Purchase this article with an account.
A. Martin-Gil, J. Pintor; P2Y2 Nucleotide Receptors Increase the Presence of Aquaporin-1 in Rabbit Non Pigmented Ciliary Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):691.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the role of P2Y2 purinergic receptors in the raise of aquaporin-1 levels in NPE cell membranes and cytoplasm.
Immortalized NPE cells, kindly provided by Dr. Coca-Prados, were seeded in high glucose DMEM until they were subconfluent. Immunocytochemical studies were performed by means of an antibody raised against aquaporin-1 (AQP1) from Santa Cruz. Immunolabeling of the cells was performed by incubating them after the corresponding treatment (see below) with a dilution of 1:100 of the primary antibody followed by a dilution 1:50 of a secondary antibody labelled with FITC. Previous to this immunostaining cells were challenged with 100 uM of the dinucleotide Ap4A, at 15, 30, 60, 90 and 120 min. To avoid Ap4A degradation, the medium was changed every 15 min. Antagonists of the P2Y receptors, suramin, reactive blue 2 and PPADS (all at 100 uM), were pre-incubated 30 min before Ap4A (100 uM) was added and antagonists were present during the dinucleotide application.
The application of Ap4A to the NPE cells demonstrated a gradual increase in the presence of AQP1 which was time-dependent from the beginning to 60 min, when the maximal expression of the protein is reached (200 % of increase when compared to control) (n=8). Times longer than 60 min, gradually returned the fluorescent values to the initial control values (at 120 min). Concerning the distribution of AQP1, although at the beginning its presence was both in the cytoplasm and in the membrane, at 60 min, there was a significant increase of AQP1 in the plasma membrane. The experiments perfomed with the antagonist of the P2Y receptors demonstrated the involvement of this receptor in the action of Ap4A. In particular reactive blue 2 was the best reversing the effect of Ap4A. In the case of this antagonist, it was able to inhibit up to 80 % of the effect depicted by Ap4A.
Diadenosine tetraphosphate, a good P2Y2 agonist, was able to mobilize AQP1 which was more visible after 60 min incubation with this substance. This increase in AQP1 expression can be the explanation to what was found in the whole rabbit in which P2Y2 agonists clearly increase intraocular pressure.
This PDF is available to Subscribers Only