Role of the Phospholipase C Pathway in Oxytocin Induced Contraction of Lacrimal Gland Myoepithelial Cells.

Abstract

Purpose : In other contractile cells, oxytocin is known to activate phospholipase C (PLC) to increase intracellular calcium concentration [Ca²⁺]_i and elicit contraction. The purpose of the present studies was to determine if oxytocin can induce contraction of lacrimal gland myoepithelial cells (MEC), cells that express alpha smooth muscle actin (SMA) and other contractile proteins, and if so, determine if oxytocin uses the PLC pathway.

Methods : MECs were isolated, by collagenase digestion, from the lacrimal glands of SMA-GFP mice, which express GFP under the control of the SMA promoter. To identify the G proteins expressed in MECs, total RNA and total proteins were prepared and used for RT-PCR and western blotting, respectively. For measurement of changes in [Ca²⁺]_i, MECs were seeded on 35 mm glass bottom dishes, loaded with the calcium indicator Fura-2, and placed on the stage of the microscope of a Ca²⁺ imaging system (InCyt Im2; Intracellular Imaging). Data were collected in real time, every 0.75 seconds. After a basal reading was obtained for 15 seconds, oxytocin 10^-9-10^-6 M or UTP (10^-5 M, used as a positive control) were added. For measurements of MEC contraction, dishes were placed on the stage of an inverted microscope (Van Guard) equipped with a lapse-time camera. After a baseline 20-minute recording, oxytocin was added (10^-7 M), and then cells were recorded for an additional 20 minutes. Changes in cell size were measured from still images taken before and after stimulation using ImageJ software.

Results : Lacrimal gland MECs express the PLC-coupling G proteins, Gα_q and Gα₁₁. Oxytocin increased [Ca²⁺]_i in a concentration dependent manner with a maximum increase of 900 nM at 10^-7 M. In addition, oxytocin, at 10^-7 M, stimulated MEC contraction resulting in an average 14.5% change in cell size. Pretreating the cells with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate (IP₃) receptor antagonist that abrogates intracellular Ca²⁺ mobilization, completely inhibited oxytocin-induced MEC contraction.

Conclusions : Our data suggest that oxytocin uses the PLC/Ca²⁺ pathway to stimulate lacrimal gland MEC contraction. Additional experiments are underway to determine the role of other signaling pathways as well as which Ca²⁺ responsive proteins are involved in oxytocin induced lacrimal gland MEC contraction.

This is a 2020 ARVO Annual Meeting abstract.