Rectification of the Water Permeability in COS-7 Cells at 22, 10 and 0°C

Citation: Peckys DB, Kleinhans FW, Mazur P ( Rectification of the Water Permeability in COS-7 Cells at 22, 10 and 06C Diana B. Peckys 0 F. W. Kleinhans 0 Peter Mazur 0 Boris Rubinsky, University of California at Berkeley, United States of America 0 1 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 2 Department of Physics, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana, United States of America, 3 Department of Biochemistry, and Cellular and Molecular Biology, University of Tennessee , Knoxville, Tennessee , United States of America The osmotic and permeability parameters of a cell membrane are essential physico-chemical properties of a cell and particularly important with respect to cell volume changes and the regulation thereof. Here, we report the hydraulic conductivity, Lp, the non-osmotic volume, Vb, and the Arrhenius activation energy, Ea, of mammalian COS-7 cells. The ratio of Vb to the isotonic cell volume, Vc iso, was 0.29. Ea, the activation energy required for the permeation of water through the cell membrane, was 10,700, and 12,000 cal/mol under hyper- and hypotonic conditions, respectively. Average values for Lp were calculated from swell/shrink curves by using an integrated equation for Lp. The curves represented the volume changes of 358 individually measured cells, placed into solutions of nonpermeating solutes of 157 or 602 mOsm/kg (at 0, 10 or 22uC) and imaged over time. Lp estimates for all six combinations of osmolality and temperature were calculated, resulting in values of 0.11, 0.21, and 0.10 mm/min/atm for exosmotic flow and 0.79, 1.73 and 1.87 mm/min/atm for endosmotic flow (at 0, 10 and 22uC, respectively). The unexpected finding of several fold higher Lp values for endosmotic flow indicates highly asymmetric membrane permeability for water in COS-7. This phenomenon is known as rectification and has mainly been reported for plant cell, but only rarely for animal cells. Although the mechanism underlying the strong rectification found in COS-7 cells is yet unknown, it is a phenomenon of biological interest and has important practical consequences, for instance, in the development of optimal cryopreservation. - Funding: Research supported by National Institutes of Health grant R01-RR018470, P. Mazur, PI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Water transport across cellular membranes is of crucial importance in animal and plant physiology. The permeability of a cell to water and the temperature coefficient of that permeability are two of its more important parameters. They, along with a cells permeability or lack thereof to solutes, determine the magnitude and kinetics of cell volume changes when the cell is subjected to conditions that depart from isotonic or isoosmotic. The permeability of a cell to water is usually referred to as the hydraulic conductivity, Lp. It has the units of volume divided by area6 pressure6time, or commonly, mm/atm. min. Water permeability is of particular importance in cryobiology, which is the main focus in our laboratory. The value of Lp is one of the chief factors determining the conditions under which ice forms or does not form in the interior of a cell [1,2]. Intracellular ice formation (IIF) is almost always lethal. If the cooling rate is low enough or if the Lp is high enough, the cell will dehydrate during cooling and will not undergo IIF. In contrast, if the cooling rate is too high or the Lp too low, the cell will not dehydrate rapidly enough to maintain osmotic or chemical equilibrium with the external ice and solution; the cell water will increasingly supercool and will eventually freeze in situ, usually with lethal consequences. For the past seven years, our laboratory has been studying various conditions under which IIF occurs or does not occur in several cell types; namely, mouse oocytes and early embryos [3,4], oocytes of the frog Xenopus [5,6] at various stages of development, V79 Chinese hamster tissue culture cells, and the yeast Saccharomyces cerevisiae [7]. Based on physical chemical equations and knowledge of certain parameters such as Lp and its temperature coefficient or activation energy, Ea, one can compute the likelihood of IIF as a function of temperature and cooling rate [2]. By comparing that likelihood with experimental observations on mouse oocytes, mouse embryos and on yeast cells, we have found the agreement to be excellent. The current paper deals with the water permeability of COS-7 fibroblasts. This tissue cell line is widely used in cell biology as a convenient protein expression system when specific proteins are to be studied. The average diameter of COS-7 cells (18.51 mm) is about two to three times that of most other mammalian cells, making its volume 8 to 27 times higher. Individual cell diameters range from 9 to 33 mm; hence, individual (isotonic) cell volumes of COS-7 cell can differ by as much as 44-fold. These facts made it of interest to determine experimentally the relation between IIF in these cells and cooling rate, to determine the temperature at which IIF occurs, and to compare the observed relation between cooling rate and IIF with the computed relation. As mentioned, that computation requires knowledge of Lp and its activation energy, Ea. Determining these parameters was the purpose of the present study. The standard way to determine Lp is to transfer cells from an isotonic solution to a hypotonic or hypertonic solution of an impermeant solute and determine the rate at which the cell swells or shrinks, respectively. Usually, the Lp that is calculated from the rate of swelling has about the same numerical value as the Lp calculated from the rate of shrinkage. But occasionally, the two values for Lp differ; i.e., the resistance of the membrane to inflow of water differs from its resistance to outflow. Such a difference is referred to as rectification. We have found that COS-7 cells exhibit extremely large apparent rectification; i.e., the value of Lp for the influx of water is as much as 18-fold larger than the value for the outflow of water. Materials and Methods Isotonic and anisotonic test solutions Test solutions were made from Tyrodes Buffered Saline (TBS). It has a measured osmolality of 0.308, a value that we define as isotonic. Hypotonic solutions were made by diluting TBS with HPLC grade water; hypertonic solutions were made by adding sucrose to the TBS (all the above were from Sigma Aldrich). All test solutions were prepared with a 20% higher or lower osmolality than the desired final concentration to compensate for two subsequent dilutions with 10% volumes of isotonic TBS. One dilution was used for adding 10 mM of the live-dead dye calcein AM (Invitrogen, Ca (...truncated)