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

Noel Da Silva, Caroline E. Herron, Kelly Stevens, Christine A. B. Jollimore, Steven Barnes, Melanie E. M. Kelly
2008 Investigative Ophthalmology and Visual Science  
PURPOSE. Metabotropic receptor agonists that signal through G q -coupled pathways increase Ca 2ϩ in mammalian Müller cells by release from intracellular stores and Ca 2ϩ influx pathways that have not been well described. The authors examined the involvement of voltage-dependent and non-voltage-dependent Ca 2ϩ channels in metabotropic muscarinic receptor-activated Ca 2ϩ increases and store-operated Ca 2ϩ influx in cultured mouse Müller cells. METHODS. Intracellular Ca 2ϩ was measured using
more » ... scence 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 M) produced increases in Ca 2ϩ that were blocked by the muscarinic receptor antagonists atropine and pirenzepine. RT-PCR confirmed mRNA for metabotropic M1 muscarinic receptors. Depletion of Ca 2ϩ stores by the sarcoplasmic/endoplasmic Ca 2ϩ ATPase (SERCA) inhibitors thapsigargin and cyclopiazonic acid or the inhibition of phospholipase C occluded the carbachol-activated increase in Ca 2ϩ . Carbachol-activated Ca 2ϩ 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 3ϩ , La 3ϩ , 2-APB, and flufenamic acid. Both muscarinic receptor activation and thapsigargin treatment depleted Ca 2ϩ stores and produced Ca 2ϩ entry that was attenuated by La 3ϩ , 2-APB, Gd 3ϩ , and flufenamic acid. mRNA and protein for TRPC1 and TRPC6 were present in mouse Müller cells, and carbachol activated a Gd 3ϩ -sensitive, TRP-like cation channel. CONCLUSIONS. Metabotropic muscarinic receptor-activated Ca 2ϩ increases in mouse Müller cells require the release of Ca 2ϩ from intracellular stores and the activation of Ca 2ϩ entry that involves TRP-like cation channels but is independent of voltage-dependent Ca 2ϩ channels. (Invest Ophthalmol Vis Sci.
doi:10.1167/iovs.07-1118 pmid:18316702 fatcat:cvt2oq4ltvbtvoc7jqnop3n4je