Regulation of Aqp1 expression by osmotic balance in fenestrated endothelial cells of the posterior lobe of the pituitary

Regulation of Aqp1 expression by osmotic balance in fenestrated endothelial cells of the posterior lobe of the pituitary | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Regulation of Aqp1 expression by osmotic balance in fenestrated endothelial cells of the posterior lobe of the pituitary Takashi Nakakura, Takeshi Suzuki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6677059/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Cell and Tissue Research → Version 1 posted 4 You are reading this latest preprint version Abstract The anterior lobe (AL) and posterior lobe (PL) of the vertebrate pituitary are richly vascularized with a dense network of fenestrated capillaries. In this study, we found that the expression of Aqp1, which encodes a plasma membrane-localized water channel protein, was significantly higher in endothelial fractions isolated from the rat PL than in those isolated from the AL. Immunohistochemistry revealed aquaporin 1 (AQP1)-positive signals in fenestrated endothelial cells of the PL. Furthermore, immunoelectron microscopy demonstrated the presence of AQP1 signals on both the luminal and abluminal plasma membranes of these cells. AQP1 plays a pivotal role in facilitating water movement across the plasma membrane in response to changes in osmotic pressure on a cell. To investigate the effect of hyperosmolarity on Aqp1 expression, we examined the expression levels of Aqp1 in the PL of water-deprived rats as well as in isolated endothelial cells of the PL cultured in a hyperosmotic medium supplemented with raffinose. Immunohistochemical analysis showed no changes in the proportion of AQP1-positive endothelial cells or in subcellular localization of AQP1 in cultured endothelial cells of the PL under hyperosmotic conditions. In contrast, analysis using quantitative real-time PCR revealed that hyperosmolar conditions significantly downregulated Aqp1 expression in the cultured endothelial cells. These findings suggest that fenestrated capillaries in the PL actively regulate Aqp1 expression by sensing osmotic pressure of the interstitial fluid. Our results indicate that AQP1 is selectively expressed in fenestrated capillaries of the PL and plays a crucial role in maintaining water homeostasis in this region. Aquaporin 1 (AQP1) Fenestrated endothelial cells Posterior lobe Pituitary Hyperosmolarity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The pituitary gland is a master endocrine organ that regulates various physiological processes in vertebrates. It is composed of anterior, intermediate, and posterior lobes. Both the anterior lobe (AL) and posterior lobe (PL) contain dense networks of fenestrated capillaries (Eurenius 1977 ; Galabov and Schiebler 1983 ; Murakami et al. 1987 ; Rinehart and Farquhar 1955 ). In the PL, two distinct layers of basement membrane (BM) surround the fenestrated capillaries (Nakakura et al. 2024 ). The outer surface of the fenestrated capillaries is enclosed by the inner BM, while axons of neurosecretory cells that are distributed throughout the hypothalamus terminate at the outer BM. A broad perivascular space exists between the inner and outer BMs (Miyata 2017 ). Our recent study demonstrated that collagen XIII is involved in the formation and maintenance of a neurovascular junction between the hypothalamic axon terminals and the outer BM (Nakakura et al. 2024 ). Arginine vasopressin (AVP) and oxytocin, secreted from the hypothalamic axonal terminals adjacent to the outer BM, reach the fenestrated capillaries via the perivascular space (Murphy et al. 2012 ). AVP is released in response to elevated blood osmolality and plays a critical role in regulating water balance of the body (Dicker and Nunn 1957 ). Furthermore, an increase in the perivascular spaces of the PL in response to hyperosmolarity has been observed (Nishikawa et al. 2017 ). However, the relationship between the fenestrated capillaries in the PL and osmolarity of blood is unclear. Aquaporins (AQPs) are a family of plasma membrane channel proteins that facilitate water transport across cell membranes in response to osmotic gradients (Verkman and Mitra 2000 ). They are found in all living organisms including bacteria, plants, and animals (Agre et al. 2002 ). In humans, 13 members of the AQP family have been identified, ranging from AQP0 to AQP12 (Ishibashi et al. 2009 ). These proteins can be categorized into three distinct subgroups: water-selective AQPs (AQP0, 1, 2, 4, 5, 6, and 8), aquaglyceroporins (AQP3, 7, 9, and 10), and superaquaporins (AQP11, 12). Their expression is widespread across mammalian tissues, including the kidneys (Su et al. 2020 ), lungs (Wittekindt and Dietl 2019 ), nervous system (Papadopoulos and Verkman 2013 ), and vasculature (Verkman 2006 ). In the rat pituitary, AQP4 has been reported to be expressed in folliculo-stellate cells of the AL, pituicytes of the PL, and marginal layer cells of Rathke's residual pouch (Kuwahara et al. 2010 ; Matsuzaki et al. 2011 ). AQP5 has also been shown to be localized on the apical membrane of marginal layer cells in Rathke’s residual pouch (Matsuzaki et al. 2011 ). Additionally, our previous research identified the expression of AQP-h3BL, a homolog of mammalian AQP3, in gonadotrophs of the tree frog pituitaries (Sato et al. 2011 ). In the present study, we found that Aqp1 expression was significantly higher in endothelial cells of the PL than in those of the AL. We investigated the localization of AQP1 in the rat pituitary gland using immunohistochemistry and immunoelectron microscopy. Next, we analyzed changes in Aqp1 expression in the PL of water-deprived rats and in cultured endothelial cells isolated from the PL under hyperosmotic conditions induced by raffinose. Our findings suggest that AQP1 is selectively expressed in fenestrated capillaries of the PL and plays an essential role in maintaining water homeostasis within this specialized neurovascular environment. Materials and Methods Animals Eight-week-old male Wistar rats were obtained from Japan SLC, Inc (Shizuoka, Japan). The animals were housed in a temperature-controlled room (22 ± 2°C) with a 12-h light/dark cycle with lights from 06:00 to 18:00. Food and water were provided ad libitum. For water deprivation experiments, rats were deprived of drinking water for 48 h. Isolation and culture of platelet endothelial cell adhesion molecule 1 (PECAM1)- positive endothelial cells from the rat pituitary Isolation of PECAM1-positive endothelial cells from rat pituitaries was performed as previously described (Nakakura et al. 2021b). Briefly, AL and PL cells were enzymatically dispersed using collagenase and trypsin, and then incubated at 25°C for 40 min in isolation buffer [phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA), and 2 mM ethylenediaminetetraacetic acid (EDTA)] with dynabeads-labeled anti-PECAM1 antibody. PECAM1-positive cells were collected using a magnetic stand and lysed directly in extraction buffer provided in the NucleoSpin RNA kit (Takara Bio Inc., Shiga, Japan) or cultured in Endothelial Cell Growth Medium MV 2 (PromoCell, Heidelberg, Germany) on human fibronectin-coated culture plates. For hyperosmotic treatment, PECAM1-positive cells isolated from the PL were preincubated in a medium containing 0.1% BSA without fetal bovine serum and growth factors for 2 h. The cells were then treated with hyperosmotic medium containing 100 mM raffinose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 20 h and subsequently lysed in extraction buffer provided in the NucleoSpin RNA kit (Takara Bio Inc.) for quantitative real-time polymerase chain reaction (qPCR). Quantitative real-time PCR (qPCR) Total RNA was extracted from rat pituitary cells using the NucleoSpin RNA kit (Takara Bio Inc.). cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio Inc.) as described previously (Nakakura et al. 2021a). qPCR was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix with Low ROX (Agilent Technologies, CA, USA). The gene-specific primers are listed in Supplemental Table 1. Relative gene expression was calculated using the 2 -(ΔCt sample-ΔCt control) method. Immunohistochemistry and immunocytochemistry For immunohistochemistry, paraffin-embedded sections were prepared as described previously (Nakakura et al. 2017). For antigen retrieval, deparaffinized sections were autoclaved at 121°C for 5 min in 1 mM EDTA. The sections were then incubated with primary antibodies in PBS containing 1% BSA at 25°C for 16 h (antibodies are listed in Supplemental Table 2). After washing with PBS, the sections were incubated with Cy3-, Alexa Fluor 488-, or Alexa Fluor 647-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) and 4’, 6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan) at 25°C for 90 min. For immunocytochemistry, cells cultured on fibronectin-coated coverslips were fixed with 4% paraformaldehyde in PBS and permeabilized with PBS containing 0.2% Triton X for 10 min at 25°C. After washing with PBS, the cells were incubated with primary antibodies at 37°C for 2 h, followed by secondary antibodies at 37°C for 1 h. All antibodies were diluted in PBS containing 1% BSA. After washing with PBS, the samples were mounted using PermaFluor (Richard-Allan Scientific, MI, USA) and analyzed using the Nikon A1 confocal laser scanning microscope (Nikon, Tokyo, Japan). Transmission electron microscopy (TEM) Ultrathin sections of rat pituitaries were prepared using the Leica EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) as previously described (Nakakura et al. 2024). Sections were stained with uranyl acetate and lead citrate, and examined using the Hitachi H-7650 transmission electron microscope (Hitachi High-Tech Corp., Tokyo, Japan) at an accelerating voltage of 80 kV. Immunoelectron microscopy Ultrastructural localization of AQP1 in the rat pituitary was analyzed using immunoelectron microscopy as previously described (Nakakura et al. 2021b). Frozen sections of rat pituitaries were incubated with rabbit anti-AQP1 antibody (1:1,000; Proteintech, IL, USA), followed by 1.4-nm nanogold-conjugated anti-rabbit immunoglobulin G (IgG; 1:100; Nanoprobe, NY, USA) diluted in HEPES buffer (30 mM HEPES, 100 mM NaCl, 2 mM CaCl 2 , pH7.4) containing 1% BSA and 0.1% Triton X-100. The sections were washed with HEPES buffer containing 0.1% Triton X-100, postfixed with 1% glutaraldehyde/HEPES buffer, and silver-enhanced for 8 min using the HQ-Silver kit (Nanoprobe). The samples were then treated with 0.05% aurochlorohydric acid and 1% chitosan in 0.5% acetic acid, fixed with osmium tetroxide, dehydrated, and embedded in Epon812. Ultrathin sections were prepared as described above and examined using the Hitachi H-7650 TEM at 80 kV. Statistical Analyses Data are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using unpaired Student’s t-test or Welch’s t-test depending on the outcome of F-tests for variance equality. A p -value of <0.05 was considered statistically significant. Results Expression and distribution of plasmalemma vesicle-associated protein (PLVAP) in fenestrated capillaries in the rat pituitary PLVAP, a type II transmembrane glycoprotein, is the only known molecular component of fenestral diaphragms present in fenestrated endothelial cells (Denzer et al. 2023 ; Stan et al. 2012 ). To characterize endothelial cells in the AL and PL of the rat pituitary, we analyzed the expression levels of Plvap in PECAM1-positive endothelial fractions isolated from the AL and PL using qPCR (Fig. 1 a). The results showed that the expression levels of Plvap were equivalent in the PECAM1-positive endothelial fractions of both the AL and PL (Fig. 1 a). Next, we examined the distribution of fenestrated capillaries expressing PLVAP in the rat pituitary using immunohistochemistry with an antibody against PLVAP (Fig. 1 b-k). PLVAP-positive signals were detected in both the AL and PL (Fig. 1 b). In the PL, PLVAP-positive signals were located on the luminal side of NG2-positive pericytes and laminin-positive BMs (Fig. 1 c-h). PLVAP-positive signals were also localized around AVP-positive axon terminals in the PL (Fig. 1 i–k). In addition, ultrastructural observations using TEM confirmed the presence of fenestrae and diaphragms within the walls of the capillaries in the PL (Fig. 2 a, b). Diameters of the fenestra ranged from 60 to 80 nm (Fig. 2 b). Expression and localization of AQP1 in fenestrated endothelial cells of the rat PL qPCR analysis revealed that the expression level of Aqp1 was approximately 80-fold higher in PECAM1-positive endothelial fraction isolated from the PL than in that isolated from the AL (Fig. 2 a). In contrast, other Aqp genes were negligibly expressed in the PECAM1-positive endothelial fractions obtained from both the lobes (Fig. 2 a). Next, the distribution of AQP1 in the rat pituitary was examined by immunohistochemistry. AQP1-positive signals were detected in both the PL and AL (Fig. 3 b). These signals disappeared in the pre-absorption assay (Fig. 3 c). In the PL, AQP1-positive signals were identified in PLVAP-positive endothelial cells (Fig. 4 a-f). In contrast, in the AL, PLVAP-positive signals were predominantly found in non-endothelial cells surrounding the PLVAP-positive capillaries (Fig. 4 g-l). Immunoelectron microscopy further demonstrated localization of AQP1 in both the luminal and abluminal plasma membranes of fenestrated endothelial cells in the PL (Fig. 5 a, b). Effect of water deprivation on Aqp1 expression in the rat PL Dehydration of the body leads to an increase in blood osmolarity (Kadekaro et al. 1992 ). To elucidate the effect of AQP1-positive endothelial cells on changes in osmolarity, we quantified the number of AQP1/PLVAP double-positive endothelial cells in the PL of rats deprived of water for 48 h and analyzed their proportion relative to the total number of PLVAP-positive cells (Fig. 6 a-g). This analysis showed that the proportion of AQP1/PLVAP double-positive endothelial cells remained unchanged in the water-deprived group than in the control group (Fig. 6 g). Next, we analyzed the expression levels of Aqp1 in the PL of rats deprived of water for 48 h, using qPCR (Fig. 6 h). The results indicated that deprivation of water significantly decreased Aqp1 expression (Fig. 6 h), whereas the expression levels of Pecam1 and Plvap remained unchanged (Fig. 6 h). Regulation of Aqp1 expression in cultured endothelial cells of the rat PL by hyperosmolarity We confirmed the characteristics of cultured PECAM1-positive endothelial cells isolated from the PL using immunocytochemistry (Fig. 7 a-f). AQP1-positive signals were observed in the cytoplasm and on plasma membrane employing an anti-PECAM1 antibody (Fig. 7 a-c). AQP1-positive signals were also detected using an anti-PLVAP antibody around sieve plates, which were observed as oval-shaped structures (Fig. 7 d-f). To investigate whether hyperosmotic conditions affect Aqp1 expression, PECAM1-positive endothelial cells isolated from the PL were cultured in a hyperosmotic medium containing raffinose, which is known to induce hyperosmolarity (Matsuzaki et al. 2001 ). After 20 h of exposure to this medium, qPCR analysis revealed a significant reduction in Aqp1 expression in the endothelial cells, whereas Pecam1 expression remained unchanged (Fig. 7 g). Discussion In the present study, we first confirmed that the capillaries distributed in the PL were of the fenestrated type. Endothelial fenestra function as a pathway for transport of peptide hormones from endocrine tissues into the bloodstream (Stucker et al. 2021 ). These fenestrae typically range from 60 to 80 nm in diameter and are divided into 5–6 nm openings by fenestral diaphragms composed of PLVAP (Herrnberger et al. 2012 ; Stan et al. 2012 ). qPCR analysis revealed no difference in the expression of Plvap between PECAM1-positive fractions of the AL and PL. Electron microscopy also confirmed that the diameters of the fenestra in endothelial cells of the PL ranged from 60 to 80 nm and revealed the presence of diaphragms within these fenestrae. Our previous study reported that the average diameter of fenestrae in endothelial cells of the AL was 71.8 ± 1.02 nm (Nakakura et al. 2021b ). Our findings indicated a remarkable similarity between the basic characteristics of the fenestrated capillaries distributed in the AL and PL. Origins of development of the PL and AL differ from each other. The PL arises from the ventral diencephalon, whereas the AL is derived from an invagination of the oral ectoderm known as Rathke's pouch (Kelberman et al. 2009 ; Nakakura et al. 2009 ). It is also known that the fenestrated capillaries in the PL and AL originate from the inferior pituitary arteries and hypophyseal portal veins, respectively (Nakakura et al. 2006 ; Page 1982 ). Although the expression levels of Plvap were similar in the PECAM1-positive fraction of the PL and AL, Aqp1 expression was markedly higher in the PECAM1-positive fraction of the PL compared to that of AL. Furthermore, immunohistochemical studies revealed that AQP1 was localized on plasma membranes of fenestrated endothelial cells in the PL, whereas in the AL, AQP1 was detected in non-endothelial cells surrounding fenestrated capillaries. These findings suggest that the distinct expression pattern of AQP1 in the pituitary gland may be associated with the distribution area and anatomical origins of fenestrated capillaries. In this study, we demonstrated that Aqp1 expression in the PL was significantly decreased by water deprivation. However, the number of AQP1-positive endothelial cells in the PL remained unchanged following water deprivation. Because water deprivation has been reported to increase blood osmolarity in rats (Kadekaro et al. 1992 ), we also investigated the effect of hyperosmolarity using a raffinose-supplemented medium. We observed that Aqp1 expression was significantly downregulated in cultured PECAM1-positive endothelial cells isolated from the PL under hyperosmotic conditions. In previous studies, changes in expression levels of the AQP1 gene were observed in rat peritoneal mesothelial cells, human renal proximal tubule epithelial cells, and mouse medullary mIMCD-3 cells in response to hyperosmolality induced by glucose, NaCl, or raffinose (Jenq et al. 1999 ; Ota et al. 2002 ; Umenishi and Schrier 2003 ). In our study, immunoelectron microscopic observation confirmed the localization of AQP1 on both the luminal and abluminal plasma membranes of endothelial cells in the PL. The localization of AQP1 also was maintained in the PL-derived endothelial cells cultured in raffinose-supplemented medium (data not shown). These results indicate that localization of AQP1 in fenestrated endothelial cells of the PL is not influenced by osmotic changes. The results also imply that functional regulation of AQP1 in fenestrated endothelial cells of the PL is controlled at the transcriptional level rather than by intracellular translocation of the protein. We found that AQP1 was localized on the plasma membrane of fenestrated endothelial cells of the PL, whereas it was not detected in fenestrated endothelial cells of the AL. Previous studies have reported that AQP1 is distributed in fenestrated capillaries of the median eminence in the brain (Wilson et al. 2010 ) but not in the continuous capillary network of the cerebral parenchyma (Wilson et al. 2010 ). The median eminence releases hypothalamic hormones into circulation through fenestrated capillaries (Miyata 2015 ). In parallel, it has been shown that under hyperosmolar conditions in the extracellular space, cell volume decreased by the movement of water from the intracellular space to extracellular space through the action of AQP1 (Jiang et al. 2021). These results suggest that a reduction in endothelial cell volume in fenestrated capillaries, caused by water efflux via AQP1, contributes not only to thinning of the capillary walls but also to development of abnormal permeability. Therefore, downregulation of Aqp1 expression in fenestrated endothelial cells distributed in these regions under hyperosmotic conditions may represent a protective mechanism that prevents excessive water loss and helps maintain capillary integrity. Based on these results, we conclude that transcriptional regulation of Aqp1 plays a pivotal role in mediating structural changes in fenestrated capillaries in response to alterations in extracellular osmolarity. Moreover, this study demonstrates that even within fenestrated capillaries, the gene expression profile of endothelial cells varies depending on the tissue in which they reside, thus contributing to tissue-specific physiological functions. To date, fenestrated capillaries have been thought to perform the same function as that by static biomolecule-passing pores throughout the body. However, our findings suggest that the functions of fenestrated capillaries may vary depending on the environment of surrounding tissue. Future investigations will focus on elucidating the extracellular osmolarity-sensing mechanisms that regulate Aqp1 expression in endothelial cells of the PL. In addition, further studies are required to clarify the localization and functional roles of AQP1 in the AL. Abbreviations AL anterior lobe AQP aquaporin AVP arginine vasopressin BM basement membrane BSA bovine serum albumin DAPI 4′, 6-diamidino-2-phenylindole GP guinea pig IgG immunoglobulin G PB phosphate buffer PBS phosphate-buffered saline PCR polymerase chain reaction PECAM1 platelet endothelial cell adhesion molecule 1 PL posterior lobe PLVAP plasmalemma vesicle-associated protein qPCR quantitative real-time polymerase chain reaction TEM transmission electron microscopy Declarations Acknowledgments :We thank Professor Yoshiharu Deguchi (Teikyo University, Tokyo, Japan) for the excellent technical advice and Editage (www.editage.com) for English language editing. Funding information : This work was supported in part by the JSPS KAKENHI (C) Grant Numbers JP22K06800 and JP25K10137, a research grant from the Takeda Science Foundation, Yamaguchi Endocrine Research Foundation, and Advanced Comprehensive Research Grants of the Teikyo University. Conflict of interest : The authors declare that they have no conflicts of interest. Ethical approva l: This study was approved by the Laboratory Animal Ethics Committee of Teikyo University (Tokyo, Japan) and conducted according to the guidelines. The document ID of approval was 23-044. This article did not include any studies involving human participants. References Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels – from atomic structure to clinical medicine. J Physiol 542:3-16 Denzer L, Muranyi W, Schroten H, Schwerk C (2023) The role of PLVAP in endothelial cells. Cell Tissue Res 392:393-412 Dicker SE, Nunn J (1957) The role of the antidiuretic hormone during water deprivation in rats. J Physiol 136:235-248 Eurenius L (1977) An electron microscope study of the differentiating capillaries of the mouse neurohypophysis. 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Supplementary Files Table1primer.docx Supplemental table 1 Table2antibody.docx Supplemental table 2 Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Cell and Tissue Research → Version 1 posted Editorial decision: Revision requested 22 May, 2025 Editor assigned by journal 16 May, 2025 Submission checks completed at journal 16 May, 2025 First submitted to journal 16 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6677059","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":460637988,"identity":"299886ff-ee95-426d-b7c2-8918679b0d8e","order_by":0,"name":"Takashi Nakakura","email":"data:image/png;base64,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","orcid":"","institution":"Teikyo University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Nakakura","suffix":""},{"id":460637989,"identity":"9906fa2c-489c-4ac7-b71a-f594b29c4ddd","order_by":1,"name":"Takeshi Suzuki","email":"","orcid":"","institution":"Sapporo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Suzuki","suffix":""}],"badges":[],"createdAt":"2025-05-16 04:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6677059/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6677059/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00441-025-03995-x","type":"published","date":"2025-07-22T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83763243,"identity":"ce80dd3e-592d-4cf8-b3f2-3b5a7fa824b5","added_by":"auto","created_at":"2025-06-02 10:13:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11111209,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression and localization of plasmalemma vesicle-associated protein (PLVAP) in rat pituitary. \u003cstrong\u003ea\u003c/strong\u003e Expression of \u003cem\u003ePlvap\u003c/em\u003e in PECAM1-positive fraction isolated from anterior lobe (AL) and posterior lobe (PL) of rat pituitary was determined using qPCR, followed by normalization with expression levels of \u003cem\u003eHprt1\u003c/em\u003e, which was used as the control housekeeping gene. \u003cstrong\u003eb\u003c/strong\u003e PLVAP-positive signals (red) were detected in the AL and the PL of the rat pituitary. \u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e Double immunofluorescence images for PLVAP (\u003cstrong\u003ec\u003c/strong\u003e, red) and NG2 (\u003cstrong\u003ed\u003c/strong\u003e, green) in the PL are shown. \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003eDouble immunofluorescence images for PLVAP (\u003cstrong\u003ef\u003c/strong\u003e, red) and laminin (\u003cstrong\u003eg\u003c/strong\u003e, green) in the PL are shown. \u003cstrong\u003ei\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003eDouble immunofluorescence images for PLVAP (\u003cstrong\u003ei\u003c/strong\u003e, red) and AVP (\u003cstrong\u003ej\u003c/strong\u003e, green) in the PL are shown. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, blue). IL, intermediate lobe. Bars:\u003cem\u003e \u003c/em\u003e100 μm (\u003cstrong\u003eb\u003c/strong\u003e), 20 μm (\u003cstrong\u003ec\u003c/strong\u003e-\u003cstrong\u003ek\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/4e8164b86116601d466358f8.png"},{"id":83763244,"identity":"0d6a8fb2-11ee-4741-be8b-10ccc15f51a2","added_by":"auto","created_at":"2025-06-02 10:13:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3394582,"visible":true,"origin":"","legend":"\u003cp\u003eElectron micrographs of PL of rat pituitary. \u003cstrong\u003ea\u003c/strong\u003eElectron micrography indicates a fenestrated capillary and a wide perivascular space (PVS) in the PL. \u003cstrong\u003eb\u003c/strong\u003e Enlarged photograph of the region surrounded by a black line in \u003cstrong\u003ea\u003c/strong\u003e is shown. Asterisks show the fenestrae of the endothelial cell. Arrows indicate the fenestral diaphragms. EC, endothelial cell; L, lumen of the fenestrated capillary; PVS, perivascular space; AT, axon terminal. Bars: 1 μm (\u003cstrong\u003ea\u003c/strong\u003e), 100 nm (\u003cstrong\u003eb\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/5e34eb2ac313fb98ff4b6db9.png"},{"id":83762625,"identity":"ebb2f472-3f7f-4a11-a6ca-36b83cdfff31","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2409009,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression and localization of aquaporin 1 (AQP1) in rat pituitary. \u003cstrong\u003ea\u003c/strong\u003e Expression of \u003cem\u003eAqp\u003c/em\u003e genes in PECAM1-positive fraction isolated from AL and PL of rat pituitary was determined using qPCR, followed by normalization with expression levels of \u003cem\u003eHprt1\u003c/em\u003e, which was used as the control housekeeping gene. \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ed\u003c/strong\u003e AQP1-positive signals (red) were detected in the PL (\u003cstrong\u003ec\u003c/strong\u003e) and AL (\u003cstrong\u003ed\u003c/strong\u003e) of the rat pituitary. Nuclei were counterstained with DAPI (blue). IL, intermediate lobe. Bars:\u003cem\u003e \u003c/em\u003e100 μm (\u003cstrong\u003eb\u003c/strong\u003e), 20 μm (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/a757ce16404719a85d8e57b5.png"},{"id":83762632,"identity":"d1422fb3-bc05-4dd3-ab0a-5cde861c1ad2","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9585996,"visible":true,"origin":"","legend":"\u003cp\u003eImmunolocalization of AQP1 in PL and AL of the rat pituitary. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e Double immunofluorescence images for AQP1 (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, red) and PLVAP (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, green) in the PL (\u003cstrong\u003ea\u003c/strong\u003e-f) and the AL (\u003cstrong\u003eg\u003c/strong\u003e-\u003cstrong\u003el\u003c/strong\u003e) are shown. AQP1 signals were observed in PLVAP-positive endothelial cells in the PL (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e). In contrast, AQP1 signals were observed in cells surrounding PLVAP-positive endothelial cells in the AL (\u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e). Nuclei were counterstained with DAPI (blue). Bars:\u003cem\u003e \u003c/em\u003e20 μm.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/04ab314791faf7a771de93a7.png"},{"id":83762630,"identity":"0c461bea-28fa-4ad6-833e-3c1d909a4641","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2947885,"visible":true,"origin":"","legend":"\u003cp\u003eImmunoelectron micrographs of AQP1 signals in PL of rat pituitary. (\u003cstrong\u003ea\u003c/strong\u003e) Electron micrography indicates that AQP1 signals were localized to the plasma membrane of an endothelial cell. Panel b shows the enlarged photograph of the region surrounded by a black line in panel a. Arrows indicate AQP1-positive signals on the plasma membrane of the endothelial cell. EC, endothelial cell; L, the lumen of the fenestrated capillary; PVS, perivascular space. Bars: 2 μm (\u003cstrong\u003ea\u003c/strong\u003e), 500 nm (\u003cstrong\u003eb\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/08316e02d4631c1ad8ec2cad.png"},{"id":83763514,"identity":"42f5f0c3-abf9-481d-8bed-b412323e8956","added_by":"auto","created_at":"2025-06-02 10:21:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6419500,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of water deprivation (WD) on the number of AQP1-positive endothelial cells and \u003cem\u003eAqp1\u003c/em\u003e expression in PL of rat pituitary. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e Double immunofluorescence images for AQP1 (\u003cstrong\u003ea\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, red) and PLVAP (\u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, green) in the PL of control group (\u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003e) and after 48 h of WD (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e) are shown. Nuclei were counterstained with DAPI (blue). Bar: 50 μm. \u003cstrong\u003eg \u003c/strong\u003eThe proportion of AQP1/PLVAP double-positive endothelial cells in total PLVAP-positive endothelial cells in the PL of control group and after 48 h of WD is shown. All experiments were performed using three animals per group. The data obtained from each animal were used for statistical analysis. \u003cstrong\u003eh\u003c/strong\u003e Expressions of \u003cem\u003eAqp1\u003c/em\u003e, \u003cem\u003ePecam1\u003c/em\u003e, and \u003cem\u003ePlvap\u003c/em\u003e in rat PL of control and after 48 h of WD were determined using qPCR, followed by normalization with expression levels of \u003cem\u003eHprt1\u003c/em\u003e, which was used as the control housekeeping gene. Statistical analyses were performed using unpaired Student’s t-test or Welch’s t-test. **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 vs. control. All experiments were performed using four animals per group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/a056a85a42ed7afa20bcbc37.png"},{"id":83762627,"identity":"7f95441a-a77f-4e77-93bc-ea7b614cd39f","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6208250,"visible":true,"origin":"","legend":"\u003cp\u003eChange in the expression level of\u003cem\u003e Aqp1 \u003c/em\u003ein PECAM1-positive endothelial cells of rat PL by treatment with raffinose. \u003cstrong\u003ea\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003eDouble immunofluorescence images for AQP1 (\u003cstrong\u003ea\u003c/strong\u003e, red) and PECAM1 (\u003cstrong\u003eb\u003c/strong\u003e, green) in PECAM1-positive endothelial cells isolated from rat PL are shown. AQP1 signals were observed on the PECAM1-positive plasma membrane and in the cytoplasm of the cells (\u003cstrong\u003ec\u003c/strong\u003e). \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e Double immunofluorescence images for AQP1 (\u003cstrong\u003ed\u003c/strong\u003e, red) and PLVAP (\u003cstrong\u003ee\u003c/strong\u003e, green) in PECAM1-positive endothelial cells isolated from rat PL are shown. AQP1 signals were observed around PLVAP-positive signals in the cells (\u003cstrong\u003ef\u003c/strong\u003e). Nuclei were counterstained with DAPI (blue). Bars:\u003cem\u003e \u003c/em\u003e10 μm. \u003cstrong\u003eg\u003c/strong\u003e Expression levels of \u003cem\u003eAqp1\u003c/em\u003eand \u003cem\u003ePecam1\u003c/em\u003e in PECAM1-positive endothelial cells of rat PL treated with raffinose for 20 h were determined using qPCR. Statistical analyses were performed using unpaired Student’s t-test or Welch’s t-test. **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01 vs. control. The results of five independent experiments are shown.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/3d7ce3924af788b20d7de26a.png"},{"id":87756775,"identity":"373c055a-0a1e-46ba-923c-b73a587533a8","added_by":"auto","created_at":"2025-07-28 16:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":58226011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/660f6420-0273-43d7-8e53-80d1bf34ca07.pdf"},{"id":83762623,"identity":"86abe995-52c5-491d-aa1b-d6b229fa4fb7","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22543,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 1\u003c/p\u003e","description":"","filename":"Table1primer.docx","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/f8b4042c4c0f1a5764a3193d.docx"},{"id":83762624,"identity":"2684bd23-6f07-4e34-a952-ed8a24240a38","added_by":"auto","created_at":"2025-06-02 10:05:28","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20132,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental table 2\u003c/p\u003e","description":"","filename":"Table2antibody.docx","url":"https://assets-eu.researchsquare.com/files/rs-6677059/v1/f59099c25aa3766d8279c2e3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulation of Aqp1 expression by osmotic balance in fenestrated endothelial cells of the posterior lobe of the pituitary","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe pituitary gland is a master endocrine organ that regulates various physiological processes in vertebrates. It is composed of anterior, intermediate, and posterior lobes. Both the anterior lobe (AL) and posterior lobe (PL) contain dense networks of fenestrated capillaries (Eurenius \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Galabov and Schiebler \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Murakami et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Rinehart and Farquhar \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1955\u003c/span\u003e). In the PL, two distinct layers of basement membrane (BM) surround the fenestrated capillaries (Nakakura et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The outer surface of the fenestrated capillaries is enclosed by the inner BM, while axons of neurosecretory cells that are distributed throughout the hypothalamus terminate at the outer BM. A broad perivascular space exists between the inner and outer BMs (Miyata \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our recent study demonstrated that collagen XIII is involved in the formation and maintenance of a neurovascular junction between the hypothalamic axon terminals and the outer BM (Nakakura et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Arginine vasopressin (AVP) and oxytocin, secreted from the hypothalamic axonal terminals adjacent to the outer BM, reach the fenestrated capillaries via the perivascular space (Murphy et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). AVP is released in response to elevated blood osmolality and plays a critical role in regulating water balance of the body (Dicker and Nunn \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1957\u003c/span\u003e). Furthermore, an increase in the perivascular spaces of the PL in response to hyperosmolarity has been observed (Nishikawa et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the relationship between the fenestrated capillaries in the PL and osmolarity of blood is unclear.\u003c/p\u003e \u003cp\u003eAquaporins (AQPs) are a family of plasma membrane channel proteins that facilitate water transport across cell membranes in response to osmotic gradients (Verkman and Mitra \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). They are found in all living organisms including bacteria, plants, and animals (Agre et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In humans, 13 members of the AQP family have been identified, ranging from AQP0 to AQP12 (Ishibashi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These proteins can be categorized into three distinct subgroups: water-selective AQPs (AQP0, 1, 2, 4, 5, 6, and 8), aquaglyceroporins (AQP3, 7, 9, and 10), and superaquaporins (AQP11, 12). Their expression is widespread across mammalian tissues, including the kidneys (Su et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), lungs (Wittekindt and Dietl \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), nervous system (Papadopoulos and Verkman \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and vasculature (Verkman \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In the rat pituitary, AQP4 has been reported to be expressed in folliculo-stellate cells of the AL, pituicytes of the PL, and marginal layer cells of Rathke's residual pouch (Kuwahara et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Matsuzaki et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). AQP5 has also been shown to be localized on the apical membrane of marginal layer cells in Rathke\u0026rsquo;s residual pouch (Matsuzaki et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, our previous research identified the expression of AQP-h3BL, a homolog of mammalian AQP3, in gonadotrophs of the tree frog pituitaries (Sato et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we found that \u003cem\u003eAqp1\u003c/em\u003e expression was significantly higher in endothelial cells of the PL than in those of the AL. We investigated the localization of AQP1 in the rat pituitary gland using immunohistochemistry and immunoelectron microscopy. Next, we analyzed changes in \u003cem\u003eAqp1\u003c/em\u003e expression in the PL of water-deprived rats and in cultured endothelial cells isolated from the PL under hyperosmotic conditions induced by raffinose. Our findings suggest that AQP1 is selectively expressed in fenestrated capillaries of the PL and plays an essential role in maintaining water homeostasis within this specialized neurovascular environment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old male Wistar rats were obtained from Japan SLC, Inc (Shizuoka, Japan). The animals were housed in a temperature-controlled room (22 ± 2°C) with a 12-h light/dark cycle with lights from 06:00 to 18:00. Food and water were provided ad libitum. For water deprivation experiments, rats were deprived of drinking water for 48 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and culture of platelet endothelial cell adhesion molecule 1 (PECAM1)- positive endothelial cells from the rat pituitary\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation of PECAM1-positive endothelial cells from rat pituitaries was performed as previously described (Nakakura et al. 2021b). Briefly, AL and PL cells were enzymatically dispersed using collagenase and trypsin, and then incubated at 25°C for 40 min in isolation buffer [phosphate-buffered saline (PBS), 0.1% bovine serum albumin (BSA), and 2 mM ethylenediaminetetraacetic acid (EDTA)] with dynabeads-labeled anti-PECAM1 antibody. PECAM1-positive cells were collected using a magnetic stand and lysed directly in\u0026nbsp;extraction buffer provided in the NucleoSpin RNA kit (Takara Bio Inc., Shiga, Japan) or cultured in Endothelial Cell Growth Medium MV 2 (PromoCell, Heidelberg, Germany) on human fibronectin-coated culture plates.\u003c/p\u003e\n\u003cp\u003eFor hyperosmotic treatment, PECAM1-positive cells isolated from the PL were preincubated in a medium\u0026nbsp;containing 0.1% BSA without fetal bovine serum and growth factors for 2 h. The cells were then treated with hyperosmotic medium containing 100 mM raffinose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 20 h and subsequently lysed in extraction buffer provided in the NucleoSpin RNA kit (Takara Bio Inc.) for quantitative real-time polymerase chain reaction (qPCR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from rat pituitary cells using the NucleoSpin RNA kit (Takara Bio Inc.). cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio Inc.) as described previously (Nakakura et al. 2021a). qPCR was performed on the 7500 Fast Real-Time PCR System (Applied Biosystems, CA, USA) using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix with Low ROX (Agilent Technologies, CA, USA). The gene-specific primers are listed in Supplemental Table 1. Relative gene expression was calculated using the 2\u003csup\u003e-(ΔCt sample-ΔCt control)\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry and immunocytochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunohistochemistry, paraffin-embedded sections were prepared as described previously (Nakakura et al. 2017). For antigen retrieval, deparaffinized sections were autoclaved at 121°C for 5 min in 1 mM EDTA. The sections were then incubated with primary antibodies in PBS containing 1% BSA at 25°C for 16 h (antibodies are listed in Supplemental Table 2). After washing with PBS, the sections were incubated with Cy3-, Alexa Fluor 488-, or Alexa Fluor 647-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) and 4’, 6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan) at 25°C for 90 min.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor immunocytochemistry, cells cultured on fibronectin-coated coverslips were fixed with 4% paraformaldehyde in PBS and permeabilized with PBS containing 0.2% Triton X for 10 min at 25°C. After washing with\u0026nbsp;PBS, the cells were incubated with primary antibodies at 37°C for 2 h, followed by secondary antibodies at 37°C for 1 h. All antibodies were diluted in PBS containing 1% BSA. After\u0026nbsp;washing with\u0026nbsp;PBS, the samples were mounted using PermaFluor (Richard-Allan Scientific, MI, USA) and analyzed using the Nikon A1 confocal laser scanning microscope (Nikon, Tokyo, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltrathin sections of rat pituitaries were prepared using the Leica EM UC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) as previously described (Nakakura et al. 2024). Sections were stained with uranyl acetate and lead citrate, and examined using the Hitachi H-7650 transmission electron microscope (Hitachi High-Tech Corp., Tokyo, Japan) at an accelerating voltage of 80 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoelectron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUltrastructural localization of AQP1 in the rat pituitary was analyzed using immunoelectron microscopy as previously described (Nakakura et al. 2021b). Frozen sections of rat pituitaries were incubated with rabbit anti-AQP1 antibody (1:1,000; Proteintech, IL, USA), followed by 1.4-nm nanogold-conjugated anti-rabbit immunoglobulin G (IgG; 1:100; Nanoprobe, NY, USA) diluted in HEPES buffer (30 mM HEPES, 100 mM NaCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, pH7.4) containing 1% BSA and 0.1% Triton X-100. The sections were washed with HEPES buffer containing 0.1% Triton X-100,\u0026nbsp;postfixed with 1% glutaraldehyde/HEPES buffer, and silver-enhanced for 8 min using the HQ-Silver kit (Nanoprobe). The samples were then treated with 0.05% aurochlorohydric acid and 1% chitosan in 0.5% acetic acid, fixed with osmium tetroxide, dehydrated, and embedded in Epon812.\u0026nbsp;Ultrathin sections were prepared as described above and examined using the Hitachi H-7650 TEM at 80 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using unpaired Student’s t-test or Welch’s t-test depending on the outcome of F-tests for variance equality. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExpression and distribution of plasmalemma vesicle-associated protein (PLVAP) in fenestrated capillaries in the rat pituitary\u003c/h2\u003e \u003cp\u003ePLVAP, a type II transmembrane glycoprotein, is the only known molecular component of fenestral diaphragms present in fenestrated endothelial cells (Denzer et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Stan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To characterize endothelial cells in the AL and PL of the rat pituitary, we analyzed the expression levels of \u003cem\u003ePlvap\u003c/em\u003e in PECAM1-positive endothelial fractions isolated from the AL and PL using qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The results showed that the expression levels of \u003cem\u003ePlvap\u003c/em\u003e were equivalent in the PECAM1-positive endothelial fractions of both the AL and PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Next, we examined the distribution of fenestrated capillaries expressing PLVAP in the rat pituitary using immunohistochemistry with an antibody against PLVAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-k). PLVAP-positive signals were detected in both the AL and PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the PL, PLVAP-positive signals were located on the luminal side of NG2-positive pericytes and laminin-positive BMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-h). PLVAP-positive signals were also localized around AVP-positive axon terminals in the PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei\u0026ndash;k). In addition, ultrastructural observations using TEM confirmed the presence of fenestrae and diaphragms within the walls of the capillaries in the PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Diameters of the fenestra ranged from 60 to 80 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExpression and localization of AQP1 in fenestrated endothelial cells of the rat PL\u003c/h2\u003e \u003cp\u003eqPCR analysis revealed that the expression level of \u003cem\u003eAqp1\u003c/em\u003e was approximately 80-fold higher in PECAM1-positive endothelial fraction isolated from the PL than in that isolated from the AL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast, other \u003cem\u003eAqp\u003c/em\u003e genes were negligibly expressed in the PECAM1-positive endothelial fractions obtained from both the lobes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Next, the distribution of AQP1 in the rat pituitary was examined by immunohistochemistry. AQP1-positive signals were detected in both the PL and AL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These signals disappeared in the pre-absorption assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). In the PL, AQP1-positive signals were identified in PLVAP-positive endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-f). In contrast, in the AL, PLVAP-positive signals were predominantly found in non-endothelial cells surrounding the PLVAP-positive capillaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg-l). Immunoelectron microscopy further demonstrated localization of AQP1 in both the luminal and abluminal plasma membranes of fenestrated endothelial cells in the PL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of water deprivation on\u003c/b\u003e \u003cb\u003eAqp1\u003c/b\u003e \u003cb\u003eexpression in the rat PL\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDehydration of the body leads to an increase in blood osmolarity (Kadekaro et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). To elucidate the effect of AQP1-positive endothelial cells on changes in osmolarity, we quantified the number of AQP1/PLVAP double-positive endothelial cells in the PL of rats deprived of water for 48 h and analyzed their proportion relative to the total number of PLVAP-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-g). This analysis showed that the proportion of AQP1/PLVAP double-positive endothelial cells remained unchanged in the water-deprived group than in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). Next, we analyzed the expression levels of \u003cem\u003eAqp1\u003c/em\u003e in the PL of rats deprived of water for 48 h, using qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). The results indicated that deprivation of water significantly decreased \u003cem\u003eAqp1\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), whereas the expression levels of \u003cem\u003ePecam1\u003c/em\u003e and \u003cem\u003ePlvap\u003c/em\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulation of\u003c/b\u003e \u003cb\u003eAqp1\u003c/b\u003e \u003cb\u003eexpression in cultured endothelial cells of the rat PL by hyperosmolarity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe confirmed the characteristics of cultured PECAM1-positive endothelial cells isolated from the PL using immunocytochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-f). AQP1-positive signals were observed in the cytoplasm and on plasma membrane employing an anti-PECAM1 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). AQP1-positive signals were also detected using an anti-PLVAP antibody around sieve plates, which were observed as oval-shaped structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-f). To investigate whether hyperosmotic conditions affect \u003cem\u003eAqp1\u003c/em\u003e expression, PECAM1-positive endothelial cells isolated from the PL were cultured in a hyperosmotic medium containing raffinose, which is known to induce hyperosmolarity (Matsuzaki et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). After 20 h of exposure to this medium, qPCR analysis revealed a significant reduction in \u003cem\u003eAqp1\u003c/em\u003e expression in the endothelial cells, whereas \u003cem\u003ePecam1\u003c/em\u003e expression remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we first confirmed that the capillaries distributed in the PL were of the fenestrated type. Endothelial fenestra function as a pathway for transport of peptide hormones from endocrine tissues into the bloodstream (Stucker et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These fenestrae typically range from 60 to 80 nm in diameter and are divided into 5\u0026ndash;6 nm openings by fenestral diaphragms composed of PLVAP (Herrnberger et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Stan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). qPCR analysis revealed no difference in the expression of \u003cem\u003ePlvap\u003c/em\u003e between PECAM1-positive fractions of the AL and PL. Electron microscopy also confirmed that the diameters of the fenestra in endothelial cells of the PL ranged from 60 to 80 nm and revealed the presence of diaphragms within these fenestrae. Our previous study reported that the average diameter of fenestrae in endothelial cells of the AL was 71.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 nm (Nakakura et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Our findings indicated a remarkable similarity between the basic characteristics of the fenestrated capillaries distributed in the AL and PL.\u003c/p\u003e \u003cp\u003eOrigins of development of the PL and AL differ from each other. The PL arises from the ventral diencephalon, whereas the AL is derived from an invagination of the oral ectoderm known as Rathke's pouch (Kelberman et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Nakakura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is also known that the fenestrated capillaries in the PL and AL originate from the inferior pituitary arteries and hypophyseal portal veins, respectively (Nakakura et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Page \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). Although the expression levels of \u003cem\u003ePlvap\u003c/em\u003e were similar in the PECAM1-positive fraction of the PL and AL, \u003cem\u003eAqp1\u003c/em\u003e expression was markedly higher in the PECAM1-positive fraction of the PL compared to that of AL. Furthermore, immunohistochemical studies revealed that AQP1 was localized on plasma membranes of fenestrated endothelial cells in the PL, whereas in the AL, AQP1 was detected in non-endothelial cells surrounding fenestrated capillaries. These findings suggest that the distinct expression pattern of AQP1 in the pituitary gland may be associated with the distribution area and anatomical origins of fenestrated capillaries.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that \u003cem\u003eAqp1\u003c/em\u003e expression in the PL was significantly decreased by water deprivation. However, the number of AQP1-positive endothelial cells in the PL remained unchanged following water deprivation. Because water deprivation has been reported to increase blood osmolarity in rats (Kadekaro et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), we also investigated the effect of hyperosmolarity using a raffinose-supplemented medium. We observed that \u003cem\u003eAqp1\u003c/em\u003e expression was significantly downregulated in cultured PECAM1-positive endothelial cells isolated from the PL under hyperosmotic conditions. In previous studies, changes in expression levels of the AQP1 gene were observed in rat peritoneal mesothelial cells, human renal proximal tubule epithelial cells, and mouse medullary mIMCD-3 cells in response to hyperosmolality induced by glucose, NaCl, or raffinose (Jenq et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ota et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Umenishi and Schrier \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In our study, immunoelectron microscopic observation confirmed the localization of AQP1 on both the luminal and abluminal plasma membranes of endothelial cells in the PL. The localization of AQP1 also was maintained in the PL-derived endothelial cells cultured in raffinose-supplemented medium (data not shown). These results indicate that localization of AQP1 in fenestrated endothelial cells of the PL is not influenced by osmotic changes. The results also imply that functional regulation of AQP1 in fenestrated endothelial cells of the PL is controlled at the transcriptional level rather than by intracellular translocation of the protein.\u003c/p\u003e \u003cp\u003eWe found that AQP1 was localized on the plasma membrane of fenestrated endothelial cells of the PL, whereas it was not detected in fenestrated endothelial cells of the AL. Previous studies have reported that AQP1 is distributed in fenestrated capillaries of the median eminence in the brain (Wilson et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) but not in the continuous capillary network of the cerebral parenchyma (Wilson et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The median eminence releases hypothalamic hormones into circulation through fenestrated capillaries (Miyata \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In parallel, it has been shown that under hyperosmolar conditions in the extracellular space, cell volume decreased by the movement of water from the intracellular space to extracellular space through the action of AQP1 (Jiang et al. 2021). These results suggest that a reduction in endothelial cell volume in fenestrated capillaries, caused by water efflux via AQP1, contributes not only to thinning of the capillary walls but also to development of abnormal permeability. Therefore, downregulation of \u003cem\u003eAqp1\u003c/em\u003e expression in fenestrated endothelial cells distributed in these regions under hyperosmotic conditions may represent a protective mechanism that prevents excessive water loss and helps maintain capillary integrity.\u003c/p\u003e \u003cp\u003eBased on these results, we conclude that transcriptional regulation of \u003cem\u003eAqp1\u003c/em\u003e plays a pivotal role in mediating structural changes in fenestrated capillaries in response to alterations in extracellular osmolarity. Moreover, this study demonstrates that even within fenestrated capillaries, the gene expression profile of endothelial cells varies depending on the tissue in which they reside, thus contributing to tissue-specific physiological functions. To date, fenestrated capillaries have been thought to perform the same function as that by static biomolecule-passing pores throughout the body. However, our findings suggest that the functions of fenestrated capillaries may vary depending on the environment of surrounding tissue. Future investigations will focus on elucidating the extracellular osmolarity-sensing mechanisms that regulate \u003cem\u003eAqp1\u003c/em\u003e expression in endothelial cells of the PL. In addition, further studies are required to clarify the localization and functional roles of AQP1 in the AL.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eanterior lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAQP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eaquaporin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eAVP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003earginine vasopressin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eBM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003ebasement membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003ebovine serum albumin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003e4\u0026prime;, 6-diamidino-2-phenylindole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eGP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eguinea pig\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eIgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eimmunoglobulin G\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003ephosphate buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003ephosphate-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003epolymerase chain reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePECAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eplatelet endothelial cell adhesion molecule 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eposterior lobe\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003ePLVAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003eplasmalemma vesicle-associated protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eqPCR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003equantitative real-time polymerase chain reaction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003eTEM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 350px;\"\u003e\n \u003cp\u003etransmission electron microscopy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e:We thank Professor Yoshiharu Deguchi (Teikyo University, Tokyo, Japan) for the excellent technical advice and Editage (www.editage.com) for English language editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e: This work was supported in part by the JSPS KAKENHI (C) Grant Numbers JP22K06800 and JP25K10137, a research grant from the Takeda Science Foundation, Yamaguchi Endocrine Research Foundation, and Advanced Comprehensive Research Grants of the Teikyo University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e: The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approva\u003c/strong\u003el: This study was approved by the Laboratory Animal Ethics Committee of Teikyo University (Tokyo, Japan) and conducted according to the guidelines. The document ID of approval was 23-044. This article did not include any studies involving human participants.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAgre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels \u0026ndash; from atomic structure to clinical medicine. J Physiol 542:3-16\u003c/li\u003e\n \u003cli\u003eDenzer L, Muranyi W, Schroten H, Schwerk C (2023) The role of PLVAP in endothelial cells. Cell Tissue Res 392:393-412\u003c/li\u003e\n \u003cli\u003eDicker SE, Nunn J (1957) The role of the antidiuretic hormone during water deprivation in rats. J Physiol 136:235-248\u003c/li\u003e\n \u003cli\u003eEurenius L (1977) An electron microscope study of the differentiating capillaries of the mouse neurohypophysis. Anat Embryol (Berl) 152:89-108\u003c/li\u003e\n \u003cli\u003eGalabov PG, Schiebler TH (1983) Development of the capillary system in the neurohypophysis of the rat. 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Cell Tissue Res 324:87-95\u003c/li\u003e\n \u003cli\u003eNishikawa K, Furube E, Morita S, Horii-Hayashi N, Nishi M, Miyata S (2017) Structural reconstruction of the perivascular space in the adult mouse neurohypophysis during an osmotic stimulation. J Neuroendocrinol 29 (2). doi: 10.1111/jne.12456.\u003c/li\u003e\n \u003cli\u003eOta T, Kuwahara M, Fan S, Terada Y, Akiba T, Sasaki S, Marumo F (2002) Expression of aquaporin-1 in the peritoneal tissues: localization and regulation by hyperosmolality. Perit Dial Int 22:307-315\u003c/li\u003e\n \u003cli\u003ePage RB (1982) Pituitary blood flow. Am J Physiol 243:E427-E442\u003c/li\u003e\n \u003cli\u003ePapadopoulos MC, Verkman AS (2013) Aquaporin water channels in the nervous system. Nat Rev Neurosci 14:265-277\u003c/li\u003e\n \u003cli\u003eRinehart JF, Farquhar MG (1955) The fine vascular organization of the anterior pituitary gland. An electron microscopic study with histochemical correlations. 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Front Physiol 12:624928\u003c/li\u003e\n \u003cli\u003eSu W, Cao R, Zhang X-y, Guan Y (2020) Aquaporins in the kidney: physiology and pathophysiology. Am J Physiol Renal Physiol 318:F193-F203\u003c/li\u003e\n \u003cli\u003eUmenishi F, Schrier RW (2003) Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. J Biol Chem 278:15765-15770\u003c/li\u003e\n \u003cli\u003eVerkman AS (2006) Aquaporins in endothelia. Kidney Int 69:1120-1123\u003c/li\u003e\n \u003cli\u003eVerkman AS, Mitra AK (2000) Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 278:F13-F28\u003c/li\u003e\n \u003cli\u003eWilson AJ, Carati CJ, Gannon BJ, Haberberger R, Chataway TK (2010) Aquaporin-1 in blood vessels of rat circumventricular organs. Cell Tissue Res 340:159-168\u003c/li\u003e\n \u003cli\u003eWittekindt OH, Dietl P (2019) Aquaporins in the lung. Pflugers Arch 471:519-532\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aquaporin 1 (AQP1), Fenestrated endothelial cells, Posterior lobe, Pituitary, Hyperosmolarity","lastPublishedDoi":"10.21203/rs.3.rs-6677059/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6677059/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe anterior lobe (AL) and posterior lobe (PL) of the vertebrate pituitary are richly vascularized with a dense network of fenestrated capillaries. In this study, we found that the expression of \u003cem\u003eAqp1,\u003c/em\u003ewhich encodes a plasma membrane-localized water channel protein, was significantly higher in endothelial fractions isolated from the rat PL than in those isolated from the AL. Immunohistochemistry revealed aquaporin 1 (AQP1)-positive signals in fenestrated endothelial cells of the PL. Furthermore, immunoelectron microscopy demonstrated the presence of AQP1 signals on both the luminal and abluminal plasma membranes of these cells. AQP1 plays a pivotal role in facilitating water movement across the plasma membrane in response to changes in osmotic pressure on a cell. To investigate the effect of hyperosmolarity on \u003cem\u003eAqp1\u003c/em\u003e expression, we examined the expression levels of \u003cem\u003eAqp1 \u003c/em\u003ein the PL of water-deprived rats as well as in isolated endothelial cells of the PL cultured in a hyperosmotic medium supplemented with raffinose. Immunohistochemical analysis showed no changes in the proportion of AQP1-positive endothelial cells or in subcellular localization of AQP1 in cultured endothelial cells of the PL under hyperosmotic conditions. In contrast, analysis using quantitative real-time PCR revealed that hyperosmolar conditions significantly downregulated \u003cem\u003eAqp1 \u003c/em\u003eexpression in the cultured endothelial cells. These findings suggest that fenestrated capillaries in the PL actively regulate \u003cem\u003eAqp1\u003c/em\u003eexpression by sensing osmotic pressure of the interstitial fluid. Our results indicate that AQP1 is selectively expressed in fenestrated capillaries of the PL and plays a crucial role in maintaining water homeostasis in this region.\u003c/p\u003e","manuscriptTitle":"Regulation of Aqp1 expression by osmotic balance in fenestrated endothelial cells of the posterior lobe of the pituitary","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 10:05:23","doi":"10.21203/rs.3.rs-6677059/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-22T21:35:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-16T12:11:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-16T12:10:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell and Tissue Research","date":"2025-05-16T04:33:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-and-tissue-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ctre","sideBox":"Learn more about [Cell and Tissue Research](https://link.springer.com/journal/441)","snPcode":"441","submissionUrl":"https://submission.springernature.com/new-submission/441/3","title":"Cell and Tissue Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9e3e5652-098e-44fe-a9b5-407429e99c0e","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:03:18+00:00","versionOfRecord":{"articleIdentity":"rs-6677059","link":"https://doi.org/10.1007/s00441-025-03995-x","journal":{"identity":"cell-and-tissue-research","isVorOnly":false,"title":"Cell and Tissue Research"},"publishedOn":"2025-07-22 15:57:58","publishedOnDateReadable":"July 22nd, 2025"},"versionCreatedAt":"2025-06-02 10:05:23","video":"","vorDoi":"10.1007/s00441-025-03995-x","vorDoiUrl":"https://doi.org/10.1007/s00441-025-03995-x","workflowStages":[]},"version":"v1","identity":"rs-6677059","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6677059","identity":"rs-6677059","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}