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.     

L-antigen and active potassium transport in HK and LK red cells of Barbary sheep (Ammotragus lervia)

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.     

Role of Nitrite, a Nitric Oxide Derivative, in K-Cl Cotransport Activation of Low-Potassium Sheep Red Blood Cells

K-Cl cotransport (COT) is the coupled movement of K and Cl, present in most cells, associated with regulatory volume decrease, susceptible to oxidation and functionally overexpressed in sickle cell anemia. The aim of this study was to characterize the effect of the oxidant nitrite (NO₂ ⁻) on K-Cl COT. NO₂ ⁻ is a stable metabolic end product of the short-lived highly reactive free radical nitric oxide (NO), an oxidant and modulator of ion channels, and a vasodilator. In some systems, the response to NO₂ ⁻ is identical to that of NO. We hypothesized that NO₂ ⁻ activates K-Cl COT. Low potassium (LK) sheep red blood cells (SRBCs) were used as a model. The effect of various concentrations (10⁻⁶ to 10⁻¹ m) of NaNO₂ was studied on K efflux in hypotonic Cl and NO₃ media, Cl-dependent K efflux (K-Cl COT), glutathione (GSH), and methemoglobin (MetHb) formation. In support of our hypothesis, K efflux and K-Cl COT were stimulated by increasing concentrations of NaNO₂. Stimulation of K efflux was dependent upon external Cl and exhibited a lag phase, consistent with activation of K-Cl COT through a regulatory mechanism. Exposure of LK SRBCs to NaNO₂ decreased GSH, an effect characteristic of a thiol-oxidizing agent, and induced MetHb formation. K-Cl COT activity was positively correlated with Methb formation. N-ethyl-maleimide (NEM), a potent activator of K-Cl COT, was used to assess the mechanism of NO₂ ⁻ action. The results suggest that NEM and NO₂ ⁻ utilize at least one common pathway for K-Cl COT activation. Since NaNO₂ is also a well known vasodilator, the present findings suggest a role of K-Cl COT in vasodilation. Received: 15 January 1998/Revised: 3 September 1998     

Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li _i ) but not external (Li _o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K _i of ∼7 mm. In Cl, Li _i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li _i reduced the V _max and increased the K _m for K efflux in Cl. Furthermore, Li _i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li _i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume. Received: 1 November 1999/Revised: 6 June 2000     

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.     

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells [1]. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithio-threitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux [1]. Maximum ³H-NEM incorporation into the DTT-reduced thiols occurred at 50 M DTT. Saturation labelling by ³H-NEM of about 2 × 10⁴ diamide-protected thiols/cell occurred at 25 M NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier [2]. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.     

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor (PDGF), apotent serum mitogen for vascular smooth muscle cells (VSMCs), plays animportant role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, isinvolved in ion homeostasis. VSMCs possess K-Cl COT activity and theKCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-ClCOT activity and mRNA expression in primary cultures of rat VSMCs. K-ClCOT was determined as the Cl-dependent Rb influx and mRNA expression bysemiquantitative RT-PCR. Twenty four-hour serum deprivation inhibitedbasal K-Cl COT activity. Addition of PDGF increased total proteincontent and K-Cl COT activity in a time-dependent manner. PDGFactivated K-Cl COT in a dose-dependent manner, both acutely (10 min)and chronically (12 h). AG-1296, a selective inhibitor of the PDGFreceptor tyrosine kinase, abolished these effects. Actinomycin D andcycloheximide had no effect on the acute PDGF activation of K-Cl COT,suggesting posttranslational regulation by the drug. Furthermore, PDGFincreased KCC1 and decreased KCC3 mRNA expression in a time-dependentmanner. These results indicate that chronic activation of K-Cl COTactivity by PDGF may involve regulation of the two KCC mRNA isoforms,with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

     

Properties of a goat anti-L antibody: further evidence for heterogeneity of the L antigen.

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L antibody has two specificities--a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L1y).     

Enzymatic modification of the L and M antigens in LK and HK sheep erythrocytes and their membranes

Summary This paper reports on the effect of two hydrolytic enzymes, neuraminidase and trypsin, on the interaction of blood group L-positive low-potassium-type (LK) and blood group M-positive high-potassium-type (HK) sheep red cells with their respective isoimmune antisera. It was found that treatment of LK and HK red cells with neuraminidase did not change the interaction of these cells with their homologous antibodies as measured by K⁺-pump flux, complement-mediated immune hemolysis and absorption of antibody. Similarly, trypsin pretreatment of LK and HK red cells did not interfere with the hemolytic action of anti-L and anti-M antibodies, respectively. In striking contrast, however, it was observed that pretreatment of LK cells with trypsin rendered these cells insensitive to the K⁺-pump stimulating antibody present in the anti-L serum.     

K-Cl cotransport in vascular smooth muscle and erythrocytes: possible implication in vasodilation

K-Cl cotransport, theelectroneutral-coupled movement of K and Cl ions, plays an importantrole in regulatory volume decrease. We recently reported that nitrite,a nitric oxide derivative possessing potent vasodilation properties,stimulates K-Cl cotransport in low-K sheep red blood cells (LK SRBCs).We hypothesized that activation of vascular smooth muscle (VSM) K-Clcotransport by vasodilators decreases VSM tension. Here we tested thishypothesis by comparing the effects of commonly used vasodilators,hydralazine (HYZ), sodium nitroprusside, isosorbide mononitrate, andpentaerythritol, on K-Cl cotransport in LK SRBCs and in primarycultures of rat VSM cells (VSMCs) and of HYZ-induced K-Clcotransport activation on relaxation of isolated porcine coronaryrings. K-Cl cotransport was measured as the Cl-dependent K efflux or Rbinflux in the presence and absence of inhibitors for other K/Rbtransport pathways. All vasodilators activated K-Cl cotransport in LKSRBCs and HYZ in VSMCs, and this activation was inhibited by calyculinand genistein, two inhibitors of K-Cl cotransport. KT-5823, a specificinhibitor of protein kinase G, abolished the sodiumnitroprusside-stimulated K-Cl cotransport in LK SRBCs, suggestinginvolvement of the cGMP pathway in K-Cl cotransport activation.Hydralazine, in a dose-dependent manner, and sodium nitroprussiderelaxed (independently of the endothelium) precontractedarteries when only K-Cl cotransport was operating and other pathwaysfor K/Rb transport, including the Ca-activated K channel, wereinhibited. Our findings suggest that K-Cl cotransport may be involvedin vasodilation.

     

Comparison of K-Cl cotransport expression in high and low K dog erythrocytes.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.     

Antibody-Induced Alterations in the Kinetic Characteristics of the Na:K Pump in Goat Red Blood Cells

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K_c), increasing K_c at first stimulates the active K influx in HK cells, but at higher K_c the pump is inhibited. These results suggest that in HK cells K_c acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na_c) at the Na translocation sites. In LK cells, K_c inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K_c. The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K_c LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells.

Binding of highly purified 125I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75-100 and 500-750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3-6 x 10(3) in HK (MM) and 1.5-4 x 10(3) in LK (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2-1.8 x 10(3) in LL and less in LM(LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na+K+ pumps measured previously in these cells by 3H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na+K+ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and /or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics, which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na+K+ pump molecules is more complex than that in goat red cells previously reported by others.     

PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells

K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.     

Binding characteristics of M and L isoantibodies to high and low potassium sheep red cells

Summary Binding of highly purified¹²⁵I labeled M and L antibodies, both belonging to the immunoglobulin G class, was studied in high potassium (HK) and low potassium (LK) sheep red cells. Anti-M and anti-L bound specifically to M and L antigen positive HK and LK red cells, respectively. Nonspecific binding was higher for anti-L to HK cells than for anti-M to LK cells. Once bound, the M and L antibodies were capable of inducing complement dependent immune hemolysis. Only 75–100 and 500–750 molecules of anti-M and anti-L immunoglobulins were required to hemolyze 50% of HK (MM) and LK (LL) red cells, respectively, suggesting that the M and L antigens may be clustered on the surfaces of these cells. Equilibrium binding studies revealed that the maximum number of M sites is 3–6×10³ in HK (MM) and 1.5–4×10³ in LM (LM) cells, respectively. In comparison, the number of L antigens is slightly lower in LK cells, about 1.2–1.8×10³ in LL and less in LM (LK) red cells. The number of M and L antigens, therefore, is more than an order of magnitude larger than that of the Na⁺K⁺ pumps measured previously in these cells by³H-ouabain binding, thus precluding a quantitative correlation between M and L antigens and the Na⁺K⁺ pumps different in the three genetic types of sheep red cells. The binding affinities of both anti-M and anti-L could not be described by a single equilibrium dissociation constant indicating heterogeneous antibody populations and/or variability in the antigenic sets of individual HK or LK cells. The pronounced heterogeneity of antigens and/or antibodies in both the M and L systems was reflected in the antibody association kinetics which also exhibited a remarkable temperature dependence. The data suggest that the correlation between the M and L antigens and the Na⁺K⁺ pump molecules is more complex than that in goat red cells previously reported by others.     

Properties of a goat anti-L antibody Further evidence for heterogeneity of the L antigen

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L anti-body has two specificities — a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L_ly.     

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.     

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.     

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 Lp and Ll 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.