Tissue Plasminogen Activator and Plasminogen Are Critical for Osmotic Homeostasis by Regulating Vasopressin Secretion Yuhki Imamura, 1 Shoko Morita, 1 Yoshihiro Nakatani, 1 Kiyotaka Okada, 2 Shigeru Ueshima, 2 Osamu Matsuo, 2 and Seiji Miyata 1 * 1 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan 2 Department of Physiology, Kinki University School of Medicine, Osakasayama, Osaka, Japan Systemic osmotic homeostasis is regulated mainly by neuroendocrine system of arginine-vasopressin (AVP) in mammalians. In the present study, we demonstrated that the immunoreactivity of tissue plasminogen activa- tor (tPA) was observed specifically at neurosecretory granules of AVP-positive magnocellular terminals and that of plasminogen was seen at astrocytes in the neu- rohypophysis (NH). Both tPA and plasminogen knock- out (KO) mice revealed higher plasma osmolarity upon water deprivation, a chronic osmotic stimulation, as compared with their wild-type (WT) animals, indicating abnormal osmotic control in these KO mice. tPA KO mice but not plasminogen ones revealed lower ability in secreting AVP into the blood circulation upon an acute osmotic stimulation. Both tPA and plasminogen KO ani- mals showed lower ability in secreting AVP into the blood circulation upon a chronic osmotic stimulation. The recombinant tPA was able to promote the release of AVP from isolated NH. Chronic osmotic stimulation decreased the laminin expression level of neurohypo- physial microvessel in WT mice but not in plasminogen KO ones. We suggest that AVP secretion is critically regulated by tPA-dependent facilitation of AVP release from terminals and plasminogen-dependent increase of AVP permeability across microvessels possibly via lami- nin degradation. V V C 2010 Wiley-Liss, Inc. Key words: protease; knockout mice; oxytocin; osmotic homeostasis Osmotic control of body fluids is one of the im- portant homeostatic mechanisms for maintaining the in- ternal environment in mammalians, which is achieved through the control of water intake and renal excretion of salt and water (Voisin and Bourque, 2002). Arginine- vasopressin (AVP) is synthesized in the somata of hypo- thalamic magnocellular neurons in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) and packed into neurosecretory granules (NSGs), which are trans- ported through their axons into the terminals in the neurohypophysis (NH) (Miyata and Hatton, 2002). Upon depolarization, AVP is secreted into the blood cir- culation after the process of exocytosis, and the circulat- ing AVP largely determines the ability of the collecting ducts in the kidney by reabsorbing water (Cunningham and Sawchenko, 1991; Schrier, 2007). Tissue plasminogen activator (tPA) is known to be a serine protease that converts plasminogen into the broad-spectrum protease plasmin which controls the proteolysis of numerous targets such as laminin, collagen IV, proteoglycans, brain-derived neurotrophic factor (BDNF), and protease activated receptor-1 (Dityatev and Schachner, 2003; Pang et al., 2004; Nagai et al., 2006). In addition to the activation of plasminogen, tPA is known to interact directly with low-density lipoprotein receptor-related protein (LRP) to trigger the phospho- rylation of mitogen-activated protein kinase kinase 1 and extracellular signal-regulated kinase-1/2 (Zhuo et al., 2000; Hu et al., 2006). tPA is also able to potentiate N- methyl-D-aspartate (NMDA) receptor through cleavage of the NMDA receptor 1 subunit (Nicole et al., 2001). Recent evidence has suggested that tPA-plasminogen system is responsible for important functions within the brain. First, tPA is stored within dense-core granules (Parmer et al., 1997; Silverman et al., 2005) and is released into the extracellular space upon neuronal Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: Scientific Research Grants from the Japan Soci- ety for the Promotion of Science; Contract grant number: 19500293 (to S.M.); Contract grant sponsor: The Salt Science Research Foundation; Contract grant number: 0838 (to S.M.); Contract grant sponsor: The High-Tech Research Center Project for Private Universities (to O.M.). *Correspondence to: Dr. Seiji Miyata, Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan. E-mail: smiyata@kit.ac.jp Received 7 October 2009; Revised 14 November 2009; Accepted 25 December 2009 Published online 19 February 2010 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.22370 Journal of Neuroscience Research 88:1995–2006 (2010) ' 2010 Wiley-Liss, Inc.