Effects of PKCalpha activation on Ca2+ pump and KCa channel in deoxygenated sickle cells

Hassana Fathallah1, Monique Sauvage1, Jose R. Romero2, Mitzy Canessadagger ,3, and Françoise Giraud1

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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 PKCalpha and PKCzeta in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The alpha -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 PKCalpha -mediated phosphorylation both of the Ca2+ pump and of the KCa channel or an auxiliary protein.

phosphorylation; dehydration; cationic fluxes; protein kinase C alpha ; protein kinase C zeta ; calcium-activated potassium channel

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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; alpha , beta I, beta II, and gamma ) and of the novel PKC (nPKC; delta , epsilon , eta , and theta ) 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).

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (partial ) 1.076 and 1.106. Unless otherwise mentioned, the SS cell fraction (1.076 < partial  <=  1.106) was washed three times in solution A [in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 NaH2PO4-Na2HPO4 (20:80), 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris) (pH 7.4 at 37°C, 300 mosmol/kgH2O)] and stored at 4°C for 12 to 36 h. Just before the experiments were performed, the cells were washed once in solution A and incubated at 15% hematocrit for 45 min at 37°C, in the same solution containing 2 mM adenine and 10 mM inosine, to replete the ATP pool. Alternatively, in deoxygenation-stimulated K+ efflux experiments, the SS cell fraction (1.076 < partial  <=  1.091) was washed twice with preservation solution [in mM: 135 KCl, 15 NaCl, and 10 Tris-3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4 at 4°C)] and stored overnight at 4°C. AA cells, recovered after centrifugation of the blood and elimination of white cells, were washed in solution A and ATP repleted under the same conditions described for SS cells. All washing steps were performed at 4°C.

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(beta -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.

Aliquots of solubilized DE 52 cytosolic fractions, membranes (equivalent to 3-6 µl of cells), or cell lysates were subjected to SDS-polyacrylamide gel electrophoresis in 8% polyacrylamide gels. Following electrophoresis, proteins were transferred from the gel to Immobilon-P membranes (Millipore). The blotted proteins were incubated with a blocking solution containing 5% skimmed milk and 0.2% Tween 20 in phosphate-buffered saline (PBS) for 1 h at 37°C and washed three times in washing buffer (2.5% skimmed milk and 0.1% Tween 20 in PBS). The blots were incubated with primary rabbit polyclonal antibodies either overnight at 4°C with anti-PKCalpha , -beta , -delta , -gamma , -epsilon , and -zeta (GIBCO-BRL), or 1 h at 20°C with anti-PKCalpha , -zeta , -eta , and -theta (Santa Cruz Biotechnology). Antibodies were diluted in washing buffer (to 5 and 0.1 µg/ml for GIBCO and Santa Cruz antibodies, respectively) in the absence or presence of competing antigens (0.5 µg/ml). The blots were washed, incubated for 1 h at room temperature with one of three secondary antibodies [1:5,000 dilution of goat alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (IgG) (Sigma-Chimie), 1:16,000 dilution of peroxidase-conjugated anti-rabbit IgG (Sigma-France), or 1.5 µCi/ml of 125I-labeled anti-rabbit IgG (Amersham-France)], and washed again. Depending on the secondary antibody, immunoreactive bands were visualized using alkaline phosphatase substrate, enhanced chemiluminescence (Amersham kit), or autoradiography for 125I detection. Data were quantified by scanning densitometry with a Hewlett-Packard DeskScan II, using the National Institutes of Health Image software (Fig. 1), or by liquid scintillation counting. Comparisons of PKC levels between different cells, between different treatments, or between total cell and membranes were done on the same immunoblots.


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Fig. 1.   Linear response of enhanced chemiluminescence (ECL) signal as a function of amount of antigen. Different volumes of membranes in SDS sample buffer were subjected to SDS-polyacrylamide gel electrophoresis. After transfer to Immobilon-P membranes and incubation with primary [anti-protein kinase C (PKC) alpha ] and secondary [peroxidase-conjugated anti-immunoglobulin G (IgG)] antibodies, immunoreactive bands were revealed by ECL. Signal intensity was quantified by scanning densitometry (inset). Area of peaks was plotted vs. volume of cell equivalent used for electrophoresis.

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 [gamma -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 [gamma -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 4alpha -phorbol 12,13-didecanoate (4alpha -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.

    RESULTS
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Materials & Methods
Results
Discussion
References

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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Fig. 2.   Effects of activators on cytosolic PKC activity. Cytosolic PKC of sickle red blood cells (SS cells; A) and normal red blood cells (AA cells; B) were partially purified by DE 52 chromatography. Activity was estimated by measuring incorporation of 32P from [gamma -32P]ATP into histone III-S in presence of 1 mM Ca2+ or 1 mM EGTA and either phosphatidylserine (PS; 50 µg/ml), diacylglycerol (DAG; 8 µg/ml), or arachidonate (ARACH; 300 µM), alone or in combination. Data (means ± SE of 6 experiments) are expressed as percent of maximal activity (in presence of Ca2+, PS, and DAG). Absolute values of maximal PKC activities were 257 ± 57 and 93 ± 10 pmol · mg protein-1 · min-1 in SS and AA cytosols, respectively (means ± SE of 8 experiments). These values differ from those we have previously published (3), which were obtained from a nonpurified diluted cytosol, in which hemoglobin was a major contaminant, and were much less reliable than the present values.


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Fig. 3.   Effect of phorbol 12-myristate 13-acetate (PMA) on membrane PKC activity. After 10 min of incubation of cells in absence or presence of 1 µM PMA, membranes were prepared by cell lysis. Activity was estimated by measuring incorporation of 32P from [gamma -32P]ATP into a synthetic peptide substrate. Data are means ± SE of 5 (SS) or 9 (AA) experiments.

Identification and Intracellular Distribution of PKC Isoforms

PKC isoforms were detected by Western blotting with antibodies raised against isoform-specific peptides alpha , beta , delta , epsilon , and zeta (Fig. 4A) and gamma , theta , and eta  (not shown). Two PKC isoforms, alpha  and zeta , were identified in both cytosol and membrane of SS cells (Fig. 4). Each PKC isoform band was specifically depleted by adsorption with the peptide against which the antibody was raised (Fig. 4A). Two immunoreactive bands, migrating at ~82 and 73 kDa, were detected when the GIBCO anti-PKCzeta was incubated overnight at 4°C with the Immobilon membranes (Fig. 4A), whereas only the 73-kDa band was revealed after 1 h of incubation at 20°C with the Santa Cruz anti-PKCzeta (Fig. 4B). On the basis of their molecular weights (17) and the effect of PMA or Ca2+ on the translocation to the membrane (see below), the 73-kDa band was identified as PKCzeta and the 82-kDa band presumably was PKCalpha , as reported in other studies with other cells (1). The possibility that this band corresponds to another yet undefined cPKC isoform, or to a recently described form of PKCzeta that responds to PMA treatment (19) cannot be totally excluded. The same PKC isoforms were found in both membranes and cytosol of AA cells. As shown in Table 1, the amounts of membrane PKCalpha (P < 0.01) and PKCzeta (P < 0.001) were higher in SS than in AA cells. A similar difference in the level of PKCalpha and PKCzeta was observed in total AA and SS cell lysates. The percentage of total cellular PKCalpha and PKCzeta associated with the membranes was also higher in SS cells (Table 1).


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Fig. 4.   Immunoblot analysis of PKC isoforms in cytosol (A) and in membranes (B) of SS cells. Cytosolic eluates, obtained after chromatography on DE 52, or membranes were electrophoresed and transferred to Immobilon P membranes. A: PKC isoforms were detected using PKC-specific antisera (from GIBCO), without (-) or with (+) antigenic peptide, and alkaline phosphatase-conjugated IgG. Arrow at left, 82-kDa band corresponding to PKCalpha ; 2 arrows at right, 2 bands (73 and 82 kDa) detected by anti-zeta antibody (see text). B: SS cells (hematocrit 5%) were incubated with 0.15% DMSO (control) for 10 min (lane 1), with 5 µM A-23187 in presence of 0.1 mM Ca2+ for 1 min (lane 2), 5 min (lane 3), or 10 min (lane 4), with 1 µM PMA for 10 min (lane 5), under oxygenated conditions for 30 min (lane 6), or under deoxygenated conditions for 30 min (lane 7). Membrane PKC isoforms were detected by ECL using anti-PKCalpha (top) or anti-PKCzeta (bottom) antibodies (from Santa Cruz). Arrows, bands corresponding to PKCalpha (top; 82 kDa) and to PKCzeta (bottom; 73 kDa).

                              
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Table 1.   PKC levels in SS and AA cells

Effects of PMA, Elevation of [Ca2+]i, and Deoxygenation on PKC Translocation to the Membrane

PKCalpha is known as a Ca2+- and DAG-dependent isoform, sensitive to PMA, whereas the zeta -isoform is independent of all these activators (17), although its translocation on PMA treatment has been reported in some studies (reviewed in Ref. 33). Treatment of erythrocytes with 1 µM PMA for 10 min resulted in the translocation to the membrane of PKCalpha but not of PKCzeta (Fig. 4B, lane 5). The 82-kDa band, but not the 73-kDa band detected by the GIBCO PKCzeta antibody, was translocated on PMA treatment (data not shown). The percentage increase in membrane-associated PKCalpha was the same in both SS and AA cells (368 ± 69 and 411 ± 99%, respectively) (Fig. 5A). However, because the basal membrane level was two times higher in SS cells (Table 1), it can be deduced that the membrane content after translocation will be two times higher than that in AA cells. In parallel, PMA treatment induced a significant increase in membrane PKC activity (from 140 ± 20 to 634 ± 80 and from 39 ± 7 to 153 ± 24 pmol · mg protein-1 · min-1, respectively, in SS and AA cell membranes, P < 0.001; Fig. 3).


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Fig. 5.   Effect of PMA, Ca2+, or both activators on PKC translocation to membrane. Cells (hematocrit 5%) were incubated for 10 min in presence of 0.1 mM CaCl2 and either 1 µM PMA or 5 µM A-23187 or for 10 min with 1 µM PMA followed by 10 min with 5 µM A-23187. Membranes were prepared by cell lysis, electrophoresed, and transferred to Immobilon P membranes. PKCalpha and PKCzeta were detected by autoradiography or by ECL and quantified by counting 125I radioactivity of relevant bands or by scanning densitometry, respectively. A: translocation after 10 min. Data are expressed as percent of control and are means ± SE of 4-9 (PMA) or 3 (Ca2+) experiments. B: time course of translocation of PKCalpha in SS cells (means ± SE of 3 experiments).

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 PKCalpha but had no effect on PKCzeta 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 PKCalpha 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 PKCalpha , 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 PKCalpha 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 PKCalpha translocation. In two separate experiments, no significant enhancement of membrane PKCalpha 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 PKCalpha 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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Fig. 6.   Effects of deoxygenation and PMA on Ca2+ efflux and Ca2+ pump maximal extrusion rate (Vmax). A: Ca2+ efflux. SS cells were loaded with quin-2-acetoxymethyl ester and incubated with 45Ca2+ for 40 min. Incubation was continued for 50 min under either oxygenated (oxy) or deoxygenated (deoxy) conditions. DMSO (0.15%) or PMA (1 µM) were added for final 10 min. Cells were rapidly washed 4 times and reincubated under oxygenated conditions. Aliquots were taken every 90 s and centrifuged. 45Ca2+ radioactivity was measured in supernatant to calculate rate of Ca2+ efflux (means ± SE of 3 experiments). B: Ca2+ pump Vmax. SS cells (hematocrit 10%) were incubated for 10 min in a medium containing 0.1 mM 45CaCl2, under either oxygenated or deoxygenated conditions and in absence or presence of 1 µM PMA. At time 0, A-23187 (10 µM) was added to each suspension, followed 2 min later by CoCl2 (200 µM) to block Ca2+ entry by ionophore. Aliquots were withdrawn every 30 s and centrifuged. 45Ca2+ radioactivity was measured in cell pellets to calculate Ca2+ pump Vmax (means ± SE of 7 experiments). C: data are expressed as percent of control (without PMA) and are means ± SE of 3 or 7 values, respectively, in Ca2+ efflux and Ca2+ pump Vmax experiments.

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-1 cotransport. Compared with oxygenated conditions, deoxygenation stimulated K+ efflux by five times (0.05 and 0.24 mmol · l cells-1 · min-1, respectively) in the experiment shown in Fig. 7A. CTX (10 nM) inhibited K+ efflux under deoxygenated conditions (0.14 mmol · l cells-1 · min-1) but not under oxygenated conditions (Fig. 7A). PMA pretreatment caused a reduction in total but not in CTX-resistant K+ efflux under deoxygenated conditions (0.18 and 0.12 mmol · l cells-1 · min-1, respectively; Fig. 7B). The means obtained in three experiments are shown in Fig. 7C. Deoxygenation-stimulated K+ efflux (difference between K+ efflux under oxygenated and deoxygenated conditions: 0.26 ± 0.04 mmol · l cells-1 · min-1) was inhibited by ~50% (0.11 ± 0.01 mmol · l cells-1 · min-1, P < 0.05) in the presence of CTX and can be attributed to KCa channel activity. In addition, PMA treatment resulted in a significant inhibition (P < 0.05, compared with control without PMA; Fig. 7C) of both deoxygenation-stimulated and CTX-sensitive K+ efflux (difference between deoxygenated-stimulated K+ efflux in the presence and absence of CTX, 0.12 ± 0 and 0.04 ± 0.02 mmol · l cells-1 · min-1, respectively). The addition of 1 mM Ca(NO3)2 in the medium did not affect the extent of inhibition induced by CTX or by PMA. The structurally related phorbol ester 4alpha -PDD, which does not activate PKC, did not affect either the total deoxygenation-stimulated K+ efflux or the CTX-sensitive K+ efflux (data not shown).


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Fig. 7.   Effect of PMA on deoxygenation-stimulated (A-C) and Ca2+-stimulated (D) K+ efflux. A-C: SS cells were preincubated in a NaNO3 medium in absence or presence of 1 µM PMA for 10 min, centrifuged, and introduced into a previously oxygenated or deoxygenated NaNO3 medium, with or without 10 nM charybdotoxin (CTX; final hematocrit 2%). A and B: time course of K+ efflux without (A) or with (B) PMA in same experiment. C: means ± SE of 3 experiments. D: SS cells were incubated for 10 min in absence or presence of 1 µM PMA in a medium containing 0.1 mM CaCl2, with or without CTX. At time 0, 1 µM A-23187 was added to each suspension (final hematocrit 1%). Data are means ± SE of 5 experiments.

To determine whether the inhibition of KCa channel activity was due to an effect of PKCalpha 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 PKCalpha (data not shown).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The principal findings of this study are that 1) SS and AA cells express only two PKC isoforms, alpha  and zeta , with a higher level in the SS cells, and 2) CTX-sensitive KCa channel and Ca2+ pump activities are regulated through a PKCalpha -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 (alpha , beta I, beta II, and gamma ), 2) the Ca2+-independent, phorbol ester-binding nPKC isoforms (delta , epsilon , eta , and theta ), and 3) the Ca2+-independent, non-phorbol-ester-binding atypical PKC isoforms (zeta , lambda , µ, iota ) (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 PKCalpha and PKCbeta has been mentioned in one report (11). We have identified two isoforms, alpha  and zeta , in both AA and SS cells, and no other isoforms among those tested (beta , gamma , delta , epsilon , theta , or eta ) were detectable. The total and the membrane-associated contents of both PKCalpha and PKCzeta 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 PKCzeta was unaffected by either PMA treatment or elevation of [Ca2+]i. In contrast, PMA induced an increase in membrane association of PKCalpha 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 PKCalpha translocation and activation. Elevation of [Ca2+]i by the ionophore A-23187 also induced the translocation of PKCalpha to the membrane, in agreement with previous data showing a stimulation of membrane protein phosphorylation under these conditions (22). PKCalpha 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 PKCalpha translocation, indicating that [Ca2+]i increase in deoxygenated SS cells is not sufficient to activate PKCalpha , 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 PKCalpha -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 PKCalpha . 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 PKCalpha 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 PKCalpha 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 PKCalpha inhibits the activity of KCa channels in SS cells. The effects of PMA are attributed to PKCalpha , because a structurally similar compound (4alpha -PDD), which has no effect on PKCalpha , also had no significant effect on KCa channel activity. Furthermore, the effect of PKCalpha 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 PKCalpha -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 PKCalpha 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 PKCalpha 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 PKCalpha (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 PKCalpha .

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

dagger Deceased 1 February 1997. 

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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Discussion
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AJP Cell Physiol 273(4):C1206-C1214
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