Rat Stomach Tissue Research Articles

Comparative study on the gastroprotective potential of some antidepressants in indomethacin-induced ulcer in rats

Clinical studies have shown that anxiolytic and antidepressant drug therapy benefits patients with ulcers. Many antidepressant drugs have been shown experimentally to produce antiulcer activity in various ulcer models. This study investigated the antiulcer activities of tianeptine, trazodone, and venlafaxine on indomethacin-induced ulcers in rats; and evaluated tianeptine's effects on oxidant and antioxidant parameters in rat stomach tissue. The results show that trazodone and venlafaxine did not prevent indomethacin-induced ulcers. Tianeptine, however, decreased indomethacin-induced ulcers significantly at all doses used (6, 12, and 25 mg/kg). Famotidine, an H 2 receptor blocker, showed the highest antiulcer activity. Tianeptine significantly prevented the decrease in glutathione (GSH) content that occurred in the indomethacin-only group's damaged stomach tissues. All doses of tianeptine, but especially the 25 mg/kg dose, significantly decreased catalase (CAT) activity in stomach tissue, compared to the control. All doses of tianeptine eliminated the decrease in superoxide dismutase (SOD) activity in the stomach tissue of rats given indomethacin. Although all doses of tianeptine significantly decreased the malondialdehyde (MDA) content, all doses of tianeptine, except 6 mg/kg, decreased myeloperoxidase (MPO) activities significantly compared to the control. Our results indicate that activating enzymatic and non-enzymatic antioxidant mechanisms and inhibiting some toxic oxidant mechanisms play a role in tianeptine's antiulcer effect mechanism.

Protective Effect of Mirtazapine on Indomethacin-Induced Ulcer in Rats and Its Relationship with Oxidant and Antioxidant Parameters

Even though there are many drugs for the treatment of gastric ulcers, these drugs sometimes cannot succeed. Since the 1950s, antidepressant drugs have been used for several non-psychiatric indications. A lot of antidepressant drugs have been shown experimentally to produce antiulcer activity in various ulcer models. This study aimed to investigate the antiulcer effects of mirtazapine and to determine its relationship with antioxidant mechanisms. The antiulcer activities of 15, 30, and 60 mg/kg mirtazapine have been investigated on indomethacin-induced ulcers in rats, and the results have been compared with that of the control group. Mirtazapine decreased the indomethacin-induced ulcers significantly at all doses used. Mirtazapine significantly increased the glutathione (GSH) level, which decreased in the control group given only indomethacin. All doses of mirtazapine significantly decreased the catalase (CAT) level in stomach tissue compared to the control. Additionally, all doses of mirtazapine reversed the decrease in the superoxide dismutase (SOD) level in the stomach tissue of control rats. And finally, all doses of mirtazapine decreased malondialdehyde (MDA) and myeloperoxidase (MPO) levels significantly compared to the control. In conclusion, the activation of enzymatic and non-enzymatic antioxidant mechanisms and the inhibition of some toxic oxidant mechanisms play a role in the antiulcer effect mechanism of mirtazapine. This new indication of mirtazapine will make it the first-choice drug in depressive patients with gastric ulcers.

Beneficial effects of vegetable oils (corn, olive and sunflower oils) and α-tocopherol on anti-inflammatory and gastrointestinal profiles of indomethacin in rats

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin are widely used in the treatment of inflammation, fever and pain. However, NSAIDs cause gastric damage as a major adverse reaction. In this study, the effects of vegetable oils (corn, olive and sunflower oils) and α-tocopherol on anti-inflammatory and gastrointestinal profiles of indomethacin were evaluated in rats. Results showed that indomethacin given with sunflower, corn and olive oils reduced paw edema induced by carrageenan by 79.5%, 74.0% and 60.5%, whereas individual indomethacin and diclofenac reduced paw edema by 56.2% and 50.7%, respectively. Furthermore, it has been found that the vegetable oils possess significant anti-inflammatory effect against paw edema when given alone. These results showed that the vegetable oils have beneficial effects on reduction paw edema induced by carrageenan. Besides, the administration of indomethacin together with the vegetable oils and α-tocopherol did not cause a statistically significant gastric damage in rats ( P > 0.05). However, indomethacin caused statistically significant gastric lesions as compared with untreated rats ( P < 0.05). Moreover, it was also found that the effects of the vegetable oils and α-tocopherol improved the levels of antioxidant defense systems in rat stomach tissues against oxidative damage. These results suggest that indomethacin as well as other NSAIDs do not have any adverse effect on the gastrointestinal tract when they are used together with vegetable oils and vitamin E or as the preparations of the oils.

Protective effects of cysteine, methionine and vitamin C on the stomach in chronically alcohol treated rats

A chronic intake of high dose alcohol may cause oxidative stress and inflammation in the stomach. It is hypothesized that cysteine-methionine and vitamin C may neutralize harmful compounds while potentiating the antioxidant capacity of the cell or tissue. The experimental animals were fed regular diets and were maintained for 90 days in the control group, the alcoholic group, which was given 2.5 g of 50% ethanol kg(-1) body wt. administered intragastrically every other day, or the alcoholic with antioxidant supplement group, to whom 2.5 g of 50% ethanol kg(-1) body wt. + a solution that contained 200 mg vitamin C, 100 mg cysteine and 100 mg methionine was administered intragastrically every other day. After the treatments, the stomach was taken for pathological and biochemical analysis. The stomach of the alcoholic group rats had higher scores of pathological findings compared with the control group, whereas the scores of the antioxidant-supplemented group were lower than the alcoholic group. In addition, the oxidized protein and lipid content in the stomachs of the alcoholic group were significantly higher than the control, but antioxidant supplementation lowered the amount of oxidation in the antioxidant supplemented group. The amount of stomach glutathione in the alcoholic group was higher than that of the control and antioxidant-supplemented groups. Interestingly, the level of total thiol in the stomach tissue of rats with antioxidant supplement was statistically higher than that of the control and alcoholic groups. In conclusion, the scores of the pathological findings in the stomach of rats with the antioxidant supplement were lower than the chronic alcohol-treated rats, albeit the amount of total thiol was increased in this group. Moreover, chronic alcohol treatment led to an increase in the level of lipid and protein oxidation in the stomach tissue of rats. A simultaneous intake of ascorbate/l-cys/l-met along with ethanol attenuated the amount of oxidation which suggested that cysteine-methionine and vitamin C could play a protective role in the stomach against oxidative damage resulting from chronic alcohol ingestion.

Upregulation of N-acetylaspartic acid alters inflammation, transcription and contractile associated protein levels in the stomach and smooth muscle contractility

N-acetylaspartic acid (NAA) is converted into aspartate and acetate by aspartoacylase. Abnormal levels of the enzyme leads to accumulation of NAA and these changes have been observed in Canavan disease and type 2 diabetes. How upregulation of NAA affect the gastrointestine protein levels and the function is not known. Incubation of rat stomach tissue with NAA 1.5 mM, 1.5 microM and 1.5 nM induced inflammatory agents TNFalpha, p38MAPK, iNOS, PKC, COX2 and ICAM3; transcription factors phospho-NF-kBp65, cjun and cfos; contractile proteins MLCK and phospho MLC; and calcium channel alpha1C and calcium channel, voltage-dependent, beta 3 subunit compared to their respective control. Incubation of circular smooth muscle cells with the above doses of NAA induced contractility compared to the control. These studies suggest that NAA alters proteins levels and smooth muscle contractility and these changes likely to contribute to gastrointestinal disorder seen in these diseases.

Effects of Onosma armeniacum root extract on ethanol-induced oxidative stress in stomach tissue of rats

This study investigated the effects of Onosma armeniacum K. (Boraginaceae) root extract (AR-1) on ethanol-induced stomach ulcers, and on some oxidant and antioxidant parameters, in stomach tissue in rats. The results obtained showed that AR-1 significantly inhibited ethanol-induced ulcers at 25, 50, 100 and 200 mg/kg doses. We found that 50, 100 and 200 mg/kg doses of AR-1 inhibited ulcers more effectively than did ranitidine. AR-1 at doses of 25, 50, 100 and 200 mg/kg significantly prevented the decrease in total glutathione (tGSH) level which occurs in damaged stomach tissues of rats given ethanol (control group). Only a 100 mg/kg dose of AR-1 significantly increased the glutathione S-transferase (GST) level in stomach tissue compared to the control. All doses of AR-1 except the 25 mg/kg dose eliminated the decrease in the superoxide dismutase (SOD) level in the stomach tissue of rats given ethanol. While all doses of AR-1 decreased malondialdehyde (MDA) levels significantly; all doses AR-1 except 25 mg/kg decreased myeloperoxidase (MPO) levels significantly compared to the control. The effect of AR-1 on catalase (CAT) activity was insignificant at all doses. AR-1 significantly increased nitric oxide (NO) levels at 50, 100 and 200 mg/kg doses compared to the control. Our results indicate that the protection of some antioxidant mechanisms and the inhibition of some oxidant mechanisms have a role in AR-1's antiulcer effect mechanism.

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1 × 10 −9 M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE–LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5–4.1 × 10 −7 M.

Effect of vanadyl sulfate on the status of lipid parameters and on stomach and spleen tissues of streptozotocin-induced diabetic rats

Diabetes mellitus is a significant risk factor for cardiovascular complications. Experimental evidence suggests that oxidative stress plays a dominant role in the pathogenesis of diabetes mellitus. This study was undertaken to investigate the effect of vanadyl sulfate on blood glucose, serum and tissue lipid profiles and on stomach and spleen tissues in STZ-induced diabetic rats. In this study, male 6–6.5-month-old Swiss albino rats were used. Rats were randomly divided into four groups. Group I: control animals (normal, nondiabetic animals) ( n = 13); Group II: vanadyl sulfate controls ( n = 5); Group III: streptozotocin (STZ)–diabetic, untreated animals ( n = 11); and Group IV: STZ diabetic animals given vanadyl sulfate ( n = 11). Experimental diabetes was induced by intraperitoneal (i.p.) injection of STZ in a single dose of 65 mg kg −1 body weight. Vanadyl sulfate was administered by gavage at a dose of 100 mg kg −1. The levels of cholesterol, phospholipid, high density lipoprotein-cholesterol (HDL-), low density lipoprotein-cholesterol (LDL-), very low density lipoprotein-cholesterol (VLDL-), triglycerides and lipid peroxidation (LPO) in serum and cholesterol in liver were assayed according to standard procedures. The levels of lipid peroxidation, glutathione (GSH) and nonenzymatic glycosylation (NEG) in stomach and lipid peroxidation and glutathione (GSH) in spleen tissues were analyzed. After 60 days of treatment, serum cholesterol, LDL-cholesterol, triglyceride, phospholipid, VLDL-cholesterol, LPO, blood glucose levels, stomach LPO and NEG, spleen LPO significantly increased, but serum HDL-cholesterol, stomach GSH and spleen GSH levels significantly decreased in the diabetic group. On the other hand, treatment with vanadyl sulfate reversed these effects. These results reveal that diabetes mellitus increased oxidative damage in stomach and spleen tissues and vanadyl sulfate has an ameliorating effect on the oxidative stress via its antioxidant property. The administration of vanadyl sulfate may be able to reduce hyperglycemia and hyperlipidemia related to the risk of diabetes mellitus.

The protective effect of thymoquinone on ethanol-induced acute gastric damage in the rat

Previous studies have indicated a pivotal role of reactive oxygen species in the pathomechanism of gastric ulcer. Recent studies demonstrated that thymoquinone (TQ) had an antioxidant effect on injuries caused by various toxic agents in different experimental models. The present study was planned to test whether TQ, the main constituent of the volatile oil of Nigella sativa seeds, was capable to exert beneficial effects on acute gastric ulcer model in rats. We examined antiulcerative and antioxidant effects of TQ on ethanol (EtOH)-induced gastric lesions in rats. The data we collected showed that gastric ulcer caused by absolute EtOH induction resulted in an increase in lipid peroxidation, represented by malondialdehyde level as well as by superoxide dismutase level, an antioxidant enzyme, whereas it resulted in a decrease in glutathione content in rat stomach tissue. Thymoquinone (20 mg/kg) administration reduced the ulcer index and the malondialdehyde level, and reversed the glutathione depletion. However, it did not statistically change the high superoxide dismutase activity induced by EtOH. These results suggest that TQ could inhibit the development of EtOH-induced gastric ulcer, and gastroprotective action of TQ might be in part dependent on its antioxidant property.

Effects of water extract of Usnea longissima on antioxidant enzyme activity and mucosal damage caused by indomethacin in rats

In this study, the antiulcerogenic effect of a water extract obtained from the lichen species Usnea longissima was investigated using indomethacin-induced ulcer models in rats. Experimental groups consisted of six rats. Antiulcerogenic activities of 50, 100 and 200mg/kg body wt. doses of the water extract were determined by comparing the negative (treated only with indomethacin) and positive (ranitidine) control groups. Although all doses of the water extract of U. longissima showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 100 mg/kg body wt. doses (79.8%). The water extract of U. longissima showed moderate antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. In addition, the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST)] were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of antioxidant enzymes on antiulcerogenic activity. SOD and GST enzymes activities in indomethacin-administrated tissues were reduced significantly by indomethacin in comparison to control groups. These enzymes were activated, however, by the water extracts of U. longissima. In contrast to SOD and GST activities, CAT activity was increased by indomethacin and reduced by all doses of U. longissima and ranitidine. The present results indicate that the water extract of U. longissima has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential.

Chronic renal failure rats are highly sensitive to aristolochic acids, which are nephrotoxic and carcinogenic agents

Aristolochic acid (AA), a component of some Chinese herbal medicines, may cause Chinese Herbs Nephropathy (CHN) and multi-systemic tumors by the formation of AA–DNA adducts. In this study, we established an animal model to further characterize the mechanisms of AA-induced diseases. Our results indicated that AA significantly inhibited rat growth in terms of weight gain. By measuring the serum creatinine levels, AA resulted in considerable damage to the rat renal system, not only for those in which chronic renal failure (CRF) was induced but also for normal healthy rats. Mutation-specific polymerase chain reaction (PCR) and XbaI restriction fragment length polymorphism (RFLP) revealed the CAA→CTA transversion mutation at codon 61 of the H- ras proto-oncogene from the stomach tissues of CRF rats fed with AA, but not from other tissues of rats in the same experimental group. In addition, no such mutations were found in the tissues of CRF rats without AA treatment or healthy rats fed with AA. Our results strongly demonstrated that AA was in fact nephrotoxic and carcinogenic, especially to those CRF rats.

Immunohistochemical Detection of the Cytoskeletal Components in Gastric Parietal Cells

Gastric parietal cells are characterized by intracellular canaliculi with many microvilli and tubulovesicles. These membranous structures are continuous and change in volume according to gastric acid secreting or resting state. Morphological changes are induced by cytoskeletal actin. Actin filaments are anchored to the cell membrane via ezrin. This study aimed to ascertain whether morphological changes were related to the nature of ezrin molecules, which were either phosphorylated or non-phosphorylated. Rat stomach tissues were rapid-frozen followed by freeze-substitution and embedding in paraffin for light microscopy. Also, the specimens were high pressure-frozen, freeze-substituted and embedded in Epon 812 or Lowicryl K4M for electron microscopy. Sections from paraffin or Lowicryl K4M were stained with anti-H+·K+-ATPase antibodies, anti-phosphorylated or non-phosphorylated ezrin antibodies, and anti-actin antibody. The H+·K+-ATPase was distributed on the intracellular canaliculi and tubulovesicles. The intracellular canaliculi were labeled with anti-phosphorylated ezrin antibodies in fed (gastric acid secreting state) rats. These were stained weakly in starved (gastric acid resting state) rats. Anti-nonphosphorylated ezrin antibodies hardly stained these organelles in either state. A possible explanation is that the phosphorylation of ezrin molecules is related to the reciprocal membranous transformation between intracellular canaliculi and tubulovesicles.

Relationship between Ghrelin and Energy Expenditure in Healthy Young Women

Ghrelin is a novel peptide that has been isolated from human and rat stomach tissues. Despite its known stimulatory effects on appetite and eating behavior, little information is available regarding its relationship with energy expenditure in normal-weight humans. To address this issue, we examined the relationship between serum ghrelin and resting metabolic rate (RMR), the thermic effect of food (TEF), fasting and postprandial respiratory quotient, physical activity level, peak aerobic capacity (VO(2 peak)), energy intake, and psychological measures of feeding behavior. We recruited 65 young healthy women and determined RMR and TEF by indirect calorimetry after a 12-h fast. Physical activity was determined by a leisure time physical activity questionnaire; VO(2 peak) was determined by bicycle ergometer test to exhaustion; energy intake was determined by a 24-h dietary recall; and food behavior was determined by a three-factor eating questionnaire. Our cohort showed a broad range of body mass index (range, 16.8-28.3 kg/m2), RMR (range, 820-1550 kcal/d), TEF (range, 74.4-136.5 kcal/d), and percent body fat (range, 14.0-37.7%). We noted significant inverse correlations between ghrelin and RMR (r = -0.350, P = 0.004) and TEF (r = -0.396, P = 0.001). These inverse correlations persisted after statistical control for both fat-free mass and fat mass (ghrelin vs. RMR partial, r = -0.284, P = 0.024; and ghrelin vs. TEF partial, r = -0.329, P = 0.01) and insulin levels (ghrelin vs. RMR partial, r = -0.255, P = 0.046; and ghrelin vs. TEF partial, r = -0.287, P = 0.024) using partial correlation analysis. We also observed a significant inverse correlation between ghrelin and daily caloric intake (r = -0.266, P = 0.032), but ghrelin levels were not significantly correlated with fasting (r = -0.002), postprandial respiratory quotient (r = -0.016), leisure time physical activity (r = 0.104), VO(2 peak) (r = 0.138), dietary disinhibition (r = -0.071), dietary restraint (r = 0.051), or feeling of general hunger (r = -0.028). These results suggest that higher levels of ghrelin are associated with low levels of resting and postprandial thermogenesis, which is independent of individual differences in fat-free mass and fat mass. Although speculative, serum ghrelin may play a role in the regulation of energy homeostasis by acting as a hormonal marker of increased energy efficiency.

Regional permeability of salmon calcitonin in isolated rat gastrointestinal tracts: Transport mechanism using Caco-2 cell monolayer

The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250-300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 microm in phosphate-buffered saline, pH 7.4, 37 degrees C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-microm pore size, 0.33 cm2 area) in a humidified 37 degrees C incubator (with 5% CO2). Transport experiments were conducted for sCT solutions (50 microm in Dulbecco's modified eagle's medium [DMEM], pH 7.4) from the apical-to-basolateral (A-to-B) direction and B-to-A direction at 37 degrees C and from the A-to-B direction at 4 degrees C. Cell monolayer integrity was monitored by mannitol permeability and transepithelial electrical resistance (TEER) measurements. The permeability coefficients (x 10(-9), cm/sec) for sCT through rat stomach, duodenum, jejunum, ileum, and colon tissues were 0.482 +/- 0.086, 0.718 +/- 0.025, 0.830 +/- 0.053, 1.537 +/- 0.32, and 0.934 +/- 0.15, respectively. The region of maximum sCT permeability is ileum followed by colon, jejunum, duodenum, and stomach. The permeability coefficients (x 10(-6), cm/sec) for sCT through Caco-2 cell monolayer were 8.57 +/- 2.34 (A-to-B, 37 degrees C), 8.01 +/- 1.22 (A-to-B, 4 degrees C), and 6.15 +/- 1.97 (B-to-A, 37 degrees C). The mechanism of its permeation is passive diffusion through the mucosa as determined from the Caco-2 monolayer permeability of sCT.

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.

Regional biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids from arachidonic acid in the rat stomach tissue

The present study was conducted to determine regional differences in the biosynthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs) in the rat stomach tissue (fundus, corpus and pyloric antrum) from radioactive arachidonic acid (AA). The radioactive metabolites were validated by RP-HPLC using non-radioactive AA as substrate. PGE 2 was the major prostanoid in the tissue . The relative ratio of PGE 2:PGF 2 α:PGD 2 in the whole stomach was 1:0.5:0.1. Regionally, the fundus biosynthesized the largest amount of all three cyclo-oxygenase products. Among the lipoxygenase metabolites, 15S-HETE was the predominant product, while 12S-HETE was found to be the lowest. The relative ratio of 15S-HETE:5S-HETE:12S-HETE in the whole stomach was 1:0.6:0.4. Interestingly, the generation of lipoxygenase products was the highest in the pyloric antrum when compared to fundus or corpus. Thus, the regional differences in the biosyntheses of gastric PGs and monohydroxy fatty acids may be relevant to our understanding of corresponding differences in mucosal resistance or susceptibility to gastric disease.

Quantitation of agmatine by liquid chromatography with laser-induced fluorescence detection

A high performance liquid chromatography (HPLC) method is described for the determination of agmatine, an endogenous neuromodulator. The method involves pre-column derivatization of the sample with a fluorescent tagging reagent, 7-fluoro-4-nitrobenzoxadiazole (NBD-F). The resulting agmatine derivative is stable and can be readily extracted into ethyl acetate at pH 8.5. The extraction enhances the quantification of low level agmatine because it eliminates chromatographic peaks caused by endogenous amino acids. The HPLC separation is carried out on a C 8 reversed phase column and completed in less than 10 min. With laser-induced fluorescence (LIF) detection, the detection limit is 5×10 −9 M agmatine. Method precision (coefficient of variation) is 5% for agmatine in human plasma at the sub-μM level. This method has been validated by determination of agmatine in biological samples including human plasma and rat brain and stomach tissues.

Discovery and mapping of ten novel G protein-coupled receptor genes

We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, ψGPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). ψGPR79 shared highest sequence identity with the P2Y 2 gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y 1 gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y 5 (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y 12 receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35–42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34–38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed in the human prostate and rat stomach and fetal tissues. GPR101 revealed mRNA transcripts in caudate putamen and hypothalamus. GPR103 mRNA signals were detected in the cortex, pituitary, thalamus, hypothalamus, basal forebrain, midbrain and pons.

Ghrelin Stimulates Gastric Acid Secretion and Motility in Rats

Ghrelin, a novel growth-hormone-releasing peptide, was discovered in rat and human stomach tissues. However, its physiological and pharmacological actions in the gastric function remain to be determined. Therefore, we studied the effects of rat ghrelin on gastric functions in urethane-anesthetized rats. Intravenous administrations of rat ghrelin at 0.8 to 20 μg/kg dose-dependently increased not only gastric acid secretion measured by a lumen-perfused method, but also gastric motility measured by a miniature balloon method. The maximum response in gastric acid secretion was almost equipotent to that of histamine (3 mg/kg, i.v.). Moreover, these actions were abolished by pretreatment with either atropine (1 mg/kg, s.c.) or bilateral cervical vagotomy, but not by a histamine H2-receptor antagonist (famotidine, 1 mg/kg, s.c.). These results taken together suggest that ghrelin may play a physiological role in the vagal control of gastric function in rats.

Characterization of a Human Digestive Tract-Specific Calpain, nCL-4, Expressed in the Baculovirus System

Human nCL-4, a digestive tract-specific calpain, was stably produced with 30K, a regulatory subunit for ubiquitous calpain as a fully active form using the baculovirus-expression system. nCL-4 showed an activity only when it was coexpressed with 30K. Expressed heterodimeric recombinant nCL-4 was purified to near homogeneity by sequential column chromatographies. Purified nCL-4 showed a calcium-dependent activity (calcium concentration at 50% maximum activity (Ka): 0.125 mM) with a sp act of 21 U/mg, which is distinct from those of ubiquitous calpains. nCL-4 exhibited calcium-dependent autolysis, but the cleavage pattern of nCL-4 was clearly different from ubiquitous calpains. Although it was inhibited by leupeptin, E-64, and calpastatin, and exhibited an optimal pH at 7.3 like other ubiquitous calpains, its optimal temperature was much lower. When overexpressed in COS-7 cells, clear asymmetric juxtanuclear, and/or nuclear staining rather than typical cytoplasmic staining was observed. Moreover, a translation product of nCL-4 was detected in rat stomach tissue by immunofluorescence analysis. In conclusion, human nCL-4 resembles ubiquitous calpain in some enzymatic properties and interacts with 30K for its activity. This is the first report on biochemical and enzymatic properties of a fully active tissue-specific calpain species expressed in the baculovirus system.