Neurochemical effects of the endocannabinoid uptake inhibitor UCM707 in various rat brain regions

Abstract

To date, UCM707, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, has the highest potency and selectivity in vitro and in vivo as inhibitor of the endocannabinoid uptake. Its biochemical, pharmacological and therapeutic properties have been intensely studied recently, but the information on its capability to modify neurotransmitter activity, which obviously underlies the above properties, is still limited. In the present study, we conducted a time-course experiment in rats aimed at examining the neurochemical effects of UCM707 in several brain regions following a subchronic administration (5 injections during 2.5 days) of this inhibitor in a dose of 5 mg/kg weight. In the hypothalamus, the administration of UCM707 did not modify GABA contents but reduced norepinephrine levels at 5 h after administration, followed by an increase at 12 h. Similar trends were observed for dopamine, whereas serotonin content remained elevated at 1 and, in particular, 5 and 12 h after administration. In the case of the basal ganglia, UCM707 reduced GABA content in the substantia nigra but only at longer (5 or 12 h) times after administration. There were no changes in serotonin content, but a marked reduction in its metabolite 5HIAA was recorded in the substantia nigra. The same pattern was found for dopamine, contents of which were not altered by UCM707 in the caudate–putamen, but its major metabolite DOPAC exhibited a marked decrease at 5 h. In the cerebellum, UCM707 reduced GABA, serotonin and norepinephrine content, but only the reduction found for norepinephrine at 5 h reached statistical significance. The administration of UCM707 did not modify the contents of these neurotransmitters in the hippocampus and the frontal cortex. Lastly, in the case of limbic structures, the administration of UCM707 markedly reduced dopamine content in the nucleus accumbens at 5 h, whereas GABA content remained unchanged in this structure and also in the ventral–tegmental area and the amygdala. By contrast, norepinephrine and serotonin content increased at 5 h in the nucleus accumbens, but not in the other two limbic structures. In summary, UCM707 administered subchronically modified the contents of serotonin, GABA, dopamine and/or norepinephrine with a pattern strongly different in each brain region. So, changes in GABA transmission (decrease) were restricted to the substantia nigra, but did not appear in other regions, whereas dopamine transmission was also altered in the caudate–putamen and the nucleus accumbens. By contrast, norepinephrine and serotonin were altered by UCM707 in the hypothalamus, cerebellum (only norepinephrine), and nucleus accumbens, exhibiting biphasic effects in some cases.

Introduction

Within the present interest in the cannabinoid signaling system as a possible target for novel therapy in several neurological disorders, the mechanism of endocannabinoid uptake has attracted special interest (for a recent review see McFarland and Barker, 2004). This mechanism is part of the process of inactivation of endocannabinoid ligands which also includes the participation of at least two degradative enzymes (Giuffrida et al., 2001, Ortega-Gutierrez, 2005). Several synthetic compounds, such as N-(4-hydroxyphenyl)-arachidonamide (AM404; for details, see Khanolkar et al., 1996, Beltramo et al., 1997), N-(4-hydroxy-2-methylphenyl)-arachidonamide (VDM11; for details, see De Petrocellis et al., 2000), or (S)-N-oleoyl-(1′-hydroxybenzyl)-2′-ethanolamine (OMDM2; for details, see Ortar et al., 2003), have been reported to block the endocannabinoid uptake and have been accordingly evaluated for their therapeutic potential. Other related compounds that have recently attracted interest are: (i) the first covalent inhibitors of anandamide cellular uptake, which might be used for the molecular characterization of the protein involved in the membrane transport of endocannabinoids (Moriello et al., 2006); (ii) the heterocyclic urea inhibitor LY2183240, a compound presented first as a transporter inhibitor (Moore et al., 2005), but that has been recently demonstrated to act as an inhibitor of the fatty acid amide hydrolase (FAAH) enzyme (Alexander and Cravatt, 2006); and (iii) the series of URB compounds, such as URB597, that act as FAAH inhibitors without blocking the transporter (Kathuria et al., 2003). All these compounds, termed “indirect agonists”, act by potentiating the action of endogenous ligands and, hence, they might be particularly recommended for diseases in which an increase in cannabinoid signaling has been postulated to be of therapeutic value (Giuffrida et al., 2001, Pertwee, 2002). The use of these compounds may make it possible to minimize the unwanted effects produced by the “direct activation” of cannabinoid CB₁ receptors with classical cannabinoids, through the control of endocannabinoid levels in a concentration range that avoids psychoactive side effects (Felder and Glass, 1998, Ortega-Gutierrez, 2005). However, some of these compounds, such as in the case of AM404, may also behave as agonists of vanilloid TRPV1 receptors (Zygmunt et al., 2000) and, then, exhibit direct effects by themselves (González et al., 1999, Beltramo et al., 2000).

We have recently designed and synthesized a series of arachidonic acid derivatives (López-Rodríguez et al., 2001, López-Rodríguez et al., 2003a) that exhibit a high potency and selectivity in vitro as inhibitors of endocannabinoid uptake, with negligible affinity for cannabinoid, CB₁ and CB₂, and vanilloid TRPV1 receptors, and the FAAH enzyme. One of these compounds, (5Z,8Z,11Z,14Z)-N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide, so-called UCM707, is the most potent and selective endocannabinoid uptake inhibitor described to date (López-Rodríguez et al., 2001, López-Rodríguez et al., 2003a, López-Rodríguez et al., 2003b). When used in vivo at low to moderate doses (0.1–1 mg/kg), this compound does not produce any relevant cannabimimetic effects, such as hypomotility and antinociception (de Lago et al., 2002). However, at these doses, it exhibited an interesting capability to enhance the hypokinetic and antinociceptive actions of subeffective doses of anandamide (de Lago et al., 2002). Higher doses of UCM707 (> 1 mg/kg) did produce hypokinetic and analgesic effects by themselves (de Lago et al., 2002), possibly due to the increase of the endogenous cannabinoid tone (de Lago et al., 2005). These observations may enable UCM707 to be used in several neurological disorders, where, as mentioned above, an elevation of the endocannabinoid tone has been postulated to have therapeutic benefits. For instance, we have extensively studied the effects of UCM707 in animal models of Huntington's disease and multiple sclerosis, where we found that this compound has potential as a symptom-alleviating agent, producing antihyperkinetic and antispastic effects, respectively (de Lago et al., 2006). It may also attenuate the progression of the disease in a rodent model of multiple sclerosis (Ortega-Gutiérrez et al., 2005). In addition, UCM707 may be also used as an analgesic agent in chronic or inflammatory pain (de Lago et al., 2002, La Rana et al., 2006).

As mentioned above, the neurobiological effects of UCM707 or the other endocannabinoid uptake inhibitors in healthy or pathological conditions would be produced by enhancing the action of endocannabinoids at their receptors (de Lago et al., 2002, de Lago et al., 2005). Through this mechanism, UCM707 might influence the activity of several classic neurotransmitters, such as GABA, serotonin or dopamine, which are under the modulatory influence of endocannabinoids (Schlicker and Kathmann, 2001). The literature published so far on UCM707 effects has covered: (i) its in vitro inhibitory action on the endocannabinoid uptake (López-Rodríguez et al., 2001, López-Rodríguez et al., 2003a), (ii) its capability to elevate endocannabinoid levels (de Lago et al., 2005), (iii) its effects, alone or in combination with anandamide, on several behavioural responses (de Lago et al., 2002), and (iv) its potential therapeutic properties in several neurological disorders (Marsicano et al., 2003, Ortega-Gutiérrez et al., 2005, de Lago et al., 2006). However, there are no relevant data on the effects of UCM707 on the activity of those neurotransmitters, such as GABA, norepinephrine, serotonin or dopamine, that underly the behavioural and therapeutic effects of UCM707 in several brain regions. This has been the main objective of the present study. To address these issues, we conducted a time-course experiment in rats, aimed at examining the effects of UCM707 on these four neurotransmitters in several brain regions following a subchronic administration (five injections during 2.5 days) of this inhibitor at a dose of 5 mg/kg weight. The conditions used for UCM707 treatment (dose, timing, route of administration) were selected from a previous study showing an enhancement by UCM707 of endocannabinoid levels (de Lago et al., 2005). In addition, this dose was revealed to produce motor inhibition and analgesia in previous dose-response analyses (de Lago et al., 2002) and to be beneficial in animal models of some motor disorders (de Lago et al., 2006). Subchronic treatment was chosed because it was expected to have more marked neurochemical effects than those found after a single administration of the same dose in a preliminary study centered in the basal ganglia (de Lago et al., 2006). In the present study, we wanted to also extend the analysis to other brain areas, selecting in each region those neurotransmitters that have been reported to be most affected by the treatment with different cannabinoid compounds. Thus, norepinephrine, serotonin, GABA and dopamine were investigated in the hypothalamus since they have been related to the effects of cannabinoids on several hypothalamic functions, such as regulation of feeding behaviour (Thiébot et al., 2006) and control of anterior pituitary hormone secretion (Fernández-Ruiz et al., 2006). The same neurotransmitters were also investigated in the frontal cortex and several limbic structures because they were involved in the mediation of some cognitive, emotional and motivational effects of cannabinoids (Thiébot et al., 2006). Serotonin, GABA and dopamine were investigated in the basal ganglia (Thiébot et al., 2006, Fernández-Ruiz et al., 2006), whereas GABA, serotonin and norepinephrine were analyzed in relation with the effects of cannabinoids in the cerebellum (Hoffman and Lupica, 2006). These last three neurotransmitters were investigated in the hippocampus because they were involved in memory effects of cannabinoids (Thiébot et al., 2006, Hoffman and Lupica, 2006).