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 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 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 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.