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. 1992 Oct;456:503–518. doi: 10.1113/jphysiol.1992.sp019349

Hyperosmotic activation of the Na(+)-H+ exchanger in a rat bone cell line: temperature dependence and activation pathways.

A Dascalu 1, Z Nevo 1, R Korenstein 1
PMCID: PMC1175694  PMID: 1338103

Abstract

1. The hyperosmotic activation of the Na(+)-H+ exchanger was studied in an osteoblast-like rat cell line (RCJ 1.20). The activation was monitored by recording the intracellular pH (pHi) changes employing double excitation of the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). 2. Exposure of the cells to a hyperosmotic HCO(3-)-free medium at 37 degrees C produced an initial cytosolic acidification of 0.05 pH units followed by a lag period and an alkalinization overshoot of about 0.2 pH units, without a concomitant change of the free cytosolic calcium [Ca2+]i by the use of Fura-2 calcium-sensitive probes. This response was completely inhibited by amiloride (0.33 mM) or by Na+ depletion from the external medium and insensitive to the extracellular Cl- replacement, indicating the involvement of a Na(+)-H+ exchanger in the hyperosmotic response. 3. Hyperosmotic stimuli (200 moSM sucrose) applied in the temperature range of 17-37 degrees C demonstrated a shortening of the lag period preceding alkalinization and an increased rate of proton extrusion upon temperature elevation. The biochemical reaction underlying the lag period and the proton extrusion resulted in apparent activation energies of 19 and 29 kcal mol-1, respectively, as calculated from the appropriate Arrhenius plots. 4. Stimulation of the exchanger under isosmotic conditions by 25 nM 4 beta-phorbol 12-myristate 13-acetate (PMA) and 0.1 mM vanadate resulted in an amiloride-sensitive pHi increase of about 0.08 pH units. The hyperosmotic stress was additive to the stimulatory effects of these agents, suggesting an independent hyperosmotic activation pathway. 5. The hyperosmotic activation of the Na(+)-H+ exchanger was independent of cAMP, cGMP, cytosolic Ca2+ and protein kinase C. Thus, none of the classical transduction mechanisms seem to be involved directly in the hyperosmotic activation of the antiporter. 6. The pHi response induced by the hyperosmotic stress was abolished by two calmodulin inhibitors, W-7 and chlorpromazine (50% inhibition, Ki at 28 and 20 microM, respectively), 20 microM cytochalasin B, but not by 10 microM colchicine. The results suggest the involvement of actin and calmodulin-like structural elements of the cytoskeleton in the transduction process leading to the activation of the Na(+)-H+ exchanger.

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Selected References

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