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De-Ann M Pillers, Michelle Chiu, Patrick Halbach, Nathan York, Bikash R Pattnaik; Oxytocinergic Signaling via GPCR in a Single HEK293 Cell with Stable Expression of the Oxytocin Receptor. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2951.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown that oxytocin (OXT) localizes to the cone photoreceptor outer segment whereas the oxytocin receptor (OXTR) localizes to the retinal pigment epithelium (RPE). OXT is a neuropeptide hormone traditionally recognized for its role in parturition. A rise in intracellular calcium ([Ca2+]i) occurs as a result of OXT binding to the oxytocin receptor (OXTR), most likely through G-protein coupled receptor (GPCR) mediated activation. We used molecular and live-cell imaging techniques to visualize OXT-OXTR signaling by real-time fluorescence.
We generated Human Embryonic Kidney (HEK293) cells stably expressing human OXTR (hOXTR-HEK). Intracellular changes in [Ca2+] in response to OXT and ATP were measured using a FURA-2AM ratiometric assay. We used a live-cell fluorescent marker (pleckstrin homology domain-fused GFP or PH-GFP) for the detection of membrane phosphatidylinositol 4,5-biphosphate (PIP2). In the hypothesized rhodopsin-type class I GPCR pathway, upon agonist binding, membrane PIP2 is hydrolyzed to inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) catalyzed by phospholipase C (PLC). hOXTR-HEK cells were transiently transfected with PH-GFP using TransIT-HEK (MirusBio, Madison, WI). ATP binding was used as a positive control. GFP positive cells were imaged within 24-72 hr post transfection. Fluorescence images were acquired every 10 sec.
In response to OXT, hOXTR-HEK cells demonstrated an average ratiometric increase of 0.0738 ± 0.0028 units (P<0.005) corresponding to an increase in ~ 75 nM of free [Ca2+]. The response to ATP was not significantly different, consistent with the involvement of a GPCR mechanism. PH-GFP has a high affinity for PIP2 but when PIP2 hydrolyzes, PH-GFP translocates with IP3 to the cytoplasm. In our live-cell imaging experiments, resting cells expressing GFP in the sub-membrane domain showed translocation of GFP fluorescence from the membrane to the cytoplasm (an average increase in cytoplasmic pixel density of 512.09 ± 54.27 units, P<0.005) when the cells were exposed to OXT. The time course of GFP translocation correlated with the increase in intracellular [Ca2+].
In a HEK293 OXTR expression cell model, we have shown that OXT-OXTR signaling uses a GPCR mechanism to mobilize intracellular [Ca2+]. These results suggest that intercellular communication may occur in the eye via OXT-OXTR mediated GPCR signaling.
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