LK Sheep Red Cells Research Articles

Effects of protein kinase and phosphatase inhibitors and anti-L antisera on K + transport in LK sheep red cells

We investigated the role of protein phosphorylation in the action of anti-L on low potassium (LK) sheep red cells. Anti-L stimulated the Na/K pump by four- to fivefold, but Na/K pump activity in anti-L-stimulated or in control cells was unaffected by protein kinase/protein phosphatase (PK/PP) inhibitors. KCl co-transport activity was inhibited by anti-L (about 50%). Co-transport was stimulated by staurosporine; and inhibited by calyculin A, okadaic acid, tyrphostin B46 and genistein; with a similar pattern in both control and anti-L-treated cells. O 2 sensitivity of KCl co-transport was similar in control and anti-L-treated cells. Neither control nor anti-L-stimulated Na/K pump activities were O 2 sensitive. Incubation with urea stimulated KCl co-transport in both control and anti-L-treated cells. Inhibition of co-transport by anti-L was unaffected by low concentrations of urea but was reduced at higher urea concentrations. Na/K pump activity of control cells was unaffected by incubation with urea, but that in cells stimulated by anti-L was reduced, though not significantly. Under high hydrostatic pressure, KCl co-transport was stimulated, and the inhibitory effects of PP inhibition (okadaic acid), anti-L or combinations of the two were reduced. Results suggest that anti-L does not affect K + transport in LK sheep red cells via protein phosphorylation.

Volume-sensitive K(+)/Cl(-) cotransport in rabbit erythrocytes. Analysis of the rate-limiting activation and inactivation events.

The kinetics of activation and inactivation of K(+)/Cl(-) cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg(2+)) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg(2+)-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0 degrees C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (E(a) approximately 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37 degrees C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg(2+), acid pH, and NEM.

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.

H2O2 activates red blood cell K-Cl cotransport via stimulation of a phosphatase.

K-Cl cotransport is involved in volume regulation in a number of cell types. Cell swelling stimulates K-Cl cotransport, probably by inhibition of a volume-sensitive kinase. K-Cl cotransport can also be activated by oxidants and thiol reagents. We investigated the effect of H2O2 on K-Cl cotransport of LK sheep red blood cells in an attempt to identify the target of oxidants. H2O2 stimulated K-Cl cotransport. The stimulation was virtually abolished by subsequent incubation with calyculin, a protein phosphatase inhibitor. This suggests that H2O2 stimulates a calyculin-sensitive phosphatase and activates K-Cl cotransport by causing a decrease in phosphorylation of the transporter or a regulatory protein. The thiol reagent N-ethylmaleimide, which stimulates K-Cl cotransport, did not stimulate cotransport further in cells with cotransport activated by staurosporine but did stimulate cotransport further in cells with cotransport activated by H2O2. These results suggest that there are at least two distinct phosphorylation sites on the transporter or a regulator. The results also suggest that the phosphatase is associated with the membrane.

Differentiation of Na(+)-K+ pumps of low-K+ sheep red blood cells is promoted by Lp membrane antigens.

Na(+)-K+ pumps of red blood cells from sheep of the low-K+ (LK) phenotype undergo differentiation during circulation, manifested in part by a striking increase in sensitivity to inhibition by intracellular K+ (Ki). Pumps of red blood cells from sheep from the allelic phenotype, high K+ (HK), do not undergo this type of maturation. The hypothesis was tested that the Lp antigen, found on LK but not HK cells, is responsible for the maturation of LK pumps. Lp antigens have been shown to inhibit LK pumps because anti-Lp antibody stimulates the pumps by relieving inhibition by the antigen. Lp antigens were recently shown to be molecular entities separate from Na(+)-K+ pumps [Xu, Z.-C., P. Dunham, J. Munzer, J. Silvius, and R. Blostein. Am. J. Physiol. 263 (Cell Physiol. 32): C1007-C1014, 1992]. The test of the hypothesis was to modify the Lp antigens of immature LK red blood cells with two kinds of treatments, anti-Lp antibody and trypsinization (which cleaves Lp), and to observe the effects of these treatments on maturation of pumps during culture of the cells in vitro. Both of these treatments prevented the maturation of the kinetics of the pumps to the Ki-sensitive pattern, supporting the hypothesis that interaction of the pumps with Lp antigens is responsible for the maturation of the pumps. Strong supportive evidence came from experiments on Na(+)-K+ pumps from rat kidney delivered into immature LK sheep red blood cells by microsome fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnesium and ATP dependence of K‐Cl co‐transport in low K+ sheep red blood cells.

1. In low K+ (LK) sheep red blood cells, depletion of adenosine triphosphate (ATP) by glycolysis inhibition induced specific effects on ouabain-resistant Cl(-)-dependent K+ transport (K-Cl co-transport), depending on the osmolarity: stimulation in isosmotic while inhibition in hyposmotic solutions. However, these effects depended upon the presence of internal Mg2+. 2. In LK sheep red blood cells, ATP constituted nearly 90% of the Mg2+ buffering capacity. As no significant reduction of total Mg2+ was observed after ATP depletion, the overall internal Mg2+ in ATP-depleted cells exists in the free form. 3. The dependence of K+ efflux on internal Mg2+ was also directly related to the presence of ATP. In control cells, Mg2+ constituted an endogenous inhibitor, inducing a 70% inhibition of K-Cl fluxes but only 30% in ATP-depleted cells. The Cl(-)-insensitive component of K+ efflux was unaffected by the divalent cation. 4. After Mg2+ removal, the rate of K+ efflux was significantly increased at all osmolarities, between 240 mosM (swollen cells) and 440 mosM (shrunken cells). Hence, Mg(2+)-depleted LK sheep red cells lose volume sensitivity of K-Cl co-transport. 5. Internal K+ or Cl- were not required for the Mg2+ inhibition, and Mg2+ did not interfere with the internal binding sites for Cl- or K+. Hence, the sites for Mg2+ or MgATP, and for K+ and Cl- are independent of each other.

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179-188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257-261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.

Voltage-activated cation permeability in high-potassium but not low-potassium red blood cells

We have recently reported that voltage-activated fluxes of Na, K, and Ca occur in human red blood cells [J.A. Halperin, C. Brugnara, M. Tosteson, T. Van Ha, and D. C. Tosteson. Am. J. Physiol. 257 (Cell Physiol. 26): C986-C996, 1989]. The cation permeability increases progressively as the membrane potential becomes more inside positive above +20 mV. In this paper we show that this effect also occurs in high-potassium (HK), but not in low-potassium (LK), sheep and dog red blood cells. This result suggests that the voltage-activated cation transport pathway is not the result of nonspecific dielectric breakdown of the lipid bilayer but, rather, relates to some membrane component, presumably a protein, that is expressed in HK human and sheep but not in LK sheep and dog red blood cells.

The effect of sodium periodate treatment on the modulation of the sodium pump in low-potassium type (LK) sheep red cells by the L antigen

1. The action of sodium periodate and neuraminidase on active and passive K + transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the L p and L 1 antigens. 2. Active K + transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K + transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl − dependent, and so differed from the increased passive K + uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K + uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K + uptake. 5. In LK sheep red cells the stimulation of active K + transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K + transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.

Anion-dependent cation transport in erythrocytes.

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.

Interaction of L antibody with low potassium-type sheep red cells: resolution of two separate functional antibodies.

Antibodies of two specificities in alloimmune sheep anti-L sera, anti-Lp and anti-L1, were separated by a new technique and characterized. Absorption of anti-L serum with trypsinized LK(LL) sheep red cells left anti-Lp antibodies; the absorbed anti-L1 antibodies were then eluted. Anti-Lp was only weakly lytic in the presence of complement; it had no effect on passive K influx, but stimulated active K influx. The stimulation could be reversed by eluting the antibody in glycine buffer at low pH. Stimulatory activity in the eluted cells could be restored by resensitization with anti-Lp. Anti-L1 was more strongly lytic than anti-Lp in the presence of complement; it had no effect on active K influx, but inhibited passive K influx. Pig anti-ruminant IgG conjugated to hemocyanin was used to visualize by electron microscopy the number of Lp and L1 antigen sites on LL sheep red cells sensitized with anti-Lp an L1 antigen sites on LL sheep red cells sensitized with anti-Lp and anti-L1. The values obtained were 590 Lp sites/cell and 847 L1 sites/cell.

Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride.

The major pathway of passive K influx (ouabain-insensitive) was characterized in low-K type (LK) red cells of sheep. 1. Passive K transport in these cells was highly sensitive to variations in cell volume; it increased threefold or more in cells swollen osmotically by 10%, and decreased up to twofold in cells shrunken 5-10%. Active K influx was insensitive to changes in cell volume. Three different methods for varying cell volume osmotically all gave similar results. 2. The volume-sensitive pathway was specific for K in that Na influx did not vary with changes in cell volume. 3. The volume-sensitive K influx was a saturable function of external K concentration. It was slightly inhibited by Na, whereas K influx in shrunken cells was unaffected by Na. 4. Passive K influx was dependent on the major anion in the medium in that replacement of Cl with any of six other anions resulted in a reduction of K influx by 50-80% (replacement of Cl by Br caused an increase in K influx). The activation of K influx by Cl followed sigmoid kinetics. 5. Passive K influx is inhibited by anti-L antibody. The antibody affected only that portion of influx which was Cl-dependent and volume-sensitve. Of the subfractions of the antibody, it is anti-L1 which inhibits passive K transport. 6. Pretreatment of cells with iodoacetamide reduced the sensitivity of K influx to cell volume in that the influx was reduced in swollen IAA-treated cells and increased in shrunken IAA-cells. 7. Intracellular Ca has no role in altering passive K transport in LK sheep cells. Therefore, the major pathway of passive K transport in LK sheep red cells is sensitive to changes in cell volume, specific for K, dependent on Cl, and inhibited by anti-L1 antibody, The minor pathway, observed in shrunken cells, has none of these properties.

Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes

The activity of the ouabain-insensitive Na +/Na + exchange system was assessed by measurements of Li + net-uptake in LK and HK sheep erythrocytes in the absence and presence of the L-antibody and various inhibitors. N-ethylmaleimide, p-chloromercuribenzoic sulfonate and phloretin inhibited the exchange by about 50%. Anti-L, while stimulating the K + pump flux in LK cells, did not alter Na +/Li + countertransport. The activity of the exchange system with fully saturated internal and external loading sites was estimated to be identical in LK and HK sheep red cells. Hence the Na +/Na + exchange system seems to be molecularly unrelated to the ouabain-sensitive Na +K + pump in these cells and not under genetic control of the HK/LK and M/L genes.

Membrane cholesterol depletion and K+ transport in high and low potassium sheep red cells.

The effect of cholesterol depletion on potassium tracer fluxes was studied in sheep red cells. Removal by the plasma incubation method (5, 12, 30) of approximately 31 and 34% membrane cholesterol from high-potassium (HK) and low-potassium (LK) sheep red cells, respectively, did not induce significant changes in the steady-state cation composition of these cells nor in their passive (leak) and active (pump) K+ influxes. In cholesterol-depleted LK sheep red cells, there was no impairment nor augmentation of the Lp an antibody stimulated K+ pump flux and L1-antibody-mediated reduction of K+ leak flux indicating that the removed cholesterol does not contribute to the activity of the Lp and L1 antigens.

The effect of anti-L on ouabain binding to sheep erythrocytes.

Binding of 3H-ouabain was studied in high potassium (HK) and low potassium (LK) sheep red cells. In particular, we investigated the effect of anti-L, an antibody raised in HK sheep against L-positive LK sheep red cells, on 3H-oubain binding and its relation to K+ -pump flux inhibition in LK cells. HK cells were found to have about twice as many 3H-ouabain binding sites and a higher association rate for 3H-ouabain than homozygous LL-type LK cells. The number of 3H-ouabain molecules bound to heterozygous LM-type LK cells is lower than that on LL cells, but the rate of ouabain binding is between that of HK and LL red cells. A close correlation was observed between the rates of 3H-oubain binding and fraction K+-pump inhibition. Exposure of LM and LL cells to anti-L did not affect the number of 3H-ouabain molecules bound at saturation, but increased the rates of glycoside binding and K+ -pump inhibition proportionately, so that for LK cells in the presence of anti-L, the rates of the two processes approximate those of HK cells. These data exclude the possibility that anti-L generates entirely new pump sites in LK sheep cells, but suggest that the antibody increases the affinity of the existing -a+ -K+ pumps for the glycoside.

Comparison of the number of anti-L-binding sites on LK goat red cells with the number of Na-K pumps.

SHEEP1, goats2, possums3 and some other species are unusual mammals in that the red cells of individual animals have either high (HK) or low (LK) concentrations of potassium, which correlate with the activity of the Na–K pump. The pump of LK cells is less active than that of HK cells4,5. When LK sheep red cells are injected into HK sheep an antibody of the IgG class (anti-L) is raised which, when reacted with LK sheep or goat red cells, stimulates the pump5,6. In goat cells the major, if not the only, effect of the antibody is to reduce the affinity of the pump for K at the inside cell surface, and that stimulation results from the relative increase in the effectiveness of Na as a substrate. It has been reported that in sheep, anti-L also increases the maximal pump rate8. The antibody might function by binding either to the pump, and so altering its kinetic characteristics, or to another component of the cell surface, indirectly altering the environment of the pump. If it combines with the pump, the number of anti-L molecules bound should be about the same as the number of pumps. We have now confirmed this using an estimate of the number of pumps of LK goat cells obtained with 3H-ouabain9, and our own estimate of the number of 131I-labelled antibody molecules bound when the pump is stimulated maximally. We used LK goat red cells as LK sheep red cells seem to contain a second antigen in addition to the antigen associated with pump stimulation5,10.

Interaction of HK and LK goat red blood cells with ouabain.

The characteristics of the interaction of Na-K pumps of high potassium (HK) and low potassium (LK) goat red blood cells with ouabain have been determined. The rate of inhibition by ouabain of the pump of HK cells is greater than the rate of inhibition of the pumps of LK cells. Treatment of LK cells with an antibody (anti-L) raised in HK sheep by injecting LK sheep red cells increases the rate of inhibition of the LK pumps by ouabain to that characteristic of HK pumps; reduction of intracellular K (K(c)) in LK cells increases the rate at which ouabain inhibits their pumps and exposure of these low K(c) cells to anti-L does not affect the rate of inhibition. There is considerable heterogeneity in the pumps of both HK and LK cells in the rate at which they interact with ouabain or the rate at which they pump or both. LK pumps which are sensitive to stimulation by anti-L bind ouabain less rapidly than the remainder of the LK pumps and exposure to antibody increases the rate at which ouabain binds to the sensitive pumps; the difference between the two types of pumps disappears if intracellular K is very low. The calculated number of ouabain molecules bound at 100% inhibition of the pump is about the same for HK and LK cells. Although exposure to anti-L increases the apparent number of ouabain binding sites in LK cells at normal K(c), it does not alter the apparent number of sites in LK cells when K(c) has been reduced.

Communication to the editors stimulation of active potassium transport in LK sheep red cells by monovalent fragments of anti-L antibody

Communication to the editors stimulation of active potassium transport in LK sheep red cells by monovalent fragments of anti-L antibody

Communication to the editors stimulation of active potassium transport in LK sheep red cells by monovalent fragments of anti-L antibody

Communication to the editors stimulation of active potassium transport in LK sheep red cells by monovalent fragments of anti-L antibody

Incubation of HK and LK sheep red cells in vitro for long periods

HK and LK sheep red cells have been maintained in vitro at 37°C for periods of up to 3 weeks. During the incubation, periodic measurements of cell Na +, K +, ouabain-sensitive and ouabain-insensitive K + influxes were made. The effects of lowering the in vitro incubation temperature and of reducing the K + concentration in the medium on cell cation content and volume were also investigated. The results of these experiments were, in general, consistent with previous measurements of the effects of these parameters on K + and Na + fluxes in short term experiments. In vitro incubation for several days was also used to show that exposure to anti-L serum produces a net uptake of K + and extrusion of Na + in LK sheep red cells. A new procedure for measuring the active and passive influxes of K + was developed. The early time course of K + influx inhibition by ouabain was also studied by this procedure.