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Noel Da Silva, Caroline E. Herron, Kelly Stevens, Christine A. B. Jollimore, Steven Barnes, Melanie E. M. Kelly; Metabotropic Receptor-Activated Calcium Increases and Store-Operated Calcium Influx in Mouse Müller Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(7):3065-3073. doi: https://doi.org/10.1167/iovs.07-1118.
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© ARVO (1962-2015); The Authors (2016-present)
purpose. Metabotropic receptor agonists that signal through Gq-coupled pathways increase Ca2+ in mammalian Müller cells by release from intracellular stores and Ca2+ influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non–voltage-dependent Ca2+ channels in metabotropic muscarinic receptor-activated Ca2+ increases and store-operated Ca2+ influx in cultured mouse Müller cells.
methods. Intracellular Ca2+ 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 μM) produced increases in Ca2+ that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca2+ stores by the sarcoplasmic/endoplasmic Ca2+ ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca2+. Carbachol-activated Ca2+ 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 Gd3+, La3+, 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca2+ stores and produced Ca2+ entry that was attenuated by La3+, 2-APB, Gd3+, and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd3+-sensitive, TRP-like cation channel.
conclusions. Metabotropic muscarinic receptor-activated Ca2+ increases in mouse Müller cells require the release of Ca2+ from intracellular stores and the activation of Ca2+ entry that involves TRP-like cation channels but is independent of voltage-dependent Ca2+ channels.
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