Study on the relationship between CFTR physiological secretion pathway and intracellular calcium signaling in rabbit cornea epithelium

Background

Researches showed that cystic fibrosis transmembrane conductance regulator protein(CFTR) is a channel secreting anion and water, and it plays an important role in tear secreting.Traditional conception thought that CFTR pathway is cAMP-PKA -dependent without the participation of intracellular calcium.However, studies disclosed that elevating intracellular cAMP could not open the CFTR channel.So whether calcium signal is associated with CFTR-related corneal epithelial secretion is controversial.

Objective

This study was to investigate the association between CFTR secretion and intracellular calcium signaling in rabbit corneal epithelium.

Methods

Sixteen New Zealand white rabbits were randomized into odd number group and even number group by computer randomized number method.The corneas were obtained under the general anesthesia and placed in Ussing Chamber for the record of short cricuit current (Isc). The right eyes of rabbits in the odd number group were stimulated with ATP and served as ATP stimulating group.The left eyes were pretreated with CFTR_inh-172 prior to ATP stimulation and served as CFTR_inh-172 pretreated group.In the even number group, the left eyes of rabbits were pretreated with BMPTA/AM before ATP stimulation and served as BMPTA/AM pretreated group, and the right eyes of the rabbits were used to isolate and culture corneal epithelial cells by explant adherent method, the level of intracellular calcium were evaluated using Leica SP5 laser scan confocal microscope.

Results

The ATP-induced ΔIsc of corneal epithelium was (5.73±1.36), (1.30±0.95) and (2.47±0.55)μA/cm² in the ATP stimulating group, CFTR_inh-172pretreated group and BMPTA/AM pretreated group, respectively, and the ΔIsc was significantly reduced in the CFTR_inh-172 pretreated group and BMPTA/AM pretreated group compared with ATP stimulating group (t=11.201, 5.508, both at P<0.001). The fluorescence intensity of intracellular calcium release after ATP stimulation was 3.25 folds more than that before ATP stimulation.

Conclusions

ATP promotes rapid short circuit current of corneal epithelium.CFTR_inh-172depresses the ATP-induced corneal epithelium ΔIsc, and BMPTA/AM suppresses intracellular calcium release.It is suggested that intracellular calcium signaling secretion probably participates in the functional CFTR activity in rabbit corneal epithelium.

Cystic fibrosis transmembrane conductance regulator/metabolism; Epithelial cells, corneal/drug effects; ATP; Cells, cultured; Short circuit current; Calcium signaling; Rabbits