Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers

doi:10.1016/0143-4160(90)90059-4

Cell Calcium

Volume 11, Issues 2–3, February–March 1990, Pages 57-62

https://doi.org/10.1016/0143-4160(90)90059-4 Get rights and content

Abstract

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facililate the use of Fura-2 in a variety of cell types.

References (16)

RY Tsien et al.
Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator
J. Cell Biol.
(1982)
P Arslan et al.
Cytosolic Ca²⁺ homeostasis in Ehrlich and Yoshida carcinomas
J. Biol. Chem.
(1985)
A Malgaroli et al.
Fura-2 measurement of cytosolic free Ca²⁺ in monolayers and suspensions of various types of animal cells
J. Cell Biol.
(1987)
SF Steinberg et al.
Fura-2 fluorescence is localized to mitochondria in endothelial cells
Am. J. Physiol.
(1987)
W Almers et al.
The Ca signal from Fura-2 loaded mast cells depends strongly on the method of dye-loading
FEBS Lett.
(1985)
F Di Virgilio et al.
Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions
J. Immunol.
(1988)
TH Steinberg et al.
Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix
J. Cell Biol.
(1987)
TH Steinberg et al.
A prelysosomal compartment sequesters membrane impermeable fluorescent dyes from the cytoplasmic matrix of J774 macrophages
J. Cell Biol.
(1988)

There are more references available in the full text version of this article.

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^∗: Present address: Washington University School of Medicine, Infectious Diseases, St Louis, Missouri, USA

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Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers

Abstract

Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator

J. Cell Biol.

Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas

J. Biol. Chem.

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

J. Cell Biol.

Fura-2 fluorescence is localized to mitochondria in endothelial cells

Am. J. Physiol.

The Ca signal from Fura-2 loaded mast cells depends strongly on the method of dye-loading

FEBS Lett.

Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions

J. Immunol.

Macrophages possess probenecid-inhibitable organic anion transporters that remove fluorescent dyes from the cytoplasmic matrix

J. Cell Biol.

A prelysosomal compartment sequesters membrane impermeable fluorescent dyes from the cytoplasmic matrix of J774 macrophages

J. Cell Biol.

Cytosolic Ca²⁺ homeostasis in Ehrlich and Yoshida carcinomas

Fura-2 measurement of cytosolic free Ca²⁺ in monolayers and suspensions of various types of animal cells