title: Different storing and processing conditions of human lymphocytes do not alter P-glycoprotein rhodamine 123 efflux.
abstract: P-glycoprotein (Pgp), a protein codified by Multi Drug Resistance (MDR1) gene, has a detoxifying function and might influence the toxicity and pharmacokinetics and pharmacodynamics of drugs. Sampling strategies to improve Pgp studies could be useful to optimize the sensitivity and the reproducibility of efflux assays. This study aimed to compare Pgp expression and efflux activity by measuring Rhodamine123 (Rh123) retention in lymphocytes stored under different conditions, in order to evaluate the potential utility of any of the storing conditions in Pgp functionality. Our results show no change in protein expression of Pgp by confocal studies and Western blotting, nor changes at the mRNA level (qRT-PCR). No differences in Rh123 efflux by Pgp activity assays were found between fresh and frozen lymphocytes after 24 hours of blood extraction, using either of the two Pgp specific inhibitors (VP and PSC833). Different working conditions in the 24 hours post blood extraction do not affect Rh123 efflux. These results allow standardization of Pgp activity measurement in different individuals with different timing of blood sampling and in different geographic areas.
authors.affiliation: Nephrology Department, IDIBELL-Hospital Universitari Bellvitge, L'Hospitalet de Llobregat, Catalonia, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Chiva-Blanch
authors.forename: Pepita
authors.initials: P
authors.lastname: Giménez-Bonafe
authors.forename: Inés
authors.initials: I
authors.lastname: Llaudó
authors.forename: Joan
authors.initials: J
authors.lastname: Torras
authors.forename: Josep M
authors.initials: JM
authors.lastname: Cruzado
authors.forename: Jordi
authors.initials: J
authors.lastname: Petriz
authors.forename: Esther
authors.initials: E
authors.lastname: Castaño
authors.forename: Marcel la
authors.initials: Ml
authors.lastname: Franquesa
authors.forename: Immaculada
authors.initials: I
authors.lastname: Herrero-Fresneda
authors.forename: Avelina
authors.initials: A
authors.lastname: Tortosa
authors.forename: Inés
authors.initials: I
authors.lastname: Rama
authors.forename: Oriol
authors.initials: O
authors.lastname: Bestard
authors.forename: Josep M
authors.initials: JM
authors.lastname: Grinyó
authors.forename: Núria
authors.initials: N
authors.lastname: Lloberas
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000328:Adult; D017304:Annexin A5 / Q000235:genetics; D017209:Apoptosis; D017252:CD3 Complex; D045744:Cell Line, Tumor; D002470:Cell Survival; D015925:Cryopreservation; D003609:Dactinomycin / Q000031:analogs & derivatives; D005260:Female; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000502:physiology*; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D020112:Rhodamine 123 / Q000378:metabolism*; D013997:Time Factors; D055815:Young Adult
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017304:Annexin A5; D017252:CD3 Complex; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D003609:Dactinomycin; D020112:Rhodamine 123; C025942:7-aminoactinomycin D