Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Computer vision analysis of mother-infant interaction identified efficient pup retrieval in V1b receptor knockout mice.

Close contact between lactating rodent mothers and their infants is essential for effective nursing. Whether the mother's effort to retrieve the infants to their nest requires the vasopressin-signaling via V1b receptor has not been fully defined. To address this question, V1b receptor knockout (V1bKO) and control mice were analyzed in pup retrieval test. Because an exploring mother in a new test cage randomly accessed to multiple infants in changing backgrounds over time, a computer vision-based deep learning analysis was applied to continuously calculate the distances between the mother and the infants as a parameter of their relationship. In an open-field, a virgin female V1bKO mice entered fewer times into the center area and moved shorter distances than wild-type (WT). While this behavioral pattern persisted in V1bKO mother, the pup retrieval test demonstrated that total distances between a V1bKO mother and infants came closer in a shorter time than with a WT mother. Moreover, in the medial preoptic area, parts of the V1b receptor transcripts were detected in galanin- and c-fos-positive neurons following maternal stimulation by infants. This research highlights the effectiveness of deep learning analysis in evaluating the mother-infant relationship and the critical role of V1b receptor in pup retrieval during the early lactation phase.

Vasopressin V1a receptor and oxytocin receptor regulate murine sperm motility differently.

Specific receptors for the neurohypophyseal hormones, arginine vasopressin (AVP) and oxytocin, are present in the male reproductive organs. However, their exact roles remain unknown. To elucidate the physiological functions of pituitary hormones in male reproduction, this study first focused on the distribution and function of one of the AVP receptors, V1a. In situ hybridization analysis revealed high expression of the Avpr1a in Leydig cells of the testes and narrow/clear cells in the epididymis, with the expression pattern differing from that of the oxytocin receptor (OTR). Notably, persistent motility and highly proportional hyperactivation were observed in spermatozoa from V1a receptor-deficient mice. In contrast, OTR blocking by antagonist atosiban decreased hyperactivation rate. Furthermore, AVP stimulation could alter the extracellular pH mediated by the V1a receptor. The results highlight the crucial role of neurohypophyseal hormones in male reproductive physiology, with potential contradicting roles of V1a and OTR in sperm maturation. Our findings suggest that V1a receptor antagonists are potential therapeutic drugs for male infertility.

Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

Agonist dependency of the second phase access of ß-arrestin 2 to the heteromeric µ-V1b receptor.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to ß-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that ß-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and ß-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, ß-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of ß-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of ß-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

Perivascular macrophages produce type I collagen around cerebral small vessels under prolonged hypertension in rats.

Hypertension leads to structural remodeling of cerebral blood vessels, which has been implicated in the pathophysiology of cerebrovascular diseases. The remodeling and progression of arteriolosclerosis under hypertension involve fibrosis along with the production of type I collagen around cerebral arterioles. However, the source and regulatory mechanisms of this collagen production remain elusive. In this study, we examined if perivascular macrophages (PVMs) are involved in collagen production around cerebral small vessels in hypertensive SHRSP/Izm rats. Immunoreactivity for type I collagen around cerebral small vessels in 12-week-old hypertensive rats tended to higher than those in 4-week-old hypertensive and 12-week-old control rats. In ultrastructural analyses using transmission electron microscopy, the substantial deposition of collagen fibers could be observed in the intercellular spaces around PVMs near the arterioles of rats with prolonged hypertension. In situ hybridization analyses revealed that cells positive for mRNA of Col1a1, which comprises type I collagen, were observed near cerebral small vessels. The Col1a1-positive cells around cerebral small vessels were colocalized with immunoreactivity for CD206, a marker for PVMs, but not with those for glial fibrillary acidic protein or desmin, markers for other perivascular cells such as astrocytes and vascular smooth muscle cells. These results demonstrated that enhanced production of type I collagen is observed around cerebral small vessels in rats with prolonged hypertension and Col1a1 is expressed by PVMs, and support the concept that PVMs are involved in collagen production and vascular fibrosis under hypertensive conditions.

Differential Localization and Invasion of Tumor Cells in Mouse Models of Human and Murine Leukemias.

Leukemias are refractory hematopoietic malignancies, for which the development of new therapeutic agents requires in vivo studies using tumor-bearing mouse models. Although several organs are commonly examined in such studies to evaluate the disease course, the effectiveness of interventions and the localization of tumor cells in the affected organs are still unclear. In this study, we histologically examined the distribution of leukemia cells in several organs using two leukemic mouse models produced by the administration of two cell lines (THP-1, a human myelomonocytic leukemia, and A20, a mouse B cell leukemia/lymphoma) to severe immunodeficient mice. Survival of the mice depended on the tumor burden. Although A20 and THP-1 tumor cells massively infiltrated the parenchyma of the liver and spleen at 21 days after transplantation, A20 cells were hardly found in connective tissues in Glisson's capsule in the liver as compared with THP-1 cells. In the bone marrow, there was more severe infiltration of A20 cells than THP-1 cells. THP-1 and A20 cells were widely spread in the lungs, but were rarely observed in the small intestine. These findings suggest that each leukemia model has a unique localization of tumor cells in several affected organs, which could critically affect the disease course and the efficacy of therapeutic agents, including cellular immunotherapies.

Immunohistochemical Study of the Laminin α5 Chain and Its Specific Receptor, Basal Cell Adhesion Molecule (BCAM), in both Fetal and Adult Rat Pituitary Glands.

Laminin, a major basement membrane protein, comprises three subunit chains: α, ß, and γ chains. Among these chains, only the laminin α chain is capable of signaling via laminin receptors. Although laminin isoforms containing the α5 chain were reported to be the first laminin produced during rat anterior pituitary gland development, the functions of these isoforms are unknown. We used immunohistochemical techniques to localize the laminin α5 chain and its specific receptor, basal cell adhesion molecule (BCAM), in fetal and adult pituitary gland. Laminin α5 chain immunoreactivity was observed in the basement membrane of the primordial adenohypophysis at embryonic days 12.5 to 19.5. Double immunostaining showed that BCAM was present and co-localized with the laminin α5 chain in the tissue. Quantitative analysis showed that the laminin α5 chain and BCAM were expressed in the anterior pituitary gland during postnatal development and in adulthood (postnatal day 60). In the adult gland, co-localization of the laminin α5 chain and BCAM was observed, and BCAM was detected in both the folliculo-stellate cells and endothelial cells. These results suggest that laminin α5 chain signaling via BCAM occurs in both the fetal adenohypophysis and adult anterior pituitary gland.

Methodological Improvements With Conductive Materials for Volume Imaging of Neural Circuits by Electron Microscopy.

Recent advancements in electron microscope volume imaging, such as serial imaging using scanning electron microscopy (SEM), have facilitated the acquisition of three-dimensional ultrastructural information of biological samples. These advancements help build a comprehensive understanding of the functional structures in entire organelles, cells, organs and organisms, including large-scale wiring maps of neural circuitry in various species. Advanced volume imaging of biological specimens has often been limited by artifacts and insufficient contrast, which are partly caused by problems in staining, serial sectioning and electron beam irradiation. To address these issues, methods of sample preparation have been modified and improved in order to achieve better resolution and higher signal-to-noise ratios (SNRs) in large tissue volumes. These improvements include the development of new embedding media for electron microscope imaging that have desirable physical properties such as less deformation in the electron beam and higher stability for sectioning. The optimization of embedding media involves multiple resins and filler materials including biological tissues, metallic particles and conductive carbon black. These materials alter the physical properties of the embedding media, such as conductivity, which reduces specimen charge, ameliorates damage to sections, reduces image deformation and results in better ultrastructural data. These improvements and further studies to improve electron microscope volume imaging methods provide options for better scale, quality and throughput in the three-dimensional ultrastructural analyses of biological samples. These efforts will enable a deeper understanding of neuronal circuitry and the structural foundation of basic and higher brain functions.

Notch Signaling and Maintenance of SOX2 Expression in Rat Anterior Pituitary Cells.

After publication of reports describing the presence of stem/progenitor cells among non-hormone-producing cells in the pituitary, the mechanism responsible for proliferation and differentiation generated considerable interest. Several studies have suggested that Notch signaling is involved. In the present study, we examined the histochemical relationship between Notch signaling molecules and the transcription factor SOX2 in rat pituitary. Combined in situ hybridization and immunohistochemistry showed that Notch2 mRNA and SOX2 were co-expressed at embryonic day 14.5 in most cells in the adenohypophyseal primordium. In adult rat pituitary, double immunohistochemistry showed that SOX2 and either Notch2 or the Notch signaling target HES1 were co-localized within cells with large oval nuclei in both the marginal cell layer and cell aggregates in the main part of the anterior lobe, which are believed to be stem cell niches. Furthermore, when the Notch signaling inhibitor DAPT was added to a primary culture of adult rat anterior pituitary cells, the proportion of SOX2-expressing cells within Notch2-positive cells was approximately 30% lower. These findings suggest that Notch signaling has a role in maintaining the stemness of precursor cells in the adult rat pituitary gland.

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10-6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Tissue inhibitors of metalloproteinase-expressing cells in human anterior pituitary and pituitary adenoma.

Extracellular matrix (ECM) is essential in tissue physiology and pathologic conditions such as tumorigenesis. It affects tumor cell behavior, proliferation, and metastasis. Pituitary adenomas differ in their clinical characteristics, including ECM deposition, and we recently reported that the characteristics of collagen-producing cells differed between control human anterior pituitary gland and pituitary adenomas. ECM deposition is not defined solely by production; degradation and maintenance are also important. Tissue inhibitors of metalloproteinases (TIMPs) help maintain ECM by inhibiting degradation caused by matrix metalloproteases. The present study attempted to characterize TIMP-expressing cells in the human anterior pituitary. Specimens of human pituitary adenomas and control pituitary were obtained during surgery, and in situ hybridization for TIMP1, TIMP2, TIMP3, and TIMP4, followed by immunohistochemistry, was used to characterize TIMP-expressing cells. TIMP expression exhibited a distinct pattern in the human anterior pituitary. Azan staining showed that fibrous matrix deposition varied among pituitary adenomas and that the area of fibrosis was associated with the number and number of types of TIMP3-expressing cells. These results suggest that TIMPs are important in the maintenance of ECM in human pituitary and that TIMP expressions are altered in fibrosis associated with pituitary adenoma.

Notch signaling-mediated cell-to-cell interaction is dependent on E-cadherin adhesion in adult rat anterior pituitary.

The rat anterior pituitary is composed of hormone-producing cells, non-hormone-producing cells (referred to as folliculostellate cells) and marginal layer cells. In the adult rat, progenitor cells of hormone-producing cells have recently been reported to be maintained within this non-hormone-producing cell population. In tissue, non-hormone-producing cells construct homophilic cell aggregates by the differential expression of the cell adhesion molecule E-cadherin. We have previously shown that Notch signaling, a known regulator of progenitor cells in a number of organs, is activated in the cell aggregates. We now investigate the relationship between Notch signaling and E-cadherin-mediated cell adhesion in the pituitary gland. Immunohistochemically, Notch signaling receptor Notch2 and the ligand Jagged1 were localized within E-cadherin-positive cells in the marginal cell layer and in the main part of the anterior lobe, whereas Notch1 was localized in E-cadherin-positive and -negative cells. Activation of Notch signaling within E-cadherin-positive cells was confirmed by immunostaining of the Notch target HES1. Notch2 and Jagged1 were always co-localized within the same cells suggesting that homologous cells have reciprocal effects in activating Notch signaling. When the E-cadherin function was inhibited by exposure to a monoclonal antibody (DECMA-1) in primary monolayer cell culture, the percentage of HES1-positive cells among Notch2-positive cells was less than half that of the control. The present results suggest that E-cadherin-mediated cell attachment is necessary for the activation of Notch signaling in the anterior pituitary gland but not for the expression of the Notch2 molecule.

Effects of retinoic acid on growth hormone-releasing hormone receptor, growth hormone secretagogue receptor gene expression and growth hormone secretion in rat anterior pituitary cells.

Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.

Folliculostellate cell interacts with pericyte via TGFß2 in rat anterior pituitary.

The anterior pituitary gland comprises five types of endocrine cells plus non-endocrine cells including folliculostellate cells, endothelial cells, and capillary mural cells (pericytes). In addition to being controlled by the hypothalamic-pituitary-target organ axis, the functions of these cells are likely regulated by local cell and extracellular matrix (ECM) interactions. However, these complex interactions are not fully understood. We investigated folliculostellate cell-mediated cell-to-cell interaction. Using S100ß-GFP transgenic rats, which express GFP in folliculostellate cells, we designed a three-dimensional cell culture to examine the effects of folliculostellate cells. Interestingly, removal of folliculostellate cells reduced collagen synthesis (Col1a1 and Col3a1). Because pericytes are important collagen-producing cells in the gland, we stained for desmin (a pericyte marker). Removal of folliculostellate cells resulted in fewer desmin-positive pericytes and less desmin mRNA. We then attempted to identify the factor mediating folliculostellate cell-pericyte interaction. RT-PCR and in situ hybridization revealed that the important profibrotic factor transforming growth factor beta-2 (TGFß2) was specifically expressed in folliculostellate cells and that TGFß receptor II was expressed in pericytes, endothelial cells, and parenchymal cells. Immunocytochemistry showed that TGFß2 induced SMAD2 nuclear translocation in pericytes. TGFß2 increased collagen synthesis in a dose-dependent manner. This action was completely blocked by TGFß receptor I inhibitor (SB431542). Diminished collagen synthesis in folliculostellate cell-deficient cell aggregates was partially recovered by TGFß2. TGFß2-mediated folliculostellate cell-pericyte interaction appears to be essential for collagen synthesis in rat anterior pituitary. This finding sheds new light on local cell-ECM interactions in the gland.

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.

Fibromodulin Expression in Folliculostellate Cells and Pericytes Is Promoted by TGFß Signaling in Rat Anterior Pituitary Gland.

Fibromodulin belongs to the family of small leucine-rich proteoglycans (SLRPs), an active component of extracellular matrix. It directly binds collagens to promote fibrillogenesis and also binds transforming growth factor-beta (TGFß) to antagonize its actions. Our previous studies of rat anterior pituitary gland revealed that fibromodulin is expressed in folliculostellate cells and pericytes. Although our recent study showed that TGFß2 secreted from folliculostellate cells induces collagen synthesis in pericytes, the involvement of fibromodulin in TGFß2-mediated collagen regulation has not been studied. The present study examined the effect of TGFß2 on fibromodulin synthesis in rat anterior pituitary gland. In situ hybridization for TGFß receptor II and immunohistological techniques revealed the presence of TGFß receptor II in folliculostellate cells and pericytes. To confirm canonical TGFß intracellular signaling, Smad2 immunocytochemistry was performed. Nuclear translocation of Smad2 was observed in folliculostellate cells and pericytes after TGFß2 treatment. TGFß2 strongly enhanced fibromodulin mRNA and protein expressions, and TGFß2-induced mRNA expression was completely blocked by TGFß receptor I inhibitor (SB431542). These results suggest that folliculostellate cells and pericytes exhibit canonical TGFß2 signaling, which is associated with fibromodulin production. Thus, this is the first report to show that TGFß signaling regulates the endogenous TGFß antagonist fibromodulin in the gland.

Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors.

Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases.

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

Expression of the heparin-binding growth factor midkine and its receptor, Ptprz1, in adult rat pituitary.

Midkine (MK) belongs to a family of secreted heparin-binding growth factors and is highly expressed in various tissues during development. MK has multiple functions, such as regulation of cell proliferation, migration, survival and differentiation. We recently reported that MK mRNA is strongly expressed in the developing rat pituitary gland. In the adult pituitary, however, expression of MK and its receptor and the characteristics of the cells that produce them, have not been determined. Therefore, in this study, we investigate whether MK and its receptor, protein tyrosine phosphatase receptor-type Z (Ptprz1), are present in the adult rat pituitary. In situ hybridization, real-time reverse transcription-PCR and immunoblotting were performed to assess MK and Ptprz1 expression. We also characterize MK- and Ptprz1-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for each pituitary hormone or S100 protein [a marker of folliculostellate (FS) cells]. MK-expressing cells were located in the anterior and posterior lobes but not in the intermediate lobe. Double-staining and immunoblotting revealed that MK mRNA and protein were only expressed in FS cells in the anterior pituitary. Regarding Ptprz1 expression, Ptprz1 mRNA was detected in adrenocorticotropic hormone (ACTH) cells and growth hormone (GH) cells but not in prolactin cells, thyroid-stimulating hormone cells, luteinizing hormone cells, or FS cells. These findings suggest that MK produced in FS cells acts locally on ACTH cells and GH cells via Ptprz1 in the adult rat anterior pituitary.