Different storing and processing conditions of human lymphocytes do not alter P-glycoprotein rhodamine 123 efflux.
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.
Chiva-Blanch
Gemma
G
Nephrology Department, IDIBELL-Hospital Universitari Bellvitge, L'Hospitalet de Llobregat, Catalonia, Spain.
Giménez-Bonafe
Pepita
P
Llaudó
Inés
I
Torras
Joan
J
Cruzado
Josep M
JM
Petriz
Jordi
J
Castaño
Esther
E
Franquesa
Marcel la
Ml
Herrero-Fresneda
Immaculada
I
Tortosa
Avelina
A
Rama
Inés
I
Bestard
Oriol
O
Grinyó
Josep M
JM
Lloberas
Núria
N
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
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