Pivotal role of reduced glutathione in oxygen-induced regulation of the Na(+)/K(+) pump in mouse erythrocyte membranes.

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na(+)/K(+) pump, K(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na(+)/K(+) pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na(+)-loaded cells, revealing no effect of O(2) on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na(+)/K(+) pump activity.

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes.

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.

Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

Copper effects on ion transport across lamprey erythrocyte membrane: Cl(-)/OH(-) exchange induced by cuprous ions.

We studied the effects of prelytic copper concentrations on cell volume, intracellular pH, and ion transport in lamprey erythrocytes. Ion fluxes and pH were measured by radioactive tracer technique, patch clamp, and flame photometry. Prelytic CuSO(4) concentration of 100 microM caused anion-dependent intracellular acidification and increase in Cl(-) influx after 2 min lag-phase. In the presence of ascorbate copper effect was amplified and lag-phase was skipped. Pretreatment of the cells with N-phenyl maleimide abolished copper-induced changes completely. Copper treatment caused an increase in Na(+) fluxes in both directions and a net Na(+) uptake. Copper-induced Na(+) transport was partially amiloride(MIA)-sensitive representing Na(+)/H(+) exchange. The nature of the amiloride-insensitive fraction of copper-activated Na(+) influx remains unknown. Cell swelling after 15 min of copper exposure induced regulatory volume decrease response involving KCl extrusion via K(+) and Cl(-) volume-sensitive channels. We suggest that the effects of copper on ion transport fit the following sequence of events: (i) cupric ions are reduced to cuprous state on the membrane surface, (ii) electroneutral pairs CuCl and CuOH mediate chloride/hydroxyl exchange, as shown before for trialkyltin, dissipating transmembrane pH gradient, and (iii) changes in intracellular pH result in the activation of the Na(+)/H(+) exchange and consecutive volume changes cause the RVD response.