Endothelins activate Ca2+-gated K+ channels via endothelin B receptors in CD-1 mouse erythrocytes

Alicia Rivera, Michelle A. Rotter, and Carlo Brugnara

Department of Laboratory Medicine Bader 7, The Children's Hospital, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell dehydration mediated by Ca2+-activated K+ channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca2+-activated K+ channel (Gardos channel) with maximal velocity (Vmax) of 0.154 ± 0.02 mmol · l cells-1 · min-1 and an affinity constant (K0.5) for Ca2+ of 286 ± 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their Vmax by 88 ± 9% (n = 8) and decreased their K0.5 for Ca2+ to 139 ± 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC50 of 75 ± 20 nM for ET-1 and 374 ± 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 ± 51 and 785 ± 143 nM and with capacity of binding sites of 298 ± 38 and 1,568 ± 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ETB) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ETB receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ETB receptors and a calphostin-sensitive mechanism.

Gardos channel; endothelin-1; sickle cell anemia; volume regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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CELL DEHYDRATION IS AN important step in the formation of sickle cells because Hb S polymerization markedly increases with small increases in cell Hb concentration. The Ca2+-gated K+ channel (Gardos channel) can be activated in vitro by oxygenation-deoxygenation cycles with resulting dehydration of sickle erythrocytes. The imidazole antimycotic clotrimazole (CLT) specifically inhibits the Gardos channel and reduces cell dehydration in vitro (7). CLT administration reduces erythrocyte dehydration in vivo in a transgenic mouse model of sickle cell disease (12) and in patients (8). It is not known whether other modalities of cell dehydration via activation of the Gardos channel could be relevant for the pathogenesis of sickle cell disease. It has been demonstrated that prostaglandin E2 (PGE2) can activate the Gardos channels of normal human erythrocytes, but it is not known whether this effect is mediated by Ca2+ entry or direct activation of the channel (29). Prostaglandins have been previously shown to alter size, deformability, and membrane structure by a Ca2+-dependent mechanism (35). In Hb S-containing cells, these effects would tend to increase cell sickling and favor erythrocyte entrapment and ultimate blockage of the microcirculation.

Recently, it has been found that levels of endothelin-1 (ET-1) in plasma are significantly elevated in sickle cell patients during painful crisis, suggesting a possible role of this endothelial cell product in the pathogenesis of the painful crisis (18, 36). Prostaglandins and endothelins have been shown to alter K+ transport in various cell types (35, 39). ET-1 was shown to activate Ca2+-gated K+ channels in vascular smooth muscle cells (40).

It has been found that the interaction of ET-1 with its receptor mediates an increase in intracellular Ca2+ that not only leads to a contractile response in myocytes but may also activate Ca2+-dependent K+ channels (38). However, it has also been reported that ET-1 inhibits Ca2+-gated K+ channels in rat basilar artery myocytes (39).

The effect of endothelins on erythrocyte ion transport has not been investigated. We postulated that endothelins could affect the function of the erythrocyte Gardos channel and could possibly affect control of cell volume in normal and sickle erythrocytes. However, there are no reports on the possible presence of endothelin receptors on mouse or human erythrocytes. Because mouse models of sickle cell disease have played an important role in the understanding of the pathophysiology of the disease and the design of new therapies, we investigated the functional characteristics of the Gardos channel in normal CD-1 mouse erythrocytes. In this report, we describe the presence of the Gardos channel in CD-1 mouse erythrocytes and its regulation by endothelins.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Drugs and chemicals. Charybdotoxin (ChTX), ET-1, BQ-788, IRL 1620, PGE2, RANTES (regulated on activation, normal T cell expressed, and secreted), interleukin-10 (IL-10), and IL-8 were purchased from RBI Signal Innovation (Natick, MA). Iberiotoxin (IbTX), Stichodactyla toxin (STX), kaliotoxin (KTX), and noxiustoxin (NxTX) were purchased from Peptide International (Louisville, KY). Dr. Maria L. Garcia (Merck Research Laboratories, Rahway, NJ) kindly provided margatoxin (MgTX). All peptides were prepared as indicated by the manufacturer and stored at -20°C for <3 mo. The A-23187 ionophore was purchased from Calbiochem-Novabiochem (La Jolla, CA). The iodinated ligand ET-1 and 86Rb were purchased from DuPont-New England Nuclear. All other reagents were purchased from Sigma (St. Louis, MO).

Animals and erythrocyte preparation. Male CD-1 mice (Charles River, MA), 5-8 wk old, were used for these studies. Blood was collected in the presence of Na+-heparin from ether-anesthetized animals. Blood was passed through cotton to decrease the number of leukocytes and then centrifuged in a Sorvall RC (Jouan) centrifuge for 4 min at 4°C and 2,000 rpm. Erythrocytes were washed four times with choline washing solution containing (in mM) 165 choline chloride, 1 MgCl2, and 10 Tris-MOPS (pH 7.4 at 4°C).

Measurement of 86Rb influx. Freshly washed erythrocytes were suspended at a hematocrit of 2% in normal influx media containing 165 mM NaCl, 2 mM KCl, 0.15 mM MgCl2, 1 mM ouabain, 10 mM Tris-MOPS (pH 7.4 at 22°C), 10 µM bumetanide, and 10 µCi/ml 86Rb in the presence or absence of an active peptide. Preincubations with endothelins or other substances were carried out for 20 min at 37°C in an isotonic saline. The same concentrations of active peptides or drugs were also added to the influx media. Free Ca2+ in the influx media was buffered to between 0 and 3.5 µM with 1 mM EGTA or citrate buffer as described by Wolff et al. (46). The Ca2+ concentration was calculated by using the dissociation constants (Kd) for EGTA or citrate and correcting for ionic strength at pH 7.4 and 0.15 mM MgCl2. The effects of ET-1 in the absence of A-23187 were tested by preincubating fresh washed mouse erythrocytes with or without the active peptide in saline solution [165 mM NaCl, 2 mM KCl, 1 mM CaCl2, 0.15 mM MgCl2, 10 mM Tris-MOPS (pH 7.4, 37°C), 1 mM ouabain, and 10 µM bumetanide] for 15 min. The fluxes were measured in the presence or absence of ET-1 at the same preincubation concentration. For experiments with A-23187, a 5 µM concentration of the ionophore was added at time 0 and aliquots at 0.33, 2, and 5 min were removed and immediately spun down through 0.8 ml of cold medium containing 5 mM EGTA buffer and an underlying cushion of n-butyl phthalate. Supernatants were aspirated, and the tube tip containing the cell pellet was cut off. The erythrocyte-associated radioactivity was counted in a gamma counter (model 41600 HE; Isomedic ICN Biomedicals, Costa Mesa, CA). K+ uptake was linear up to 5 min, and fluxes were calculated from the slope of the linear regression as described by Brugnara et al. (4).

ET-1 binding assay. Erythrocytes were washed with choline washing solution and suspended at 10% hematocrit for 1 h at 4°C in a binding solution containing 165 mM NaCl, 2 mM KCl, 0.15 mM MgCl2, 10 mM Tris-MOPS (pH 8.0, 4°C), and 1 mg/ml BSA. Cells were centrifuged and added to a final concentration of 1 × 106 cells/ml into binding media without BSA containing 125I-labeled ET-1 in the absence or presence of unlabeled ET-1 as described in the figure legends. In experiments using the antagonist BQ-788 or BQ-123, unlabeled ET-1 was replaced by these two peptides. Cell suspensions were incubated for up to 1 h at 4°C, unless otherwise stated. At specific time points, aliquots of 0.25 ml were pelleted in filters (Microfiber GF/B; Whatman) and washed with 5 vol of the binding media at 4°C. The filters were presoaked for 1 h at room temperature in BSA-binding solution (0.1%). The cell-containing filters were counted in a gamma counter. All linear or nonlinear curve fittings were performed as described in the figure legends with Enzyme Fitter (version 1.05; Elsevier-Biosoft), unless otherwise stated.

PKC activity measurements. Blood was centrifuged at 1,500 rpm for 10 min at 4°C to remove plasma and buffy coat (white blood cells). Erythrocytes were washed four times at 1,500 rpm for 5 min at 4°C with washing medium A containing (in mM) 145 NaCl, 5 KCl, 10 HEPES-Tris (pH 7.4), and 0.1 sodium phosphate. Membranes were prepared by hemolysis of cells in a 20-fold excess of lysis medium B containing 10 mM Tris · HCl (pH 7.4), 0.1 mM EDTA, 5 mM dithiothreitol (DTT), 0.01 mM phenylmethylsulfonyl fluoride, and 1 mg/ml leupeptin. The membrane pellet was centrifuged at 18,000 rpm (Sorvall SS34 rotor) for 20 min, the supernatant (cytosol) was removed and stored on ice, and the ghosts were washed four times in lysis medium B. Membranes were resuspended to a final concentration of ~1 mg protein/ml lysis buffer. Protein determination was performed by the bicinchoninic acid method (Pierce). The Hb-free membrane suspensions were stored in Eppendorf tubes at -70°C until ready for assay.

Protein kinase C (PKC) activity was assayed by measuring the rate of 32P incorporation into a peptide that is specific for PKC (kit RPN77; Amersham, Arlington Heights, IL). The assay mixture contained 12 mM calcium acetate, 30 mM DTT, 50 mM Tris · HCl (pH 7.5), 900 µM peptide, 0.3 mg/ml L-phosphatidylserine, and 24 µg/ml phorbol 12-myristate 13-acetate. An aliquot (25 µl) of the membrane suspension was added to this mixture (25 µl), and the reaction was initiated by the addition of [32P]ATP. After 30 min of incubation at 37°C, the reaction was stopped by the addition of orthophosphoric acid. A 40-µl aliquot of the reaction medium was spotted on a 2.5 × 2.5 cm square of phosphocellulose paper. The papers were washed in 75% (vol/vol) phosphoric acid to eliminate any nonspecific binding and dried, and the 32P level was determined by scintillation counting. PKC activity was expressed as picomoles of 32P incorporated into the peptide per microgram of protein per minute.


    RESULTS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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Ionized Ca2+ activation curve of K+ influx via the Gardos channel. The activation of the K+ influx by cellular Ca2+ was studied in the presence of A-23187 to clamp intracellular Ca2+ at desired values as shown by Escobales and Canessa (14). Figure 1A shows the dependence of K+ influx on extracellular free Ca2+ in CD-1 mouse erythrocytes, in the presence or absence of 50 nM ChTX. K+ influx increased rapidly and saturated at ~0.166 mmol · l cells-1 · min-1 when Ca2+ was increased up to 3.5 µM. The nonlinear fitting of the experimental points for a sigmoidal function gave a maximal velocity (Vmax) of 0.158 ± 0.01 mmol · l cells-1 · min-1 (n = 8). In the presence of ChTX (50 nM), the K+ influx was inhibited to 0.02 ± 0.001 mmol · l cells-1 · min-1. The difference between the two curves (Fig. 1B; ChTX-sensitive flux) gave a Vmax of 0.154 ± 0.02 mmol · l cells-1 · min-1 and affinity constant (K0.5) for Ca2+ of 286 ± 83 nM (n = 8). These data demonstrate the presence of Ca2+-activated K+ channels (Gardos channels) in CD-1 mouse erythrocytes as previously described for human erythrocytes (6). We tested the effects of other venom toxins on Ca2+-activated K+ influx in CD-1 mouse erythrocytes in a low-ionic-strength medium containing 18 mM NaCl, 2 mM KCl, 230 mM sucrose, 10 mM Tris · HCl (pH 8.0), 0.01 mM bumetanide, 5 µM A-23187, 54 µM CaCl2, 1 mM ouabain, and 10 µCi/ml 86Rb. A significant inhibition of the Ca2+-activated K+ influx was observed with KTX (95 ± 5%; 50 nM), MgTX (92 ± 10%; 50 nM), and NxTX (87 ± 1%; 50 nM). IbTX showed partial inhibitor potency (55 ± 10%; 50 nM). We also tested the effect of CLT, a potent and specific inhibitor of the Gardos channel in human erythrocytes (5, 7). The Ca2+-activated K+ influx in the presence of A-23187 was completely inhibited by 10 µM CLT in a low-ionic-strength medium (97 ± 5%) and was 92 ± 8% inhibited in normal saline (n = 3). In addition, the CLT metabolites 2-chlorophenyl-diphenylmethanol and 2-chlorophenylmethane (10 µM) were also effective as inhibitors of the Gardos channel in mouse erythrocytes. Although less potent than CLT, 2-chlorophenyl-4-hydrophenyl-phenyl-methane and/or 2-chlorophenyl-4-hydrophenyl-phenyl-methanol at 10 µM produced significant inhibition (59 ± 5 and 42 ± 7%, respectively) of K+ influx mediated by the mouse erythrocyte Gardos channel (8).


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Fig. 1.   Activation curve of K+ influx by ionized Ca2+ in the absence () and presence (open circle ) of charybdotoxin (ChTX) in CD-1 mouse erythrocytes. K+ influx was measured in media containing Ca2+ concentrations between 0 and 3.5 µM. Ca2+-activated K+ influx was calculated from the total K+ influx minus the influx at nominal free external Ca2+. A: the 2 curves were fit with a nonlinear regression analysis for sigmoidal kinetics. B: difference between curves shown in A. Nonlinear regression analysis gave an affinity constant (K0.5) of 286 ± 83 nM and a maximal velocity (Vmax) of 0.154 ± 0.012 mmol · l cells-1 · min-1 (n = 8). Experimental points are means ± SE.

Vasoactive mediators activate the Gardos channels of mouse erythrocytes. Active agents such as RANTES, platelet activator factor, PGE2, and interleukins have been shown to mobilize Ca2+ causing an increase in cytosolic Ca2+ in white blood cells (2, 3, 27). Therefore, we hypothesized that these peptides could also activate the Gardos channels in erythrocytes, as had been previously shown for eosinophils (13, 37). We tested the effect of these peptides on the ChTX-sensitive K+ influx in CD-1 mouse erythrocytes as a function of ionized extracellular Ca2+. As shown in Table 1, RANTES and IL-10 significantly increased by 1.5- to 2-fold the Vmax of the channel at 10 and 20 ng/ml, respectively. Whereas IL-10 significantly increased the K0.5 for Ca2+ from 286 ± 83 to 703 ± 17 nM (P < 0.05, n = 3), PGE2 and IL-8 significantly decreased it to 129 ± 51 and 130 ± 32 nM, respectively. A Hill plot analysis indicated that the Hill coefficients for Ca2+-activated K+ influx significantly increased (2-to 3-fold) in cells treated with IL-10, IL-8, and RANTES.

                              
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Table 1.   Modulators of Ca2+-activated K+ channels in CD-1 mouse erythrocytes

Endothelins activate the Gardos channels of CD-1 mouse erythrocytes. We measured the ChTX-sensitive fraction of the K+ influx in CD-1 mouse erythrocytes after 20-min pretreatments with and without ET-1 (500 nM). The time course of K+ uptake was measured at 1 µM free extracellular Ca2+ in the presence of A-23187 with or without ChTX (50 nM) as shown in Fig. 2. Under these experimental conditions, intracellular Ca2+ was clamped at its electrochemical equilibrium. In control cells, K+ uptake was linear up to 5 min (r2 = 0.98) and was significantly inhibited by 50 nM ChTX. In ET-1-pretreated cells, K+ uptake doubled in 5 min and was completely inhibited by ChTX. The effect of ET-3 was tested under similar conditions. From Fig. 2, the Vmax in cells pretreated with ET-1 was 0.32 ± 0.04 mmol · l cells-1 · min-1; it was 0.28 ± 0.02 mmol · l cells-1 · min-1 in cells pretreated with ET-3. Both fluxes were significantly higher than the control (0.16 ± 0.04 mmol · l cells-1 · min-1; n = 8, P < 0.03) under similar conditions.


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Fig. 2.   Effects of endothelin-1 (ET-1) and ET-3 on K+ uptake. Time courses of K+ uptake in presence of 1 µM Ca2+ and 5 µM A-23187 and in absence (solid symbols) or presence (open symbols) of ChTX (50 nM) are shown. Cells were preincubated with either 500 nM ET-1 (black-triangle, triangle ) or 500 nM ET-3 (, ) or in normal saline (, open circle ) at 37°C for 20 min. ChTX-sensitive flux was calculated from difference of linear regression curves in presence or absence of ChTX. Graph represents 1 of 3 similar experiments.

Mouse erythrocytes were incubated with and without ET-1 (500 nM) for up to 15 min at room temperature in the absence of A-23187. 86Rb (K+) uptake was measured during that time in a saline solution containing 1 mM CaCl2 in the presence or absence of ChTX (50 nM). As seen in Fig. 3, the basal ChTX-sensitive K+ influx (0.015 ± 0.002 mmol · l cells-1 · min-1) was enhanced by the presence of ET-1 to a value of 0.055 ± 0.004 mmol · l cells-1 · min-1 (n = 3). These findings suggest that ET-1 could mediate regulation of the Gardos channel in vivo.


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Fig. 3.   Time course of ET-1-induced K+ uptake in absence of A-23187. ChTX-sensitive fraction was calculated from difference between fluxes in presence or absence of ChTX (50 nM). Influx media contained 1 mM CaCl2 with (open circle ) or without () 500 nM ET-1. Graph represents 1 of 3 similar experiments.

To study the specific effect of ET-1 on the kinetic properties of the Gardos channel, the dependence of the channel activity on free Ca2+ was determined in cells pretreated with and without ET-1 (500 nM). As shown in Fig. 4, the Vmax of the system after ET-1 treatment increased twofold and the K0.5 decreased from 286 ± 83 to 139 ± 63 nM (n = 4; P < 0.05). A dose-response curve for the activation of ChTX-sensitive K+ influx by ET-1 is shown in Fig. 5. The estimated EC50 for ET-1 was 75 ± 20 nM (n = 3). Similar experiments with ET-3 yielded an EC50 of 374 ± 97 (n = 3).


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Fig. 4.   Ca2+ activation curve of K+ influx with (black-triangle) or without (open circle ) ET-1 in CD-1 mouse erythrocytes. Cells were pretreated with or without ET-1 (500 nM) for 20 min at 37°C. K+ influx was measured in presence of A-23187 (5 µM) and various Ca2+ concentrations as described in MATERIALS AND METHODS. The ChTX-sensitive fraction was calculated from difference between K+ influxes in presence and absence of ChTX (50 nM) under similar conditions. A nonlinear fitting for sigmoidal kinetics gave a K0.5 for ET-1-treated cells of 139 ± 63 nM and Vmax of 0.305 ± 50 mmol · l cells-1 · min-1. Control value for Vmax is 0.154 ± 0.02 mmol · l cells-1 · min-1 and for K0.5 is 286 ± 83 nM. Values are means ± SE of 3 or more experiments.



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Fig. 5.   Dose-response curves for ChTX-sensitive K+ influx activated by ET-1 and ET-3 in mouse erythrocytes. K+ influx was measured in cells pretreated with various concentrations of ET-1 (triangle ) or ET-3 () in presence or absence of ChTX (50 nM). ChTX-sensitive fraction was estimated as described in legend for Fig. 2. Curves were estimated from best fit by a nonlinear regression analysis. Calculated EC50 values for ET-1 and ET-3 were 75 ± 20 and 374 ± 97 nM, respectively. Data points are means ± SE of 3 or more experiments.

ET-1 binds specifically to CD-1 mouse erythrocytes. Because ET-1 receptors have not been described in erythrocytes, we tested whether ET-1 specifically binds to an endothelin receptor in intact erythrocytes. As shown in Fig. 6, 125I-ET-1 binding to intact erythrocytes reached a plateau in 30 min at 4°C. The presence of 1 µM ET-1 significantly decreased (80%) the total binding. The specific binding of 125I-ET-1 to mouse erythrocytes was a saturable process consistent with a specific receptor interaction as shown in Fig. 7A. A Scatchard plot analysis revealed the presence of a class of high-affinity binding sites with an apparent association constant of 155 ± 23 pM and a maximal binding capacity of 390 ± 35 sites/cell (0.163 ± 0.01 fmol/2.5 × 105 cells). A competition assay of the radiolabeled ET-1 (100 pM) with ET-1 revealed a maximal inhibition at ~1.5 µM. Analysis of these experiments showed two distinct binding sites with Kd values of 167 ± 51 and 787 ± 143 nM, and maximal binding values of 298 ± 38 and 1,568 ± 211 sites/cell, respectively.


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Fig. 6.   Time course of 125I-ET-1 binding to intact mouse erythrocytes. Binding of iodinated ET-1 (100 pM) was assayed at 4°C in presence (open circle ) or absence () of ET-1 (1 µM). Specific binding was calculated from difference between total binding values in absence and presence of ET-1. Data points are means ± SE of 3 experiments in duplicate determinations.



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Fig. 7.   Specific binding of 125I-ET-1 in presence of various concentrations of ET-1. A: specific binding was calculated from difference between total binding of 125I-ET-1 and binding in presence of 1 µM ET-1. Scatchard plot of specific binding of iodinated ET-1 gave an association constant of 155 ± 23 pM and 390 ± 35 sites/cell by single-ligand binding site analysis (Enzyme Fitter; Elsevier-Biosoft). B: dose-dependent inhibition of 125I-ET-1 binding by cold ET-1. Modification of Scatchard plot (125I-ET-1 binding vs. 125I-ET-1 binding × [ET-1]) showed dissociation constant (Kd) values of 167 ± 51 and 787 ± 143 nM, with maximal binding values of 298 ± 38 and 1,568 ± 211 sites/cell, respectively. Line represents nonlinear regression of experimental points. Data represent 1 of 3 or more similar experiments performed in triplicate determinations.

A comparison between levels of 125I-ET-1 binding in the presence of ET-1, BQ-788 (a selective antagonist of ETB receptors), and BQ-123 (a selective antagonist of ETA receptors) is shown in Fig. 8. ET-1 displaced labeled ET-1 with an IC50 of 600 ± 125 nM, which was lower affinity than that for BQ-788 (IC50: 200 ± 53 nM) and higher than that for BQ-123 (IC50: 750 ± 42 nM). These data suggest that in mouse erythrocytes the specific binding of ET-1 can be accounted for by the presence of both ETA and ETB receptor subtypes.


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Fig. 8.   Dose-dependent inhibition curves of 125I-ET-1 binding by ET-1 (), BQ-788 (), and BQ-123 (black-triangle). Erythrocytes were incubated with 50 pM 125I-ET-1 and different concentrations of unlabeled ET-1 or antagonists for 1 h at 4°C. Data are inhibitory percentages of specific binding observed at each peptide concentration. Points are means for 1 of 3 similar experiments.

ETB receptors mediate endothelin effect on Gardos channels in mouse erythrocytes. To test whether endothelin's action on the Gardos channel was mediated by an ETB receptor, we measured the effect of the ETB receptor agonist IRL 1620 on the Gardos channel activity in CD-1 erythrocytes (Fig. 9). IRL 1620 (500 nM) elicited a significant increase in the Gardos-mediated 86Rb influx from 0.156 ± 0.01 to 0.223 ± 0.003 mmol · l cells-1 · min-1 (P < 0.05; n = 3), which was not significantly different from that induced by 500 nM ET-1 (0.220 ± 0.01 mmol · l cells-1 · min-1). Thus the effect of ET-1 on Gardos channel activity could be mediated by ETB receptors. The effect of ETB receptor antagonist BQ-788 on the ET-1-induced activation of the Gardos channel was also studied. Preincubation of erythrocytes with both ET-1 (500 nM) and BQ-788 (1 µM) for 20 min at 37°C significantly decreased the ET-1-induced activation of the Gardos channel by 85%. Similarly, Gardos channel activation by the ETB receptor agonist IRL 1620 was significantly suppressed by BQ-788. As shown in Fig. 9, BQ-788 by itself did not induce inhibition of the Gardos channel.


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Fig. 9.   Effect of selective endothelin B (ETB) agonist and antagonist on ChTX-sensitive K+ influx. Erythrocytes were preincubated with 500 nM ET-1, 500 nM IRL 1620, and/or 1 µM BQ-788 for 20 min. K+ influx was measured in a medium containing 1 µM Ca2+ and 5 µM A-23187 with or without ChTX (50 nM) in presence or absence of peptides. Values are means ± SE of 3 or more experiments in duplicate. Statistical significance was determined by t-test (* P < 0.05 compared with control; ** P < 0.05 compared with ET-1- and IRL 1620-stimulated fluxes).

Endothelin-induced activation of the Gardos channel is blocked by the PKC inhibitor calphostin C. Human erythrocytes express only two isoforms of PKC, xi  and alpha . It has been shown that the Gardos channel is modulated by PKCalpha under low-oxygen conditions in human sickle cells (15). Elevation of intracellular Ca2+ by A-23187 has been shown to increase the translocation of PKCalpha to the cell membrane. Because ET-1 has been demonstrated to activate PKC in vascular smooth muscle (19), we have investigated the effect of a PKC inhibitor, calphostin C, on the ET-1-induced Gardos channel in CD-1 mouse erythrocytes. Calphostin C is an inhibitor of PKC that binds to the phorbol/diacylglycerol site and has little effect on other protein kinase activity (9). Erythrocytes were pretreated with calphostin C (10 µM) and 500 nM ET-1 for 20 min at 37°C. As shown in Fig. 10A, calphostin C significantly decreased (86 ± 11%; n = 3) the ET-1-induced activation of the Gardos channel.


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Fig. 10.   ET-1 effects on Gardos channel and protein kinase C (PKC) activity. A: erythrocytes were pretreated with 10 µM calphostin C for 20 min at 37°C. K+ influx was measured in presence of 1 µM Ca2+ and 5 µM A-23187. ChTX-sensitive fractions were calculated from difference between K+ influxes with and without ChTX (50 nM). Bars represent means ± SE of 3 or more experiments in duplicate. Statistical significance was determined by t-test (*, ** P < 0.03). B: erythrocytes were incubated with and without ET-1 (300 nM) for 15 min at 37°C. Cells were placed on ice rapidly and lysed as described in materials and methods, and PKC activity was calculated. Membranes were treated with and without calphostin C (1 µM) at 4°C for 30 min. Data points represent 1 of 3 similar experiments. *, ** P < 0.05. *Control vs. ET-1-treated cells; **ET-1-treated cells vs. ET-1 and calphostin C-treated cells.

PKC activity is increased in ET-1-treated cells. PKC enzymatic activity in membranes from cells treated with 300 nM ET-1 for 15 min at 37°C or not treated was measured. Membranes were prepared as described in MATERIALS AND METHODS. As shown in Fig. 10B, the basal activity of PKC increased by 84 ± 3% (n = 3) in ET-1-treated cells. This increase was significantly inhibited (66 ± 2%; n = 3) by calphostin C, suggesting that ET-1 increased specifically the activity of PKC.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that CD-1 mouse erythrocytes express Ca2+-activated K+ influx that is specifically inhibited by ChTX. This toxin has been shown to be a highly specific inhibitor of the Ca2+-activated K+ channels (Gardos channels) in human erythrocytes and other mammalian cells (6, 32). In mouse erythrocytes, activation kinetics of the Gardos channel by extracellular Ca2+ in the presence of A-23187 indicated a very high affinity for Ca2+. Kinetic analysis indicated a K0.5 for Ca2+ of <300 nM (286 ± 83 nM; n = 8) and a Hill coefficient of 2.7 ± 0.3. It is possible that in mouse erythrocytes the activation of the Gardos channel by Ca2+ may involve more than one active site as previously described for human erythrocytes (46).

The Gardos channels of mouse erythrocytes display a sensitivity for peptide toxins similar to that displayed by human erythrocytes (6, 17). Venom peptide toxins ChTX, MgTX, and KTX are highly effective in inhibiting the mouse channel in normal saline solution. MgTX is much less potent in human erythrocytes (4). IbTX, a specific inhibitor of the high-conductance Gardos channel in excitable cells (11), shows only partial inhibitory effect at 50 nM on the mouse Gardos channel (55%), as well as in human erythrocytes. STX displayed significant inhibition of the Gardos channel and displacement of bound ChTX in human erythrocytes (6). Likewise, STX toxin can also inhibit the CD-1 Gardos channel under similar conditions. Three different types of the Gardos channel in human erythrocytes have been reported (28), which could account for the different inhibitory potencies of these toxins in mouse and human erythrocytes. We speculate that the lack of inhibition of MgTX in human erythrocytes at physiological conditions may represent the absence of a subtype Gardos channel present in mouse erythrocytes. Vandorp et al. (43) recently cloned the cDNA encoding the Gardos channel from a murine erythroleukemia cell line and showed that the amino acid sequence is 88% identical to that of the human Gardos channel. This difference could also explain the pattern of sensitivity to peptide toxins of mouse erythrocytes (43).

Previous studies have indicated that activation of ET-1 receptors mobilizes intracellular Ca2+ stores in nonerythroid cells (1, 30, 42). The ET-1- and ET-3-induced relaxation of trachea smooth muscle cells is mediated by ChTX-sensitive K+ channels (20). Also, patch-clamp techniques have been used to describe the activation of the Ca2+-gated K+ channel by endothelins in isolated coronary artery smooth muscle cells (23). In mouse erythrocytes, we observed that ET-1 increased both the Gardos channel activity and, by twofold, the affinity for internal Ca2+, indicating a positive modulation of this channel by this peptide. The threefold increase in the Vmax of the Gardos channel by endothelins suggests that the active peptides either increase the number of active channels by recruiting quiescent units or, alternatively, increase the open time of the active units.

The displacement of 125I-ET-1 by unlabeled ET-1 at 4°C demonstrates the presence of endothelin receptors in mouse erythrocytes (Fig. 6). Saturation of the receptor by labeled ET-1 was obtained in <1 h at 4°C. A Scatchard analysis of radiolabeled ET-1 binding indicated a binding site with a Kd of 156 ± 23 pM and 390 ± 35 sites/cell (Fig. 7A). These results are in agreement with the Kd and binding kinetics described for endothelin receptors in other cell types (41, 48). However, the effects of ET-1 on the K+ channels are seen at much higher concentrations, suggesting the involvement of a receptor with lower affinity. Competition of unlabeled ET-1 with 100 pM 125I-ET-1 indicated that the radiolabeled ligand was maximally displaced at 1.5 µM ET-1. Analysis of Fig. 7B using a modified version of the Scatchard plot as shown by Bylund (10), demonstrates the presence of at least two other sites, one with a Kd of 167 ± 51 nM and the other with a Kd of 787 ± 143 nM, with maximal binding values of 289 ± 38 and 1,598 ± 211 sites/cell, respectively. These data suggest that the effect of ET-1 on the channel might be mediated by these two low-affinity ET-1 receptors.

Unlabeled ET-1 and endothelin receptor antagonists (BQ-788 and BQ-123) displaced 125I-ET-1 with an order of potency of BQ-788 > ET-1 > BQ-123. The strong inhibition of 125I-ET-1 binding by BQ-788 may suggest that ETB receptors are mainly present in mouse erythrocytes. BQ-788 also blocks the transport effect of ET-1 (Fig. 9). A concentration of IRL 1620 (ETB-selective agonist) equivalent to that of ET-1 can induce similar ChTX-sensitive K+ fluxes in mouse erythrocytes, which are inhibited by BQ-788. It is not clear why BQ-788 significantly reduces the K+ influx below control values in the presence of the ETB agonist IRL 1620 (Fig. 9). We can speculate that when there is complete blockage of the ETB receptor by 1 µM BQ-788, IRL 1620 might be interfering with another receptor that significantly inhibits the Gardos channels or possibly that the presence of IRL 1620 and BQ-788 in the cell suspension blocks the channel directly (Fig. 9).

The intracellular signaling mechanisms that mediate ET-1 actions in nonerythroid cells include phospholipase C, diacylglycerol, and PKC (44). In single-channel studies, it has been observed that the sensitivity of the Gardos channel to Ca2+ is dependent on the phosphorylation state of the protein (25). The ET-1 effect on the mouse erythrocyte Gardos channel can be blocked by calphostin C (Fig. 10). This is consistent with a specific effect on the channel's activity and suggests that the channel or an associated regulatory protein or proteins are required to be phosphorylated to be active. Although phosphorylation events are required for ET-1-induced Gardos activity, phosphorylation by PKC seems not to be essential for channel activation (Fig. 10). Furthermore, we found that the activity of PKC significantly increased by 84% in the presence of ET-1 and that this increase was inhibited by calphostin C. Recent studies indicated that human erythrocytes express only two isoforms of PKC, xi  and alpha , and that the Gardos channel is modulated by PKCalpha under low-oxygen conditions in human sickle cells (15). Furthermore, elevation of intracellular Ca2+ by A-23187 increased the translocation of PKCalpha to the cell membrane (15). Therefore, we can speculate that the effect of ET-1 on the Gardos channel might be mediated by PKCalpha .

We have also shown that cytokines and chemokines can activate the Gardos channels in mouse erythrocytes. These active peptides are well known to act on a variety of immune cells via receptor-ligand interactions (33). Recently, chemokine receptors in erythrocytes were described (22). Among the active peptides tested in mouse erythrocytes, IL-10 and RANTES significantly increased the Gardos channel Vmax (Table 1). In addition, IL-10 as well as IL-8 alters the affinity of the Gardos channel for intracellular Ca2+ (Table 1). Because some of these ligands specifically interact with the chemokine receptor, it is possible that the Gardos channel and chemokine receptors are functionally coupled in CD-1 mouse erythrocytes. In addition, under pathological conditions, these active peptides may positively or negatively regulate the Gardos channel, with possible changes in the hydration and deformability of the erythrocytes. Recently, Kumar et al. (26) reported that IL-8 can also promote adherence to the endothelium of sickle, but not normal, erythrocytes. The events leading to the overexpression of adhesion molecules such as integrins and glycoproteins in sickle cells are not completely understood. Elevation of intracellular Ca2+ and PKC activation causing phosphorylation of integrins have been postulated as possible physiological mechanisms for the enhancement of adhesion molecules (16, 45). It has been shown that integrin alpha 4beta 1 and glycoprotein IV are expressed on circulating reticulocytes from sickle cell patients (24). Because we found that normal erythrocytes express ET-1 receptors that can increase PKC activity, the relationship of ET-1 with integrin and glycoprotein expression on sickle reticulocytes and erythrocytes should also be investigated.

Our data suggest that erythrocyte dehydration may take place via activation of the Gardos channel by vasoactive peptides, in the absence of deoxygenation. Thus the modulation of Gardos channel activity by ET-1 might play an important role in the dehydration of sickle erythrocytes. It is possible that, on ET-1 receptor activation, intracellular Ca2+ and activation of PKC induce the opening of the Gardos channel, resulting in K+ and water loss and possibly formation of denser erythrocytes. This is in agreement with the activation of the Gardos channel by ET-1 in the absence of A-23187 (Fig. 3) and could suggest the coupling of the Gardos channel to the ET-1 receptor. In addition, sickle erythrocytes have been shown to interact with vascular endothelial cells, stimulating the release of active peptides and regulating the expression of the ET-1 gene and protein in cultured endothelial cells (34). This effect seems to be specific for sickle cells, because induction of other genes, such as those for actin and platelet-derived growth factor-beta , was not regulated by sickle cells. This is consistent with elevated plasma ET-1 levels during the painful-crisis episode and during acute chest syndrome observed in sickle cell patients (21, 31, 34, 47).

Furthermore, it has been shown that the level of ET-1 in plasma correlates with the state of the disease (21). However, we do not know whether increased plasma ET-1 levels can modulate the Gardos channel in vivo. There is no evidence for erythrocyte dehydration in normal CD-1 mice. This is not unexpected, because normal plasma ET-1 levels fail to induce significant activation of the Gardos channel in vitro.

The concentration of ET-1 necessary to stimulate the Gardos channel exceeds the levels documented in plasma during painful crisis. However, local ET-1 levels in the microvasculature are likely to be much higher than those measured systematically. Therefore, these local interactions between active peptides, PKC, and K+ channels might be relevant to the pathophysiology of sickle cell disease in the course of steady-state or developing acute crisis. If the role of ET-1 in sickle cell dehydration can be confirmed in vivo in either the transgenic sickle mouse model or patients, this interaction could offer new potential therapeutic approaches to sickle cell disease.


    ACKNOWLEDGEMENTS

We thank Dr. Seth Alper for his excellent critique of the manuscript and Tammy Nguyen for excellent technical assistance.


    FOOTNOTES

This work was supported by National Institutes of Health Grants P604L15157 and DK-50422.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Rivera, The Children's Hospital, Dept. of Laboratory Medicine Bader 7, 300 Longwood Ave., Boston, MA 02115 (E-mail: rivera_a{at}a1.tch.harvard.edu).

Received 12 January 1999; accepted in final form 16 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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