Thiol-dependent passive K:Cl transport in sheep red blood cells: X. A hydroxylamine-oxidation induced K:Cl flux blocked by diethylpyrocarbonate

Summary Hydroxylamine, a potent oxidizing agent used to reverse carbethoxylation of histidine by diethylpyrocarbonate, activated Cl-dependent K flux (KCl cotransport) of low K sheep red blood cells almost sixfold. When KCl cotransport was already stimulated by N-ethylmaleimide, hydroxylamine caused an additional twofold activation suggesting modification of sites different from those thiol alkylated. This conclusion was supported by the finding that hydroxylamine additively augmented also the diamide-induced KCl flux (Lauf, P.K. 1988.J. Membrane Biol. 101:179–188) with dithiothreitol fully reversing the diamide but not the hydroxylamine effect. Stimulation of KCl cotransport by hydroxylamine was completely inhibited by treatment with diethylpyrocarbonate also known to prevent KCl cotransport stimulation by N-ethylmaleimide, both effects being independent of the order of addition. Hence, although the effect of carbethoxy modification on KCl flux cannot be reversed by hydroxylamine and thus excludes histidine as the target for diethylpyrocarbonate, our finding reveals an important chemical determinant of KCl cotransport stimulation by both hydroxylamine oxidation and thiol group alkylation.     

Effect of peroxynitrite on passive K+ transport in human red blood cells.

Peroxynitrite is generated in vivo by the reaction between nitric oxide, from endothelial and other cells, and the superoxide anion. It is therefore pertinent to examine its effects on the membrane permeability of red blood cells. Treatment of human red blood cells with peroxynitrite (nominally 1 mM) markedly stimulated passive K+ permeability. The main effect was on a Cl(-)-independent K+ pathway, which remains unidentified. Although K+-Cl- cotransport (KCC) was stimulated, this was dependent on saline composition, being inhibited by physiological levels of glucose (IC50 4 mM), and also by sucrose and MOPS. Effects on the Cl(-)-independent K+ pathway were less dependent on saline composition, and were not inhibited by amiloride, ethylisopropylamiloride, dimethylamiloride or gadolinium. Na+-K+-2Cl- cotransporter was inhibited whilst there was little effect on the Gardos channel (Ca2+-activated K+ channel). Peroxynitrite was markedly more effective in oxygenated cells than deoxygenated ones. Treatment with peroxynitrite per se did not affect initial cell volume. Anisotonic swelling modestly increased the Cl(-)-independent K+ influx, but did not affect peroxynitrite-stimulated KCC. Decreasing extracellular pH from 7.4 to 7.2 or 7.0 increased KCC stimulation, whilst the Cl(-)-independent component of K+ transport was lowest at pH 7.2. Finally, protein phosphatase inhibition with calyculin A (100 nM) inhibited KCC, implying that, as with other KCC stimuli, peroxynitrite acts via decreased protein phosphorylation; pre-treatment with calyculin A also inhibited the Cl(-)-independent component of K+ transport. These findings are relevant to the actions of peroxynitrite in vivo.     

Thiol-dependent passive K/Cl transport in sheep red cells: VII. Volume-independent freezing by iodoacetamide,and sulfhydryl group heterogeneity

Summary The sulfhydryl (SH) reagent iodoacetamide (IAAM) inhibits stimulation of Cl-dependent K transport in low K (LK) sheep red cells by another SH reagent, N-ethylmaleimide (NEM), without itself activating this transport pathway (J. Membrane Biol., 1983,73:257–261). We now report that IAAM alone, acting with a kinetic slower than NEM, sharply reduced the capability of the Cl-dependent K transport system to regulate its activity in response to cell volume changes. This effect of IAAM did not depend on the cell volume maintained during chemical treatment, a fact ruling out that the reactivity of the SH groups with IAAM was a function of the volume-dependent turnover rate of the transporter. On the other hand, the prevention of the NEM-stimulatory effect on Cl-dependent K transport was found to be volume-dependent since 1) the rate with which IAAM blocked the subsequent NEM action was twice as fast in cells swollen in 250 mOsm as opposed to cells shrunken in 370 mOsm media, and 2) the dose response of the IAAM effect was different in swollen and shrunken cells. The action of IAAM with or without subsequent treatment with NEM seemed to be independent of cellular ATP which is required for full expression of the stimulatory modification of Cl-dependent K transport by NEM (Am. J. Physiol., 1983,245:C445–C448). Clusters of SH groups on the Cl-dependent K transporter apparently react differently with IAAM and NEM when separately applied but, used in combination, reflect a complex volume-dependent effect that may reveal a volume-sensing component of the transport molecule.     

Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide

The sulfhydryl (SH) oxidant diamide activated in a concentration-dependent manner ouabain-resistant (OR), Cl-dependent K flux in both low potassium (LK) and high potassium (HK) sheep red cells as determined from the rate of zero-trans K efflux into media with Cl or Cl replaced by NO3 or methane sulfonate (CH3SO3). Diamide did not alter the OR Na efflux into choline Cl. The diamide effect on K efflux appeared after 80% of cellular glutathione (GSH) was oxidized to GSSG, its disulfide. The stimulation of K efflux was completely reversed during metabolic restitution of GSH, a process that depended on the length of exposure to and the concentration of diamide. The action of diamide on both the K:Cl transporter and GSH was also fully reversed by the reducing agent dithiothreitol (DTT). Diamide apparently oxidized the same SH groups alkylated by N-ethylmaleimide (NEM) (Lauf, P.K. 1983. J. Membrane Biol. 73:237-246). Like NEM, diamide activated K:Cl transport several-fold more in LK cells than in HK cells, and the effect on LK cells was partially inhibited by anti-L1, the allo-antibody known to inhibit OR K fluxes.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells

Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+.     

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.     

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.     

Secondary delayed type hypersensitivity to sheep red blood cells in mice: Dependence on long-lived memory cells

Secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is a long-lived memory phenomenon which is characterized by the accelerated reappearance of the state of DTH after a booster injection of the antigen. In this paper the nature of the DTH-related T memory cells accounting for secondary DTH was investigated. Parabiosis of primed and nonprimed mice for a period of 4 weeks resulted in an equally large secondary DTH responsiveness in both partners. This ability was maintained in both members for at least 6 months after termination of the parabiosis. These results indicate that (a) DTH-related T memory cells are potentially circulating cells, and (b) the persistence of these memory cells is not dependent on the presence of the antigen which induced their generation. Subcutaneous (sc) injection of intravenously (iv) primed mice with a small dose of antithymocyte serum before boosting did prevent the development of secondary DTH responsiveness in sc boosted mice, but not in iv boosted mice. Treatment of primed mice with vinblastine or azathioprine did not decrease the capacity of adoptive transfer of secondary DTH by means of spleen cells, but passive transfer of secondary DTH was completely abolished by this treatment. These results suggest that (a) SRBC-induced DTH-related T memory cells are nonproliferating, partially sessile, partially recirculating cells, and (b) these memory cells proliferate before they become DTH-related effector cells.     

Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells

Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump.     

K(+) transport in red blood cells from human umbilical cord

The current study was designed to characterise K(+) transport in human fetal red blood cells, containing mainly haemoglobin F (HbF, and termed HbF cells), isolated from umbilical cords following normal parturition. Na(+)/K(+) pump activity was comparable to that in normal adult human red cells (which contain HbA, and are termed HbA cells). Passive (ouabain-resistant) K(+) transport was dominated by a bumetanide (10 microM)-resistant component, inhibited by [(dihydroxyindenyl)oxy]alkanoic acid (100 microM), calyculin A (100 nM) and Cl(-) removal, and stimulated by N-ethylmaleimide (1 mM) and staurosporine (2 microM) - all consistent with mediation via the K(+)-Cl(-) cotransporter (KCC). KCC activity in HbF cells was also O(2)-dependent and stimulated by swelling and urea, and showed a biphasic response to changes in external pH. Peak activity of KCC in HbF cells was about 3-fold that in HbA cells. These characteristics are qualitatively similar to those observed in HbA cells, notwithstanding the different conditions experienced by HbF cells in vivo, and the presence of HbF rather than HbA. KCC in HbF cells has a higher total capacity, but when measured at the ambient PO(2) of fetal blood it would be similar in magnitude to that in fully oxygenated HbA cells, and about that required to balance K(+) accumulation via the Na(+)/K(+) pump. These findings are relevant to the mechanism by which O(2) regulates membrane transporters in red blood cells, and to the strategy of promoting HbF synthesis as a therapy for patients with sickle cell disease.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.