Light activation of the phosphoinositide cycle in intrinsically photosensitive chicken retinal ganglion cells.
OBJECTIVE
In vertebrates, intrinsically photosensitive retinal ganglion cells (ipRGCs) acting as nonvisual photoreceptors transmit environmental illumination information to the brain, regulating diverse non-image-forming tasks. The phototransduction cascade in chicken ipRGCs has been shown to resemble that of rhabdomeric photoreceptors and involves phospholipase C (PLC) activation. The current work was an investigation of the participation of the phosphoinositide (PIP) cycle in this mechanism and of whether changes in activities of inositol 1,4,5-trisphosphate (IP(3)) and PIP kinase are triggered by light.


METHODS
Primary cultures of Thy-1 immunopurified chicken embryonic RGCs were exposed to bright light pulses or kept in the dark, to assess intracellular Ca(2+) mobilization by Fluo-3 AM fluorescence microscopy, IP(3) levels, and enzymatic activities of diacylglycerol, phosphatidylinositol, and phosphatidylinositol phosphate kinases (DAGK, PIK, and PIPK, respectively), by radioactive assays. The presence of different melanopsins (Opn4m and Opn4x) and other photopigments was determined by RT-PCR and immunochemistry.


RESULTS
Cultured RGCs expressing different nonvisual photopigments displayed a significant and rapid increase in IP(3) levels (1.3-fold) and Ca(2+) mobilization by light, which was reversed 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 compared with that in the dark control.


CONCLUSIONS
The results indicate for the first time that light stimulation of chicken RGC cultures activates the PIP cycle, causing an increase in intracellular levels of IP(3), changes in levels of phosphatidic acid, PIP, and PIP(2); and mobilization of Ca(2+).
Contín
Maria Ana
MA
Departamento de Química Biológica, Facultad de Ciencias Químicas, Centro de Investigaciones en Química Biológica de Córdoba, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Córdoba-Córdoba.
Verra
Daniela M
DM
Salvador
Gabriela
G
Ilincheta
Monica
M
Giusto
Norma M
NM
Guido
Mario E
ME
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism*; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D017931:DNA Primers; D003623:Dark Adaptation; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D008027:Light; D008856:Microscopy, Fluorescence; D010716:Phosphatidylinositols / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010775:Photic Stimulation; D011760:Pyrrolidinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D012165:Retinal Ganglion Cells / Q000378:metabolism / Q000528:radiation effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000235:genetics; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D014966:Xanthenes / Q000378:metabolism; D000099210:Melanopsin
D019870:1-Phosphatidylinositol 4-Kinase; D000814:Aniline Compounds; D002118:Calcium; D017931:DNA Primers; D019852:Diacylglycerol Kinase; D004963:Estrenes; D015544:Inositol 1,4,5-Trisphosphate; D010716:Phosphatidylinositols; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012333:RNA, Messenger; D017299:Rod Opsins; D010738:Type C Phospholipases; D014966:Xanthenes; D000099210:Melanopsin; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C059715:Fluo-3