Inner Blood–Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig

Abstract

The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood–retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood–brain barrier (BBB), and blood–cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/μg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/μg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/μg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/μg protein), and sodium–potassium adenosine triphosphatase (Na + /K + -ATPase; 53.7 fmol/μg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB ( R 2 = 0.8906), followed by outer BRB ( R 2 = 0.7988) and BCSFB ( R 2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6 ) and multidrug resistance-associated protein 1 (MRP1, ABCC1 ) were expressed at the outer BRB (0.378 and 1.03 fmol/μg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.