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Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation

M. V. Espelt¹, P. N. Mut¹, G. Amodeo², G. Krumschnabel³ and P. J. Schwarzbaum^1,*

¹ Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, C1113AAD Buenos Aires, Argentina
² Laboratorio de Biomembranas (Facultad de Medicina), Universidad de Buenos Aires, C1121ABG Buenos Aires, Argentina
³ Institut für Zoologie, Abteilung für Ökophysiologie, Universität Innsbruck, A-6020, Austria

View larger version (14K): [in a new window]   Fig. 2. Time course of volume changes in hepatocytes from goldfish, trout and rat, assessed by quantitative phase-contrast microscopy. (A) Total volume (V) changes of hepatocytes from goldfish exposed to 180 mosmol l-1 medium at pH 7.45. Hepatocytes were isolated in collagenase-containing medium (filled circles) or in collagenase-free medium (open circles). (B) Relative volume (Vr) changes of hepatocytes of rat (triangles) and trout (squares) exposed to 180 mosmol l-1 medium at pH 7.45 and of goldfish hepatocytes (circles) exposed to 180 mosmol l-1 at pH 7.8. Values are means ± S.E.M. (N=4). Lines represent the empirical fit to data, with values of the parameters given in Table 1 and in the Results section.

View larger version (23K): [in a new window]   Fig. 1. Time course of total volume (V) change in goldfish hepatocytes, assessed by quantitative phase-contrast microscopy. Cells were exposed to media of the various osmolarities indicated at pH 7.45 and to L-alanine, aminooxyacetic acid (ALA-AOA). Results are means ± S.E.M. (N=4; in certain points, the error bars lie within the symbol). Lines represent the exponential fit to experimental data with parameters of best fit given in Table 1. The inset shows phase-contrast micrographs of goldfish hepatocytes in 300 mosmol l-1 (top panels) and 120 mosmol l-1 media (bottom panels).

View larger version (23K): [in a new window]   Fig. 3. Relative volume (Vr) versus time in goldfish hepatocytes incubated in control medium A or in medium with (A) iodoacetic acid (IAA) or (B) cyanide plus iodoacetic acid (CN-+IAA). Calibration was performed with anisosmotic media of 264 mosmol l-1 (medium H) and 308 mosmol l-1 (medium J). Results are means ± S.E.M. (N=4).

View larger version (22K): [in a new window]   Fig. 4. Relative volume (Vr) versus time in hepatocytes from (A) goldfish and (B) rat incubated in control media (denoted as A for goldfish cells and C for rat cells) or in medium with cyanide (CN-). Calibration was performed with anisosmotic media as follows: (A) 264 mosmol l-1 (medium H) and 308 mosmol l-1 (medium J); (B) 225 mosmol l-1 (medium H) and 293 mosmol l-1 (medium J). Results are means ± S.E.M. (N=4).

View larger version (19K): [in a new window]   Fig. 5. Rates of 86Rb+ influx in goldfish hepatocytes during 44 min of exposure to control medium (A), 180 mosmol l-1 medium (HY), isosmotic media with cyanide (CN-), iodoacetic acid (IAA) and cyanide plus iodoacetic acid (CN-+IAA). Fluxes were measured at 4 min, 14 min, 24 min and 44 min after the start of the experiment. Results are means + S.E.M. (N=4). An asterisk indicates P<0.05 with respect to isosmotic control values.

View larger version (19K): [in a new window]   Fig. 6. Rates of 86Rb+ efflux in goldfish hepatocytes during 44 min of exposure to control medium (A), 180 mosmol l-1 medium (HY), isosmotic media with cyanide (CN-), iodoacetic acid (IAA), and cyanide plus iodoacetic acid (CN-+IAA). Fluxes were measured at 4 min, 14 min, 24 min and 44 min after the start of the experiment. Results are means + S.E.M. (N=4).

View larger version (17K): [in a new window]   Fig. 7. Rates of 86Rb+ efflux in goldfish hepatocytes exposed to 180 mosmol l-1 medium and pH 7.45, pH 7.8 or pH 7.45 + N-ethylmaleimide (NEM). Results are means + S.E.M. (N=4). An asterisk indicates P<0.05 with respect to isosmotic values.

View larger version (11K): [in a new window]   Fig. 8. Intracellular Na+ (nmol 10-6 cells-1) versus time in hepatocytes of goldfish exposed to control medium (A), cyanide (CN-), ouabain (OB) and isotonic 100 mmol l-1 MgCl2 (Na+-free medium). Results are means ± S.E.M. (N=4).

View larger version (18K): [in a new window]   Fig. 9. Loss of intracellular K+ (as a percentage of control values) in trout and goldfish hepatocytes exposed to N-ethylmaleimide (NEM), hyposmotic medium (HY) and cyanide (CN-). For trout hepatocytes, loss of intracellular K+ was derived from data of Bianchini et al. (1988, 1991) and Krumschnabel et al. (1996).