Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK IIÎ±) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK IIÎ±, there was no increase in PI-4,5-P 2 measured by liquid chromatography-mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina.
|State||Published - Jun 26 2014|
Bibliographical noteFunding Information:
This study was supported by grants from the National Institutes of Health (EY016507, EY00871, and NEI Core grant EY12190); and an unrestricted grant from Research to Prevent Blindness, Inc., to the department of Ophthalmology. The authors also thank the NCRR COBRE grant (8P20GM103527-05) that provided partial support for these studies.
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