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Aims Long-term or recreational use of ketamine affects the urinary system and can result in ketamine-induced cystitis (KIC). Rodent models of KIC are important to study KIC pathophysiology and are paramount to the future development of therapies for this painful condition. This review aims to provide a summary of rodent models of KIC, focusing on disease induction, experimental methods, and pathological features of the model. Method A literature search was performed using the National Center for Biotechnology Information (NCBI) Pubmed database up to March 2021. 20 articles met the inclusion criteria and were finally selected. Results There are considerable variations in the rodent models used for studying KIC in terms of the strain of the animal being used; dose, duration, and route of ketamine administration to induce KIC, and assessment of pathological features. Conclusion KIC remains difficult to fully recapitulate in humans. Improved characterization of KIC models and the experimental parameters and meticulous discussion on translational limitations are required to improve the translational value of research using rodent models of KIC.

The cannabinoid type 1 (CB1 ) receptor and the dopamine type 2 (D2 ) receptor are co-localized on medium spiny neuron terminals in the globus pallidus where they modulate neural circuits involved in voluntary movement. Physical interactions between the two receptors have been shown to alter receptor signaling in cell culture. The objectives of the current study were to identify the presence of CB1 /D2 heteromers in the globus pallidus of C57BL/6J male mice, define how CB1 /D2 heteromer levels are altered following treatment with cannabinoids and/or antipsychotics, and determine if fluctuating levels of CB1 /D2 heteromers have functional consequences. Using in situ proximity ligation assays, we observed CB1 /D2 heteromers in the globus pallidus of C57BL/6J mice. The abundance of the heteromers increased following treatment with the nonselective cannabinoid receptor agonist, CP55,940. In contrast, treatment with the typical antipsychotic haloperidol reduced the number of CB1 /D2 heteromers, whereas the atypical antipsychotic olanzapine treatment had no effect. Co-treatment with CP55,940 and haloperidol had similar effects to haloperidol alone, whereas co-treatment with CP55,940 and olanzapine had similar effects to CP55,940. The observed changes were found to have functional consequences as the differential effects of haloperidol and olanzapine also applied to γ-aminobutyric acid release in STHdhQ7/Q7 cells and motor function in C57BL/6J male mice. This work highlights the clinical relevance of co-exposure to cannabinoids and different antipsychotics over acute and prolonged time periods.

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.

We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs β-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.

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

The cannabinoid 1 receptor (CB1R) is one of the most widely expressed metabotropic G protein-coupled receptors in brain, and its participation in various (patho)physiological processes has made CB1R activation a viable therapeutic modality. Adverse psychotropic effects limit the clinical utility of CB1R orthosteric agonists and have promoted the search for CB1R positive allosteric modulators (PAMs) with the promise of improved drug-like pharmacology and enhanced safety over typical CB1R agonists. In this study, we describe the synthesis and in vitro and ex vivo pharmacology of the novel allosteric CB1R modulator GAT211 (racemic) and its resolved enantiomers, GAT228 (R) and GAT229 (S). GAT211 engages CB1R allosteric site(s), enhances the binding of the orthosteric full agonist [3H]CP55,490, and reduces the binding of the orthosteric antagonist/inverse agonist [3H]SR141716A. GAT211 displayed both PAM and agonist activity in HEK293A and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in mouse-bra...

Proliferative vitreoretinopathy (PVR) can develop after ocular trauma or inflammation and is a common complication of surgery to correct retinal detachment. Currently, there are no pharmacological treatments for PVR. Cannabinoids acting at cannabinoid 2 receptor (CB2R) can decrease inflammation and fibrosis. The objective of this study was to examine the anti-inflammatory actions of CB2R as a candidate novel therapeutic target in experimental PVR. PVR was induced by intravitreal injection of dispase in wild-type (WT) and CB2R genetic knockout (CB2R −/− ) mice. Ocular pathology was studied at 24 h or one week after dispase injection. CB2R modulation was examined in WT mice, using the CB2R agonist, HU308, and the CB2R antagonist, AM630. Histopathological scoring and quantification of microglia was used to evaluate tissue pathology. Quantitative PCR and multiplex assays were used to assess changes in proinflammatory cytokines. Intravital microscopy (IVM) was used to visualize and quantify leukocyte-endothelial adhesion to the iridial microcirculation. Activation of CB2R with HU308 in WT mice with PVR decreased mean histopathological scores, the number of microglia, and leukocyte adhesion compared to vehicle-treated animals. Conversely, an increase in histopathological scores and activated microglia was observed in PVR animals after treatment with AM630. CB2R −/− mice with PVR exhibited exacerbated ocular histopathology, increased microglia numbers, and elevated protein levels of cytokines as compared to WT mice. In conclusion, our results indicate that intervention at early stage PVR with CB2R agonists reduces ocular inflammation and disease severity. CB2R may represent a therapeutic target to prevent PVR progression and vision loss. This article is part of the Special Issue entitled ‘Lipid Sensing G Protein-Coupled Receptors in the CNS’.

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.

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.

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

G protein-coupled receptors (GPCRs) interact with multiple intracellular effector proteins such that different ligands may preferentially activate one signal pathway over others, a phenomenon known as signaling bias. Signaling bias can be quantified to optimize drug selection for preclinical research. Here, we describe moderate-throughput methods to quantify signaling bias of known and novel compounds. In the example provided, we describe a method to define cannabinoid-signaling bias in a cell culture model of Huntington's disease (HD). Decreasing type 1 cannabinoid receptor (CB

Uveitis is inflammation of the uvea which consists of the iris, ciliary body and the choroid of the eye. Uveitis can lead to impaired vision and is responsible for 10% of all cases of blindness globally. Using an endotoxin-induced uveitis (EIU) rodent model, our previous data implicated the endogenous cannabinoid system (ECS) in the amelioration of many of the components of the inflammatory response. Here, we test the hypothesis that the reduction in inflammatory mediators in the EIU model by the CB

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.

Cannabinoids are known to modulate cardiovascular functions including heart rate, vascular tone, and blood pressure in humans and animal models. Essential components of the endocannabinoid system, namely, the production, degradation, and signaling pathways of endocannabinoids have been described not only in the central and peripheral nervous system but also in myocardium, vasculature, platelets, and immune cells. The mechanisms of cardiovascular responses to endocannabinoids are often complex and may involve cannabinoid CB

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.

INTRODUCTION Impairment of the intestinal microcirculation in endotoxemia may cause a deterioration of the mucosal barrier function thus releasing intraluminal bacteria and their toxins into the systemic circulation. In clinical sepsis this mechanism may influence disease severity and outcome. The aim of the study was to investigate the impact of cannabinoid receptor 1 (CB1R) modulation within the intestinal microcirculation with regard to leukocyte activation and capillary perfusion, and on intestinal histology in experimental endotoxemia in rats. METHODS Endotoxemia was induced by intravenous lipopolysaccharide (LPS) administration. We studied 5 groups of animals: controls (CON), LPS, LPS + CB1R agonist (ACEA, 2.5 mg/kg), LPS + CB1R antagonist (AM281, 2 mg/kg) and LPS + CB1R agonist (ACEA, 2.5 mg/kg) + CB1R antagonist (AM281, 2 mg/kg). Intestinal intravital microscopy (IVM) was performed two hours following LPS/placebo administration. Intestinal leukocyte adhesion in submucosal venules and functional capillary density (FCD) of the intestinal wall was quantified using IVM. Histological changes were assessed using a standardized injury score. RESULTS After two hours of endotoxemia, we observed a significant increase of leukocyte adhesion in intestinal submucosal venules. Administration of the CB1R antagonist in endotoxemic animals significantly reduced the number of adhering leukocytes (p < 0.05). The CB1R agonist did not further increase leukocyte adhesion. FCD was significantly improved by the CB1R antagonist (p < 0.05). Administration of the CB1R agonist, ACEA, reversed the beneficial effect of the CB1R antagonist, AM281. CONCLUSIONS CB1R inhibition significantly improved intestinal microcirculation by reducing leukocyte adhesion and increasing FCD in acute endotoxemia in rats. The data supports the involvement of the CB1R signaling in leukocyte activation during sepsis. Drugs targeting the CB1R may have therapeutic potential in systemic inflammation, such as sepsis.

The type 1 cannabinoid receptor (CB1) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB1 increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB1 mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB1 expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh(7/7) cells with the cannabinoids ACEA, mAEA, and AEA produced a CB1receptor-dependent increase in CB1 promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB1 expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB1 expression in STHdh(7/111) and (111/111) cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB1 mRNA levels approximately 10-fold in STHdh(7/111) and (111/111) cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB1 levels could reduce the severity of some molecular pathologies observed in HD.

The endocannabinoid, N-arachidonoylethanolamine (AEA), is degraded by the enzyme fatty acid amide hydrolase (FAAH). This study examined whether the FAAH inhibitor, URB597, increases retinal ganglion cell (RGC) survival following optic nerve axotomy in young and aged animals. URB597 alone, or together with either a CB1 or CB2 receptor antagonist, was administered daily for 1 or 2 weeks post-axotomy. Histological assessment of retinas indicated that URB597 increased RGC survival in young retina at 1 and 2 weeks post-axotomy. The increase in RGC survival at 2 weeks was accompanied by a reduction in phagocytic microglia. The CB1 antagonist, AM281, but not the CB2 antagonist, AM630, ablated URB597-mediated RGC neuroprotection. CB1 or CB2 antagonism increased phagocytic microglia in URB597 and vehicle-treated animals. In aged animals, URB597 increased RGC survival at 1 week, but not at 2 weeks post-axotomy and had no effect on microglia. Retinal Iba-1 positive microglia were also decreased in URB597-treated axotomized young animals and this decrease was mitigated by CB1 but not CB2 antagonism. As seen with phagocytotic microglia, the CB2 antagonist, AM630, increased Iba-1 positive microglia in the absence of URB597 treatment. Measurement of retinal endocannabinoid levels in URB597-treated animals at 2 weeks post-axotomy revealed a significant increase in AEA levels, accompanied by a decrease in the AEA metabolite, N-arachidonoyl glycine, in young animals but not aged animals. 2-arachidonoylglycerol levels were similar across all experimental groups. These data demonstrate that URB597-mediated retinal neuroprotective effects are mediated primarily through CB1 receptors and that URB597 neuroprotective efficacy declines with age.

Leukocyte-endothelial interactions within the microvasculature represent a hallmark of inflammation regardless of whether the inflammation results from non-infectious or infectious triggers. In this review, we highlight features of leukocyte recruitment in ocular disease and postulate mechanisms by which the infiltrating cells may lead to the progression of the ocular inflammatory response, including cytokine and chemokine production, T cell or non-T cell responses. Additionally, ex-vivo and in vivo methods used to study the general features of the immune response are discussed, with a specific focus on intravital imaging, which allows real-time non-invasive examination of leukocyte-endothelial interactions in the ocular microvasculature. At the present time there are still significant gaps in our understanding of the process of leukocyte recruitment in vivo in different microvascular beds. Further studies using non-invasive imaging approaches, such as intravital microscopy, provide an opportunity to study dynamic tissue-specific leukocyte-endothelial interactions in vivo and identify novel targets for early intervention in the inflammatory process. This knowledge is essential to the rational use of therapeutics to resolve inflammation in ocular disease.

Endothelin-1 (ET-1) is a vasoconstrictor implicated in age-related retinal pathologies. This study determined whether responses to ET-1 differed in retinal arterioles isolated from adult (2–3 months) and aged (>20 months) Fischer 344 rats of both sexes. Risk factors for retinal disease (retinal perfusion pressure, intraocular pressure, blood glucose) were not affected by age. However, sensitivity to ET-1 declined with age, especially in females. Vasoconstrictor responses to 50 mM KCl and Ca2+ release by caffeine (10 mM) were similar in all groups. Retinal ETA and ETB receptor expression also was similar in young and aged rats, regardless of sex. Contractions elicited by 10 nM ET-1 were inhibited by the ETA antagonist BQ-123 (1 μM) in all groups. In contrast, the ETB antagonist BQ-788 (1 μM) restored ET-1-induced contractions in aged female vessels, but had no effect in any other group. Removal of the endothelium also restored contractions in vessels from aged females but not males. Thus, responsiveness to ET-1 declines with age in retinal microvasculature. In males, this is likely mediated by age-related changes in the ETA receptor signaling pathway. By contrast, effects of ET-1 on endothelial ETB receptors attenuate vasoconstrictor responses in aged females.

The objective of our study was to examine the pharmacology of the intraocular pressure (IOP)-lowering actions of the behaviorally inactive cannabinoids, abnormal cannabidiol (abn-CBD), and a cannabigerol analog, cannabigerol-dimethyl heptyl (CBG-DMH), in comparison to that of the nonselective cannabinoid 1 receptor (CB(1)R) and CB(2)R agonist, WIN55,212-2, in Brown Norway rats. The IOP was measured noninvasively using a hand-held tonometer in nonanesthetized animals. The IOP measurements were taken every 15 min for a period of 2 h after drug administration. All drugs were administered via intraperitoneal (i.p.) injections, and abn-CBD and CBG-DMH were also given topically. Both abn-CBD and CBG-DMH reduced IOP when administrated i.p. at doses of ≥2.5 mg/kg or topically at concentrations of 1%-2%. The IOP-lowering effects of abn-CBD and CBG-DMH were reduced by i.p. administration of O-1918 (2.5 mg/kg), a selective antagonist of the abn-CBD-sensitive cannabinoid-related receptor (CBx), but were unaffected by the CB(1)R antagonist, AM251 (2.5 mg/kg), or the CB(2)R antagonist, AM630 (2.5 mg/kg). In contrast, the IOP-lowering action of WIN55,212-2 was completely blocked by the CB(1)R-selective antagonist, AM251, and was unaffected by the CBx receptor antagonist, O-1918. However, similar to the nonpsychotropic cannabinoids, the ocular hypotensive actions of WIN55,212-2 were also insensitive to block by the CB(2)R antagonist, AM630. Consistent with this, the selective CB(2)R agonist, HU-308 (2 mg/kg) failed to reduce IOP in Brown Norway rats. Concurrent application of a dose of WIN55,212-2 that was subthreshold to reduce IOP (0.25 mg/kg), together with a topical dose of either abn-CBD (0.5%) or CBG-DMH (0.25%), respectively, potentiated the ocular hypotensive effect of either compound applied alone. This study demonstrates that the atypical cannabinoid, abn-CBD, and the cannabigerol analog, CBG-DMH, decrease IOP in the normotensive Brown Norway rat eye independent of CB(1)R or CB(2)R activation, via activation of CBx receptors. The enhanced decrease in IOP seen after coapplication of the CB(1)R agonist, WIN55,212-2, together with either abn-CBD or CBG-DMH, respectively, further suggests that the ocular pharmacodynamics of abn-CBD and CBG-DMH are mediated by receptor targets distinct from CB(1)R. These results indicate that both CBG-DMH and abn-CBD have the potential for further investigation as novel ocular hypotensive cannabinoids devoid of CB(1)R-mediated side-effects.

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 (CB 1 ) 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 CB 2 (−/−) mice. In contrast, none of these compounds were found to reduce IOP in βAR(−/−) or CB 1 (−/−) 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, CB 1 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.

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.

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.

The observation that marijuana reduces intraocular pressure was made by Hepler and Frank in the 1970s. Since then, there has been a significant body of work investigating cannabinoids for their potential use as therapeutics. To date, no endocannabinoid system (ECS)-modulating drug has been approved for clinical use in the eye; however, recent advances in our understanding of the ECS, as well as new pharmacological tools, has renewed interest in the development of ocular ECS-based therapeutics. This review summarizes the current state-of-affairs for the use of ECS-modulating drugs for the treatment of glaucoma and ocular inflammatory and ischemic disease.

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.

In the present study, we compared the in vivo neuroprotective efficacy of intraperitoneally administered tetracycline and minocycline to enhance the survival of retinal ganglion cells (RGCs) following unilateral axotomy of the adult rat optic nerve. We also examined the effects of the tetracycline drugs on the activation of retinal microglia. RGCs in retinal whole-mounts were visualized by retrograde labeling with fluorogold. The presence of activated microglia was confirmed immunohistochemically using OX-42 monoclonal antibodies. Optic nerve axotomy produced RGC death and increased activation of microglia. No significant RGC loss was seen prior to 5 days and approximately 50% and 80-90% cell loss occurred at 7 and 14 days, respectively. Examination of the effects of tetracycline and minocycline on RGC survival at 7 days post-axotomy, revealed increased numbers of RGCs in minocycline-treated animals (75% of non-axotomized control) compared with vehicle-only (52% of control) and tetracycline-treated (58% of control) animals. The densities of RGCs (RGCs/mm2+/-S.D.) for control, vehicle-, tetracycline- and minocycline-treated axotomized animals were 1996+/-81, 1029+/-186, 1158+/-190 and 1497+/-312, respectively. The neuroprotective effect of minocycline seen at 7 days was transient, since RGCs present in minocycline-treated animals at 14 days post-axotomy (281+/-43, 14% of control) were not significantly different to vehicle-treated animals (225+/-47, 11% of control). OX-42 staining of activated retinal microglia was reduced in tetracycline- and minocycline-treated axotomized animals compared with axotomized animals receiving vehicle-only. These results demonstrate that systemic administration of the second-generation tetracycline derivative, minocycline, delays the death of axotomized RGCs by a mechanism that may be associated with inhibition of microglia activation. The neuroprotective efficacy of minocycline following optic nerve axotomy was superior to that of tetracycline.

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl– conductance (ICl,swell), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the ICl,swell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl– current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as ICl,swell. Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of ICl,swell. Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with ICl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to ICl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.Key words: ClC-3, NPE, cell swelling, membrane trafficking, ciliary body epithelium.

The purpose of this study was to determine the efficacy and putative mechanisms of action of tetracycline and minocycline in inhibiting retinal cell apoptosis after glutamate-induced excitotoxicity and trophic factor deprivation in a retinal cell line (E1A-NR.3) and in primary mixed retinal cell cultures. In addition, a differentiated PC-12 cell line was used to determine whether minocycline was neuroprotective after trophic withdrawal in a pure neuronal cell line devoid of glia. Results from this study demonstrated that minocycline, but not tetracycline, is protective in in vitro models of excitotoxicity-induced retinal cell apoptosis. Moreover, the protective effects provided by minocycline in retinal cells seemed independent of actions on N -methyl-d-aspartate receptors (NMDARs) and glutamate receptor-mediated Ca 2+ influx. Doses of the NMDAR antagonist MK-801 (dizocilpine) and minocycline that alone provided no significant neuroprotection resulted in enhanced retinal cell survival when applied concurrently, suggestive of distinct signaling pathways, and minocycline was without effect on glutamate-induced Ca 2+ influx, as assessed by calcium imaging. Minocycline was also neuroprotective after trophic factor withdrawal, producing a decrease in apoptosis and caspase-3 activation in both retinal cells and the PC-12 neuronal-like cell line. These results support a role for minocycline as a retinal neuroprotectant and demonstrate that the antiapoptotic actions of minocycline in retinal cells do not arise from the blockage of NMDARs or glutamate receptor-mediated Ca 2+ influx but do involve inhibition of caspase-3 activation. In addition, the survival-promoting actions of minocycline may arise via actions on both neuronal and non-neuronal cell targets.

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.

The benzothiazepine, diltiazem, is commonly used as an inhibitor of vascular L-type Ca channels, and is a clinically important anti-anginal and antihypertensive medication. In the retina, diltiazem also inhibits cyclic-nucleotide gated (CNG) channels, including the cGMP-gated channels in photoreceptors, and has been suggested to be a neuroprotectant in an animal model of retinitis pigmentosa, a degenerative disease of photoreceptors. In contrast to CNG channels, the actions of diltiazem on photoreceptor Ca channels have not been studied. We show that D-cis-diltiazem can block Ca channels in cone photoreceptors and that the potency and efficacy of cone photoreceptor Ca channel inhibition by this drug is unconventional. Over the concentration range of 5-500 microM diltiazem, the dose response curve was biphasic with a high affinity saturation level of approximately 30% block in the 20-50 microM range (IC(50)=4.9 microM) and a low affinity saturation block (near 100%) with concentrations up to 500 microM (IC(50)=100.4 microM). The degree of block was found to be equivalent when Bay K 8644 was used to increase Ca channel current, indicating that the levels of block do not result from multiple Ca channel subtypes having differing sensitivities to diltiazem. Calcium imaging showed that the relatively low efficacy of the high-affinity Ca channel block was not due to the species of charge-carrying divalent cation nor that it was associated with dialysis of cellular contents. These data contribute to an emerging perspective that the photoreceptor Ca channel has properties unique from other L-type channels, an important consideration should these channels become a target for testing putative neuroprotective therapies.

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.

Purpose Topical beta-blockers, such as timolol, have been used extensively in the medical treatment of glaucoma to lower intraocular pressure (IOP). Recently, these drugs have been shown to have effects on the retinal and optic nerve circulation as well as potential neuroprotective properties. In the current study, the concentration of timolol attained in the cornea, iris-ciliary body, retina, vitreous, and plasma was measured after topical or intraperitoneal administration in rats to determine the relative contributions of each route to intraocular timolol concentrations. Materials and methods One group of rats received one drop of commercially available 0.5% timolol in the right eye and two drops in the left eye for 3 to 12 days. Another group of rats received one drop of 0.5% timolol in one eye only and concentrations were studied in the ocular tissues at 15, 30, 60, 120, and 240 minutes after instillation. The final group of rats received a single intraperitoneal injection of timolol ranging in concentration from 5 to 75 mg/kg after which tissue and plasma concentrations were measured 30 minutes after injection. All tissue and plasma concentrations were measured by high performance liquid chromatography. Results Rats that received topical timolol daily for 3 to 12 consecutive days accumulated timolol concentrations of 2.3 to 4.4 microg/g in cornea, 198 to 326 microg/g in iris, 0.05 to 0.11 microg/ml in vitreous, and 0.17 to 0.77 microg/g in retina. In rats that received a single drop of timolol in one eye, the tissue concentrations were higher in the treated eye than in the untreated eye in all cases except for vitreous. In these experiments, timolol levels in plasma were either low or not detectable. Increasing timolol doses administered intraperitoneally resulted in corresponding increased tissue and plasma concentrations. Conclusions Absorption of drug into the systemic circulation plays a significant role in delivering timolol to the retina and vitreous in addition to a local ocular route. A clear dose-response relationship exists in all ocular tissues studied after an intraperitoneal dose of timolol. High doses of timolol were required to achieve measurable concentrations of drug in the ocular tissues via our high performance liquid chromatography assay suggesting that a significant hepatic first-pass effect may be involved after an intraperitoneal injection of timolol.

Betaxolol, a beta 1-selective adrenoceptor antagonist, is widely used in the treatment of glaucoma. In addition to its ocular hypotensive effects, betaxolol has been suggested to act as a retinal neuroprotective agent (Osborne et al., 1997). To investigate possible mechanisms underlying the neuroprotective effects, we tested the actions of betaxolol on ion channels and calcium signaling in isolated retinal ganglion cells. Betaxolol (50 microM) reduced by about 20% the high-voltage-activated (HVA) Ca channel currents in ganglion cells isolated from tiger salamander retina. In contrast, the beta 1-adrenoceptor antagonists propranolol (10 microM) and timolol (50 microM) had no inhibitory actions on HVA Ca channel currents. The L-type Ca channel antagonist, nisoldipine, blocked the HVA Ca channel current partially and the remaining current was not inhibited by betaxolol. Outward current was inhibited in the presence of betaxolol. Both iberiotoxin (IBTX; 10 nM), a selective inhibitor of large-conductance Ca-activated K channels, and Cd2+ (100 microM), which suppresses Ca-activated K channels subsequent to its block of Ca channels, reduced outward current and the remaining current was not blocked significantly with betaxolol. In the presence of betaxolol, Na channel currents were reduced by about 20%, as were currents evoked by glutamate (10 mM) and GABA (1 mM). Current clamp recordings from isolated ganglion cells showed that betaxolol had several effects on excitability: spike height decreased, repetitive spike activity was suppressed, spike width increased and hyperpolarization following spikes was reduced. Calcium imaging in isolated rat retinal ganglion cells revealed that betaxolol inhibited glutamate-induced increases in [Ca2+]i. These results suggest that betaxolol has a diversity of suppressive actions on ganglion cell ion channels and that, as a consequence, one of the net actions of the drug is to reduce Ca2+ influx. The subsequent reduction in [Ca2+]i may contribute to the apparent neuroprotective actions of betaxolol in promoting ganglion cell survival following ischemic insult, as may occur in glaucoma and retinal disease.

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.

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.

Transcriptional dysregulation is a major pathological feature of Huntington's disease (HD). The goal of this study was to understand how p65/RelA co-regulated genes, specifically those of the cytokine and endocannabinoid systems, were affected in HD. p65/RelA levels were lower in human HD tissue and R6/2 HD mice, as were the levels of the type 1 cannabinoid receptor (CB1), IL-1β, IL-8, CCL5, GM-CSF, MIP-1β, and TNFα, all of which may be regulated by p65/RelA. Activation of p65/RelA restored CB1 and CCL5 expression in STHdh cell models of HD. Therefore, p65/RelA activation may normalize the expression of some genes in HD.

The endocannabinoid system plays a role in regulation of vasoactivity in the peripheral vasculature; however, little is known about its role in regulation of the CNS microvasculature. This study investigated the pharmacology of cannabinoids and cannabimimetic lipids in the retinal microvasculature, a CNS vascular bed that is autoregulated. Vessel diameter (edge detector) and calcium transients (fura-2) were recorded from segments of retinal microvasculature isolated from adult, male Fischer 344 rats. Results showed that abnormal cannabidiol (Abn-CBD), an agonist at the putative endothelial cannabinoid receptor, CBe, inhibited endothelin 1 (ET-1) induced vasoconstriction in retinal arterioles. These actions of Abn-CBD were independent of CB1/CB2 receptors and were not mediated by agonists for GPR55 or affected by nitric oxide synthase (NOS) inhibition. However, the vasorelaxant effects of Abn-CBD were abolished when the endothelium was removed and were inhibited by the small Ca(2+)-sensitive K channel (SKCa) blocker, apamin. The effects of the endogenous endocannabinoid metabolite, N-arachidonyl glycine (NAGly), a putative agonist for GPR18, were virtually identical to those of Abn-CBD. GPR18 mRNA and protein were present in the retina, and immunohistochemistry demonstrated that GPR18 was localized to the endothelium of retinal vessels. These findings demonstrate that Abn-CBD and NAGly inhibit ET-1 induced vasoconstriction in retinal arterioles by an endothelium-dependent signaling mechanism that involves SKCa channels. The endothelial localization of GPR18 suggests that GPR18 could contribute to cannabinoid and lipid-mediated retinal vasoactivity.

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.

The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system.

Background Changes in leukocyte-endothelial and microvascular perfusion are hallmark events in inflammation. Thus, protection of the intestinal microcirculation represents a pivotal therapeutic target in systemic inflammation and sepsis. The endocannabinoid system (ECS) modulates a number of critical homeostatic functions and has been associated with anti-inflammatory responses. Our study aimed to examine intestinal leukocyte adhesion and capillary perfusion following selective inhibition of the endocannabinoid degradation enzyme, fatty acid amide hydrolase (FAAH), in experimental sepsis (endotoxemia). Methods Five groups of rats were used: controls, endotoxemia [lipopolysaccharide (LPS)], FAAH inhibitor URB597 (0.3 mg/kg)+LPS, URB597 (0.6 mg/kg)+LPS, and URB597 (0.6 mg/kg)+cannabinoid 2 receptor (CB2R) antagonist (AM630)+LPS. After 2 h, intravital microscopy was performed to quantify intestinal leukocyte recruitment and functional capillary density (FCD), as well as macrohemodynamic monitoring and histological examinations. Results LPS induced a significant increase in leukocyte adhesion in collecting and postcapillary submucosal venules and a decrease in intestinal FCD. URB597 pretreatment prevented the LPS-induced increase in leukocyte adhesion in intestinal venules and a decrease in intestinal FCD. The administration of the CB2R inhibitor, AM630, with URB597 reversed the protective effects of URB597 on the LPS-induced increase in leukocyte adhesion in intestinal venules, but not URB597's effect on the intestinal FCD. Conclusions FAAH inhibition prevents the LPS-induced increase in leukocyte adhesion and improves the capillary perfusion of the gut. This might be mediated in part by CB2R activation. Our study encourages further investigation into the therapeutic potential of drugs targeting the ECS in sepsis.

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.

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was −50 ± 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+ inward current and both sustained and transient outward K+ currents. Sustained outward K+ current was reduced (55–77%) by 5 mM tetraethylammonium and to a lesser extent (42–46%) by superfusion with nominally Ca2+ free external solution. Transient outward current was blocked by 100 μm 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to −50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+ and K+ currents giving rise to action potentials.Key words: fluorescent tracer, patch clamp, neuron, ciliary ganglion.

The main function of antibiotics is related to their capacity to eliminate a microorganism. In addition to the anti-microbial function of antibiotics, they are known to have anti-inflammatory and vasomodulatory effects on the microcirculation.The ability of non-antimicrobial derivatives of antibiotics to control inflammation illustrates the distinct anti-microbial and anti-inflammatory roles of antibiotics. In this review, we discuss the impact of antibiotics on leukocyte recruitment and the state ofthe microcirculation. Literature reporting the effect of antibiotics in non-infectious inflammatory conditions is reviewed as wellas the studies demonstrating the anti-inflammatory effects of antibiotics in animal models of infection. In addition, the effect ofthe antibiotics on the immune system is summarized in this review, in order to postulate some mechanisms of action for the pro-and anti-inflammatory contribution of antibiotics. Literature reported the effect of antibiotics on the production of cytokines,chemotaxis and recruitment of leukocytes, production of reactive oxygen species, process of phagocytosis and autophagy, andapoptosis of leukocytes. Yet, all antibiotics may not necessarily exert an anti-inflammatory effect on the microcirculation. Thus,we suggest a model for spectrum of anti-inflammatory and vasomodulatory effects of antibiotics in the microcirculation ofanimals in local and systemic inflammation. Although the literature suggests the ability of antibiotics to modulate leukocyterecruitment and microperfusion, the process and the mechanism of action are not fully characterized. Studying this process willexpand the knowledge base that is required for the selection of antibiotic treatment based on its anti-inflammatory functions,which might be particularly important for critically ill patients.Keywords: Antibiotics, microcirculation, leukocyte recruitment, inflammation, immune system