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RED CELLS
From the Departments of Veterinary Pathobiology and
Chemistry, Purdue University, West Lafayette, IN.
Calcium entry into mature erythrocytes (red blood cells; RBCs) is
associated with multiple changes in cell properties. At low
intracellular Ca2+, efflux of potassium and water
predominates, leading to changes in erythrocyte rheology. At higher
Ca2+ content, activation of kinases and phosphatases,
rupture of membrane-to-skeleton bridges, stimulation of a phospholipid
scramblase and phospholipase C, and induction of
transglutaminase-mediated protein cross-linking are also observed.
Because the physiologic relevance of these latter responses depends
partially on whether Ca2+ entry involves a regulated
channel or nonspecific leak, we explored mechanisms that initiate
controlled Ca2+ influx. Protein kinase C (PKC) was
considered a prime candidate for the pathway regulator, and phorbol-12
myristate-13 acetate (PMA), a stimulator of PKC, was examined for its
influence on erythrocyte Ca2+. PMA was found to stimulate a
rapid, dose-dependent influx of calcium, as demonstrated by the
increased fluorescence of an entrapped Ca2+-sensitive dye,
Fluo-3/AM. The PMA-induced entry was inhibited by
staurosporine and the PKC-selective inhibitor chelerythrine chloride,
but was activated by the phosphatase inhibitors okadaic acid and
calyculin A. The PMA-promoted calcium influx was also inhibited by
Although erythrocytes (red blood cells; RBCs) have
historically been considered inert to regulatory signals from other
cells, RBCs are surprisingly well equipped with the machinery required for intercellular communication. Thus, in addition to a plethora of
hormone receptors, mature RBCs contain substantial numbers of cyclases,
phospholipases, kinases, phosphatases, and both ligand-gated and
mechanically activated ion channels.1 There is even
mounting evidence that RBCs play an active role in regulating both
blood rheology2-5 and hemostasis,6-10
responding to such diverse stimuli as Although the responsiveness of RBCs to regulators of circulatory health
and homeostasis is becoming more apparent, the signaling pathways that
mediate the changes in RBC properties remain unresolved. One of the
more common intermediates implicated in regulating RBC behavior is an
influx of extracellular calcium. Although activation of the Gardos
channel and the consequent loss of cell water have been the only
responses demonstrated to occur in vivo,15,16 there is
still abundant in vitro evidence to suggest that elevated intracellular
calcium can do the following: (1) activate a scramblase that
translocates phosphatidylserine to the outer leaflet,17,18 (2) induce changes in membrane skeletal architecture via association with calmodulin,19-21 (3) promote protein
cross-linking by activation of a transglutaminase,22 (4)
stimulate membrane phospholipases C23,24 and
A,25 (5) activate various protein kinases and phosphatases,26,27 and (6) stimulate calpains that can
cleave membrane proteins.22 Although the magnitudes and
frequencies of these latter responses to calcium entry remain
unresolved, calcium clearly constitutes a reasonable candidate for
mediating communication between erythrocytes and other cells of the
circulatory system.
Because mature RBCs lack intracellular calcium stores, elevation
in intracellular calcium must stem from calcium influx. Although increased cytosolic calcium has been commonly ascribed to breaches in
the permeability barrier of the membrane, calcium fluxes that mediate
signal transduction would seem more likely to derive from a controlled
pathway for Ca2+ entry. Pharmacologic evidence, in fact,
already suggests that some type of calcium channel may operate in the
mature RBC membrane,28-33 but its identity has never been
established. We report here the identification of an
Materials
Red blood cells
Preparation of Fluo-3/AM-loaded RBCs Fluo-3/AM was loaded into RBCs according to an established protocol34 using one of the following buffer solutions: PBS-G (NaCl 125 mM, KCl 2.7 mM, Na2HPO4 8 mM, K2H2PO4 1.5 mM, glucose 10 mM, pyruvate 10 mM, pH 7.4) or HEPES-G (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-glucose) (NaCl 123 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, HEPES 25 mM, glucose 10 mM, pyruvate 10 mM, pH 7.4). Briefly, 20 µL of packed RBCs (hematocrit [HCT] approximately 80%) were placed in 10 mL HEPES-G buffer (0.16% HCT) in a 50-mL conical tube and mixed gently by hand. The tube containing the RBCs was then covered in aluminum foil to exclude light, and 10 µL of 2.0 mM Fluo-3/AM was added to the dilute RBC suspension. The RBC-Fluo-3/AM suspension was then incubated at 37°C for 15 minutes with vigorous shaking. An additional 10 µL Fluo-3/AM (2.0 mM stock) was added, with incubation carried out for an additional 25 minutes (final Fluo-3/AM concentration 3.5 µM). The shaking was then decreased to a gentle motion for an additional 20 minutes. All subsequent manipulations were performed with the Fluo-3/AM-loaded RBCs carefully protected from light. Fluo-3/AM-loaded RBCs were centrifuged at 1000g for 3 minutes at 22°C, and the incubation buffer was carefully removed. The Fluo-3/AM-loaded packed RBCs were then washed 2 times with PBS-G plus 0.5% BSA (Sigma) and one time in HEPES buffer.Fluorescence spectroscopy The Fluo-3/AM-loaded RBCs were resuspended in 2 mL HEPES-G buffer and allowed to equilibrate for 15 minutes at 22°C. One hundred microliters of Fluo-3/AM-loaded RBCs (0.8% HCT) was added to 1400 µL HEPES-G buffer in a stirred quartz cuvette in a fluorimeter (Aminco Bowman Series 2 Luminescence Spectrometer; SLM-AMINCO, Rochester, NY). Kinase and phosphatase inhibitors, when desired, were added individually to the diluted Fluo-3/AM-loaded RBCs and incubated for 10 minutes at 22°C before transfer to the spectrofluorimeter. Fifty seconds of baseline fluorescence was initially recorded, and then PMA (dissolved in DMSO) was added at the desired final concentration. The same concentration of DMSO was used in all control experiments, with the final DMSO concentration never exceeding 0.1% of the total volume. RBCs used in the phosphatase inhibition experiments were diluted in PBS-G instead of HEPES buffer to avoid the phosphatase inhibitor activity present in the HEPES.35 Fluo-3/AM-loaded cells were excited at 506 nm with a 4-nm bandpass, and the emission was set at 530 nm with an 8-nm bandpass. Calcium influx was monitored by recording the change in fluorescence intensity over time.Flow cytometry For flow cytometry, Fluo-3/AM-loaded RBCs were resuspended in 5 mL HEPES buffer (0.32% HCT) and allowed to equilibrate for 15 minutes at 22°C. PMA was then added, and the cells were incubated for an additional 3 minutes before analysis. For calcium inhibition experiments, Fluo-3/AM-loaded RBCs (50 µL) were added to 450 µL HEPES buffer and incubated for approximately 10 minutes with the calcium channel antagonists or control solvents. These antagonist-pretreated, Fluo-3/AM-loaded RBCs were then stimulated with PMA or A23187 and placed in a Coulter ELITE Flow Cytometer for evaluation of fluorescence (Beckman Coulter, Hiahleah, FL). The excitation source for Fluo-3/AM was a 488-nm air-cooled argon laser, and the emission was measured using a 525-nm bandpass filter.Western blotting Washed RBCs were lysed using 30 volumes of ice-cold lysing buffer (Na2HPO4, 5 mM; ethylenediaminetetraacetic acid [EDTA], 1 mM; pepstatin A, leupeptin, and aprotinin, 1 µg/mL; phenylmethylsulfonyl fluoride, 0.2 mM; benzamidine, 0.1 mg/mL; and calpain inhibitor I and II, 8 µg/mL each; pH 8.0) and then washed in the same buffer until the membranes were white. The protein concentration of treated ghosts was determined using a premixed bicinchonicic acid (BCA) assay (Pierce, Rockford, IL). RBC membranes (50-80 µg) were loaded onto an 8% gel and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were then transferred to nitrocellulose for 2 hours at 4°C using 200 mV. The nitrocellulose membranes were blocked for a minimum of 2 hours at 22°C with 10% powdered skim milk in PBS plus 0.2% Tween-20. The primary 1A antibody (produced in rabbits against rat calcium
channel 1A peptide; accession number P54282; sequence
SSPERAPGREGPYGRE) was incubated at 1:200 dilution for 2 hours at
22°C. For competition of the 1A antibody with the
above specific peptide on Western blots, 10 µg of the
1A antibody was preincubated with 10 µg peptide in
Tris (tris[hydroxymethyl]aminomethane)-buffered saline
(TBS)/Tween-20/BSA buffer (20 mM Tris-OH, 137 mM NaCl, 0.05%
Tween-20, 2% BSA, pH 7.6) for 1 hour at 22°C and then centrifuged at
10 000g for 5 minutes. The antigen-peptide solution was
then diluted 1:200 in the same TBS/Tween-20/BSA buffer and incubated
for 2 hours at 22°C. The secondary antibody (GAR-HRP) was diluted
1:10 000 and incubated for 1 hour at 22°C. Reactive bands were
revealed by chemiluminescence using ECL Western blot detection reagents
on Kodak X-OMAT film.
Osmotic fragility test Buffer A (125 mM NaCl, 3 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 16 mM HEPES, 10 mM glucose, pH 7.4) was mixed with buffer B (99 mL 2 mM CaCl2 plus 1 mL 120 mM NaHPO4, pH 7.2-7.4) to generate solutions of 0, 30, 60, 75, 90, 105, 120, 135, 150, and 300 mOsm. Nine hundred microliters of each osmotic solution was then placed in a 1.5-mL Eppendorf tube and cooled to 4°C. Fresh, washed RBCs (HCT 10% in buffer A) were incubated with 3 µM PMA (or DMSO as control) for 40 minutes at 37°C in a shaking water bath. These samples were staggered at 20-minute intervals to allow time for processing. After the appropriate incubation period, 100 µL of RBCs was added to the first 10 Eppendorf tubes, mixed, and incubated at 4°C for 5 minutes. The cells were then centrifuged for 5 minutes. Five hundred microliters of supernatant was immediately placed in 500 µL Drabkin solution (Sigma), and absorbance at 540 nm was recorded using an ultraviolet spectrophotometer.Forward light scattering detected by flow cytometry Forward light scattering data of PMA-treated RBCs were obtained by flow cytometry as a means to detect changes in RBC size on an individual cell basis. Fresh whole blood was collected and washed as described above, and 20 µL of packed RBCs was loaded with Fluo-3/AM. An additional 20 µL of packed RBCs were subjected to the same experimental conditions as the Fluo-3/AM loading without any Fluo-3/AM present to compare size changes in RBCs loaded or not loaded with Fluo-3/AM. The Fluo-3/AM-loaded and nonloaded cells were equilibrated in HEPES buffer and treated with 3 µM PMA, 2 µM A23187, or 5 µM 1,2-didecanoyl-rac-glycerol (or DMSO as control) for 40 minutes at 22°C. Forward light scattering data of RBCs treated with PMA, DMSO, 1,2-didecanoyl-rac-glycerol, and A23187 were collected using a Coulter Elite Flow Cytometer.
Activation of protein kinase C induces a rapid influx of calcium into RBCs After considering a variety of potential signaling components, we selected protein kinase C (PKC) as a leading candidate for regulation of erythrocyte Ca2+ because of the following: (1) It is known to modulate Ca2+ homeostasis in other cells36-39; (2) it is highly active in mature human erythrocytes40,41; and (3) it influences both erythrocyte morphology and ion transport.40-44 To test directly whether PKC can modulate erythrocyte calcium homeostasis, we stimulated RBCs with PMA, a well-known activator of PKC, and examined them for changes in cytosolic Ca2+ using the Ca2+-sensitive fluorescent dye, Fluo-3/AM, as a reporter molecule. As shown in Figure 1, RBCs treated with 3 µM PMA displayed a Ca2+ influx that was slower than that catalyzed by the Ca2+ ionophore, A23187, but more rapid than that promoted by the PMA solvent DMSO. The influx was concentration dependent, and it reached a maximum near 3 µM (data not shown).
Although Fluo-3/AM is commonly used to monitor changes in cell calcium and although Fluo-3/AM becomes stably entrapped within a cell following its hydrolysis to the free acid, 2 artifacts can still arise that require additional controls. First, there was concern that the Ca2+-sensitive dye might have leaked out of the cell and become fluorescent as it encountered extracellular calcium. Therefore, calcium influx was monitored by flow cytometry because the flow cytometer can be adjusted to detect fluorescence only from intact cells. As seen in Figure 1B, PMA indeed stimulated calcium influx into intact erythrocytes. However, the cation is found to enter only a subpopulation of PMA-treated cells. Thus, incubation with 3 µM PMA for more than 3 minutes at 22°C induced approximately 47% ± 18% (n = 11, P < .001) of the RBCs to take up Ca2+ in an "all or none" fashion. In contrast, incubation with 1 µM A23187, as expected, caused approximately 99% (n = 11, P < .001) of the cells to exhibit increased fluorescence. Further, RBCs treated with the solvent DMSO as a negative control predictably displayed only background numbers of RBCs with equivalent fluorescent intensity (3.9% ± 4.7%, n = 11). Second, intracellular hydrolysis of acetoxymethyl esters (eg, Fluo-3/AM) releases formaldehyde, which can block glycolysis if present in sufficient quantities.45,46 Because blockade of glycolysis can lead to depletion of adenosine triphosphate (ATP) and a concomitant passive influx of extracellular calcium, we decided to measure cellular ATP content at multiple time points during loading of Fluo-3/AM. It is important to note that erythrocyte ATP concentration was found to decrease by no more than 10%, even at the longest incubation times (data not shown). These data dismiss the possibility that ATP depletion was responsible for the PMA-stimulated Ca2+ fluxes. The observation that the Ca2+ influx occurred within a minute of PMA stimulation (Figure 1, also confirmed by flow cytometry) also argues that its entry was not a consequence of ATP depletion. To ensure that the PMA-stimulated rise in Fluo-3 fluorescence was
directly caused by calcium entry, we also treated the
Fluo-3/AM-loaded RBCs with A23187, PMA, or DMSO in the
absence of calcium (Figure 2). Under
these conditions, no increase in Fluo-3/AM fluorescence was
detected. However, when calcium was reintroduced into the extracellular
buffer of the same cell suspensions 5 minutes after PMA
stimulation, the previously identified calcium influx was once again
observed in the A23187- and PMA-treated RBCs. Thus, PMA opens a calcium
influx pathway whether calcium is present or not, and the activated
Ca2+ channel remains open for several minutes.
PKC regulates calcium channel activity in PMA-treated RBCs Diacylglycerol (DAG) is well established as the natural activator of PKC, although it is weaker and shorter acting than PMA.47 Synthetic DAG isoforms such as 1,2-didecanoyl-rac-glycerol also activate PKC,48,49 and because they are more water soluble than DAG, they were used in these studies to explore whether the calcium influx seen in PMA-treated RBCs could be replicated by a physiologic activator. For this purpose, Fluo-3/AM-loaded RBCs were stimulated with either 5 µM or 10 µM 1,2-didecanoyl-rac-glycerol, and fluorescence intensity was monitored as a function of time using a fluorescence spectrophotometer. The results showed that 1,2-didecanoyl-rac-glycerol stimulated calcium influx into Fluo-3/AM-loaded RBCs similar to PMA, although the magnitude of the fluorescence change was somewhat less (Figure 3). It is important to note that 4-phorbol, an inactive analog of PMA, induced a
Ca2+ influx no different from the solvent control, DMSO.
These data demonstrate that the PMA-promoted calcium influx requires
the correct stereochemistry of the PMA-PKC interaction.
Although calcium influx was shown to be stimulated by both PMA and 1,2 didecanoyl-rac-glycerol, it was still necessary to establish
directly the possible participation of PKC in this signaling pathway.
In this effort, we initially confirmed that PKC-
Because phosphatases generally provide the "off" switch for
proteins activated by PKC phosphorylation, we hypothesized that the
PMA-induced calcium influx might be augmented in response to one or
more phosphatase inhibitors. As seen in Figure
5, both okadaic acid and calyculin A
enhanced the calcium uptake by PMA-treated RBCs.
PMA-mediated calcium influx occurs through an
 -agatoxin-TK, a specific
P-type calcium channel blocker,50 was found to inhibit the
PMA-induced influx in a dose-dependent manner. In fact, RBCs incubated
with 1 µM -agatoxin-TK before PKC stimulation displayed more than
95% inhibition of Ca2+ entry (Figure
6). Even at a lower -agatoxin-TK
concentration of 0.5 µM, only background levels of PMA-treated RBCs
responded with increased fluorescence, compared with approximately 70%
of control RBCs from the same donor. In a dose-response study, the 50%
inhibitory concentration (IC50) of -agatoxin-TK was
determined to be 100 nM. Inhibition of the PMA-mediated calcium influx
was minimally detectable at 10 nM. These inhibitory concentrations of
-agatoxin are similar to those used to inhibit calcium influx in
native P-type calcium channels found in rat neurons.51 It is important to note that -agatoxin-TK did not inhibit
A23187-induced calcium influx (data not shown).
L-type calcium channel blockers were also examined for their inhibition of the PMA-induced calcium influx. However, only at concentrations well above levels that normally inhibit L-type calcium channels in classic electrically excitable cells52 were these inhibitors able to reduce influx (data not shown). Thus, IC50 values for blockade of PMA-induced calcium entry into erythrocytes were determined to be approximately 100 µM for both nifedipine and verapamil. Because pharmacologic evidence suggested that a Cav2.1
(P/Q-type) calcium channel may function in the RBC membrane, we further explored the specific identity of the calcium channel. Erythrocyte membranes separated by SDS-PAGE and transferred to nitrocellulose were
blotted with a commercial anti-
Physiologic changes in PMA-stimulated RBCs To determine whether activation of PKC might affect erythrocytes in a physiologically meaningful manner, we investigated the impact of PMA-initiated calcium fluxes on erythrocyte volume. The mechanistic motivation for this study stems from the fact that one of the earliest consequences of Ca2+ entry is activation of the Gardos channel and the resulting efflux of cell water. To test whether PKC stimulation indeed leads to cell shrinkage, we incubated PMA (3 µM)-treated RBCs for 40 minutes at 37°C and evaluated them for a change in volume using forward scatter flow cytometry. As seen in Figure 8A, PMA-treated RBCs were significantly smaller than DMSO-treated negative controls, although not as small as A23187-treated positive controls.54 Similarly, erythrocytes stimulated with 1,2-didecanoyl-rac-glycerol showed an analogous decline in cell size (Figure 8B). Further, chelation of intracellular calcium by Fluo-3 had no effect on the size change of the treated RBCs (data not shown). These data suggest that PMA can induce influx of sufficient calcium to promote a change in cell volume.
To further document this change in surface-to-volume ratio, we
suspended PMA-treated RBCs in salt solutions of varying tonicities ranging from 0 to 300 mOsm to determine their resistance to hypotonic lysis. PMA-treated RBCs exhibited increased resistance to hypotonic lysis when compared with DMSO-treated controls in osmotic fragility tests (Figure 9).
We have presented 3 lines of evidence to demonstrate that
a regulatable channel can mediate the influx of Ca2+ across
the human erythrocyte membrane. First, both phorbol myristate acetate
(PMA) and diacylglycerol, 2 activators of protein kinase C (PKC), were
found to stimulate calcium entry into RBCs, whereas 4 Cav2.1 calcium channels from brain cells are well known to
be voltage gated, activating at membrane potentials of In a related study, Lijnen et al61 observed that PMA activates the human erythrocyte Na+/H+ exchanger. During their studies, these authors also noted that PMA stimulated calcium influx into the treated cells, in close agreement with our observations. Although activation of the 2 influx pathways may arise independently, it is also conceivable that activation of Na+/H+ exchange and stimulation of Ca2+ influx might be mechanistically related. Thus, PMA activation of PKC could trigger stimulation of Na+ influx via the Na+/H+ exchanger, and the elevation of intracellular Na+ could promote Ca2+ entry via induction of Na+/Ca2+ exchange. Further analysis of the linkage between Ca2+ influx and Na+/Ca2+ exchange activity will obviously be necessary before this hypothesis can be adequately evaluated. Earlier, our laboratory reported a calcium influx pathway initiated by
lysophosphatidic acid (LPA), a product of platelet activation.34 Although there are clear similarities
between the influx pathways, differences also exist to suggest that 2 separate signal transduction pathways might be converging on a common calcium permeability pathway. Thus, the LPA influx pathway was
not inhibited by the specific PKC inhibitor chelerythrine chloride,
whereas the PMA-mediated pathway was. Further, PMA was observed to
activate roughly twice as many cells to influx anywhere from 2- to
10-fold more calcium per cell than LPA, assuming that Fluo-3/AM fluorescence intensity is proportional to
intracellular calcium concentration. Nevertheless, the 2 calcium
permeability pathways are both blocked by Finally, calcium entry into RBCs is of more than academic interest because a variety of diseases are now characterized by elevated erythrocyte Ca2+. Although in vitro experiments have documented a number of changes in RBCs with increased intracellular calcium, the physiologic consequences of such calcium entry have not been demonstrated in vivo other than in sickle cells,62,63 where at least part of the dehydration appears to be a consequence of Ca2+ activation of the Gardos channel.64-67 We suggest that the Cav2.1-like calcium permeability pathway described here might contribute to this Ca2+-stimulated process. Further, because activation of PKC by PMA causes RBCs to expose phosphatidylserine on their outer leaflets,68 Ca2+ entry could enhance the thrombotic tendency of sickle cells by enriching their cell surfaces as an established procoagulant. Elevated erythrocyte calcium has also been associated with essential hypertension,69,70 primary hyperparathyroidism,71 idiopathic hypercalciuria,72 diabetes,73 and sepsis.74,75 Although these are clearly syndromes that are primarily manifested in other tissues, the impact of elevated cytosolic Ca2+ on erythrocyte structure and rheology may contribute to the pathologies of the diseases. And where such linkages become established, pharmacologic inhibition of RBC Ca2+ permeability may contribute to an eventually optimal therapy.
We thank Kathy Ragheb of the Purdue University Flow Cytometer Laboratory for her advice and expertise with the flow cytometer and Dr Jiazhen Wang for her excellent technical assistance in Western blotting.
Submitted November 6, 2001; accepted June 21, 2002.
Supported in part by National Institutes of Health grants GM24417 and K08HL03350.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dina A. Andrews, Department of Veterinary Pathobiology, Purdue University, 1243 Veterinary Pathology Bldg, West Lafayette, IN 47907-1243; e-mail: andrews{at}purdue.edu.
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B. A. Klarl, P. A. Lang, D. S. Kempe, O. M. Niemoeller, A. Akel, M. Sobiesiak, K. Eisele, M. Podolski, S. M. Huber, T. Wieder, et al. Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion Am J Physiol Cell Physiol, January 1, 2006; 290(1): C244 - C253. [Abstract] [Full Text] [PDF]
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J. F. Hoffman, W. Joiner, K. Nehrke, O. Potapova, K. Foye, and A. Wickrema The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells PNAS, June 10, 2003; 100(12): 7366 - 7371. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-adrenergic agonists, nitric
oxide, oxygen tension, platelet releasates, and hypertensive agents in
their participation in the functions of the circulatory system. Not
surprisingly, modulation of RBC properties has become a viable
pharmacologic target for the treatment of certain circulatory
diseases.
45 to