Department of Laboratory Medicine Bader 7, The Children's Hospital, Boston, Massachusetts 02115
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
cells1 · 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
cells1 · 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).
|
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.
|
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
cells1 · 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.
|
|
|
|
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.
|
|
|
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
cells1 · 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.
|
Endothelin-induced activation of the Gardos channel is blocked by
the PKC inhibitor calphostin C.
Human erythrocytes express only two isoforms of PKC, and
. It
has been shown that the Gardos channel is modulated by PKC
under
low-oxygen conditions in human sickle cells (15). Elevation of
intracellular Ca2+ by A-23187 has
been shown to increase the translocation of PKC
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.
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, and
, and that the Gardos channel is modulated by PKC
under low-oxygen conditions in human sickle cells (15). Furthermore, elevation of intracellular Ca2+ by A-23187 increased the
translocation of PKC
to the cell membrane (15). Therefore, we can
speculate that the effect of ET-1 on the Gardos channel might be
mediated by PKC
.
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 4
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-, 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Araki, S.,
Y. Kawahara,
K. Kariya,
M. Sunako,
H. Fukuzaki,
and
Y. Takai.
Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells.
Biochem. Biophys. Res. Commun.
159:
1072-1079,
1989[Medline].
2.
Bacon, K. B.
Calcium mobilization and phosphoinositide turnover as measure of chemokine receptor function in lymphocytes.
Methods Enzymol.
288:
362-383,
1997[Medline].
3.
Barker, M. D.,
and
P. N. Monk.
Structure-function relationships of leucocyte chemoattractant receptors.
Biochem. Soc. Trans.
25:
1027-1031,
1997[Medline].
4.
Brugnara, C.,
C. C. Armsby,
L. De Franceschi,
M. Crest,
M. F. Euclaire,
and
S. L. Alper.
Ca2+-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins.
J. Membr. Biol.
147:
71-82,
1995[Medline].
5.
Brugnara, C.,
C. C. Armsby,
M. Sakamoto,
N. Rifai,
S. L. Alper,
and
O. Platt.
Oral administration of clotrimazole and blockade of human erythrocyte Ca2+-activated K+ channel: the imidazole ring is not required for inhibitory activity.
J. Pharmacol. Exp. Ther.
273:
266-272,
1995[Abstract].
6.
Brugnara, C.,
L. De Franceschi,
and
S. L. Alper.
Ca2+-activated K+ transport in erythrocytes. Comparison of binding and transport inhibition by scorpion toxins.
J. Biol. Chem.
268:
8760-8768,
1993
7.
Brugnara, C.,
L. De Franceschi,
and
S. L. Alper.
Inhibition of Ca2+-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives.
J. Clin. Invest.
92:
520-526,
1993[Medline].
8.
Brugnara, C.,
B. Gee,
C. C. Armsby,
S. Kurth,
M. Sakamoto,
N. Rifai,
S. L. Alper,
and
O. S. Platt.
Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.
J. Clin. Invest.
97:
1227-1234,
1996
9.
Bruns, R. F.,
F. D. Miller,
R. L. Merriman,
J. J. Howbert,
W. F. Heath,
E. Kobayashi,
I. Takahashi,
T. Tamaoki,
and
H. Nakano.
Inhibition of protein kinase C by calphostin C is light-dependent.
Biochem. Biophys. Res. Commun.
176:
288-293,
1991[Medline].
10.
Bylund, D. B.
Graphic presentation and analysis of inhibition data from ligand-binding experiments.
Anal. Biochem.
159:
50-57,
1986[Medline].
11.
Candia, S.,
M. L. Garcia,
and
R. Latorre.
Mode of action of iberiotoxin, a potent blocker of the large conductance Ca2+-activated K+ channel.
Biophys. J.
63:
583-590,
1992[Abstract].
12.
De Franceschi, L.,
N. Saadane,
M. Trudel,
S. L. Alper,
C. Brugnara,
and
Y. Beuzard.
Treatment with oral clotrimazole blocks Ca2+-activated K+ transport and reverses erythrocyte dehydration in transgenic SAD mice. A model for therapy of sickle cell disease.
J. Clin. Invest.
93:
1670-1676,
1994[Medline].
13.
Diserbo, M.,
M. Fatome,
and
J. Verdetti.
Activation of large conductance Ca2+-activated K+ channels in N1E-115 neuroblastoma cells by platelet-activating factor.
Biochem. Biophys. Res. Commun.
218:
745-748,
1996[Medline].
14.
Escobales, N.,
and
M. Canessa.
Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system.
J. Membr. Biol.
90:
21-28,
1986[Medline].
15.
Fathallah, H.,
M. Sauvage,
J. R. Romero,
M. Canessa,
and
F. Giraud.
Effects of PKC activation on Ca2+ pump and KCa channel in deoxygenated sickle cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1206-C1214,
1997
16.
Fitzgerald, L. A.,
and
D. R. Phillips.
Calcium regulation of the platelet membrane glycoprotein IIb-IIIa complex.
J. Biol. Chem.
260:
11366-11374,
1985
17.
Galvez, A.,
G. Gimenez-Gallego,
J. P. Reuben,
L. Roy-Contancin,
P. Feigenbaum,
G. J. Kaczorowski,
and
M. L. Garcia.
Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.
J. Biol. Chem.
265:
11083-11090,
1990
18.
Graido-Gonzalez, E.,
J. C. Doherty,
E. W. Bergreen,
G. Organ,
M. Telfer,
and
M. A. McMillen.
Plasma endothelin-1, cytokine, and prostaglandin E2 levels in sickle cell disease and acute vaso-occlusive sickle crisis.
Blood
92:
2551-2555,
1998
19.
Griendling, K. K.,
T. Tsuda,
and
R. W. Alexander.
Endothelin stimulates diacylglycerol accumulation and activates protein kinase C in cultured vascular smooth muscle cells.
J. Biol. Chem.
264:
8237-8240,
1989
20.
Hadj-Kaddour, K.,
A. Michel,
and
C. Chevillard.
Different mechanisms involved in relaxation of guinea-pig trachea by endothelin-1 and -3.
Eur. J. Pharmacol.
8:
145-148,
1996.
21.
Hammerman, S. I.,
S. Kourembanas,
T. J. Conca,
M. Tucci,
M. Brauer,
and
H. W. Farber.
Endothelin-1 production during the acute chest syndrome in sickle cell disease.
Am. J. Respir. Crit. Care Med.
156:
280-285,
1997
22.
Horuk, R.,
T. J. Colby,
W. C. Darbonne,
T. J. Schall,
and
K. Neote.
The human erythrocyte inflammatory peptide (chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay for the soluble receptor.
Biochemistry
32:
5733-5738,
1993[Medline].
23.
Hu, S.,
H. S. Kim,
R. W. Lappe,
and
R. L. Webb.
Coupling of endothelin receptors to ion channels in rat glomerular mesangial cells.
J. Cardiovasc. Pharmacol.
22, Suppl. 8:
S149-S153,
1993[Medline].
24.
Joneckis, C. C.,
D. D. Shock,
M. L. Cunningham,
E. P. Orringer,
and
L. V. Parise.
Glycoprotein IV-independent adhesion of sickle red blood cells to immobilized thrombospondin under flow conditions.
Blood
87:
4862-4870,
1996
25.
Klaerke, D. A.,
H. Wiener,
T. Zeuthen,
and
P. L. Jorgensen.
Regulation of Ca2+-activated K+ channel from rabbit distal colon epithelium by phosphorylation and dephosphorylation.
J. Membr. Biol.
151:
11-18,
1996[Medline].
26.
Kumar, A.,
J. R. Eckmam,
R. A. Swerlick,
and
T. M. Wick.
Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin.
Blood
88:
4348-4358,
1996
27.
Lee, J.,
R. Horuk,
G. C. Rice,
G. L. Bennett,
T. Camerato,
and
W. I. Wood.
Characterization of two high affinity human interleukin-8 receptors.
J. Biol. Chem.
267:
16283-16287,
1992
28.
Leinders, T.,
R. G. van Kleef,
and
H. P. Vijverberg.
Single Ca2+-activated K+ channels in human erythrocytes: Ca2+ dependence of opening frequency but not of open lifetimes.
Biochim. Biophys. Acta
1112:
67-74,
1992[Medline].
29.
Li, Q.,
V. Jungmann,
A. Kiyatkin,
and
P. S. Low.
Prostaglandin E2 stimulates a Ca2+-dependent K+ channel in human erythrocytes and alters cell volume and filterability.
J. Biol. Chem.
271:
18651-18656,
1996
30.
Loffler, B. M.,
and
W. Lohrer.
Different endothelin receptor affinities in dog tissues.
J. Recept. Res.
11:
293-298,
1991[Medline].
31.
Longenecker, G. L.,
and
V. Mankad.
Decreased prostacyclin levels in sickle cell disease.
Pediatrics
71:
860-861,
1983[Medline].
32.
Miller, C.,
E. Moczydlowski,
R. Latorre,
and
M. Phillips.
Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle.
Nature
313:
316-318,
1985[Medline].
33.
Mita, S.,
A. Tominaga,
Y. Hitoshi,
K. Sakamoto,
T. Honjo,
M. Akagi,
Y. Kikuchi,
N. Yamaguchi,
and
K. Takatsu.
Characterization of high-affinity receptors for interleukin 5 on interleukin 5-dependent cell lines.
Proc. Natl. Acad. Sci. USA
86:
2311-2315,
1989[Abstract].
34.
Phelan, M.,
S. P. Perrine,
M. Brauer,
and
D. V. Faller.
Sickle erythrocytes, after sickling, regulate the expression of the endothelin-1 gene and protein in human endothelial cells in culture.
J. Clin. Invest.
96:
1145-1151,
1995[Medline].
35.
Rasmussen, H.,
W. Lake,
and
J. E. Allen.
The effect of catecholamines and prostaglandins upon human and rat erythrocytes.
Biochim. Biophys. Acta
411:
63-73,
1975[Medline].
36.
Rybicki, A. C.,
and
L. J. Benjamin.
Increased levels of endothelin-1 in plasma of sickle cell anemia patients.
Blood
92:
2594-2596,
1998
37.
Saito, M.,
R. Sato,
I. Hisatome,
and
T. Narahashi.
RANTES and platelet-activating factor open Ca2+-activated K+ channels in eosinophils.
FASEB J.
10:
792-798,
1996
38.
Salter, K. J.,
and
R. Z. Kozlowski.
Endothelin receptor coupling to potassium and chloride channels in isolated rat pulmonary arterial myocytes.
J. Pharmacol. Exp. Ther.
279:
1053-1062,
1996[Abstract].
39.
Salter, K. J.,
and
R. Z. Kozlowski.
Differential electrophysiological actions of endothelin-1 on Cl and K+ currents in myocytes isolated from aorta, basilar and pulmonary artery.
J. Pharmacol. Exp. Ther.
284:
1122-1131,
1998
40.
Salter, K. J.,
J. L. Turner,
S. Albarwani,
L. H. Clapp,
and
R. Z. Kozlowski.
Ca2+-activated Cl and K+ channels and their modulation by endothelin-1 in rat pulmonary arterial smooth muscle cells.
Exp. Physiol.
80:
815-824,
1995[Abstract].
41.
Stanimirovic, D. B.,
T. Yamamoto,
S. Uematsu,
and
M. Spatz.
Endothelin-1 receptor binding and cellular signal transduction in cultured human brain endothelial cells.
J. Neurochem.
62:
592-601,
1994[Medline].
42.
Sugiura, M.,
T. Inagami,
G. M. Hare,
and
J. A. Johns.
Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols.
Biochem. Biophys. Res. Commun.
158:
170-176,
1989[Medline].
43.
Vandorpe, D. H.,
B. E. Shmukler,
L. Jiang,
B. Lim,
J. Maylie,
J. P. Adelman,
L. De Franceschi,
M. D. Cappellini,
C. Brugnara,
and
S. L. Alper.
cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles In regulatory volume decrease and erythroid differentiation.
J. Biol. Chem.
273:
21542-21553,
1998
44.
Van Renterghem, C.,
P. Vigne,
J. Barhanin,
A. Schmid-Alliana,
C. Frelin,
and
M. Lazdunski.
Molecular mechanism of action of the vasoconstrictor peptide endothelin.
Biochem. Biophys. Res. Commun.
157:
977-985,
1988[Medline].
45.
Van Willigen, G.,
and
J. W. Akkerman.
Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets.
Biochem. J.
273:
1-20,
1991.
46.
Wolff, D.,
X. Cecchi,
A. Spalvins,
and
M. Canessa.
Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: individual differences in the number of channels.
J. Membr. Biol.
106:
243-252,
1988[Medline].
47.
Yang, Y. M.,
C. Hoff,
C. Hamm,
V. Mankad,
R. C. Boerth,
and
L. Friedrich.
Pharmacokinetics of meperidine in sickle cell patients.
Am. J. Hematol.
49:
357-358,
1995[Medline].
48.
Yue, T. L.,
P. Nambi,
H. L. Wu,
and
G. Feuerstein.
Endothelin receptor binding and cellular signal transduction in neurohybrid NG108-15 cells.
Neuroscience
44:
215-222,
1991[Medline].