Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance

C. Cao, T. H. Steinberg, H. C. Neu, D. Cohen, Susan Band Horwitz, S. Hickman, S. C. Silverstein

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Macrophages possess organic anion transporters that carry membrane- impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxyfluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug- resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical. In addition, Northern blot analysis showed much lower levels of mdr mRNA in PBR J774 cells than in J7.C1 cells. These observations indicate that organic anion transporter activity and multiple drug resistance are regulated independently of each other in J774 cells; they also suggest that organic anion transport in macrophages is mediated by protein(s) other than P-glycoprotein.

Original languageEnglish (US)
Pages (from-to)193-200
Number of pages8
JournalInfectious Agents and Disease
Volume2
Issue number4
StatePublished - 1993
Externally publishedYes

Fingerprint

Probenecid
Multiple Drug Resistance
Anions
Organic Anion Transporters
P-Glycoprotein
lucifer yellow
Macrophages

Keywords

  • Macrophages
  • Multiple drug resistance
  • Organic anion transporters
  • Probenecid

ASJC Scopus subject areas

  • Microbiology (medical)

Cite this

Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance. / Cao, C.; Steinberg, T. H.; Neu, H. C.; Cohen, D.; Band Horwitz, Susan; Hickman, S.; Silverstein, S. C.

In: Infectious Agents and Disease, Vol. 2, No. 4, 1993, p. 193-200.

Research output: Contribution to journalArticle

Cao, C. ; Steinberg, T. H. ; Neu, H. C. ; Cohen, D. ; Band Horwitz, Susan ; Hickman, S. ; Silverstein, S. C. / Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance. In: Infectious Agents and Disease. 1993 ; Vol. 2, No. 4. pp. 193-200.
@article{ab2f03d7f7924e36bb97531bbd8f2e2d,
title = "Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance",
abstract = "Macrophages possess organic anion transporters that carry membrane- impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxyfluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug- resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical. In addition, Northern blot analysis showed much lower levels of mdr mRNA in PBR J774 cells than in J7.C1 cells. These observations indicate that organic anion transporter activity and multiple drug resistance are regulated independently of each other in J774 cells; they also suggest that organic anion transport in macrophages is mediated by protein(s) other than P-glycoprotein.",
keywords = "Macrophages, Multiple drug resistance, Organic anion transporters, Probenecid",
author = "C. Cao and Steinberg, {T. H.} and Neu, {H. C.} and D. Cohen and {Band Horwitz}, Susan and S. Hickman and Silverstein, {S. C.}",
year = "1993",
language = "English (US)",
volume = "2",
pages = "193--200",
journal = "Infectious Agents and Disease",
issn = "1056-2044",
publisher = "Raven Press",
number = "4",

}

TY - JOUR

T1 - Probenecid-resistant J774 cell expression of enhanced organic anion transport by a mechanism distinct from multidrug resistance

AU - Cao, C.

AU - Steinberg, T. H.

AU - Neu, H. C.

AU - Cohen, D.

AU - Band Horwitz, Susan

AU - Hickman, S.

AU - Silverstein, S. C.

PY - 1993

Y1 - 1993

N2 - Macrophages possess organic anion transporters that carry membrane- impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxyfluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug- resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical. In addition, Northern blot analysis showed much lower levels of mdr mRNA in PBR J774 cells than in J7.C1 cells. These observations indicate that organic anion transporter activity and multiple drug resistance are regulated independently of each other in J774 cells; they also suggest that organic anion transport in macrophages is mediated by protein(s) other than P-glycoprotein.

AB - Macrophages possess organic anion transporters that carry membrane- impermeant fluorescent dyes, such as lucifer yellow (LY) and carboxyfluorescein, from the cytoplasm into endosomes and out of the cells. Probenecid, an organic anion transport inhibitor, blocks these processes. Prolonged incubation of J774 cells in medium containing 2.5 mM probenecid eventually kills most of these cells. To identify J774 variants that express increased organic anion transport activity, we selected probenecid-resistant (PBR) J774 cells by growing them in medium containing increasing concentrations of probenecid. When PBR and unselected J774 cells were loaded with LY by ATP4- permeabilization, the amount of LY accumulated by the PBR cells was about half that in the unselected cells. This difference was abolished by adding 10 mM probenecid to the medium in which the cells were loaded, suggesting that the diminished LY accumulation in PBR cells was due to enhanced LY secretion and that the PBR cells expressed increased organic anion transport activity. Direct comparison of LY efflux from J774 and PBR J774 cells showed a faster initial rate of secretion of LY from PBR J774 cells than from unselected J774 cells. To determine whether LY efflux is mediated by P-glycoprotein, we compared LY efflux in unselected J774 cells, PBR J774 cells, and multidrug-resistant J774 cells (J7.C1). LY efflux from J7.C1 cells was not sensitive to verapamil, which inhibits multidrug- resistance transporters, and reverses the multidrug-resistant phenotype of J7.C1 cells. The rates of LY efflux from unselected J774 and J7.C1 cells were virtually identical. In addition, Northern blot analysis showed much lower levels of mdr mRNA in PBR J774 cells than in J7.C1 cells. These observations indicate that organic anion transporter activity and multiple drug resistance are regulated independently of each other in J774 cells; they also suggest that organic anion transport in macrophages is mediated by protein(s) other than P-glycoprotein.

KW - Macrophages

KW - Multiple drug resistance

KW - Organic anion transporters

KW - Probenecid

UR - http://www.scopus.com/inward/record.url?scp=0027853073&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027853073&partnerID=8YFLogxK

M3 - Article

VL - 2

SP - 193

EP - 200

JO - Infectious Agents and Disease

JF - Infectious Agents and Disease

SN - 1056-2044

IS - 4

ER -