Melanie E. M. Kelly

Ligand- and Heterodimer-Directed Signaling of the CB1 Cannabinoid Receptor

Seven-transmembrane G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors. Signaling through these receptors is controlled by changes in the conformation of the receptor from inactive to active conformations, which in turn lead to the activation of multiple downstream signaling pathways. To facilitate greater diversity in signaling responses, many of these receptors are capable of adopting several distinct active conformations, in which each couples preferentially to its own set of downstream signaling partners. Because these unique signaling responses result from specific receptor active conformations, GPCR signaling may be directed toward these selective responses through either strength-of-signal effects resulting from partial agonism or through biased agonism and functional selectivity, resulting from the selective stabilization of one active conformation over the others. This review uses the CB1 cannabinoid receptor as a specific example to highlight the contribution of two important aspects of GPCR function—orthosteric ligand binding and receptor heterodimerization—toward directed GPCR signaling.

Calcium Channels at the Photoreceptor Synapse

Presynaptic Ca2+ channels mediate early stages of visual information processing in photoreceptors by facilitating the release of neurotransmitter and by receiving modulatory input that alters transmission. Two types of L-type Ca2+ channels, composed of alpha1F and alpha1D subunits and having similar biophysical andpharmacological properties, appear to form the principle voltage-dependent Ca2+ influx pathways in rods and cones, respectively. The role played by these channels in neurotransmitter release at these graded potential, non-spiking synapses, has been well described. The channels mediate sustained glutamate release in darkness where the cells rest at potentials near -40 mV, and signal increases in light intensity as the cells hyperpolarize negative to this value. Synaptic modulation and integration mediated by these channels has not yet been as fully described but appears to involve GABA, nitric oxide (NO), glutamate, and dopamine. Ca2+ permeable cyclic nucleotide gated (CNG) channels appear to have supporting roles at the photoreceptor output synapse and may transduce NO signals from other cells by either directly permitting Ca2+ influx or by providing depolarizing influences that gate voltage dependent Ca2+ channels.

PURINERGIC REGULATION OF CATION CONDUCTANCES AND INTRACELLULAR CA2+ IN CULTURED RAT RETINAL PIGMENT EPITHELIAL CELLS

1 We used whole‐cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5′‐triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 μM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2 The ATP‐activated inward current was a non‐selective cation (NSC) current that showed inward rectification, reversed at −1.5 ± 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3 The outward current activated by ATP reversed at −68 ± 3 mV (equilibrium potential for potassium (EK) =−84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi‐KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+‐activated K+ channel in rat RPE cells. The Ca2+‐activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4 In fluo‐3 or fluo‐4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+‐ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5 These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non‐selective cation conductance and G protein‐coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium‐activated K+ current.

Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells

Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM).

Structural and functional neuroprotection in glaucoma: role of galantamine-mediated activation of muscarinic acetylcholine receptors

Glaucoma is the leading cause of irreversible blindness worldwide. Loss of vision due to glaucoma is caused by the selective death of retinal ganglion cells (RGCs). Treatments for glaucoma, limited to drugs or surgery to lower intraocular pressure (IOP), are insufficient. Therefore, a pressing medical need exists for more effective therapies to prevent vision loss in glaucoma patients. In this in vivo study, we demonstrate that systemic administration of galantamine, an acetylcholinesterase inhibitor, promotes protection of RGC soma and axons in a rat glaucoma model. Functional deficits caused by high IOP, assessed by recording visual evoked potentials from the superior colliculus, were improved by galantamine. These effects were not related to a reduction in IOP because galantamine did not change the pressure in glaucomatous eyes and it promoted neuronal survival after optic nerve axotomy, a pressure-independent model of RGC death. Importantly, we demonstrate that galantamine-induced ganglion cell survival occurred by activation of types M1 and M4 muscarinic acetylcholine receptors, while nicotinic receptors were not involved. These data provide the first evidence of the clinical potential of galantamine as neuroprotectant for glaucoma and other optic neuropathies, and identify muscarinic receptors as potential therapeutic targets for preventing vision loss in these blinding diseases.

Physical and functional interaction between CB1 cannabinoid receptors and β2-adrenoceptors

Background and purpose:  The CB1 cannabinoid receptor and the β2‐adrenoceptor are G protein‐coupled receptors (GPCRs) co‐expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.

Background: To understand the differential response to cannabinoids, we examined the functional selectivity of type 1 cannabinoid receptor (CB1) agonists in a cell model of striatal neurons. Results: 2-Arachidonylglycerol, Δ9-tetrahydrocannabinol, and CP55,940 were arrestin2-selective; endocannabinoids and WIN55,212-2 activated Gαi/o, Gβγ, and Gαq; and cannabidiol activated Gαs independent of CB1. Conclusion: Cannabinoids displayed functional selectivity. Significance: CB1 functional selectivity may be exploited to maximize therapeutic efficacy. 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 Δ9-tetrahydrocannabinol (THC)) on arrestin2-, Gαi/o-, Gβγ-, Gαs-, and Gαq-mediated intracellular signaling in the mouse STHdhQ7/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 Gαi/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 Gαq-dependent pathways. CP55,940 and CBD both signaled through Gαs. CP55,940, but not CBD, activated downstream Gαs 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.

Effects of minocycline and tetracycline on retinal ganglion cell survival after axotomy

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.

Timolol Concentrations in Rat Ocular Tissues and Plasma After Topical and Intraperitoneal Dosing

PurposeTopical &bgr;-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 MethodsOne 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. ResultsRats that received topical timolol daily for 3 to 12 consecutive days accumulated timolol concentrations of 2.3 to 4.4 &mgr;g/g in cornea, 198 to 326 &mgr;g/g in iris, 0.05 to 0.11 &mgr;g/ml in vitreous, and 0.17 to 0.77 &mgr;g/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. ConclusionsAbsorption 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.

A GPR18-based signalling system regulates IOP in murine eye

GPR18 is a recently deorphaned lipid receptor that is activated by the endogenous lipid N‐arachidonoyl glycine (NAGly) as well the behaviourally inactive atypical cannabinoid, abnormal cannabidiol (Abn‐CBD). The presence and/or function of any GPR18‐based ocular signalling system remain essentially unstudied. The objectives of this research are: (i) to determine the disposition of GPR18 receptors and ligands in anterior murine eye, (ii) examine the effect of GPR18 activation on intraocular pressure (IOP) in a murine model, including knockout mice for CB1, CB2 and GPR55.