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Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids

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Abstract

Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green–dextran–tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.

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Abbreviations

BAF:

Bafilomycin A1

CAD:

Cationic amphiphilic drug

CLSM:

Confocal laser scanning microscopy

CPZ:

Chlorpromazine

dex:

Dextran

DIPL:

Drug-induced phospholipidosis

HPLC:

High-performance liquid chromatography

LAMP2:

Lysosomal-associated membrane protein 2

MALDI-TOF:

Matrix-assisted laser desorption-time of flight

MON:

Monensin sodium salt

NHE:

Na+/H+ exchanger

OG:

Oregon Green 488

PMSF:

Phenylmethylsulfonyl fluoride

TMR:

Tetramethylrhodamine

v-ATPase:

Vacuolar ATPase

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Acknowledgments

This work was financially supported by JSPS KAKENHI Grant Number 21790051. This public sponsor had no role in the design of the study, in its execution or in the decision to submit the manuscript for publication. The authors extend appreciation to Dr Ken-ichi Akagi and Dr Sumie Katayama at the National Institute of Biomedical Innovation for obtaining the transmission electron microscopic images.

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  1. School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11-68, Koshien Kyuban-cho, Nishinomiya, Hyogo, 663-8179, Japan

    Ryohei Hamaguchi, Jun Haginaka, Toshiko Tanimoto & Yukihiro Kuroda

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  1. Ryohei Hamaguchi
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  2. Jun Haginaka
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  3. Toshiko Tanimoto
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  4. Yukihiro Kuroda
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Correspondence to Yukihiro Kuroda.

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Hamaguchi, R., Haginaka, J., Tanimoto, T. et al. Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids. Cell Biol Toxicol 30, 67–77 (2014). https://doi.org/10.1007/s10565-014-9269-2

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