EXPERIMENTAL CELL RESEARCH 227, 277–284 (1996) ARTICLE NO. 0277 Hydrostatic Pressure to 400 atm Does Not Induce Changes in the Cytosolic Concentration of Ca 2/ in Mouse Fibroblasts: Measurements Using Fura-2 Fluorescence HUGH C. CRENSHAW 1,2 AND E. D. SALMON Department of Biology, CB 3280, University of North Carolina, Chapel Hill, North Carolina 27599-3280 INTRODUCTION Hydrostatic pressure has a pronounced effect on the Extreme hydrostatic pressures (bathymetric pres- morphology and cytoskeletal organization of mamma- sures) profoundly affect the physiology of surface- lian tissue cells. At pressures of about 300 atm (30 dwelling organisms [1]. These include such diverse ef- MPa), cells ‘‘round up’’ — they withdraw their lamellar fects as a positive inotropic effect on muscle contraction extensions and greatly rearrange actin, tubulin, and at pressures greater than 10 atm (1 MPa) [2], high several other cytoskeletal proteins. It has been pro- pressure nervous syndrome which affects humans at posed that these changes are caused by a pressure- pressures exceeding 20 atm (2 MPa) [3], and disruption induced elevation of cytosolic Ca 2/ concentrations. To of the cytoskeleton of vertebrate tissue cells at pres- test this hypothesis we constructed a miniature optical sures above 250 atm (25 MPa) [4, 5]. pressure chamber for fluorescent light microscopy to allow measurement of cytosolic Ca 2/ concentrations The cytological and ultrastructural analyses of Mars- with the intracellular fluorescent indicator fura-2. land, Landau, Zimmerman, and Salmon (for reviews This chamber and fura-2 were used to measure the see [4, 6, 7]) document that many cytoskeletal elements concentrations of Ca 2/ in a mouse fibroblast line (C 3 H are either depolymerized or reorganized by pressures 10T1/2) at pressures up to 400 atm (40 MPa). Controls ranging from 200 to 400 atm. At pressures exceeding included in vitro tests with standard buffers to deter- about 300 atm, accompanying this disruption of cy- mine the effect of pressure on fura-2 fluorescence. toskeletal organization, many mammalian tissue cells These controls detected a change in fura-2 fluores- ‘‘round up’’ — they go from being flattened against their cence with increasing pressure, but the data indicated substrate to being nearly spherical. that pressure affects fura-2 fluorescence indirectly, by Several mechanisms can possibly explain pressure’s altering the pH of the solution via pressure-induced alteration of cytoskeletal organization. One possible changes in the ionization of the pH buffer. These in mechanism is that pressure directly alters the dynam- vitro changes in fura-2 fluorescence, nevertheless, ics of cytoskeletal assembly. Cytoskeletal elements are were small relative to changes in fura-2 fluorescence polymeric proteins. Inside the cell these proteins are produced by elevation in intracellular Ca 2/ concentra- in a steady-state equilibrium between monomer and tions in response to physiological stimulation of the polymer. Pressure can affect the kinetics of polymer cells (serum feeding after serum starvation). The assembly, driving equilibrium toward depolymeriza- mouse fibroblasts rounded at pressures of 275 atm or tion when the molar volume of polymer is greater than greater, as expected. However, no changes in cytosolic the molar volume of monomer [8, 9]. For example, in- Ca 2/ concentrations were detected at any pressure, at creases in molar volume of approximately 100 the onset of pressure, during periods of high pressure mlrmol 01 have been measured in vitro for the polymer- (up to 10 min), or at the release of pressure. These ization of myosin filaments [10 – 14], microtubules [15], results strongly suggest that the mechanism by which pressure alters cell morphology and cytoskeletal orga- and actin microfilaments [16 – 19]. Pressure-induced nization must, at least in these cells, be something depolymerization of tubulin in the meiotic spindle of other than elevation of cytosolic Ca 2/ concentrations. an invertebrate egg closely follows the predictions of a  1996 Academic Press, Inc. simple equilibrium model based on changes in the mo- lar volume [15]. In other cells, however, pressure ef- fects on the cytoskeleton are complex and are not di- rectly predictable from in vitro analyses [4, 20]. 1 Current address: Department of Zoology, Box 90325, Duke Uni- A second possible mechanism is that pressure alters versity, Durham, NC 27708-0325. some cellular control system that regulates the dynam- 2 To whom correspondence and reprint requests should be sent. Fax: (919) 684-6168; E-mail: crenshaw@acpub.duke.edu. ics of cytoskeletal assembly. Otter et al. [4] proposed 277 0014-4827/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.