Role of CFTR in Lacrimal Gland Ductal Fluid Secretion in Mouse

Abstract

Purpose : Cystic fibrosis transmembrane conductance regulator (CFTR) has been shown to play a key role in fluid secretion from many tissues. Accumulating evidence, most of them were descriptive by determining the expression patterns and/or changes of mRNA and protein of CFTR in the lacrimal gland (LG) suggest CFTR also plays a key role in LG secretion, particularly in the duct system where it is most abundant. Transgenic mouse models carrying genetic defects in CFTR offers a unique opportunity to explore the role of CFTR in LG secretion. Therefore the aim of the present study was to investigate fluid secretion of isolated LG duct segments from wild type (WT) and CFTR knockout (KO) mice.

Methods : Immunofluorescence was used to confirm the presence and localization of CFTR protein. LG interlobular/intralobar ducts were isolated from WT and KO mice as previously described by us. During overnight incubation, ends of the ducts seal, forming a closed luminal space. Fluid secretion into this intraluminal space was analyzed using bright field video-microscopy. Effects of forskolin (10 µM) and carbachol (100 µM) stimulation on duct segments from WT and KO mice were investigated. Intracellular Ca²⁺ homeostasis underlying cholinergic stimulation was measured in ducts from WT and KO mice using microfluorometry. Data was presented as means ± SEM.

Results : Immunofluorescence confirmed the presence of CFTR in apical membranes of ductal cells. Forskolin (10 µM) stimulation caused a rapid fluid secretion in ducts from WT mice (149.51±13.50 pl/min/mm²), in contrast to ducts from KO animals which secretion remained unchanged (1.47±4.38 pl/min/mm²). Cholinergic agonist carbachol (100 µM) caused a biphasic secretory response consisting of a pulse-like secretion in the first 5 min, followed by a plateau phase with similar secretory patterns both in WT and in KO ducts (secterory rates in WT ducts: 218.90±15.03 in the first 5 min of stimulation and 109.59±18.97 pl/min/mm² calculated for the first 10 min; secretory rates in KO ducts: 201.90±12.02 in the first 5 min of stimulation and 101.50±15.89 pl/min/mm² calculated for the first 10 min). However, no significant differences were observed in carbachol-stimulated cytosolic Ca²⁺ signaling in ducts from WT and KO mice.

Conclusions : These data suggest the key role CFTR may play in LG ductal secretion, and the independence of secretory mechanisms activated by Ca²⁺ mobilizing stimuli from functionally active CFTR.

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