Cannabinoid CB1 and Cholecystokinin CCK2 Receptors Modulate, in an Opposing Way, Electrically Evoked [H]GABA Efflux from Rat Cerebral Cortex Cell Cultures: Possible Relevance for Cortical GABA Transmission and Anxiety

The effects of treatments with cannabinoid (CB)1 and cholecystokinin (CCK)2 receptor agonists and antagonists, as well as compounds that enhance endocannabinoid signaling by inhibiting degradation, e.g., the fatty acid amide hydrolase inhibitor 3 -carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) or the endocannabinoid reuptake inhibitor (5Z,8Z,11Z,14Z)-N-(3furanylmethyl)-5,8,11,14-eicosatetraenamide (UCM707), were studied both on spontaneous and electrically evoked [H]GABA efflux from rat cerebral cortex cell cultures. The CCK2 receptor agonist CCK-8S, the CB1 receptor agonist (R)-( )-[2,3-dihydro5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2), URB597, UCM707, the CB1 receptor antagonist N-piperidino-5-(4chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide (SR141716A), and the CCK2 receptor antagonist 2-[2-(5-Br-1H-indol-3-yl)ethyl]-3-[3-(1-methylethoxy)phenyl]-4(3H)-quinazolinone (LY225910) did not affect spontaneous [H]GABA efflux. CCK-8S concentration-dependently increased electrically evoked [H]GABA overflow, and this effect was prevented by LY225910. WIN55,212-2, URB597, and UCM707 induced a reduction of electrically evoked [H]GABA overflow. This reduction was counteracted by SR141716A. When CCK-8S and one of cannabinoid-interfering compounds were simultaneously added, at concentrations by themselves ineffective, to the superfusion medium, an enhancement in electrically evoked [H]GABA efflux was observed. This increase was counteracted by either SR141716A or LY225910 as well as by the inhibitor of protein kinase C, (1R)-2-[12-[(2R)-2(benzoyloxy)propyl]-3,10-dihydro-4,9-dihydroxy-2,6,7,11tetramethoxy-3,10-dioxo-1-perylenyl]-1-methylethylcarbonic acid 4-hydroxyphenyl ester (calphostin C). These results indicate that CB1 and CCK2 receptors modulate, in an opposing way, electrically evoked [H]GABA efflux from rat cerebral cortex cell cultures. The existence of a CB1/CCK2 receptor heteromer on cortical GABA terminals, with a possible relevance for cortical GABA transmission and anxiety, is postulated. The involvement of endocannabinoid (eCB) system in the regulation of physiopathological processes has been generally accepted. Thus, the interest for potential therapeutic applications of cannabinoids is increasing. Among the pharmacological actions of cannabinoids, there is emerging evidence for a possible involvement of the eCB signaling in the control of mood and anxiety (Chhatwal and Ressler, 2007; Mangieri and Piomelli, 2007). However, despite the fact that numerous studies have been focused on this issue, the anxiogenic/anxiolytic profile of action of cannabinomimetics has not been This work has been supported by Ministero dell’Istruzione, dell’Università e della Ricerca COFIN (Cofinanziamento Programmi di Ricerca di Interesse Nazionale) 2004 [Grant 2004057025] and Fondazione Cassa di Risparmio di Ferrara, Italy. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.150649. ABBREVIATIONS: eCB, endocannabinoid; CB, cannabinoid; CCK, cholecystokinin; FAAH, fatty acid amide hydrolase; URB597, 3 -carbamoylbiphenyl-3-yl-cyclohexylcarbamate; UCM707, (5Z,8Z,11Z,14Z)-N-(3-furanylmethyl)-5,8,11,14-eicosatetraenamide; SKF 89976A hydrochloride, 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride; WIN55,212-2 mesylate, (R)-( )-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone; SR144528, [N-(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]heptan-2-yl] 5-(4chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide); LY225910, 2-[2-(5-Br-1H-indol-3-yl)ethyl]-3-[3-(1-methylethoxy)phenyl]-4(3H)-quinazolinone; St1, first stimulation; St2, second stimulation; ANOVA, analysis of variance; PKC, protein kinase C; AM404, N-(4hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide. 0022-3565/09/3292-708–717$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 150649/3459528 JPET 329:708–717, 2009 Printed in U.S.A. 708 at A PE T Jornals on Jauary 5, 2018 jpet.asjournals.org D ow nladed from fully elucidated. For example, some studies demonstrated that CB1 receptor antagonists or genetic disruption of CB1 receptors enhances anxiety in several paradigms in rodents (Kathuria et al., 2003; Haller et al., 2004), suggesting the existence of an anxiolytic eCBs tone in the central nervous system. This hypothesis is supported by studies indicating that inhibitors of eCB reuptake or degradation display anxiolytic effects (Kathuria et al., 2003; Moreira et al., 2008; Rubino et al., 2008a; Micale et al., 2009). In addition, at selected doses, CB1 receptor agonists reduce anxiety-related behavioral responses (Rubino et al., 2008b). Opposite results have been obtained in other studies. Degroot and Nomikos (2004) demonstrated that either genetic deletion or pharmacological blockade of CB1 receptors reduces anxiety, whereas cannabinoid agonists displayed anxiogenic responses, generally at doses higher than those showed to be anxiolytic (Rubino et al., 2008b). Taken together, these results indicate that cannabinoids can both suppress and worsen anxiety-like behaviors depending on a set of variables such as the drug dose, genetic background, environmental context, and the behavioral test used. Moreover, it cannot be excluded that the interaction with other neuronal systems such as GABA or cholecystokinin (CCK) by cannabinoids (Katona et al., 1999; Rotzinger and Vaccarino 2003; Ali 2007) may be state-dependent, which can contribute to these variable results. Among the endogenous compounds supposed to have a crucial role in anxiety, CCK, a gut-brain peptide, has been postulated to be one of the most important (Rotzinger and Vaccarino 2003). The sulfated carboxyl-terminal octapeptide (CCK-8S) is the predominant active form of the peptide with a high brain distribution. CCK2 receptor is the main CCK receptor subtype presents in the central nervous system, and its distribution is consistent with the functions attributed to neural CCK, including regulation of feeding, control of learning and memory, behavioral expression of anxiety and mediation of pain (Chandra and Liddle, 2007). The presence of CCK receptors in brain regions assumed to control emotions such as the cerebral cortex and the limbic system, the responsiveness of cortical and amygdala endogenous CCK system to anxiety and other stressful stimuli, as well as the functional interactions among CCK, monoaminergic and GABAergic neurones, provide evidence that CCK may have a direct or mediated involvement in anxiety-like behaviors. CB1 receptor high levels have been found on the terminals of CCK-positive GABAergic interneurons in several brain areas (Katona et al., 1999). Hippocampal CB1 receptor activation inhibits potassium-evoked CCK release (Beinfeld and Connolly, 2001), and eCBs can function as retrograde messengers, modulating CCK release (Valverde, 2005; Chhatwal et al., 2009). These findings raised the hypothesis for the existence of a reciprocal interaction between eCBs and CCK (Valverde, 2005; Kurrikoff et al., 2008; Chhatwal et al., 2009), which could represent the first step of a cascade of events that may regulate the functional activity of other neuronal systems related to anxiety processes, such as the GABAergic process. On this background, in this study the possible existence of a functional interaction between eCBs and CCK systems in controlling GABAergic neuronal transmission in the rat cerebral cortex, a brain region that is important in regulating a variety of emotion-related behaviors, has been evaluated. Thus, the effects of pharmacological treatments with CB1 and CCK2 receptors agonists/antagonists, as well as compounds that enhance eCB signaling by inhibiting degradation, e.g., the fatty acid amide hydrolase (FAAH) inhibitor URB597, or reuptake (UCM707), were studied both on spontaneous and electrically evoked [H]GABA efflux from rat cerebral cortex cell cultures. Materials and Methods Animal Care. All experiments were performed in accordance with the guidelines issued by the Italian Ministry of Health, the Declaration of Helsinki, and the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Bethesda, MD). Cortical Cell Cultures Preparation. Primary cultures of cortical neurons were prepared from embryonic day 18 Sprague-Dawley rats. Removed cortices were dissected free of meninges and dissociated in 0.025% (w/v) trypsin. The tissue fragments were dissociated mechanically by gentle pipetting through wideand narrow-bore fire-polished Pasteur pipettes in culture medium (Neurobasal medium with supplements of 0.1 mM glutamine, 10 g/ml gentamicin, and 2% B27). The cells were counted and then plated on poly-L-lysine (5 g/ml)-coated dishes at a density of 2.5 10 cells/dish. Cultures were grown at 37°C in a humidified atmosphere of 5% CO2, 95% air. Cytosine arabinoside (1 M) was added at 5 days in vitro to prevent glial cell proliferation. The cultures were maintained for 8 days in vitro before experiments. Experimental Protocol. Preloaded [H]GABA was used as a tracer of endogenous GABA. Experiments were performed as described previously (Tomasini and Antonelli, 1998). Before starting the release experiment, the dishes containing the cells were rinsed three times by replacing the culture medium with 1 ml of warmed (37°C) Krebs-Ringer bicarbonate buffer consisting of 118.5 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3, 1.2 mM KaH2PO4, and 11 mM glucose, pH 7.4). Thereafter, the cells were preloaded, for 30 min at 37°C in a humidified atmosphere 5% CO2, air, with [ H]GABA (40 nM; 32.9 Ci/mmol). The dishes containing the cell cultures preloaded with [H]GABA, were rinsed as described above and subsequently placed in eight small chambers (0.5 ml) of a thermostated superfusion system. Each superfusion chamber was closed with a cylinder equipped with two U-shaped platinum wire electrodes to allow, when required, the electrical stimulation of the neurons. The cell monolayer were continuously superfused with Krebs-Ringer bicarbonate buffer (flow rate, 0.6 ml/min) bubbled previously with carbogen (95% O2 and 5% CO2) and containing 30 M SKF 89976-A to inhibit the high-affinity GABA carrier. After 45 min of superfusion, the steady state of [H]GABA efflux was reached ( 8–10% variation of tritium content between consecutive 5-min samples), and collection of the perfusate (5 min/sample) was initiated