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Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid

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Abstract

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

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Acknowledgements

This work was funded by FAPEMIG (Fundação de Amparo a Pesquisa do Estado de Minas Gerais) APQ-00290-16, PPM-00307-18, APQ-00855-19; CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) Finance Code 01, and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) 305173/2018-9, 409436/2016-0.

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  1. Laboratório de Bioquímica Celular, Universidade Federal de São João del Rei, Campus Centro-Oeste Dona Lindu, Av Sebastião Gonçalves Coelho, 400, Divinópolis, MG, Zip Code: 35501-296, Brazil

    Lilian N. D. Silva, Israel J. P. Garcia, Jessica M. M. Valadares, Marco Tulio C. Pessoa, Marina Marques Toledo, Herica L. Santos, Vanessa F. Cortes & Leandro A. Barbosa

  2. Laboratório de Bioquímica de Membranas e ATPases, Universidade Federal de São João del Rei, Campus Centro-Oeste Dona Lindu, Divinópolis, MG, Brazil

    Lilian N. D. Silva, Israel J. P. Garcia, Jessica M. M. Valadares, Marco Tulio C. Pessoa, Marina Marques Toledo, Herica L. Santos, Vanessa F. Cortes & Leandro A. Barbosa

  3. Laboratório de Síntese Orgânica e Nanoestruturas, Universidade Federal de São João del Rei, Campus Centro-Oeste Dona Lindu, Divinópolis, MG, Brazil

    Matheus V. Machado & José Augusto F. P. Villar

  4. Laboratório de Bioquímica de Lipídios, Instituto de Bioquímica Médica Leopoldo de Meis, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

    Mileane Souza Busch, Isabella Rocha & Georgia C. Atella

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  1. Lilian N. D. Silva
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Contributions

LNDS, IJPG wrote the main manuscript text. LNDS and IJPG performed the lipids and filipin experiments, JMMV and MMT performed the caveolae blotting, MTCP performed MTT experiments, MVM performed the 21-BD synthesis, MSB and IR performed the quantification of phospholipids. JAFPV, GCA, VFC, HLS, and LAB reviewed all the experiments, mentored the students, and reviewed the manuscript. GCA and LAB supported the paper with grants.

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Correspondence to Vanessa F. Cortes or Leandro A. Barbosa.

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Silva, L.N.D., Garcia, I.J.P., Valadares, J.M.M. et al. Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid. J Membrane Biol 254, 499–512 (2021). https://doi.org/10.1007/s00232-021-00203-z

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