Oxygen‐dependent K⁺ fluxes in sheep red cells

Abstract

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). 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. 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. 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. Intracellular [Mg2+] ([Mg2+]i) was altered by incubating sheep red cells with A23187 (20 μM) 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. 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 [Mg2+‐ATP]i.

Oxygen‐dependent K+ influxes in Mg2+‐clamped equine red blood cells

“…The K¤ influx stimulated by Mg¥ depletion was abolished by Cl¦ substitution and also by treatment with calyculin A. Oµ-dependent K¤-Cl¦ cotransport was also observed by Motais and coworkers after Mg¥ clamping trout red cells (Borgese et al 1991), and we have previously shown that the same occurs in LK sheep red cells (Campbell & Gibson, 1998a), a system commonly used to study K¤-Cl¦ cotransport. Subsequently, however, we found that changes in free [Mg¥]é of a magnitude similar to that occurring physiologically during oxygenation-deoxygenation cycles (a few hundred micromolar), had very little effect on the cotransporter.…”

“…In most experiments, samples were deoxygenated for 60 min and then either held in Nµ or incubated with Oµ for 15 min before dilution into saline at low haematocrit (about 5%) pre-equilibrated and maintained at the requisite POµ for influx measurement, alteration of [Mg¥]é or pH determinations, as appropriate (see Campbell & Gibson, 1998a, for details). In most experiments, samples were deoxygenated for 60 min and then either held in Nµ or incubated with Oµ for 15 min before dilution into saline at low haematocrit (about 5%) pre-equilibrated and maintained at the requisite POµ for influx measurement, alteration of [Mg¥]é or pH determinations, as appropriate (see Campbell & Gibson, 1998a, for details).…”

Erythrocyte Signal Transduction Pathways, their Oxygenation Dependence and Functional Significance

“…Unlike the complete inactivation and volume-and pH-insensitivity of K-Cl cotransport in trout [81] and equine [68] RBCs under deoxygenation, K-Cl cotransport in deoxygenated sheep erythrocytes is active and volume-and pH-sensitive [49]. Nevertheless, oxygenation of sheep RBCs affects K-Cl cotransport by increasing both its rate and the degree of its volume-and pH-dependence [49]. It is noteworthy that K-Cl cotransport in sheep erythrocytes seems to be controlled by two signals of cell swelling, a reduction in macromolecular crowding and a putative mechanical signal [112].…”

“…Similar evidence is provided by sheep Hb that exhibits low DPG binding and hence may not be expected to bind to negatively charged tails of proteins. Nevertheless, KCl cotransport in sheep RBCs has been shown to be O 2 -sensitive [49].…”

“…In this context, increased mean cell Hb concentration is a major cause of decreased deformability of DPG-enriched erythrocytes [135]. However, regulation of K-Cl cotransport, which in contrast to Na-K-Cl cotransport, is inhibited by free Mg 2+ [140,141] is independent of the oxygenation-induced fluctuations of free Mg 2+ [10,49]. Concerning DPG-mediated mechanisms modulating membrane properties, spectrin has binding sites for both ATP [142] and DPG [143] (Fig.…”