Christine A. B. Jollimore

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).

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.

Regulation of α1G T-type calcium channel gene (CACNA1G) expression during neuronal differentiation

Down‐regulation of T‐type Ca channel current and mRNA occurs following differentiation of Y79 retinoblastoma cells. To understand how the decrease in expression is linked to cell differentiation, we examined transcriptional regulation of the Cav3.1 Ca channel gene, CACNA1G. We identified two putative promoters (A and B) in 1.3 kb of cloned genomic DNA. Reverse transcriptase‐polymerase chain reaction and 5′ rapid amplification of cDNA ends‐polymerase chain reaction analyses demonstrated that two transcripts with different 5′ untranslated regions are generated by different transcription start sites, with promoter A favoured in undifferentiated cells and promoter B favoured in differentiated cells. Functional analyses of the promoter sequence revealed that both promoters are active. Enhancer and repressor sequences were identified upstream of promoter A and B, respectively. These results suggest that the down‐regulation of α1G mRNA in differentiated Y79 cells is mediated primarily by decreased activity of promoter A, which could occur in conjunction with repression of the activity of promoter B. The decrease in T‐type Ca channel expression in Y79 cells may be an essential signal affecting phenotypic maturation and expression of other ion channel subtypes in the differentiated cells.

Metabotropic Receptor-Activated Calcium Increases and Store-Operated Calcium Influx in Mouse Muller Cells

PURPOSE Metabotropic receptor agonists that signal through G(q)-coupled pathways increase Ca(2+) in mammalian Müller cells by release from intracellular stores and Ca(2+) influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non-voltage-dependent Ca(2+) channels in metabotropic muscarinic receptor-activated Ca(2+) increases and store-operated Ca(2+) influx in cultured mouse Müller cells. METHODS Intracellular Ca(2+) was measured using fluorescence imaging with the ratiometric dye fura-2. Currents were recorded using the whole-cell patch-clamp recording method: mRNA and protein were identified using reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical approaches. RESULTS The muscarinic receptor agonist carbachol (3-20 microM) produced increases in Ca(2+) that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca(2+) stores by the sarcoplasmic/endoplasmic Ca(2+) ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca(2+). Carbachol-activated Ca(2+) increases in Müller cells were enhanced by the diacylglycerol derivative 1-oleyl-2-acetyl-sn-glycerol and were blocked by transient receptor potential (TRP) channel blockers Gd(3+), La(3+), 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca(2+) stores and produced Ca(2+) entry that was attenuated by La(3+), 2-APB, Gd(3+), and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd(3+)-sensitive, TRP-like cation channel. CONCLUSIONS Metabotropic muscarinic receptor-activated Ca(2+) increases in mouse Müller cells require the release of Ca(2+) from intracellular stores and the activation of Ca(2+) entry that involves TRP-like cation channels but is independent of voltage-dependent Ca(2+) channels.

A3 adenosine and CB1 receptors activate a PKC-sensitive Cl− current in human nonpigmented ciliary epithelial cells via a Gβγ-coupled MAPK signaling pathway

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

High‐voltage‐activated calcium channels in Müller cells acutely isolated from tiger salamander retina

Müller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole‐cell patch clamp recording, we describe a high‐voltage‐activated, slowly inactivating Ca channel current in isolated salamander Müller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of ∼−8 mV and an inactivation midpoint of ∼−26 mV in 10 mM Ba2+. The time constant for inactivation is ∼380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 μM) blocks ∼50%, while nifedipine (1 μM), diltiazem (20 μM), and verapamil (50 μM) each block one‐third of the current. In contrast to its typical actions, BayK 8644 blocks the current by ∼ 25%. Blockers of other Ca channel subtypes were also tested: ω‐agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while ω‐conotoxin GVIA (1 μM) blocked 84% of the current. Immnohistochemistry supported the presence of α1A, α1B, α1C, and α1D Ca channel subunits. Mapping of dihydropyridine‐binding sites with DM‐BODIPY revealed a distribution of channels over the entire membrane of the Müller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L‐ and N‐type Ca channels or a single, unconventional HVA Ca channel subtype sharing L‐ and N‐type Ca channel characteristics.

Cav3.1 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.