Abstract
Purpose::
Physiological imbalances in Zn2+ concentration and synaptic transmission are contributing factors in neuronal cell death. Zn2+ treatment is beneficial for slowing the development of macular degeneration. Two solute-linked carrier gene families, Znt and Zip, encode transmembrane proteins that function as Zn2+ transporters. There are at least 24 Zn2+ transporters with opposing functions in zinc availability and homeostasis. Of these, 9 are Znts, the zinc efflux transporters and 15 are Zips, the zinc influx transporters. The purpose of this study was to examine the expression of these Zn2+ transporters in the RPE and retina.
Methods::
Gene array analysis was performed using RNA from A-RPE19 cells. Cy3 and Cy9 labeled cDNA were hybridized to Human OHU28K oligo array slides and data collected with a GenePix 4000A scanner and analyzed by GenePix software V4.0, Cluster v2.20, TreeView v1.60, and LocuLink. The expression of Zn2+ transporters in RPE cells was confirmed by RT-PCR. Expression of Znts and Zips was measured in C57 B/L6 mouse retina at development stages between E13 and P21 and in adult and fetal human retinas. Zinc uptake by RPE cells was measured after treatment with 0-10 mM free Zn2+ between 0-60 min. Intracellular Zn2+ concentration was measured using two fluorescent Zn2+ indicators, zinquin ethyl ester (Ex/Em 364/385 nm) and Newport Green (Ex/Em 505/535 nm).
Results::
6/9 Znt and 9/15 Zip transporters were expressed in A-RPE19 cells with Znt 1, 7 and Zip 4,6,7,9,10,13 being the most abundant. All Znts and Zips were expressed in the retina but varied with developmental stages and between fetal and adult human retina. At PN3, Zip5, Znt 2, and Znt8 expression peaked while Zip8 expression decreased. The expression of Znt 2, 8 and Zip11 varied between fetal and adult human retina. Zn2+ uptake in the RPE cells was dose and time dependent.
Conclusions::
Fluctuations in Zn2+ availability and Zn2+ transporter expression are contributing factors to neurological disorders including epilepsy and Alzheimer’s disease. Since treatment with high levels of zinc slows the development of macular degeneration, the distribution, expression, and function of zinc transporters in the aging RPE and retina are worthy of further investigation.
Keywords: age-related macular degeneration • cell survival • ion transporters