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10th International Congress "Cell Volume Regulation: Novel Therapeutic Targets and Pharmacological Approaches"
TEMPERATURE DEPENDENCE OF VOLUME CHANGES TRIGGERED BY ANISOSMOTIC MEDIA IN INTACT AND PERMEABILIZED A549 CELLS
Platonova, A.A.1'2, Grygorczyk, R.1, and Orlov, S.N.1'2,
1 Research Centre, University of Montreal Hospital, Montreal, Canada
2 Department of Biophysics and Laboratory of Physical Chemistry of Biological Membranes, M.V. Lomonosov Moscow State University, Moscow, Russian Federation
Differing strikingly from aqueous solution, the gellike cytoplasm of all cells shows enhanced viscosity, slowed diffusion and an ability to retain water. However, the gel-like character of the cytoplasm has not been fully documented for mammalian cells, and its impact on cellular processes, such as volume sensing and regulation, remains unclear. Previously, we employed the dual-image, surface reconstruction (DISUR) technique based on phase-contrast, digital video microscopy [1] and revealed that gentle perforation of the surface membrane with digitonin preserves the gel-like character of the whole-cell cytoplasm and cytoplasm of permeabilized cells swells or shrinks in response to alterations of external osmolality [2]. In mammalian erythrocytes, regulation of volumesensitive ion transporter such as Na+,K+,2Cl- cotransport, Na+/H+ exchanger and K+,Cl- cotransport by anisosmotic medium was completely abolished by 10 min incubation at 48-50 °C [3, 4]. Keeping this in mind, we compared cell volume adjustment of intact and permeabilized A549 cells subjected to 10-min preincubation at 37 °C, 44 °C and 48 °C. In intact cells, 2-fold decrease of medium os-molality resulted in transient elevation of cell volume by 50% followed by it almost complete restoration in 30 min via regulatory volume decrease (RVD). Consistently with previous reports, shrinkage evoked by 30 min elevation of medium osmolality was not accompanied by regulatory volume increase (RVI). As predicted, plasma
membrane permeabilization completely abolished RVD. Ten min preincubation of intact, as well as permeabilized cells at 48 °C but not at 44 °C, completely blocked volume changes triggered by cell exposure to anisosmotic medium. Our results strongly suggest that temperature-dependent inhibition of volume-sensitive ion carries documented in early studies [3] is caused by inactiva-tion of osmosensing behaviour of cytoplasmic biogel rather than biochemical signalling involved in RVD and RVI.
References
1. Boudreault, F. and Grygorczyk, R. Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imaging. J. Microscopy, 2004, 215, pp. 302312.
2. Fels, J., Orlov, S.N., and Grygorczyk, R. The hydrogel nature of mammalian cytoplasm contributes to osmosensing and extracellular pH sensing. Biophys.J., 2009, 96, pp. 4276-4285.
3. Orlov, S.N., Kolosova, I.A., Cragoe, E.J., Gurlo, T.G., Mongin, A.A., Aksentsev, S.L., and Konev, S.V. Kinetics and peculiarities of thermal inactivation of volume-dependent Na/H exchange, Na,K,2Cl cotransport and K,Cl cotransport in rat erythrocytes. Biochim. Biophys. Acta, 1993, 1151, pp. 186-192.
4. Parshina, E.Yu., Yusipovich, A.I., Platonova, A.A., Grygorczyk, R., Maksimov, G.V., and Orlov, S.N. Thermal inac-tivation of volume-sensitive K+,Cl- cotransport and plasma membrane relief changes in human eyrthrocytes. Pfluger Arch. Eur. J. Physiol., 2013, in press.
Бюллетень сибирской медицины, 2013, том 12, № 4, с. 24-68
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