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N. Da Silva, C.E. Herron, K. Stevens, S. Barnes, M.E. M. Kelly; Müller Cell Calcium Signaling in the Mammalian Retina: Potential Role of Trp Channels in Metabotropic Receptor Activation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5894.
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© ARVO (1962-2015); The Authors (2016-present)
Activation of metabotropic receptors in non–excitable cells and heterologous systems has been shown to result in activation of G–protein signaling cascades that can lead to calcium influx through TRP channels. To investigate the possible contribution of TRP channels to calcium signaling in the retina, we established a mouse Müller cell culture and examined the actions of muscarinic and purinergic receptor activation on calcium signaling and TRP channel activation.
Purified mouse Müller cell cultures were prepared as previously described (Da Silva et al., 2004 ARVO E–Abstr). RT–PCR was used to identify TRP channel isoforms present and intracellular calcium signals were recorded using fluorescent imaging techniques with Fura–2 AM.
RT–PCR identified mRNA for TRPC6, TRPM7, and TRPV4 in purified mouse Müller cell cultures. Carbachol, a muscarinic receptor agonist, and ATP, a purinergic receptor agonist, both produced increases in intracellular calcium. Bath application of carbachol resulted in the downstream activation of a Gq–coupled signaling pathway, leading to stores calcium release and influx of calcium across the membrane. This response was independent of voltage–gated calcium channels. The muscarinic receptor antagonist, atropine, PLC enzyme blockers, and depletion of intracellular calcium stores with thapsigargin or cyclopiazonic acid abolished the carbachol–induced calcium response. The carbachol–induced calcium influx was attenuated with TRP channels blockers La3+, 2–APB, and Gd3+. Depletion of internal stores with thapsigargin resulted in Ba2+ or Sr2+ influx, which was blocked by Gd3+ or La3+, consistent with a contribution of TRP channels to refilling of internal stores.
Our results suggest that cultured mouse Müller cells express TRPC6, TRPM7, and TRPV4 channel isoforms. Calcium imaging indicates that TRP channels contribute to store refilling in Müller cells following metabotropic muscarinic receptor activation. We propose that metabotropic receptor–mediated TRP channel activation can contribute to cytosolic calcium signaling in Müller cells.
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