Regulation of intracellular pH in anoxia-tolerant and anoxia-intolerant teleost hepatocytes
Gerhard Krumschnabel1,*,
Claudia Manzl1 and
Pablo J. Schwarzbaum2
1 Institut für Zoologie und Limnologie, Abteilung für Ökophysiologie, Universität Innsbruck, Technikerstraße 25, A-6020 Innsbruck, Austria and
2 Instituto de Química y Fisicoquímica Biológicas (Facultad de Farmacia y Bioquímica), Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina
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Fig. 1. Intracellular pH (pHi) as a function of extracellular pH (pHe) in goldfish (A) and trout (B) hepatocytes. BCPCF-loaded hepatocytes (3x106 ml–1) were incubated in standard saline, titrated to the specified pH with HCl or NaOH, until a constant fluorescent signal was obtained. Each measurement was subsequently calibrated as described in Materials and methods. Data are from 21 (goldfish) and 15 (trout) hepatocyte preparations. The linear regression analysis equation is shown for trout hepatocytes (continuous line).
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Fig. 2. The effect of 30 mmol l–1 sodium propionate on pHi in goldfish hepatocytes incubated in standard saline (A, Controls) and in nominally Cl–-free medium, Cl– being replaced by gluconate (B, Cl–-free). Arrows indicate addition of sodium propionate.
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Fig. 3. Rates of proton extrusion by goldfish hepatocytes in response to acidification with 30 mmol l–1 sodium propionate (see Fig. 2) in standard saline (Cont), with 1 mmol l–1 amiloride (Am), in low Na+ medium (low Na), with 0.5 mmol l–1 of the anion exchanger blocker SITS (SITS), in Cl–-free medium (Cl–-free), with both amiloride and SITS (Am+SITS), and with 0.5 mmol l–1 iodoacetic acid (IAA). Rates shown were determined from the change of pHi over the first 5 min after the lowest measured pHi was observed. Values are means + S.E.M. of the number of hepatocyte preparations, given in parentheses. * indicates a significant difference from controls and IAA-treated cells (P<0.05, one-way ANOVA followed by Tukey’s procedure).
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Fig. 4. Intracellular pH (pHi) during chemical anoxia, created by addition of 1 mmol l–1 CN at time zero, and after washout of CN at 60 min, in hepatocytes from goldfish (A) and trout (B). Values are means ± S.E.M. of 4 (goldfish, 3 values at 15 min) and 6 (trout) hepatocyte preparations. * indicates a significant difference compared to the initial value (repeated-measures ANOVA followed by Tukey’s procedure).
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Fig. 5. Effect of chemical anoxia and transport inhibitors on acid secretion (% of control rate) by goldfish (A) and trout (B) hepatocytes, as estimated from the rate of acidification of the external medium measured with a cytosensor microphysiometer. At the times indicated, the perfusion medium was changed. Closed symbols, cells subjected to CN-treatment only; open symbols, cells treated with CN and transport inhibitors. Values are means ± S.E.M. of 9 (goldfish) and 7 (trout) experiments. For abbreviations, see legend to Fig. 3.
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Fig. 6. Production and export of lactate by goldfish hepatocytes exposed to chemical anoxia in the presence or absence of 0.5 mmol l–1 quercetin (Que). Ic, intracellular [lactate]; ec, extracellular [lactate]. Values are means ± S.E.M. of 6 (CN) and 3 (CN+Que) hepatocyte preparations.
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Fig. 7. Effect of 0.5 mmol l–1 quercetin (Que) on acid secretion determined with a cytosensor microphysiometer of goldfish hepatocytes in the absence and presence of CN. Perfusion medium was changed as indicated in the figure. Values (% of control rate) are means ± S.E.M. of 8 experiments.
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© The Company of Biologists Ltd 2001
