© 1995 American Heart Association, Inc.
Articles |
Platelet-Derived Growth Factor Causes Sustained Depletion of Both Inositol Trisphosphate-Sensitive and Caffeine-Sensitive Intracellular Calcium Stores in Vascular Smooth Muscle Cells
From the Department of Biomedical Engineering (S.A.L., R.D.P.) and the Department of Physiology (R.D.P.), The Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Dr Robert D. Phair, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205.
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Abstract |
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Abstract Since the platelet-derived growth factor (PDGF)–induced increase in cellular inositol 1,4,5-trisphosphate (InsP3) has been found to decay to basal levels soon after the onset of PDGF exposure, it has been argued that activation of Ca2+ release from intracellular stores must be similarly transient. The possibility remains, however, that PDGF-induced release of stored Ca2+ is initiated and sustained by other second-messenger systems. To test the hypothesis that PDGF-BB initiates sustained Ca2+ release from cellular stores, we performed 4-hour 45Ca effluxes on monolayers of A7r5 vascular smooth muscle cells in small, continuously perfused chambers. Isoform PDGF-BB (5 ng/mL for 30 minutes or 30 ng/mL for 15 minutes) was added to the perfusate beginning at 30 minutes of efflux. A dose-related increase in 45Ca release was sustained as long as PDGF-BB was present. Detailed kinetic analysis and nonlinear least-squares fitting of the experimental data revealed that (1) PDGF-BB induced sustained increases of 2.86-fold (5 ng/mL) and 6.50-fold (30 ng/mL) in the rate constant governing Ca2+ release from intracellular stores, (2) the apparent Km for this effect was 13.4±1.31 ng PDGF-BB/mL, and (3) the entire agonist-releasable Ca2+ store (presumably sarcoplasmic reticulum) is sensitive to PDGF-BB. These data indicate that PDGF-BB causes a sustained depletion of intracellular Ca2+ stores by means of sustained activation of Ca2+ release and suggest that intraorganellar Ca2+ may be one of the signals that mediates long-term smooth muscle responses to PDGF.
Key Words: calcium • 45Ca • A7r5 cells • PDGF • tracer kinetics • vascular smooth muscle cells • SAAM
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Introduction |
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Vascular smooth muscle cells (VSMCs) respond to platelet-derived growth factor (PDGF) by initiating a multitude of second-messenger pathways culminating in mitogenesis.1 Two additional long-term responses to PDGF are cell migration and increased secretion of extracellular matrix.2 3 All these effects are prominent features of VSMCs in atherosclerotic plaque,4 5 6 which lends support to the hypothesis that PDGF plays a causal role in some aspects of atherogenesis.
Since these responses are sustained, often for many hours, there must be intracellular signals that are initiated by PDGF and that remain at increased levels for the duration of growth-factor exposure. PDGF triggers transient production of inositol 1,4,5-trisphosphate (InsP3),7 8 thereby releasing calcium from the InsP3-sensitive intracellular store. However, the degradation of InsP3 is apparently rapid, with InsP3 concentration returning to basal levels after 5 minutes of PDGF exposure.8 Therefore, the InsP3-induced increase in cytosolic calcium [Ca2+]i cannot be the mediator of long-term PDGF responses unless the temporary increase in [Ca2+]i reflects triggering of a cellular "switch."
A candidate switch is the calcium content of intracellular organellar Ca2+ stores. Because these stores are membrane bounded and their Ca2+ content is regulated by integral Ca2+ transport proteins, such as Ca2+ ATPase pumps and InsP3 receptors, it is entirely possible to achieve sustained changes in Ca2+ stores accompanied by only transient changes in [Ca2+]i concentration. Under these circumstances, sustained changes in organellar Ca2+ content could invoke sustained cellular responses even though [Ca2+]i concentration has returned to control. For example, sarcoplasmic reticular (SR) Ca2+ (intraluminal) is potentially an important signal regulating protein secretion9 ; decreased SR luminal Ca2+ reportedly causes resident proteins to be secreted.10 In addition, recent work suggests that Ca2+ content of intracellular stores is linked to control of cell growth.11
PDGF is a dimeric protein,2 and all three possible combinations of two homologous chains, A and B, have been isolated.7 12 Recent studies have shown that the three isoforms have different activities.7 8 13 14 15 16 In all cases it has been shown that the PDGF-BB isoform is most potent for stimulating intracellular Ca2+ mobilization. In addition, the PDGF-B type receptor has been found to dominate in VSMCs.14 Therefore, in this study we have used the PDGF-BB isoform.
A recent article17 reports that 2-hour stimulation of VSMCs with PDGF-BB results in sustained activation of Ca2+ entry and extrusion across the plasma membrane. Studies treating cells with PDGF for 24 hours or more demonstrate alterations in protein expression. For example, Masuo et al18 suggest that PDGF negatively regulates functional expression of voltage-dependent, InsP3-mediated, and Ca2+-induced Ca2+ release (CICR) channels in VSMCs. An additional study19 reports PDGF-BB–mediated upregulation of the sarcoendoplasmic reticulum Ca2+-ATPases in VSMCs. A recent report by Cattaneo et al20 provides strong evidence that PDGF discharges and maintains discharge of InsP3-sensitive Ca2+ stores in Swiss 3T3 cells but also suggests that further work is required to distinguish between PDGF actions on organellar Ca2+ uptake and organellar release. The study presented here was designed to quantify acceleration of inhibition of organellar Ca2+ uptake or release that is sustained for the duration of PDGF-BB exposure and that therefore may cause sustained changes in VSMC Ca2+ signaling by producing sustained changes in SR Ca2+ content.
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Methods |
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Cell Culture
Cells from the A7r5 VSMC line, obtained from the American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 9% iron-enriched calf serum (GIBCO catalog No. 16201-022), 4 mmol/L L-glutamine, 1% (vol/vol) penicillin-streptomycin (2.47 g/mL, stock solution concentration), and 1% (vol/vol) Fungizone (0.123 g/mL, stock solution concentration). Every 7 days, the cells were dispersed with medium containing 0.25% trypsin and 1 mmol/L ethylenediaminetetraacetic acid (EDTA) for passage or subculture into the lumen of the efflux chambers. Experiments were performed with a 55% to 85% confluent monolayer of cells between the 5th and 14th days after subculturing into the chamber. For uptake experiments, cells were subcultured into six-well dishes (35-mm wells), and the experiments were carried out on confluent monolayers.
45Ca Efflux Experiments
Cells in the efflux chamber, 30-mm-long segments of glass tubing
with an inside diameter of 4 mm, were made quiescent before the
experiment by replacing the growth medium with DMEM containing 0.5%
iron-enriched calf serum (GIBCO catalog No. 16201-022). After 24-hour
incubation in this medium, the cells were loaded for 1 or 18 hours in
the same medium with 90 Ci/mL 45Ca2+ at
37°C. Extracellular, unbound 45Ca2+
was removed by washing (for 10 seconds) the lumen of the efflux chamber
with 25 mL modified Krebs' solution of the following composition
(mmol/L): 120 NaCl; 4 KCl; 1 KH2PO4; 1
MgSO4; 1.3 CaCl2; 9.22
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid] (HEPES); and 5 glucose (pH 7.4, 20°C). The efflux chamber was
then perfused with this Krebs-HEPES buffer at a constant flow rate of
1.5 mL/min with a peristaltic pump. For the pulse of agonist exposure,
the perfusate was changed to Krebs-HEPES buffer with agonist at the
desired concentration for the duration of the pulse and then back to
Krebs-HEPES buffer without the agonist. The agonists used were PDGF-BB
(UBI), [Arg8]-vasopressin (Sigma Chemical Co),
ryanodine (Calbiochem), and caffeine (Sigma). Effluent was collected
with a fraction collector set to collect 1-minute samples. Efflux
fractions and aliquots of loading and wash solutions were prepared for
liquid scintillation counting. At the end of each experiment, cells
were trypsinized from the efflux chamber and counted with a
hemacytometer. The efflux data were normalized for cell count and
loading solution radioactivity.
45Ca Uptake Experiments
A7r5 cells were grown in six-well dishes and were transferred to
medium containing 0.5% serum 24 hours before the experiment began. At
the start of the experiment, cells were washed three times with
Krebs-HEPES buffer solution (same content as described above). Then
each well was loaded with 1 mL Krebs-HEPES solution containing 21
µCi/mL 45Ca. Designated wells contained 30 ng/mL PDGF-BB
in addition to 45Ca and the Krebs-HEPES buffer; there were
control wells for each experimental time point. The loading solution
was removed at 30, 60, or 120 minutes; then the cells were washed with
Krebs-HEPES buffer for 15 minutes. Finally, the buffer was removed, and
the cells were solubilized with a solution containing 10 mg/mL sodium
dodecyl sulfate and 4 mg/mL EDTA. The solubilized mixture was
transferred to a 20-mL scintillation vial and prepared for
scintillation counting. The data were normalized for cell number on the
basis of confluence of the monolayer and for loading solution
activity.
Kinetic Modeling
We analyzed the data and tested models by using the Simulation
Analysis and Modeling (SAAM) software21 and
its interactive counterpart CONSAM22 running
on an Intel 386 platform with a math coprocessor installed. The model
developed in this report is a modification of our previously published
model23 for rabbit aortic smooth muscle. Since the
present work was carried out on a monolayer of cultured cells, the
compartment representing interstitial space in the published
model was removed.
Experimental protocols, including the 45Ca loading period, were simulated by using each hypothesized model, and the simulated results were compared directly with the experimental data. Modeling the loading period was important both because this determined the starting point for the efflux experiment in terms of 45Ca content of the cells and because the model needed to be general enough to account for experiments with loading periods of different durations.
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Results |
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PDGF-mediated regulation of cellular Ca2+ transport was first examined by measuring PDGF stimulation of 45Ca effluxes. Fig 1
shows experimental data
from two different protocols. The top part of Fig 1 shows a 30-minute
pulse with 5 ng/mL PDGF-BB beginning 30 minutes after the start of the
efflux. The bottom part of Fig 1 shows a 15-minute pulse with 30 ng/mL
PDGF-BB beginning 30 minutes after the start of the efflux experiment.
PDGF-BB caused an increase in Ca2+ release from
intracellular stores; this is observed in the data as the increase in
efflux that begins shortly after the start of PDGF exposure. This
change was not instantaneous; rather, the peak increase in efflux from
the SR was attained after 6 minutes of exposure to PDGF-BB. In
addition, after the removal of PDGF, almost 30 minutes was required for
the release of 45Ca to return to its prestimulus rate. This
is reflected by the slope of the efflux curve returning to its
prestimulus value. These dynamics are consistent with the hypothesis
that a second messenger is produced in response to PDGF exposure and
that this messenger is degraded to allow the Ca2+
release to return to its unstimulated rate. Ca2+
release from A7r5 cells in response to PDGF is dose dependent, as
summarized in Table 1. An off-response observed as a
steep decrease in efflux was evident upon removal of PDGF in every
experiment but is more prominent in experiments performed with lower
doses of PDGF (see Fig 1). This suggests that prolonged exposure to
PDGF causes sustained release from intracellular
Ca2+ stores.
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, n=3) from a 30-minute pulse with 5
ng/mL PDGF-BB beginning 30 minutes after start of efflux. Fractional
SEM is <0.197 of the measured value for 79 of the analyzed time points
and <0.284 for all 86 analyzed points (only two time points were
omitted from least-squares analysis; t=62 minutes and
t=180 minutes). Data are representative of seven
independent experiments. Bottom, Mean data (
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To examine the consequences of prolonged exposure to PDGF-BB,
45Ca uptake experiments were performed. Fig 2 shows the averaged results of total 45Ca
accumulation in the cells. These experiments reinforced the results of
the pulse protocol efflux experiments, which are consistent with the
conclusion that PDGF exposure increases the rate of release of
Ca2+ from internal stores (SR
Ca2+) for the duration of its presence. Even after 2
hours of PDGF exposure, the accumulation of 45Ca in the
PDGF-BB–treated cells was significantly less than in control
cells.
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To gain quantitative insight into the specific calcium transport
processes affected by PDGF-BB, we constructed a compartmental model
describing these processes (Fig 3). The differential
equations corresponding to this model were solved by using
SAAM, as described in "Methods." Putative
compartmental identities are as indicated in Fig 3. Compartments (3),
(4), and (7) are intracellular Ca2+ compartments;
compartment (3) represents cytosolic Ca2+, commonly
designated [Ca2+]i. Compartments
(1), (5), and (6) are the extracellular Ca2+
compartments; compartments (5) and (6) represent
Ca2+ that is bound outside the plasma membrane but
still associated with the cell. Note that the circular compartment is
not a Ca2+ compartment; rather, it
represents a putative second messenger produced in response to
PDGF binding. This messenger was hypothesized to increase the rate
coefficient k(3,4) that governs release of
Ca2+ from the SR [compartment (4)]. To fit the
on- and off-responses of the calcium efflux to PDGF, we modeled the
production of second messenger as having saturable dependence on the
concentration of PDGF-BB. The equation for these saturable kinetics and
its relation to k(3,4) appear in the "Appendix." These
parameters yielded an effective half-life of 11.3 minutes for the
putative second messenger. A simpler model that postulates
instantaneous changes in k(3,4) on addition or removal of
PDGF failed to account for the dynamics observed in the
45Ca efflux curves.
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The rate coefficient k(3,4), which governs the release of
Ca2+ from the SR, thus undergoes a PDGF-dependent
increase that begins with the start of PDGF exposure. Fig 4C and 4D shows the dynamics of this rate coefficient. At its
peak, k(3,4) had increased 2.86-fold in the experiments with
5 ng/mL PDGF-BB and 6.50-fold in the experiments with 30 ng/mL PDGF-BB.
As can be seen in Fig 4, the value of k(3,4) is still
increasing at the end of PDGF exposure but gradually returns to its
prestimulus value on PDGF removal.
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The solid lines in each panel of Fig 1
are the least-squares fit of the
experimental data using the compartmental model shown in Fig 3. Data in
both panels of Fig 1 were fitted simultaneously so that a single model
accounts for both data sets. An important advantage of this approach is
that the same set of model parameters accounts for responses to
different doses of PDGF as well as different durations of PDGF
exposure. Least-squares fitting of the data in Fig 1 resulted in
uniquely defined rate constants with coefficients of variation (CVs)
that are all <0.18 (Table 2). Steady-state prestimulus
Ca2+ content of the individual stores was calculated
by using the control rate constants, and the known extracellular fluid
[Ca2+]; typical values are given in the legend
to Fig 3. The dynamics of compartment (4) (SR Ca2+)
are shown in Fig 4E and 4F to illustrate the net effect of PDGF-BB on
the mass of this Ca2+ store. Note that Fig 4A and 4B
shows the 45Ca content of this same store. Fig 4G and 4H
illustrates the model predictions of
[Ca2+]i content in response to
PDGF-BB.
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It is widely believed that the SR of VSMCs is composed of two Ca2+ stores.26 One store is InsP3-sensitive, and the other is a CICR store that is releasable by caffeine and blocked by high (micromole per liter) concentrations of ryanodine.27 Variable overlap between these stores has also been reported.28 For the compartmental model to be consistent with the 45Ca data, however, it was necessary that both stores be equally sensitive to PDGF. Any model that retained a PDGF-insensitive store predicted 45Ca effluxes that systematically deviated from the experimental data, and the parameters were ill defined, with CV>100%.
To substantiate this interesting theoretical result,
45Ca-loaded cells were pretreated with vasopressin,
ryanodine, or caffeine to release or block the corresponding
Ca2+ stores before pulsing with 30 ng/mL PDGF-BB for
15 minutes, starting at 30 minutes. The results are summarized in Table 1 as the percentage increase in 45Ca peak efflux in
response to PDGF-BB for each protocol. The dose of vasopressin (10
nmol/L) was chosen on the basis of the dose-response curve reported for
A7r5 cells by Blatter and Wier.28 The dose of caffeine (20
mmol/L) was chosen on the basis of preliminary experiments, and the
dose of ryanodine (2 µmol/L) was chosen to inactivate the ryanodine
receptor channel.27
Vasopressin pretreatment blunted the PDGF response compared with
control cells (Table 1). This is consistent with (but does not prove)
the hypothesis that a portion of the Ca2+ released
by PDGF originates in the InsP3-sensitive store since
vasopressin is reported to release Ca2+ by the
InsP3 mechanism.28 29 30 Interestingly, when the
cells were exposed to PDGF-BB in the presence of ryanodine, there was a
significantly increased 45Ca release compared with the PDGF
response with no ryanodine present. This may be due to the
ryanodine block causing a relative accumulation of 45Ca in
the SR and PDGF overriding this block, similar to reports of the
ability of caffeine to override a ryanodine block.31
Pretreatment with caffeine also had the surprising effect of
significantly increasing the subsequent PDGF-induced 45Ca
release; this could be explained if one of the effects of caffeine was
to sensitize CICR channels to Ca2+. However, prior
PDGF exposure significantly reduced (by 37%) the peak increase in
45Ca release when a caffeine pulse was initiated 60 minutes
after the start of efflux (Table 3). These results thus
support the 45Ca efflux results (Fig 1) and suggest that
both the InsP3-sensitive and the CICR intracellular
Ca2+ stores are PDGF-releasable.
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Discussion |
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This study has provided evidence suggesting (1) that PDGF-BB–induced release of Ca2+ from intracellular stores of VSMCs is sustained for the duration of growth-factor exposure, (2) that Ca2+ release is dose dependent and has unique, relatively slow dynamics that are consistent with mediation by a second messenger with an effective half-life of 11.3 minutes, and (3) that the InsP3-sensitive and CICR Ca2+ stores are equally sensitive to release by PDGF-BB. These findings raise the interesting possibility that intraorganellar Ca2+ content plays a role in signaling the long-term cellular responses elicited by PDGF-BB.
The combination of compartmental modeling with least-squares fitting of
the data shown in Fig 1 resulted in a working hypothesis that
PDGF-induced second-messenger production, stimulating SR
Ca2+ release, remains activated for the duration of
growth-factor exposure. This hypothesis is quantitatively consistent
with the data, and the parameters are well defined. The reduced net
uptake of 45Ca shown in Fig 2 after 30, 60, and 120 minutes
of both PDGF-BB and 45Ca exposure is also consistent with
this hypothesis. Although there are only a few studies of the altered
SR Ca2+ content in response to PDGF, one
report20 provides evidence that in Swiss 3T3 cells PDGF
depletes the InsP3-sensitive Ca2+ store
continuously.
Many recent reports12 14 16 20 32 33 34 35 have measured
[Ca2+]i during exposure to PDGF-BB
and have reported a transient increase in
[Ca2+]i, in a variety of
cultured cell lines. Our experiments were performed on A7r5 cells, an
established VSMC line derived from embryonic rat aorta.36
Although the hormonal responses of an established cell line may differ
from whole tissue or primary cells, similar results are reported when
contractile force is measured as an index of
[Ca2+]i,37 38 39
which suggests that our observations are not unique to the A7r5 cell
line. As can be seen in Fig 4G and 4H, a transient increase in
[Ca2+]i as a result of PDGF
treatment is also consistent with our model and our experimental
results. In fact, since the plasma membrane is not the rate-limiting
step in our compartmental model, any increase in SR
Ca2+ release that is resolved in our efflux data
must cause a transient increase in
[Ca2+]i.
Some, but not all, reports demonstrate that the PDGF-induced increase
in [Ca2+]i has two phases: one that
is transient and a second that is sustained, presumably a consequence
of Ca2+ entry from the extracellular
space.20 40 41 Cirillo et al17 report finding
increased calcium fluxes across the plasma membrane in VSMCs exposed to
PDGF for as long as 2 hours, part of which is attributed to
Na-Ca2+ exchange stimulation by PDGF. Another
study42 shows that continuous occupancy of the PDGF
receptor is required for continuous Ca2+ influx
across the plasma membrane. All these studies are consistent with our
premise that for PDGF to elicit long-term responses in cells, there
must be continuous signaling of some kind for the duration of the PDGF
presence. However, our experiments were designed to focus on
intracellular Ca2+ so we have no direct evidence
concerning PDGF-mediated Ca2+ flux across the plasma
membrane. In addition, as shown in Fig 2, we found decreased net uptake
of 45Ca in cells exposed to PDGF-BB. It is possible that
the influx across the plasma membrane is increased, but since PDGF is
continuously releasing calcium from the SR, the ability of this store
to sequester Ca2+ has been reduced, thus resulting
in a lower net uptake of 45Ca for PDGF-treated cells versus
control cells. Another important possibility is that the
"capacitative Ca entry" pathway,43 the increase in
plasma membrane Ca2+ influx signaled by SR
Ca2+ depletion, has been activated by the
PDGF-induced depletion of the SR.
The PDGF dose dependence that we report here is in general agreement
with that reported by others. Cirillo et al17 report a
dose-response curve that is in agreement with our finding of
Km=13.4 ng PDGF-BB/mL. Even studies in
isolated aortic strips37 report effective concentrations
for PDGF-induced contraction (EC50=0.25 nmol/L) and lag
time until the peak effect is reached (6 to 8 minutes) that are in
agreement with our results. Several studies reporting
[Ca2+]i describe the rise in
[Ca2+]i as having a long lag time
relative to other hormones and Ca2+ mobilizing
agents and that the peak increase is not reached until after several
minutes of exposure.32 33 35 This, too, is in close
agreement with our model, which suggests that there is a delay
associated with the accumulation of putative second messenger.
Interestingly, our model predictions for
[Ca2+]i (Fig 4G and 4H) resemble the
data in these published studies.14 20 32 34 An interesting
possibility is that PDGF activates the production of cADP-ribose, thus
initiating release of Ca2+ by means of CICR
channels.44 Another possibility that may account for the
slow time course of the PDGF response may be the time required for the
receptor tyrosine kinase to phosphorylate other targets in a
second-messenger cascade.45 Yet another alternative is
that there is cross talk between the Ca2+
mobilization and the mitogen-activated protein–kinase
cascade44 activated by PDGF.
Another provocative finding of our kinetic analysis was that PDGF
releases Ca2+ from both the InsP3 and
CICR stores. This conclusion has been substantiated by the experimental
data summarized in Tables 1 and 3. Pretreatment with vasopressin
clearly shows a subsequent blunted effect of PDGF. This result
corroborates the results of Powis et al,32 who report no
vasopressin response when fibroblasts are pretreated with PDGF.
The results of caffeine pretreatment are always more difficult to interpret. It is possible that caffeine causes direct sensitization of CICR channels to Ca2+. This might result in PDGF releasing more calcium from the SR in the presence of caffeine. Nevertheless, a pulse with caffeine after a treatment with PDGF results in a reduced peak increase in Ca2+ efflux, which indicates that both agonists are affecting the same store and thus supports the hypothesis that PDGF affects both the InsP3 and CICR stores.
A simpler but less detailed approach to analysis of
45Ca efflux experiments has been developed and applied to
the interpretation of PDGF responses.17 47 48 We have
chosen to model our data with the compartmental model that is
represented in Fig 3 for a number of reasons. First, since
we are interested in discerning between Ca2+
movement across an organellar membrane and Ca2+
movement across the plasma membrane, we explicitly include a cytosolic
compartment. In the approach developed in the laboratory of
Borle,47 48 organellar and cytosolic compartments are
lumped. In addition, since our experiments are carried out for
prolonged periods (4 hours), the very slowly turning over calcium
compartment (presumably the mitochondria) affects the kinetics of the
45Ca efflux. This compartment is absent from the Borle
model because that model was developed for short-term studies. We think
that the ability to differentiate between Ca2+
movement across organellar membranes versus movement across the plasma
membrane is pivotal to the study reported presently. By using
physiological and pharmacological agents, such as vasopressin and
ryanodine, we verify that the compartments we are investigating are the
intracellular Ca2+ stores since extracellular
Ca2+ compartments are unresponsive to these
agents.
This study has demonstrated that PDGF depletes intracellular Ca2+ for the duration of its presence in A7r5 VSMCs. Since there is a growing body of evidence that decreased Ca2+ content of intracellular stores provides signals for increased plasma membrane Ca2+ entry,43 49 50 51 for stimulated release of secretory proteins,9 10 and for control of cell growth,11 our result identifies a new mechanism that may mediate the long-term responses of VSMCs to PDGF.
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Acknowledgments |
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The authors wish to acknowledge Brian M. Gates for collecting some of the data presented in Table 1
and Dr Lynn E. Dobrunz for
critical reading of the manuscript. The authors are also grateful to
Amir Fayazi and Bill K. Huang for helping with the maintenance of cell
cultures and for suggesting the uptake experimental protocol.
The model solutions presented in Figs 1 and 4 were obtained by
postulating a second messenger that is produced in response to PDGF
binding, depicted in Fig 3 as the circular compartment. The dynamics of
this second-messenger subsystem are governed by the following equation,
in which all parameter values were obtained by least-squares fitting of
the experimental data in Fig 1.
 |
where dS/dt is the rate of accumulation of putative second
messenger, S. Vmax, the rate of production of S in the
presence of a saturating concentration of PDGF-BB, is 0.011±0.001.
Km, the Michaelis-Menten constant, is
13.4±1.31 ng PDGF-BB/mL. n, the Hill coefficient, was fixed at 1.5 to
provide the dynamic range required by the data. [PDGF-BB] is the
concentration of growth factor in the perfusate, and
kSM is the first-order rate constant governing
degradation of the putative second messenger (see Table 2).
Ca2+ release from the SR, k(3,4), was
regulated by this putative second messenger as follows.
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where G is a constant of proportionality accounting for the
changes in units; kbasal(3,4), the unstimulated
value of the rate constant (see Table 2). All rate constants have units
of min-1.
Received May 17, 1994; accepted November 3, 1994.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Williams LT. Signal transduction by the platelet-derived growth factor receptor. Science. 1989;243:1564-1570.
2. Ross R, Raines EW, Bowen-Pope DF. The biology of platelet-derived growth factor. Cell. 1986;46:155-169. [Medline] [Order article via Infotrieve]
3. Ross R, Vogel A. The platelet-derived growth factor. Cell. 1978;14:203-210. [Medline] [Order article via Infotrieve]
4.
Stary HC, Blankenhorn DH, Chandler AB, Glagov S,
Richardson M, Rosenfeld ME, Shaffer SA, Schwartz CJ, Wagner WD, Wissler
RW. A definition of the intima of human arteries and of its
atherosclerosis-prone regions. Arterioscler
Thromb. 1992;12:120-134.
5. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
6. Schwartz SM, Liaw L. Growth control and morphogenesis in the development and pathology of arteries. J Cardiovasc Pharmacol. 1993;21(suppl I):S31-S48.
7.
Block LH, Emmons LR, Vogt E, Sachinidis A, Vetter W, Hoppe J.
Ca-channel blockers inhibit the action of recombinant platelet-derived
growth factor in vascular smooth muscle cells. Proc Nat Acad Sci
U S A. 1989;86:2388-2392.
8.
Kondo T, Konishi F, Inui H, Inagami T. Differing signal
transductions elicited by three isoforms of platelet-derived growth
factor in vascular smooth muscle cells. J Biol Chem. 1993;268:4458-4464.
9.
Schwaninger R, Beckers CJM, Balch WE. Sequential
transport of protein between the endoplasmic reticulum and successive
golgi compartments in semi-intact cells. J Biol Chem. 1991;266:13055-13063.
10. Sambrook JF. The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum. Cell. 1990;61:197-199. [Medline] [Order article via Infotrieve]
11.
Short AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, Gill DL.
Intracellular Ca2+ pool content is linked to control
of cell growth. Proc Natl Acad Sci U S A. 1993;90:4986-4990.
12. Szollosi J, Feuerstein BG, Vereb G, Pershadsingh HA, Marton LJ. Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent. Cell Calcium. 1991;12:477-491. [Medline] [Order article via Infotrieve]
13.
Sachinidis A, Locher R, Vetter W, Tatje D, Hoppe J. Different
effects of platelet-derived growth factor isoforms on rat vascular
smooth muscle cells. J Biol Chem. 1990;265:10238-10243.
14.
Diliberto PA, Gordon G, Herman B. Regional and mechanistic
differences in platelet-derived growth factor-isoform-induced
alterations in cytosolic free calcium in porcine vascular smooth muscle
cells. J Biol Chem. 1991;266:12612-12617.
15. Sachinidis A, Locher R, Hoppe J, Vetter W. The platelet-derived growth factor isomers, PDGF-AA, PDGF-AB and PDGF-BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms. FEBS Lett. 1990;275:95-98. [Medline] [Order article via Infotrieve]
16.
Diliberto PA, Gordon GW, Yu C, Earp S, Herman B.
Platelet-derived growth factor (PDGF) alpha-receptor activation
modulates the calcium mobilizing activity of the PDGF beta-receptor in
Balb/c3T3 fibroblasts. J Biol Chem. 1992;267:11888-11897.
17.
Cirillo M, Quinn SJ, Romero JR, Canessa ML. Regulation of
Ca2+ transport by platelet-derived growth factor-BB
in rat vascular smooth muscle cells. Circ Res. 1993;72:847-856.
18.
Masuo M, Toyo-oka T, Shin WS, Sugimoto T. Growth-dependent
alterations of intracellular Ca2+-handling
mechanisms of vascular smooth muscle cells: PDGF negatively regulates
functional expression of voltage-dependent, IP3-mediated,
and Ca2+-induced Ca2+ release
channels. Circ Res. 1991;69:1327-1339.
19.
Magnier C, Papp B, Corvazier E, Bredoux R, Wuytack F,
Eggermont J, Maclouf J, Enouf J. Regulation of sarco-endoplasmic
reticulum Ca2+-ATPases during platelet-derived
growth factor-induced smooth muscle cell proliferation. J Biol
Chem. 1992;267:15808-15815.
20. Cattaneo MG, Magrini L, Sparber SB, Vicentini LM. Interaction between mitogens upon intracellular Ca2+ pools in murine fibroblasts. Cell Calcium. 1992;13:603-614. [Medline] [Order article via Infotrieve]
21. Boston RC, Greif PC, Berman M. Conversational SAAM—an interactive program for kinetic analysis of biological systems. Comput Programs Biomed. 1981;13:111-119. [Medline] [Order article via Infotrieve]
22. Berman M, Weiss MF. SAAM Manual (Simulation Analysis and Modeling). Washington, DC; 1978:78-180.
23.
Phair RD, Hai C-M. Resolution of intracellular calcium
metabolism in intact aortic segments. Circ
Res. 1986;59:74-84.
24.
Nelson MT, Patlak JB, Worley JF, Standen NB. Calcium channels,
potassium channels, and voltage dependence of arterial smooth muscle
tone. Am J Physiol. 1990;259:C3-C18.
25. Robertson SP, Johnson JD, Potter JD. The time-course of Ca-2+ exchange with calmodulin, troponin, parvalbumin and myosin in response to transient increases in Ca-2+. Biophys J. 1981; 34:559-569.
26. Ghosh TK, Mullaney JM, Tarazi FI, Gill DL. GTP-activated communication between distinct inositol 1,4,5-trisphosphate-sensitive and -insensitive calcium pools. Nature. 1989;340:236-239. [Medline] [Order article via Infotrieve]
27. Herrmann-Frank A, Darling E, Meissner G. Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle cell sarcoplasmic reticulum. Eur J Physiol. 1991;418:353-359.[Medline] [Order article via Infotrieve]
28.
Blatter LA, Wier WG. Agonist-induced
[Ca2+]i waves and
Ca2+-induced Ca2+ release in
mammalian vascular smooth muscle cells. Am J Physiol. 1992;263:H576-H586.
29.
Lopez-Rivas A, Mendoza SA, Nanberg E, Sinnett-Smith J.
Ca2+-mobilizing actions of platelet-derived growth
factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse
cells. Proc Natl Acad Sci U S A. 1987;84:5768-5772.
30. Vigne P, Breittmayer J-P, Lazdunski M, Frelin C. The regulation of the cytoplasmic free Ca2+ concentration in aortic smooth muscle cells (A7r5 line) after stimulation by vasopressin and bombesin. Eur J Biochem. 1988;176:47-52. [Medline] [Order article via Infotrieve]
31.
Marban E, Wier WG. Ryanodine as a tool to determine the
contributions of calcium entry and calcium release to the calcium
transient and contraction of cardiac Purkinje fibers.
Circ Res. 1985;56:133-138.
32.
Powis G, Seewald MJ, Sehgal I, Iaizzo PA, Olsen RA.
Platelet-derived growth factor stimulates non-mitochondrial
Ca2+ uptake and inhibits mitogen-induced
Ca2+ signaling in Swiss 3T3 fibroblasts. J
Biol Chem. 1990;265:10266-10273.
33. Bochkov V, Tkachuk V, Buhler F, Resink T. Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF. Biochem Biophys Res Commun. 1992;188:1295-1304. [Medline] [Order article via Infotrieve]
34.
Jacobs LS, Kester M. Sphingolipids as mediators of effects of
platelet-derived growth factor in vascular smooth muscle cells.
Am J Physiol Cell Physiol. 1993;265:C740-C747.
35. Tucker RW, Chang DT, Meade-Cobun K. Effects of platelet-derived growth factor and fibroblast growth factor on free intracellular calcium in mitogenesis. J Cell Biochem. 1988;39:139-151.
36. Kimes BW, Brandt BL. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp Cell Res. 1976; 98:349-366.
37.
Sauro MD, Thomas B. Tyrphostin attenuates platelet-derived
growth factor-induced contraction in aortic smooth muscle through
inhibition of protein tyrosine kinase(s). J Pharmacol Exp
Ther. 1993;267:1119-1125.
38. Bendhack LM, Sharma RV, Bhalla RC. Altered signal transduction in vascular smooth muscle cells of spontaneously hypertensive rats. Hypertension. 1992;19(suppl II):II-142-II-148.
39. Berk BC, Brock TA, Webb RC, Taubman MB, Atkinson WJ, Gimbrone MA, Alexander RW. Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction. J Clin Invest. 1985;75:1083-1086.
40. Mogami H, Kojima I. Stimulation of calcium entry is prerequisite for DNA synthesis induced by platelet-derived growth factor in vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;196:650-658. [Medline] [Order article via Infotrieve]
41. Nishimura J, Kobayashi S, Shikasho T, Kanaide H. Platelet derived growth factor induces c-fos and c-myc mRNA in rat aortic smooth muscle cells in primary culture without elevation of intracellular Ca2+ concentration. Biochem Biophys Res Commun. 1992;188:1198-1204. [Medline] [Order article via Infotrieve]
42.
Huang C-L, Takenawa T, Ives HE. Platelet-derived growth
factor-mediated Ca2+ entry is blocked by antibodies
to phosphatidylinositol 4,5-bisphosphate but does not involve
heparin-sensitive inositol 1,4,5-trisphosphate receptors. J Biol
Chem. 1991;266:4045-4048.
43. Putney JW Jr. Capacitative calcium entry revisited. Cell Calcium. 1990;11:611-624. [Medline] [Order article via Infotrieve]
44.
Galione A. Cyclic ADP-ribose: a new way to control calcium.
Science. 1993;259:325-326.
45. Gips SJ, Kandzari DE, Goldschmidt-Clermont PJ. Growth factor receptors, phospholipases, phospholipid kinases and actin reorganization. Semin Cell Biol. 1994;5:201-208. [Medline] [Order article via Infotrieve]
46.
Pelech SL, Sanghera JS. MAP kinases: charting the regulatory
pathways. Science. 1992;257:1355-1356.
47. Borle AB, Uchikawa T, Anderson JH. Computer simulation and interpretation of 45-Ca efflux profiles. J Membr Biol. 1982;68:37-46. [Medline] [Order article via Infotrieve]
48. Borle AB. Measurement of calcium movement across membranes: kinetic analysis and conceptualization. In: Bronner F, ed. Intracellular Calcium Regulation. New York, NY: Alan R. Liss: 1990;19-75.
49. Randriamampita C, Tsien RY. Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature. 1993;364:809-814. [Medline] [Order article via Infotrieve]
50. Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature. 1992;355:353-356. [Medline] [Order article via Infotrieve]
51. Putney JW Jr, Bird G St J. The signal for capacitative calcium entry. Cell. 1993;75:199-201. [Medline] [Order article via Infotrieve]
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