Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells.

BACKGROUND AND PURPOSE: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM). EXPERIMENTAL APPROACH: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis. KEY RESULTS: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 microM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Galpha(q/11) subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations>or=15 microM, and PTX-sensitive increases in ERK1/2 phosphorylation. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.

Ca(v)3.1 splice variant expression during neuronal differentiation of Y-79 retinoblastoma cells.

A decrease in transient-type calcium channel current, Ca(v)3.1 protein and the mRNA encoding these channels has been reported during differentiation of human retinoblastoma cells. In this study, we examined splice variants of Ca(v)3.1 before and after neuronal differentiation of the Y-79 retinoblastoma cell line to investigate the potential contribution of Ca(v)3.1 to Y-79 differentiation. In Ca(v)3.1, alternative splicing induces variations in three cytoplasmic regions, e.g. the link between domains II and III (producing isoforms e+ and e-), the link between domains III and IV (producing isoforms a, b, ac and bc) and the carboxy terminal region (producing isoforms f and d). Our results demonstrate that Ca(v)3.1e was not expressed in either undifferentiated or differentiated retinoblastoma cells. Splice variants Ca(v)3.1ac; Ca(v)3.1bc and Ca(v)3.1b were all identified in undifferentiated retinoblastoma cells, while expression of these variants in differentiated cells was restricted to the Ca(v)3.1bc isoform. The carboxy terminal variant Ca(v)3.1f is expressed independently of the differentiation status of retinoblastoma cells with or without Ca(v)3.1d. Examination of the functional contribution of Ca(v)3.1 protein to Y-79 cell differentiation revealed that in Y-79 cells transfected with Ca(v)3.1 antisense oligodeoxynucleotides, knockdown of Ca(v)3.1 did not alter the time-course of differentiation or neuritogenesis. The changes in Ca(v)3.1 splice variants were not required for the initiation of differentiation but may be associated with tissue-specific expression or localized alterations in Ca(2+) signaling that are essential for establishment of the mature differentiated phenotype.

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.

Mammalian retinal pigment epithelial cells in vitro respond to the neurokines ciliary neurotrophic factor and leukemia inhibitory factor.

We examined whether primary cultures of rat retinal pigment epithelial (RPE) cells and RPE cells of an immortalized rat cell line, BPEI-1, would be responsive to the neurokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), which are known to be potent trophic factors for neuronal cells. Primary RPE cell cultures were characterized by indirect immunofluorescence and exhibited positive immunoreactivity for RET-PE2, a monoclonal antibody that recognizes RPE cells, and for the intermediate filaments cytokeratin and vimentin. The survival of cultured RPE cells in serum-free defined medium in the presence of CNTF or LIF was investigated during a 0- to 5-day period. Both CNTF and LIF, at concentrations of 1-50 ng/mL (4-200 pM), markedly enhanced RPE cell survival. Bromodeoxyuridine labelling of RPE cells revealed an increased mitotic activity in cell cultures treated with either CNTF or LIF in comparison to untreated serum-free cultures. Increases in cell survival and proliferation after neurokine treatment were also observed with the BPEI-1 cell line. However, in comparison to the primary RPE cultures, LIF was more effective than CNTF in promoting survival of the cell line over a 5-day treatment period. These studies demonstrate that the neurokines CNTF and LIF are potent trophic factors for mammalian RPE cells in vitro and may serve as candidate therapeutic agents in degenerative conditions that affect the retina and RPE.

Beta-oxidation of [1-14C]palmitic acid by mouse astrocytes in primary culture: effects of agents implicated in the encephalopathy of Reye's syndrome.

beta-Oxidation of [1-14C]palmitic acid was examined in homogenates of astrocytes cultured from neonatal mouse brain. Under optimal reaction conditions (< or = 50 micrograms protein, 10 min at 37 degrees C), oxidation increased as a function of palmitate concentration (15 microM to 2 mM) and reached a maximum rate of 1.98 +/- 0.29 nmol/min/mg protein (mean +/- SEM) at 0.2 mM substrate. Eadie-Hofstee analysis of data from four experiments yielded apparent values for Vmax of 1.87 nmol/min/mg protein, and for Km, 35-40 microM. There were no dramatic changes in the oxidation rate in cells between 10 and 36 days in culture. During the 10-min assays, less than 0.05% of the radioactivity was converted to 14CO2 by the astrocytes; water-soluble products accounted for 1-2% of the total substrate added. Studies with KCN indicated that 60-70% of the total activity occurred in the mitochondria. We have been studying the structural and functional changes associated with the cerebral encephalopathy of Reye's syndrome (RS). Three-week-old astrocytes exposed to serum from RS children for the final 7 days of culture exhibited minor mitochondrial pleomorphism and had increased numbers of other intracellular organelles. Examination of the effects of agents implicated in RS indicated that oxidation of [1-14C]palmitate was not altered by Na+ salicylate (1-3 mM), but was inhibited by the industrial surfactant, Toximul MP-8 (> or = 10 micrograms/ml), 4-pentenoic acid (> or = 0.1 microM), or with 4 days' exposure to ammonia (10 nM). The latter treatment also resulted in an increase in protein synthesis, cell volume, and malondialdehyde formation. These results suggest that some of the "toxins" implicated in RS inhibit fatty-acid oxidation in the astrocytes and produce other lipid-related abnormalities that could be related to encephalopathy.