br Conflict of interest br Acknowledgments Dr
Conflict of interest
Acknowledgments Dr. Hauger was supported by a BLR&D Merit Review grant from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development. Dr. Hauger also received support from the VA Center of Excellence for Stress and Mental Health (CESAMH), and NIH/NIA (AG018386) and NIH/NIMH RO1 (MH074697) grants. Dr. Oakley received support from the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, and completed a portion of the confocal microscopy experiments while he was employed at Xsira Pharmaceuticals (Morrisville, NC). Dr. Olivares-Reyes was supported by CINVESTAV-IPN, a UC MEXUS-CONACYT grant for collaborative projects, and a Grant for Research on Health from Fundacion Miguel Aleman 2010. Receptor internalization and cell surface receptor expression measurements were performed at the Flow Cytometry Research Core of the San Diego VA Research Service that is supported by DVA and a NIH/NIAID RO1 (AI36214). We also gratefully acknowledge Dr. Dimitri Grigoriadis and Neurocrine Biotechnology (La Jolla, CA) for generously providing NBI-30775 under an approved material transfer agreement. Dr. Robert Lefkowitz (HHMI, Duke University) kindly provided the βarrestin KO and wild-type MEF Chidamide under a material transfer agreement.
Materials and methods
Discussion In the present study, etifoxine attenuated the anxiogenic-like behavioral and autonomic responses to i.c.v. injection of CRF in the rodent. Thus, in the open field, rats receiving CRF responded with an increased sensitivity to the stressful aspects of a new situation. They remained in one of the corners of the arena, grooming or moving forwards and backwards. Increased grooming behavior has been related to fear or increased emotional-like response (Morley and Levine, 1982, Kalueff and Tuohimaa, 2004, Kalueff and Tuohimaa, 2005). The decrease by etifoxine of the CRF-induced grooming is interpreted as a manifestation of its anxiolytic effect and is consistent with the activity previously exhibited in classical anxiety animal models (Schlichter et al., 2000, Verleye and Gillardin, 2004). Etifoxine also reversed the CRF-induced delay in gastric emptying of a solid meal. It is well known that exposure to stressful stimuli, mediated by the endogenous release of CRF, alters gastrointestinal motility resulting in decreased gastric secretion and emptying in experimental animals and humans (Coskun et al., 1997, Taché et al., 2001). In the present study, α-h-CRF, a competitive antagonist of CRF1 and CRF2 receptors (Rivier et al., 1984) exhibited similar effects except that α-h-CRF reversed the CRF-induced hypo-locomotion in the open field. The hypothesis that etifoxine might act as an antagonist at the CRF1 and CRF2 receptors was not supported by two sets of results: (i) the failure of etifoxine to reproduce the effects of α-h-CRF on CRF-induced hypo-locomotion and (ii) the results of binding and in vitro functional studies. Etifoxine did not exhibit affinity for CRF1 and CRF2 binding sites (IC50>10μM), suggesting that this compound does not act directly at CRF receptors. The CRF1 and CRF2 receptors belong to the class II G-protein-coupled receptor super-family (Hillhouse et al., 2002). Functionally, in most tissues (e.g. brain), stimulation of these receptors by CRF and CRF-related peptides triggers activation of adenyl cyclase and increases cAMP levels (Grammatopoulos et al., 1994, Grammatopoulos and Chrousos, 2002). To assess the potential antagonistic activity of etifoxine, the in vitro neuroblastoma cell system was suitable to investigate the functional consequences mediated through the CRF1 and CRF2 receptors. The fact that etifoxine inhibited the CRF-induced cAMP stimulation with a weak potency supports the notion that it does not behave as a functional antagonist of the CRF receptors. There is precedent for anxiolytics with various mechanisms of action to counteract CRF-induced anxiety-like behavior in rodents without a real antagonistic activity on the CRF1 and CRF2 receptors (Lazosky and Britton, 1991, To et al., 1999). In vitro and in vivo pharmacological studies have suggested that drugs acting through the GABA/benzodiazepine/chloride channel receptor complex (such as benzodiazepines) may exert, at least in part, their anxiolytic and/or antidepressant effects via suppression of central CRF secretion (Calogero et al., 1988, Patchev et al., 1994, Serra et al., 1999, Cullinan, 2000). The well established etifoxine-induced enhancing of the GABAergic transmission by a positive allosteric effect via the chloride channel site (Verleye et al., 1999, Verleye et al., 2002, Schlichter et al., 2000), may be linked to its inhibitory effect on the CRF system.