Background and purpose: The CB₁cannabinoid receptor and the β₂‐adrenoceptor are G protein‐coupled receptors (GPCRs) co‐expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.Experimental approach: Physical interactions between CB₁receptors and β₂‐adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal‐regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.Key results: Physical interactions between CB₁receptors and β₂‐adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co‐expression of β₂‐adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁receptors. Co‐expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i‐dependent ERK phosphorylation, but decreased non‐Gα_i‐mediated CREB phosphorylation. The CB₁receptor inverse agonist AM251 (N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) attenuated β₂‐adrenoceptor‐pERK signalling in cells expressing both receptors, while the CB₁receptor neutral antagonist O‐2050 ((6aR,10aR)‐3‐(1‐methanesulfonylamino‐4‐hexyn‐6‐yl)‐6a,7,10,10a‐tetrahydro‐6,6,9‐trimethyl‐6H‐dibenzo[b,d]pyran) did not. The actions of AM251 and O‐2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co‐expressing CB₁receptors and β₂‐adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O‐2050, altered the β₂‐adrenoceptor–pERK response.Conclusion and implications: A complex interaction was demonstrated between CB₁receptors and β₂‐adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co‐expressed.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visithttp://dx.doi.org/10.1111/j.1476‐5381.2010.00831.x

Glaucoma is an irreversible blinding eye disease which produces progressive retinal ganglion cell (RGC) loss. Intraocular pressure (IOP) is currently the only modifiable risk factor, and lowering IOP results in reduced risk of progression of the disorder. The endocannabinoid system (ECS) has attracted considerable attention as a potential target for the treatment of glaucoma, largely due to the observed IOP lowering effects seen after administration of exogenous cannabinoids. However, recent evidence has suggested that modulation of the ECS may also be neuroprotective. This paper will review the use of cannabinoids in glaucoma, presenting pertinent information regarding the pathophysiology of glaucoma and how alterations in cannabinoid signalling may contribute to glaucoma pathology. Additionally, the mechanisms and potential for the use of cannabinoids and other novel agents that target the endocannabinoid system in the treatment of glaucoma will be discussed.

Interstitial cystitis (IC) is a chronic bladder disorder with unclear etiology. The endocannabinoid system has been identified as a key regulator of immune function, with experimental evidence for the involvement of cannabinoid receptors in bladder inflammation. This study used intravital microscopy (IVM) and behavioral testing in lipopolysaccharide-induced IC, to investigate the anti-inflammatory analgesic effects of a natural dietary sesquiterpenoid, beta-caryophyllene (BCP), which is present in cannabis among other plants, and has reported agonist actions at the cannabinoid 2 receptor (CB₂R). BCP’s anti-inflammatory actions were compared to the synthetic CB₂R-selective cannabinoid, HU308, and to an FDA-approved clinical treatment (dimethyl sulfoxide: DMSO). IVM data revealed that intravesical instillation of BCP and/or HU308 significantly reduces the number of adhering leukocytes in submucosal bladder venules and improves bladder capillary perfusion. The effects of BCP were found to be comparable to that of the selective CB₂R synthetic cannabinoid, HU308, and superior to intravesical DMSO treatment. Oral treatment with BCP was also able to reduce bladder inflammation and significantly reduced mechanical allodynia in experimental IC. Based on our findings, we believe that CB₂R activation may represent a viable therapeutic target for IC, and that drugs that activate CB₂R, such as the generally regarded as safe (GRAS) dietary sesquiterpenoid, BCP, may serve as an adjunct and/or alternative treatment option for alleviating symptoms of inflammation and pain in the management of IC.

Intraocular pressure (IOP) is the primary risk factor for glaucoma, a blinding eye disease. Cannabinoid agonists have long been known to decrease IOP, suggesting they may be useful in glaucoma treatment. However, the specific mechanism by which cannabinoids generate this ocular hypotensive effect remains unknown. The current evidence suggests the cannabinoids reduce IOP through actions at cannabinoid 1 (CB₁) receptors within the eye, and adrenergic receptors (ARs) may also contribute to this action of cannabinoids. Considering this, the present study aimed to elucidate the mechanism behind the ocular hypotensive properties of cannabinoids through the use of mice genetically lacking either cannabinoid receptors or βARs. Cannabinoid agonists, βAR antagonists, and βAR agonists decreased IOP in wild-type mice and CB₂(−/−) mice. In contrast, none of these compounds were found to reduce IOP in βAR(−/−) or CB₁(−/−) mice. Desensitization of the βARs and depletion of catecholamines in wild-type mice also eliminated the ability of the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2) to reduce IOP, strongly implicating a role for both βARs and catecholamines in the ocular hypotensive properties of cannabinoids. Finally, CB₁ receptors were shown to colocalize with tyrosine hydroxylase, a marker for adrenergic neurons. Taken together, these findings suggest that βARs are required for the ocular hypotensive properties of cannabinoids, and cannabinoids reduce IOP by acting as indirect sympatholytics and inhibiting norepinephrine release within the eye.

(1) Background: The cannabinoid 2 receptor (CB₂R) is a promising anti-inflammatory drug target and development of selective CB₂R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB₂R ligands: CB₂R agonists, RO6871304, and RO6871085, as well as a CB₂R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB₂R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB₂R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB₂R^-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB₂R^-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB₂R^-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB₂R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB₂R and selectivity for CB₂R > CB₁R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC₅₀s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB₂R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB₂R^-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB₂R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB₂R agonists support selective targeting of CB₂R for treating ocular inflammatory diseases.

Purpose. Metabotropic receptor agonists that signal through G_q-coupled pathways increase Ca²⁺ in mammalian Müller cells by release from intracellular stores and Ca²⁺ influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non–voltage-dependent Ca²⁺ channels in metabotropic muscarinic receptor-activated Ca²⁺ increases and store-operated Ca²⁺ influx in cultured mouse Müller cells. methods. Intracellular Ca²⁺ was measured using fluorescence imaging with the ratiometric dye fura-2. Currents were recorded using the whole-cell patch-clamp recording method. mRNA and protein were identified using reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical approaches. results. The muscarinic receptor agonist carbachol (3–20 μM) produced increases in Ca²⁺ that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca²⁺ stores by the sarcoplasmic/endoplasmic Ca²⁺ ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca²⁺. Carbachol-activated Ca²⁺ increases in Müller cells were enhanced by the diacylglycerol derivative 1-oleyl-2-acetyl-sn-glycerol and were blocked by transient receptor potential (TRP) channel blockers Gd³⁺, La³⁺, 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca²⁺ stores and produced Ca²⁺ entry that was attenuated by La³⁺, 2-APB, Gd³⁺, and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd³⁺-sensitive, TRP-like cation channel. conclusions. Metabotropic muscarinic receptor-activated Ca²⁺ increases in mouse Müller cells require the release of Ca²⁺ from intracellular stores and the activation of Ca²⁺ entry that involves TRP-like cation channels but is independent of voltage-dependent Ca²⁺ channels.