title: Organic cation rhodamines for screening organic cation transporters in early stages of drug development.
abstract: The aim of this study was to investigate the suitability of rhodamine-123, rhodamine-6G and rhodamine B as non-radioactive probes for characterizing organic cation transporters in respiratory cells. Fluorescent characteristics of the compounds were validated under standard in vitro drug transport conditions (buffers, pH, and light). Uptake/transport kinetics and intracellular accumulation of the compounds were investigated. Uptake/transport mechanisms were investigated by comparing the effect of pH, temperature, concentration, polarity, OCTs/OCTNs inhibitors/substrates, and metabolic inhibitors on the cationic dyes uptake in Calu-3 cells. Fluorescence stability and intensity of the compounds were altered by buffer composition, light, and pH. Uptake of the dyes was concentration-, temperature- and pH-dependent. OCTs/OCTNs inhibitors significantly reduced intracellular accumulation of the compounds. Whereas rhodamine-B uptake was sodium-dependent, pH had no effect on rhodamine-123 and rhodamine-6G uptake. Transport of the dyes across the cells was polarized: (AP→BL>BL→AP transport) and saturable: {Vmax=14.08±2.074, Km=1821±380.4 (rhodamine-B); Vmax=6.555±0.4106, Km=1353±130.4 (rhodamine-123) and Vmax=0.3056±0.01402, Km=702.9±60.97 (rhodamine-6G)}. The dyes were co-localized with MitoTracker®, the mitochondrial marker. Cationic rhodamines, especially rhodamine-B and rhodamine- 6G can be used as organic cation transporter substrates in respiratory cells. During such studies, buffer selection, pH and light exposure should be taken into consideration.
authors.affiliation: Biopharmaceutics and Drug Delivery Lab, College of Pharmacy, 5968 College Street, PO Box 15000, Halifax, NS B3H 4R2, Canada; Department of Pharmaceutical Microbiology & Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Near NAFDAC Zonal Lab, Agulu, Anambra State, Nigeria.
authors.forename: Malachy C
authors.initials: MC
authors.lastname: Ugwu
authors.affiliation: Biopharmaceutics and Drug Delivery Lab, College of Pharmacy, 5968 College Street, PO Box 15000, Halifax, NS B3H 4R2, Canada; Department of Pharmaceutical Microbiology & Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Near NAFDAC Zonal Lab, Agulu, Anambra State, Nigeria.
authors.forename: Angus
authors.initials: A
authors.lastname: Oli
authors.affiliation: Department of Pharmaceutical Microbiology & Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Near NAFDAC Zonal Lab, Agulu, Anambra State, Nigeria.
authors.forename: Charles O
authors.initials: CO
authors.lastname: Esimone
authors.affiliation: Biopharmaceutics and Drug Delivery Lab, College of Pharmacy, 5968 College Street, PO Box 15000, Halifax, NS B3H 4R2, Canada. Electronic address: Remigius.agu@dal.ca.
authors.forename: Remigius U
authors.initials: RU
authors.lastname: Agu
mesh_terms: D001692:Biological Transport; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000493:pharmacokinetics*; D006801:Humans; D008954:Models, Biological; D027701:Organic Cation Transport Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism; D020112:Rhodamine 123 / Q000737:chemistry / Q000493:pharmacokinetics; D012235:Rhodamines / Q000737:chemistry / Q000493:pharmacokinetics*; D019810:Sodium Azide / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D027701:Organic Cation Transport Proteins; D012235:Rhodamines; C026188:rhodamine 6G; D020112:Rhodamine 123; D019810:Sodium Azide; C029773:rhodamine B
keywords: Calu-3 cells; Carnitine/organic cation transporter (OCTN1, OCTN2); Fluorescence; Organic cation transporters (OCT1–3); Rhodamines