Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing

CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs -coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1 hour after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.post-print1,81 M

CB1 Receptor Negative Allosteric Modulators as a Potential Tool to Reverse Cannabinoid Toxicity

Peer reviewe

Revisiting cannabinoid receptor 2 expression and function in murine retina.

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-print8285 K

Evidence that cannabinoid CB1 receptors regulate intraocular pressure via two opposing mechanisms

The cannabinoid signaling system regulates intraocular pressure (IOP) in the mouse via a complex system that includes three receptors: CB1, GPR18 and GPR119. In each case, activating the receptor lowers IOP, but CB1 receptors are found both at sites of aqueous humor inflow and outflow. As such, knockout mice for any of these receptors would be expected to have higher-than average, or at least unchanged, intraocular pressure. The current study investigates the unexpected observation that CB1 knockout mice have lower pressure than wild type counterparts by testing various regulators of cannabinoid signaling in murine models of IOP. We now report that a CB1 antagonist has differential effects on IOP: SR141716 raises IOP in standard light cycle (SLC) but lowers IOP in reverse light cycle (RLC). This is mimicked by ABD1085, a negative allosteric modulator of CB1. CB1 inhibitors lower IOP in both normotensive and hypertensive mouse eyes. The pressure-lowering effect is absent in CB1 knockout mice. IOP rebounds after the end of treatment but shows no sign of desensitization with daily treatment for a week. Unlike the positive cannabinoid effect, antagonist effects are not sex-dependent. We propose that there are two mechanisms of action for CB1, one that lowers IOP upon activation and a second with inverse sign that lowers IOP when CB1 is antagonized. The relatively lower pressure in CB1 knockout mouse eyes suggests that this second negative regulation of IOP is dominant

Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

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

An Evaluation of Understudied Phytocannabinoids and Their Effects in Two Neuronal Models

Cannabis contains more than 100 phytocannabinoids. Most of these remain poorly characterized, particularly in neurons. We tested a panel of five phytocannabinoids—cannabichromene (CBC), cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), and Δ9-tetrahydrocannabivarin (THCV) in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons expressed a form of CB1-dependent retrograde plasticity while DRGs expressed a variety of transient receptor potential (TRP) channels. CBC, CBDA, and CBDVA had little or no effect on neuronal cannabinoid signaling. CBDV and THCV differentially inhibited cannabinoid signaling. THCV inhibited CB1 receptors presynaptically while CBDV acted post-synaptically, perhaps by inhibiting 2-AG production. None of the compounds elicited a consistent DRG response. In summary, we find that two of five ‘minor’ phytocannabinoids tested antagonized CB1-based signaling in a neuronal model, but with very different mechanisms. Our findings highlight the diversity of potential actions of phytocannabinoids and the importance of fully evaluating these compounds in neuronal models

Requiem for Rimonabant: Therapeutic Potential for Cannabinoid CB₁ Receptor Antagonists after the Fall

The endocannabinoid system is found throughout the CNS and the body where it impacts many important physiological processes. Expectations were high that targeting cannabinoid receptors would prove therapeutically beneficial; pharmaceutical companies quickly seized on the appetitive and metabolic effects of cannabinoids to develop a drug for the treatment of weight loss. Alas, the experience with first-in-class cannabinoid type-1 receptor (CB1R) antagonist rimonabant is a now-classic cautionary tale of the perils of drug development and the outcome of rimonabant’s fall from grace dealt a blow to those pursuing therapies involving CB1R antagonists. And this most commercially compelling application of rimonabant has now been partially eclipsed by drugs with different mechanisms of action and greater effect. Still, blocking CB1 receptors causes intriguing metabolic effects, some of which appear to occur outside the CNS. Moreover, recent years have seen a startling change in the legal status of cannabis, accompanied by a popular embrace of ‘all things cannabis’. These changes combined with new pharmacological strategies and diligent medicinal chemistry may yet see the field to some measure of fulfillment of its early promise. Here, we review the story of rimonabant and some of the therapeutic niches and strategies that still hold promise after the fall

Cannabinoid CB1 Receptor-Dependent Long-Term Depression in Autaptic Excitatory Neurons

Long-term depression (LTD) of synaptic signaling—lasting from tens of minutes to hours or longer—is a widespread form of synaptic plasticity in the brain. Neurons express diverse forms of LTD, including autaptic LTD (autLTD) observed in cultured hippocampal neurons, the mechanism of which remains unknown. We have recently reported that autaptic neurons express both endocannabinoid-mediated depolarization-induced suppression of excitation (DSE) and metabotropic suppression of excitation (MSE). We now report that activating cannabinoid CB1 receptors is necessary for the induction of autLTD. Most surprisingly, CB1 does not induce autLTD via the Gi/o proteins typically activated by this receptor nor with Gs. Rather, the requirements of presynaptic phospholipase C and filled calcium stores suggest Gq. In autLTD, a 3- to 4-min activation of the receptor by the endocannabinoid 2-arachidonoyl glycerol leads to prolonged inhibition while leaving short-term inhibition (e.g., DSE) intact. autLTD requires activation of both metabo- and ionotropic glutamate receptors. autLTD also requires MEK/ERK activation. Under certain conditions, one or more DSE stimuli will elicit autLTD. It is becoming evident that cannabinoids mediate multiple forms of plasticity at a single synapse, stretching temporally from tens of seconds (DSE/MSE) to tens of minutes (autLTD) to hours (CB1 desensitization). Our findings imply a remarkable flexibility for the cannabinoid signaling system whereby discrete mechanisms of CB1 activation within a single neuron yield temporally and mechanistically distinct forms of plasticity

Fmr1 deletion enhances and ultimately desensitizes CB1 signaling in autaptic hippocampal neurons

Fragile X Syndrome (FXS) is a heritable form of mental retardation caused by a non-coding trinucleotide expansion of the FMR1 gene leading to loss of expression of this RNA binding protein. Mutations in this gene are strongly linked to enhanced Group I metabotropic glutamate receptor (mGluR) signaling. A recent report found that mGluR5-dependent endogenous cannabinoid signaling is enhanced in hippocampal slices from fmr1 knockout mice, suggesting a link between FXS and cannabinoid signaling. Alterations in cannabinoid signaling have an impact on learning and memory and may therefore be linked to some aspects of the FXS phenotype.We have used autaptic hippocampal neurons cultured from fmr1 knockout mice to further explore the interaction between endocannabinoid signaling and FMRP. These neurons express several robust forms of retrograde endocannabinoid signaling including depolarization induced suppression of excitation (DSE) and a metabotropic form (MSE) that results from Group I mGluR activation.We now report that young fmr1 neurons exhibit considerably enhanced DSE, likely via increased production of 2-AG, rather than enhanced mGluR-MSE. We find that depolarizations as brief as 50ms, which do not ordinarily produce DSE, routinely inhibited glutamate release. Furthermore, as neuronal cultures mature, CB1-receptor signaling strongly desensitizes. Our results suggest that loss of FMRP broadly affects the endocannabinoid signaling system, possibly through local 2-AG over production. Furthermore, the net effect of the loss of FMRP may actually be diminished cannabinoid signaling due to receptor desensitization as an adaptation to 2-AG overproduction.close