Oxygen-dependent K+ fluxes in sheep red cells

Abstract

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.

Figure 8. Effect of variations in free intracellular magnesium concentration ([Mg²⁺]_i) on K⁺ influx in LK sheep red cells

Free [Mg²⁺]_i, calculated as described in Fig. 7, was altered using A23187 (20 μM) and different extracellular [Mg²⁺]. Ionophore was removed by washing cells with saline containing BSA (50 mg ml⁻¹) and K⁺ influxes were then measured in O₂ (○) or N₂ (•) in cells swollen by 5 %. K⁺ influxes in oxygenated and deoxygenated control cells, i.e. handled in the same way except that they were untreated with A23187, were 1.03 ± 0.02 (n = 4) and 0.45 ± 0.02 mmol (l cells)⁻¹ h⁻¹ (n = 4), respectively, and are indicated by the dashed lines. Points represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 1. Time course for the effect of oxygen tension on K⁺ influx in LK sheep red cells

K⁺ influx (mmol (l cells)⁻¹ h⁻¹) was measured at the times given in cells shrunken (○) or swollen (•) by 10 %. Cells were incubated in tonometers equilibrated with N₂ or O₂, as indicated, and K⁺ influxes measured under this same gas atmosphere. *Cells in which K⁺ influx was measured in saline of unknown P_O2 without pre-incubation in tonometers; **cells incubated in N₂ for the entire duration of the experiment, including during influx measurement. Points represent means ±s.d. for quadruplicate measurements on a single sample.

Figure 2. Effect of anion replacement on volume-sensitive K⁺ influx in LK sheep red cells

K⁺ influx was measured in N₂ or O₂ in the presence or absence of Cl⁻ (Cl⁻ replaced with NO₃⁻). Samples were shrunken (□) or swollen () by 20 %. Bars represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 3. Effect of oxygen tension on H⁺-sensitive K⁺ influx in LK sheep red cells

K⁺ influx was measured in cells at pH 7.4 () or pH 7.0 (□) in N₂ or O₂. Bars represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 4. Effect of anion replacement on H⁺-sensitive K⁺ influx in LK sheep red cells

K⁺ influx was measured at pH 7.0 in N₂ or O₂ in the presence (□) or absence () of Cl⁻ (Cl⁻ replaced with NO₃⁻). Bars represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 5. Effect of oxygen tension on H⁺-stimulated K⁺ influx in LK sheep red cells

Cells were incubated for 15 min at pH 7.4 at the P_O2 indicated before measurement of K⁺ influx in saline of pH 7, pre-equilibrated with the same P_O2. Points represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 6. Effect of oxygen tension on volume-sensitive K⁺ influx in HK sheep red cells

K⁺ influx was measured in cells shrunken (□) or swollen () by 10 % in N₂ or O₂. Bars represent means ±s.d. for quadruplicate determinations on a single sample.

Figure 7. Effect of variations in free intracellular magnesium concentration ([Mg²⁺]_i) on total [Mg²⁺]_i

Total [Mg²⁺]_i was determined in oxygenated (○) and deoxygenated (•) cells, incubated with A23187 (20 μM) and different concentrations of extracellular Mg²⁺ ([Mg²⁺]_o). Free [Mg²⁺]_i was calculated from the Donnan ratio and [Mg²⁺]_o, as explained in the Methods. Total [Mg²⁺]_i measured in control cells in the absence of ionophore was 1.29 ± 0.08 mM (n = 5) and is indicated by the dashed line. Points represent means ±s.e.m., n=5.