title: Metabotropic receptor-activated calcium increases and store-operated calcium influx in mouse Müller cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Noel
authors.initials: N
authors.lastname: Da Silva
authors.forename: Caroline E
authors.initials: CE
authors.lastname: Herron
authors.forename: Kelly
authors.initials: K
authors.lastname: Stevens
authors.forename: Christine A B
authors.initials: CA
authors.lastname: Jollimore
authors.forename: Steven
authors.initials: S
authors.lastname: Barnes
authors.forename: Melanie E M
authors.initials: ME
authors.lastname: Kelly
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018721:Muscarinic Agonists / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D015220:Calcium Channels; D004791:Enzyme Inhibitors; D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D050052:TRPC Cation Channels; D019284:Thapsigargin; D002217:Carbachol; D010738:Type C Phospholipases; D002118:Calcium