Search

The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ETB receptor knock-out rats and mounted the vessels in plexiglass pressure myograph chambers to measure myogenic tone in response to increasing pressure and targeted pharmacological treatments. We used an ETA receptor antagonist (BQ-123), an ETB receptor antagonist (BQ-788), endothelin-1, an endothelin-1 synthesis inhibitor (phosphoramidon), and vessel denudation to dissect the roles of each receptor in aging vasculature. Aged rats exhibited a higher myogenic tone than young rats, and the tone was sensitive to the ETA antagonist, BQ-123, but insensitive to the ETB antagonist, BQ-788. By contrast, the tone in the vessels from young rats was raised by BQ-788 but unaffected by BQ-123. When the endothelial layer that is normally enriched with ETB1 receptors was removed from young vessels, myogenic tone increased. However, denudation of the endothelial layer did not influence vessels from aged animals. This indicated that endothelial ETB1 receptors were not functional in the vessels from aged rats. There was also an increase in ETA receptor expression with age, whereas ETB receptor expression remained constant between young and aged animals. These results demonstrate that in young vessels, ETB1 receptors maintain a lower myogenic tone, but in aged vessels, a loss of ETB receptor activity allows ETA receptors in vascular smooth muscle cells to raise myogenic tone. Our findings have potentially important clinical implications for treatments to improve cerebral perfusion in older adults with diseases characterized by reduced cerebral blood flow.

Huntington’s disease (HD) is an inherited progressive neurodegenerative disease characterized by motor, cognitive, and behavioural changes. One of the earliest changes to occur in HD is a reduction in cannabinoid 1 receptor (CB(1)) levels in the striatum, which is strongly correlated with HD pathogenesis. CB(1) positive allosteric modulators (PAM) enhance receptor affinity for, and efficacy of activation by, orthosteric ligands, including the endocannabinoids anandamide and 2-arachidonoylglycerol. The goal of this study was to determine whether the recently characterized CB(1) allosteric modulators GAT211 (racemic), GAT228 (R-enantiomer), and GAT229 (S-enantiomer), affected the signs and symptoms of HD. GAT211, GAT228, and GAT229 were evaluated in normal and HD cell models, and in a transgenic mouse model of HD (7-week-old male R6/2 mice, 10 mg/kg/d, 21 d, i.p.). GAT229 was a CB(1) PAM that improved cell viability in HD cells and improved motor coordination, delayed symptom onset, and normalized gene expression in R6/2 HD mice. GAT228 was an allosteric agonist that did not enhance endocannabinoid signaling or change symptom progression in R6/2 mice. GAT211 displayed intermediate effects between its enantiomers. The compounds used here are not drugs, but probe compounds used to determine the potential utility of CB(1) PAMs in HD. Changes in gene expression, and not protein, were quantified in R6/2 HD mice because HD pathogenesis is associated with dysregulation of mRNA levels. The data presented here provide the first proof of principle for the use of CB(1) PAMs to treat the signs and symptoms of HD.

The human brain requires adequate cerebral blood flow to meet the high demand for nutrients and to clear waste products. With age, there is a chronic reduction in cerebral blood flow in small resistance arteries that can eventually limit proper brain function. The endothelin system is a key mediator in the regulation of cerebral blood flow, but the contributions of its constituent receptors in the endothelial and vascular smooth muscle layers of cerebral arteries have not been well defined in the context of aging. We isolated posterior cerebral arteries from young and aged Fischer 344 rats, as well as ET

Background Proton pump inhibitors (PPI) are commonly prescribed medications which are indicated in various different gastrointestinal (GI) diseases, including peptic ulcer disease, gastroesophageal reflux disorder and upper GI bleeding. There is some evidence to suggest that PPI use in cirrhosis may predispose to the development of hepatic encephalopathy (HE) and spontaneous bacterial peritonitis (SBP), albeit with some controversy. Aims We aim to conduct a retrospective epidemiological analysis of the association between PPI use in hospitalized patients with cirrhosis, and prevalence of HE and SBP. Methods This was a retrospective cohort study of 600 adult patients (mean age 61.4 (SD=12.2)) admitted the Ottawa Hospital between January 1, 2011 and December 31, 2015 with cirrhosis. A chart review was conducted and relevant information extracted. Results Average MELD-Na on admission was 16.2 (sd=6.7). 14.8% of patients had a history of HE, 5.0% SBP, 7.8% with history of hepatocellular carcinoma and 1.0% with history of hepatorenal syndrome. 28.5% of patients had a history of varices, of which 11.0% had previous variceal bleeding. 69.3% of patients were on a PPI during their hospitalization. Not surprisingly, patients admitted with variceal bleeding were more likely to be exposed to PPI in hospital (97.2% vs 63.2%, p Conclusions We did not observe a significant difference in HE and SBP among this cohort of cirrhotic patients by in-hospital PPI use. We did however note significantly higher PPI use in patients with previous diagnosis of cirrhosis as compared to those who were newly diagnosed, as well as those whose admissions were related to bleeding. Patients with ascites had lower prevalence of PPI use. Prescribing patterns for PPIs in patients with cirrhosis warrant further attention, including clinical utility and longer-term risks and benefits of this therapy. Funding Agencies None

Background and purpose We sought to understand why (-)-cannabidiol (CBD) and (-)-cannabidiol-dimethylheptyl (CBD-DMH) exhibit distinct pharmacology, despite near identical structures. Experimental approach HEK293A cells expressing either human type 1 cannabinoid (CB1 ) receptors or CB2 receptors were treated with CBD or CBD-DMH with or without the CB1 and CB2 receptor agonist CP55,940, CB1 receptor allosteric modulator Org27569 or CB2 receptor inverse agonist SR144528. Ligand binding, cAMP levels and βarrestin1 recruitment were measured. CBD and CBD-DMH binding was simulated with models of human CB1 or CB2 receptors, based on the recently published crystal structures of agonist-bound (5XRA) or antagonist-bound (5TGZ) human CB1 receptors. Key results At CB1 receptors, CBD was a negative allosteric modulator (NAM), and CBD-DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist-bound model of CB1 receptors (5TGZ) but shared a binding site with CP55,940 in the agonist-bound model of CB1 receptors (5XRA). The binding site for CBD-DMH in the CB1 receptor models overlapped with CP55,940 and Org27569. At CB2 receptors, CBD was a partial agonist, and CBD-DMH was a positive allosteric modulator of cAMP modulation but a NAM of βarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB2 receptor models that was separate from CBD-DMH. Conclusion and implications The pharmacological activity of CBD and CBD-DMH in HEK293A cells and their modelled binding sites at CB1 and CB2 receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB1 and CB2 receptors. Linked articles This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Background Portal vein thrombosis (PVT) has a reported prevalence ranging from 0.6 to 26% in cirrhotic patients and yet optimal management in these patients remains unclear [1]. PVT can lead to poor outcomes including increased risk of bleeding, intestinal injury, and deterioration in liver function. Conversely, treatment of PVT in cirrhotic patients increases their risk of bleeding complications, particularly in patients with known varices. Aims The aim of this study is to better characterize the prevalence and impact of PVT in cirrhotic inpatients. Methods We conducted a retrospective cohort study based on data collected on adult patients admitted to the Ottawa Hospital between January 1, 2011 and December 31, 2015. We included patients with a diagnosis of cirrhosis either before or during index admission. Patients with a radiology report indicating a PVT were compared to those without PVT. Non-Ontario residents were excluded and where there were multiple admissions per patient one admission was randomly selected to be used. Ethics approval was obtained from the Research Ethics Board at the University of Ottawa. Results This study found 34 patients with cirrhosis diagnosed with PVT during their hospitalization (3.73%). Of the patients with PVT, 23 were acute and 11 were chronic based on radiologic appearance. Mean age was similar between groups (PVT: 61.7, SD=9.8; No PVT: 62.3, SD=12.3). The mean Na-MELD was also similar (PVT: 17.6, no PVT: 17.3, p=0.82). Among patients with PVT, 11 patients presented with ascites, 10 with hepatic encephalopathy (HE), 5 with abdominal pain and 5 with an upper GI bleed. Spontaneous bacterial peritonitis (SBP) occurred in 11.76% of patients with PVT as compared to 3% of patients without PVT (p value= 0.006). There also seemed to be a trend towards more HE in the cohort with PVT (20.6% vs 10.7%, p value= 0.07). With regards to screening for varices, 2 patients had an EGD in the 6 months prior to admission, 11 had an EGD on admission, 1 after anticoagulation due to bleeding, and 18 had no screening in the 6 months prior to admission. Twelve patients were treated for PVT, 17 were untreated and 5 did not have documentation about treatment. Of the patients that were not treated, 9 were due to palliative goals of care, 1 due to bleeding, 1 due to thrombocytopenia, 2 due to chronicity on imaging and 4 did not have reasons documented. Conclusions PVT is a known complication of cirrhosis, however the clinical significance and optimal management of patients with PVT is poorly understood. Although prevalence of PVT was low in this cohort, our data suggests some possible association between liver related complications and PVT, including SBP and HE. Further research is needed to determine how to best manage patients with PVT. 1. Garcia-Pagan JC, Valla DC. Portal vein thrombosis: a predictable milestone in cirrhosis? Journal of hepatology. 2009 Oct 1;51(4):632–4. Funding Agencies None

Cannabinoid receptor 1 (CB1) activation has been reported to reduce transient receptor potential cation channel subfamily V member 1 (TRPV1)-induced inflammatory responses and is anti-nociceptive and anti-inflammatory in corneal injury. We examined whether allosteric ligands, can modulate CB1 signaling to reduce pain and inflammation in corneal hyperalgesia. Corneal hyperalgesia was generated by chemical cauterization of cornea in wildtype and CB2 knockout (CB2&minus, /&minus, ) mice. The novel racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229 were examined alone or in combination with the orthosteric CB1 agonist &Delta, 8-tetrahydrocannabinol (&Delta, 8-THC). Pain responses were assessed following capsaicin (1 &micro, M) stimulation of injured corneas at 6 h post-cauterization. Corneal neutrophil infiltration was also analyzed. GAT228, but not GAT229 or GAT211, reduced pain scores in response to capsaicin stimulation. Combination treatments of 0.5% GAT229 or 1% GAT211 with subthreshold &Delta, 8-THC (0.4%) significantly reduced pain scores following capsaicin stimulation. The anti-nociceptive effects of both GAT229 and GAT228 were blocked with CB1 antagonist AM251, but remained unaffected in CB2&minus, mice. Two percent GAT228, or the combination of 0.2% &Delta, 8-THC with 0.5% GAT229 also significantly reduced corneal inflammation. CB1 allosteric ligands could offer a novel approach for treating corneal pain and inflammation.

(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 (&szlig, 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 &gt, CB1R, with both ligands acting as full agonists in cAMP and &szlig, 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.

One of the most abundant G-protein coupled receptors (GPCRs) in brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic target for treating diverse psychobehavioral and somatic disorders. Adverse on-target effects associated with small-molecule CB1R orthosteric agonists and inverse agonists/antagonists have plagued their translational potential. Allosteric CB1R modulators offer a potentially safer modality through which CB1R signaling may be directed for therapeutic benefit. Rational design of candidate, druglike CB1R allosteric modulators requires greater understanding of the architecture of the CB1R allosteric endodomain(s) and the capacity of CB1R allosteric ligands to tune the receptor's information output. We have recently reported the synthesis of a focused library of rationally designed, covalent analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative allosteric modulators (NAMs). Among the novel, pharmacologically active CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead by virtue of its exceptional potency in the [(35)S]GTPγS and β-arrestin signaling assays and its ability to label CB1R as a covalent allosteric probe with significantly reduced inverse agonism in the [(35)S]GTPγS assay as compared to Org27569. We report here a comprehensive functional profiling of GAT100 across an array of important downstream cell-signaling pathways and analysis of its potential orthosteric probe-dependence and signaling bias. The results demonstrate that GAT100 is a NAM of the orthosteric CB1R agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2 phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A cells overexpressing CB1R and in Neuro2a and STHdh(Q7/Q7) cells endogenously expressing CB1R. Distinctively, GAT100 was a more potent and efficacious CB1R NAM than Org27569 and PSNCBAM-1 in all signaling assays and did not exhibit the inverse agonism associated with Org27569 and PSNCBAM-1. Computational docking studies implicate C7.38(382) as a key feature of GAT100 ligand-binding motif. These data help inform the engineering of newer-generation, druggable CB1R allosteric modulators and demonstrate the utility of GAT100 as a covalent probe for mapping structure-function correlates characteristic of the druggable CB1R allosteric space.

Central nervous system (CNS) injury, such as stroke or trauma, is known to increase susceptibility to various infections that adversely affect patient outcomes (CNS injury-induced immunodepression—CIDS). The endocannabinoid system (ECS) has been shown to have immunoregulatory properties. Therefore, the ECS might represent a druggable target to overcome CIDS. Evidence suggests that cannabinoid type 2 receptor (CB2R) activation can be protective during the early pro-inflammatory phase after CNS injury, as it limits neuro-inflammation and, therefore, attenuates CIDS severity. In the later phase post CNS injury, CB2R inhibition is suggested as a promising pharmacologic strategy to restore immune function in order to prevent infection.

Challenges in the management of ocular pain are an underappreciated topic. Currently available therapeutics lack both efficacy and clear guidelines for their use, with many also possessing unacceptable side effects. Promising novel agents would offer analgesic, anti-inflammatory, and possibly neuroprotective actions; have favorable ocular safety profiles; and show potential in managing neuropathic pain. Growing evidence supports a link between the endocannabinoid system (ECS) and a range of physiological and disease processes, notably those involving inflammation and pain. Both preclinical and clinical data suggest analgesic and anti-inflammatory actions of cannabinoids and ECS-modifying drugs in chronic pain conditions, including those of neuropathic origin. This review will examine existing evidence for the anatomical and physiological basis of ocular pain, specifically, ocular surface disease and the development of chronic ocular pain. The mechanism of action, efficacy, and limitations of currently available treatments will be discussed, and current knowledge related to ECS-modulation of ocular pain and inflammatory disease will be summarized. A perspective will be provided on the future directions of ECS research in terms of developing cannabinoid therapeutics for ocular pain.

Background Exogenous endophthalmitis is a potential complication of intraocular surgery and frequently results in visual impairment. Current treatment involves administration of intravitreal (IVT) antibiotics with or without vitrectomy surgery. Evidence for the use of adjunctive anti-inflammatory agents is conflicting. We set out to determine if bevacizumab, a humanized monoclonal IgG1 antibody targeted against vascular endothelial growth factor (VEGF), has anti-inflammatory properties in experimental models of Gram-positive and Gram-negative inflammation. Methods BALB/c mice were subjected to lipopolysaccharide- (LPS) or peptidoglycan- (PGN) induced ocular inflammation and treated with IVT bevacizumab. Iris microvasculature was imaged 6 hours following irritant/treatment using intravital microscopy (IVM) before the mice were euthanized and the eyes were enucleated immediately post-mortem. Following enucleation, levels of VEGF and 23 cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, TNF, KC, G-CSF, GM-CSF, Eotaxin, INF-γ, MCP-1, MIP-1α, MIP-1β, RANTES) were quantified using a multiplex assay. Results Levels of VEGF were significantly increased during the inflammatory response, triggered by either PGN or LPS. Both the adherence of leukocytes to the iris vascular endothelium and the levels of pro-inflammatory cytokines and chemokines were significantly increased following administration of either irritant. Treatment with bevacizumab decreased levels of leukocyte adherence in LPS-treated eyes, however, not in PGN-treated eyes. Conversely, bevacizumab treatment decreased levels of cytokines and chemokines (TNF, IL-6, MCP-1, MIP-1α, MIP-1β, RANTES, KC) in PGN-treated eyes, however, not in LPS-treated eyes. Conclusions Within a 6-hour window bevacizumab had anti-inflammatory actions that were distinct in both Gram-positive (PIU) and Gram-negative (EIU) models, respectively. Given our findings, this would suggest that bevacizumab may have utility as an adjunctive therapy to IVT antibiotics and vitrectomy in the management of exogenous endophthalmitis.

The cannabinoid receptor CB2 plays a significant role in the regulation of immune function whereas neuronal expression remains a subject of contention. Multiple studies have described CB2 in retina and a recent study showed that CB2 deletion altered retinal visual processing. We revisited CB2 expression using immunohistochemistry and a recently developed CB2-eGFP reporter mouse. We examined the consequence of acute vs. prolonged CB2 deactivation on the electroretinogram (ERG) responses. We also examined lipidomics in CB2 knockout mice and potential changes in microglia using Scholl analysis. Consistent with a published report, in CB2 receptor knockout mice see an increased ERG scotopic a-wave, as well as stronger responses in dark adapted cone-driven ON bipolar cells and, to a lesser extent cone-driven ON bipolar cells early in light adaptation. Significantly, however, acute block with CB2 antagonist, AM630, did not mimic the results observed in the CB2 knockout mice whereas chronic (7 days) block did. Immunohistochemical studies show no CB2 in retina under non-pathological conditions, even with published antibodies. Retinal CB2–eGFP reporter signal is minimal under baseline conditions but upregulated by intraocular injection of either LPS or carrageenan. CB2 knockout mice see modest declines in a broad spectrum of cannabinoid-related lipids. The numbers and morphology of microglia were unaltered. In summary minimal CB2 expression is seen in healthy retina. CB2 appears to be upregulated under pathological conditions. Previously reported functional consequences of CB2 deletion are an adaptive response to prolonged blockade of these receptors. CB2 therefore impacts retinal signaling but perhaps in an indirect, potentially extra-ocular fashion. post-print 8285 KB

Background and Purpose: Corneal injury can result in dysfunction of corneal nociceptive signaling and corneal sensitization. Activation of the endocannabinoid system has been reported to be analgesic and anti-inflammatory. The purpose of this research was to investigate the antinociceptive and anti-inflammatory effects of cannabinoids with reported actions at cannabinoid 1 (CB1R) and cannabinoid 2 (CB2R) receptors and/or noncannabinoid receptors in an experimental model of corneal hyperalgesia. Methods: Corneal hyperalgesia (increased pain response) was generated using chemical cauterization of the corneal epithelium in wild-type (WT) and CB2R knockout (CB2R−/−) mice. Cauterized eyes were treated topically with the phytocannabinoids Δ8-tetrahydrocannabinol (Δ8THC) or cannabidiol (CBD), or the CBD derivative HU-308, in the presence or absence of the CB1R antagonist AM251 (2.0 mg/kg i.p.), or the 5-HT1A receptor antagonist WAY100635 (1 mg/kg i.p.). Behavioral pain responses to a topical capsaicin challenge at 6 h postinjury were quantified from video recordings. Mice were euthanized at 6 and 12 h postcorneal injury for immunohistochemical analysis to quantify corneal neutrophil infiltration. Results: Corneal cauterization resulted in hyperalgesia to capsaicin at 6 h postinjury compared to sham control eyes. Neutrophil infiltration, indicative of inflammation, was apparent at 6 and 12 h postinjury in WT mice. Application of Δ8THC, CBD, and HU-308 reduced the pain score and neutrophil infiltration in WT mice. The antinociceptive and anti-inflammatory actions of Δ8THC, but not CBD, were blocked by the CB1R antagonist AM251, but were still apparent, for both cannabinoids, in CB2R−/− mice. However, the antinociceptive and anti-inflammatory actions of HU-308 were absent in the CB2R−/− mice. The antinociceptive and anti-inflammatory effects of CBD were blocked by the 5-HT1A antagonist WAY100635. Conclusion: Topical cannabinoids reduce corneal hyperalgesia and inflammation. The antinociceptive and anti-inflammatory effects of Δ8THC are mediated primarily via CB1R, whereas that of the cannabinoids CBD and HU-308, involve activation of 5-HT1A receptors and CB2Rs, respectively. Cannabinoids could be a novel clinical therapy for corneal pain and inflammation resulting from ocular surface injury.

Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.

BACKGROUND AND PURPOSE: We sought to understand why (−)‐cannabidiol (CBD) and (−)‐cannabidiol‐dimethylheptyl (CBD‐DMH) exhibit distinct pharmacology, despite near identical structures. EXPERIMENTAL APPROACH: HEK293A cells expressing either human type 1 cannabinoid (CB(1)) receptors or CB(2) receptors were treated with CBD or CBD‐DMH with or without the CB(1) and CB(2) receptor agonist CP55,940, CB(1) receptor allosteric modulator Org27569 or CB(2) receptor inverse agonist SR144528. Ligand binding, cAMP levels and βarrestin1 recruitment were measured. CBD and CBD‐DMH binding was simulated with models of human CB(1) or CB(2) receptors, based on the recently published crystal structures of agonist‐bound (5XRA) or antagonist‐bound (5TGZ) human CB(1) receptors. KEY RESULTS: At CB(1) receptors, CBD was a negative allosteric modulator (NAM), and CBD‐DMH was a mixed agonist/positive allosteric modulator. CBD and Org27569 shared multiple interacting residues in the antagonist‐bound model of CB(1) receptors (5TGZ) but shared a binding site with CP55,940 in the agonist‐bound model of CB(1) receptors (5XRA). The binding site for CBD‐DMH in the CB(1) receptor models overlapped with CP55,940 and Org27569. At CB(2) receptors, CBD was a partial agonist, and CBD‐DMH was a positive allosteric modulator of cAMP modulation but a NAM of βarrestin1 recruitment. CBD, CP55,940 and SR144528 shared a binding site in the CB(2) receptor models that was separate from CBD‐DMH. CONCLUSION AND IMPLICATIONS: The pharmacological activity of CBD and CBD‐DMH in HEK293A cells and their modelled binding sites at CB(1) and CB(2) receptors may explain their in vivo effects and illuminates the difficulties associated with the development of allosteric modulators for CB(1) and CB(2) receptors. LINKED ARTICLES: This article is part of a themed section on 8(th) European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc

Medical access to cannabis was first granted in Canada in 1999, with several amendments leading to the current Access to Cannabis for Medical Purposes Regulations .[1][1] Patients have the right to access cannabis for medical purposes if they have the support of a health care practitioner. Three

Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

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&rsquo, 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.

Free fatty acids (FFAs) are metabolic intermediates that may be obtained through the diet, synthesized endogenously, or produced via fermentation of carbohydrates by gut microbiota. In addition to serving as an important source of energy, FFAs are known to produce a variety of both beneficial and detrimental effects on metabolic and inflammatory processes. While historically, FFAs were believed to produce these effects only through intracellular targets such as peroxisome proliferator-activated receptors, it has now become clear that FFAs are also agonists for several GPCRs, including a family of four receptors now termed FFA1-4. Increasing evidence suggests that FFA1-4 mediate many of the beneficial properties of FFAs and not surprisingly, this has generated significant interest in the potential of these receptors as therapeutic targets for the treatment of a variety of metabolic and inflammatory disorders. In addition to the traditional strategy of developing small-molecule therapeutics targeting these receptors, there has also been some consideration given to alternate therapeutic approaches, specifically by manipulating endogenous FFA concentrations through alteration of either dietary intake, or production by gut microbiota. In this review, the current state of knowledge for FFA1-4 will be discussed, together with their potential as therapeutic targets in the treatment of metabolic and inflammatory disorders. In particular, the evidence in support of small molecule versus dietary and microbiota-based therapeutic approaches will be considered to provide insight into the development of novel multifaceted strategies targeting the FFA receptors for the treatment of metabolic and inflammatory disorders.

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

UNLABELLED The type 1 cannabinoid receptor (CB(1) ) is an integral component of the endocannabinoid system that modulates several functions in the CNS and periphery. The majority of our knowledge of the endocannabinoid system involves ligand-receptor binding, mechanisms of signal transduction, and protein-protein interactions. In contrast, comparatively little is known about regulation of CB(1) gene expression. The levels and anatomical distribution of CB(1) mRNA and protein are developmental stage-specific and are dysregulated in several pathological conditions. Moreover, exposure to a variety of drugs, including cannabinoids themselves, alters CB(1) gene expression and mRNA levels. As such, alterations in CB(1) gene expression are likely to affect the optimal response to cannabinoid-based therapies, which are being developed to treat a growing number of conditions. Here, we will examine the regulation of CB(1) mRNA levels and the therapeutic potential inherent in manipulating expression of this gene. 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.2012.167.issue-8.

Background and purpose: The CB1 cannabinoid 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 CB1 receptors 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 CB1 receptors 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 CB1 receptors. Co-expression altered the signalling properties of CB1 receptors, resulting in increased Gαi-dependent ERK phosphorylation, but decreased non-Gαi-mediated CREB phosphorylation. The CB1 receptor 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 CB1 receptor 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 CB1 receptors 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 CB1 receptors 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 visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x

This review examines the function of calcium-activated chloride currents (I(Cl(Ca))) in the retina with an emphasis on their physiological role in photoreceptors. Although found in a variety of neurons and glial cells of the retina, I(Cl(Ca)) has been most prominently studied in cones, where it activates in response to depolarization-evoked Ca(2+) influx. The slow and complex gating kinetics of the chloride current have been considered to reflect the changing submembrane concentration of intracellular calcium. It is likely that the role of I(Cl(Ca)) is to stabilize the membrane potential of cones during synaptic activity and presynaptic Ca channel modulation. Several candidates in the molecular identification of the channel have been put forward but the issue remains unresolved.

Activation of dopamine receptor 2 long (D2L) switches the signaling of type 1 cannabinoid receptor (CB1) from Gαi to Gαs, a process thought to be mediated through CB1-D2L heteromerization. Given the clinical importance of D2 antagonists, the goal of this study was to determine if D2 antagonists could modulate CB1 signaling. Interactions between CB1 and D2L, Gαi, Gαs, and β-arrestin1 were studied using bioluminescence resonance energy transfer 2 (BRET(2)) in STHdh(Q7/Q7) cells. CB1-dependent extracellular regulated kinase (ERK)1/2, CREB phosphorylation, and CB1 internalization following cotreatment of CB1 agonist and D2 antagonist were quantified. Preassembled CB1-Gαi complexes were detected by BRET(2) Arachidonyl-2'-chloroethylamide (ACEA), a selective CB1 agonist, caused a rapid and transient increase in BRET efficiency (BRETEff) between Gαi-Rluc and CB1-green fluorescent protein 2 (GFP(2)), and a Gαi-dependent increase in ERK phosphorylation. Physical interactions between CB1 and D2L were observed using BRET(2) Cotreatment of STHdh(Q7/Q7) cells with ACEA and haloperidol, a D2 antagonist, inhibited BRETEff signals between Gαi-Rluc and CB1-GFP(2) and reduced the EMax and pEC50 of ACEA-mediated Gαi-dependent ERK phosphorylation. ACEA and haloperidol cotreatments produced a delayed and sustained increase in BRETEff between Gαs-Rluc and CB1-GFP(2) and increased the EMax and pEC50 of ACEA-induced Gαs-dependent cAMP response element-binding protein phosphorylation. In cells expressing CB1 and D2L treated with ACEA, binding of haloperidol to D2 receptors switched CB1 coupling from Gαi to Gαs In addition, haloperidol treatment reduced ACEA-induced β-arrestin1 recruitment to CB1 and CB1 internalization. D2 antagonists allosterically modulate cannabinoid-induced CB1 coupling, signaling, and β-arrestin1 recruitment through binding to CB1-D2L heteromers. These findings indicate that D2 antagonism, like D2 agonists, change agonist-mediated CB1 coupling and signaling.

Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM). Experimental approach: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis. Key results: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10–100 μM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Gαq/11 subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations ≥15 μM, and PTX-sensitive increases in ERK1/2 phosphorylation. Conclusions and implications: This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP. British Journal of Pharmacology (2007) 152, 1111–1120; doi:10.1038/sj.bjp.0707495; published online 8 October 2007

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.

Activation of the type 1 cannabinoid receptor (CB1) may be a means of treating neuropathic pain, glaucoma and neurodegenerative disorders where CB1 levels decline, such as Huntington's diseasse. Po...

Cannabidiol has been reported to act as an antagonist at cannabinoid CB1 receptors. We hypothesized that cannabidiol would inhibit cannabinoid agonist activity through negative allosteric modulation of CB1 receptors.Internalization of CB1 receptors, arrestin2 recruitment, and PLCβ3 and ERK1/2 phosphorylation, were quantified in HEK 293A cells heterologously expressing CB1 receptors and in the STHdh(Q7/Q7) cell model of striatal neurons endogenously expressing CB1 receptors. Cells were treated with 2-arachidonylglycerol or Δ(9)-tetrahydrocannabinol alone and in combination with different concentrations of cannabidiol.Cannabidiol reduced the efficacy and potency of 2-arachidonylglycerol and Δ(9)-tetrahydrocannabinol on PLCβ3- and ERK1/2-dependent signalling in cells heterologously (HEK 293A) or endogenously (STHdh(Q7/Q7)) expressing CB1 receptors. By reducing arrestin2 recruitment to CB1 receptors, cannabidiol treatment prevented internalization of these receptors. The allosteric activity of cannabidiol depended upon polar residues being present at positions 98 and 107 in the extracellular amino terminus of the CB1 receptor.Cannabidiol behaved as a non-competitive negative allosteric modulator of CB1 receptors. Allosteric modulation, in conjunction with effects not mediated by CB1 receptors, may explain the in vivo effects of cannabidiol. Allosteric modulators of CB1 receptors have the potential to treat CNS and peripheral disorders while avoiding the adverse effects associated with orthosteric agonism or antagonism of these receptors.

We show that carbenoxolone, a drug used to block hemichannels in the retina to test the ephaptic model of horizontal cell inhibitory feedback, has strong inhibitory effects on voltage-gated Ca channels. Carbenoxolone (100 μM) reduced photoreceptor-to-horizontal cell synaptic transmission by 92%. Applied to patch-clamped, isolated cone photoreceptors, carbenoxolone inhibited Ca channels with an EC50 of 48 μM. At 100 μM, it reduced cone Ca channel current by 37%, reduced depolarization-evoked [Ca2+] signals in fluo-4 loaded retinal slices by 57% and inhibited Ca channels in Müller cells by 52%. A synaptic transfer model suggests that the degree of block of Ca channels accounts for the reduction in synaptic transmission. These results suggest broad inhibitory actions for carbenoxolone in the retina that must be considered when interpreting its effects on inhibitory feedback.

We examined A3 adenosine and CB1 cannabinoid receptor-coupled signaling pathways regulating Cl− current in a human nonpigmented ciliary epithelial (NPCE) cell line. Whole-cell patch-clamp recordings demonstrated that the A3 receptor agonist, IB-MECA, activates an outwardly rectifying Cl−current (ICl,Aden) in NPCE cells, which was inhibited by the adenosine receptor antagonist, CGS-15943 or by the protein kinase C (PKC) activator, phorbol 12,13 dibutyrate (PDBu). Treatment of NPCE cells with pertussis-toxin (PTX), or transfection with the COOH-terminus of β-adrenergic receptor kinase (ct-βARK), inhibited ICl,Aden. The phosphatidyl inositol 3-kinase (PI3K) inhibitor, wortmannin, had no effect on ICl,Aden; however, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, inhibited ICl,Aden. Reverse transcription–polymerase chain reaction experiments and immunocytochemistry confirmed mRNA and protein expression for the CB1 receptor in NPCE cells, and the CB1 receptor agonist, Win 55,212-2, activated a PDBu-sensitive Cl− current (ICl,Win). Transfection of NPCE cells with the human CB1 (hCB1) receptor, increased ICl,Win, consistent with increased receptor expression, and ICl,Win in hCB1 receptor-transfected cells was decreased after application of a CB1 receptor inverse agonist, SR 141716. Constitutive activity for CB1 receptors was not significant in NPCE cells as transfection with hCB1 receptors did not increase basal Cl− current, nor was basal current inhibited by SR 141716. ICl,Win was inhibited by PTX preincubation, by transfection with ct-βARK and by the MEK inhibitor, PD98059, but unaffected by the PI3K inhibitor, wortmannin. We conclude that both A3 and CB1 receptors activate a PKC-sensitive Cl− current in human NPCE cells via a Gi/o/Gβγ signaling pathway, in a manner independent of PI3K but involving MAPK. British Journal of Pharmacology (2003) 139, 475–486. doi:10.1038/sj.bjp.0705266

We report on seizures during anesthesia induction in animals treated with a cannabinoid receptor 1 (CB1R) antagonist for experimental sepsis. Animals received surgery for colon ascendens stent peritonitis-induced sepsis or sham surgery followed by treatment of CB1R antagonist, CB1R agonist, or placebo. Fourteen hours later, animals received pentobarbital or ketamine for anesthesia induction and animal behavior was observed. Tonic-clonic seizures were observed in 5 of 12 septic animals (42%) treated with CB1R antagonist after induction of anesthesia with pentobarbital. The data suggest that CB1R inhibition in combination with pentobarbital may increase the incidence of anesthetic-induced seizures in the case of sepsis.

Levels of the type 1 cannabinoid receptor (CB1) decline prior to Huntington’s disease (HD) symptom onset in the striatum and this decline is correlated with disease progression. Consequently, strat...

We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl− channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. Under conditions designed to isolate Cl− currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl− selective. The disulphonic stilbene derivatives 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0·5 mM), 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS, 0·5 mM) and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS, 0·5 mM) produced a voltage-sensitive block of HOS-activated Cl− current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl− current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0·05). The non-specific kinase inhibitor H-7 (100 μM), upregulated HOS-activated Cl− current amplitude in all cells tested (P < 0·05). Noise analysis of whole-cell Cl− current indicated that cell swelling activated a high density of small conductance Cl− channels (< 1 pS). We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl− channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation. The ciliary body epithelium (CBE) forms the inner surface covering of the ciliary processes of the eye and is composed of two different epithelial cell layers: a non-pigmented ciliary epithelial (NPCE) cell layer and a pigmented ciliary epithelial (PCE) cell layer (Caprioli, 1992). Both PCE and NPCE cells are involved in the production of aqueous humour, an isotonic solution composed primarily of water, Na+, Cl− and HCO3−. The balance between the rate and quantity of aqueous humour produced and aqueous humour escape from the eye, via drainage pathways, is the primary determinant of intraocular pressure (IOP), and is subject to autonomic modulation (Caprioli, 1992). Transport data from intact and dispersed ciliary epithelial tissue suggest that PCE cells have solute uptake properties and are functionally coupled to the NPCE cells which have solute efflux properties (Wiederholt et al. 1991; Edelman et al. 1994). In this cell coupled model, ions and water from the stroma are taken up by PCE cells and passed to the NPCE cells via apical gap junctions (Raviola & Raviola, 1978), where they are secreted at the basolateral membrane into the posterior chamber as aqueous humour. Despite our understanding of this functional coupling between CE cells, the exact cellular transport mechanisms involved in fluid and ion secretion remain unresolved. However, it has now been shown that Na+, K+ and Cl− enter PCE cells via a furosemide- (frusemide-) and bumetamide-sensitive Na+-K+-2Cl− symport and diffuse from PCE to NPCE cells via the apical gap junctions. Na+, K+ and Cl− ions are then secreted from NPCE cells through Na+-K+ exchange pumps and via basolateral K+ and Cl− channels, and this is accompanied by paracellular Na+ movement. A HCO3−-dependent transepithelial potential of approximately 1 mV, aqueous humour negative, provides a net electrochemical driving force (for review see Krupin & Civan, 1995; Jacob & Civan, 1996). In addition, the activity of volume-regulated K+ and Cl− channels in NPCE cells probably contributes to regulatory volume decrease (RVD) and transepithelial salt transport in the CBE following alterations in cellular osmotic gradients (Farahbakhsh & Fain, 1987; Yantorno et al. 1989, 1992; Civan et al. 1992, 1994; Adorante & Cala, 1995). In support of this, NPCE cells in the intact ciliary process have been shown to respond to hypotonic media with cell swelling accompanied by ion and water efflux (Farahbakhsh & Fain, 1987). Chloride channels in the NPCE cells of the ciliary body epithelium have been suggested to be critical to the formation of aqueous humour, as well as in volume regulation of these cells (for review see Jacob & Civan, 1996). Several candidates for the volume-activated Cl− channel/channel regulator in NPCE cells have now been presented. These include the multidrug resistance gene product (MDR1) in native bovine ciliary epithelial cells (Wu et al. 1996; Wang et al. 1998), CIC-3 Cl− channel in a cultured transformed human NPCE cell line (Coca-Prados et al. 1996), and pICln in the transformed human NPCE cell line (Coca-Prados et al. 1995, 1996) and in acutely isolated NPCE cells from rabbit (Chen et al. 1998). To date, despite extensive investigation, none of these candidates have yet been unequivocably linked to the volume-activated Cl− channel(s) in NPCE cells. In addition, various mechanisms have also been suggested to be involved in linking cell swelling and activation of Cl− channels in NPCE cells. These signalling pathways include protein kinase C (PKC), Ca2+-calmodulin (CaM) and an epoxide (Civan et al. 1994; Coca-Prados et al. 1995, 1996). The purpose of this study was to identify the electrophysiological and pharmacological properties of a hyposmotic (HOS)-activated Cl− channel in SV40-transformed rabbit NPCE cells, using whole-cell patch-clamp recordings and noise analysis. The rabbit CBE has been used extensively for studies of transepithelial ion transport and aqueous humour production, thus information from isolated cell studies can be correlated with existing data and transport models in this species (Farahbakhsh & Fain, 1987; Sears et al. 1991; for review see Jacob & Civan, 1996). Our results demonstrate that rabbit NPCE cells activate volume-sensitive Cl− channels in response to hyposmotic shock. These Cl− channels share some properties with other volume-activated Cl− channels, being sensitive to specific Cl− channel blockers and modulated by Ca2+ and phosphorylation. Noise analysis revealed that the HOS-activated Cl− channels in NPCE cells had a small unitary conductance. Since Cl− channel activation represents a rate-limiting step in fluid secretion, understanding the mechanisms of Cl− channel and cell volume regulation in NPCE cells can provide information about the physiology of aqueous humour secretion.

The retinal pigment epithelium (RPE) carries out a number of roles that are essential for the maintenance and viability of the neurosensory retina. These roles include phagocytosis of shed rod and cone outer segments, melanin synthesis and recycling and regulation of subretinal volume via ion-coupled fluid absorption (Steinberg & Miller, 1979; Zinn & Benjamin-Henkind, 1979; Clark, 1986). In order to carry out these diverse functions, the RPE must be able to detect and respond to paracrine signals coming from the adjacent choroidal and/or neural retinal tissue and/or via systemic sources. A number of metabotropic receptors have been identified on the RPE including those for dopamine, acetylcholine, adrenaline (epinephrine) and adenosine (Friedman et al. 1988; Dearry et al. 1990; Frambach et al. 1990). Activation of these receptors by their respective signalling molecules has been linked to changes in light-evoked responses (Dearry et al. 1990; Gallemore & Steinberg, 1990), phagocytic ability (Gregory et al. 1994) and ion and fluid transport across the RPE (Edelman & Miller, 1991; Joseph & Miller, 1992). Recently, in monolayers of bovine and rat RPE, extracellular adenosine 5′-triphosphate (ATP) and uridine triphosphate (UTP) were demonstrated to induce changes in intracellular Ca2+ and transepithelial ion and fluid movement (Stalmans & Himpens, 1997; Peterson et al. 1997). A role for intracellular ATP has also recently been demonstrated for the activation of a delayed inwardly rectifying K+ current (IK(IR)) in isolated bovine RPE cells (Hughes & Takahira, 1998). These findings support the presence of metabotropic purinoceptors and suggest that ATP may act as an important paracrine signal in the RPE. Purinoceptors are divided into two main classes, P1 and P2, based on their selectivity for adenosine and ATP, respectively (Burnstock & Kennedy, 1985). Adenosine or P1 receptors are G protein-coupled receptors that respond only to purine nucleosides and nucleoside monophosphates. ATP or P2 receptors are activated primarily by purine or pyrimidine nucleotide triphosphates, and are organized into two major subclasses: P2X purinoceptors and the P2Y purinoceptors (Barnard et al. 1997). P2X receptor subtypes comprise a novel family of ligand-gated cation channels (Barnard, 1992). Seven P2X receptor subtypes have been identified and classified on the basis of sequence homology, agonist/antagonist sensitivity and kinetics of receptor desensitization. The seven subtypes of P2Y receptors thus far identified belong to the larger superfamily of seven transmembrane proteins that are G protein-coupled (metabotropic) receptors. Although signalling molecules downstream from P2Y receptors have not been fully classified, all subtypes to date have been linked by heterotrimeric G proteins to phospholipase C (PLC) activation, generation of inositol trisphosphate (IP3) and diacylglycerol (DAG), and subsequent release of Ca2+ from internal stores (Barnard et al. 1994). Pharmacological and functional data support the presence of P2Y purinoceptors in rat and bovine RPE (Stalmans & Himpens, 1997; Peterson et al. 1997). More recently, P2Y2 purinoceptors were found to be expressed in cultured human RPE cells (Sullivan et al. 1997). In the present study we report that extracellular ATP, UTP and other nucleotides, but not adenosine, activate two distinct cation conductances. The rapid activation of an inward cation current suggests for the first time, the presence of functional P2X purinoceptors in the rat RPE. We also provide evidence for the presence of G protein-coupled P2Y purinoceptors linked to increases in [Ca2+]i and activation of a Ca2+-sensitive outward K+ conductance. Our findings that extracellular ATP (or UTP) mediates cation channel activation and [Ca2+]i mobilization in the RPE support a role for ATP and/or UTP as paracrine signals in the RPE and suggest that they may act to regulate RPE function under physiological and/or pathological conditions.

1. The effects of adrenergic agonists on K+ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells. 2. Outward K+ current (IK) was reduced by tetraethylammonium chloride, the Ca2+-activated K+ (K(Ca)) channel blocker iberiotoxin (IbTX), or Ca2+-free external Ringer solution. The calcium ionophore ionomycin increased an IbTX-sensitive IK in PCE cells. 3. The adrenergic agonists adrenaline and phenylephrine increased IK in PCE cells. The induced current was blocked by IbTX and the alpha1-antagonist prazosin, suggesting that adrenergic agonists activate IK(Ca) via alpha1-adrenoreceptors. 4. Internal dialysis of D-myo-inositol 1,4, 5-trisphosphate (IP3) increased IK, whilst pre-incubation of PCE cells with thapsigargin or the phospholipase C (PLC) inhibitor U-73122 reduced phenylephrine-induced increases in IK(Ca). Adrenergic increases in IK(Ca) were mediated by a pertussis toxin-insensitive G protein. 5. These results demonstrate that IK(Ca) channels in rabbit PCE cells are coupled to alpha1-adrenergic receptors and a PLC/IP3 signalling pathway. Activation of these channels may modulate fluid secretion by the ciliary epithelium.

1. Whole-cell patch-clamp recording techniques were used to investigate the G protein subtype and related signalling molecules involved in activation of a nonspecific cation (NSC) current in rat cultured retinal pigment epithelial (RPE) cells. 2. Under control conditions, in 130 mM NaCl with K+ aspartate in the pipette, cytosolic dialysis with guanosine-5'-O-(3-triphosphate) (GTPgammaS, 0.1 mM) activated a large non-inactivating NSC current in 80% of the cells recorded from. 3. Loading RPE cells with antibodies (10 microg-ml(-1)) against the alpha subunit of all PTX-sensitive G proteins (G(alpha i/o/t/z)) reduced NSC current activation to 11%, while loading RPE cells with antibodies directed specifically against the alpha subunits of the Gi subclass (G(alpha i-3)) completely abolished current activation. In RPE cells loaded with anti-G(alpha s) activation of the NSC current was unaffected. 4. Investigation of the potential downstream mediators in the G(alpha i) NSC channel pathway revealed that activation of the cation conductance was unaffected by treatment of RPE cells with the selective protein kinase C inhibitor GF 109203X (3 microM) or the selective CaM kinase II inhibitor KN-93 (50 microM). However, NSC current activation was delayed and the current amplitude reduced in the presence of the nonselective kinase inhibitor H-7 (100 microM) or the selective inhibitor of MAPKK (MEK) activation, PD 98059 (50 microM). 5. In the absence of GTPgammaS, the NSC current was not activated by superfusion of the cells with the cyclic GMP kinase activator dibutyryl-cyclic GMP or with the adenylate cyclase activator forskolin. 6. These results support the involvement of a G protein of the G(alpha i) subclass in the activation of a NSC current in rat RPE cells, and suggest a potential modulatory role for MAP kinase-dependent phosphorylation in current regulation.

Neuropeptides are known to modulate the excitability of mammalian sympathetic neurons by their actions on various types of K+ and Ca2+ channels. We used whole cell patch-clamp recording methods to study the actions of substance P (SP) on dissociated adult guinea pig stellate ganglion (SG) neurons. Under current-clamp conditions, SG neurons exhibited overshooting action potentials followed by afterhyperpolarizations (AHP). The K+ channel blocker tetraethylammonium (1 mM), the Ca2+ channel blocker Cd2+ (0.1–0.2 mM), and SP (500 nM) depolarized SG neurons, decreased the AHP amplitude, and increased the action potential duration. In the presence of Cd2+, the effect of SP on membrane potential and AHP was reduced. Under voltage-clamp conditions, several different K+ currents were observed, including a transient outward K+ conductance and a delayed rectifier outward K+ current ( I K) consisting of Ca2+-sensitive [ I K(Ca)] and Ca2+-insensitive components. SP (500 nM) inhibited I K. Pretreatment with Cd2+ (20–200 μM) or the high-voltage-activated Ca2+ channel blocker ω-conotoxin (10 μM) blocked SP’s inhibitory effects on I K. This suggests that SP reduces I K primarily through the inhibition of I K(Ca) and that this may occur, in part, via a reduction of Ca2+ influx through voltage-dependent Ca2+ channels. SP’s actions on I K were mediated by a pertussis toxin-insensitive G protein(s) coupled to NK1 tachykinin receptors. Furthermore, we have confirmed that 500 nM SP reduced an inward Cd2+- and ω-conotoxin-sensitive Ba2+ current in SG neurons. Thus the actions of SP on I K(Ca) may be due in part to a reduction in Ca2+influx occurring via N-type Ca2+channels. This study presents the first description of ionic currents in mammalian SG neurons and demonstrates that SP may modulate excitability in SG neurons via inhibitory actions on K+ and Ca2+ currents.

Free fatty acids (FFAs) are metabolic intermediates that may be obtained through the diet, synthesized endogenously, or produced via fermentation of carbohydrates by gut microbiota. In addition to serving as an important source of energy, FFAs are known to produce a variety of both beneficial and detrimental effects on metabolic and inflammatory processes. While historically, FFAs were believed to produce these effects only through intracellular targets such as peroxisome proliferator-activated receptors, it has now become clear that FFAs are also agonists for several GPCRs, including a family of four receptors now termed FFA1-4. Increasing evidence suggests that FFA1-4 mediate many of the beneficial properties of FFAs and not surprisingly, this has generated significant interest in the potential of these receptors as therapeutic targets for the treatment of a variety of metabolic and inflammatory disorders. In addition to the traditional strategy of developing small-molecule therapeutics targeting these receptors, there has also been some consideration given to alternate therapeutic approaches, specifically by manipulating endogenous FFA concentrations through alteration of either dietary intake, or production by gut microbiota. In this review, the current state of knowledge for FFA1-4 will be discussed, together with their potential as therapeutic targets in the treatment of metabolic and inflammatory disorders. In particular, the evidence in support of small molecule versus dietary and microbiota-based therapeutic approaches will be considered to provide insight into the development of novel multifaceted strategies targeting the FFA receptors for the treatment of metabolic and inflammatory disorders.

Biased agonism describes the ligand-dependent selectivity of different signal transduction pathways by molecules that act as agonists at the same receptor. This functional selectivity may be exploi...

The endocannabinoid system represents a potential therapeutic target in sepsis due to the presence of cannabinoid receptors (CB2) on immune cells. In this review we discuss how various targets within the endocannabinoid system can be manipulated to treat the immune consequences of sepsis. One of the targets outlined are the endocannabinoid receptors and modulation of their activity through pharmacological agonists and antagonists. Another therapeutic target covered in this review is the modulation of the endocannabinoid degradative enzyme’s activity. Modulation of degradative enzyme activity can change the levels of endogenous cannabinoids thereby altering immune activity. Overall, activation of the CB2 receptors causes immunosuppression and can be beneficial during the hyperactivated immune state of sepsis, while suppression of the CB2 receptors may be beneficial during a hypoimmune septic state.

Mutations in genes that code for components of the Norrin-FZD4 ligand-receptor complex cause the inherited childhood blinding disorder familial exudative vitreoretinopathy (FEVR). Statistical evidence from studies of patients at risk for the acquired disease retinopathy of prematurity (ROP) suggest that rare polymorphisms in these same genes increase the risk of developing severe ROP, implying that decreased Norrin-FZD4 activity predisposes patients to more severe ROP. To test this hypothesis, we measured the development and recovery of retinopathy in wild type and Fzd4 heterozygous mice in the absence or presence of ocular ischemic retinopathy (OIR) treatment. Avascular and total retinal vascular areas and patterning were determined, and vessel number and caliber were quantified. In room air, there was a small delay in retinal vascularization in Fzd4 heterozygous mice that resolved as mice reached maturity suggestive of a slight defect in retinal vascular development. Subsequent to OIR treatment there was no difference between wild type and Fzd4 heterozygous mice in the vaso-obliterated area following exposure to high oxygen. Importantly, after return of Fzd4 heterozygous mice to room air subsequent to OIR treatment, there was a substantial delay in retinal revascularization of the avascular area surrounding the optic nerve, as well as delayed vascularization toward the periphery of the retina. Our study demonstrates that a small decrease in Norrin-Fzd4 dependent retinal vascular development lengthens the period during which complications from OIR could occur.