Hypolocomotor effects in rats of capsaicin and two long chain capsaicin homologues

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Abstract

Capsaicin and its analogue N-arachidonoyl-vanillyl-amine (arvanil) are agonists of vanilloid VR1 receptors, and suppress spontaneous activity in mice through an unknown mechanism. Here, we tested in rats the effect on motor behavior of: (1) capsaicin; (2) N-linoleoyl-vanillyl-amine (livanil) and N-α-linolenoyl-vanillyl-amine (linvanil), which, unlike arvanil, have very little affinity for cannabinoid CB1 receptors; and (3) the endocannabinoid anandamide (N-arachidonoyl-ethanolamine), which is a full agonist at both cannabinoid CB1 and vanilloid VR1 receptors. All compounds, administered i.p., dose-dependently (0.1–10 mg/kg) inhibited ambulation and stereotypic behavior and increased inactivity in the open field test. The rank of potency observed in vivo (livanil>capsaicin>linvanil>anandamide) bore little resemblance with the relative potencies in a functional assay for rat vanilloid VR1 receptors (livanil=linvanil>capsaicin>anandamide) and even less with the relative affinities in rat CB1 receptor binding assays (anandamide>livanil>linvanil>capsaicin). The vanilloid VR1 receptor antagonist capsazepine (10 mg/kg, i.p.) blocked the effect of capsaicin but not of livanil or anandamide, whereas the CB1 receptor antagonist (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A, 3 mg/kg, i.p.) antagonized the actions of the CB1 receptor agonist Δ9-tetrahydrocannabinol, but not of livanil, anandamide or capsaicin. Anandamide occluded the effects of livanil on locomotion, possibly suggestive of a common mechanism of action for the two compounds. Finally, stimulation with capsaicin of cells expressing rat vanilloid VR1 receptors led to anandamide formation. These data suggest that motor behavior can be suppressed by the activation of: (1) vanilloid receptors, possibly via the intermediacy of anandamide; or (2) capsazepine- and SR141716A-insensitive sites of action for anandamide, livanil and linvanil, possibly the same that were previously suggested to mediate arvanil hypokinetic effects in mice.

Introduction

Anandamide (N-arachidonoyl-ethanolamine) was the first endogenous ligand of cannabinoid receptors to be discovered (Devane et al., 1992). This compound binds with moderate affinity to cannabinoid CB1 and CB2 receptors (Pertwee, 1997), but is a preferential functional agonist only for the CB1 subtype. Anandamide exhibits a pharmacological profile (see Mechoulam et al., 1998, Di Marzo, 1998) similar, but not identical, to that of the psychotropic component of Cannabis sativa, (−)-Δ9-tetrahydro-cannabinol (Gaoni and Mechoulam, 1964). Differences in the pharmacology of anandamide and Δ9-tetrahydrocannabinol may be due to the facile degradation of the former compound in vivo (Willoughby et al., 1997), or also to its interactions with targets other than cannabinoid CB1 or CB2 receptors. Indeed, a selective antagonist of CB1 receptors, (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.HCl (SR141716A, Rinaldi-Carmona et al., 1994), does not block the typical cannabimimetic neurobehavioral effects of anandamide in mice (Adams et al., 1998), which consist of suppression of spontaneous activity and induction of immobility, analgesia and hypothermia, and are known as the mouse “tetrad” of tests. Furthermore, some of these effects of anandamide are still observed in mutant mice where the CB1 receptor gene has been disrupted (“CB1 knockouts”) (Di Marzo et al., 2000a). Recent experiments Zygmunt et al., 1999, Smart et al., 2000 showed that anandamide is also a full agonist at the capsaicin receptor, a ligand and heat-activated non-selective cation channel named “vanilloid” VR1 receptor (Caterina et al., 1997). By acting in part through vanilloid receptors, anandamide relaxes both the buffer-perfused small artery preparations (Zygmunt et al., 1999) and the electrically contracted mouse vas deferens (Ross et al., 2001), and induces apoptosis of cancer cells (Maccarrone et al., 2000). Although vanilloid VR1 receptors have been identified in several areas of the rodent brain, e.g. the basal ganglia, hypothalamus and brainstem (Mezey et al., 2000), there is still no evidence for their participation either in central nervous system (CNS) functions other than body temperature control (Szallasi and Di Marzo, 2000). The involvement of vanilloid VR1 receptors in the control of spontaneous activity, body temperature and (supra)spinal nociception is supported by the observation that capsaicin exhibits moderate to strong activity in the four tests of the mouse “tetrad”, even though it was not possible to counteract these effects of capsaicin with the vanilloid VR1 receptor antagonist, capsazepine (Di Marzo et al., 2000b). Apart from VR1 receptors, anandamide interacts also with non-CB1, non-CB2 G-protein-coupled receptors in mouse brain (Di Marzo et al., 2000a) which are activated also by the cannabinoid receptor ligand R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN 55212-2) Sagan et al., 1999, Breivogel et al., 2001. Therefore, it is possible that the effects of anandamide in the mouse ‘tetrad’ are partly mediated by VR1 receptors or novel cannabinoid receptors, although these possibilities have not yet been fully investigated.

Arvanil (N-[3-methoxy-4-hydroxy-benzyl]-arachidonamide) is a recently developed anandamide/capsaicin structural “hybrid” (Melck et al., 1999) with an affinity for cannabinoid CB1 receptors comparable to that of anandamide, and an activity at vanilloid VR1 receptors that is stronger than that of capsaicin De Petrocellis et al., 2000, Ross et al., 2001. Arvanil is also a potent inhibitor of anandamide-facilitated transport into cells (Melck et al., 1999). Its actions at these multiple sites, and its higher metabolic stability as compared to anandamide, may explain in part why arvanil is more potent than either anandamide or capsaicin in the mouse ‘tetrad’, even though its effects, like for anandamide, are insensitive to SR141716A (Di Marzo et al., 2000b). Although the time-course of arvanil actions in mice seem to differ from that observed with capsaicin, it is possible that this novel compound acts in the CNS via VR1 receptors. Alternatively, since it induces potent analgesia in the tail-flick test in a manner insensitive to either SR141716A or capsazepine (Di Marzo et al., 2000b), arvanil may also activate non-CB1, non-CB2, and non-VR1 receptors.

Here, we have addressed the question of whether vanilloid VR1 receptors or non-CB1, non-VR1 sites of action are involved in the regulation of motor behavior in rats. We have studied the effect on locomotion of capsaicin, anandamide and of two arvanil analogues having weak activity, if any, at rat CB1 receptors. We report that the activation of either VR1 receptors or putative novel anandamide sites of action leads to suppression of motor behavior in rats, and suggest that vanilloid receptor stimulation may affect motor behavior also through the intermediacy of anandamide.

Section snippets

Compounds

Capsaicin and capsazepine were purchased from Alexis Biochemicals or Sigma-Aldrich, and anandamide for in vivo studies from Tocris. Anandamide, N-linoleoyl-vanillyl-amine (livanil) and N-α-linolenoyl-vanillyl-amine (linvanil) (Fig. 1) were synthesized from the condensation of the corresponding fatty acids and amines as described previously (Melck et al., 1999). SR141716A was a kind gift from Sanofi Recherche (Montpellier, France). Δ9-Tetrahydrocannabinol was kindly provided by the National

Affinity of the compounds for cannabinoid CB1 receptors

The two long chain, polyunsaturated capsaicin analogues synthesized here were chosen instead of arvanil because of their lower affinity for mouse cannabinoid CB1 receptors and less potent inhibitory effect on the anandamide membrane transporter in rat cells (Melck et al., 1999). In a binding assay carried out with rat brain membranes, and, therefore, more relevant to the in vivo studies described here, we found that livanil and linvanil were very weak ligands of these receptors (Table 1). In

Discussion

Vanilloid VR1 receptors have been identified in several nuclei of rat brain, including the substantia nigra compacta and the striatum, which are involved in the control of motor behavior (Mezey et al., 2000). Furthermore, it was also shown that i.v. administration of capsaicin to mice caused inhibition of spontaneous activity (ED50=0.3 mg/kg) and, to a lesser extent, immobility on a ring (ED50∼1 mg/kg) (Di Marzo et al., 2000b). In mice, it was not possible to counteract capsaicin effects with

Acknowledgements

The authors wish to thank A. Schiano-Moriello for assistance. This work was partly funded by the MURST (grant 3933 and Fondi Strutturali) to VDM and by CAM-PRI (08.5/0029/98) to JJFR.

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