Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes Olfa Masmoudi, Pierrick Gandolfo, Tursonjan Tokay, Je ´ro ˆme Leprince, Aure ´lia Ravni, Hubert Vaudry and Marie-Christine Tonon European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, Mont-Saint-Aignan, France Abstract Endozepines, a family of regulatory peptides related to diazepam-binding inhibitor (DBI), are synthesized and released by astroglial cells. Because rat astrocytes express various subtypes of somatostatin receptors (sst), we have investigated the effect of somatostatin on DBI mRNA level and endozepine secretion in rat astrocytes in secondary culture. Somatostatin reduced in a concentration-dependent manner the level of DBI mRNA in cultured astrocytes. This inhibitory effect was mimicked by the selective sst4 receptor agonist L803-087 but not by the selective sst1, sst2 and sst3 receptor agonists L779-591, L779-976 and L797-778, respectively. Somatostatin was unable to further reduce DBI mRNA level in the presence of the MEK inhibitor U0126. Somatostatin and the sst1, sst2 and sst4 receptor agonists induced a concentration-dependent inhibition of endozepine release. Somatostatin and the sst1, sst2 and sst4 receptor agonists also inhibited cAMP formation dose-dependently. In addition, somatostatin reduced forskolin-induced endozepine release. H89 mimicked the inhibitory effect of somatostatin on endozepine secretion. In contrast the PLC inhibitor U73122, the PKC activator PMA and the PKC inhibitor calphostin C had no effect on somatostatin- induced inhibition of endozepine release. The present data demonstrate that somatostatin reduces DBI mRNA level mainly through activation of sst4 receptors negatively coupled to the MAPK pathway, and inhibits endozepine release through activation of sst1, sst2 and sst4 recep- tors negatively coupled to the adenylyl cyclase/PKA pathway. Keywords: astrocytes, diazepam-binding inhibitor, mitogen- activated protein kinase, octadecaneuropeptide, protein kinase A, somatostatin. J. Neurochem. (2005) 94, 561–571. The term endozepines designates a family of neuropeptides originally isolated from the rat brain as endogenous ligands of benzodiazepine receptors (Guidotti et al. 1983). All endozepines identified so far derive from diazepam-binding inhibitor (DBI), a 10-kDa polypeptide that can generate through proteolytic cleavage several biologically active fragments, including the triakontatetraneuropeptide DBI[17–50] (TTN) and the octadecaneuropeptide DBI[33– 50] (ODN) (Ferrero et al. 1986; Slobodyansky et al. 1994). DBI has been initially characterized as an inverse agonist of central-type benzodiazepine receptors (Bormann 1991). Subsequently, endozepines were also found to interact with peripheral-type benzodiazepine receptors (Papadopoulos et al. 1991; Slobodyansky et al. 1994). More recently, it has been shown that endozepines can activate a metabotropic receptor positively coupled to phospholipase C (PLC) Resubmitted manuscript received September 15, 2004; accepted January 4, 2005. Address correspondence and reprint requests to Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr Abbreviations used: BSA, bovine serum albumin; DBI, diazepam- binding inhibitor; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GRE, glucocorticoid-response element; HPLC, high-performance liquid chro- matography; IBMX, isobutylmethylxanthine; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NF-Y, nuclear factor-Y; ODN, octadecaneuropeptide DBI [33–50]; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; RIA, radioimmunoassay; SRE, sterol regulatory element; sst, somatostatin receptor subtype; TCA, trichloro- acetic acid; TFA, trifluoroacetic acid; TTN, triakontatetraneuropeptide DBI [17–50]. Journal of Neurochemistry , 2005, 94, 561–571 doi:10.1111/j.1471-4159.2005.03076.x Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 561–571 561