Oxygen-dependent K+ fluxes in sheep red cells.
1. This study was designed to investigate the O2 dependence of K+ influx in sheep red cells. Influx was determined using 86Rb+ as a tracer for K+; glass tonometers coupled to a gas mixing pump were used to equilibrate cell samples to the requisite oxygen tension (PO2). 2. Both volume- and H(+)-stimulated K+ influxes in low potassium-containing (LK) sheep red cells were approximately doubled on equilibration with O2 relative to influxes measured in N2.O2-dependent influxes were abolished when Cl- was replaced with NO3-, consistent with mediation by the KCl cotransporter. At pH 7, PO2 required for half-maximal stimulation was 56 +/- 1 mmHg (mean +/- S.E.M., 3 sheep) for the O2-dependent component of K+ influx: thus PO2 values over the physiological range affected K+ influx. 3. K+ influx in fully deoxygenated sheep red cells showed substantial volume and H+ sensitivity. These residual components in N2 were also Cl- dependent, indicating that the KCl cotransporter of LK sheep red cells was active in the absence of O2. 4. Volume-sensitive K+ influxes in high potassium-containing (HK) sheep red cells responded in a similar way to those in cells from LK sheep, although much smaller in magnitude, showing that intracellular [K+] had no significant effect on the O2 dependence of the cotransporter. 5. Intracellular [Mg2+] ([Mg2+]i) was altered by incubating sheep red cells with A23187 (20 microM) and different values of extracellular [Mg2+] ([Mg2+]o). Total [Mg2+]i was determined by atomic absorption spectroscopy and free [Mg2+]i from [Mg2+]o and the Donnan ratio. Total [Mg2+]i was 1.29 +/- 0.08 mM (mean +/- S.E.M., n = 5), similar to that reported in the literature. Estimates of free [Mg2+]i showed an increase from 0.39 +/- 0.05 in oxygenated cells to 0.52 +/- 0.04 mM (mean +/- S.E.M., n = 5; P < 0.05) in deoxygenated ones. 6. Finally, although K+ influxes were altered by pharmacological loading or depletion of cells with Mg2+, the free [Mg2+]i required to affect influxes significantly was outside the physiological range. Results are difficult to reconcile with PO2 modulating KCl cotransport activity directly via changes in free [Mg2+]i or [Mg(2+)-ATP]i.
Campbell
E H
EH
Department of Veterinary Preclinical Sciences, University of Liverpool, UK.
Gibson
J S
JS
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D048429:Cell Size; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008274:Magnesium / Q000097:blood; D010100:Oxygen / Q000097:blood / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000097:blood; D012756:Sheep
D007476:Ionophores; D015221:Potassium Channels; D000001:Calcimycin; D011189:Potassium Chloride; D008274:Magnesium; D010100:Oxygen