Irregular spiking in free calcium concentrationin single, human platelets Regulation by modulation of the inositol trisphosphate receptors Roosje M.. Heemskerk1 1 Departments of Biochemi
Trang 1Irregular spiking in free calcium concentration
in single, human platelets
Regulation by modulation of the inositol trisphosphate receptors
Roosje M A van Gorp1, Marion A H Feijge1, Wim M J Vuist1, Martin B Rook2
and Johan W M Heemskerk1
1 Departments of Biochemistry and Human Biology, University of Maastricht, the Netherlands; 2 Department of Medical Physiology, University Medical Centre Utrecht, the Netherlands
Fluorescence ratio imaging indicates that immobilized,
aspirin-treated platelets, loaded with Fura-2, respond to
inositol 1,4,5-trisphosphate- (InsP3)-generating agonists
such as thrombin by high-frequency, irregular rises in
cytosolic [Ca2+]i with spikes that vary in peak level and
peak-to-peak interval This differs from the regular [Ca2+]i
oscillations observed in other, larger cells We found that the
thiol-reactive compounds thimerosal (10 lM) and U73122
(10 lM) evoked similar irregular Ca2+responses in platelets,
but in this case in the absence of InsP3generation
Throm-bin-induced spiking was acutely abolished by inhibiting
phospholipase C or elevating intracellular cAMP levels,
while spiking with sulfhydryl reagents was only partially
blocked by cAMP elevation Confocal laser scanning
microscopy using fluo-3-loaded platelets indicated that, with
all agonists or conditions, the irregular spikes were almost
instantaneously raised in various regions within a single
platelet When using saponin-permeabilized platelets, we
found that InsP3-induced Ca2+ release from stores was
stimulated by modest Ca2+concentrations, pointing to a
mechanism of InsP3-dependent Ca2+-induced Ca2+release
(CICR) This process was completely inhibitable by heparin
The Ca2+release by InsP3, but not the CICR sensor, was
negatively regulated by cAMP elevation Thimerosal treat-ment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3 In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3 Taken together, these results demonstrate that InsP3 recep-tor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such
as thimerosal or U73122 which do not stimulate InsP3 for-mation The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3channel sensitization by sulfhydryl (thim-erosal) modification, (d) InsP3channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation The observation that individual Ca2+peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process
Keywords: Ca2+-induced Ca2+ release; cyclic AMP; cytosolic Ca2+; inositol trisphosphate; platelets
Most vertebrate cells respond to specific agonists by
repetitive spiking or oscillation in cytosolic [Ca2+]i as a
consequence of regenerative release of Ca2+from stores
into the cytosol through inositol 1,4,5-trisphosphate (InsP3)
or ryanodine receptor channels, located in the membrane of
the endoplasmic or sarcoplasmic reticulum, respectively [1]
For large cells such as oocytes and HeLa cells, evidence has
been collected that local clusters of InsP3 receptors in the
reticular membrane function as discrete Ca2+release sites Such local spots, being spaced at intervals of tens of micrometers apart, are taken responsible for so-called elementary Ca2+ release events [2–4] At low concentra-tions, InsP3 may trigger individual release sites, which results in the appearance of local Ca2+ÔpuffsÕ, i.e of brief
Ca2+release events of usually low amplitude Higher InsP3 concentrations cause a summation in amplitude or fre-quency mode of these release events, and lead to recruitment
of neighbouring release sites As a consequence, global increases in [Ca2+]ican develop that propagate through the entire cell as Ca2+oscillations or waves These whole-cell
Ca2+ responses are usually regular in shape, such in contrast to the local Ca2+puffs which are heterogeneous in both amplitude and time of appearance
In a variety of cells, the InsP3 receptor channels play crucial roles in eliciting [Ca2+]ioscillations and puffs [1–4] Three different InsP3 receptor isoforms are presently recognized with subtle differences in the regulation of
Ca2+channel opening Characteristic for the type 1 InsP3 receptors is a biphasic effect of cytosolic Ca2+ on the channel activity, with Ca2+stimulating the Ca2+ release
Correspondence to J W M Heemskerk, Departments of
Biochemis-try/Human Biology, University of Maastricht, PO Box 616,
6200 mD Maastricht, the Netherlands.
Fax: + 31 43 3884160, Tel.: + 31 43 3881671,
E-mail: JWM.Heemskerk@bioch.unimaas.nl
Abbreviations: CICR, Ca2+-induced Ca2+release; InsP 3 , inositol
1,4,5-trisphosphate; PGE 1 , prostaglandin E 1
Note: Part of this paper appears in the PhD Thesis of
R M A Beisser-van Gorp (University of Maastricht,
the Netherlands).
(Received 21 September 2001, revised 21 December 2001, accepted
22 January 2002)
Trang 2facilitated by the sensitizing mechanism of Ca2+-induced
Ca2+ release (CICR), whereas at higher Ca2+ levels the
InsP3 receptors become desensitized Other factors
deter-mining the open probability of the receptor channels are the
luminal Ca2+concentration in the endoplasmic reticulum
[9,10], modulation or oxidation of the receptor sulfhydryl
groups [11–14], and phosphorylation by cAMP-dependent
protein kinase [15,16]
Platelets are among the smallest cellular entities in the
mammalian body (diameter of about 2 lm with estimated
volume of 6 fL) They acutely respond to InsP3-forming
agonists by regenerative Ca2+release [17–20] The [Ca2+]i
spiking pattern of platelets is remarkably irregular in shape
in comparison to that of larger cells, e.g of the smoothly
oscillating megakaryocytes [21,22] All three InsP3receptor
isoforms have been identified in platelets, i.e mostly type 1
and type 2 receptors in addition to some type 3 receptors
[23–26] In platelet membrane preparations it is shown that
the InsP3receptors are susceptible to sulfhydryl
modifica-tion and cAMP-dependent phosphorylamodifica-tion [24,25] There
is, however, little evidence that such modulation influences
InsP3receptor functioning also in intact platelets [27,28] In
particular, it is controversial whether cAMP-dependent
protein kinase may stimulate InsP3-induced Ca2+release
[29], cause modest inhibition [30,31], or is without effect [32]
on the release process
In this report we consider the nature and subcellular
organization of the regenerative Ca2+ release in platelets
triggered by InsP3-mobilizing receptor agonists and
non-InsP3-mobilizing sulfhydryl reagents We investigated the
importance of InsP3 receptor-dependent CICR in the
irregular Ca2+signal generation by these agents, and the
sensitivity of this signal toward cAMP elevation We found
that the irregular spiking Ca2+signal of platelets contains
several but not all characteristics of local, InsP3
receptor-dependent Ca2+puffs described for other, larger cells
E X P E R I M E N T A L P R O C E D U R E S
Materials
H-Arg-Gly-Asp-Ser-OH (RGDS) was purchased from
Bachem (Bubendorf, Switzerland), and ultra-pure
calcium-free water from Baker (Phillipsburg, NJ, USA) Fura-2,
Fluo-3 and Indo-1 acetoxymethyl esters as well as
noneste-rified Fluo-3 were bought from Molecular Probes (Leiden,
the Netherlands) Manoalide, U73122, U73343 and InsP3
came from Biomol (Plymouth Meeting, PA, USA), and
thimerosal (sodium ethylmercuri-thiosalicylate) was from
Janssen (Beerse, Belgium) Other chemicals were obtained
from Sigma (St Louis, MO, USA) or Merck (Darmstadt,
Germany)
Platelet preparation and loading with Ca2+probes
Blood was collected from healthy volunteers, who had not
taken medication for at least two weeks Platelet-rich plasma
was prepared by centrifugation [18] It was incubated with
acetoxymethyl ester of Fura-2 (3 lM) or Fluo-3 (7 lM) in
(pH 7.45), which was composed of 136 mMNaCl, 10 mM
glucose, 5 mMHepes, 5 mMKCl, 2 mMMgCl2, 0.1% (v/v) bovine serum albumin and apyrase (0.2 U ADPaseÆmL)1) The suspension was adjusted to 1· 108plateletsÆmL)1 Measurement of [Ca2+]iin single, immobilized platelets Aspirin-treated, Fura-2-loaded platelets were immobilized
on fibrinogen-coated glass coverslips, as described previ-ously [18] Briefly, the platelets were allowed to bind to the surface, and bathed in 0.5 mL buffer A supplemented with
10 lM RGDS, apyrase (0.2 U ADPaseÆmL)1) and CaCl2 (2 mM) at 23°C Agonists and antagonists were given as freshly prepared solutions in bathing medium (0.1 mL) Changes in Fura-2 fluorescence were recorded in individual cells using an inverted Nikon microscope (Tokyo, Japan), equipped with a dichroic mirror, computer-driven excitation and emission filter wheels, and an intensified charge-coupled device camera working at standard video rate (Photonic Sciences, Robertsbridge, UK) A 100-W Xenon lamp was used for illumination The excitation wavelength was alternated between 340 and 380 nm, and fluorescent light was detected at 505 nm The light was collected with a 40· oil objective (Fluor Nikon, numerical aperture 1.3) Final image resolution was 1.0 pixelsÆlm)1, while confocality giving half-maximal intensity in the x–y plane was deter-mined at 2.3 lm.QUANTICELL700 software (Visitech, Sun-derland, UK) was used to control the filter wheels and capture the images [33] Four-times averaged, background-subtracted fluorescence ratio images were obtained every second Calibration of 340/380 nm fluorescence ratio to [Ca2+]i, using lysed platelets, was as described elsewhere [20] Fluorescence measurements with suspensions of Fura-2- or Fluo-3-loaded platelets were carried out as described [20] High-resolution, confocal images were collected with a Nikon RCM 8000 real-time confocal laser scanning system, equipped with an Argon laser Light was collected with a
60· oil objective (Apo Nikon, numerical aperture 1.4) Fluo-3-loaded platelets were visualized at a laser power of 87–91 lW, and excitation and emission wavelengths of
488 nm and 500–550 nm, respectively Using a small pinhole, confocality in the x–y plane was experimentally determined at 0.2 lm (matching the final image resolution
of 6.0 pixelsÆlm)1), while confocality along the z axis was 0.5 lm Because of the limited fluorescence levels in the platelets, image frames were eightfold averaged to give a final temporal resolution of 10 Hz The Fluo-3 fluorescence level was expressed as a pseudo-ratio value (F/Fo) of the actual fluorescence intensity (F) relative to the basal intensity
of the platelet at rest (Fo), as described elsewhere [3,4] Calibration was performed as described by Yao et al [2] The same confocal system was also used to monitor Indo-1-labelled platelets, at settings described elsewhere [34]
Measurement of [Ca2+] in suspensions
of saponin-permeabilized and intact platelets Aspirin-treated platelets were suspended at a concentration
of 1–1.5· 109mL)1 in buffer B (pH 7.45), composed of
Trang 3136 mM NaCl, 20 mM glucose, 5 mM Hepes, 5 mM KCl,
2 mMMgCl2and 0.1 mMEGTA prepared in calcium-free
water The platelets were permeabilized with saponin at
23°C, basically as described elsewhere [31] Immediately
before start of a measurement, a sample of 0.4 mL was
added to 1.6 mL of Hepes/KCl buffer pH 7.4 (buffer C),
composed of 100 mMKCl, 100 mMsucrose, 20 mMHepes,
1.4 mMMgCl2and 1.25 mMNaN3(prepared in
calcium-free water) The mixture, in a fluorescence cuvette, was
supplemented with 7.5 mM phosphocreatine, 1 mM ATP,
1 mM KH2PO4, 30 lgÆmL)1 creatine kinase, 0.6 lgÆmL)1
oligomycin and 1 lM Fluo-3 Permeabilization of the
platelets was achieved by addition of 15–20 lgÆmL)1
saponin After 10 min of stirring, fluorescence was
mea-sured and the free Ca2+level was titrated to 110 nM by
stepwise additions from a 0.05-mMCaCl2 solution InsP3
and other agents were given during the fluorescence
recording Part of the experiments were carried out with
0.75 mM phosphocreatine and 0.1 mMATP In that case,
apyrase (2 UÆmL)1) was added after 6 min of
permeabili-zation to degrade ATP Free Ca2+was then adjusted to the
desired level, after which InsP3 was added Ultra-pure,
calcium-free water was used for preparation of all buffers,
supplements and agonists
Fluo-3 fluorescence intensities (F) were continuously
recorded at 488 nm excitation and 526 nm emission
wave-lengths (slits of 4 nm), using an SLM-Aminco DMX-1100
spectrofluorometer (Rochester, NY, USA) Calibrations
were performed by adding excess amounts of CaCl2 and
EGTA/Tris (1 : 1, mol/mol) to obtain Fmaxand Fminvalues,
respectively Level of [Ca2+] in the suspension was
calcu-lated from the binding equation [Ca2+]¼ Kd· b (F–Fmin)/
(Fmax–F) The same fluorometer was also used to measure
changes in [Ca2+]iin intact platelets loaded with Fura-2 or
Fluo-3 [34]
Measurement of InsP3
InsP3 levels were determined in samples of resting and
activated platelets (180 lL, 3.5· 108cells) Cellular activity
was stopped by addition of 75 lL ice-cold 10% (w/v)
HClO4 After standing on ice for 30 min and centrifuging at
2000 g for 10 min (strictly at 4°C), supernatants were
collected and neutralized to pH 7 with a solution of 1.7M
KOH and 75 mMHepes After 30 min on ice, the precipi-tated KClO4was removed by another centrifugation step (4°C) The supernatants were used to measure mass amounts of InsP3 with a Biotrak radioreceptor assay system (Amersham-Pharmacia, UK) Freshly dissolved InsP3was taken as a standard
Statistics Paired data were compared for significance of difference using a Student t-test Unpaired data were compared by
ANOVA
R E S U L T S
Irregular spiking in [Ca2+]iin single platelets independently of InsP3formation
Fura-2-loaded platelets immobilized on fibrinogen often exhibit ÔspontaneousÕ, spiking increases in [Ca2+]i, which can partially be prevented by treatment of the platelets with aspirin and apyrase (blocking the effects of released thromboxane A2and ADP, respectively) [35] Using plate-lets treated with aspirin and apyrase, we compared the effects of various Gq/phospholipase C-b stimulating recep-tor agonists on Ca2+signal generation Extracellular CaCl2 was present to allow physiological, store-regulated influx of
Ca2+ Both platelet-activating factor (400 nM) and the thromboxane A2analogue, U46619 (1 lM), caused repetit-ive increases in [Ca2+]iin single platelets for up to 3 min In these traces, individual Ca2+spikes varied in peak levels and occurred after short but variable time intervals (Fig 1A,B) The strong agonist thrombin (4 nM) also elicited irregular, spiking rises in [Ca2+]i, but the signal now persisted for more than 5 min (Fig 1C) These responses differ markedly from the quite regular and symmetric oscillations in [Ca2+]i, which have been reported for larger cells such as rat megakaryocytes [9,10] To determine the involvement of cytosolic InsP3in the irregular spiking process in platelets, we used the phospholipase C-inhibiting agents manoalide [36,37] and U73122 [38,39] Addition of manoalide (10 lM) or a low dose of U73122
Fig 1 Irregular spiking in [Ca2+] i induced by
phospholipase C-activating agonists
Aspirin-treated, Fura-2-loaded platelets on a
fibrin-ogen surface were stimulated with 0.4 l M
platelet-activating factor (PAF) (A), 1 l M
U46619 (B) or 4 n M thrombin (Thr) (C–F) in
the presence of 1 m M CaCl 2 and apyrase (0.1
U ADPaseÆmL)1) Where indicated, 10 l M
manoalide (D), 2 l M U73122 (E), or 10 l M
PGE 1 (F) was added after stimulation.
Fluorescence ratio images were collected from
microscopic fields using a camera-based
sys-tem Traces are Ca 2+ responses of single
platelets, representative for 50–100 cells from
at least four independent experiments.
Trang 4was also annulled by addition of the cAMP-elevating agent,
prostaglandin E1 (PGE1, Fig 1F) Thus, the irregular
spiking process with thrombin apparently depends on
continuous generation of InsP3 and is down-regulated
by elevation of the cAMP concentration (see also below)
Note that similar, irregular Ca2+ responses were also
obtained when using platelets loaded with Fluo-3 instead of
Fura-2
Membrane-permeable sulfhydryl reagents provide an
alternative way of evoking Ca2+ responses, although
occurring in the apparent absence of phospholipase C
activation [40,41] We used thimerosal, a compound that
sensitizes the platelet InsP3receptor channels [28,42], and a
high dose of U73122 which acts as an N-ethylmaleimide
derivative thus affecting other enzymes than only
phospho-lipase C [38,39,43] When aspirin-treated platelets on
fibri-nogen were treated with thimerosal (10 lM) or U73122
(10 lM), this resulted in prolonged, irregular spiking in
[Ca2+]iafter a lag time of one or more minutes (Fig 2A,B)
As U73122 inhibits phospholipase C activity already at
2 lM (see below), the spiking with U73122 is unlikely to
result from phospholipase C activation and InsP3
genera-tion This conclusion was also drawn for thimerosal, as
neither pretreatment with manoalide (Fig 3A) nor
postad-dition of manoalide (Fig 3B) or a low dose of U73122 (not
shown) influenced the spiking induced by thimerosal In
quantitative trms, after 5 min of stimulation with
thimero-sal, peak amplitudes were 667 ± 80 nM [Ca2+]i in the
absence of manoalide pretreatment and 586 ± 48 nMafter
manoalide pretreatment (mean ± SEM, n¼ 22 cells,
P¼ 0.38) In contrast, preincubation of the platelets with
10 lM PGE1 lowered the amplitude of the
thimerosal-induced peaks to 390 ± 70 (n¼ 24 platelets, P ¼ 0.009)
(Fig 3C) PGE1, when added after thimerosal, gradually
inhibited the appearance of new [Ca2+]ispikes, although it
did not restore [Ca2+]ito the basal level (compare Fig 3A
and D) When added after U73122, PGE1 had a similar
effect on the spiking process (Fig 2C)
In experiments with aspirin-treated platelets in
suspen-sion, we verified the effects of these platelet-activating agents
on phospholipase C stimulation Levels of InsP3levels were
measured at time points where the Ca2+signal was still
maximal Thrombin, but not thimerosal, had a potent
InsP3-elevating effect that was largely abolished by a
preincubation with PGE1 (Table 1) This is in agreement
with earlier data [44] U73122 blocked the thrombin-induced
increase in InsP3level at concentrations that also suppressed
the thrombin-induced Ca2+response Together these results
indicate that both InsP3-generating (thrombin) and
non-InsP3-generating (sulfhydryl reagents) agents cause irregular
[Ca2+]ispiking in platelets The thrombin-induced spiking
and to a lesser extent the thimerosal/U73122-induced
spiking appears to be sensitive to cAMP modulation
Regulation of InsP3receptor function and store
depletion by Ca2+, cAMP and sulfhydryl reagents
To better understand the effects of these agents on the
spiking process, we directly measured the Ca2+ release
through the InsP3receptor channels Therefore, platelets in suspension were permeabilized with saponin under low
Ca2+-buffering and ATP-regenerating conditions using Fluo-3 as a Ca2+probe [31] In this experimental system, InsP3 caused a (nonlinear) dose-dependent increase in [Ca2+] from stores, which was completely suppressed by the InsP3 receptor antagonist heparin (Fig 4A) A low con-centration of InsP3 (50 nM) caused a Ca2+ release of
27 ± 6 pmol per 108platelets (mean ± SEM, n¼ 7) at a medium free Ca2+concentration of 110 nM
The Ca2+-dependency of the InsP3-evoked Ca2+release was evaluated by permeabilization experiments designed as
to prevent changes in the Ca2+ store content Platelets were thus permeabilized at 110 nM [Ca2+], after which apyrase was added (to block Ca2+re-uptake), followed by different amounts of Ca2+and 50 nMInsP3(see Materials and methods) Under these conditions, the Ca2+ release
Fig 2 Irregular spiking in [Ca 2+ ] i induced by sulfhydryl-reactive agents Aspirin-treated, Fura-2-loaded platelets on a surface were sti-mulated with 10 l M thimerosal (TMS) (A) or 10 l M U73122 (B, C) CaCl 2 and apyrase were present (see Fig 1); PGE 1 (10 l M ) was given
as indicated Calcium responses are shown of single platelets, and are representative for > 50 cells.
Trang 5increased about tenfold when the [Ca2+] was raised from 50
to 200 nM, whereas it declined at [Ca2+] above 400 nM
(Fig 4B) This result thus resembles the biphasic effect of
Ca2+ on InsP3-dependent CICR, previously observed in
preparations from cerebellum, synaptosomes and A7r5
smooth muscle cells [5–7,45], although in the latter
sys-tems higher levels of InsP3 were needed to achieve Ca2+
release Preincubation of platelets with 10 lMPGE1before
permeabilization resulted in a 50% suppression of the
Ca2+-mobilizing effect of InsP3but was without influence
on the biphasic effect of Ca2+ (Fig 4B and Table 2)
Control experiments indicated that PGE1treatment did not
influence the slow Ca2+release evoked by the
endomem-brane Ca2+-ATPase inhibitor thapsigargin (data not
shown) Thus, cAMP elevation seems to partially block
the InsP3receptor channel opening, but not to affect the
sensitization mechanism by Ca2+
Further experiments with permeabilized platelets were
performed under conditions where the Ca2+release process
was most sensitive to modulation, i.e at InsP3 and Ca2+ concentrations of 50 and 110 nM, respectively Thrombin activation of the platelets prior to permeabilization signifi-cantly increased the amount of Ca2+ released by InsP3 (Table 2) This is possibly due to a decrease in the platelet cAMP level caused by this Gi-stimulating agonist [46] Thimerosal and U73122 had very different effects Thim-erosal (10 lM) did not elicit Ca2+ release, but strongly stimulated InsP3-induced Ca2+release (Fig 5A), as repor-ted for hepatocytes and other cells [13,40,41] On the other hand, a high dose of U73122 (10 lM) caused strong release
of Ca2+by itself (Fig 5A), which process was insensitive to pretreatment with heparin or PGE1(Table 2) This U73122 reaction was of little effect on subsequent InsP3-induced
Ca2+release Control experiments showed that the inhib-itory effects of heparin and PGE1on InsP3-evoked Ca2+ release were not influenced by U73122 (Table 2) These data thus suggest that InsP3and U73122 have additive effects on
Ca2+release from intracellular stores
To confirm this, InsP3was applied at a higher, saturating concentration With 1 lM InsP3, increasing [Ca2+] from
100 to 200 nMresulted in a 1.7–fold (± 0.2, n¼ 3) increase
in Ca2+ release; PGE1 pretreatment reduced the Ca2+ release by 45% When given after high InsP3 (1 lM), U73122 (10 lM) still caused a rapid phase of Ca2+release (Fig 5B) This suggested that its effect was mediated by
Ca2+-leak channels different from the InsP3 receptors Thapsigargin was used to determine the possible effect of U73122 on (thapsigargin-releasable) Ca2+ stores [18,27]
In permeabilized platelets, thapsigargin (1 lM) caused a slow but progressive Ca2+ release, when applied either before or after InsP3 However, the release by U73122 was not reduced, but even proceeded faster, after InsP3 /thaps-igargin application (Fig 5B) When applied to suspensions
of intact platelets in EGTA-containing medium, thapsigar-gin caused slow and partial Ca2+release In this system, preincubation with U73122 accelerated and potentiated the
Ca2+release in a similar way as did the InsP3-generating agonist thrombin (Fig 5C) A synergism of thapsigargin-and thrombin-evoked Ca2+mobilization in platelets has
Fig 3 Regulation of thimerosal-induced
spiking in [Ca2+] i Immobilized platelets were
stimulated with 10 l M thimerosal (TMS)
under conditions, as described for Fig 2.
Manoalide (10 l M ) was added at either 5 min
before (A) or 1.5 min after (B) thimerosal In
other experiments, PGE 1 (10 l M ) was added
at 5 min before (C) or 1.5 min after (D)
thimerosal Traces are typical responses from
a single platelet, representative for 50–75
analysed cells.
Table 1 Levels of InsP 3 activated platelets Aspirin-treated platelets
(1 · 10 9 mL)1) in 1 m M CaCl 2 and apyrase remained unstimulated or
were activated with thrombin (10 n M ) or thimerosal (10 l M ) The
platelets were preincubated with U73122 (2 l M ) and/or PGE 1 (10 l M )
for 5 min, where indicated Mass amounts of InsP 3 were determined
after 5 s (thrombin) or 60 s (thimerosal) of activation, i.e when
maximal rises in [Ca2+] i were reached, as measured in parallel
incu-bations Data are mean values ± SEM (n ¼ 4–6) ND, not
deter-mined.
Agonist
InsP 3 (pmol/108platelets)
No pretreatment PGE 1 pretreatment
Thrombin 1.40 ± 0.15 a 0.75 ± 0.04 b
U73122 + thrombin 0.51 ± 0.08c ND
Thimerosal 0.58 ± 0.14 0.56 ± 0.10
a P < 0.005; b P < 0.05 compared to the control condition, i.e no
agonist (t-test, two-sided);cn ¼ 3.
Trang 6been described earlier [18], but this can now be extended to
thapsigargin- and U73122-evoked responses From these
experiments we concluded that InsP3, Ca2+(via CICR) and
U73122 cause additional amounts of Ca2+release both in
intact and permeabilized platelets The sulfhydryl reagent
U73122 seems to release Ca2+from stores that differ from
those used by InsP3, in a way insensitive to heparin and cAMP
Puff-like characteristics of [Ca2+]ispiking in single platelets
To determine the involvement of different Ca2+stores in the [Ca2+]ispiking process in single platelets, we monitored this at higher spatial and temporal resolution A fast confocal fluorescence laser system was used to produce fluorescent images from immobilized Fluo-3-loaded plate-lets at an image resolution of 6.0 pixels per micrometer and
a scanning rate of 10 Hz Because platelets spread on fibrinogen increase in surface area from about 2–4 lm in diameter (thickness of 0.5 lm), this set-up gave image series of 250–450 pixels per platelet We first monitored the characteristics of the Ca2+release events at low agonist conditions, i.e the ÔspontaneousÕ [Ca2+]ispikes that are due
to autocrine produced ADP [35] Quite similar fluctuating patterns in fluorescence were detected in different sub-cellular regions (80–100 pixels) within a single platelet (Fig 6A-B) The fluorescence pattern was completely different in the adjacent region of a nearby platelet, proving that the optical resolution was sufficiently high to detect differences between the selected regions The high temporal resolution allowed precise analysis of the [Ca2+]ispikes The ÔspontaneousÕ peaks arose after long but variable intervals of 15.1 ± 1.6 s (mean ± SEM, n¼ 63) (Fig 6C) The ampli-tudes of the individual peaks were highly variable, but rela-ted to the total peak duration (Fig 6D) When compared to the usual criteria for low-amplitude Ca2+puffs (maximal
Fig 4 InsP 3 -induced CICR in permeabilized platelets (A) Traces of
InsP 3 -induced mobilization of Ca2+ from stores Aspirin-treated
platelets (3 · 10 8 ÆmL)1) permeabilized with saponin in the presence of
Fluo-3, as described in Materials and methods The Ca 2+ level of the
medium was adjusted to 110 n M , InsP 3 was added at 50 or 200 n M
concentrations, heparin (20 lgÆmL)1) was given at 2 min before InsP 3
where indicated (B) InsP 3 –induced Ca 2+ release as a function of
[Ca2+] of the medium Platelets were permeabilized with saponin
at 110 n M Ca2+, after which ATP generation was abolished with
apyrase, and Ca 2+ in the medium was changed to the indicated level
(x axis) The release of Ca2+by 50 n M InsP 3 was measured (y axis).
Before permeabilization, the platelets were treated with 10 l M PGE 1
(open circles) or remained untreated (closed circles) Vertical line is at
standard [Ca2+] of 110 n M Data are from three or more experiments
(mean ± SEM).
(40 n M , 2 min) The platelets were permeabilized with saponin, and [Ca 2+ ] in the medium was adjusted to 110 n M Thimerosal (10 l M ), U73122 (10 l M ) and/or heparin (2 min, 20 lgÆmL)1) were added at
8 min after saponin, as indicated InsP 3 (50 n M ) was given at 10 min after saponin Increases in [Ca 2+ ] were measured in response to the agonist (thrombin, thimerosal or U73122) and InsP 3 The Ca 2+ release
by 50 n M InsP 3 under control conditions (no pretreatment) was taken
as 100% (82 ± 17 n M , equivalent to 27 ± 6 pmol per 108platelets) Data are mean values ± SEM (n ¼ 3–5) ND, not determined.
Agonist
Ca2+release (% of control) Agonist,
no heparin
InsP 3 ,
no heparin
InsP 3
with heparin
Thimerosal 8 ± 4 217 ± 30 a,b 3 ± 2 a
a
P < 0.001 compared to the release by InsP 3 under control con-ditions, i.e no pretreatment/no other agonist (t-test, two-sided).
b Effect of thimerosal was 145 ± 17% of control with 500 instead
of 50 n M InsP 3 c Relative to corresponding control value at
550 n M [Ca2+].dP < 0.01 compared to control conditions.
Trang 7amplitude of < 200 nMand total duration of 1–2 s) [2–4],
many of the low-amplitude Ca2+release events in platelets
(< 200 nM) appear to be of longer duration
The high-resolution confocal scanning revealed irregular
trains of [Ca2+] spikes when the Fluo-3-loaded platelets
were stimulated with thrombin (Fig 7A) Again, no more than minor differences in peak generation were found between different subcellular regions The average peak-to-peak interval was now decreased to 4.8 ± 0.3 s (mean ± SEM, n¼ 67 peaks of 20 cells) This is similar to the highest oscillation frequency reported for ATP-stimulated rat megakaryocytes (peak-to-peak interval per cell varying from 5 to 30 s) [22] After platelet stimulation with thimerosal, again trains of [Ca2+]i peaks started almost simultaneously in various subcellular parts (Fig 7B) With thimerosal, the average peak-to-peak interval was 8.9 ± 0.8 s (mean ± SEM, n¼ 50; P < 0.001 compared
to thrombin) Thus, regardless of the peak generation frequency, individual Ca2+-release events seemed to be generated in various parts of a platelet at quite similar intervals and amplitudes
D I S C U S S I O N
Here we describe that InsP3-mobilizing agonists (thrombin, U46619 and platelet-activating factor) as well as agents acting independently of InsP3 formation (thimerosal and U73122 at 10 lM) evoke irregular [Ca2+]ispiking in aspirin-treated platelets The thrombin-induced spiking appears to
be strictly dependent on InsP3 formation, because it is abolished by manoalide or low U73122 It is also inhibited
by cAMP elevation with PGE1, in part due to reduced InsP3 formation (probably by phospholipase C inhibition) and in part due to decreased InsP3-mediated Ca2+release from intracellular stores On the other hand, the sulfhydryl reagent thimerosal elicits [Ca2+]ispiking not by increasing the InsP3level but by potentiating InsP3receptor-mediated
Ca2+release This may explain why the Ca2+response with thimerosal is only partially inhibitable by PGE1 The N-ethyl maleimide derivative U73122, at a high dose of
10 l , yet acts in a still different manner In permeabilized
Fig 6 Confocal monitoring of ÔspontaneousÕ spiking in [Ca2+] i in spread
platelets Fluorescence changes were monitored by confocal laser
scanning microscopy in aspirin-treated, Fluo-3-loaded platelets spread
on fibrinogen Apyrase was omitted from the incubation medium
(nominally Ca 2+ -free) High-resolution images of 250–450 pixels/
platelet were collected at 10 Hz (A) Fluorescence recordings from
three selected regions of one spread platelet (a-c); and from a region of
interest of an adjacent platelet (r) (initial value of each trace, F/F o ¼ 1).
Insert shows expanded part of curves a–c (B) Selection of regions of
interest of the platelets (areas 0.8 · 2.5 lm) (C) Histogram of
variation in peak-to-peak interval of 15 responsive platelets (D) Plot
of total duration of individual peaks (90% decay) vs peak amplitude.
Data are mean values plus SEM of analysis results from the three
regions per platelet Regression analysis of all data: y ¼ 0.83 + 0.98 x
(R 2
¼ 0.68, P < 0.001).
Fig 5 Calcium mobilization from stores in permeabilized and intact platelets (A,B) Aspirin-treated platelets were permeabilized with saponin in Fluo-3-containing medium After [Ca 2+ ] adjustment to 110 n M , thimerosal (TMS, 10 l M ), U73122 (10 l M ) and thapsigargin (TG, 1 l M ) were given, as indicated (A) InsP 3 was added at a low concentration of 50 n M with 3 · 10 8 plateletÆmL)1(B) InsP 3 was given at a higher concentration (1 l M ), while the platelet concentration was 2 · 10 8
plateletsÆmL)1 Note that U73122-evoked Ca2+release leads to a higher medium [Ca2+], which potentiates the InsP 3 -evoked release (C) Intact, aspirin-treated platelets in suspension (1 · 10 8 plateletsÆmL)1), loaded with Fura-2, were stimulated with thrombin (Thr, 4 n M ), thapsigargin (1 l M ) and/or U73122 (10 l M ) in the presence of 1 m M EGTA.
Trang 8platelets, it causes a cAMP/heparin-insensitive Ca2+leak
that seems to be independent of the InsP3receptor-mediated
Ca2+ release It can thus be envisioned that, in intact
platelets, the Ca2+release evoked by U73122 stimulates the
process of InsP3receptor-mediated CICR, and thereby the
generation of [Ca2+]ispikes
In a variety of cells, thimerosal is known to react with
critical thiol groups controlling InsP3-receptor channel
opening, which results in repetitive Ca2+release at basal
levels of InsP3[13,14,40,41] In platelets sulfhydryl groups
may similarly control InsP3receptor functioning [42] This
agrees with our finding that, in permeabilized platelets,
heparin completely inhibits the thimerosal-enhanced Ca2+
release by InsP3 Taken together, the present work thus
indicated that the platelet InsP3receptors play a key role in
the regenerative, spiking Ca2+release evoked by
phospho-lipase C-stimulating and InsP3receptor-modulating agents,
similarly as established for other cell types
Using saponin-permeabilized platelets, we found that the
InsP3-evoked Ca2+-mobilizing potency changed with the
cytosolic Ca2+concentration in a biphasic way (Fig 6),
similarly as firstly described for neuronal cells [5–7] and later
for pancreatic acinar cells, hepatocytes and smooth muscle
cells [45,47,48] Whereas in many cell types micromolar
concentrations of InsP3were needed to detect a stimulating
effect of Ca2+ on InsP3 receptor-mediated Ca2+release
[5–8,45,47], this could be demonstrated in platelets already
low levels of 50–200 nMInsP3 It is noted that platelets are
relatively rich in type 1 InsP3receptors [26], which are quite
sensitive to Ca2+modulation
For rabbit and mouse pancreatic acinar cells, it has been
shown that U73122 evokes [Ca2+]ioscillations by
potenti-ating the release of Ca2+ from a InsP3-sensitive store
compartment [39,43] This release may lead to increased
Ca2+influx from the external medium and to subsequent
overloading of InsP3-insensitive stores, which in turn can
trigger regenerative Ca2+release [1,43] A similar
mechan-ism, i.e cooperation of store compartments in [Ca2+]i
spiking, may also apply to platelets
Typical for platelets is that the amount of Ca2+released
by a suboptimal InsP3 concentration, but not the Ca2+
sensitivity of the release, is suppressed upon cAMP
eleva-tion There is little doubt that most or all cAMP-mediated
effects in platelets are due to cAMP-dependent protein phosphorylation, and that the platelet InsP3receptors are targets of cAMP-dependent protein kinase [27] Earlier, we have reported that thrombin- and thapsigargin-induced
Ca2+responses in platelets are down-regulated by cAMP analogues and inhibitors of cAMP phosphodiesterase, and that cAMP-dependent protein kinase was important in this effect These cAMP-elevating interventions also suppressed the InsP3-induced Ca2+mobilization in saponin-permeabi-lized platelets [46] Together with the new evidence it thus becomes clear that cAMP-dependent phosphorylation ren-ders the InsP3 receptor less active as a Ca2+ channel [30,31,49], and also that the phosphorylated receptor remains sensitive to changes in [Ca2+]i(this paper) In this respect, platelets differ from other cells such as hepatocytes, where activation of cAMP-dependent kinase was found to increase the amount of Ca2+released by InsP3[41] The confocal laser scanning experiments with Fluo-3-loaded platelets, permitting a simultaneously high tem-poral and spatial resolution of the Ca2+ signal, clearly indicated that the [Ca2+]i release events in platelets are highly irregular in shape, amplitude and frequency, regard-less of whether they are raised by InsP3-generating receptor agonists or sulfhydryl-reactive compounds The experi-ments show that the irregular traces detected in Fura-2-loaded platelets by camera-based microfluorometry are most probably not artefacts of the ratio imaging procedure
In addition, they detect similar Ca2+ release events at distant sites within a platelet: this holds not only for single [Ca2+]i spikes, but also for complex series of consecutive spikes (Figs 6,7) Calcium puffs as recorded in larger cells are commonly defined as single Ca2+ release events that arise due to the action of multiple InsP3receptor channels clustered in functional units [2–4] The operating definitions
of a Ca2+puff vary somewhat, but congregate as a local
Ca2+release event (diameter about 1 lm) with a maximal amplitude of < 200 nM, a rising time of < 0.35 s and total duration of 1–2 s The Ca2+spikes of platelets resemble the puffs seen in larger cells in local appearance, but differ from these in at least two aspects First, the platelet spikes appear
at a variable frequency (0.02–0.3 Hz), regardless of whether CaCl2or EGTA is externally present (see [35]) Second, they are rather broad and do not sum up, i.e the individual
Fig 7 Uniform [Ca2+] i transients within acti-vated, spread platelets Fluo-3-loaded platelets were stimulated with (A) thrombin (4 n M , given at t ¼ 8 s) or (B) thimerosal (10 l M , given at t ¼ 0 s) in the presence of 1 m M
CaCl 2 and apyrase High-resolution images were collected by confocal laser scanning microscopy, as described for Fig 6 Fluores-cence recordings are shown from three non-overlapping regions of one platelet (initial value of each trace, F/F o ¼ 1) Inserts give extended parts Data are representative for 3
or more experiments.
Trang 9events do not seem to be subjected to frequency or
amplitude recruitment, such as described for HeLa cells [4]
Because of the small size of platelets with nearby Ca2+
-ATPases throughout the cell, it is likely that the rate of
Ca2+pumping rather than the diffusion of released Ca2+
into the cytosol (as in bigger cells) determines the duration
of the platelet spikes
In many cell types, the global release of Ca2+ is
controlled by an intimate interplay between
thapsigargin-and InsP3-sensitive Ca2+store compartments For instance,
in rabbit pancreatic acinar cells the (thapsigargin-inhibited)
compensatory Ca2+ pumping by endomembrane Ca2+
-ATPases restricts the Ca2+-store depletion by InsP3 [47]
In mouse lacrimal cells, the thapsigargin-induced Ca2+
mobilization is dependent on the basal level of InsP3and the
InsP3-receptor function [50] Such a situation may also
exists in platelets, where both the InsP3- and
thapsigargin-sensitive Ca2+store compartments are likely to contribute
to the [Ca2+]i spiking [18,27] In the present paper, we
describe that regardless of the type of agonist, stimulating
(thrombin) or sensitizing (thimerosal) InsP3 receptors or
acting primarily independently of InsP3receptors (U73122),
and regardless of the type of stores used by these agonists,
the spiking process was always irregular in amplitude and
frequency and occurred with no more than little subcellular
heterogeneity This situation however, differs from that of
pancreatic acinar cells, where even within the voxel of a
Ca2+Ôhot spotÕ quite different patterns of spike-like events
can be observed [51] This apparently points to a high
cooperation of Ca2+mobilization from the various stores in
platelets to generate smaller as well as larger Ca2+-release
events
In summary, the small platelets forms an attractive model
to study the function of InsP3receptors, even when induced
by agents such as U73122 and thimerosal that do not cause
InsP3formation The platelet InsP3receptors are subjected
to extensive regulation by at least four factors: (a) local
InsP3levels; (b) the potentiating effect of moderate increases
in [Ca2+]i on InsP3 action via CICR; (c) InsP3 receptor
channel sensitization (thimerosal) and desensitization
(mediated by cAMP); and (d) InsP3 channel-independent
Ca2+leak with U73122 Given the importance of the Ca2+
signal for the process of platelet activation, it is likely that
the highly regulated nature of the Ca2+ signal plays an
important role in ensuring rapid platelet deposition at the
right physiological sites during hemostasis and at
athero-sclerotic sites during thrombosis
A C K N O W L E D G E M E N T S
We acknowledge grants from the Netherlands Heart Foundation and
the Netherlands Organization for Scientific Research.
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