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M.C. Isoldi, M. Rollag, A.M. Castrucci, I. Provencio; Melanopsin Signals Through a Phosphoinositide Pathway . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1723.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Melanopsin is the photopigment that confers light sensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). Mammalian ipRGCs are involved in the photic sychronization of circadian rhythms to the day:night cycle. Here we report the identification of the molecular components involved in melanopsin signaling using the cultured Xenopus dermal melanophore system. Methods: Xenopus melanophores respond to light by dispersing their melanin granules, thereby increasing their optical density. Melanophores were seeded in 96–well plates and kept in the dark for a minimal of 3 days. Prior to assay, cells were incubated in 1 nM melatonin to fully aggregate the melanin granules. To test the inhibitory effects of various pharmacological agents on the light response, one of a series of signaling pathway inhibitors (KT 5823 and DT–2 [protein kinase G], SQ 22,536 [adenylyl cyclase], H–89 [protein kinase A], U–73122 [phospholipase C; PLC], Ro 31–8220 [protein kinase C; PKC], or BAPTA–AM [Ca2+ chelator]) was added to the medium. Experimental plates and controls without inhibitors were then exposed to light; additional controls were kept in the dark. Optical density was measured and the responses were expressed as a percentage of the maximal response obtained after 60 min of light exposure in the absence of inhibitors. For cyclic nucleotide assays, cells were light exposed for 1 min, or 15 min for inositol trisphosphate (IP3) and PKC assays and Western blot analysis. Kit reagents and protocols were followed per the manufacturer for the measurement of total cAMP, total cGMP, IP3 and PKC activity (all Amersham Biosciences). For Western blot analysis, 25 µg of protein were loaded in a 10% NuPAGE Tris–Bis gel (Invitrogen). After transfer to PVDF membranes, the blot was developed according to the WesternBreeze chromogenic immunodetection kit instructions (Invitrogen). Membranes were incubated in a mixture of phospho–(Ser) PKC substrate antibody and an antibody against b–actin (for normalization). Results: Photoactivated melanopsin initiates a phosphoinositide signaling pathway. In melanophores, light increases intracellular IP3 and disperses melanosomes. Inhibitors of PLC and PKC, and chelation of intracellular Ca2+ block the effect of light on melanophores. At least 4 proteins, of molecular weights 43, 74, 90 and 134 kDa, are phosphorylated by PKC upon light stimulation. Conclusions: This is the first evidence of an invertebrate–like light–activated signaling cascade within vertebrate cells.
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