International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2.

There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

Cannabinoid receptor signaling.

The cannabinoid receptor family currently includes two types: CB1, characterized in neuronal cells and brain, and CB2, characterized in immune cells and tissues. CB1 and CB2 receptors are members of the superfamily of seven-transmembrane-spanning (7-TM) receptors, having a protein structure defined by an array of seven membrane-spanning helices with intervening intracellular loops and a C-terminal domain that can associate with G proteins. Cannabinoid receptors are associated with G proteins of the Gi/o family (Gi1, 2 and 3, and Go1 and 2). Signal transduction via Gi inhibits adenylyl cyclase in most tissues and cells, although signaling via Gs stimulates adenylyl cyclase in some experimental models. Evidence exists for cannabinoid receptor-mediated Ca2+ fluxes and stimulation of phospholipases A and C. Stimulation of CB1 and CB2 cannabinoid receptors leads to phosphorylation and activation of p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK and Jun N-terminal kinase (JNK) as signaling pathways to regulate nuclear transcription factors. The CB1 receptor regulates K+ and Ca2+ ion channels, probably via Go. Ion channel regulation serves as an important component of neurotransmission modulation by endogenous cannabinoid compounds released in response to neuronal depolarization. Cannabinoid receptor signaling via G proteins results from interactions with the second, third and fourth intracellular loops of the receptor. Desensitization of signal transduction pathways that couple through the G proteins probably entails phosphorylation of critical amino acid residues on these intracellular surfaces.

International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (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 CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) 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 CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, 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.

Cannabinoid receptor agonist and antagonist effects on motor function in normal and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-treated non-human primates.

RATIONALE: Although cannabinoid effects on motor function have been extensively studied in rodents, the role of cannabinoids in regulating behavior in primates is relatively unknown. OBJECTIVES: We compared the effects of cannabinoid agonists and dopamine antagonists on unconditioned behaviors in cynomolgus monkeys (Macaca fascicularis). We further investigated the therapeutic potential of cannabinoid antagonists in a primate model of Parkinson's disease. METHODS: Drugs were administered i.m., and sessions were videotaped and rated by a "blind" observer using a rating scale. RESULTS: The dopamine antagonist haloperidol decreased locomotor activity and increased bradykinesia in three subjects. Haloperidol also produced a dose-dependent increase in freezing and catalepsy in two out of the three subjects. The cannabinoid agonist levonantradol dose-dependently decreased general and locomotor activity and increased bradykinesia. In contrast to haloperidol, levonantradol failed to produce freezing or catalepsy. At the dose range studied, tetrahydrocannabinol did not affect general or locomotor activity, but increased bradykinesia. In view of the psychomotor slowing induced by cannabinoid agonists, we investigated the therapeutic potential of the cannabinoid receptor antagonist SR141716A in an early and advanced stage of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine-induced parkinsonism. In both models of Parkinson's disease, SR141716A failed to alleviate the motor deficits of parkinsonism. CONCLUSIONS: Cannabinoid agonists do not induce catalepsy in primates, a finding that differs from their effects in rodents. The primate may be more suitable than rodents for predicting the effects of cannabinoids and their therapeutic potential on select primate behaviors.

Nitric oxide regulates adenylyl cyclase activity in rat striatal membranes.

The regulation of adenylyl cyclase activity by nitric oxide (NO) was studied in rat (Sprague-Dawley) striatal membranes. Three chemically distinct NO donors attenuated forskolin-stimulated activity but did not alter basal activity. Maximum inhibition resulted in a 50% decrease in forskolin-stimulated activity, consistent with the presence of multiple isoforms of adenylyl cyclase and our previous findings that only the forskolin-stimulated activity of the type-5 and -6 isoform family of enzymes is inhibited by NO. To monitor primarily the type-5 isoform, we examined the ability of NO donors to attenuate D(1)-agonist-stimulated adenylyl cyclase activity. Under those conditions, complete inhibition was observed. The data indicate that NO attenuates neuromodulator-stimulated cAMP signaling in the striatum.

Signal transduction interactions between CB1 cannabinoid and dopamine receptors in the rat and monkey striatum.

Signal transduction interactions between the CB1 cannabinoid and 1 and D2 dopamine receptor systems were studied in rat (Sprague Dawley) and monkey (Macaca fascilaris) striatal membranes. The D2 agonist quinelorane inhibited forskolin (10 microM)-stimulated adenylyl cyclase in a dose-dependent manner (26% and 20% maximal inhibition; EC50 = 2 and 0.5 microM, in rats and monkeys, respectively) and maximal inhibition was completely blocked by the D2 antagonist sulpiride (10 microM). The CB1 agonist desacetyllevonantradol inhibited forskolin-stimulated adenylyl cyclase (18% and 36% maximal inhibition; EC50 = 160 and 73 nM, in rats and monkeys, respectively) and the CB1 antagonist SR141716A (10 microM) completely blocked the maximal inhibition. Combined addition of > EC(90) concentrations of quinelorane (10, 30 microM) and desacetyllevonantradol (1 microM) resulted in no greater inhibition than that produced by either drug alone, indicative of signal transduction convergence between the D2 and CB1 receptor systems. The D1 agonist 6-Br-APB (3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin) produced a dose-dependent stimulation of adenylyl cyclase (45% and 26% stimulation; EC50 = 24 and 32 nM, in rat and monkey, respectively), and maximal stimulation was completely blocked by the D1 antagonist SCH23390 (1 microM). D1 agonist-stimulated activity could be inhibited to basal levels with desacetyllevonantradol (1 microM), indicative of D1 and CB1 signal transduction convergence. The data suggest that CB1 receptors are co-localized with D1 or D2 receptors on the same population of striatal membranes and can interact at the level of G-protein/adenylyl cyclase signal transduction. Similar results obtained with both rat and monkey membranes indicate that striatal dopamine and cannabinoid interactions are conserved for these two species.

CB1 receptor-G protein association. Subtype selectivity is determined by distinct intracellular domains.

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C-terminal domain (CB(1)401-417). Guanosine 5'-O-(3-thio)triphosphate binding to G proteins can be stimulated by both peptide CB(1)401-417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol. 53, 504-510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry 38, 3447-3455]. In Chaps-solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB(1) receptor-G alpha(i) association and attenuated the CB(1)401-417-induced inhibition of adenylate cyclase. CB(1)401-417 significantly reduced the CB(1) receptor association with G alpha(i3), but not with G alpha(i1) or G alpha(i2). In contrast, third intracellular loop peptides significantly reduced the CB(1) receptor association with G alpha(i1) and G alpha(i2), but not G alpha(i3). These interactions are specific for the CB(1) receptor because a peptide corresponding to the juxtamembrane C-terminal domain of the CB(2) receptor failed to compete for the association of the CB1 receptor with any of the Gi alpha subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB(1) receptor direct the interaction with specific G protein subtypes.

Cellular signal transduction by anandamide and 2-arachidonoylglycerol.

Anandamide (arachidonylethanolamide) and 2-arachidonoylglycerol mediate many of their actions via either CB(1) or CB(2) cannabinoid receptor subtypes. These agonist-receptor interactions result in activation of G proteins, particularly those of the G(i/o) family. Signal transduction pathways that are regulated by these G proteins include inhibition of adenylyl cyclase, regulation of ion currents (inhibition of voltage-gated L, N and P/Q Ca(2+)-currents; activation of K(+) currents); activation of focal adhesion kinase (FAK), mitogen activated protein kinase (MAPK) and induction of immediate early genes; and stimulation of nitric oxide synthase (NOS). Other effects of anandamide and/or 2-arachidonoylglycerol that are not mediated via cannabinoid receptors include inhibition of L-type Ca(2+) channels, stimulation of VR(1) vanilloid receptors, transient changes in intracellular Ca(2+), and disruption of gap junction function. Cardiovascular regulation by anandamide appears to occur by a variety of receptor-mediated and non-receptor-mediated mechanisms. This review will describe and evaluate each of these signal transduction pathways and mechanisms.

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor.

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.

Azido- and isothiocyanato-substituted aryl pyrazoles bind covalently to the CB1 cannabinoid receptor and impair signal transduction.

3-Azidophenyl- and 3-isothiocyanatophenyl-and 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were synthesized to determine whether these compounds could behave as covalently binding ligands for the CB1 cannabinoid receptor in rat brain membranes. Heterologous displacement of [3H]CP55940 indicated that the apparent affinity of these compounds for the CB1 receptor was similar to that of the parent compound, SR141716A, with the exception of the 3-isothiocyanato derivatives, which showed a 10-fold loss of affinity. The 3-azidophenyl and 3-isothiocyanatophenyl compounds behaved as antagonists against the cannabinoid agonist desacetyllevonantradol in activation of G proteins [guanosine 5'-O-(y-[35S]thio)triphosphate ([35S]GTPgammaS) binding] and regulation of adenylyl cyclase. The 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were poor antagonists for [35S]GTPgammaS binding, and both compounds failed to antagonize the cannabinoid regulation of adenylyl cyclase. After incubation with the isothiocyanato analogues or UV irradiation of the azido analogues, the 3-substituted aryl pyrazoles formed covalent bonds with the CB1 receptor as evidenced by the loss of specific binding of [3H]CP55940. In the case of the isothiocyanato analogues, the log concentration-response curve for cannabinoid-stimulated [35S]GTPgammaS binding was shifted to the right, indicating that loss of receptors compromised signal transduction capability. These irreversibly binding antagonists might be useful tools for the investigation of tolerance and receptor down-regulation in both in vitro and in vivo studies.

Development of a novel class of monocyclic and bicyclic alkyl amides that exhibit CB1 and CB2 cannabinoid receptor affinity and receptor activation.

CB1 and CB2 cannabinoid receptors can be activated by several different classes of agonists, including cannabinoids such as delta9-tetrahydrocannabinol and 9-nor-9beta-hydroxyhexahydrocannabinol, and eicosanoids such as arachidonylethanolamide. Structure-activity relationship studies have identified potential pharmacophoric elements for binding to cannabinoid receptors by both cannabinoids and eicosanoids. Molecular models have hypothesized conformational, spatial, and pharmacophoric distance requirements based upon radioligand binding data whereby overlap of pharmacophoric elements of the two classes disclose a low energy conformation of arachidonylethanolamide that can occupy the same receptor space as cannabinoid ligands. To test this model, we have developed a novel class of monocyclic and bicyclic alkyl amide cannabinoid receptor ligands. Further, we predicted a spatial conformation for these compounds in a molecular model based on the pharmacophoric and structural requirements for binding to the CB1 cannabinoid receptor.

D(2), but not D(1) dopamine receptor agonists potentiate cannabinoid-induced sedation in nonhuman primates.

In primates, CB(1) cannabinoid receptor agonists produce sedation and psychomotor slowing, in contrast to behavioral stimulation produced by high doses of dopamine receptor agonists. To investigate whether dopamine agonists attenuate the sedative effects of a cannabinoid agonist in monkeys, we compared the effects of D(1) or D(2) dopamine receptor agonists on spontaneous behavior in three to six cynomolgus monkeys (Macaca fasicularis) alone and after administration of a low dose of the CB(1) agonist levonantradol. Alone, the CB(1) cannabinoid receptor agonist levonantradol (0.01-0. 3 mg/kg) induced sedation, ptosis, and decreased locomotor and general activity. Alone, D(2)-type dopamine agonists quinelorane (0. 001-1.0 mg/kg; n = 4) or pergolide (0.01-1.0 mg/kg) or a D(1) dopamine agonist 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-3-allyl-[1H]-3-benzazepine (0.3-3.0 mg/kg) produced either no effect or promoted hyperactivity. Thirty minutes after administration of a threshold dose of levonantradol (0.03 mg/kg), D(2)-type agonists, but not the D(1) agonist, precipitated marked sedation, ptosis, and decreased general activity and locomotor activity. These data inducate the following: 1) D(2,) but not D(1) dopamine agonists, potentiate sedation in monkeys treated with a CB(1) cannabinoid agonist, at doses of agonists that alone do not produce sedation; 2) the threshold dose for cannabinoid-induced sedation is reduced by D(2) agonists, but not by a D(1) dopamine agonist, differentiating D(1) and D(2) dopamine receptor linkage to cannabinoid receptors; and 3) modulation of D(2) dopamine receptor activity by a nonsedating dose of a cannabinoid agonist has implications for the pathophysiology and treatment of dopamine-related neuropsychiatric disorders and drug addiction. Cannabinoid agonists and D(2) dopamine agonists should be combined with caution.

The CB(1) cannabinoid receptor juxtamembrane C-terminal peptide confers activation to specific G proteins in brain.

Under reducing conditions of SDS-polyacrylamide gel electrophoresis, the CB(1) receptor exists in its monomeric form as well as in an SDS-resistant high molecular weight form that appears to be devoid of G proteins. The CB(1) cannabinoid receptor was immunoprecipitated from 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate-solubilized rat brain membranes using an antibody against the CB(1) receptor N terminus. The CB(1) receptor was coimmunoprecipitated with its associated G proteins, specifically those of the Galpha(i/o) family, but not Galpha(s), Galpha(q), or Galpha(z). The CB(1) receptor-Galpha(i/o) complex existed in the absence of exogenous agonists, and the cannabinoid receptor agonist desacetyllevonantradol failed to alter the stoichiometry of the receptor-Galpha(i/o) interaction. Guanosine-5'-O-(3-thio)triphosphate could disrupt the interaction. A peptide derived from the CB(1) receptor juxtamembrane C-terminal domain, peptide CB(1)401-417, autonomously activates G(i/o) proteins. Peptide CB(1)401-417 competitively disrupted the CB(1) receptor association with Galpha(o) and Galpha(i3) but not Galpha(i1) or Galpha(i2). This G protein specificity was also observed in detergent extracts from membranes of the frontal cortex, striatum, and cerebellum. Alternative peptides, including peptides from the CB(1) receptor third intracellular loop and the G protein activating peptide mastoparan-7, failed to promote uncoupling from Galpha(o). A CB(2) receptor juxtamembrane C-terminal peptide failed to disrupt the CB(1) receptor-Galpha(o) complex. These studies illustrate that the CB(1) receptor can exist as an SDS-resistant multimer. In 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate detergent, the CB(1) receptor exists in a complex with G proteins of the G(i/o) family in the absence of exogenous agonists. Furthermore, this study provides the first description of domain specificity for interaction with a selective set of G proteins.

Cannabinoid and dopamine interaction in rodent brain: effects on locomotor activity.

We investigated interactions between cannabinoid and dopamine receptor systems in ICR mice. Mice were treated with the cannabinoid agonist levonantradol, the D(1) dopamine agonist 6-Br-APB, or the D(2) dopamine agonist quinelorane, or with combinations of these drugs. In addition, the D(1) antagonist SCH23390 was administered both alone and in combination with levonantradol. Two tests were used to evaluate changes in motor function: the immobility (ring stand) test and the catalepsy (bar) test. Levonantradol increased immobility and catalepsy in a dose-dependant manner. Both the D(2) agonist quinelorane and the D(1) agonist 6-Br-APB were able to attenuate the motor dysfunction caused by levonantradol. Administration of the D(1) antagonist SCH23390 enhanced the effects of levonantradol, producing a leftward shift of the log dose-response curve. These results differ from the augmentation by D(2) agonists of the hypoactivity induced by levonantradol in non-human primates [Meschler JP, Clarkson FA, Mathew PJ, Howlett AC, Madras BK. D(2), but not D(1) dopamine receptor agonists potentiate cannabinoid-induced sedation in nonhuman primates. J Pharmacol Exp Ther 2000;292:952-959], suggesting that conclusions about the interactions between the dopamine and cannabinoid receptor motor systems in rodents may not extend to primates.

Signal transduction of eicosanoid CB1 receptor ligands.

The eicosanoid ligand, arachidonylethanolamide (anandamide), interacts with the CB1 cannabinoid receptor in the brain to signal its response. Pharmacophoric points of interaction between this agonist and the receptor have been proposed based upon structure-activity relationship studies of ligand binding to the receptor. Three dimensional quantitative structure-activity relationship (3D-QSAR) models have been constructed based upon the corresponding pharmacophoric points predicted for cannabinoid ligands delta9-tetrahydrocannabinol and 9-nor-9beta-hydroxyhexa-hydrocannabinol. A novel data set has been used to test the statistical validity of these models. Once the ligand interacts with the CB1 receptor, signal transduction occurs via G-proteins of the Gi/o family which are shown to be associated with the receptor. Evidence suggests that the juxtamembrane region of the C-terminal of the CB1 receptor is critical for activation of these G-proteins.

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis.

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.

Thujone exhibits low affinity for cannabinoid receptors but fails to evoke cannabimimetic responses.

Absinthe, an abused drug in the early 1900s, has been speculated to activate the receptors responsible for marijuana intoxication (the CB1 cannabinoid receptor) (Nature 253:365-356; 1975). To test this hypothesis, we investigated oil of wormwood (Artemisia absinthium) the active plant product found in absinthe, and thujone, the active compound found in oil of wormwood. Radioligand receptor binding assays employing membrane preparations from rat brains containing CB1 cannabinoid receptors, and human tonsils containing CB2 receptors, demonstrated that thujone displaced [3H]CP55940, a cannabinoid agonist, only at concentrations above 10 microM. HPLC analysis of oil of wormwood revealed that only the fractions having mobility close to thujone displaced [3H]CP55940 from the CB1 cannabinoid receptor. [35S]GTPgammaS binding assays revealed that thujone failed to stimulate G-proteins even at 0.1 mM. Thujone failed to inhibit forskolin-stimulated adenylate cyclase activity in N18TG2 membranes at 1 mM. Rats administered thujone exhibited different behavioral characteristics compared with rats administered a potent cannabinoid agonist, levonantradol. Therefore, the hypothesis that activation of cannabinoid receptors is responsible for the intoxicating effects of thujone is not supported by the present data.

Effect of environmental estrogens on IL-1beta promoter activity in a macrophage cell line.

Environmental estrogens or estrogen disrupters have recently received a great deal of attention because of their potential health impact on reproductive tissues. Few, if any, studies have been made on the impact of these compounds on the immune system. We sought to determine the activities of various environmental estrogens on the modulation of the interleukin-1beta (IL-1beta) gene in a model monocytic cell line, hER + IL-1beta-CAT+. This cell line stably transfected with the human estrogen receptor, and an IL-1beta promoter construct fused to the CAT reporter gene allows us to monitor the effect of estrogenic compounds on IL-1beta promoter activity. 17beta-estradiol (E2) markedly enhanced lipopolysaccharide- (LPS) induced IL-1beta promoter-driven CAT activity in a dose-dependent manner. The mycotoxins alpha-zearalenol and zearalenone both exhibited full agonist activity, but at lower potencies, with EC50 values of 1.8 and 54 nM, respectively, compared with E2 at 0.5 nM. In addition, genistein was a very low-potency agonist, having an EC50 of 1.5 microM. Similar to the E2 response, the slope factors for alpha-zearalenol, zearalenone, and genistein were close to 3.0, suggesting positive cooperativity in the estrogenic response. The activity of the mycotoxins appeared to be mediated through the estrogen receptor, since both the antiestrogens H1285 and ICI 182,780 effectively inhibited their agonist activity in a dose-dependent manner. Representative environmental estrogenic compounds both from plant and industrial sources were also tested. Unlike the mycoestrogens, none of the compounds, with the exception of genistein, synergized with LPS to enhance IL-1beta promoter activity. When tested for antiestrogenic activity, the industrial compound 4-octylphenol was able to antagonize the response to E2; however, the response was three orders of magnitude less potent than H 1285. Naringenin, a plant flavonoid, showed little or no ability to antagonize the response to E2. Overall, the results show that some environmental estrogens that display agonist activity in reproductive tissue also have an effect on IL-1 gene expression in hemopoietic-derived tissue.

Three-dimensional quantitative structure-activity relationship study of the cannabimimetic (aminoalkyl)indoles using comparative molecular field analysis.

The present study describes the implementation of comparative molecular field analysis (CoMFA) to develop two 3D-QSAR (quantitative structure-activity relationship) models (CoMFA models 1 and 2) of the cannabimimetic (aminoalkyl)indoles (AAIs) for CB1 cannabinoid receptor binding affinity, based on pKi values measured using radioligand binding assays that displace two different agonist ligands, [3H]CP-55940 and [3H]WIN-55212-2. Both models exhibited a strong correlation between the calculated steric-electrostatic fields and the observed biological activity for the respective training set compounds. In light of the basicity of the morpholine nitrogen in the AAIs, separate CoMFA models were built for the AAIs as unprotonated and protonated species. Comparison of the statistical parameters resulting from these CoMFA models failed to provide unequivocal evidence as to whether the AAIs are protonated or neutral as receptor-bound species. Although the training sets of CoMFA model 1 and CoMFA model 2 differed with respect to composition and to the choice of displacement radioligand in each biological assay, their CoMFA StDevCoeff contour plots reveal similarities in terms of identifying those regions around the AAIs that are important for CB1 cannabinoid receptor binding such as the sterically favored region around the C3 aroyl group and the sterically forbidden region around the indole ring. When the experimental pKi values for the training set compounds to displace the AAI radioligand [3H]WIN-55212-2 were plotted against the pKi values as predicted for the same compounds to displace the cannabinoid radioligand [3H]CP-55940, the correlation was moderately strong (r = 0.73). However, the degree of correlation may have been lowered by the structural differences in the compounds comprising the training sets for CoMFA model 1 and CoMFA model 2. Taken together, the results of this study suggest that the binding site region within the CB1 cannabinoid receptor can accommodate a wide range of structurally diverse cannabimimetic analogues including the AAIs.