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 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.