Behavioral, Pharmacological, and Molecular Characterization of an Amphibian Cannabinoid Receptor

Abstract

Investigation of cannabinoid pharmacology in a vertebrate with a phylogenetic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granulosa, the roughskin newt, was used here to characterize an amphibian cannabinoid receptor. Behavioral experiments demonstrated that the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor activity and courtship clasping behavior. Inhibition of clasping was dose-dependent and potent (IC 50 ϭ 1.2 g per animal). Radioligand binding studies using [ 3 H]CP-55940 allowed identification of a specific binding site (K D ϭ 6.5 nM, B max ϭ 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (K D , nM): CP-55940 (3.8) Ͼ levonantradol (13.0) Ͼ WIN55212-2 (25.7) Ͼ Ͼ anandamide (1,665) Ϸ anandamide ϩ 100 M phenylmethylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stably expressing the newt CB1 cannabinoid receptor was prepared that allowed demonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity. This inhibition was dose-dependent and occurred at concentrations consistent with affinities determined through radioligand binding experiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand binding affinity, ligand binding specificity, and amino acid sequence to mammalian CB1. The high degree of evolutionary conservation of cannabinoid signaling systems implies an important physiological role in vertebrate brain function. Key Words: CannabinoidAmphibian-G protein-coupled receptor-Adenylate cyclase -Locomotor behavior-Sex behavior. J. Neurochem. 75, 413-423 (2000).The active constituents of Cannabis sativa have been used for centuries for their medicinal and psychoactive properties. Despite this long history of cannabinoid use an understanding of cannabinoid pharmacology has only recently begun to emerge. This progress was initiated with evidence that cannabinoids inhibit neuronal adenylate cyclase activity through activation of G proteins (Howlett and Fleming, 1984;Howlett, 1985). Cannabinoid activation of G proteins with an appropriate pharmacological signature implied the presence of specific cannabinoid receptors. Two subtypes of specific cannabinoid receptors have been identified: CB1, CNS-associated [although peripheral expression has been detected (Devane et al., 1988;Matsuda et al., 1990)], and CB2, which may be exclusively expressed within tissues of the immune system (Munro et al., 1993).Both cannabinoid receptor subtypes alter cellular activity through activation of G proteins of the G i/o subtype, although reports sugges...

Effects of Cannabinoids on Synaptic Transmission in the Frog Neuromuscular Junction

“…However, the presence of the CB 1 -selective antagonist AM281 at 1 M (Howlett et al, 2002) blocked the effects of WIN on the frequency and amplitude of the MEPPs, indicating that WIN acts through activation of the CB 1 receptors. These results are accord with only the CB 1 receptor ortholog having reported in amphibians (Soderstrom et al, 2000;McPartland et al, 2006).…”

“…These effects are produced by the interaction of these compounds with the cannabinoid membrane receptors named CB 1 and CB 2 (Howlett et al, 2002). Both subtypes of cannabinoid receptors alter cellular activity through activation of G proteins of the G i/o subtype, which are sensitive to pertussis toxin (Soderstrom et al, 2000), although some effects are thought to occur through G s (Bayewitch et al, 1995;Glass and Felder, 1997). Activation of G i/o proteins by CB 1 and CB 2 receptors decreases adenylate cyclase activity, leading to decreased levels of intracellular cyclic AMP and protein phosphorylation.…”

“…Therefore, in amphibians, only the CB 1 receptor ortholog has been reported to date (Soderstrom et al, 2000;McPartland et al, 2006). The endocannabinoids (endogenous cannabis-like substances) are fatty acids derived from arachidonic acid, and they notably include anandamide and 2-arachidonylglycerol.…”

The evolution and comparative neurobiology of endocannabinoid signalling

“…Furthermore, consistent with the notion that CB 1 has an evolutionarily ancient role in neural pathways that control movement, Valenti et al [97] have reported that the CB 1 receptor antagonist AM 251 (1 mg g 21 body mass) causes a reduction in locomotor activity in the goldfish Carassius auratus. Behavioural effects of drugs that bind to the CB 1 receptor have also been investigated in an amphibian species, the roughskinned newt T. granulosa, revealing an inhibitory effect on spontaneous locomotor activity and courtship clasping behaviour [98]. Likewise, the cannabinoid WIN 55,212-2 causes inhibition of locomotor activity in the zebra finch [99].…”

Pharmacological Properties of Cannabinoid Receptors in the Avian Brain: Similarity of Rat and Chicken Cannabinoid1 Receptor Recognition Sites and Expression of Cannabinoid2 Receptor‐Like Immunoreactivity in the Embryonic Chick Brain

“…It is important to determine whether cannabinoid receptors behave like their mammalian equivalents in other species. Thus, for example, the cannabinoid 1 receptor in the roughskin newt (Taricha granulosa) showed considerable structural homology and affinity for cannabinoid ligands to its rodent equivalent (Sö -derströ m et al 2000). In contrast, however, the cannabinoid receptor identified in Drosophila melanogaster in binding studies using [ 3 H]CP 55,940 could not be blocked by SR 141716A or SR 144528 (Howlett et al 2000).…”