Light-activation of the Phosphoinositide Cycle in Intrinsically Photosensitive Chicken Retinal Ganglion Cells

MARIA A CONTIN; DANIELA M VERRA; GABRIELA SALVADOR; MONICA ILINCHETA; NORMA M GIUSTO; MARIO E GUIDO

INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE

ASSOC RESEARCH VISION OPHTHALMOLOGY INC

Lugar: Rockville, MD ; Año: 2010 vol. 51 p. 5491 - 5498

0146-0404

Abstract Purpose: In vertebrates, intrinsically photosensitive retinal ganglion cells (ipRGCs) acting as non-visual photoreceptors transmit environmental illumination information to the brain, regulating diverse non-image-forming tasks. We have demonstrated that the phototransduction cascade occurring in chicken ipRGCs resembles that of rhabdomeric photoreceptors and involves phospholipase C (PLC) activation. In the current work we investigate the participation of the phosphoinositide (PIPs) cycle in this mechanism and probe whether changes in inositol 1,4,5-trisphosphate (IP3) and PIP kinase activities are triggered by light. Methods: We used primary cultures of Thy-1 immunopurified chicken embryonic RGCs exposed to bright light pulses or kept in the dark to assess intracellular Ca+2 mobilization by Fluo-3 AM fluorescence microscopy, IP3 levels and enzymatic activities of diacylglicerol-, phosphatidylinositol-, and phosphatidylinositol phosphate kinases (DAGK, PIK, PIPK respectively) by radioactive assessments. The presence of different melanopsins (Opn4m and Opn4x) and other photopigments was determined by RT-PCR and immunochemistry. Results: Our results show that cultured RGCs expressing different non-visual photopigments display a significant and rapid increase in IP3 levels (1.3-fold) and Ca+2 mobilization by light which was reverted by administration of the PLC inhibitor U73122 (5 µM). Brief light pulses also caused a very rapid and transient activation of DAGK, PIK and PIPK activities compared with dark controls. Conclusions: The results indicate for the first time that light stimulation of chicken RGC cultures activates the PIPs cycle causing an increase in intracellular levels of IP3, changes in phosphatidic acid, PIP and PIP2 levels and Ca2+ mobilization. Key Words: ganglion cell ? inositol ? phospholipids ? photoreceptor transduction ? phospholipase ? cell culture