595
Intracellular Calcium Storage and Release in the
Human Platelet
Chlorotetracycline as a Continuous Monitor
Wenche Jy and Duncan H. Haynes
From the Department of Pharmacology, University of Miami Medical School, Miami, Florida
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SUMMARY, t h e calcium-sensitive fluorescent probe chlorotetracycline was used to monitor
calcium movement in human platelets. The chlorotetracycline fluorescence signal is a linear
measure of the level of free calcium in the dense tubules and in the mitochondria, with probe
sensitivity in the millimolar range. Experiments perturbing the system with the calcium ionophore
A23187 shows that the level of free internal calcium in the organelle depends upon the cytoplasmic
level, which, in turn, depends upon the passive permeability of the plasma membrane. Chlorotetracycline in the cytoplasmic compartment does not respond to changes in the cytoplasmic
calcium concentration, which is held in the micromolar to submicromolar range by an extrusion
system. The calcium concentration in the cytoplasmic compartment can be directly manipulated
by the calcium ionophore A23187 and is measured in parallel experiments with Quin 2, a highaffinity indicator. The calcium transport systems of the organelles are shown to be less susceptible
to short circuit by A23187. Analysis shows that mitochondrial uptake is slow (ti/2 = 20 minutes),
produces a large increase in chlorotetracycline fluorescence, and is inhibited by sodium azide plus
oligomycin. Uptake by the dense tubules is more rapid (t]/2 = 2 minutes), produces a smaller
increase in chlorotetracycline fluorescence, is inhibited by trifluoperazine, and is less sensitive to
A23187. The Km is estimated as 1 /*M or lower. Studies show that the chlorotetracycline technique
is useful for the monitoring of calcium uptake and release by the platelet organelles, and suggests
that the Quin-2/chlorotetracycline technique will be useful as a diagnostic of both physiological
and pathological activation mechanisms. (CircRes 55: 595-608, 1984)
THE platelet plays a central role in the maintenance
of the integrity of the vascular system. Exposure of
the platelet to stimuli such as collagen, adenosine
diphosphate (ADP), or thrombin results in a series
of reactions leading to clot formation. Initial responses include the following: (1) a shape change
which increases the surface area and provides a
surface capable of adhering to the subendothelium,
other platelets, and red blood cells (Frojmovic and
Milton, 1982), (2) the release of secretory granules
containing ADP, serotonin, adenosine triphosphate
(ATP), calcium (Ca++), platelet factor 4, acid hydrolase, fibrinogen, etc. (Holmsen et al., 1969), and (3)
massive aggregation.
For platelets in the unactivated or resting state,
the intracellular free Ca ++ concentration is maintained at the 10~7 M range (Rink et al., 1982). An
increase in cytoplasmic Ca++ is sufficient to induce
both the shape change and the release reaction
(LeBreton et al., 1976; Feinstein, 1980; Knight and
Scrutton, 1980; Lyons and Shaw, 1980). The aggregation reaction requires both external membranebound Ca++ (Hepstinstall 1976; Peerscheke et al.,
1980; Taylor and Hepstinstall, 1980; Ganguly and
Bradford, 1982) and increases in cytoplasmic Ca++
(Massini and Luscher, 1976; Owen and LeBreton,
1980; Rink et al., 1982). Stimulation elevates cyto-
plasmic Ca++ concentrations, either by releasing
Ca++ from intracellular stores or by increasing Ca
influx across the plasma membrane (Feinman and
Detwiler, 1974; Massini and Luscher, 1974; White
et al., 1974). The sources of intracellular sequestered
Ca++, and the regulation of its release and uptake
are not well understood. Biochemical studies have
identified and characterized an ATP-dependent
Ca++ uptake mechanism in human platelet membrane fractions (Robblee et al., 1973; Kaser-Glanzman et al., 1977; Fox et al., 1979; Javors et al., 1982).
A fraction of the membranes was purified and identified as the dense tubular system (Meriashi et al.,
1981) and active uptake by this fraction was shown
to be inhibited by trifluoperazine (TFP). In this
paper, we investigate intracellular Ca ++ sequestration in situ using fluorometric methods.
The Ca++-sensitive fluorescent probe chlorotetracycline (CTC)* has been applied widely in biological
* Abbreviations used in this paper are as follows: CTC, chlorotetracycline; ECTA
ethyleneglycol-bis-(-aminoethylether)-N,N'tetraacetic acid; TFP, trifluoperazine; Quin 2 AM, 2-[[2-[bis(ethoxycarbonyl)-methyl]amino]-5-methylphenoxy]methyl]-6methoxy-8-[bis-[ethoxycarbonylmethyl]amino]quinoline
acetoxymethyl ester; Tris, tris(hydroxyl)aminomethane. The free cytoplasmic
Ca** concentration is designated [Ca**]^, the free Ca** concentration in the organelles is designated [Ca**]j, and the external Ca** is
designated [Ca**]c.
Circulation Research/Vo/. 55, No. 5, November 1984
596
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FIGURE 1. Schematic
representation
showing how CTC reports changes in the
free internal Ca+* concentration in the
organelles ([Ca**]J. The relative concentrations of the various Ca** and CTC
species are represented by their size. The
charge of CTC and its complex (cf. Methods) is not given for the sake of clarity of
the diagram. The free concentrations of
Ca** in the aqueous phase of the organelle
lumena and cytoplasmic and external medium are equal. The concentrations of(CaCTQ* in these phases are governed by
their free Ca** concentrations. Both CTC
and(Ca-CTQ+ can bind to membrane surfaces, with the latter making the largest
contribution to the fluorescence. The contribution of the former is shown to be
small by measuring fluorescence in the
Ca**-depleted state. The system can be
treated as a multiple equilibrium (Millman et al., 1980) with communication
between the species shown by the arrows.
EXTERNAL MEDIUM
systems (Caswell and Hutchison, 1971; Caswell,
1972; Caswell and Warren, 1972; Chandler and
Williams, 1978a, 1978b; Gershergorn and Thaw,
1982) to monitor active Ca++ transport. A large
increase in fluorescence accompanies active Ca
transport. Our previous work with isolated sarcoplasmic reticulum (SR) has shown that CTC fluorescence can be used to report the free internal Ca++
concentration obtained by the Ca++,Mg++-ATPase
pump (Millman et al., 1980). We have modeled the
phenomenon showing that the fluorescence increase
is proportional to the free internal Ca++ concentration ([Ca++]i) in the SR lumen. The mechanism is
based on accumulation of the Ca-CTC complex in
aqueous compartments having high Ca ++ concentrations and binding of the complex to the membrane
to produce a large increase in CTC fluorescence. In
the SR, the CTC response is detectable when the
internal free Ca++ concentration exceeds 1 X 10~4 M.
Figure 1 shows the application of these principles to
the platelet, with high levels of (Ca-CTC)+ bound
to the outer surface of the plasma membrane and
on the inner surface of the organelle membranes.
The former contribution is constant for constant
external Ca++ concentration ([Ca++]o). The latter contribution varies directly with Ca++ uptake and release by the organelles. Figure 1 also shows that
changes in the cytoplasmic Ca++ concentrations are
not reported by the technique, since the cytoplasmic
free Ca ++ concentration is usually less than 10~6 M.
Further details are given in Methods, and in the
references cited. This mechanism is implicit in earlier
studies of Ca++ handling using CTC fluorescence as
a probe. LeBreton et al. (1976) interpreted decreases
in CTC fluorescence as a release of Ca++ from internal membrane compartments and correlated these
with the platelet shape change. Feinstein (1980) also
used the CTC technique to demonstrate that the
release of intracellularly sequestered Ca++ precedes
the onset of stimulus-induced exocytosis.
The present communication extends these observations. By using the Ca++ ionophore A23187 to
increase the cytoplasmic free Ca++ concentration,
we show that net Ca++ uptake (or release) by the
organelles is easily followed by monitoring the CTC
signal. We also show that the technique can be used
to differentiate between Ca ++ accumulation by the
mitochondria and by the dense tubular system, laying the groundwork for quantitative study of Ca++
uptake and release with physiological activators.
The present study will also develop experimental
approaches which may be useful in pinpointing
disorders in abnormal platelets.
Methods
Apyrase, A23187, oligomycin, EGTA, bovine serum
albumin, antimycin A, 2-deoxyglucose, and heparin were
purchased from Sigma. Other indicators and drugs were
597
Jy and Haynes/Ca** Homeostasis in Platelet: CTC as a Monitor
obtained as follows: chlorotetracycline, ICN Pharmaceuticals; Quin 2 AM, Lancaster Synthesis Ltd.; trifluoperazine, Smith, Kline & French; and NaN3, Alfa Products.
The remaining reagents for Tyrode's solution were obtained from Mallinckrodt.
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Washed Platelet Suspension
Freshly prepared platelet concentrate was purchased
from a local commercial blood bank and was worked up
and studied on the same day. The blood bank procedure
was as follows: 250 ml of blood was drawn into ACD
(acid-citrate-dextrose) collection bags, and the red cells
were removed by centrifugation at 150 g for 15 minutes.
The maximum time from blood drawing to receipt of the
platelet concentrate was 3 hours. In our laboratory, the
platelets were separated from the plasma by a centrifugation at 1100 g for 7 minutes in a clinical centrifuge
(International, model CL). The platelets were washed
twice and suspended in calcium and magnesium-free Tyrode's solution containing 138 mM NaCl, 3 mM KG, 5.5
mM glucose, 12 DIM NaHCO3, 0.4 mM NaH2PO4, with the
pH adjusted to 7.35 with HC1 and checked on an hourly
basis according to the method of Mustard et al. (1972).
This medium was used in all experimentation. The platelet
concentration was adjusted to give 20% transmittance for
aggregation experiments and 40% transmittance for CTC
fluorescence experiments. This corresponded to 0.1 mg/
ml protein concentration. Quin 2 experiments were carried
out at the same concentration of platelets as in CTC
experiments. Storage was at room temperature and experimentation was at 37°C.
Fluorescence Measurements
Fluorescence was measured in a Perkin Elmer (model
MPF-3L) fluorometer equipped with a thermostatically
controlled cell holder (T = 37°C). Reactions were carried
out in a 1-cm plastic cuvette. Mixing and stirring was
achieved by a top-mounted motor-driven plastic stirring
rod. The excitation beam was polarized horizontally by a
single polarizer (Chen, 1966) to reduce the light-scattering
effects to negligible values. The fluorometer collects emitted light over a wide angle, and control experiments
showed that inner filter effects were negligible. Repetition
of the fluorescence experiments at lower platelet concentrations (1/2 or 1/4 of the normal concentration) resulted
in identical fluorescence amplitude ratios and kinetics. The
absolute amplitudes are proportional to the platelet concentration. Independence of the amplitude ratios and kinetics on platelet concentration was observed both in
experiments involving aggregation (A23187 addition) and
in experiments where aggregation was not elicited. This,
together with the lack of an inner filter effect, is good
evidence that aggregation per se is not affecting our measurements.
Chlorotetracycline Fluorescence as a Quantitative
Measure of [Ca++]i
CTC fluorescence was measured at 380-nm excitation
and 520-nm emission. Previous work with sarcoplasmic
reticulum has shown that the probe reports the free internal Ca++ concentration in the organelle lumen by the
following mechanism: CTC can permeate in the neutral
uncomplexed form and the internal and external CTC
concentrations will be equal at equilibrium. The aqueous
CTC° also exits as an anion (CTC") with a pKa of 6.8 for
the transition (Millman et al., 1980). Within the lumen,
Ca++ complexes with CTC to give a small increase in
fluorescence:
Ca++i + CTCr <-> (Ca-CTC)+;.
(1)
The complex can bind to the inner surface of the membrane with a large increase in fluorescence:
(Ca-CTC)+i <-> (Ca-CTC)*b
(2)
It is this species which makes the largest contribution to
CTC fluorescence in the membrane-containing system.
We have shown that when the membrane concentration
is low enough to allow the bulk of the CTC to remain at
constant concentration in the aqueous phase, the amount
of (Ca-CTC)i!b is proportional to [Ca++]i. In the platelet,
the external Ca++ concentration, [Ca++]o and the Ca++
concentrations in the organelles ([Ca++]i) are much higher
than the Ca++ concentrations in the cytoplasm ([Ca"1"1"],^).
Thus, the largest contributions are made by (Ca-CTC)+
bound to the inside surface of the Ca++-sequestering organelles and by (Ca-CTC)+ bound to the outside surface
of the plasma membrane. These relationships are shown
schematically in Figure 1. The contribution of (Ca-CTC)+
bound to the outside surface of the platelet can easily be
determined, since it occurs instantaneously upon addition
of CTC or upon the addition of Ca++ in the presence of
CTC (cf. Fig. 2). The organelle contribution occurs with a
ti/2 of 4 ~ 5 minutes due to the necessity of CTC permeation across the membranes. The Results section shows
that, under most conditions, the cytoplasmic Ca++ concentration is held in the less-than-micromolar range. Thus,
there is little contribution from cytoplasmic (Ca-CTC)+ or
from the cytoplasmically oriented ("p faces') of the platelet
membranes. CTC is also able to make complexes with
endogenous Mg++ (cf. Caswell and Hutchison, 1971; Caswell, 1972; Caswell and Warren, 1972). These make a
constant contribution to the CTC signal, since the internal
Mg++ concentration does not vary. Their contributions can
be shown to be small by comparison of experiments with
and without metabolic inhibition. The affinity of CTC for
Ca++ is also dependent on pH. Internal alkalinization
produces a higher affinity, which results in increased
fluorescence at constant [Ca++]i. Experiments with Tris, a
penetrating buffer which reduces pH gradients across
membranes (Haynes, 1982), suggest that our results are
not influenced by this factor.
Quin 2 Experiments
Free cytoplasmic calcium concentrations were measured
by the Quin 2 method. Quin 2 AM loading of the platelets
was accomplished using the procedure of Rink et al.
(1982). The platelets were washed until the internally
trapped Quin 2 represented over 99% of the cell-associated indicator. Control experiments showed that Quin did
not leak from the platelets during the course of the experiment. Quin 2 fluorescence was recorded at 340 nm (excitation) and 490 nm (emission). The free internal calcium
concentrations were calculated from the extent of Ca++
complexation of the Quin 2 and from the published values
of the equilibrium constant of the reaction [K<j = 115 riM
(Tsien et al., 1982)]. The extent of Quin 2 complexation
with Ca++ is determined readily from comparison of the
sample fluorescence with the fluorescence observed in the
Ca++-saturated and Ca++-free medium. In our experiments, the latter two quantities were determined in situ
by Ca++-saturation and Ca++-depletion (excess EGTA).
Ca++ permeability was facilitated by digitonin. Calibrations were carried out for every platelet sample studied.
Circulation Research/Vo/. 55, No. 5, November 1984
598
FIGURE 2. Response of fluorescence change due
to the addition of platelets, CTC, Ca++ and EGTA.
Curve A, normal platelet; curve B, platelet pretreated with sodium azide (0.5%), oligomycin (4 ng/
ml) and trifluoperazine (15 fiM) for 1 hour; curve C,
response with no platelets. Abbreviations and concentrations are: CTC, 10 HM; Ca+*, 2 mM; EGTA.
3 mM; P, platelet suspension; S, saline. Curve A
represents one of 40 such experiments. Curves B
and C represent one of six experiments. The above
manipulations did not result in platelet aggregation.
B
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C
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Platelet Aggregation Measurement
The platelet aggregation was measured by the turbidimetric method (Born, 1962). The extent of platelet aggregation was determined by monitoring transmittance
change in a Beckman spectrophotometer (model DB-G) at
600 nm. This instrument was also equipped with a plastic
cuvette and motor-driven stirring rod.
Control of Extracellular Ca++ Concentration
Activation experiments were carried out at several external Ca++ concentrations. In the range of 0.1 to 20 mM,
the calcium concentration was set by simple Ca++ addition.
In the range of 10"4 to 10"8 M, the Ca++ concentration was
controlled by a Ca++-EGTA buffer ([EGTA], = 2 mM). The
Ca++:EGTA ratio was set, and the free Ca++ concentration
was calculated using the published equilibrium constants
(Martell and Smith 1974; cf. Haynes and Mandveno,
1983).
Quality Control and Reproducibility
The quality of the platelet sample was determined
routinely before experimentation. No spontaneous aggregation was found upon stirring. All samples had to show
normal aggregation kinetics. The ED50 for collagen and
ADP-induced aggregation was 15-25 /ig/ml and 1-3 /m,
respectively. This compares with literature values of 30
/ig/ml (Chesney et al., 1972) and 3 HM (MacMillan, 1966),
respectively. Occasional samples that did not meet these
criteria or that showed abnormal rates or extents of reaction were discarded. The CTC assay itself was also used
as a criterion of quality. The responses shown in Figure 2
were observed to be more sensitive measures of platelet
quality than the above-cited tests. After approximately 24
hours of storage, the slow amplitudes shown in Figure 2
would increase, while the size of the response to A23187
would decrease. This pattern was also observed in our
testing of platelets from patients suffering from arterial or
50
60
venous thrombosis. In the present study, all of the samples
were from normal donors, all had passed the abovementioned criteria, and all were studied in the first 24
hours after blood drawing.
All of the phenomena reported here were observed at
least three times with three separate preparations. Figures
2, 3, 4, 7, and 8 are recorder traces of highly reproducible
experiments which have been repeated at least five times
with five separate preparations. Figures 5, 6, and 9 are the
average of three or four separate experiments with an
equal number of preparations. The standard errors of
measurements are shown.
Results
CTC as a Measure of Ca++ Homeostasis
Figure 2 is a typical experiment showing that
platelet-associated CTC responds to changes in
Ca++. Curve A shows the results of serial addition
of platelets, CTC and Ca ++ . The platelet addition
gives rise to only a small light-scattering artifact.
Addition of CTC results in a rapid rise in fluorescence, followed by a slow increase with a ti /2 of 45 minutes. The rapid change is due to the fluorescence of CTC in the aqueous phase and CTC bound
to the outer surface of the plasma membrane. At the
low ambient external Ca ++ concentration, the corresponding Ca ++ complexes make only a small contribution (cf. Fig. 1). The slow fluorescence increase
will be shown to arise primarily in the binding of
CTC and its Ca++ complexes to the inner surfaces
of the organelles. The control experiments (curve C)
in the absence of platelets show that only 20-25%
of the rapid response is due to CTC in the aqueous
phase. No slow phase was observed. Information
Jy and Haynes/Ca** Homeostasis in Platelet: CTC as a Monitor
++
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about Ca handling by the platelets is obtained by
observing the response to Ca ++ addition. Curve A
shows that the addition of Ca++ to a final concentration of 2 mM gives rise to two phases of increase:
(1) an instantaneous phase corresponding to the
binding of the (Ca-CTC)+ complex on the outside
surface, and (2) a slower phase (ti/2 = 6-8 minutes)
due to increases in internal Ca++, which, in turn,
give rise to increased (Ca-CTC)+ binding to the
internal membranes. The slow phase amplitude is
11 units. Addition of EGTA to reduce the free external Ca++ concentration to the 10~8 M range returns
the fluorescence signal to its pre-Ca++ levels. The
reversal also occurs in a biphasic manner. Comparison with curve B shows that the slow phase amplitude can be blocked by inhibitors of energy metabolism in Ca++ transport. The platelet sample of curve
B was pretreated with NaN3, oligomycin, and trifluoperazine (TFP) for 1 hour and the procedure of
curve A was repeated. The comparison shows that
pretreatment with metabolic inhibitors lowers the
amplitudes and shortens the half-times of the slow
phases observed in both the CTC and Ca++ jumps.
The fast phase amplitudes were not affected. Such
behavior would be expected if the slow phase amplitude of curve A depended on actively accumulated Ca++ in the organelles. Furthermore, the lack
of effect of inhibitors on the fast phases is also in
line with our interpretation; metabolic inhibitors
would not be expected to affect the plasma membrane surface area or CTC affinity. The lack of effect
of inhibitors on the amplitudes assigned to the outside surface is readily seen by comparison of the
fast amplitudes of curve A and B.
599
Influence of A23187
Further experimentation was carried out to demonstrate that the CTC signal indicates the free Ca++
concentrations in two or more intracellular compartments, and that each of these is, in turn, influenced by the extracellular Ca++ concentrations. Figure 3 shows the CTC response of metabolically
competent platelets to elevation of cytoplasmic Ca++
concentrations brought about by addition of the
Ca++ ionophore A23187. The platelets were preincubated with 2 mM Ca++ and 10 /XM CTC to reach
the state identical to the plateau phase in Figure 2.
Next, 2.5 HM A23187 was added. The figure shows
that ionophore addition in the presence of 2 mM
Ca++ brings about an increase in fluorescence of 23
units resulting from influx of Ca++ and uptake by
the organelles. In contrast, Ca++ addition in the
absence of ionophore (Figure 2) results in no aggregation and a much smaller fluorescence increase (11
units for the slow phase). The increase in plasma
membrane Ca++ permeability is sufficient to explain
this difference. The ionophore-induced fluorescence
increase occurs in two phases, a fast phase with a
ti/2 of 2 minutes, and a slower phase with a ti/2 of
20 minutes. The slow phase is complete in 60 minutes. Both of these phases are slower than the exocytotic release events which occur in the first 5-10
seconds after activation with ionophores (Feinstein,
1980). The next section will show that the A23187elicited increases in CTC fluorescence are sensitive
to metabolic inhibition. Figure 3 shows that ionophore addition in the presence of 2 mM EGTA
([Ca++]j < 1 X 10"7 M) brings about a decrease in
fluorescence and no aggregation.
FIGURE 3. Fluorescence and
transmittance
changes during platelet activation induced by
A23187. The experiment was done with the same
preparation as in Figure 2. The platelet suspension
was preincubated with CTC, 2 mMCa**, or 2 mM
EGTA for 20 minutes, and a fluorescence level
equivalent to the plateau level in Figure 2 was
achieved. A new baseline was set, and A23187 was
added as indicated. The A23187 (2.5 /IA<) was added
as indicated. Upper panel: transmittance change
measured in a parallel experiment with the spectrophotometer. Downward deflection represents an
increase in transmittance. The lower panel is the
fluorescence change.
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The results observed in Figure 2 and 3 are in
accord with the well-accepted role of the plasma
membrane as a regulator of the cytoplasmic Ca++
concentration and the established ability of intracellular organelles to accumulate Ca ++ actively from
the cytoplasmic pool. Stochastic analysis shows that
the balance between the pumping activity of the
plasma membrane and organelle membranes which
accumulate Ca++ from the cytoplasm is expected to
be an important determinant of the platelet-associated Ca++. The levels of organelle-associated Ca++
are moderately sensitive to the external Ca ++ concentration under normal conditions (Figure 2). The
balance is shifted in favor of the organelles when
the plasma membrane is made Ca++ permeable by
A23187. This is supported by experiments described
in a later section, in which the plasma membrane is
made leaky by treatment with digitonin. The two
phases of organelle-associated CTC fluorescence
change and Ca ++ uptake are separated through the
use of metabolic inhibitors, as discussed in the next
section.
Influence of Metabolic Inhibitors
The protocol of Figure 3 was repeated with platelets incubated with several metabolic inhibitors. Figure 4 shows that two organelle systems can be
identified on the basis of sensitivity to the inhibitors.
Curve A is identical to the experiment of Figure 3
(no inhibitors). The figure shows that NaN 3/ an
inhibitor of mitochondrial electron transport, inhibits 60 ± 3% (mean ± SE, n = 4) of the response
(curve D). An identical effect was observed with 10
fiM antimycin A (56 ± 4% inhibition; data not
shown). No additional effect was observed when
azide and antimycin was added together. Azide and
antimycin A have been shown to inhibit mitochondrial electron transport (Stannard and Horecker,
1948; Lardy and Ferguson, 1969) and are adequate
to inhibit electron transport-dependent mitochondrial Ca++ uptake (Brierley, 1963; Lehninger et al.,
A CONTROL
B OLIGOMYCIN
C TFP
D AZIDE
E AZIDE
+ OLIGOMYCIN
F AZIDE * T F P
+OLIGOMYCIN
60
FIGURE 4. Effect of different inhibitors on the CTCfluorescencechange
induced by A23187 in the presence of 2 JHM Ca** and CTC The
platelets were preincubated with indicated inhibitors for 30 minutes.
The inhibitor concentrations were oligomycin (4 iig/ml); trifluoperazine (TFP) 15 IIM; sodium azide (0.5%;. Platelet aggregation was
induced by A23187 addition (2.5 IIM).
1967; Mela, 1977; Luthra and Olson, 1978; Nicholis
and Akerman, 1982), and have been used for this
purpose in platelets (Muerer et al., 1967; Mills,
1973). Also, control experiments in which the inhibitor concentrations were varied showed that this
represents complete inhibition of the mitochondrial
contribution to the Ca ++ uptake-associated CTC signal. Oligomycin has been shown to inhibit mitochondrial use of ATP to support ion transport
(Moore and Pressman, 1964; Kagawa and Racker,
1966; cf. Cockrell et al., 1966) and has also seen
application in platelets (Muerer et al., 1967; Mills,
1973). Application of oligomycin + azide (curve D)
does not produce significantly greater inhibition
than azide alone (curve D). Since azide + oligomycin
blocks both mitochondrial energy production and
mitochondrial use of ATP, the difference between
the control and azide + oligomycin curves must
represent the uptake characteristics of the mitochondria. The difference representing the mitochondrial
contribution is characterized by a slow uptake (ti/2
= 20 minutes) and a large extent of fluorescence
increase.
Trifluoperazine (TFP) has been shown to inhibit
ATP-energized Ca++ uptake in the isolated dense
tubular system (White et al., 1982). Figure 4 shows
that 15 fitA TFP produces a substantial inhibition
(27% ± 4%; mean ± SE, n = 4) of the CTC fluorescence associated with Ca++ uptake. Additional experiments (not shown) established 15 /iM as the
concentration giving optimal effect.f The difference
between the control and the TFP curves gives the
uptake characteristics of the TFP-inhibitable dense
tubular Ca++ uptake system. The system is characterized by a rapid uptake (ti/2 ~ 2 minutes) and a
small extent of fluorescence increase.
The lowest curve in Figure 4 represents the CTC
fluorescence change observed in the presence of
NaN3, oligomycin, and trifluoperazine (mitochondrial and dense tubular inhibition). This residual
corresponds to the (Ca-CTC)+ associated with the
cytoplasmic pool and uptake by any remaining organelles which are not influenced by this system.
The latter includes storage granules.:}:
•f Increasing the TFP concentration from 0 to 15 IIM gives maximal
inhibition of calcium uptake, as measured in the protocol of Figure 3.
However, 15 IIM TFP does not affect the behavior of Figure 2
(ionophore-unassisted uptake). Increasing the concentration from 15
to 30 IIM gives no change in the ionophore-stimulated uptake. Increasing the TFP concentration from 30 to 50 IIM increases the slow
amplitude seen in the protocol of Figure 2, suggesting that the
permeability of the plasma membrane is increased and the mitochondria are taking up availabile calcium from the cytoplasm. We conclude
that 15 iiM TFP is optimal for eliciting specific effects on the dense
tubular transport system, and that effects on membrane integrity are
minimal at this concentration. Our experiments are not in disagreement with studies showing TFP effects on the transport ATPases of
the plasma membrane or on protein kinase or the secretory response
(Sanchez et al., 1983); these effects are simply not encompassed in the
protocol of Figure 3.
X Storage granules contain a high concentration ofCa** and ATP.
However much of the Ca** is thought to exist as an ADP, ATP, or
5-hydroxytryptamine complex which would not contribute to Ca-CTC
Jy and Haynes/Ca.** Homeostasis in Platelet: CTC as a Monitor
An important test for the correctness of our analysis can be made by comparison of amplitudes of the
curves in Figure 4. If the mitochondrial and dense
tubular inhibitors work only on their respective organelles, then the effects of the inhibitors should be
additive. Figure 4 shows that this expectation is
fulfilled, since curve A = curve C + curve E — curve
F. This indicates that the dense tubular and mitochondrial uptakes occur independently of one another and do not compete for metabolic energy
under the conditions of the experiments. The experiments clearly identify the rapid phase of uptake
(ti/2 = 2 minutes) with the dense tubular system and
the slower phase with the mitochondrial system.
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Sources of Energy for Mitochondrial and Dense
Tubular Uptake
With the dense tubular and mitochondrial uptake
differentiated on the basis of TFP and NaN3 +
oligomycin, respectively, we can make further deductions about the source of energy for uptake by
the two organelles. Oligomycin can block both ATP
production and usage by the mitochondria. As noted
above, oligomycin alone shows little inhibition of
uptake (curve B vs. curve A). This shows that the
dense tubules do not require ATP produced by
oxidative phosphorylation. Comparison of curves D
and E shows that the mitochondria cannot use energy generated by glycolysis to take up Ca++. Curve
E (NaN3 + oligomycin) and the above-mentioned
additivity relationships demonstrate that glycolysis
is sufficient to drive dense-tubular Ca++ uptake. To
test the effect of inhibition of glycolysis, platelets
were preincubated with 30 rrtM 2-deoxyglucose. Experiments (not shown) revealed no effect of this
preincubation on the CTC response observed after
A23187 addition. However, preincubation with 2deoxyglucose plus azide or antimycin A for 1 hour
totally inhibited the CTC response to A23187. The
comparison shows that when energy production by
glycolysis is largely inhibited, oxidative phosphorylation is still able to provide sufficient energy to meet
both the demands of resting Ca++ homeostasis and
complex formation on the membrane surface. The bulk of the Ca** is
stored in an inert form, and is not thought to equilibrate rapidly with
the cytoplasm. The upper limit of the fluorescence contribution of
Ca** and Ca-ADP from the storage granules is given in the lowest
curve of Figure 4. It has been suggested to us that A23187 might
liberate Mg** from the cells and make a negative contribution to CTC
fluorescence. Curve F shows this is not the case. Another possible
source of artifact could arise from exocytosis which results in expulsion of the vesicle and vesicle contents. It is conceivable that this
reaction could increase the exposed surface of the outer plasma
membrane to such an extent as to affect the CTC signal (ca. 10% of
instantaneous phase of Figure 2). This would be expected to contribute
not more than two fluorescence units. Comparison of the instantaneous amplitudes observed upon Ca** addition before activation and
observed with Ca** removal after activation shows that the two are
identical, and no such contribution is detected. Also, the exocytotic
events occur in the first 5 seconds to 3 minutes (ti/i = 20-25 seconds)
after activation (Detwiler and Feinman, 1973), whereas the increases
in Ca** uptake occur on the time scale of 2-20 minutes.
601
TABLE 1
Energy Dependence of Mitochondrial and Dense Tubular
Uptake
Energy source
Oxidative
phosphorylation
Uptake by
Glycolysis
Mitochondria
Dense
tubules
Availability of energy and ability to take up Ca++ are denoted
by +. Nonavailability of energy and inability to take up Ca++ are
denoted by —
post-activational Ca++ uptake by the dense tubules
and mitochondria. These relationships are summarized in Table 1.
External Ca++ Concentration Dependence of
Ca++ Uptake by Organelles
Figure 5, curve A, shows the dependence of the
amplitude of the A23187-induced organelle uptake
reactions on the external Ca++ concentration for
metabolically competent platelets. The phenomenon
encompasses both dense tubular and mitochondrial
uptake. The next section will give evidence that at
the A23187 concentration chosen (2.5 /XM) the Ca++
expulsion mechanism of the plasma membrane has
been seriously compromised, while the Ca ++ accumulation mechanisms of the dense tubules and mitochondria are relatively unaffected. Figure 5, curve
A, shows that A23I87 addition at low [Ca++]o results
in a loss of organelle-associated Ca++. No net change
in the CTC signal is observed for [Ca++]o = 2-3 X
10~6 M. For [Ca++]o greater than this range, net
P Ca 0 (M)
FIGURE 5. Effect of external Ca** concentration on the A23187induced CTC fluorescence change. Platelets were preincubated for 30
minutes with CTC and Ca** at the stated concentrations, either in
the absence of inhibitors (A) or in presence of 4 fig/ml oligomycin, 15
fiM trifluoperazine, and 0.5% sodium azide. The baseline was reset
and 2.5 HMA23187 was added to induce Ca** influx. The change in
fluorescence was recorded and the net fluorescence change observed
in the plateau phase after 30 minutes is presented as the y axis.
External Ca** concentrations (x axis) were controlled as described in
Methods. Mean ± se, n = 4.
Circulation Research/Vol. 55, No. 5, November 1984
602
uptake is observed with a maximal extent for
[Ca++]o > 5 X 10"3 M.
Curve B of Figure 5 shows the results for platelets
which were pretreated with NaN3/ oligomycin, and
TFP. For [Ca++]o < 10"7 M, addition of A23187 does
not cause further Ca++ release. At high [Ca++]o, the
A23187-induced increase was also largely abolished.
The low amplitudes of curve B are expected, since
the experiment of the previous figure shows that
active uptake by the organelles is blocked by this
combination of inhibitors. We consider the greatly
diminished amplitudes plotted in curve B to be
representative of equilibrium of the stated Ca ++
concentrations with the cytoplasmic compartment
and with the lumena of the dense tubules, mitochondria, and other membraneous structures. The
difference between curves A and B of Figure 5
represents active transport of Ca++ into the dense
tubules and mitochondria.
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A23187 Concentration Dependence of Calcium
Uptake by Organelles
Figure 6 shows the A23187 concentration dependence of the Ca++ uptake by the organelles at a
constant external Ca++ concentration of 2 mM. The
figure shows that at a low A23187 concentration,
the extent of Ca ++ uptake increases with increasing
A23187 concentration. This is expected if the steady
state level in the cytoplasm increases with increasing
rate of A23187-induced leakage rate. Increases in
the cytoplasmic Ca ++ concentration would, in turn,
increase the levels of organelle-sequestered Ca++.
The observation of maximal uptake at 20 HM
A23187, and the decline of active accumulation at
A23187 concentrations above this value, can be
readily explained in terms of this model. At higher
A23187 levels, the passive Ca++ permeabilities of
75 •
10
20
30
40
A23I87(/>M>
FIGURE 6. Response of CTC fluorescence to the different concentrations of A23187 in the presence of 2 m\t Ca+*. The final amplitude
of the A23181-stimulated process is tabulated. Experimental protocols
were as in Figure 3. Curve A: normal platelets. Curve B: platelets
preincubated with NaN3 (0.5%), 4 fig/ml oligomycin, and 15 IIM TFP
for 30 minutes. Mean ± se, n = 4. The net change of fluorescence
was recorded after 30 minute.
the organelle membranes become comparable to the
rates of their correponding active Ca++ uptake systems, and their ability to accumulate Ca++ decreases.
Curve B shows that after treatment with NaN3,
oligomycin, and TFP, the fluorescence increase is
smaller. It reaches a plateau at 15 /*M A23187, a
concentration which is adequate to short-circuit the
plasma membrane and any accumulation of systems
which might be unaffected by our inhibitors. The
data obtained under inhibition at high A23187 concentration may provide a basis for quantification of
the active uptakes.
Our interpretations of the ionophore effect are
supported by experiments in which the plasma
membrane was made leaky by other means. An
extensive study with rat hepatocytes (Murphy et al.,
1980) showed that digitonin can make the plasma
membrane leaky to Ca++ without disrupting the
membranes of the organelles. The selectivity was
derived from the specificity of digitonin for cholesterol and from the high mole fraction of cholesterol
in the plasma membrane. Using the hepatocyte experimentation as an example, we treated platelets
(0.1 mg/ml) with digitonin (1 ng/n\l) and observed
the CTC fluorescence response. Addition of 1 X 10~7
to 1 X 10~5 M Ca++ gave rise to an ATP-dependent
increase of CTC fluorescence responses similar to
those observed upon the addition of A23187 (data
not shown). The fluorescence increase observed
with Ca++ addition was similar to that shown in
Figure 3 for ionophore addition, except that the
response was somewhat larger and more rapid. This
is what is expected for selective or semi-selective
permeabilization of the plasma membrane. Thus,
our results with digitonin seem to reinforce our
interpretation of the effects of A23187. More extensive manipulation of the cytoplasm of the platelet
will be described in a future publication.
Dependence of Organelle Uptake Reactions on
[C + + ]
The Quin 2 technique (Tsien et al., 1982) was used
to obtain information about the free cytoplasmic
Ca++ concentration achieved in the manipulation
described above. Our initial experiments, carried out
as described in Methods, showed that the resting
level of Ca++ was around 100-130 nM, in agreement
with the results of Rink et al. (1982). Upon addition
of high concentrations of A23187 with millimolar
external Ca++ concentrations, the dye indicated cytoplasmic levels of greater than 2 ^M. This shows
that the cytoplasmic Ca++ concentration can be radically influenced by the ionophore, given an adequate external Ca++ concentration. In further quantitative studies of this effect (Fig. 7), conditions had
to be chosen which would perturb the cytoplasmic
Ca++ concentration within the working range of the
dye (0.1-2 HM). In the experiments described in
Figure 7, a small concentration of A23187 (0.5 HM)
was chosen in order to produce a large effect on the
Jy and Haynes/Ca** Homeostasis in Platelet: CTC as a Monitor
603
CJ.- M
2Dxl6
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1.5x10
8
12
TIME(MIN)
20
16
-2.0 JJM
-1.5 JJM
-I.OjiM
FIGURE 7. Response of CTC fluorescence and the
cytoplasmic free Ca** concentrations (Quin 2 signal) to the low concentration of A23187 (0.5 IIM)
with addition of different concentrations of external
calcium. Panel A: CTC fluorescence change measured as in figure 3. Panel B: Quin 2 fluorescence
change measured in parallel experiments according
to the same protocol. The righthand scale gives the
cytoplasmic Ca*+ concentrations reported by the
probe. The external concentration in the experiments were: Curve A, 2 X W~3 \c curve B, 1 X
10~3 AC curve C, 2.5 x 10~* w curve D, 5 X W's
H curve £, 5 x 10~6 M; curve F, 5 X W'7 w curve
G, 5 X 10~* M; curve H, 1.5 x 10"* M. The external
Ca ++ concentrations were set in the manner described in Methods.
-0.6 M M
-0.4 M M
-o.au M
-O.I jiM
A23I87
0
B
8
12
TIME(MIN)
16
plasma membrane but to leave the Ca++ uptake
reactions of intracellular organelles relatively intact.
The external Ca++ concentration was varied, and
corresponding variations in the cytoplasmic Ca++
concentration were thus brought about.
Figure 7A shows the CTC response achieved after
A23187 addition at several external Ca++ concentrations. In parallel, the level of cytoplasmic free calcium was monitored with Quin 2 (Fig. 7B). The CTC
response was either an increase or decrease in fluorescence, depending on the external Ca++ concentration. At high [Ca++]o, the organelle-associated
CTC fluorescence increases after a lag. At low external Ca++ levels, a decrease is observed. At intermediate levels, Ca++ release is observed, followed
by reuptake. In all of the parallel Quin 2 experiments, the A23187 addition was observed to in-
20
crease [Ca++]cyt (all conditions). The [Ca++]cyt reached
a maximal level and then decreased slightly. When
the experiment is carried out at [Ca++]o < 0.25 mM,
the rapid increase in [Ca++]cyt was also observed, but
the overshoot effect was eliminated. The plateau
values of [Ca++]cyt decrease with decreasing [Ca++]o.
For [Ca++]o < 5 X 10"7 M, the plateau phase does
not seem to be maintained, and there is some indication of reversal of the increase in [Ca++]cyt. The
phenomenon of overshoot and subsequent decrease
is interpreted as massive Ca++ influx from the external medium and subsequent uptake by the organelles.
Figure 7 showed that upon addition of 0.5 /*M
A23187 in the presence of external Ca++ concentrations in the intermediate range (5 X 10"7 to 0.25
mM), and initial decrease in CTC fluorescence is
Circulation Research/Vo/. 55, No. 5, November 1984
604
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observed. This is followed by an increase representing reuptake of Ca++. This suggests release by one
organelle and uptake by the other. The experiment
shown in Figure 8 tests the inhibitor sensitivity of
the reuptake phenomenon under identical conditions, and shows that the reuptake is due to the
dense tubules. The platelets were preincubated with
5 X 10"5 M Ca++, and the release phase was initiated
by adding 0.5 HM A23187. In the middle of the
reuptake phase, NaN3 and oligomycin were added
(curve A) to inhibit the mitochondrial contribution.
Only slight attenuation of the rate was observed,
showing that the mitochondria were not contributing to the reuptake phase. Next, the reuptake was
challenged with TFP, the inhibitor of dense Ca++
uptake. Curve A shows that the reuptake process
was immediatly reversed and inhibited. Panel B
shows the effect of the inverse order of addition.
Reuptake is promptly reversed and inhibited by TFP
addition; mitochondrial inhibition produces little additional effect. We conclude from this that, at low
values of [Ca++]o and [Ca++]cyt (5 X 10"5 and 5 X
10~7 M, respectively), 0.5 fiM A23187 short-circuits
the mitochondrial system but has little effect on the
dense tubular system. This correlates with observations of low rates of mitochondrial Ca ++ uptake and
high rates by the dense tubular system.
The information contained in Figures 7 and 8
allows further characterization of the kinetics of
Ca++ uptake by the dense tubules in situ. Figure 8
TFP
i
0
1
1
4
1
1
1
1
8
12
TIME(MIN)
1
1
1—
16
FIGURE 8. Effect of different inhibitors on the Ca** uptake in the
presence of low concentration of A23187. The platelets were preincubated with CTC and approximately 5 x 10~5 M Ca**for30 minutes.
The baseline was reset and 0.5 HMA23187 was added to induce Ca**
influx. The different inhibitors were added as indicated in the figure.
Their concentrations are: 1% NaN3; 4 ng/ml oligomycin (oligo); 15
IIM trifluoperazine (TFP). The figure represents one of four highly
reproducible experiments.
2.0-
\
1.5-
=3
UJ
i.o-
0.5-
FIGURE 9. The relationship between rate of Ca** uptake and the
cytoplasmic free Ca** concentration in the platelet. The initial rate
of the increase phase of CTC fluorescence was plotted vs, [Ca**]^, (at
the same external [Ca**]0 as shown in Figure 7, panels A and B).
Mean ±SE,n = 3. The dotted line shows the maximal rate of increase
of CTC fluorescence observed at higher [A23187] and [Ca**], = 2.0
mu The fitted line shows the dependence calculated according to
Rate = 2.3 ([Ca^p^/d.07
mJ2 + [Ca**]\,), which assumes a
second power dependence on [Ca**]^, and a Km of 1.07 HM.
and our analysis showed that only dense tubules
contribute to the reuptake process seen at low
[Ca++]o and [Ca++]cy,. Figure 4 showed that the initial
rate observed at high [Ca++]o (and high [Ca++]cyt) is
dominated by the dense tubular contribution. Thus,
the rates of the uptake seen in Figure 7 are characteristic of the dense tubular system. Figure 9 shows
the dependence of the initial rate of uptake on [Ca++]
cyt. These quantities are taken directly from Figures
7, A and B, respectively. The plot shows the saturation characteritics of the dense tubular system in
situ. It shows that the rate increases from approximately 7% to 70% of its maximal value (shown by
the dotted line) when [Ca++]cyt is increased between
0.2 and 2 AIM. Half-maximal rates are observed at
approx. 1 fiM. This agrees with an apparent Km of
0.5-1 /iM determined for isolated dense tubules
(Robblee et al., 1973; Kaser-Glanzman et al., 1977).
The line through the points was fitted according
to a second-power dependence of the rate on
Discussion
The most important contribution made in the
present study is the development of methods by
which Ca++ movements in the human platelet can
be observed rapidly and continuously. The technique depends on our observations that CTC fluorescence is a linear measure of [Ca++]j in the dense
tubules and mitochondria and makes use of Quin 2
as a cytoplasmic indicator. The characteristics of the
Ca++ handling systems of the platelet were probed
Jy and Haynes/Ca** Homeostasis in Platelet: CTC as a Monitor
by the Ca++ ionophore A23187. The Ca++ extrusion
mechanisms of the plasma membrane are easily
short-circuited by low concentrations of A23187,
resulting in higher [Ca++]cyt and net accumulation
by the organelles. The Ca++ uptake function of the
organelles is less sensitive, and requires higher
A23187 concentrations for attenuation. With the
help of metabolic inhibitors, the two types of uptake
were analyzed. Mitochondrial Ca++ uptake is slow
and results in large increases in fluorescence. Dense
tubular Ca++ uptake is rapid and results in a smaller
increase in fluorescence. We have shown that oxidative phosphorylation can provide sufficient energy to power both types of uptake; that glycolysis
can provide sufficient energy to power dense tubular
uptake, and that energy produced by glycolysis cannot be used to power mitochondrial uptake. The
implications of our methodology and findings will
be discussed below under their respective headings.
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CTC Fluorescence is a Linear Measure of [Ca++]i
in the Dense Tubules and the Mitochondria
Our experiments show that the fluorescence increase of CTC serves as an indicator of active accumulation of Ca++ by the organelles of the platelet.
Our previous experiments have shown that it is an
indicator of the free Ca++ concentration contained
within the lumen of organelles (Millman et al., 1980;
Dixon and Haynes, submitted for publication). The
neutral form of CTC crosses membrane, eventually
achieving equality of concentrations on both sides.
The (Ca-CTC)+ complex does not cross membranes,
and thus accumulates in compartments with high
internal free Ca++ concentration. The mechanism of
Figure 1 has been supported by mathematical analysis (Millman et al., 1980), experiment (Dixon and
Haynes, submitted for publication), and by the results of the present study. According to the mechanism, the concentration of (Ca-CTC)+ complex
bound to the membrane is proportional to the (CaCTC)+ concentration in the aqueous phase. The
former is proportional to the fluorescence increase
observed; the latter is proportional to the free Ca++
concentration (e.g., calcium activity) in that compartment. The fluorescence increase is thus proportional to the free Ca++ concentration. We believe
that it may be possible to make use of the surface
area and volume relationships of the organelles to
quantify the free internal Ca++ concentration in the
dense tubules and the mitochondria, using the CTC
technique. From our data in Figure 5, we can state
that the organelles can accumulate Ca++ to an internal [Ca++]j level of 4 HIM or greater. This is based on
our observation that the CTC fluorescence change
with active accumulation is twice that observed with
the passive equilibration of 2 mM Ca++ in the metabolically inhibited case. It is possible that the total
Ca++ associated with the dense tubules is much
larger than the free internal Ca++ concentration estimated here, since the Ca++ can bind to acidic
605
phospholipids on the membrane, and since the
dense tubules may contain Ca++-binding proteins.
For mitochondria, Ca++ binding to matrix elements
is an established fact. Rossi et al. (1963) showed that
large amounts of Ca++ and phosphate can be taken
up by mitochondria in a co-precipitation mechanism. The inner membrane contains large amounts
of cardiolipids (Lehninger, 1964) which are expected
to bind Ca++. Recently, Coll et al. (1982) have estimated that over 99.9% of the total Ca++ is bound.
It is also possible to compare CTC fluorescence
increases with the total platelet-associated Ca++ or
exchangeable 45Ca++ as an aid to quantification. We
note that the reactions of interest (physiological
activation and changes in sequestered Ca++ as a
function of disease or age) the CTC/Quin 2 method
is much more convenient and reliable than the
45
Ca++ techniques which require serial additions and
special assumptions about relative permeabilities of
the plasma and organelle membranes.
Resting Levels of Organelle-Associated Ca++
Depend on Cytoplasmic Ca++ Levels
Our study using the combined methods of CTC
and Quin 2 fluorescence has produced a number of
observations about Ca++ handling by the intact
platelet. Cytoplasmic levels are kept low (100-130
run) (Rink et al., 1982; Fig. 7B, this study), presumably by active transport systems facing the cytoplasm. Our study has shown that, under resting
conditions, the free concentrations of Ca ++ in the
organelles respond to the cytoplasmic Ca ++ concentration ([Ca++]cyt), which, in turn, responds to the
external Ca++ concentration (fCa++]o). This gives
moderate levels of internal Ca and responses to
changes in [Ca++]o with half-times of approximately
6-8 minutes (Fig. 2). Although it is perhaps intuitively obvious that this should be the case, our study
is the first to demonstrate this. In a separate study,
we have used the CTC technique (Haynes et al.,
1983) to show that platelets from patients suffering
from thrombosis have higher resting levels of Ca ++
in their dense tubules. The platelets also showed a
greater amount of releasable Ca++ than normal controls. Both the Ca++ handling abnormality and the
disease were reversed by medication with nifedipine
(Haynes et al., 1983). The success of both the CTC
technique in detecting this abnormality and the drug
in treating this hyperaggregable' condition seems to
be based on the direct coupling of sequestered and
cytoplasmic Ca++ levels.
Cytoplasmic Ca++ Levels can be Manipulated by
A23187
Our experiments show that the free internal Ca++
concentrations within the organelle can be manipulated by the calcium ionophore A23187. This action
is presumably due to its ability to move Ca++, although effects on Na+, K+, and H + permeability
have not been tested in this study. We have observed
Circulation Research/Vol. 55, No. 5, November 1984
606
++
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that, with our test system, the Ca -regulating function of the plasma membrane is the most sensitive
locus of A23187 action. Extremely low concentrations of A23187 (0.5 MM) are able to overwhelm the
Ca++ removal system of the plasma membrane, raising [Ca++]cy, approximately 10-fold to the 2 MM level
and resulting in high levels of Ca++ acumulation in
the organelles. We observed large increases in the
rate of uptake by the dense tubules for [Ca++]cyt
values in the 0.2-2.0 fiM range (Fig. 9). Addition of
ionophore to higher concentrations (0.5-15 MM
A23187) in the presence of 2.0 ITIM external Ca ++
gives rise to much higher [Ca++]j levels. For A23187
levels of 15 ^M or larger, the organelle membranes
are increasingly short-circuited, and at 40 MM
A23187, [Ca++]i is probably equal to [Ca++]o (cf. Fig.
6B).
Since the resting cytoplasmic Ca++ level determines the Ca ++ level in the dense tubule, and since
an increase in the cytoplasmic Ca++ level is a common denominator to almost all types of platelet
activation, manipulation with A23187 can be useful
in a large number of experiments designed to elucidate fundamental mechanisms. For example, resting
levels of dense tubular [Ca++]j and/or [Ca++]cyt can
be set by A23187 before stimulation with physiological agents. This, together with the ability to visualize
Ca++ movements upon activation, should help to
answer questions which events are triggers and
which processes are effectors of the release reaction
and aggregation.
Dense Tubular vs. Mitochondrial Ca++ Uptake
Our study has differentiated between the mitochondrial and dense tubular contributions of Ca ++
uptake which occurs when [Ca++]cy, is elevated by
ionophore addition. The mitochondrial uptake is of
large capacity as judged by the CTC signal. It is slow
and is 90% inhibited by NaN3 and 100% inhibited
by NaN3 plus oligomycin. In keeping with its lower
rate, it is more susceptible to the action of ionophores than are the dense tubules. The uptake by
the dense tubules is smaller, as judged by CTC, but
is more rapid. It is inhibited by TFP (White et al.,
1980). Since the dense tubular uptake is more rapid,
it is less susceptible to short-circuit by ionophores
than is the mitochondrial uptake (Figure 8). We
stress that the foregoing observations relate to the
role of the organelles in reducing cytoplasmic Ca++
concentration. In our A23187 experiments, the ionophore addition caused the platelets to aggregate.
This is the direct result of Ca++ influx to raise
[Ca^cy,. With true physiological mechanisms,
[Ca++]cyt is elevated by influx, by dense tubular
release, or by a combination of both. The dense
tubular uptake occurring during A23187-induced
aggregation is not part of the physiological activation
mechanism. On the contrary, it is a manifestation of
the normal homeostatic mechanisms which attempt
to reduce [Ca++]cyt to a quiescent level and to guard
against spurious activation through random events.
Our findings on the energetics of the Ca++ removal system can be given a similar interpretation.
Our finding that glycolysis can produce sufficient
energy to maintain normal Ca++ homeostasis for at
least 30 minutes and to support active uptake by the
dense tubules upon Ca++ influx into the cell is
significant. This finding is not surprising, since it
would be undesirable for hemostasis and local ischemia to activate aggregation. A similar interpretation can be given to our finding that oxidative
phosphorylation can power both mitochondrial uptake and dense tubular uptake, whereas glycolysis
can power only dense tubular uptake. Under conditions of adequate energization it is expeditious to
use all means at hand to lower [Ca++]cyt; under
conditions of energy dearth, it is prudent to supply
only the most rapid, efficient, and specific Ca++
removal system. Seen in this context, the mitochondrion would seem to be a secondary line of defense
against super-micromolar cytoplasmic Ca++ concentrations.
Another implication of our study is that deficiencies in energy metabolism can raise [Ca++]cyt and
[Ca++]j and thus predispose platelets to triggering of
the aggregation response. As noted above, the CTC
technique represents a most convenient means of
investigating such relationships. The CTC plus Quin
2 technique is a diagnostic for both physiological
and abnormal activation mechanisms.
Utility of the Method
Our method has resolved the kinetics of uptake
and release from the mitochondria and dense tubules. It has also proven capable of monitoring the
release from one pool and the uptake by the other
(Figure 7A). This capability, together with the Quin
2 method, represents a powerful method of real time
analysis of the Ca++ handling systems in platelets.
As an example of diagnosis of physiological mechanisms, quantitative comparisons of Figure 7, A and
B, (curves D-G) suggest that the cytoplasm has an
appreciable Ca++-buffering capacity. Our findings
of elevated [Ca++]i in platelets from patients suffering from thrombosis point to a useful role of the
technique in diagnosing and treating abnormal activation mechanisms. As noted above, the capability
of continuous monitoring allows one to approach
many problems which were difficult or impossible
to solve by 4SCa++ methods. Finally, we note that
the CTC/Quin 2 technique represents a means of
following intracellular Ca++ movements resulting
from activation by physiological agents. In a future
communication, we wul show that different patterns
of Ca ++ movement can be observed on activation
with collagen, thrombin, ADP, and epinephrine.
This work was supported by National Institutes of Health Grant
GM 23990, and by a grant from the Florida Heart Association.
Address for reprints: Dr. Duncan H. Haynes, University of Miami
Medical School, Department of Pharmacology, P.O. Box 016189,
Miami, Florida 33101.
]y and Haynes/Ca** Homeostasis in Platelet: CTC as a Monitor
Received October 20, 1983; accepted for publication August 2,
1984.
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INDEX TERMS: Platelet • Ca++ uptake and release • Dense
tubule • Mitochondria • Chlorotetracycline • Quin 2
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Intracellular calcium storage and release in the human platelet. Chlorotetracycline as a
continuous monitor.
W Jy and D H Haynes
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Circ Res. 1984;55:595-608
doi: 10.1161/01.RES.55.5.595
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