Mary E. Abood

Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves.

1 Activation of CB1 receptors by plant cannabinoids or the endogenous ligand, anandamide, causes hypotension via a sympathoinhibitory action in anaesthetized rats. In mouse isolated vas deferens, activation of CB1 receptors inhibits the electrically evoked twitch response. To determine if these effects are related to presynaptic inhibition of noradrenaline (NA) release, we examined the effects of Δ9‐tetrahydrocannabinol (‡9‐THC), anandamide and the CB1 antagonist, SR141716A, on exocytotic NA release in rat isolated atria and vasa deferentia. 2 In isolated atria and vasa deferentia preloaded with [3H]‐NA, electrical field stimulation caused [3H]‐NA release, which was abolished by tetrodotoxin 0.5 μm and concentration‐dependently inhibited by ‡9‐THC or anandamide, 0.3–10 μm. The inhibitory effect of ‡9‐THC and anandamide was competitively antagonized by SR141716A, 1–10 μm. 3 Tyramine, 1 μm, also induced [3H]‐NA release, which was unaffected by tetrodotoxin, ‡9‐THC or anandamide in either atria or vasa deferentia. 4 CB1 receptor mRNA is present in the superior cervical ganglion, as well as in whole brain, cerebellum, hypothalamus, spleen, and vas deferens and absent in medulla oblongata and atria, as demonstrated by reverse transcription‐polymerase chain reaction. There was no evidence of the presence of CB1A receptor mRNA in ganglia, brain, or cerebellum. These results suggest that activation of presynaptic CB1 receptors located on peripheral sympathetic nerve terminals mediate sympathoinhibitory effects in vitro and in vivo.

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ8-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1,1-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1,1-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1,1-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).

Evaluation of the cannabinoid CB2 receptor-selective antagonist, SR144528: further evidence for cannabinoid CB2 receptor absence in the rat central nervous system.

The aim of this study was to characterize the activity of the cannabinoid CB2 receptor selective antagonist, N-[(1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le-3-carboxamide] (SR144528) in a number of biochemical assays and to look for evidence of cannabinoid CB2 receptors in the rat central nervous system. SR144528 displaced [3H]CP 55,940 ((-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxyprop yl]cyclohexan-1-ol) from binding sites in CB2- and CB1-transfected cells (Ki = 0.67+/-0.30 and 33.0+/-5.09 nM) and from rat cerebellum and whole brain membrane homogenates (Ki = 54.7+/-9.70 and 54.8+/-7.86 nM). In the GTPgammaS binding assay, SR144528 antagonized a number of cannabinoid receptor agonists (K(B) values ranging from 26.3 to 76.6 nM) in rat cerebellar membranes and in rat whole brain membranes (K(B) = 50.8 nM). SR144528 also antagonized CP 55,940-stimulated GTPgammaS binding in a CB2-expressing cell line (K(B) = 6.34 nM). In Xenopus oocytes co-expressing the CB1 receptor and G-protein coupled inwardly rectifying K+ channels (GIRK 1/4), SR144528 antagonized WIN 55212-2((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrolo [1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone) -stimulated K+ currents (K(B) = 558 nM). In summary, this report characterizes the cannabinoid CB2 receptor-selective cannabinoid antagonist, SR144528, and additionally suggests an absence of cannabinoid CB2 receptors in the rat central nervous system, an observation confirmed by Northern blot.

Cannabinoid receptor down-regulation without alteration of the inhibitory effect of CP 55,940 on adenylyl cyclase in the cerebellum of CP 55,940-tolerant mice

The objective of this study was to determine whether the development of tolerance to CP 55,940, a potent cannabinoid agonist, was due to changes in the receptor or second messenger system. ICR mice treated with CP 55,940 (2 mg/kg) twice a day for 6 and one-half days developed a high degree of tolerance to the pharmacological effects of CP 55,940. The ability of CP 55,940 to produce motor hypoactivity, hypothermia and immobility was reduced 163-, 97- and 19-fold, respectively. Evaluation of 3H-CP 55,940 binding to rat brain membranes indicated no difference in receptor affinity between the vehicle- and CP 55,940-treated animals. However, these binding studies revealed a 50% decrease in receptor number in the cerebellum of the CP 55,940-tolerant mice. Although cAMP is generally considered to be the second messenger for cannabinoid receptors, little difference was observed in the inhibitory effects of CP 55,940 on adenylyl cyclase activity in cerebellum between vehicle and drug-treated mice. However, there was an increase in receptor mRNA which suggests a compensation for receptor loss. There are several possible explanation for these results. There may be sufficient spare receptors such that CP 55,940-tolerant mice are capable of producing a maximal effect on the second messenger system. On the other hand, one could conclude that cannabinoid receptor down-regulation does not account for the development of tolerance to all of the effects of CP 55,940 in mice.

Development of behavioral tolerance to Δ9-THC without alteration of cannabinoid receptor binding or mRNA levels in whole brain

The effect of repetitive administration of delta-9-tetrahydrocannabinol (delta 9-THC) in mice on behavioral and biochemical tolerance was determined in this study. Mice were injected twice daily with 10 mg/kg delta 9-THC for 6.5 days. On day 8, spontaneous activity was assessed or whole-brain homogenates were prepared for the cannabinoid receptor binding and mRNA studies. Although a twenty-sevenfold tolerance to delta 9-THC was observed in the behavioral assay, there was no significant alteration in receptor binding or mRNA levels.

Molecular Neurobiology of The Cannabinoid Receptor

Marijuana is currently the most widely abused street drug. However, the functional significance of the cannabinoid receptor system in health and disease includes the use of cannabinoids as analgesics, antiemetics in cancer patients, anticonvulsants for epilepsy, and as antiglaucoma agents as well as immunomodulatory agents. Our knowledge of the mechanisms of action of cannabinoids has increased greatly in the past several years. Two cannabinoid receptors have been identified to date: one is located predominantly in the central nervous system (CBI), whereas the other is expressed in peripheral tissues (CB2). Both are members of the G-protein-coupled receptor family and couple to inhibition of adenylyl cyclase (as well as additional second messenger systems), in transfected cells expressing these receptors, and in the nervous system. An endogenous ligand has been isolated for the CBI receptor; it is arachidonic acid ethanolamide, or anandamide. Candidate endogenous ligands for the CB2 receptor have also been described. Another development is the discovery of a selective antagonist for the CBI receptor. The distribution of the cannabinoid receptor subtypes has been mapped by receptor autoradiography, RT-PCR and in situ hybridization. These new research tools will aid in the elucidation of the physiological role of the endogenous cannabinoid system.

Cannabinoid receptors in developing rats : detection of mRNA and receptor binding

Despite a large body of research directed at assessing the effects of perinatal cannabinoid exposure, little is known about the development of the cannabinoid receptor. Recent advances, including the cloning of the cannabinoid receptor, have afforded us the opportunity to plot the postnatal ontogeny of the cannabinoid receptor and its mRNA in whole brain using the methods of receptor binding and RNA blot hybridization, respectively. Our results indicate that cannabinoid receptor mRNA is present at adult levels as early as postnatal day 3. The Bmax, on the other hand, increases almost fifty percent with increasing postnatal age, while the affinity does not change. The Hill coefficients for all ages studied were approximately 1. These findings suggest the possibility of a developmental progression for cannabinoid receptor development with receptor mRNA appearing first, followed by a period of rapid proliferation of the receptors themselves.

Developmental expression of cannabinoid receptor mRNA

Cloning of the cannabinoid receptor affords the opportunity to examine its developmental expression. Other G-protein-coupled receptor systems, those for the opioids for example, exhibit distinct ontogenies. For the initial study, therefore, cannabinoid receptor mRNA expression was assessed in rat pups postnatal days 3, 5, 8, 10, 12, 15, 18 and 21. The brains were grossly dissected into cerebellum/brainstem and forebrain, and total RNA was extracted by a modified acid-extraction method. Expression of the cannabinoid receptor was analyzed by two methods: polymerase chain reaction (PCR) and Northern blot analysis. Oligonucleotide primers based on bp 1-21 and bp 824-843 on the opposite strand were chosen for use in the PCR. The probe used in the Northern blot analysis was a full length cDNA corresponding to the rat cannabinoid receptor and was cloned in our lab based on published sequence information. Our results indicate that by postnatal day 3, cannabinoid receptor mRNA can be detected in the brain. Our results further indicate that cannabinoid mRNA expression steadily increases in the cerebellum/brainstem until postnatal days 18-21, while expression in the forebrain does not change. The findings from the present study indicate that cannabinoid receptor mRNA is present in very young rats. Our data also suggest, however, regional differences in the relative expression of message which may parallel cerebellar proliferation and organization.

Antisense oligodeoxynucleotides to the κ-1 receptor block the antinociceptive effects of Δ9-THC in the spinal cord

Abstract Intrathecal pretreatment of mice with an antisence oligodeoxynucleotide directed against the κ-1 receptor significantly reduced the antinociceptive effects of the kappa receptor agonist U50,488 as well as Δ 9 -THC, the major psychoactive ingredient found in cannabis. A mismatched oligodeoxynucleotide which contained four switched bases did not block the antinociception produced by U50,488 orΔ 9 -THC. Furthermore, κ-1 antisense did not alter the antinociceptive effects of either the mu receptor-selective opioid DAMGO, or the delta receptor-selective opioid DPDPE. By using κ-1 antisense, we were able to demonstrate that an interaction occurs between the cannabinoids and opioids in the spinal cord.

Progress toward understanding the cannabinoid receptor and its second messenger systems.

Publisher Summary This chapter discusses the progress toward the understanding of cannabinoid receptor and its second-messenger system. There are now overwhelming data demonstrating that cannabinoids interact with their own distinct receptor in the central nervous system to produce some or all of the pharmacological effects. The chapter describes a plethora of interactions between cannabinoids and second messenger systems. However, in the absence of evidence for multiple receptors, it is logical to speculate that heterogeneity occurs at the level of the second-messenger systems. Strategy for probing cannabinoid receptors and their second-messenger systems involves the opioids. Cannabinoids and opioids share some common pharmacological features, while diverging on several others. The discovery of putative endogenous ligands sets the stage for finally realizing the role that cannabinoid receptors play in the central nervous system.