Billy R. Martin

Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors.

In this study, we report the isolation from canine intestines of 2-arachidonyl glycerol (2-Ara-Gl). Its structure was determined by mass spectrometry and by direct comparison with a synthetic sample. 2-Ara-Gl bound to membranes from cells transiently transfected with expression plasmids carrying DNA of either CB1 or CB2--the two cannabinoid receptors identified thus far--with Ki values of 472 +/- 55 and 1400 +/- 172 nM, respectively. In the presence of forskolin, 2-Ara-Gl inhibited adenylate cyclase in isolated mouse spleen cells, at the potency level of delta 9-tetrahydrocannabinol (delta 9-THC). Upon intravenous administration to mice, 2-Ara-Gl caused the typical tetrad of effects produced by THC: antinociception, immobility, reduction of spontaneous activity, and lowering of the rectal temperature. 2-Ara-Gl also shares the ability of delta 9-THC to inhibit electrically evoked contractions of mouse isolated vasa deferentia; however, it was less potent than delta 9-THC.

International Union of Pharmacology. XXVII. Classification of Cannabinoid Receptors

Two types of cannabinoid receptor have been discovered so far, CB1 (2.1: CBD:1:CB1:), cloned in 1990, and CB2(2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB1 and CB2 exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB1 receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB1 agonists probably serve as retrograde synaptic messengers. CB2 receptors are present mainly on immune cells. Such cells also express CB1receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB1) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB1 ligand anandamide. Although anandamide binds and activates the CB1 receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH−/−) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB1-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH−/−-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB1 antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.

Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain

Abstract: Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB1), yet some of the neurobehavioral effects of this compound in mice are unaffected by a selective CB1 antagonist. We studied the levels, pharmacological actions, and degradation of AEA in transgenic mice lacking the CB1 gene. We quantified AEA and the other endocannabinoid, 2‐arachidonoyl glycerol, in six brain regions and the spinal cord by isotope‐dilution liquid chromatography‐mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB1 distribution only in part. In CB1 knockout homozygotes (CB1‐/‐), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild‐type (CB1+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2‐Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB1‐/‐ with respect to CB1+/+ mice in all regions. AEA and Δ9‐tetrahydrocannabinol (THC) were tested in CB1‐/‐ mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB1‐/‐ mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB1‐/‐ mice, and this stimulation was insensitive to CB1 and CB2 antagonists. We suggest that non‐CB1, non‐CB2 G protein‐coupled receptors might mediate in mice some of the neuro‐behavioral actions of AEA.

Behavioral, Biochemical, and Molecular Modeling Evaluations of Cannabinoid Analogs

Numerous cannabinoids have been synthesized that are extremely potent in all of the behavioral assays conducted in our laboratory. An important feature in increasing potency has been the substitution of a dimethylheptyl (DMH) side chain for the pentyl side chain. Our previous studies have shown that (-)-11-OH-delta 8-THC-dimethylheptyl was 80-1150 times more potent than delta 9-THC. Stereospecificity was demonstrated by its (+)-enantiomer which was more than 1400-7500 times less potent. A related series of DMH cannabinoid analogs has recently been synthesized and preliminary evaluations reported here. (-)-11-OH-delta 9-THC-DMH was found to be equipotent with (-)-11-OH-delta 8-THC-DMH. The aldehyde (-)-11-oxo-delta 9-THC-DMH was 15-50 times more potent than delta 9-THC. Surprisingly, (-)-11-carboxy-delta 9-THC-DMH was also active, being slightly more potent than delta 9-THC. In the bicyclic cannabinoid series, the length and bulk of the side chain were found to be equally important. Aminoalkylindoles, which are structurally dissimilar from classical cannabinoids, have been found to exhibit a pharmacological profile similar to delta 9-THC. Though not extremely potent in vivo, they appear to represent an entirely new approach to studying the actions of the cannabinoids. The structural diversity and wide-ranging potencies of the analogs described herein provide the opportunity to develop a pharmacophore for the cannabinoids using molecular modeling techniques.

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana,. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

Systemic or intrahippocampal cannabinoid administration impairs spatial memory in rats

The purpose of the present study was to investigate the disruptive effects of cannabinoids on working memory as assessed in the eight-arm radial-maze. Systemic administration of Δ9-THC, WIN-55,212-2, and CP-55,940 increased the number of errors committed in the radial-maze. CP-55,940 was the most potent cannabinoid in impairing memory (ED50=0.13 mg/kg). Δ9-THC and WIN-55,212-2 disrupted mazechoice accuracy at equipotent doses (ED50 values =2.1 and 2.2 mg/kg, respectively). In addition, systemic administration of each of these agents retarded completion time. Whereas the doses of Δ9-THC and CP-55,940 required to retard maze performance were higher than those needed to increase error numbers, WIN-55,212-2 was equipotent in both of these measures. On the other hand, neither anandamide, the putative endogenous cannabinoid ligand, nor cannabidiol, an inactive naturally occurring cannabinoid, had any apparent effects on memory. A second aim of this study was to elucidate the neuroanatomical substrates mediating the disruptive effects of cannabinoids on memory. Intrahippocampal injections of CP-55,940 impaired maze performance in a dose-dependent manner (ED50=8 µg/rat), but did not retard the amount of time required to complete the maze. The effects of intrahippocampal CP-55,940 were apparently specific to cognition because no other cannabinoid pharmacological effects (e.g., antinociception, hypothermia, and catalepsy) were detected. This dissociation between choice accuracy in the radial-maze and other cannabinoid pharmacological effects suggests that the working memory deficits produced by cannabinoids may be mediated by cannabinoid receptors in the hippocampus.

Delta 9-tetrahydrocannabinol impairs spatial memory through a cannabinoid receptor mechanism.

The purpose of the present study was to investigate whether the cannabinoid and cholinergic systems impair working memory through a common mechanism. This hypothesis was tested by examining whether the cannabinoid antagonist SR141716A would ameliorate radial-arm performance deficits caused by either the naturally occurring cannabinoid, Δ9-THC, or scopolamine, a muscarinic antagonist. In addition, we evaluated whether the cholinesterase inhibitor, physostigmine, would prevent Δ9-THC-induced impairment of spatial memory. Finally, because the locomotor suppressive effects of cannabinoids may decrease radial arm choice accuracy independent of a direct effect on memory, we examined the impact of increasing the intertrial error on radial arm choice accuracy. As previously reported, Δ9-THC impaired maze performance (ED50=3.0 mg/kg). Increasing the intertrial interval from 5 s to 30 s resulted in a three-fold increase in the amount of time required to complete the maze without affecting choice accuracy. Importantly, SR141716A prevented Δ9-THC-induced deficits in radial-arm choice accuracy in a dose-dependent manner (AD50=2.4 mg/kg); however, the cannabinoid antagonist failed to improve the disruptive effects of scopolamine. Conversely, physostigmine failed to improve performance deficits produced by Δ9-THC. These data provide strong evidence that Δ9-THC impairs working memory through direct action at cannabinoid receptors. Moreover, these results suggest that scopolamine and Δ9-THC do not impair spatial memory in a common serial pathway, though they may converge on a third neurochemical system.

Do functional relationships exist between 5-HT1A and 5-HT2 receptors?

To investigate the possible functional relationship between 5-HT1 and 5-HT2 receptors, we studied the effects of a nonselective 5-HT agonist (5-MeO DMT), a 5-HT1A-selective (8-OH-DPAT) and a 5-HT1B/5-HT1C-selective (TFMPP) agonist on the head-twitch behavior induced by the putative 5-HT2-selective receptor agonist (+/-)-DOI. In the mouse (+/-)-DOI produced the head-twitch response in a dose-dependent manner and (-)-DOI was twice as potent as the (+) isomer. Selective 5-HT2 antagonists, ketanserin and spiperone, dose-dependently inhibited the (+/-)-DOI-induced head-twitch response. The nonselective and the 5-HT1A-selective agonists also dose-dependently reduced the behavior, whereas 5-HT1B/5-HT1C-selective agonist (TFMPP) failed to affect the (+/-)-DOI-induced response. Taken together with previously published literature data, we propose a 5-HT1A inhibitory action on the 5-HT2 receptor-mediated response when induced by its selective agonist (+/-)-DOI.

Novel antagonist implicates the CB1 cannabinoid receptor in the hypotensive action of anandamide

In anaesthetised rats, the endogenous cannabinoid anandamide has potent cardiovascular effects that include a brief pressor effect and a more prolonged depressor response. The depressor response is attenuated after transection of the cervical spinal cord or blockade of alpha-adrenergic receptors by phentolamine, and is dose-dependently inhibited by a selective antagonist of the CB1 cannabinoid receptor. The pressor component is not affected by any of these interventions. This suggests that the depressor response is due to inhibition of sympathetic tone mediated by CB1 receptors, whereas the pressor component is due to a peripheral action that does not involve the same receptors or the sympathetic nervous system.