Do HbSS erythrocytes lose KCl in physiological conditions?

Abstract

Summary. KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in 'simple' physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both V max and K m for K + of the transporter. The plasma factor(s) responsible was heat-stable and dialysable (i.e. size < 10 kD).Adding magnesium, calcium, inorganic phosphate or bicarbonate to 'simple' saline to mimic the effect of plasma revealed that Mg 2+ and Ca 2+ had no significant effect at physiological concentrations. P i was not effective at 1 . 1 mM , but did inhibit significantly (42Ϯ2%Þ at 5 . 6 mM . HCO ¹ 3 had a major inhibitory effect on K + influx when added to saline, and was identified as the principal candidate for the plasma effect.We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo. It acts by altering the set point of the transporter via the signalling systems involved in its regulation.

Does Plasma Inhibit the Activity of KCl Cotransport in Red Cells From LK Sheep?

“…Reduction in the component of KCC activity altered by aniostonicity, defined as the difference in K + influx in red cells swollen compared with shrunken ones (= volume-sensitive K + flux), was about 25% comparing BBS with MBS and about 50% comparing plasma with MBS ( Figure 1 ). Although percentage inhibition was more modest in the bicarbonate-buffered media, these findings were similar to those described previously in red cells from SCD patients whose volume was altered similarly by an anisotonicity of 15% but at high O 2 tension, about 700 mmHg ( Godart et al, 1997 ), and in which the corresponding inhibition of volume-sensitive K + fluxes was about 80% (MBS to BBS) and 75% (MBS to plasma). Inhibition was not due to altered red cell volume in the different media ( Godart et al, 1997 and see above).…”

“…This finding is consistent with modulation by intracellular pH and anisotonicity having the main effects on red cell volume. KCC activity was also compared in the different media equilibrated with an O 2 tension of either 100 mmHg O 2 or 677 mmHg, the latter value used by Godart et al (1997) . The magnitude of fluxes was reduced by about 25% at the lower O 2 tension—for example at pH 7.4 K + influx reduced from 2.20 ± 0.24 mmol (l cells.h) −1 to 1.62 ± 0.32 ( n = 3)—but otherwise the pattern of responses to different modalities of stimulus (volume, pH and urea) and the effect of the incubation in the two other incubation media, BBS or plasma, were similar and unaffected by the two O 2 tensions tested.…”

“…This report focuses on the effect of plasma, as almost exclusively, studies investigating red cell KCC have used artificial media—simple salt solutions with synthetic buffers like HEPES, TRIS and MOPS—in which to suspend red cells whilst measuring K + transport. A paper published towards the end of the 1990s, however, provocatively suggested that more physiological solutions—a bicarbonate-buffered saline (BBS) mimicking plasma, or plasma itself—contain constituents which markedly inhibit red cell KCC activity when compared to that measured in simple artificial saline ( Godart et al, 1997 ). Red cell swelling was used as the stimulus for KCC activity, with cells suspended in MOPS-buffered saline (MBS), BBS or autologous plasma.…”

“…An inhibition of KCC activity of over 80%, elicited by the presence of bicarbonate/CO 2 , rather than by that of some other component of plasma. The authors provokingly considered whether high levels of KCC activity may actually occur in circulating red cells in vivo ( Godart et al, 1997 ). Their report, however, focussed on abnormal red cells from patients with sickle cell disease—which have several differences to those from normal individuals, not least a high reticulocyte count - whilst only anisotonic swelling was tested—which is unlikely to occur to a significant extent in vivo —and, in addition, very high O 2 tensions (about 700 mmHg O 2 ) were used—which might be problematical in an O 2 -sensitive system ( Gibson et al, 2000 ).…”

“… Red cells from LK sheep represent an important paradigm for control of KCl cotransport activity, as well as being important to sheep erythroid function. A previous report ( Godart et al, 1997 ) suggested that autologous plasma markedly inhibits red cell KCC activity and identified the presence of the bicarbonate/CO 2 buffer system as the probable cause. Findings were restricted, however, to red cells from patients with sickle cell disease (SCD) swollen anisotonically and carried out at a very high O 2 tension (c.700 mmHg).…”