RPE fractionation method and EM images of isolated RPE fractions.

<p>A) In addition to melanosomes, the crude melanosomal fraction contains a large quantity of other membranous structures. B) In the crude lysosomal fraction, only few melanosomes were observed among non-pigmented vesicles. C) The purified melanosomal fraction contains mainly round or ellipsoidal melanosomes. Scale bar 2 μm, 6000x magnification.</p

Size analysis of isolated melanosomes.

<p>(A) At lower melanosomal concentration 0.01 μg/μl, one population with an average diameter of 934.5 nm was observed whereas at higher melanosomal concentration (B) 0.1 μg/μl, two populations with average diameters 1533 and 5577 nm were observed.</p

Melanosomes retain their biological activity after isolation.

<p>(A) In ATP hydrolysis assay, purified melanosomes were incubated with ATP and concentration of generated free phosphate (P_i) was measured. Assay was carried out with or without V-ATPase inhibitor bafilomycin A1 (BAF). Values are shown as mean +/- S.E.M (n = 3). (B) Statistically significant difference in P_i formation with BAF is shown as 95% Confidence Interval (indicated by dashed lines, *P < 0.001, unpaired t-test).</p

Na⁺K⁺ATPase (112 kDa), HSP60 (60 kDa), V-ATPase (96 kDa) and Rab27a (27 kDa) expression in the porcine RPE melanosomal fractions and in the porcine RPE crude lysosomal fraction.

<p>The mitochondrial marker HSP60 is absent in the purified melanosomal fraction whereas the V-ATPase as well as the Na^<b>+</b>K^<b>+</b>ATPase are expressed in all fractions. Rab27a is enriched in the purified melanosomes.</p

Purified melanosomes display intact membrane structure.

<p>Staining of isolated porcine RPE melanosomes with Vybrant^™DiO (A-C), a fluorescent membrane dye (green) showed that melanosomal membrane remains intact after isolation process. (D-E) represent brightfield images. Scale bar 5 μm.</p

LC–MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line

The retinal pigment epithelium (RPE) forms the outer blood–retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na⁺/K⁺ ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood–retinal barrier