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Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells

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Summary

The membrane electric effects of N,N′-dicyclohexylcarbodiimide (DCCD) and vanadate were studied in murine erythroleukemia cells (MELC), comparing the patch-clamp technique and the accumulation ratio (AR exp) of [3H]-tetraphenylphosphonium (TPP+). Electrophysiological measurements showed that both these inhibitors produce, at micromolar concentrations, a 20–30 mV hyperpolarization of resting potential (Δψ p ) of MELC, which is abolished when the electrochemical equilibrium potential of K+ (E K) is brought close to zero.

DCCD and vanadate turned out to have distinct targets on the plasma membrane of MELC (an H+ pump and the Na+,K+-ATPase, respectively).

Measurements of AR exp showed that: (i) patch-clamp measurements of Δψ p were equivalent to those based on ARexp of antimycin-pretreated cells (AR ANT); (ii) DCCD produced a strong increase in AR ANT, that was antagonized by carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and diethylstilbestrol (DES); (iii) vanadate determined a marked increase in AR ANT that was insensitive to FCCP, but antagonized by ouabain; (iv) incubation in high K+ medium (HK) brought ARANT to 1.0 in the controls, but did not lower this ratio below 3.0 in the presence of DCCD or vanadate; (v) the total amount of TPP+ taken up by the cells was in any case water extractable by a freezing and thawing procedure.

On the whole, our data indicate that DCCD and vanadate hyperpolarize the MELC by increasing the K+ conductance and, at the same time, enhance the TPP+ binding, probably by changing the electrostatic potential profile of the plasma membrane. These effects seem to involve functional modifications of the target pumps, apparently related to the ion-occluding state of these enzymes.

We gratefully acknowledge the revision of the English by Dr. R. Manning of the Department of Biological Sciences, University of Durham, Durham, UK.

A. Arcangeli was supported by a fellowship from the Associazione Italiana contro le Leucemie (AIL); A. Becchetti and M.R. Del Bene were supported by fellowships from the Italian Association for Cancer Research (AIRC).

This work was supported by grants from the AIRC, from the Consiglio Nazionale delle Ricerche (CNR) (Special project “Ion channels”) and from the Ministero Pubblica Istruzione.

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Authors and Affiliations

  1. Institute of General Pathology, University of Florence, 50134, Florence, Italy

    A. Arcangeli, M. R. Del Bene & M. Olivotto

  2. Department of General Physiology and Biochemistry, University of Milan, 20133, Milan, Italy

    A. Becchetti & E. Wanke

Authors
  1. A. Arcangeli
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  2. M. R. Del Bene
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  3. A. Becchetti
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  4. E. Wanke
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  5. M. Olivotto
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Additional information

The authors are indebted to Prof. A. Fonnesu, Chairman of the Institute of General Pathology of the University of Florence, for his support and advice. Thanks are also due to Prof. A. Ferroni, for the critical reading of the manuscript and to Prof. S. Capaccioli for his contribution to ATP determination. We are grateful to M. Cutrì for technical assistance.

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Arcangeli, A., Del Bene, M.R., Becchetti, A. et al. Effects of inhibitors of ion-motive ATPases on the plasma membrane potential of murine erythroleukemia cells. J. Membarin Biol. 126, 123–136 (1992). https://doi.org/10.1007/BF00231911

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  • DOI: https://doi.org/10.1007/BF00231911

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