Signaling pathways elicited by light in photoreceptor nuclei from bovine retina

NATALINI, P.M.; MATEOS, M.V.; GIUSTO, N.M.; ILINCHETA DE BOSCHERO, M.G.

Orlando

Congreso; ARVO 2014 Annual Meeting; 2014

The Association for Research in Vission and Ophthalmology

 Phototransduction occurs in the outer segments of photoreceptor cells, and the signaling mechanisms elicited by light stimulation, such as Insulin receptor (IR) activation, have been well characterized. However, much less is known about the possible consequences of these mechanisms of signal transduction at the nuclear level in photoreceptor cells. The aim of this study was to evaluate whether ex-vivo light exposure of bovine retinas modulates different signaling pathways in nuclei from photoreceptor cells. The activity and localization of diacylglycerol kinases (DAGK) and the phosphorylation of extracellular signal-regulated kinase (ERK), protein kinase C (PKC) and Akt as well as the presence of the IR in the photoreceptor nuclei were studied. Bovine eyes were adapted to darkness (2 h, 0° C), and then eye cups were exposed to light (280 cd, 30 min) or to darkness. To obtain photoreceptor nuclei, isolated retinas were homogenized in 0.25 M sucrose, filtrated and purified by centrifugation onto a discontinuous sucrose gradient. To evaluate the purification protocol, immunofluorescence (IF), electronic microscopy (EM) and western blot (WB) techniques were employed. Light-induced damage was evaluated by IF (DNA damage) and by measuring the generation of thiobarbituric acid reactive substances (TBARS). DAGKs (alpha, beta, gamma, epsilon and dseta isoforms), PKC alpha, Akt and ERK1/2 were detected by WB. IR presence was detected by WB and IF. DAGKs activity was measured using radioactive substrates. WB and IF revealed the presence of IR in photoreceptor nuclei. Light exposure neither affected DNA fragmentation nor increased TBARS levels in retinal homogenates. However, light induced a differential localization and activation of DAGK isoforms in photoreceptor nuclei with respect to the darkness condition. In addition, an increase in the contents of phospho-PKC alpha (2.5 fold) and phospho-Akt (3 fold) without changes in total PKCa and Akt was seen in nuclei from light exposed retinas. A significant increase in response to light in the content of total and phosphorylated forms of ERK1/2 (2 fold) was also observed. Our results demonstrate that light activates ERK and signaling pathways linked to G-protein coupled receptors and tyrosine kinase receptors in photoreceptor nuclei. Light-dependent activation of IR, which is present in these nuclei, might be mediating these effects.