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doi: 10.1242/10.1242/jcs.00202

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Cell swelling increases intracellular calcium in Necturus erythrocytes

Department of Biology, Ripon College, Ripon, WI 54971, USA

View larger version (15K): [in a new window]   Fig. 1. Chelating extracellular Ca2+ increased osmotic fragility. The control was diluted amphibian Ringer solution (n=10), low Ca2+ was diluted Ringer containing 5 mM EGTA (n=10), and gramicidin (gram, 5 µM) was used in the presence of a low Ca2+ choline Ringer (n=10). Calcium ionophore A23187 (0.5 µM) was added to the control solution (n=10). Values are means±s.e.m. of optical density, obtained at concentrations of Ringer with approximately 50% lysed cells for the control. **P<0.01, ***P<0.001.

View larger version (18K): [in a new window]   Fig. 2. Inhibition of P2 receptors increased osmotic fragility. Cells were incubated for 1 minute in isosmotic Ringer solution with hexokinase (hexo, 2.5 U/ml, n=10) or suramin (sur, 100 µM, n=10) prior to dilution. The control was diluted (0.5x) amphibian Ringer. Gramicidin (gram, 5 µM, n=10) and A23187 (A23, 0.5 µM, n=10) were used in the presence of either hexokinase or suramin. Values are means±s.e.m of optical density. **P<0.01, ***P<0.001.

View larger version (78K): [in a new window]   Fig. 3. Necturus erythrocytes loaded with fluo-4 (10 µM) and exposed to UV light emitted from a mercury vapor bulb and filtered through a FITC cube (400x). (A) Cells display little fluorescence under isosmotic conditions (n=6). (B) Addition of A23187 (0.5 µM) to the extracellular medium increased fluorescence under isosmotic conditions (n=6). (C) Exposure to a hypotonic (0.5x) Ringer solution increased fluorescence compared to basal conditions (n=6). (D) A low Ca2+ hypotonic Ringer solution (5 mM EGTA) did not display the level of fluorescence normally observed following hypotonic swelling (n=6).

View larger version (64K): [in a new window]   Fig. 4. Necturus erythrocytes loaded with fluo-4 (10 µM), bathed in a hypotonic (0.5x) Ringer solution and exposed to FITC filtered UV light. (A,C) Hexokinase (2.5 U/ml, n=6) and suramin (100 µM, n=6) inhibited the level of fluorescence normally observed following hypotonic shock. (B,D) Addition of A23187 (0.5 µm0 to the extracellular medium increased fluorescence in the presence of hexokinase (n=6) or suramin (n=6).

View larger version (25K): [in a new window]   Fig. 5. Buffering free Ca2+ inhibited cell volume recovery following hypotonic challenge. At time 0, cells were abruptly exposed to a hypotonic (0.5x) amphibian Ringer, which caused a rapid initial increase in volume followed by a gradual recovery toward basal values. The rate of cell volume recovery was reduced with a low Ca2+ hypotonic Ringer solution (5 mM EGTA, n=8, P<0.05 after 40 minutes compared to control values) and by buffering intracellular Ca2+ with BAPTA (100 µM, n=8, P<0.05 after 40 minutes compared to control). By contrast, the inhibitory effect of BAPTA was prevented by adding gramicidin (5 µM, n=8) to the extracellular medium (added at 5 minutes, P<0.05 within 5 minutes following addition of ionophore compared to the other three groups). Values are means±s.e.m.

View larger version (25K): [in a new window]   Fig. 6. Calcium ionophore potentiated cell volume recovery following hypotonic shock. At time 0, cells were abruptly exposed to a hypotonic (0.5x) amphibian Ringer solution. Cell volume recovery was enhanced with A23187 (0.5 µM, n=8), regardless of whether it was added to the extracellular medium at 5, 40 and 70 minutes after hypotonic challenge (indicated by arrows, P<0.05 compared to control values immediately following addition of ionophore, regardless of when it was added). Values are means±s.e.m.

View larger version (23K): [in a new window]   Fig. 7. An ATP scavenger inhibited cell volume recovery following hypotonic shock. At time 0, cells were abruptly exposed to a hypotonic (0.5x) amphibian Ringer solution. The rate of cell volume recovery was reduced with hexokinase (hexo, 2.5 U/ml, n=8), regardless of whether it was added to the extracellular medium at 5, 40 and 70 minutes after hypotonic challenge (indicated by arrows, P<0.05 compared to the control immediately following addition of enzyme at the 5 minute mark, and P<0.05 after 10 and 5 minutes when hexokinase was added at the 70 and 40 minute marks, respectively. There was no significant difference between the three hexokinase groups once this enzyme exhibited an inhibitory effect that was significantly different from control values.). Values are means±s.e.m.

View larger version (19K): [in a new window]   Fig. 8. Chelating extracellular Ca2+ inhibited whole-cell currents in swollen erythrocytes. Cells were maintained at a holding potential of -15 mV and stepped to potentials between -100 to +100 mV in 20 mV intervals. (A) Whole-cell currents for an erythrocyte exposed to a hypotonic (0.5x) KCl Ringer solution and inhibition of these currents by adding EGTA (5 mM) to the bath. (B) Corresponding current-voltage (I-V) relationship for control (hypotonic Ringer) and low Ca2+ solutions (n=5). Values are means±s.e.m.