title: Mitochondrial localization of P-glycoprotein and peptide transporters in corneal epithelial cells--novel strategies for intracellular drug targeting.
abstract: This study was designed to investigate functional localization of both efflux (P-glycoprotein, P-gp) and influx (peptide) transporters in the mitochondrial membrane of cultured rabbit primary corneal epithelial cells (rPCECs). Isolation and purification of mitochondria was performed by optimized cell fractionation method. Mitochondrial integrity was measured by JC-1 uptake experiment. The efflux activity of P-gp was assessed by performing in vitro uptake studies on isolated mitochondria with Rhodamine 123 (Rho-123) alone and in the presence of P-gp inhibitors (quinidine and cyclosporine A) using fluorimetry and flow cytometry analysis. Functional activity of peptide transporter was assessed by performing in vitro uptake studies of [3H] Gly-sar on isolated mitochondria in the presence or absence of peptide transporter substrate (Val-Val). Molecular characterization of P-gp and peptide transporter was assessed by western blot and confocal analysis. Enhanced JC-1 accumulation in the isolated fraction confirmed mitochondrial membrane integrity. Significantly higher uptake of Rho-123 on isolated mitochondria was observed in the presence of quinidine (75 and 100 μM) and cyclosporine A (10 μM). Significantly lower uptake of [3H] Gly-sar was observed in the presence of val-val due to competitive inhibition of peptide transporter on isolated mitochondria. Western blot and confocal analysis further confirmed the presence of P-gp and peptide transporter on the mitochondrial membrane of rPCECs. The present study demonstrates the functional and molecular characterization of P-gp and peptide transporters in the mitochondrial membranes of rPCECs. This knowledge of mitochondrial existence of P-gp and peptide transporter will aid in the development of subcellular ocular drug delivery strategies.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri - Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA.
authors.forename: Megha
authors.initials: M
authors.lastname: Barot
authors.forename: Mitan R
authors.initials: MR
authors.lastname: Gokulgandhi
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D015153:Blotting, Western; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D016503:Drug Delivery Systems; D019573:Epithelium, Corneal / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D000074011:Peptide Transporter 1; D011802:Quinidine / Q000494:pharmacology; D011817:Rabbits; D020112:Rhodamine 123 / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001562:Benzimidazoles; D002232:Carbocyanines; D004151:Dipeptides; D005456:Fluorescent Dyes; D000074011:Peptide Transporter 1; D027981:Symporters; D020112:Rhodamine 123; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C004194:glycylsarcosine; C034792:valylvaline; D016572:Cyclosporine; D011802:Quinidine