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P. Senanayake, J. Drazba, R. Desnoyer, S. Karnik, J. G. Hollyfield; Evaluation of a Major Intracellular Phosphates Regulator I2PP2A in Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):683.
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Aim of this study was to evaluate I2PP2A and the AT2 receptor in human, rat and mouse retina. High glucose increases Angiotensin II (Ang II) in Müller cells by ten fold. In experimental diabetes Ang II and Ang II, AT2 receptors are significantly increased. Ang II is also increased in the human diabetic retina. AT2 receptor activation leads to stimulation of tyrosine and threonine phosphatases. I2PP2A is a major regulator of intracellular phosphates activity.
Donor human eyes were obtained from the Cleveland Eye Bank within 12 hours postmortem and fixed in PBS-paraformaldehyde. C57BL/6 12 week old mice were purchased from Jackson Laboratory. WKY 11 week old male rats were purchased from Charles River Lab. Mice and rats were sacrificed by carbon dioxide asphyxiation. The eyes were enucleated and fixed in PBS- paraformaldehyde. The eyes were then frozen in OCT and stored at -80º C. AT2 receptor and I2PP2A was localized by immunohistochemistry and fluorescence imaging. Retinal sections were labeled with polyclonal antibodies against AT2 receptor (1:100, Alpha Diagnostics, San Antonio, TX) and I2PP2A (B13008, 1:400, Stratagene, La Jolla , CA) and resolved with goat anti-rabbit Alexa 488 or 594 conjugated labeled secondary antibodies. Slides were mounted in anti-fade medium containing DAPI for labeling nuclei (Vectorshield Laboratories, Inc., Burlingame, CA). Images were taken with a Leica confocal laser-scanning microscope (Heidelberg, Germany).
I2PP2A was detected in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). The I2PP2A has a nuclear and perinuclear distribution. The AT2 was also detected in the GCL, INL and ONL and has nuclear and perinuclear localization. In addition, AT2 IR was detected in the inner limiting membrane, Müller cell endfeet and in the Müller cell processes.
AT2 receptor and I2PP2A may be colocalized. The link between I2PP2A and the AT2 receptor in retina remains to be established.
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