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

PURPOSE. Metabotropic receptor agonists that signal through G q -coupled pathways increase Ca 2+ in mammalian Muller 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 Muller 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 μM) produced increases in Ca 2+ that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic Ml muscarinic receptors. Depletion of Ca 2+ stores by the sarcoplasmic/endoplasmic Ca 2+ ATPase (SERCA) inhibitors thapsigargin and cyclopia-zonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca 2+ . Carbachol-activated Ca 2+ increases in Miiller 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 Miiller cells, and carbachol activated a Gd 3+ -sensitive, TRP-like cation channel. CONCLUSIONS. Metabotropic muscarinic receptor-activated Ca 2+ increases in mouse Muller 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.