1 Laboratoire des Biomembranes et Messagers Cellulaires, Unité de Recherches Associée 1116, Centre National de la Recherche Scientifique, Université Paris XI, 91405 Orsay, France; 2 Endocrine-Hypertension Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; and 3 Facultad de Ciencas, Universidad de Chile, Santiago-7, Chile
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ABSTRACT |
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We have previously shown that a pretreatment with phorbol
12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC),
reduced deoxygenation-induced K+
loss and Ca2+ uptake and prevented
cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007,
1995). The present study explores the detailed mechanism of this
PMA-induced inhibition. The main findings are, first, the detection of
PKC and PKC
in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The
-isoform only is translocated to the membrane and activated by PMA
and by elevation of cytosolic
Ca2+. Second, PMA is demonstrated
to activate Ca2+ efflux in
deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits
deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This
inhibition is partly indirect and explained by the reduced
deoxygenation-induced rise in cytosolic
Ca2+ resulting from
Ca2+ pump stimulation. However, a
significant inhibition of the
Ca2+-activated
K+ channels
(KCa channels) by PMA can also be
demonstrated when the channels are activated by
Ca2+ plus ionophore, under
conditions in which the Ca2+ pump
is operating near its maximal extrusion rate, but swamped by
Ca2+ plus ionophore. The data thus
suggest a PKC
-mediated phosphorylation both of the
Ca2+ pump and of the
KCa channel or an auxiliary
protein.
phosphorylation; dehydration; cationic fluxes; protein kinase C
; protein kinase C
; calcium-activated potassium channel
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INTRODUCTION |
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SICKLE RED BLOOD CELLS (SS cells) contain a subpopulation of abnormally dense cells. These cells are dehydrated and have altered monovalent cation content (4). The cellular dehydration appears to contribute to the physiopathology of the sickling syndrome (13). The mechanisms of SS cell dehydration are not entirely elucidated. They differ among patients and among SS cell subpopulations in the same patient.
Three pathways are generally considered to be involved in cation loss
(4, 13): 1) deoxygenation-induced
changes in membrane permeability to
Na+,
K+,
Ca2+, and
Mg2+ that result in an imbalance
between K+ efflux and
Na+ influx,
2) a
Ca2+-activated
K+ channel
(KCa channel) activated by
deoxygenation-induced rise in cytosolic
Ca2+ concentration
([Ca2+]i),
and 3) the
K+-Cl
cotransporter, activated by intracellular acidification or cell swelling.
We have reported recently (9) that deoxygenation of SS cells results in a nonspecific membrane protein dephosphorylation, which can be prevented by a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) (20), or by okadaic acid, an inhibitor of phosphoprotein phosphatases PP1 and PP2A (5). In addition, both agents abolished the deoxygenation-induced SS cell dehydration by slightly reducing the loss of K+ and increasing the gain of Na+ (9). Deoxygenation-induced Ca2+ uptake was also reduced by both drugs, an effect that was attributed to an activation of Ca2+ efflux because the drugs did not alter the Ca2+ influx (9).
The purpose of the present study was to investigate the mechanisms by
which PMA could inhibit SS cell dehydration. PMA is known to activate
the isoforms of the classical PKC (cPKC; ,
I,
II, and
) and of the novel
PKC (nPKC;
,
,
, and
) families (17, 20). However, the PKC
isoforms present in red blood cells are unknown. Their identification
and the characteristics of their activation by PMA and
Ca2+ in both SS cells and normal
red blood cells (AA cells) are presented in the first part of this
study.
The second part of this work was undertaken to determine whether the PMA-induced inhibition of SS cell dehydration could result from an inhibition of KCa channel activity. PKC activation could lead indirectly to KCa channel inhibition by stimulating Ca2+ pump activity and thus preventing the deoxygenation-induced [Ca2+]i increase. Indeed, erythrocyte membrane Ca2+-ATPase is phosphorylated by PKC when assayed using purified enzymes (29). In cell preparations, incubation with PMA increases both Ca2+-ATPase phosphorylation and Ca2+ pump activity (32). Alternatively, inhibition of the KCa channel activity could be due to a direct effect of PKC on the channel, as demonstrated in other cell types (25).
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MATERIALS AND METHODS |
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Preparation of Cells
Blood was collected in heparinized tubes, after informed consent, either from normal donors or from sickle cell anemia patients homozygous for hemoglobin S, and stored at 4°C for no more than 3 h before processing. Part of the plasma was removed after centrifugation of SS blood, and the resulting suspension (~50% hematocrit) was layered onto a discontinuous gradient of Percoll (Pharmacia) with densities (Treatment With PMA or Ca2+ Plus Ionophore: Preparation of Cytosol, Membranes, and Total Cell Lysates for PKC Detection and Activity Assays
Cells washed twice in solution B [in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES-Tris (pH 7.4 at 37°C, 300 mosmol/kgH2O)] were incubated at 10% hematocrit for 10 min at 37°C, with 1 µM PMA (Sigma-Chimie), added as 400 µM stock solutions in 60% dimethyl sulfoxide (DMSO). The same volume of 60% DMSO was added to control samples (final solvent concentration, 0.15% in all samples). Incubations were performed under either oxygenated (air) or deoxygenated conditions. In the latter case, cell suspensions were preincubated under humidified nitrogen, at a flow rate of 8-10 ml/min, in capped tubes under continuous magnetic stirring. To increase [Ca2+]i, cells suspended at 5% hematocrit in solution C [in mM: 140 NaCl, 0.2 MgCl2, 0.1 CaCl2, 10 glucose, and 10 HEPES-Tris (pH 7.4 at 37°C)] were incubated for 10 min with 5 µM Ca2+ ionophore A-23187 (Sigma-Chimie) added as 2 mM stock solution in DMSO.Unincubated or incubated cells were washed twice and suspended in 310 mM sucrose (or in 150 mM NaCl for membrane preparation after ionophore
treatment), 10 mM Tris · HCl (pH 7.4), 2 mM ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA), 2 mM EDTA, 5 mM 1,4-dithiothreitol (DTT; Sigma-Chimie),
0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Chimie) and 10 µg/ml leupeptin (Sigma-Chimie). Membranes were prepared by lysis of packed cells in 15-fold excess of the same solution without sucrose or
NaCl (lysis buffer). After centrifugation at 18,000 g for 10 min, the supernatant
(cytosol) was collected and the pellet (membranes) was washed once in
the lysis solution and solubilized in sodium dodecyl sulfate (SDS)
sample buffer [final concentrations, 70 mM DTT, 5% SDS, and 7 mM
Tris · HCl (pH 6.8 at 20°C)]. Membranes used for PKC activity assays were washed again in the lysis buffer without EGTA and EDTA. To prepare total cell lysates, the washed cell
pellets were frozen and thawed three times in liquid nitrogen and
solubilized in SDS sample buffer.
Detection of PKC Isoforms and Enzyme Activity Assays
Immunodetection of PKC isoforms.
Cytosolic PKC was partially purified by chromatography on DE 52 cellulose (Whatman). The column was equilibrated with (in mM) 20 Tris · HCl (pH 7.4 at 4°C), 0.5 EDTA, 0.5 EGTA, 1 DTT, and 2% glycerol. The column was loaded with the cytosol and
washed with equilibration buffer containing 30 mM NaCl. Proteins were eluted with a linear NaCl gradient (30-300 mM). Fractions eluting between 80 and 140 mM NaCl, containing the PKC peak, were solubilized in SDS sample buffer for electrophoresis and Western blot or stored overnight at 80°C in 30% glycerol (wt/vol) for
determination of kinase activity by using histone III-S as a substrate.
Protein concentration was determined by the Bradford method.
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Assays of PKC activity.
Partially purified cytosolic PKC activity was assayed in the absence or
presence of Ca2+,
phosphatidylserine (PS; Sigma-Chimie), diacylglycerol (DAG; diolein,
Sigma-Chimie), or arachidonic acid (Sigma-Chimie), by measuring the
incorporation of 32P into histone
type III-S (Sigma-Chimie), as described by Apovo et al. (2). DE 52 eluate fractions were incubated for 5 min at 30°C in a final volume
of 100 µl containing 50-100 µg proteins and (in mM) 20 Tris · HCl (pH 7.4), 5 MgCl2, 1 CaCl2 or 1 EGTA, and 200 µg/ml
histone III-S. When required, 300 µM arachidonic acid, 50 µg/ml PS,
or 8 µg/ml DAG was added alone or in combination. Reactions were
initiated by the addition of 25 µM
[-32P]ATP
(Amersham-France). Membrane PKC activity was determined with the PKC
enzyme assay system RPN 77 (Amersham International). In brief,
membranes were incubated for 15 min at 25°C with a synthetic peptide substrate (Arg-Lys-Arg-Thr-Leu-Arg-Arg-Leu) in the presence of
50 µM
[
-32P]ATP, 1 mM
CaCl2, and a detergent-dispersed
PS-PMA mixture.
Measurements of Ca2+ Pump Transport Activity
Ca2+ efflux. The transport activity of the Ca2+ pump was measured at physiological [Ca2+]i (Ca2+ efflux) after a 45Ca2+-loading period under either oxygenated or deoxygenated conditions. Washed cells, suspended at 30% hematocrit in solution B, were preincubated for 45 min with quin-2-acetoxymethyl ester (200 µmol/l cells; Molecular Probes) to increase the intracellular exchangeable Ca2+ pool (16), washed, and resuspended at 10% hematocrit in the same medium. Suspensions were then incubated in solution B containing 45Ca2+ (Amersham-France) for 40 min, followed by 50 min under either oxygenated or deoxygenated conditions. DMSO (0.15%) or 1 µM PMA was added for the last 10 min. Aliquots were taken for measurement of the specific radioactivity of 45Ca2+ (SR). Cells were washed rapidly four times, to remove extracellular 45Ca2+, resuspended in solution B at ~5% hematocrit, and reincubated under oxygenated conditions. Aliquots were taken every 90 s up to 6 min, diluted in ice-cold solution B (in which CaCl2 was replaced with 1 mM EGTA), and centrifuged for 10 s through dibutyl phthalate. 45Ca2+ radioactivity of the supernatants (Rs) was measured in aliquots. Ca2+ efflux was calculated from the plot of Rs/SR vs. time. Cell volume was estimated in each assay from the hemoglobin content, measured by absorbance at 540 nm, to calculate 45Ca2+ content per liter of original cells.
Maximal extrusion rate of the Ca2+ pump. The transport activity of the Ca2+ pump was determined at high [Ca2+]i to measure its maximal extrusion rate (Ca2+ pump Vmax). The method used was essentially similar to that described by Dagher and Lew (6). Cells were washed twice in solution D [in mM: 70 NaCl, 80 KCl, 0.2 MgCl2, 0.1 EGTA, 10 glucose, and 10 HEPES-Tris (pH 7.4 at 37°C)] and twice in solution D without EGTA. They were resuspended at 10% hematocrit in solution D containing 0.1 mM 45CaCl2 instead of EGTA and preincubated for 10 min at 37°C, under either oxygenated or deoxygenated conditions and in the presence of 0.15% DMSO or 1 µM PMA. At time zero, 10 µM A-23187 was added to each suspension, followed 2 min later by 200 µM CoCl2 to block Ca2+ entry by the ionophore. Aliquots were withdrawn every 20 or 30 s, mixed with 80 vol of ice-cold solution D, and centrifuged. Pellets were lysed, and the lysate was used to measure the hemoglobin content and the cell radioactivity (Rc; counted by liquid scintillation of the supernatants of trichloroacetic acid-precipitated lysates). Ca2+ pump Vmax was calculated from the plot of Rc/SR vs. time. Cell volume was calculated as described above.
Measurements of K+ Efflux
Deoxygenation-stimulated
K+
efflux.
SS cells suspended at 30% hematocrit in
solution
E [in mM: 140 NaNO3, 10 glucose, 0.15 MgCl2, 10 Tris-MOPS (pH 7.4 at
37°C), 0.1 ouabain, and 0.1 bumetanide] were preincubated for
10 min at 37°C with 0.15% DMSO or with 1 µM PMA or 1 µM
4-phorbol 12,13-didecanoate (4
-PDD; Sigma-Chimie). The nitrate
medium was used to eliminate a possible contribution of
K+-Cl
cotransport to the measured K+
efflux. In some experiments, solution
E also contained 1 mM
Ca(NO3)2. The cell suspensions were injected into sealed flasks containing solution
E that had been previously
deoxygenated for 20 min at 37°C with bubbling nitrogen (final
hematocrit 2%). The flasks were gently mixed by hand. Incubations were
performed at 37°C, with or without 10 nM charybdotoxin (CTX;
Latoxan, France), under a continuous supply of nitrogen, although it
was not bubbling the medium. Initial rates of
K+ efflux were measured by
sampling aliquots of the cell suspensions at various time points for 16 min. The samples were centrifuged for 10 s through dibutyl phthalate.
The supernatants were removed for
K+ concentration measurement by
flame emission spectroscopy. To measure the
K+ efflux under control
conditions, cells were treated in a similar manner, except that the
medium was kept oxygenated. K+
efflux was calculated from the slope of the regression line of K+ concentration vs. time. Cell
volume was estimated as described above.
KCa channel activity. Cells suspended at 10% hematocrit in solution C were preincubated for 10 min at 37°C with 0.15% DMSO or 1 µM PMA. The transport reaction was initiated by adding the cell suspensions to prewarmed (37°C) solution C containing A-23187, with or without CTX (final concentrations, 1 µM A-23187 and 10 nM CTX; final hematocrit 1%) (31). Control samples were incubated in solution C containing 1 mM EGTA. Duplicate samples were taken every 1 min for 5 min and centrifuged for 10 s through dibutyl phthalate. The supernatants were removed for K+ concentration measurement. K+ efflux was calculated as described above.
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RESULTS |
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Cytosolic and Membrane PKC Activities
Cytosolic PKC from SS and AA cells was partially purified by DE 52 cellulose chromatography. The fraction eluted between 80 and 140 mM NaCl was used either directly, to determine the effect of several activators on PKC activity, or concentrated, to identify PKC isoforms after electrophoresis and immunoblotting. Under maximal activation conditions (presence of Ca2+, PS, and DAG), PKC activity, measured using histone III-S as a substrate, was nearly threefold higher in SS than in AA cytosol (P < 0.01; Fig. 2). The data shown in Fig. 2 are expressed as percent of maximal activity. PS alone and PS + DAG were virtually equipotent in the presence of Ca2+ in AA cytosol, but in SS cytosol PS alone was not able to induce the maximal activity (P < 0.05). DAG-induced activation was smaller in SS than in AA cytosol either with Ca2+ (41 ± 5 and 73 ± 9%, respectively, P < 0.02) or without Ca2+ (20 ± 5 and 44 ± 7%, respectively, P < 0.02). Arachidonate-induced activation in the presence of Ca2+ was nearly maximal in AA cytosol but not in SS cytosol (96 ± 30 and 31 ± 8%, respectively, P < 0.05). In the absence of Ca2+, arachidonate was almost unable to elicit a measurable activation of both SS and AA cytosol kinase. As illustrated in Fig. 3 (control conditions), membrane PKC activity, measured under maximal activation conditions, was significantly higher in SS cells than in AA cells (P < 0.01). These marked differences in the activity and in the properties of PKC in both types of cells reflected differences in the levels of expression of the various isoforms, as shown below.
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Identification and Intracellular Distribution of PKC Isoforms
PKC isoforms were detected by Western blotting with antibodies raised against isoform-specific peptides
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Effects of PMA, Elevation of [Ca2+]i, and Deoxygenation on PKC Translocation to the Membrane
PKC
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A rise in
[Ca2+]i,
produced by incubating the cells with the ionophore A-23187 in the
presence of 0.1 mM CaCl2, induced
a translocation of PKC but had no effect on PKC
distribution
(Fig. 4B,
lanes 2, 3,
4, and Fig.
5A). As reported before,
[Ca2+]i
increase activates a polyphosphoinositide-specific phospholipase C in
red blood cells, resulting in DAG formation responsible for PKC
translocation (7, 27). The percentage of translocation after 10 min of
ionophore treatment was lower in SS than in AA cells (337 ± 16 and
546 ± 37, respectively, P < 0.01; Fig. 5A). Translocation of
PKC
, in SS cells, was detected as soon as 1 min after ionophore
addition and increased up to 10 min (Figs. 4B and
5B). Pretreatment with 1 µM PMA
for 10 min before ionophore addition promoted a maximal PKC
translocation that was not increased by the further addition of the
ionophore (Fig. 5B).
Because deoxygenation of SS cells increases
Ca2+ influx (3, 8, 9), we
investigated whether it could induce PKC translocation. In two
separate experiments, no significant enhancement of membrane PKC
levels was detected after deoxygenation of SS cells (Fig. 4B, compare lanes 6 and
7). Presumably, the deoxygenation-induced increase in
[Ca2+]i is too low to activate the
phospholipase C (see DISCUSSION). The effect of PMA on
PKC
translocation occurred to a similar extent under either
oxygenated or deoxygenated conditions (data not shown).
Effect of PMA on Ca2+ Pump Activity
We have previously observed that PMA treatment prevented deoxygenation-induced SS cell dehydration (9). This effect could be due to an inhibition of KCa channel activation, mediated by a stimulation of Ca2+ pump activity by phosphorylation by PKC (32), leading to the prevention of deoxygenation-induced rise in [Ca2+]i. We thus investigated whether PMA could affect Ca2+ efflux from quin-2-loaded SS cells. The loading period with 45Ca2+ was performed under either oxygenated or deoxygenated conditions, and in both cases cells were rapidly washed and reincubated under oxygenated conditions to measure Ca2+ efflux. As expected, the total exchangeable intracellular Ca2+ concentration was significantly higher after preloading under deoxygenated conditions than after preloading under oxygenated conditions (25.4 ± 3.1 and 14.1 ± 1.4 µmol/l cells, respectively, n = 8, P < 0.01), resulting in a two times higher Ca2+ extrusion rate in the former case (P < 0.001; Fig. 6A). Treatment with PMA for the last 10 min of deoxygenation induced a small increase (~35%, P < 0.02) in the rate of Ca2+ efflux, whereas PMA had no significant effect under oxygenated conditions (Fig. 6C).
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The effect of PMA was also tested on Ca2+ pump Vmax. Ca2+ pump Vmax was determined under either oxygenated or deoxygenated conditions, in cells loaded in the presence of ionophore and 0.1 mM 45CaCl2, and after addition of CoCl2 to block Ca2+ entry by the ionophore. Deoxygenation was reported to induce a small inhibition of Ca2+ pump Vmax (8, 28). In our experiments, this effect was not consistently observed, as indicated by the lack of statistical significance of the data shown in Fig. 6B. PMA had no effect on Ca2+ pump Vmax under either oxygenated or deoxygenated conditions (Fig. 6C).
Effect of PMA on Deoxygenation-Stimulated and Ca2+-Activated K+ Efflux
To investigate whether PMA could inhibit the activation of KCa channel induced by deoxygenation, K+ efflux was measured in the absence or presence of CTX, a specific inhibitor of this channel in red blood cells (31), and in a NaNO3 medium to eliminate the contribution of K+-Cl
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To determine whether the inhibition of
KCa channel activity was due to an
effect of PKC on the channel, rather than resulting only from an activation of Ca2+
efflux, the effect of PMA on KCa
channel was investigated after its stimulation with the ionophore and
0.1 mM CaCl2. Under these conditions, PMA had no effect on
Ca2+ pump activity (Fig.
6C), but still caused a significant
inhibition of the CTX-sensitive K+
efflux (P < 0.02; Fig.
7D). Total
K+ efflux was slightly but not
significantly reduced. In AA cells, a similar treatment with PMA did
not induce any inhibition of KCa
channel activity, presumably due to the lower level of membrane PKC
(data not shown).
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DISCUSSION |
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The principal findings of this study are that
1) SS and AA cells express only two
PKC isoforms, and
, with a higher level in the SS
cells, and 2)
CTX-sensitive KCa channel and
Ca2+ pump activities are regulated
through a PKC
-dependent mechanism in deoxygenated SS cells.
PKC is composed of a family of at least twelve isoforms that can be
divided into three groups: 1) the
Ca2+-dependent, phorbol
ester-binding cPKC isoforms (,
I,
II, and
),
2) the
Ca2+-independent, phorbol
ester-binding nPKC isoforms (
,
,
, and
), and
3) the
Ca2+-independent,
non-phorbol-ester-binding atypical PKC isoforms (
,
, µ,
)
(17). Although PKC subtypes have been characterized in many cells,
identification of the isoenzymes expressed in human red blood cells is
poorly documented. The presence of PKC
and PKC
has been mentioned
in one report (11). We have identified two isoforms,
and
, in
both AA and SS cells, and no other isoforms among those tested (
,
,
,
,
, or
) were detectable. The total and the
membrane-associated contents of both PKC
and PKC
were higher in
SS than in AA cells, in agreement with the higher membrane PKC activity
found here and in other studies (2, 21). This difference is likely
related to the younger age of the cell population in the patient blood,
as reflected by the high reticulocyte count usually reported (3), and
is consistent with the age-dependent decrease in PKC activity observed
in rabbit and human red blood cells (12). These findings are also
consistent with previous data showing that, during commitment to
erythroid differentiation of erythroleukemia cells, activity and
content of PKC progressively decline (18) and most isoforms are
downregulated during the maturation process leading to hemoglobin
synthesis (15, 24).
To further characterize the PKC isoforms expressed in SS and in AA
cells, the effects of PMA and
[Ca2+]i
increase on PKC translocation and activation were studied. Intracellular distribution of PKC was unaffected by either PMA treatment or elevation of
[Ca2+]i.
In contrast, PMA induced an increase in membrane association of PKC
and in membrane PKC activity. Both increases, in absolute values, were
higher in SS cells. The increase in PKC phosphorylation activity can
thus be attributed to PKC
translocation and activation. Elevation of
[Ca2+]i
by the ionophore A-23187 also induced the translocation of PKC
to
the membrane, in agreement with previous data showing a stimulation of
membrane protein phosphorylation under these conditions (22). PKC
translocation and activation require both an increase in
[Ca2+]i
and a production of DAG, the latter resulting from the hydrolysis of
polyphosphoinositides by a
Ca2+-activated phospholipase C
present in red blood cells (7). Activation of this enzyme was
previously demonstrated in both SS and AA cells, under similar
conditions of ionophore treatment, by the increase in inositol
phosphate production (23, 27). Deoxygenation of SS cells did not cause
any PKC
translocation, indicating that
[Ca2+]i
increase in deoxygenated SS cells is not sufficient to activate PKC
,
a finding consistent with the lack of phospholipase C activation reported previously (23).
It has been shown that PKC phosphorylates the erythrocyte
Ca2+ pump (29) and stimulates
Ca2+ uptake into inside-out
vesicles from erythrocytes by a direct effect on the pump protein (26).
In addition, PMA was shown to stimulate
Ca2+ efflux from erythrocytes
loaded with a low Ca2+
concentration by the ionophore A-23187 and to increase the
phosphorylation state of the Ca2+
pump (32). We have indeed observed that
Ca2+ efflux was stimulated by PMA
under deoxygenated conditions. This stimulation is likely to result
from PKC-mediated phosphorylation of the pump. Under these
conditions, Ca2+ pump, as well as
other membrane proteins, would be dephosphorylated (9), permitting its
phosphorylation and activation by PKC
. Such a stimulation of
Ca2+ efflux by PMA is consistent
with our previous data showing that the phorbol ester inhibited the
deoxygenation-induced net Ca2+
uptake, without affecting the deoxygenation-induced
Ca2+ influx (9). However, we have
not observed a stimulation of Ca2+
efflux under oxygenated conditions, although PKC
was similarly translocated. This discrepancy between our data and that of Wright et
al. (32) can be explained by the different experimental conditions used
by those authors (slight activation of the
Ca2+ pump with the ionophore and
measurements of changes in intracellular 45Ca2+
on a very short time scale, <2 min). In contrast to the effect of PMA
on Ca2+ efflux in deoxygenated SS
cells, PMA had no effect on Ca2+
pump Vmax. The conditions used in
those experiments (elevation of
[Ca2+]i)
led to PKC
translocation and presumably resulted in a maximal phosphorylation of the pump, which could not be enhanced by a pretreatment with PMA. Likewise, Wright et al. (32) have reported that
the PKC effect on Ca2+ efflux
could be observed only when the
Ca2+ pump was operating at
submaximal levels of activity.
The results reported here demonstrate that activation of PKC
inhibits the activity of KCa
channels in SS cells. The effects of PMA are attributed to PKC
,
because a structurally similar compound (4
-PDD), which has no effect
on PKC
, also had no significant effect on
KCa channel activity. Furthermore,
the effect of PKC
on KCa
channel activity did not simply result from an activation of the
Ca2+ pump. Thus PMA-induced
inhibition of the KCa channel was
observed after stimulation by either deoxygenation or ionophore
treatment. Although the phorbol ester stimulates
Ca2+ efflux under deoxygenated
conditions, it had no effect on
Ca2+ pump
Vmax, under conditions prevailing
when the channel was activated by ionophore. Our proposal
is thus that KCa channel is
inhibited as a result of PKC
-mediated phosphorylation of the channel
itself or of a regulatory associated protein. Paradoxically, the
ionophore-induced elevation of
[Ca2+]i,
which activates KCa channel, also
results in PKC
translocation, which would lead to an inhibition of
KCa channel. In fact, the rate of
CTX-sensitive K+ efflux was
maximal only up to 4-5 min following ionophore addition (data not
shown), suggesting that the channel could be inhibited from that time.
The delayed inhibition of the channel following PKC
translocation
may be part of a feedback mechanism.
Ca2+- and voltage-dependent
K+ channels of the maxi type can
be either upregulated or downregulated by adenosine
3',5'-cyclic monophosphate-dependent protein kinase or
PKC-mediated phosphorylation (14, 25). Less is known about the
regulation of the mini type KCa
channels. Those present in hepatoma cells are apamin-sensitive and
activated by PKC (30). The red blood cell
KCa channel belongs to the mini
type, is sensitive to CTX and not to apamin (14), and, as shown in SS
cells in the present study, is inhibited by PKC
.
The reduction of KCa channel activity, resulting both from Ca2+ pump activation and inhibition of the channel, may not be the sole mechanism responsible for the effect of PMA in preventing the deoxygenation-induced cell dehydration (9). Activation of the Na+/H+ exchanger by PKC-mediated phosphorylation (10) represents another possibility that deserves study.
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ACKNOWLEDGEMENTS |
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We thank Dr. Driss Zoukhri for expert assistance in initiating PKC detection experiments and Drs. Dora Bachir and Frédéric Galactéros (Centre de la Drépanocytose et des Thalassémies, Hôpital Henri Mondor, Créteil, France) and Dr. Mary Fabry (Albert Einstein College of Medicine, Bronx, New York) for providing blood samples from patients.
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FOOTNOTES |
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This work was supported by the Centre National de la Recherche Scientifique (URA 1116), the Université Paris XI, and the Fondation pour la Recherche Médicale (Grant 40000 187 S.05 to F. Giraud). M. Canessa was supported by Fondecyt 1940339 from Conycit, Chile.
Address for reprint requests: F. Giraud, Biomembranes et Messagers Cellulaires, URA 1116, Bat 440, Université Paris XI, 91405 Orsay Cedex, France.
Received 3 April 1997; accepted in final form 16 June 1997.
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