Agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors.

This study examined the ability of the endocannabinoids 2-arachidonoyl glycerol (2-AG) and noladin ether as well as the synthetic cannabinoid CP-55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] to regulate three intracellular effectors via CB2 receptors in transfected Chinese hamster ovary cells. Although the three agonists regulate all effectors with equivalent efficacy, the rank order of potencies differs depending on which effector is evaluated. Noladin ether and CP-55,940 most potently inhibit adenylyl cyclase, requiring higher concentrations to stimulate the extracellular signal-regulated kinase subgroup of the mitogen-activated protein kinases (extracellular signal-regulated kinase-mitogen-activated protein kinase; ERK-MAPK) and Ca(2+)-transients. In contrast, 2-AG most potently activates ERK-MAPK, necessitating greater concentrations to inhibit adenylyl cyclase and even higher amounts to stimulate Ca(2+)-transients. Endocannabinoids also seem to be more "efficient" agonists at CB2 receptors relative to synthetic agonists. 2-AG and noladin ether require occupancy of less than one-half the number of receptors to produce comparable regulation of adenylyl cyclase and ERK-MAPK, relative to the synthetic cannabinoid CP-55,940. The CB2 antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)-ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone (AM630) reverses the actions of all agonists except Ca(2+)-transient stimulation by 2-AG. However, the effect of 2-AG on Ca(2+)-transients is attenuated by a second CB2 antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-1-pyrazole-3-carboxamide (SR144528). This suggests that 2-AG stimulates Ca(2+)-transients by binding to sites on CB2 receptors distinct from those occupied by AM630 and the other cannabinoids examined. Agonists produce no effects in pertussis toxin-treated cells. In summary, cannabinoid agonists distinctly bind to CB2 receptors and display different rank order of potencies and fractional receptor occupancies for regulation of intracellular effectors. These data provide direct evidence for agonist-directed trafficking of response by endocannabinoids acting at CB2 receptors.

The endocannabinoid noladin ether acts as a full agonist at human CB2 cannabinoid receptors.

Noladin ether (NE) is a putative endogenously occurring cannabinoid demonstrating agonist activity at CB1 receptors. Because of reported selective affinity for CB1 receptors, the pharmacological actions of NE at CB2 receptors have not been examined. Therefore, the purpose of this study was to characterize the binding and functional properties of NE at human CB2 receptors stably expressed in Chinese hamster ovary (CHO) cells as well as in HL-60 cells, which express CB2 receptors endogenously. Surprisingly, in transfected CHO cells, NE exhibits a relatively high nanomolar affinity for CB2 receptors (K(i) = 480 nM), comparable to that observed for the endocannabinoid 2-arachidonoyl glycerol (2-AG) (K(i) = 1016 nM). Furthermore, NE activates G proteins and inhibits the intracellular effector adenylyl cyclase with equivalent efficacy relative to the full cannabinoid agonists 2-AG and CP 55,940 (CP) [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]. The rank order of potency for G protein activation and effector regulation by the three agonists is similar to their apparent affinity for CB2 receptors; CP > NE > or = 2-AG. Regulation of adenylyl cyclase activity by all agonists is inhibited by pertussis toxin pretreatment or by coincubation with AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone], a CB2 antagonist. Chronic treatment with NE or CP results in CB2 receptor desensitization and down-regulation. All agonists also inhibit adenylyl cyclase activity in HL-60 cells. Together, these data indicate that NE acts as a full agonist at human CB2 receptors and thus might have important physiological functions at peripheral cannabinoid receptors.

Agonist, antagonist, and inverse agonist characteristics of TIPP (H-Tyr-Tic-Phe-Phe-OH), a selective delta-opioid receptor ligand.

Recent evidence indicates that the well established delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) also displays agonist activity in several cellular models. Therefore, it is possible that TIPP, and structurally related compounds, might represent a novel class of opioid agonists exhibiting unique characteristics. The purpose of this study was to examine the properties of TIPP at selected points of the signal transduction pathway (i.e., receptor binding, G-protein activation, and effector regulation) in GH(3)DORT cells (GH(3) cells expressing delta-opioid receptors) and compare them with that of an established delta-opioid agonist, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE). DPDPE exhibited properties of an agonist in all assays. In contrast, TIPP demonstrated characteristics of an agonist, antagonist, or inverse agonist, depending on the step in the signal transduction cascade examined and the assay conditions employed. In receptor binding assays, the addition of guanine nucleotides and sodium ions increased the affinity of TIPP for delta-opioid receptors in both membrane preparations and digitonin-permeabilized cells, which is characteristic of an inverse agonist. In assays measuring G-protein activation, TIPP failed to stimulate guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in membrane preparations, which is consistent with an antagonist profile. However, when using cells semi-permeabilized with digitonin, TIPP exhibited properties of an agonist, producing concentration-dependent, antagonist-reversible stimulation of [(35)S]GTPgammaS binding. Finally, in assays examining regulation of the intracellular effector adenylyl cyclase, TIPP exhibited characteristics of an agonist, producing inhibition of enzyme activity in both membrane preparations and whole cells. Therefore, although DPDPE and TIPP act similarly as agonists to regulate the intracellular effector adenylyl cyclase, they demonstrate significant differences in the signal transduction cascade preceding this final point of convergence.