Age-related alterations of gastric mucosa and estrogen synthesis in rat parietal cells.

The stomach has diverse functions other than gastric acid secretion. Multifaceted studies have investigated age-related changes of the gastrointestinal tract. Nevertheless, little is known about estrogen production changes in gastric parietal cells in rats aged over 3 months. We investigated age-related changes in gastric estrogen synthesis and the accompanying changes in liver estrogen receptor from 3 to 24 months. Weights of the body, stomach, and liver increased linearly from 3 to 18 months, then maintained a constant proportion up to 24 months. The gastric mucosa area (in mm2/1 mm muscularis mucosa) showed a constant proportion throughout the rats' life. The population of parietal cells immunostained area with H+/K+-ATPase decreased gradually with advancing age. Cells that were immunopositive to aromatase antibody were observed at 3-24 months. The expressions of aromatase mRNA and its protein were somewhat lower at 18 and 24 months than at 3 months. The portal venous estradiol concentration at 12 months was 1.5 times higher than that at 3 months, and that at 18 months was a half of that at 3 months. The expression of estrogen receptor mRNA in the liver at 18 and 24 months was about 80% of that at 3 months. Results suggest that the gastric estrogen production declines with aging, and the liver estrogen receptor is also affected accordingly. Simultaneously, the gastric mucosa continues to express aromatase to maintain liver function(s) throughout the animal's life.

Expression and localization of aromatase in human gastric mucosa : Immunohistochemical study using biopsy materials.

Parietal cells in the gastric mucosa are known not only as cells playing major roles in food digestion but also as cells bearing endocrine function. In addition to their production of gastrin and ghrelin, it has been recently revealed that these cells are also involved in the synthesis and secretion of estrogens with their expression of aromatase in experimental animals. Although aromatase activity has been detected in human gastric cancer cells and related cell lines, much less study has been done to ascertain the expression of the enzymatic activity in normal gastric mucosa. It has not been established which cell type is responsible for estrogen production in human gastric glands consisting of epithelial cells of several types. The aim of this study is to define the expression of aromatase by parietal cells in human gastric glands using immunohistochemical techniques. We retrieved formalin-fixed paraffin embedded materials of gastric biopsies from 16 patients (nine men, seven women). Colocalization of aromatase and H+/K+-ATPase ß-subunit indicated that positive cells are parietal cells, but not chief cells and mucous cells. Furthermore, immunoreactivity of aromatase was detected within gastric glands irrespective of age or sex. These results suggest that human parietal cells synthesize estrogens within gastric mucosa and subsequently secrete them to the portal vein via gastric vein, as they do in rats. These estrogens might influence liver functions in humans. The estrogenic effects related to liver dysfunction might also be attributed to them.

Estrogen synthesis in the stomach of Sprague-Dawley rats: comparison to Wistar rats.

Aromatase, an estrogen synthase, exists in the gastric parietal cells of Wistar rats. The stomach synthesizes large amounts of estrogens and secretes them into the portal vein. We have been particularly studying gastric estrogen synthesis using Wistar rats. However, estrogen synthesis in the stomach of Sprague-Dawley (SD) rats, which are used as frequently as those of the Wistar strain, has not been clarified. We examined steroid synthesis in the stomach of SD rats using immunohistochemistry, in situ hybridization, Western blotting, real-time PCR, and LC-MS/MS. Aromatase also exists in the stomach of SD rats. Its distribution was not found to be different from that of Wistar rats. Results show that H+/K+-ATPase ß-subunit and aromatase colocalized in double immunofluorescence staining. Each steroid synthase downstream from progesterone was present in the gastric mucosa. These results suggest that steroid hormones are synthesized in the parietal cells in the same pathway as Wistar rats. Although mRNA expression of steroid synthases were higher in SD, no significant difference was found in the amount of protein and each steroid hormone level in the portal vein. Although differences between strains might exist in steroid hormone synthesis, results show that SD rats are as useful as Wistar rats for gastric estrogen synthesis experimentation.

Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells.

Nanoparticles are prevalent in both commercial and medicinal products; however, the contribution of nanomaterials to carcinogenesis remains unclear. We therefore examined the effects of nano-sized titanium dioxide (TiO(2)) on poorly tumorigenic and nonmetastatic QR-32 fibrosarcoma cells. We found that mice that were cotransplanted subcutaneously with QR-32 cells and nano-sized TiO(2), either uncoated (TiO(2)-1, hydrophilic) or coated with stearic acid (TiO(2)-2, hydrophobic), did not form tumors. However, QR-32 cells became tumorigenic after injection into sites previously implanted with TiO(2)-1, but not TiO(2)-2, and these developing tumors acquired metastatic phenotypes. No differences were observed either histologically or in inflammatory cytokine mRNA expression between TiO(2)-1 and TiO(2)-2 treatments. However, TiO(2)-2, but not TiO(2)-1, generated high levels of reactive oxygen species (ROS) in cell-free conditions. Although both TiO(2)-1 and TiO(2)-2 resulted in intracellular ROS formation, TiO(2)-2 elicited a stronger response, resulting in cytotoxicity to the QR-32 cells. Moreover, TiO(2)-2, but not TiO(2)-1, led to the development of nuclear interstices and multinucleate cells. Cells that survived the TiO(2) toxicity acquired a tumorigenic phenotype. TiO(2)-induced ROS formation and its related cell injury were inhibited by the addition of antioxidant N-acetyl-l-cysteine. These results indicate that nano-sized TiO(2) has the potential to convert benign tumor cells into malignant ones through the generation of ROS in the target cells.

Peroxiredoxin 4 knockout results in elevated spermatogenic cell death via oxidative stress.

Prx (peroxiredoxin) is a multifunctional redox protein with thioredoxin-dependent peroxidase activity. Prx4 is present as a secretory protein in most tissues, whereas in sexually mature testes it is anchored in the ER (endoplasmic reticulum) membrane of spermatogenic cells via an uncleaved N-terminal hydrophobic peptide. We generated a Prx4 knockout mouse to investigate the function of Prx4 in vivo. Prx4(-/y) mice lacking Prx4 expression in all cells were obtained by mating Prx4(flox/+) female mice with Cre-transgenic male mice that ubiquitously expressed Cre recombinase. The resulting Prx4(-/y) male mice were fertile, and most organs were nearly normal in size, except for testicular atrophy. The number of deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive spermatogenic cells was higher in Prx4(-/y) mice than in Prx4(+/y) mice and increased remarkably in response to warming the lower abdomen at 43 degrees C for 15 min. Cells reactive to antibodies against 4-hydroxynonenal and 8-hydroxyguanine were high in the Prx4(-/y) mice and concomitant with elevated oxidation of lipid and protein thiols. The cauda epididymis of Prx4(-/y) mice contained round spermatocytes, which were not found in Prx4(+/y) mice, and displayed oligozoospermia. However, mature spermatozoa from the epididymis of Prx4(-/y) mice exhibited normal fertilization In vitro. Taken together, these results indicate that spermatogenic cells lacking Prx4 are more susceptible to cell death via oxidative damage than their wild-type counterparts. Our results suggest that the presence of Prx4, most likely the membrane-bound form, is important for spermatogenesis, but not an absolute requisite.

Testis-specific peroxiredoxin 4 variant is not absolutely required for spermatogenesis and fertility in mice.

PRDX4, a member of peroxiredoxin family, is largely concentrated in the endoplasmic reticulum (ER) and plays a pivotal role in the redox relay during oxidative protein folding as well as in peroxidase reactions. A testis-specific PRDX4 variant transcript (PRDX4t) lacks the conventional exon 1, which encodes the signal peptide that is required for entry into the ER lumen, but instead carries alternative exon 1, which is transcribed from the upstream promoter in a testis-specific manner and results in the PRDX4t protein being localized in the cytosol. However, the potential roles of PRDX4t in male genital action remain unknown. Using a CRISPR/Cas9 system, we first disrupted the testis-specific promoter/exon 1 and generated mice that were specifically deficient in PRDX4t. The resulting PRDX4t knockout (KO) mice underwent normal spermatogenesis and showed no overt abnormalities in the testis. Mating PRDX4t KO male mice with wild-type (WT) female mice produced normal numbers of offspring, indicating that a PRDX4t deficiency alone had no effect on fertility in the male mice. We then generated mice lacking both PRDX4 and PRDX4t by disrupting exon 2, which is communal to these variants. The resulting double knockout (DKO) mice were again fertile, and mature sperm isolated from the epididymis of DKO mice exhibited a normal fertilizing ability in vitro. In the meantime, the protein levels of glutathione peroxidase 4 (GPX4), which plays an essential role in the disulfide bond formation during spermatogenesis, were significantly increased in the testis and caput epididymis of the DKO mice compared with the WT mice. Based on these results, we conclude that the disruption of the function of PRDX4t in the spermatogenic process appears to be compensated by other factors including GPX4.

[Magnetic resonance imaging findings on the eyelids of Japanese cadavers for anatomical studies and a comparative examination of their histological pictures].

OBJECTIVE: To clarify ambiguous areas in interpreting MR images of Japanese eyelids, a histological examination was conducted on cadavers after the MRI for a comparative evaluation. SUBJECTS AND METHODS: Orbital sections including the unilateral upper and lower palpebrae of two Japanese cadavers (an 87-year-old woman and a 49-year-old man) were examined. Following MRI, the specimens of the same cadavers were examined histologically for a comparative evaluation. RESULTS: In both cadavers, a high signal intensity area with a hazy appearance unlike the orbital fat--fibroadipose tissue rich with connective tissue--was recognized between the orbicularis muscle and orbital septum. The same high signal intensity area that appeared to encase the posterior section of the descending orbital fat was also composed of fibroadipose tissue. Because of the presence of this intervening fibroadipose tissue, the posterior surface of the orbicularis muscle and the orbital septum could not come into contact with each other. CONCLUSION: Although limited to only two subjects, the current observation proved that fibroadipose tissue exists not only in the superficial layer of the orbital fat but that it further descends to surround the lowest portion of the orbital fat. As already reported, it was mainly the fibroadipose tissue, but not the orbital fat, that descends into the palpebral space.

Elevated ER stress exacerbates dextran sulfate sodium-induced colitis in PRDX4-knockout mice.

BACKGROUND AND AIMS: Peroxiredoxin 4 (PRDX4), a secretory protein that is preferentially retained in the endoplasmic reticulum (ER), is encoded by a gene located on the X chromosome and highly expressed in colonic tissue. In this study, we investigated the role of PRDX4 by means of male PRDX4-knockout (PRDX4-/y) mice in the development of intestinal inflammation using a dextran sulfate sodium (DSS)-induced colitis model. MATERIALS AND METHODS: Acute colitis was induced with DSS (2.5% in drinking water) in wild-type (WT) and PRDX4-/y male C57BL/6 mice. Histological and biochemical analyses were performed on the colonic tissues. RESULTS: PRDX4 was mainly localized in the colonic epithelial cells in WT mice. The disease activity index (DAI) scores of PRDX4-/y mice were significantly higher compared to those of WT mice. Specifically, PRDX4-/y mice showed marked body weight loss and shortening of colon length compared to WT mice, whereas the myeloperoxidase levels were increased in PRDX4-/y compared to WT mice. In addition, the mRNA expression levels of TNF-α and IFN-γ were significantly higher in the colonic mucosa of PRDX4-/y compared to WT mice. Moreover, the levels of CHOP and activated caspase 3 were higher in the colonic tissues of PRDX4-/y compared to WT mice following treatment with DSS. The ER also showed greater expansion in PRDX4-/y than WT mice, which was consistent with severe ER stress under PRDX4 deficiency. CONCLUSION: Our results demonstrated that the lack of PRDX4 aggravated the colonic mucosal damage induced by DSS. Because PRDX4 functions as an ER thiol oxidase as well as an antioxidant, DSS induced oxidative damage and ER stress to a greater degree in PRDX4-/y than WT mice. These findings suggest that PRDX4 may represent a novel therapeutic molecule in intestinal inflammation.

Electron microscopic observations of the anterior pituitary gland. Part I. The neurons in the "transitional zone" of the anterior pituitary gland.

Since [Westlud, K.N., Chils, G.V., 1982. Localization of serotonin fibers in the rat adenohypophysis. Endocrinology 111, 1761-1763] initially identified the serotonin nerve fibers in the anterior pituitary gland, attention has been paid to the rostral zone of the anterior lobe into which nerve fibers enter and subsequently spread to deeper regions of the lobe. The rostral zone is the trifurcated junction of the partes tuberalis, intermedia and distalis, and has the important role(s) for hormone secretion via the "transitional zone" [Sato, G, Shirasawa, N, Sakuma, E, Sato, Y, Asai, Y, Wada, I, Horiuchi, O, Sakamoto, A, Herbert, DC, Soji, T, 2005a. Intercellular communications within the rat anterior pituitary. XI: An immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat. Tissue and Cell 37, 269-280.]. The objective of this study was to focus on the ultrastructure of this "zone." All of the animals studied were fixed by perfusion with glutaraldehyde via the left ventricle of the heart and examined by electron microscopy. In the "transitional zone," a cluster of neuronal elements was observed between the folliculo-stellate cell-rich area and the anterior lobe. This cluster consisted of myelinated fibers, unmyelinated fibers, neuroendocrine fibers, large cells, and supporting cells. The large cells were perikarya of neurons which made a "ganglion-like" structure with associated satellite cells. Agranular, folliculo-stellate cells were intermingled among the elements. This is the first report that neuronal elements form clusters in the "transitional zone." A relationship of the unmyelinated and neuroendocrine fibers in the basal layer and in the "transitional zone" is discussed.

5-aminolevulinic acid (ALA) deficiency causes impaired glucose tolerance and insulin resistance coincident with an attenuation of mitochondrial function in aged mice.

In vertebrates, the initial step in heme biosynthesis is the production of 5-aminolevulinic acid (ALA) by ALA synthase (ALAS). ALA formation is believed to be the rate-limiting step for cellular heme production. Recently, several cohort studies have demonstrated the potential of ALA as a treatment for individuals with prediabetes and type-2 diabetes mellitus. These studies imply that a mechanism exists by which ALA or heme can control glucose metabolism. The ALAS1 gene encodes a ubiquitously expressed isozyme. Mice heterozygous null for ALAS1 (A1+/-s) experience impaired glucose tolerance (IGT) and insulin resistance (IR) beyond 20-weeks of age (aged A1+/-s). IGT and IR were remedied in aged A1+/-s by the oral administration of ALA for 1 week. However, the positive effect of ALA proved to be reversible and was lost upon termination of ALA administration. In the skeletal muscle of aged A1+/-s an attenuation of mitochondrial function is observed, coinciding with IGT and IR. Oral administration of ALA for 1-week brought about only a partial improvement in mitochondrial activity however, a 6-week period of ALA treatment was sufficient to remedy mitochondrial function. Studies on differentiated C2C12 myocytes indicate that the impairment of glucose metabolism is a cell autonomous effect and that ALA deficiency ultimately leads to heme depletion. This sequela is evidenced by a reduction of glucose uptake in C2C12 cells following the knockdown of ALAS1 or the inhibition of heme biosynthesis by succinylacetone. Our data provide in vivo proof that ALA deficiency attenuates mitochondrial function, and causes IGT and IR in an age-dependent manner. The data reveals an unexpected metabolic link between heme and glucose that is relevant to the pathogenesis of IGT/IR.

Gastric 17ß-estradiol in portal vein and liver Esr1 make a circadian rhythm in systemic circulation in male rats.

The hemodynamics of 17ß-estradiol (E2) synthesized and secreted from the stomach has been revealed gradually. This study aimed to clarify the circadian rhythm of E2 synthesis and secretion in the stomach, and the relationship between the expression of hepatic estrogen receptor (ER) α and serum E2 levels in systemic circulation. Wistar male rats were maintained in a room with a 12-h light and 12-h dark cycle (lights on from 0700 to 1900 h), and were sacrificed at every 4-hour interval starting at 0800 h. The results showed that the expression of gastric Cyp19a1 was higher in nighttime than in daytime, and that the portal venous E2 level was 2.2 times higher at 2400 h than that at 1200 h. The arterial E2 level was also the highest at 2400 h, and showed an apparent circadian rhythm positively correlated with portal venous E2 levels. Conversely, the expression of liver Esr1 peaked at 1200 h and turned to decrement at 2400 h. The population of immunoreactive nuclei with ERα antibody decreased at 2400 h compared with that at 1200 h. The regression analysis showed that the liver Esr1 mRNA was negatively correlated to portal venous and arterial E2 levels. It could be concluded that the circadian rhythm of the systemic E2 level depended both on the amounts of gastric E2 in the portal vein and on the Esr1 expression in the liver.

The kinase domain of death-associated protein kinase is inhibitory for tubulointerstitial fibrosis in chronic obstructive nephropathy.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that is thought to mediate apoptosis. We have shown that the kinase domain of DAPK is crucial for the induction of renal tubular cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. DAPK-mutant mice, generated by deletion of 74 amino acids from the catalytic kinase domain, were used to investigate the role of the DAPK kinase domain in renal fibrosis following COU. Interstitial collagen and alpha-smooth muscle actin (alpha-SMA) expressions in situ were compared between obstructed kidneys in wild-type and mutant mice. As a result, tubulointerstitial fibrosis, as quantified by interstitial collagen expression, was significantly augmented in mutant kidneys compared with wild-type kidneys following COU. Furthermore, deletion of the kinase domain from DAPK significantly increased the appearance of alpha-SMA-positive myofibroblasts in the renal interstitium during COU. Thus, our results suggest that the kinase domain deleted by gene targeting plays a suppressive role for the development of renal fibrosis through inhibition of the tubular epithelial-to-mesenchymal transition in a mouse model of COU.

Intercellular communications within the rat anterior pituitary XII: immunohistochemical and physiological evidences for the gap junctional coupling of the folliculo-stellate cells in the rat anterior pituitary.

Since Farquhar [1957. "Corticotrophs" of the rat adenohypophysis as revealed by electron microscopy. Anat. Rec. 127, 291] was the first to report the presence of agranular folliculo-stellate cells as corticotrophs in the anterior pituitary gland, there were no reports about electro-physiological characteristics of the folliculo-stellate cells because of its no hormonal activity and the chaotic distribution of the parenchyma cells. Male Wistar rats, aged 7 weeks with weighing 250--300 g, were separated into two groups. One group was used for immunohistochemical and light microscopical studies to detect S-100 protein and connexin 43. The other group was used for the electro-physiological study and then for the electron microscopical study to know the fine structural character of folliculo-stellate cells after the electro-physiological experiment. Clusters of S-100 protein cells (agranulated folliculo-stellate cells) and numerous connexin 43 positive sites on S-100 protein cells were clear in the "transitional zone" at which the pituitary tissue made the transition from the pars tuberalis to the proximal part of the anterior lobe. Penetration of electrodes to the cells distributed in the transitional zone showed stable membrane potential ranged between--27 and--67mV with no spontaneous activity. Random penetration of electrode showed that larger populations of cell ( approximately 80%) had membrane potentials with -55.6+/-5.1 mV, and less than 20% of cells had the resting membrane potential with -36.0+/-4.4 mV. There were two types of cell couplings; one major group for the recordings from cells with similar deep resting membrane potentials and the other for the recordings from cells with different resting membrane potentials. The former indicated that two cells were electrically coupled while the latter no electrical couples were observed. Carbenoxolone depolarized the membrane by 12.3+/-5.5 mV and reduced the amplitude of electrotonic potentials, and the response recovered by removal of carbenoxolone by the superfusate. The transitional zones of the pituitary glands examined the electrical coupling were observed by an electron microscopy. Almost cytological profiles were observed as intact. The results clearly indicated that the folliculo-stellate cell system deeply participated in the regulation of the anterior pituitary parallel with the portal vessel system, which was the main regulatory system for pituitary hormone secretion.

Intercellular communications within the rat anterior pituitary XI: an immunohistochemical study of distributions of S-100 positive cells in the anterior pituitary of the rat.

The distribution of the S-100 protein cell (folliculo-stellate cell) is very important to our understanding of the regulation of the anterior pituitary. In this study, 10 intact 60-day-old male Wistar-Imamichi rats, were separated equally into two groups. One was used for immunohistochemical study, and the other for electron microscopic analysis. Immunostained pituitary sections with S-100 protein antibody were photographed using a CCD camera equipped with a computer. The S-100 protein cells were then measured using NIH image software, and the three-dimensional distribution of the cells was analyzed. The distribution of the cells observed in each serial section showed that S-100 protein cells were dense at the basal zone of the gland and at the "transitional zone" where the pars tuberalis adjoined the anterior and intermediate lobes, where they represented over 50% of the total cell population. They then decreased in number with distance from this region to mid-way towards the sagittal axis before increasing again in the tail of the gland. The population of cells also decreased with increasing distance from the "transitional zone" to the wing and with distance from the basal zone. Portal vessels entered the anterior lobe through the "transitional zone" as thick capillaries, ran through the basal surface and penetrated into the central area of the anterior lobe. In all planes, S-100 protein cells encircled the capillaries. Ultrastructural observations confirmed the light microscopic findings indicating that clusters of agranular cells were densely located at the "transitional zone" and in the pars tuberalis. The distribution pattern of the folliculo-stellate cells and the capillaries showed good agreement and the spatial relationship between these two is detailed so as to better understand hypophyseal histophysiology.

Gastric parietal cells: potent endocrine role in secreting estrogen as a possible regulator of gastro-hepatic axis.

Estrogen, if it is produced in the gastrointestinal tract, may overflow into the systemic circulation in the case of increased portal-systemic shunting. This idea is in accord with a significant step-up in serum estradiol (E2) concentration in the portal vein of rats, compared with that in the artery. Gene expression of aromatase, estrogen synthetase, was demonstrated by RT-PCR in the gastric mucosa of male and female adult rats, equivalent to that in the ovary. Aromatase activity and production of E2 in the gastric mucosa were demonstrated by (3)H(2)O assay and gas chromatography-mass spectrometry, and they were inhibited by aromatase inhibitor, 4-hydroxyandrostenedione. Conversion of (14)C-androstenedione to (14)C-E2 through (14)C-testosterone in cultured gastric mucosa was also demonstrated. Parietal cells exhibited strong signals for aromatase mRNA and immunoreactive protein by in situ hybridization histochemistry and immunohistochemistry. Estrogen receptor alpha mRNA and immunoreactive protein were demonstrated in hepatocytes by RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Total gastrectomy reduced portal venous E2 concentration, without changing systemic E2 concentration, together with down-regulation of estrogen receptor alpha mRNA level in the liver. These findings indicate that gastric parietal cells play a potent endocrine role in secreting estrogen that may function as a regulator of the gastro-hepatic axis.

Intercellular communication within the rat anterior pituitary gland: X. Immunohistocytochemistry of S-100 and connexin 43 of folliculo-stellate cells in the rat anterior pituitary gland.

Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells were connected; the cells were identified as S-100 cells. Double immunohistochemical staining of the connexin 43 and growth hormone (GH) or connexin 43 and luteinizing hormone (LH) was also performed, demonstrating no direct relationship between the connexin 43 and either the GH or LH cells. These findings indicate that there are two kinds of messages necessary for the hormone release in the pituitary gland. One is via the portal vein system, the other is through the gap junction-mediated networks of folliculo-stellate cells. The granulated cells directly associate with cell membrane of folliculo-stellate cells are able to discharge secretory granules through communication via gap junctions, while those granulated cells that are more distant from the folliculo-stellate cells are only able to discharge hormones via the pituitary hormone-releasing hormone from the portal vein system.

Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa.

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.

Deletion of the kinase domain in death-associated protein kinase attenuates renal tubular cell apoptosis in chronic obstructive uropathy.

Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-dependent serine/threonine kinase that has been implicated as a positive mediator of apoptosis. However, little is known about the involvement of DAPK in the apoptosis associated with several pathological states, except for cancer. Here, DAPK-mutant mice were used in order to examine the role of DAPK in renal cell apoptosis in chronic obstructive uropathy (COU) created by unilateral ureteral ligation. These mice express mutant DAPK with a deletion of 74 amino acids from the catalytic kinase domain. Obstructed kidneys in wild-type and mutant mice were examined for both DAPK protein levels and renal cell apoptosis during the course of COU. Obstructed kidneys in wild-type and mutant mice showed a marked increase in the DAPK and mutant DAPK protein levels, respectively, at day 14 after ureteric ligation. The obstructed kidneys in DAPK-mutant mice displayed a significant attenuation of tubular cell apoptosis, compared with wild-type mice. In contrast, no significant difference in interstitial cell apoptosis was observed between the obstructed kidneys from wild-type and mutant mice. Thus, these results indicate that the part of the kinase domain deleted by the gene targeting is crucial for the execution of tubular cell apoptosis, but is not essential for interstitial cell apoptosis in a COU model in mice.

Death-associated protein kinase localization to human renal tubule cells, and increased expression of chronic obstructive uropathy in rats.

BACKGROUND: Death-associated protein kinase (DAP kinase) is a Ca2+/calmodulin-dependent serine/threonine kinase implicated as a positive apoptosis mediator. However, little is known about DAP kinase involvement with apoptosis in renal diseases. METHODS: In order to determine whether DAP kinase has a role in renal cell apoptosis in kidney diseases, we performed an immunohistochemical study using a monoclonal antibody to DAP kinase. Firstly, by examining the cellular distribution of DAP kinase in normal human renal tissues and cultured proximal tubule cells. We then used western blotting and immunohistochemical analysis to examine directly whether DAP kinase protein levels could be modulated in rat kidneys with chronic obstructive uropathy created by unilateral ureteric ligation. RESULTS: Immunohistochemistry of normal human kidney tissues showed that DAP kinase was exclusively localized in the cytoplasm of renal tubule cells. Expression analysis of DAP kinase using cultured cells confirmed DAP kinase mRNA and protein presence in human renal tubule cells. Immunocytochemical analysis directly visualized DAP kinase in the cytoplasm of the renal tubule cells in culture. Finally, DAP kinase was found up-regulated in renal tubule cells of rat kidneys with chronic obstructive uropathy. CONCLUSIONS: Our study demonstrates that DAP kinase is localized to renal tubule cells, implying a crucial role for DAP kinase in renal tubular cell apoptosis in progressive renal diseases.

Changes of gastric aromatase and portal venous 17ß-estradiol during the postnatal development and estrus cycle in female rats.

Gastric parietal cells synthesize and secrete a large amount of 17ß-estradiol into the portal vein. However, there are few studies on the gastric 17ß-estradiol during the postnatal development and estrus cycle. The purpose of this study is to clarify the onset and the prepubertal change of gastric 17ß-estradiol synthesis; and the effect of gastric 17ß-estradiol on the estrus cycle. Wistar female rats aged from 15 to 40 days and 10 weeks were used in the study. The expression of aromatase and estrogen receptor (ER) α mRNAs and proteins was analyzed in the stomach, ovary, and liver by RT-PCR, immunohistochemistry, and Western blotting methods; and 17ß-estradiol levels in the artery and portal vein were assayed by the ELISA method. During postnatal development, aromatase protein and aromatase cells in gastric mucosa and portal venous 17ß-estradiol levels started increasing after 20 days, and then these subjects reached nearly the same levels as mature female rats at 40 days. In the estrus cycle, the arterial 17ß-estradiol level in proestrus was the highest, and the value was 60 % of the portal venous level. Gastric aromatase protein and portal venous 17ß-estradiol levels did not change during the estrus cycle. Ovarian ERα levels fluctuated in the same pattern of arterial 17ß-estradiol; however, hepatic ERα levels went unchanged. These results showed that gastric aromatase in females expresses earlier than the sexual maturation, and the gastric aromatase protein reaches the same levels as mature rats at 40 days. Furthermore, 17ß-estradiol synthesis and secretion in the stomach is not related to those in the ovary.