Washing out of lipophilic compounds induces a transient increase in the passive Ca2+ leak in permeabilized A7r5 cells

doi:10.1016/S0143-4160(02)00051-9

Cell Calcium

Volume 31, Issue 5, May 2002, Pages 229-233

https://doi.org/10.1016/S0143-4160(02)00051-9 Get rights and content

Abstract

We have investigated how the immunosuppressant drug FK506 affected the basal Ca²⁺ leak in permeabilized A7r5 cells. Non-mitochondrial Ca²⁺ stores loaded to steady state with Ca²⁺ slowly lost their accumulated Ca²⁺ during incubation in a Ca²⁺-free efflux medium. FK506 up to 100 μM had no effect on the basal Ca²⁺ leak. In contrast, the rate of Ca²⁺ release proceeded much faster immediately after washing out FK506. The increase in rate of Ca²⁺ release after washing out of this compound depended on both its initial concentration and on the time of pre-incubation. A similar effect was also observed after removing another immunosuppressant drug (rapamycin) and after removing the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin C. Since all these substances have a high octanol/H₂O partition coefficient and accumulate in the endoplasmic reticulum membrane, we suggest that the transient increase in the basal Ca²⁺ leak is due to the sudden removal of these lipophilic substances from the membrane.

Section snippets

INTRODUCTION

The immunophilins are a family of proteins that mediate the actions of immunosuppressant drugs like FK506 (tacrolimus) 1., 2., 3.. FK506, a macrocyclic lactone antibiotic and potent immunosuppressive agent, binds to the immunophilin FKBP12. The FK506–FKBP12 complex then inhibits calcineurin, an essential enzyme for T-cell activation [4].

It recently became apparent that FKBP12 also associates with the three different isoforms of the ryanodine receptor (RyR) 5., 6., 7.. This association between

MATERIALS AND METHODS

A7r5 cells, an established cell line derived from embryonic rat aorta, were used between the 7th and the 17th passage after receipt from the American Type Culture Collection (Bethesda, MD, USA). The cells were cultured at 37 °C in a 9% CO₂ incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 3.8 mM l-glutamine, 0.9% (v/v) non-essential amino acids, 85 IU/ml penicillin and 85 μg/ml streptomycin. The cells were seeded in 12-well dishes (Costar, MA, USA; 4 cm²) at a

RESULTS AND DISCUSSION

Permeabilized monolayers of A7r5 smooth-muscle cells form a useful model system for investigating unidirectional Ca²⁺ release out of the intracellular stores 15., 16.. Since the passive Ca²⁺ leak from the internal Ca²⁺ stores can accurately be determined [12], we have used these cells to measure the effects of FK506 and of other lipophilic compounds on the passive Ca²⁺ leak. Permeabilized A7r5 cells loaded to steady state with Ca²⁺ slowly lost their accumulated Ca²⁺ during incubation in a Ca²⁺

Acknowledgements

This research was financed by the Interuniversity Poles of Attraction Program—Belgian State, Prime Minister’s Office—Federal Office for Scientific, Technical and Cultural Affairs IUAP P4/23 and by Grant 3.0207.99 of the Foundation for Scientific Research—Flanders. G.B. was supported by the “Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie (I.W.T.)” and has now a postdoctoral fellowship of the “Onderzoeksraad—K.U.Leuven.” We thank Dr. A.

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Washing out of lipophilic compounds induces a transient increase in the passive Ca2+ leak in permeabilized A7r5 cells

Abstract

Section snippets

INTRODUCTION

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Acknowledgements

Cell

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J. Biol. Chem.

Cell Calcium

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

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Biophys. J.

J. Biol. Chem.

Neuron

Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation

Nature

FK506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin

Nature

Washing out of lipophilic compounds induces a transient increase in the passive Ca²⁺ leak in permeabilized A7r5 cells