin receptors of the parietal cells and indirectly on gastrin receptors of the enterochromaffin-like (ECL) cells to produce histamine which, in turn, stimulates the gastric acid production by parietal cells. Somatostatin is known to influence gastrin production in rat stomach through a paracrine action (
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Materials and Methods |
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Animals and Tissue Preparation
Male Wistar rats (Charles River Laboratory; Wilmington, MA) weighing 100150 g were housed on a 12-hr light/12-hr dark cycle with free access to food and water. Cisplatin at a clinical dosage of 9 mg/kg in physiological saline was injected (IP) in five divided doses over 5 days. The controls received only the injection vehicle. The rats were anesthetized with equithesin and either perfused with buffered 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4) or stomach tissues were excised and frozen (-70C) 1, 6, 10, or 15 days after last cisplatin treatment. Each interval had a minimum of three animals. Perfused stomach tissues were postfixed with buffered 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4) for 12 hr, dehydrated through ethanol gradients, and embedded in paraffin at 56C. Sections (10 (µm) were cut and placed on gelatin-coated slides for in situ hybridization (ISH) and immunocytochemical studies.
Immunocytochemistry
Immunocytochemical study for gastrin was performed using the avidinbiotinperoxidase complex (ABC) (
Oligonucleotide Probes
5'-GACCTTGGGGCCCCAGCTGTCTCCGAT-3', a 27-mer hybridization sequence complementary to the coding sequence of rat gastrin mRNA (position 212238) (
In Situ Hybridization
The protocols for in situ hybridization were followed according to
Statistical Analysis
Numbers of gastrin and its mRNA-staining positive cells were counted in 15 random visual fields of five different tissue sections from each group. The data were averaged and plotted. All data were statistically analyzed by the Student's t-test when comparison between control and cisplatin treatment was made (
Northern Blot Analysis
RNA was extracted from the stomach tissues by a guanidinium isothiocyanate solution and purified by the CsCl cushion method (
The RNA (20 µg) was size-separated by electrophoresis in a 1.3% agarose gel containing formaldehyde and electroblotted onto nylon membrane (GeneScreen; New England Nuclear, Boston, MA). The blots were prehybridized with the hybridization buffer without probes for 3 hr at 42C. Blots were incubated with fresh hybridization buffer in the presence of labeled probes (40 ng/ml) at 42C for 2 days. The hybridization buffer for the gastrin mRNA contained 50% formamide, 10% dextran sulfate, 50 mM Tris (pH 6.8), 3 x SSC, 100 µg/ml sonicated salmon sperm DNA, and 5 x Denhardt's solution. The buffer for the somatostatin was the same as that for gastrin except without dextran sulfate. The blots were washed with two changes of 2 x SSC/0.1% SDS at 42C for 30 min each and 0.1 x SSC/0.1% SDS at 42C for 15 min. The probes were detected by the same kit for in situ hybridization and following the same procedure.
Dot-blot Analysis
Stomach tissues were homogenized (
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Results |
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Immunocytochemistry and In Situ Hybridization
Using a gastrin-specific primary antibody, immunocytochemical study showed that gastrin-positive cells were mostly localized in the basal portion of the pylorus of rat stomach (Figure 1A). Control sections stained only with Vector Red substrate showed no significant endogenous AP activity. The liver tissue was also negative. At 1 and 6 days after the last cisplatin treatment, no gastrin-positive cells were observed in the gastric mucosa (Figure 1B). However, 10 days after cisplatin treatment, gastrin-positive cells were evident (Figure 1C). The intensity and distribution of staining were back to normal on Day 15 after the last cisplatin treatment.
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Gastrin-specific primary antibody crossreacts with cholecystokinin (CCK) octopeptide, so ISH and Northern blot tests were applied to the adjacent tissues for immunocytochemical and dot-blot studies to confirm the results from these studies. The intensity and distribution of gastrin mRNA-positive cells were similar to those observed after immunocytochemical study, mostly in the basal portion of the pylorus of the rat stomach, with intense positive cytoplasmic staining (Figure 1D). The number of positively stained cells was less than that after the immunocytochemical study. This is probably because the CCK-positive cells did not stain after ISH. No gastrin mRNA-positive cells were noted 1 and 6 days after cisplatin treatment in the gastric mucosa (Figure 1E). However, 15 days after cisplatin treatment, the intensity and distribution of gastrin-positive cells were similar to those of the normal tissues (Figure 1F). The numbers of gastrin and gastrin mRNA-positive cells in each group were significantly different (Figure 2), p<0.01.
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Northern Blot and Dot-blot Analysis
Total RNA from rat stomach tissues was isolated and size-separated by electrophoresis on formaldehyde1.3% agarose gels. The RNA blot was hybridized with DIG-labeled oligonucleotide probes for either gastrin mRNA or somatostatin mRNA. At the corresponding RNA molecular marker level, gastrin mRNA was detected (Figure 3). At 1 and 6 days after cisplatin treatment the gastrin mRNA bands were almost negative. However, 10 days after the last cisplatin treatment the gastrin mRNA levels became significant, reaching normal levels 15 days after treatment. Somatostatin (Figure 4) mRNA followed similar patterns as described for gastrin mRNA.
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For dot-blot studies, 100-µg protein samples were dot-blotted onto the nitrocellulose membrane. The membrane was incubated with rabbit anti-gastrin antibody and detected by the ABC method (Figure 5). At 1 and 6 days after cisplatin treatment, gastrin levels were undetectable, as in case of gastrin mRNA levels described above. However, 1015 days after cisplatin treatment the gastrin levels showed a gradual increase. The rats in the same groups demonstrated a similar change in Northern and dot-blot tests.
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Discussion |
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Gastrin regulates the secretion of gastric acid through its action on its receptors on parietal cells and ECL cells. ECL cells produce histamine, which is a potent stimulant of gastric acid production. The half-life for rat gastrin in circulation is around 10 min, so the circulating gastrin level is basically maintained by the continuous production and secretion of gastrin from G-cells (
The cellular toxicity of cisplatin is mainly caused by its ability to bind covalently to DNA to form intrastrand and/or interstrand crosslinks, which in turn prevents DNA replication and transcription (
Ulceration of the stomach is probably caused by an imbalance between gastric protection against injury and the erosive acid/peptic factors that exist in the normal stomach (
Normal stomach motility involves contraction of the stomach smooth muscle and relaxation of the pyloric sphincter, both of which are controlled by the release of acetylcholine from the nerve terminals. Acetylcholine release is dependent on calciumcalmodulin. Under the low chloride ion concentrations inside the cell, cisplatin hydrolyzes into monoaqua (monovalent) and diaqua (divalent) species. The diaquatic form interferes with binding of calmodulin to calcium, which is further affected by hypocalcemia induced by cisplatin (
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Acknowledgments |
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Our sincere thanks to Drs Neal Band and Will Kopachik for their kind guidance during the conduct of our experiments. Cisplatin was a gift from Andrulis Pharmaceutical Corporation, Beltsville, Maryland.
Received for publication January 15, 1999; accepted March 30, 1999.
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