Ukr.Biochem.J. 2015; Volume 87, Issue 5, Sep-Oct, pp. 61-71

doi: https://doi.org/10.15407/ubj87.05.061

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

Yu. V. Danylovych, S. A. Karakhim, H. V. Danylovych, O. V. Kolomiets, S. O. Kosterin

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: danylovych@biochem.kiev.ua

We demonstrated using Ca2+-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes’ inner mitochondrial membrane by a protonophore carbonyl cyanide m-chlorophenyl hуdrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3′-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane’s electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hуdrazone causes substantial increase in fluorescence of Ca2+-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca2+-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

Keywords: , , , ,


References:

  1. Kosterin SA, Burdyga ThV, Fomin VP. et al. “Mechanism of Ca2+ transport in myometrium”. Control of Uterine Contractility, Chap. 6, Garfield R. and Tabb T. (eds.), CRC Press Boca Raton, N. Y., 1994: 129-153.
  2. Kostyuk PG., Kostyuk OP, Lukyanets EA. Intracellular calcium signaling: structures and functions. Kiev: Naukova dumka, 2010. 175 p. (In Ukrainian).
  3. Burdyga T, Poul RJ. Calcium homeostasis and signaling in smooth muscle. Muscle: Fundamental Biology and Mechanisms of Disease: chapter 86. 2012: 1155-1172 с.
  4. Csordás G, Várnai P, Golenár T, Sheu SS, Hajnóczky G. Calcium transport across the inner mitochondrial membrane: molecular mechanisms and pharmacology. Mol Cell Endocrinol. 2012 Apr 28;353(1-2):109-13. Epub 2011 Nov 22. Review. PubMed, PubMedCentral, CrossRef
  5. Kosterin SO. Calcium transport in smooth muscle. Kiev: Naukova dumka, 1990. 216 p. (In Russian).
  6. Kolomiets ОV, Danylovych YuV, Danylovych HV, Kosterin SO. Ca2+/H+-exchange in myometrium mitochondria. Ukr Biochem J. 2014 May-Jun;86(3):41-8. Ukrainian. PubMed, CrossRef
  7. Vovkanych LS, Dubytsky LO. Kinetical properties of the H+-stimulated rat liver mitochondria Ca2+ efflux. Exp Clin Physiol Biochem. 2001; 3(5): 34-37. (In Ukrainian).
  8. Kucherenko ME, Voytsitskyy VM. Bioenergy. Kiyv, Vyscha shcola, 1982. 272 p. (In Russian).
  9. Mollard P, Mironneau J, Amedee T, Mironneau C. Electrophysiological characterization of single pregnant rat myometrial cells in short-term primary culture. Am J Physiol. 1986 Jan;250(1 Pt 1):C47-54. PubMed
  10. Danylovych YuV, Danylovych HV, Kolomiets ОV, Kosterin SO, Karakhim SA, Chunikhin AYu. Investigation of nitrosactive compounds influence on polarization of the mitochondrial inner membrane in the rat uterus myocytes using potential sensitive fluorescent probe DiOC6(3). Ukr Biochem J. 2014 Jan-Feb;86(1):42-55. Ukrainian. PubMed, CrossRef
  11. Kolomiiets’ OV, Danylovych IuV, Danylovych HV, Kosterin SO. Ca2+ accumulation study in isolated smooth muscle mitochondria using fluo-4 AM. Ukr Biokhim Zhurn. 2013 Jul-Aug;85(4):30-9. Ukrainian. PubMed, CrossRef
  12. Kucherenko ME, Babenuk YuD, Voytsitskyy VM. Modern methods of biochemical studies: tutorials. Kiyv: Fitosotsiotsentr, 2001. 424 p. (In Russian).
  13. Garcia Fernandez MI, Ceccarelli D, Muscatello U. Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models. Anal Biochem. 2004 May 15;328(2):174-80. PubMedCrossRef
  14. Evron Y, McCarty RE. Simultaneous measurement of deltapH and electron transport in chloroplast thylakoids by 9-aminoacridine fluorescence. Plant Physiol. 2000 Sep;124(1):407-14. PubMed, PubMedCentral, CrossRef
  15. Marchetti C, Jouy N, Leroy-Martin B, Defossez A, Formstecher P, Marchetti P. Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility. Hum Reprod. 2004 Oct;19(10):2267-76. PubMed, CrossRef
  16. Kalbácová M, Vrbacký M, Drahota Z, Melková Z. Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry. Cytometry. 2003 Apr;52A(2):110-6. PubMedCrossRef
  17. Danylovych HV, Danylovych YuV, Gorchev VF. Comparative investigation by spectrofluorimetry and flow cytometry of plasma and inner mitochondrial membranes polarization in smooth muscle cell using potential-sensitive probe DiOC6(3). Ukr Biokhim Zhurn. 2011 May-Jun;83(3):99-105. (In Ukrainian). PubMed
  18. Babich LG, Shlykov SG, Borisova LA, Kosterin SA. Energy-dependent Ca2+-transport in intracellular smooth muscle structures. Biokhimiia. 1994 Aug;59(8):1218-29. Russian. PubMed
  19. Karapetyants MH, Drakyn SI. General and nonorganic chemistry. Moscow: Chemistry, 1981. 630 p. (In Russian).
  20. Chang S, Lamm SH. Human health effects of sodium azide exposure: a literature review and analysis. Int J Toxicol. 2003 May-Jun;22(3):175-86. Review. PubMed, CrossRef
  21. Ji D, Kamalden TA, del Olmo-Aguado S, Osborne NN. Light- and sodium azide-induced death of RGC-5 cells in culture occurs via different mechanisms. Apoptosis. 2011 Apr;16(4):425-37. PubMed, CrossRef
  22. Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes. Cell Calcium. 2000 Feb;27(2):97-106. PubMedCrossRef
  23. Iakovenko IN, Zhirnov VV. Sodium azide as indirect nitric oxide donor: researches on the rat aorta isolated segments. Ukr Biokhim Zhurn. 2005 Jul-Aug;77(4):120-3. Russian. PubMed

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License.