P-glycoprotein inhibitors modulate accumulation and efflux of xenobiotics in extra and intracellular Toxoplasma gondii.
We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.
Sauvage
Virginie
V
Laboratoire de Parasitologie-Mycologie, UFR de Médecine, UPRES EA 2070, IFR53 CHU, 51 rue Cognacq Jay, Reims 51096, France.
Aubert
Dominique
D
Bonhomme
Annie
A
Pinon
Jean-Michel
JM
Millot
Jean-Marc
JM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002232:Carbocyanines / Q000378:metabolism; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology*; D003630:Daunorubicin / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D014122:Toxoplasma / Q000187:drug effects* / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology*; D015262:Xenobiotics / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D015262:Xenobiotics; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D016572:Cyclosporine; D014700:Verapamil; D003630:Daunorubicin