Cellular localization of group IIA phospholipase A2 in rats.

It has been known that group II phospholipase A2 (PLA2) mRNA and protein are present in the homogenates of the spleen, lung, liver, and kidney in normal rats, but the cellular origin of this enzyme has not been yet identified. At present, five subtypes of group II PLA2 have been identified in mammals. Antibodies or mRNA probes previously used for detecting group II PLA2 need to be evaluated to identify the subtypes of group II PLA2. In this study we tried to identify group IIA PLA2-producing cells in normal rat tissues by in situ hybridization (ISH) using an almost full-length RNA probe for rat group IIA enzyme. Group IIA PLA2 mRNA was detected in megakaryocytes in the spleen and Paneth cells in the intestine by ISH. These cells were also immunopositive for an antibody raised against group IIA PLA(2) isolated from rat platelets. Group IIA PLA2 mRNA-positive cells were not detected in lung, liver, kidney, and pancreas. Under normal conditions, group IIA PLA2-producing cells are splenic megakaryocytes and intestinal Paneth cells in rats.     

Ontogenic pattern of nucleobindin-2/nesfatin-1 expression in the gastroenteropancreatic tissues and serum of Sprague Dawley rats

Nesfatin-1 is a novel metabolic hormone that has glucose-responsive insulinotropic actions. Islet β-cells and gastrointestinal tissues have been reported as abundant sources of nesfatin-1 and its precursor hormone nucleobindin-2 (NUCB2). While nesfatin-1 is emerging as a multifunctional hormone, there are no reports on the developmental expression of NUCB2/nesfatin-1. The main objective of this study was to examine the ontogenic expression of NUCB2 mRNA, and NUCB2/nesfatin-1 immunoreactivity in the pancreas, stomach and duodenum, and the circulating levels NUCB2/nesfatin-1 in Sprague Dawley rats. In addition, we also determined the co-localization of NUCB2/nesfatin-1 and insulin immunoreactivity during development. NUCB2/nesfatin-1 immunoreactivity was found in the rat stomach from postnatal days 13-27. Furthermore, NUCB2/nesfatin-1 immunoreactivity was also detected in the enteroendocrine cells of the duodenum at postnatal days 13 and 27. Duodenal NUCB2 mRNA expression at postnatal day 27 was highest. Serum NUCB2/nesfatin-1 levels on embryonic day 21 and postnatal day 1 were lower than serum NUCB2/nesfatin-1 levels of adults and neonates at postnatal days 13, 20 and 27, gradually increasing with growth, suggesting an increase in its production and secretion from tissues including the gastrointestinal tract and pancreas. Our findings indicate that NUCB2/nesfatin-1 colocalizes with insulin in the islet β-cells at all developmental stages, but the percentage of colocalization varies in an age-dependent manner. These findings suggest that NUCB2/nesfatin-1 has potential age- and tissue-specific role in the developmental physiology of rats during growth.     

Ontogenetic expression of chromogranin A and its derived peptides, WE-14 and pancreastatin, in the rat neuroendocrine system

 The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the posttranslational processing of the molecule is developmentally controlled. Accepted: 18 October 1996     

Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells

Using a specific polyclonal antibody raised against rat pancreatic phospholipase A2 (PLA2), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA2 reactive with the anti-pancreatic PLA2 antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA2 and PAS was observed in the same cell, it is suggested that PLA2 could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA2 in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA2 secreted from the pancreas. Other possible roles of the PLA2 in the stomach are discussed.     

Maternal methamphetamine administration during pregnancy influences on fetal rat heart development. [corrected

Methamphetamine (MAP) is one of the most abused drugs in Japan. The rate of MAP abuse by young women has recently reached more than 50 percent in adolescents. A major health concern is that these women will continue to use MAP during pregnancy. The purpose of this study was to investigate whether MAP administered to the mother during pregnancy would change the expression of α- and β- myosin heavy chain (MHC) mRNA in rat neonatal hearts, as detected by quantitative RT-PCR. In addition, morphological changes in the rat neonatal ventricles were examined. Pregnant rats were injected intraperitoneally with MAP (1 mg/kg/day) starting at day 0 of gestation and ending at day 21. There was a significant increase in α-MHC mRNA expression in the neonatal ventricular muscle in the experimental group compared with the control at postnatal day (P) 0 and 5. α-MHC mRNA expression in both groups was similar after P9. β-MHC mRNA expression was similar in both groups at P0. Postnatal β-MHC mRNA expression decreased rapidly, but significant alteration was not detected. Neonatal rats at P0 exhibited some cardiac changes, including hypertrophy, degeneration, and disarrangement of myofibers, but these lesions disappeared by P14. We conclude that chronic maternal administration of MAP changes the α- and β-MHC mRNA expression pattern in fetal and neonatal hearts, correlating with abnormal development, plasma level of hormones, and myocardial damage. At the same time, it is indicated that neonatal cardiomyocytes have reversibility.     

Ghrelin in the gastrointestinal tract and blood circulation of perinatal low and normal weight piglets

Ghrelin, the ‘hunger’ hormone, is an endogenous growth hormone secretagogue that exerts a wide range of physiological functions. Its perinatal presence suggests that ghrelin might be involved in growth and metabolism processes during intrauterine and postnatal life. Intrauterine growth-restricted (IUGR) neonates have altered endocrine and metabolic pathways because of malnutrition during foetal development. These changes might include an altered gastrointestinal presence of ghrelin cells (GCs). As ghrelin is mainly secreted by the stomach, this altered presence might be reflected in its serum concentrations. Small-for-gestational age (SGA) pigs appear to be a natural occurring model for IUGR children. Therefore, the first aim of this study was to investigate the presence of gastrointestinal GCs expressing active ghrelin in normal weight (NW) foetal and postnatal piglets compared with their SGA littermates using immunohistochemical analysis in combination with stereological methods. Second, total ghrelin serum concentrations of these piglets were analysed with a porcine radioactive immunoassay. In addition, the growth of the gastric pars fundica in the NW and SGA piglets was analysed stereologically. Corresponding with humans and rats, it was shown that opened- and closed-type immunoreactive GCs are distributed along the entire gastrointestinal tract of the perinatal NW and SGA piglets. However, in contrast to the rat’s stomach, the porcine GCs do not disperse from the glandular base to the glandular neck during perinatal development. Furthermore, stereological analysis demonstrated that the NW neonates have a higher amount of gastric cells expressing active ghrelin compared with the SGA piglets that could result in higher milk consumption during the neonatal period. This finding is, however, not reflected in total serum ghrelin levels, which showed no difference between the NW and SGA piglets. Moreover, the stereological volume densities of the fundic layers demonstrate a similar growth pattern in the SGA and NW piglets.     

Maternal nicotine exposure increases oxidative stress in the offspring

Fetal and neonatal nicotine exposure causes β-cell apoptosis and loss of β-cell mass, but the underlying mechanisms are unknown. The goal of this study was to determine whether maternally derived nicotine can act via the pancreatic nicotinic acetylcholine receptor (nAChR) during fetal and neonatal development to induce oxidative stress in the pancreas. Female Wistar rats were given saline or nicotine (1 mg/kg/day) via subcutaneous injection for 2 weeks prior to mating until weaning (postnatal day 21). In male offspring, nAChR subunit mRNA expression was characterized in the developing pancreas and various oxidative stress markers were measured at weaning following saline and nicotine exposure. The nAChR subunits 2-4, 6, 7, and β2–β4 were present in the pancreas during development. Fetal and neonatal exposure to nicotine significantly increased pancreatic GPx-1 and MnSOD protein expression, as well as islet ROS production. Furthermore, protein carbonyl formation was higher in nicotine-exposed offspring relative to controls, particularly within the mitochondrial fraction. There was also a nonsignificant trend toward higher serum 8-isoPG levels. These data suggest that β-cell apoptosis in the fetal and neonatal pancreas may be the result of a direct effect of nicotine via its receptor and that this effect may be mediated through increased oxidative stress.     

磷脂酶PLA2-Ⅰ B在胃癌组织中的表达下降

目的:明确磷脂酶PLA2-ⅠB在正常小鼠胃组织中的细胞表达类型,检测PLA2-ⅠB在人胃癌组织中的表达情况。方法:利用Northern印迹验证PLA2-ⅠB在小鼠胃组织中的表达;利用原位杂交明确PLA2-ⅠB在正常小鼠胃组织中的细胞表达类型;利用反转录PCR检测14例人胃癌组织中PLA2-ⅠB的表达情况。结果:PLA2-ⅠB主要表达在小鼠腺胃的主细胞;与癌旁相比,PLA2-ⅠB的表达在93%的人胃癌标本中显著下降。结论:磷脂酶PLA2-ⅠB在胃癌组织中的表达下降。     

Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas

BACKGROUND: Ghrelin and obestatin are encoded by the preproghrelin gene and originate from posttranslational processing of the preproghrelin peptide. The fetal rat pancreas contains acylated and desacylated ghrelin peptides, as well as growth hormone secretagogue receptor -1a mRNA. Acylated ghrelin inhibits insulin secretion. We investigated the plasma and tissue ontogeny of ghrelin and obestatin in the rat. METHODS: We measured obestatin and acylated and total ghrelin concentrations in plasma, pancreas and stomach from rat fetuses (F20) and neonates at postnatal day (PN) 1, 6, 12 and 21). RESULTS: Overall, obestatin concentrations were markedly lower than total ghrelin concentrations. In plasma, total ghrelin concentrations decreased abruptly after birth (p < 0.05), contrasting with a 3 times increase in the concentration of acylated ghrelin between F20 and PN1 (p < 0.05). In pancreas, total ghrelin and obestatin concentrations decreased progressively from PN1 to PN21 but acylated ghrelin concentrations increased 6-7 times from F20 (18 [6] pg/ml) to PN6 (122 [59] pg/ml). The percent of acylated ghrelin increased from 1.8 (0.6) at F20 to 39.7 (13.0) % of total ghrelin immunoreactivity at PN12 (p < 0.05). There were significant positive correlations between postnatal obestatin, acylated or total ghrelin and insulin concentrations in the pancreas (all p < 0.02, r(2) > 0.21) and between postnatal total ghrelin and obestatin (in pancreas, r(2) = 0.37) or acylated ghrelin (in stomach, r(2) = 0.27) (p < 0.001). CONCLUSION: Ghrelin and obestatin are present in the perinatal pancreas where they could potentially affect insulin secretion.     

Differential development of aldehyde dehydrogenase in fore- and glandular stomach in postnatal rats

Many foods contain the unsaturated aldehyde, hexadienal (HX). Human exposure is thus unavoidable. HX feeding to rodents caused cancers only in the forestomach. Aldehyde dehydrogenases (ALDH) are key enzymes in the metabolism of aldehydes. We examined the distribution of ALDH using HX as the substrate (HXDH) along the GI tract of adolescent rats and found that their stomachs have high levels of HXDH activity and the enzyme preferred HX > 9-cis-retinal > acetyl aldehyde > formyl aldehyde. We also followed the postnatal development of the stomach. At birth, the forestomach represented 40-50% of the total stomach weight. Both fore- and glandular stomach gained weight, with the glandular portion gaining at a faster rate. By 21 days, the forestomach was 24-28% of the total weight and decreased slightly to an adult level of 22-24%. Gastric HXDH is low from birth to 14 days of age. HXDH activity increased thereafter, reaching higher levels at 21 days and peaking around 30-36 days of age. The activity then decreased to the adult level. The fore- and glandular stomach had the same level of HXDH activity in the newborn and at 7 and 14 days of age. At weaning, HXDH activity was higher (3x) in the forestomach than in the glandular stomach. In adults, the forestomach still had 2x the HXDH activity compared to the glandular stomach. Zymograms showed similar isozyme patterns of HXDH but with different ratios of the three major forms between the forestomach and the glandular stomach. Results indicate a differential development of HXDH between the fore- and glandular stomach that might be related to the higher sensitivity of the forestomach to HX feeding.     

Developmental regulation of somatostatin gene expression in the brain is region specific

Developmental regulation of somatostatin (SRIF) gene expression was studied in five regions of rat brain and in rat stomach. Total RNA was isolated from hypothalamus, cortex, brainstem, cerebellum, and olfactory bulb, as well as stomach at eight stages of development from prenatal day 16 to postnatal day 82. Hybridization of a 32P-labeled rat SRIF cDNA probe to Northern blots of total RNA from the above tissues during development demonstrated a single hybridizing band approximately 670 base pairs in length. When SRIF mRNA levels from each stage of development were quantified and normalized by the amount of poly (A)+ RNA present at that stage of development, a unique pattern of SRIF gene expression was seen in each region. In brainstem and cerebellum, SRIF mRNA levels peaked early in development between prenatal day 21 and postnatal day 8 and then declined until postnatal day 82. Hypothalamus and cortex, on the other hand, showed a progressive increase during development with peak levels occurring between postnatal days 13 and 82. In contrast, stomach and olfactory bulb showed SRIF mRNA levels which were low during early development and which rose late in development (postnatal days 13 to 82). Marked differences in the amount of SRIF mRNA within each region were present as well. These data suggest that there is differential expression of the SRIF gene in different regions of the brain and in the stomach during development. Further study of this phenomenon may provide insight into the in vivo control of SRIF gene expression and the role of SRIF in the developing brain.     

Accumulation and localization of two adult acinar cell secretory proteins during development of the rat submandibular gland.

The seromucous acinar cells of the adult rat submandibular gland secrete a characteristic mucin glycoprotein and a family of unusual glutamine/glutamic acid-rich proteins (GRP). Monoclonal antibodies to the mucin and GRP localized in a very few Type III cells in glands of newborn and 1 day-old rats, using light and electron microscopic immunocytochemistry. Both mucin and GRP reactivities were present in the polymorphic Type IIIP granules during the 1st postnatal week. By 9 days after birth, the granules contained both mucin and GRP and were mucous-like in appearance. At earlier stages, however, cells containing only GRP or mucin could be found, indicating that the initiation of GRP and mucin biosynthesis may not be coordinately regulated. No reactivity was seen in the neonatal Type I cells or in duct cells at any age. Northern and Western blot analysis showed GRP mRNA and protein levels to be barely detectable at birth, with marked increases during the first 2 postnatal weeks. In contrast, Western blots of B1-immunoreactive proteins (B1-IP) showed levels highest in the 1st week and markedly decreased in the adult. Immunocytochemical colocalization, using gold particles of different sizes, showed that the B1-IP, mucin, and GRP colocalized in the granules. These results strengthen the hypothesis that the adult acinar cells develop from the neonatal Type III cells. No evidence was obtained for the involvement of Type I cells in the pathway of acinar cell development.     

Endometrial expression of cellular protooncogene c-ski and its regulation by estradiol-17beta.

The expression of the cellular protooncogene c-ski was examined in the rat uterus. In situ hybridization revealed that c-ski mRNA was expressed in the uterus of the adult rat on the day of estrous and localized mainly in the luminal and glandular epithelia. To test the possibility that the expression of c-ski mRNA is induced by estrogen, rats were ovariectomized and estradiol-17beta (E2) was injected. The expression of c-ski mRNA was upregulated 3 h after E2 treatment, reaching the highest level at 6 h and this persisted until 24 h; the E2-induced expression of c-ski mRNA was restricted to the luminal and glandular epithelia. These results suggest that the c-ski gene plays a role in uterine epithelial cell proliferation and mediates the proliferative action of E2.     

Rat lung peroxiredoxins I and II are differentially regulated during development and by hyperoxia

Peroxiredoxin I (Prx I) and peroxiredoxin II (Prx II) are found in abundance in the cytoplasm of cells and catalyze the reduction of hydrogen peroxide with the use of electrons provided by thioredoxin. Here we examined Prx I and Prx II expression in rat lung during perinatal development and in response to hyperoxia. Prx I protein increased during late gestation and after birth fell to adult levels; conversely, Prx I mRNA increased after birth. Prx II protein concentration was unchanged in the perinatal period, but Prx II mRNA increased after birth. In response to hyperoxia begun on postnatal day 4, there was no change in Prx II expression; however, Prx I mRNA, protein, and enzymatic activity increased significantly. These data show that 1) Prx I and Prx II are developmentally regulated at the level of translational efficiency and 2) Prx I, but not Prx II, is inducible and is upregulated during the late-gestational preparation for the oxidative stress experienced by the lung at birth and during exposure to hyperoxia in the neonatal period.     

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue. An analysis of procedural variables

Methodological variables for in situ hybridization using 32P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42 degrees C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that 32P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.     

Expression of cholecystokinin type A receptor gene correlates with DNA demethylation during postnatal development of rat pancreas.

Cholecystokinin stimulates pancreatic amylase secretion, gallbladder contraction, and pancreatic growth, etc. by binding with high affinity to a cholecystokinin type A receptor (CCKAR). To better understand the expression of CCKAR mRNA in terms of tissue specificity and postnatal development, we determined the methylation status of BssHII sites (5'-B sites) in the rat CCKAR gene promoter. The 5'-B sites in adult pancreas expressing CCKAR mRNA were much less extensively methylated than those in fetal pancreas not expressing the mRNA. In brain, liver, and kidney of adult rats not expressing CCKAR mRNA, the 5'-B sites were methylated. In pancreas, the demethylation level of the sites increased at 21 days after birth. Concomitant with the DNA demethylation level in the 5'-B sites, the mRNA level rose rapidly in 21 days. These results demonstrate that methylation and expression of the CCKAR gene reveal a good inverse correlation.     

Genotoxicity and cell proliferative activity of omeprazole in rat stomach mucosa.

Induction of unscheduled DNA synthesis (UDS) as a marker of genotoxicity and induction of ornithine decarboxylase (ODC) activity as a marker of cell proliferative activity by omeprazole were determined in the glandular stomach mucosa of male F344 rats after oral administration. Commercial enteric-coated omeprazole (Losec) at doses of 30 and 100 mg/kg body weight induced a dose-dependent increase in UDS but not replicative DNA synthesis in the pyloric mucosa of rat stomach 4 h after its administration. Dose-dependent significant induction of ODC activity was observed in fundic and pyloric mucosa with a maximum 8 h after administration of omeprazole at doses of 37.5-100 mg/kg body weight. These results show that omeprazole has genotoxicity and cell proliferative activity in the rat glandular stomach mucosa.