Contribution of TRPV1 to microglia-derived IL-6 and NFkappaB translocation with elevated hydrostatic pressure.
OBJECTIVE
The authors investigated the contributions of the transient receptor potential vanilloid-1 receptor (TRPV1) and Ca(2+) to microglial IL-6 and nuclear factor kappa B (NFkappaB) translocation with elevated hydrostatic pressure.


METHODS
The authors first examined IL-6 colocalization with the microglia marker Iba-1 in the DBA/2 mouse model of glaucoma to establish relevance. They isolated microglia from rat retina and maintained them at ambient or elevated (+70 mm Hg) hydrostatic pressure in vitro and used ELISA and immunocytochemistry to measure changes in the IL-6 concentration and NFkappaB translocation induced by the Ca(2+) chelator EGTA, the broad-spectrum Ca(2+) channel inhibitor ruthenium red, and the TRPV1 antagonist iodo-resiniferatoxin (I-RTX). They applied the Ca(2+) dye Fluo-4 AM to measure changes in intracellular Ca(2+) at elevated pressure induced by I-RTX and confirmed TRPV1 expression in microglia using PCR and immunocytochemistry.


RESULTS
In DBA/2 retina, elevated intraocular pressure increased microglial IL-6 in the ganglion cell layer. Elevated hydrostatic pressure (24 hours) increased microglial IL-6 release, cytosolic NFkappaB, and NFkappaB translocation in vitro. These effects were reduced substantially by EGTA and ruthenium red. Antagonism of TRPV1 in microglia partially inhibited pressure-induced increases in IL-6 release and NFkappaB translocation. Brief elevated pressure (1 hour) induced a significant increase in microglial intracellular Ca(2+) that was partially attenuated by TRPV1 antagonism.


CONCLUSIONS
Elevated pressure induces an influx of extracellular Ca(2+) in retinal microglia that precedes the activation of NFkappaB and the subsequent production and release of IL-6 and is at least partially dependent on the activation of TRPV1 and other ruthenium red-sensitive channels.
Sappington
Rebecca M
RM
Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0654, USA.
Calkins
David J
DJ
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D045604:Extracellular Fluid / Q000378:metabolism; D005901:Glaucoma / Q000378:metabolism; D006874:Hydrostatic Pressure; D015850:Interleukin-6 / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D007429:Intraocular Pressure; D051379:Mice; D008811:Mice, Inbred DBA; D017628:Microglia / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism / Q000502:physiology*; D012165:Retinal Ganglion Cells / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*
D015220:Calcium Channels; D015850:Interleukin-6; D016328:NF-kappa B; D050916:TRPV Cation Channels; C493921:TRPV1 protein, mouse; D012430:Ruthenium Red; D002118:Calcium