Molecular Mechanisms of Oxytocinergic Signaling and its Inhibition of Kir7.1 in the RPE

Abstract

Purpose : Oxytocin (OXT) is a neuropeptide that activates the oxytocin receptor (OXTR), a rhodopsin family G-protein coupled receptor. We have localized OXTR to the retinal pigment epithelium (RPE) and OXT has been found in the adjacent cone photoreceptors. We hypothesize that there is OXTR signaling in the retina and sought to characterize this signaling in the RPE and explore the downstream effects of OXT on cellular signaling, focusing on the regulation of inwardly rectifying K⁺ channel Kir7.1.

Methods : Ca²⁺ response to OXT was measured in cultured human fetal RPE cells (hfRPE) using Fura-2AM in the presence of 2-APB and nifedipine, pharmacological inhibitors of Ca2+ signaling pathways. HEK-293 cells where used to establish stable expression of human OXTR and signaling was visualized using live cell imaging following transient expression of PH-GFP and PKC-GFP, monitors of GPCR metabolites PIP2 and DAG. Whole cell patch clamp electrophysiology was performed on HEK-OXTR cells transfected with GFP-fused Kir7.1 as well as freshly isolated mouse RPE cells to monitor Kir7.1 current.

Results : OXT treatment of RPE cells in culture resulted in a transient increase in cytoplasmic Ca²⁺ that was reduced by 95% in the presence of the IP₃R antagonist, 2-APB (P<0.001). Upon bathing the cells in Ca²⁺ free extracellular solution or nifedipine, the Ca²⁺ response to OXT was not altered. While the amplitude of responses was not altered, time to recover from the rise in [Ca²⁺]_i peak was faster, with time constants (τ) of 0.53 (r² = 0.983), 0.93 (r² = 0.975), and 1.7 (r² = 0.987) min for Ca²⁺-free, nifedipine, and Ringer’s solution, respectively. We also demonstrate that OXTR activation blunted Kir7.1 channel current, which has a physiologic role in RPE function. In isolated mouse RPE, we observed an average 61.81 ± 4.77 % decrease in K⁺ inward current amplitude and an average 11.4 ± 3.2 mV depolarization in resting membrane potential.

Conclusions : We propose that OXTR utilizes multiple capacitative Ca²⁺ entry (CCE) mechanisms to sustain an increase in Ca²⁺ driven by intracellular signaling molecules coupled to OXTR/G-protein in the RPE. This OXT-OXTR signaling in the RPE cell also integrated mobilization of intracellular Ca²⁺ and the parallel modulation of the Kir7.1 channel. We suggest that novel OXT-OXTR signaling pathways in the outer retina will be of fundamental importance for eye development, health and visual function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.