METABOTROPIC RECEPTOR-ACTIVATED CALCIUM INCREASES AND STORE-OPERATED CALCIUM INFLUX IN MOUSE MUELLER CELLS
Noel Jochaim Da Silva 1,
Caroline Eileen Herron 2,
Kelly Stevens 1,
Christine A.B. Jollimore 3,
Steven Barnes 1,
and
Melanie E. M. Kelly 4*
1 Physiology and Biophysics, Dalhousie University, Halifax, Canada
2 Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Dublin 4, Ireland
3 Pharmacology, Dalhousie University, Halifax, Canada
4 Department of Pharmacology, Dalhousie University, Halifax, Canada
* To whom correspondence should be addressed. E-mail: melanie.kelly{at}dal.ca.
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Abstract |
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PURPOSE. Metabotropic receptor agonists that signal via Gq-coupled pathways increase Ca2+ in mammalian Mueller cells via release from intracellular stores and Ca2+ influx pathways that have not been well described. We 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 Mueller cells.
METHODS: Intracellular Ca2+ was measured using fluorescent 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 sarco/endoplasmic Ca2+ ATPase (SERCA) inhibitors, thapsigargin and cyclopiazonic acid, or inhibition of phospholipase C, occluded the carbachol-activated increase in Ca2+. Carbachol-activated Ca2+ increases in Mueller cells were enhanced by the di-acylglycerol 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 was present in mouse Mueller cells and carbachol activated a Gd3+-sensitive TRP-like cation channel.
CONCLUSIONS: Metabotropic muscarinic receptor-activated Ca2+ increases in mouse Mueller cells require release of Ca2+ from intracellular stores and activation of Ca2+ entry that involves TRP-like cation channels, but is independent of voltage-dependent Ca2+ channels.
Key Words:
Mueller cell, Ca2+ channels, muscarinic receptors, Store-operated Ca2+ entry
