Background: G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret.Results: To bridge this gap, we have used microarrays to measure the mRNA levels of a comprehensive profile of non-chemosensory GPCRs and over a hundred GPCR signaling related gene products in four cell lines frequently used for GPCR research: HEK293, AtT20, BV2, and N18.Conclusions: This study provides researchers an easily accessible mRNA profile of the endogenous signaling repertoire that these four cell lines possess. This will assist in choosing the most appropriate cell line for studying GPCRs and related signaling proteins. It also provides a better understanding of the potential interactions between GPCRs and those signaling proteins.

Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE—tens of seconds—is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and α/β-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl)[1,1′-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB₁ receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.

Purpose: It has been known for nearly 50 years that cannabis and the psychoactive constituent Δ⁹-tetrahydrocannabinol (THC) reduce intraocular pressure (IOP). Elevated IOP remains the chief hallmark and therapeutic target for glaucoma, a major cause of blindness. THC likely acts via one of the known cannabinoid-related receptors (CB1, CB2, GPR18, GPR119, GPR55) but this has never been determined explicitly. Cannabidiol (CBD) is a second major constituent of cannabis that has been found to be without effect on IOP in most studies. Methods: Effects of topically applied THC and CBD were tested in living mice by using tonometry and measurements of mRNA levels. In addition the lipidomic consequences of CBD treatment were tested by using lipid analysis. Results: We now report that a single topical application of THC lowered IOP substantially (∼28%) for 8 hours in male mice. This effect is due to combined activation of CB₁ and GPR18 receptors each of which has been shown to lower ocular pressure when activated. We also found that the effect was sex-dependent, being stronger in male mice, and that mRNA levels of CB1 and GPR18 were higher in males. Far from inactive, CBD was found to have two opposing effects on ocular pressure, one of which involved antagonism of tonic signaling. CBD prevents THC from lowering ocular pressure. Conclusions: We conclude that THC lowers IOP by activating two receptors—CB₁ and GPR18—but in a sex-dependent manner. CBD, contrary to expectation, has two opposing effects on IOP and can interfere with the effects of THC.

Background and Purpose: GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N‐arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn‐CBD). The presence and/or function of any GPR18‐based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB₁, CB₂ and GPR55.Experimental Approach: IOP was measured in mice following topical application of Abn‐CBD, NAGly or the GPR55/GPR18 agonist O‐1602, alone or with injection of the GPR18 antagonist, O‐1918. GPR18 protein localization was assessed with immunohistochemistry. Endocannabinoids were measured using LC/MS‐MS.Key Results: GPR18 protein was expressed most prominently in the ciliary epithelium and the corneal epithelium and, interestingly, in the trabecular meshwork. The GPR18 ligand, NAGly, was also detected in mouse eye at a level comparable to that seen in the brain. Abn‐CBD and NAGly, but not O‐1602, significantly reduced IOP in all mice tested. The antagonist, O‐1918, blocked the effects of Abn‐CBD and NAGly.Conclusions and Implications: We present evidence for a functional GPR18‐based signalling system in the murine anterior eye, including receptors and ligands. GPR18 agonists, Abn‐CBD and NAGly, reduce IOP independently of CB₁, CB₂ or GPR55. These findings suggest that GPR18 may serve as a desirable target for the development of novel ocular hypotensive medications.Linked Articles: This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue‐4 & http://dx.doi.org/10.1111/bph.2012.167.issue‐8

Δ⁹-Tetrahydrocannabinol (THC) and cannabidiol (CBD) are two main cannabinoid constituents of marijuana and hashish. The pharmacology of Δ⁹-THC has been extensively studied, whereas our understanding of the pharmacology of CBD has remained limited, despite excitement in CBD’s potential role in treating certain pediatric epilepsies and its reputation for attenuating some Δ⁹-THC–induced effects. It was established early on that CBD binds poorly to the orthosteric site of CB₁ or CB₂ cannabinoid receptors, and its actions were commonly attributed to other noncannabinoid receptor mechanisms. However, recent evidence suggests that CBD does indeed act at cannabinoid CB₁ receptors as a negative allosteric modulator (NAM) of CB₁ signaling. By altering the orthosteric signaling of a G protein–coupled receptor, allosteric modulators greatly increase the richness of G protein–coupled receptor pharmacology. We have recently surveyed candidate CB₁ NAMs in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and have now tested CBD in this model. We find that although CBD has no direct effect on excitatory transmission, it does inhibit two forms of endogenous cannabinoid-mediated retrograde synaptic plasticity: depolarization-induced suppression of excitation and metabotropic suppression of excitation, while not affecting signaling via GABA-B receptors. These results are consistent with the recently described NAM activity of CBD and suggest interesting possible mechanisms for CBD’s therapeutic actions.