Hydrostatic pressure to 400 atm does not induce changes in the cytosolic concentration of Ca2+ in mouse fibroblasts: measurements using fura-2 fluorescence.
Hydrostatic pressure has a pronounced effect on the morphology and cytoskeletal organization of mammalian tissue cells. At pressures of about 300 atm (30 MPa), cells "round up"-they withdraw their lamellar extensions and greatly rearrange actin, tubulin, and several other cytoskeletal proteins. It has been proposed that these changes are caused by a pressure-induced elevation of cytosolic Ca2+ concentrations. To test this hypothesis we constructed a miniature optical pressure chamber for fluorescent light microscopy to allow measurement of cytosolic Ca2+ concentrations with the intracellular fluorescent indicator fura-2. This chamber and fura-2 were used to measure the concentrations of Ca2+ in a mouse fibroblast line (C3H 10T1/2) at pressures up to 400 atm (40 MPa). Controls included in vitro tests with standard buffers to determine the effect of pressure on fura-2 fluorescence. These controls detected a change in fura-2 fluorescence with increasing pressure, but the data indicated that pressure affects fura-2 fluorescence indirectly, by altering the pH of the solution via pressure-induced changes in the ionization of the pH buffer. These in vitro changes in fura-2 fluorescence, nevertheless, were small relative to changes in fura-2 fluorescence produced by elevation in intracellular Ca2+ concentrations in response to physiological stimulation of the cells (serum feeding after serum starvation). The mouse fibroblasts rounded at pressures of 275 atm or greater, as expected. However, no changes in cytosolic Ca2+ concentrations were detected at any pressure, at the onset of pressure, during periods of high pressure (up to 10 min), or at the release of pressure. These results strongly suggest that the mechanism by which pressure alters cell morphology and cytoskeletal organization must, at least in these cells, be something other than elevation of cytosolic Ca2+ concentrations.
Crenshaw
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Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA. crenshaw@acpub.duke.edu
Salmon
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D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D051379:Mice; D008809:Mice, Inbred C3H
D002021:Buffers; D005456:Fluorescent Dyes; D002118:Calcium; D016257:Fura-2