© 1996 American Heart Association, Inc.
Articles |
Thrombin Receptor Actions in Neonatal Rat Ventricular Myocytes
From the Departments of Medicine (S.F.S.) and Pharmacology (T.J., V.K., E.P., H.Z., R.B.R., S.F.S.), Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Medicine and Pharmacology, Department of Medicine, Columbia University, College of Physicians and Surgeons, 630 West 168 St, New York, NY 10032.
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Abstract |
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Abstract Previous studies established that thrombin stimulates phosphoinositide hydrolysis and modulates contractile function in neonatal rat ventricular myocytes. The present study further defines the signaling pathways activated by the thrombin receptor and their role in thrombin's actions in cardiac myocytes. The thrombin receptor–derived agonist peptide (TRAP, a portion of the tethered ligand created by thrombin's proteolytic activity) stimulates the rapid and transient accumulation of inositol bis- and tris-phosphates (IP2 and IP3, respectively), which is followed by the more gradual and sustained accumulation of inositol monophosphate (IP1). TRAP elicits a larger and more sustained accumulation of IP1 than does thrombin. Thrombin and TRAP also activate mitogen-activated protein kinase (MAPK) in cultured neonatal rat ventricular myocytes. Differences in the kinetics and magnitude of thrombin- and TRAP-dependent inositol phosphate (IP) accumulation are paralleled by differences in the kinetics and magnitude of thrombin- and TRAP-dependent activation of MAPK. Pretreatment with phorbol 12-myristate 13-acetate (PMA) to downregulate protein kinase C (PKC) attenuates thrombin- and TRAP-dependent activation of MAPK, although small and equivalent effects of thrombin and TRAP to stimulate MAPK persist in PMA-pretreated cells. These results support the notion that the thrombin receptor activates MAPK through PKC-dependent and PKC-independent pathways and that the incremental activation of MAPK by TRAP over that induced by thrombin is the consequence of enhanced activation through the PKC limb of the phosphoinositide lipid pathway. TRAP also increases the beating rate of spontaneously contracting ventricular myocytes and elevates cytosolic calcium in myocytes electrically driven at a constant basic cycle length. The effects of TRAP to modulate contractile function and elevate intracellular calcium are not inhibited by tricyclodecan-9-yl-xanthogenate (D609, to block TRAP-dependent IP accumulation) or pretreatment with PMA (to downregulate PKC). The TRAP-dependent rise in intracellular calcium also is not inhibited by verapamil or removal of extracellular calcium but is markedly attenuated by depletion of sarcoplasmic reticular calcium stores by caffeine. Patch-clamp experiments demonstrate that TRAP elevates intracellular calcium in cells held at a membrane potential of -70 mV. Taken together, these results support the conclusion that the thrombin receptor modulates contractile function by mobilizing intracellular calcium through an IP3-independent mechanism and that this response does not require activation of voltage-gated ion channels.
Key Words: thrombin receptors • inositol phosphates • mitogen-activated protein kinase • Ca2+ • automaticity
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Thrombin is a multifunctional serine protease that is formed at the site of vascular injury and activates a variety of cellular events important in hemostasis as well as the inflammatory and proliferative responses. Thrombin evokes multiple biological responses in many cell types, including platelets, endothelial cells, smooth muscle cells, and fibroblasts.1 2 3 4 5 6 7 These responses result from activation of the cell surface thrombin receptor, which couples via heterotrimeric G proteins to traditional intracellular effector response mechanisms, including PLC, PLA2, and adenylyl cyclase.5 8 9 10 Thrombin also increases tyrosine phosphorylation,2 5 11 activates MAPK,12 13 and stimulates AP-1 transcriptional activity.14 However, the precise mechanism by which thrombin receptors couple to these responses remains unknown.
Recent studies by Coughlin and colleagues15 have provided critical insights into the mechanism for thrombin-dependent cellular activation. Using molecular cloning techniques, these investigators have identified a receptor for thrombin that is a single polypeptide chain having seven predicted membrane-spanning domains and a long extracellular amino-terminal extension that contains a thrombin cleavage site.15 16 These structural features suggested that the thrombin receptor resembles other G protein–coupled receptors but has a unique activation mechanism. Studies in several cell systems support a model in which thrombin binds to and cleaves the N-terminal ectodomain of its receptor, thereby revealing a new N-terminal sequence that serves as a "tethered ligand." In particular, thrombin's cellular actions require a proteolytically active form of thrombin,17 and synthetic peptides corresponding to the N-terminal tetradecapeptide newly exposed by thrombin mimic thrombin's actions in many cell types.5 8 17
We previously demonstrated that thrombin stimulates phosphoinositide hydrolysis, elevates cytosolic calcium ion concentration, increases automaticity, prolongs repolarization, and enhances afterdepolarizations in cardiac myocytes.18 Thrombin also induces several of the characteristics of the cardiac hypertrophic phenotype, including increased cell size, enhanced sarcomeric organization, and increased atrial natriuretic factor expression.19 In view of recent insights into the mechanism by which thrombin activates cells, we initiated studies with TRAP. The goals of the present study were (1) to determine whether the thrombin receptor can be implicated in the rapid contractile response to thrombin in cardiac myocytes, (2) to determine whether the hypertrophic stimuli thrombin and TRAP activate MAPK in cardiac myocytes, and (3) to examine the role of second messenger molecules generated via the phosphoinositide pathway in thrombin receptor–dependent responses in cardiomyocytes.
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Materials and Methods |
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Preparation of Cultured Neonatal Rat Ventricular Myocytes
Cardiac myocytes were isolated from the ventricles of 2-day-old Wistar rats by a trypsin dispersion procedure and were cultured for 5 days in the presence of 10% fetal calf serum according to a protocol described previously.20 For experiments measuring phosphoinositide hydrolysis, MAPK activation, and intracellular cAMP accumulation, cells were grown in multiwell tissue culture dishes. Measurements of cell shortening and cytosolic calcium were performed on myocytes grown on round fibronectin-coated glass coverslips (0.1-mm thickness, 31-mm diameter; Biophysica Technologies, Inc) in 35-mm culture dishes. Although the culture technique includes a preplating step that effectively decreases fibroblast contamination, it is well known that a small number of cells with proliferative capability such as cardiac fibroblasts persist in the myocardial cell cultures. Proliferation of these cells was further curtailed with an irradiation protocol.18
Measurement of IP Accumulation
IP accumulation was measured
according to standard techniques as
described previously.18 Briefly, neonatal myocytes were
cultured in multiwell (6x35-mm) dishes in the presence of 3 µCi
[3H]myoinositol for 72 hours to label membrane
phosphoinositides. The monolayer culture was washed
extensively with HEPES-buffered saline to remove unincorporated
radioisotopes. After a 10-minute preincubation in the presence of 10
mmol/L LiCl, experimental protocols were initiated by the addition of 1
mL HEPES-buffered saline containing 10 mmol/L LiCl and the indicated
test agents. After incubation for the indicated time intervals at room
temperature, the buffer was rapidly aspirated, and 1.5 mL acidified
chloroform/methanol/6 mol/L HCl (500:1000:3) was added to the cell
monolayer. Cells were harvested with a rubber policeman, and lipids
were extracted for 30 minutes at room temperature. Chloroform (0.5 mL)
and H2O (1 mL) were added, and the mixture was vortexed and
then centrifuged at 1000g for 2 minutes to separate
the phases. The aqueous phase was transferred to Dowex
anion-exchange columns, and IPs were eluted sequentially according
to standard methods.21 Results are reported as counts per
minute (with background radioactivity subtracted).
Measurements of MAPK Activity in SDS-Polyacrylamide Gels
Containing MBP
Cells were maintained in serum-free medium (1:1
DMEM/F-12
medium, GIBCO BRL) supplemented as described by Mohamed et
al22 for 4 days for studies of MAPK activation. After
exposure of cells to agonists for the times indicated, cells were
washed three times in ice-cold calcium/magnesium-free
Dulbecco's PBS and then scraped into ice-cold extraction buffer
(20 mmol/L ß-glycerophosphate, 20 mmol/L NaF, 2 mmol/L EDTA, 0.2
mmol/L Na3VO4, 10 µg/mL aprotinin, 25
µg/mL leupeptin, 50 µg/mL phenylmethanesulfonyl fluoride,
and 0.3% [vol/vol] ß-mercaptoethanol, pH 7.5).
Homogenates were centrifuged for 10 minutes at
10 000g, and the supernatants were diluted in SDS-PAGE
sample buffer, boiled, and stored at -80°C for subsequent assay
of MAPK activity. Cell extracts were resolved on 10%
SDS-polyacrylamide gels containing 0.5 mg/mL MBP. After
electrophoresis, SDS was removed from the gel by washing the gels three
times for 20 minutes each with 20% (vol/vol) 2-propanol in 50 mmol/L
Tris-HCl, pH 8.0, followed by three additional washes (20 minutes each)
with 5 mmol/L ß-mercaptoethanol in 50 mmol/L Tris-HCl, pH 8.0.
Proteins were denatured by treating the gels twice (30 minutes each)
with 50 mL of 6 mol/L guanidine HCl, 5 mmol/L ß-mercaptoethanol,
and 50 mmol/L Tris-HCl, pH 8.0, and then renatured by washing overnight
at 4°C in five changes of 50 mmol/L Tris-HCl, pH 8.0, containing
0.04% (vol/vol) Tween 40 and 5 mmol/L ß-mercaptoethanol. After
preincubation of the gels at 20°C for 1 hour in 40 mmol/L HEPES, 2
mmol/L dithiothreitol, and 10 mmol/L MgCl2, pH 8.0,
in situ phosphorylation of MBP was carried out by
incubating the gels at 20°C for 1 hour in 15 mL of 40 mmol/L HEPES,
0.5 mmol/L EGTA, 10 mmol/L MgCl2, and 50 µmol/L
[
-32P]ATP (5 µCi/mL, 25 µCi per gel), pH
8.0. The
reaction was stopped by washing the gels in a 5% trichloroacetic acid
solution containing 10 mmol/L sodium pyrophosphate. The buffer was
changed repeatedly until the radioactivity of the solution was
equivalent to background, and the gel was dried and then subjected to
autoradiography. The combined intensities of the
signals derived from the 42- and 44-kD MAPK species were quantified
using a PhosphorImager 445SI (Molecular Dynamics).
Measurement of cAMP Accumulation
Intracellular cAMP was
measured essentially as described
previously.18 Briefly, neonatal myocytes grown in 22.1-mm
multiwell dishes were preincubated for 60 minutes at room temperature
with 10 mmol/L theophylline. Where indicated, 100 µmol/L D609 was
added to the cells 12 hours before the agonist. Assays were performed
for 5 minutes at room temperature and were terminated by removal of the
incubation buffer and addition of 1 mL ethanol. Each condition was
performed on two wells and was assayed for cAMP in quadruplicate. The
alcohol-fixed cell extract was boiled for 3 minutes, cooled,
brought to original volume with ethanol, and stored at -80°C.
Aliquots of the supernatant were dried under a stream of nitrogen, and
cAMP in the residue was determined using a radioimmunoassay (New
England Nuclear/Du Pont Co).
Measurement of Cytosolic Free Calcium and Cell
Shortening
Methods for the photometric measurement of cytosolic
calcium in
fura 2–loaded cultured neonatal ventricular myocytes have
been previously published.18 In brief, neonatal myocyte
monolayer cultures on a coverslip were loaded with fura 2 by incubation
in Tyrode's solution containing 3 µmol/L of the acetoxymethyl ester
form of fura 2 (fura 2-AM) and 1.5 µL/mL of 25% (wt/vol in dimethyl
sulfoxide) Pluronic F-127 (BASF Wyandotte Corp) for 20 minutes at room
temperature. Myocytes were rinsed with fresh Tyrode's solution and
maintained for at least 15 minutes at room temperature to allow for
deesterification of the dye.
The device used for monitoring the fluorescence of intracellular fura 2 (Photon Technologies, Inc) alternately illuminates the cells with 340- and 380-nm light while measuring emission at 520 nm. The sampling rate for collection of ratio values was 100 Hz. Cytosolic free calcium ion concentration theoretically can be calculated from the fura 2 fluorescence ratio at the two excitation wavelengths. Although numerous approaches to calibrate intracellular fura 2 have been presented, it is extremely difficult to entirely circumvent uncertainties in the calibration because of the potential compartmentalization of the dye in fura 2-AM–loaded cells, differences in the spectral properties of fura 2 in cells and in buffer solutions, and fluorescence changes due to cell contracture during calibration protocols.23 24 Because of these unavoidable uncertainties and because fura 2 fluorescence provides an extremely sensitive indicator of changes in intracellular calcium during experimental protocols (the primary focus of these studies), we have reported intracellular calcium as the fura 2 fluorescence ratio.
Glass beads (2.1±0.5 µm, Duke Scientific Corp) were added to the neonatal monolayer cultures to provide high-contrast spots for tracking cell motion. To monitor cell motion, the cells were simultaneously illuminated with red light, and a dichroic mirror (630-nm cutoff) in the emission path deflected the cell image to a video optical system (Crescent Electronics), which tracked motion of the high-contrast microsphere attached to the myocyte surface along a raster line segment of the image during electrically stimulated contractions. The analogue voltage output from the motion detector was calibrated to convert to microns of motion. The motion signal was obtained at a rate of 60 Hz and reflected the motion of the same myocyte simultaneously monitored with fura 2 for calcium. The signal was digitized and stored along with the fluorescence data. Measurements of changes in cell length are not possible in neonatal monolayer cultures, because these cells lack a single axis of myofibrillar alignment. Therefore, these studies on individual cultured neonatal myocytes report cell shortening.
Studies
were performed using a three-compartment superfusion
chamber modified so that a coverslip formed the bottom of the chamber.
The chamber was placed on the stage of a Zeiss inverted microscope, and
the cells were visualized with a x40 oil immersion Neofluor objective.
The cells were superfused with Tyrode's solution gassed with 95%
O2/5% CO2 at a rate of 1 mL/min.
Experiments were performed at room temperature. Where indicated,
myocytes were paced by electrical field stimulation at 1 Hz using
platinum wires embedded in the walls of the superfusion chamber
throughout the experimental protocol to avoid changes in cell calcium
or shortening due to the chronotropic actions of TRAP. Myocytes were
exposed to a bolus of agonist by introducing 50 µL of superfusion
buffer containing five times the desired final concentration of drug
into the initial prechamber. The fluid rapidly mixes with the inflow
stream and thereby is diluted as it enters the main 1x1-cm central
compartment of the chamber, which is designed to maintain a constant
volume of 
250 µL. In preliminary experiments, we established that
vehicle, delivered in an identical fashion, does not affect the rate,
calcium transient, or twitch. This protocol does not permit
measurements under steady state conditions but provides the means to
rapidly deliver agonist to the myocytes in the experimental
chamber.
Electrophysiological
Recording Methods
For combined patch-clamp and fura 2 measurements,
5-day-old monolayer cultures were resuspended by a 1- to 3-minute
exposure to 0.1% trypsin, preplated for 45 minutes to reduce
fibroblast contamination, and then replated onto protamine
sulfate–coated round coverslips. They were studied 2 to 8 hours
later, by which time they had reattached and formed single isolated
spheres. A coverslip of cells was loaded with fura 2 by incubation in
Tyrode's solution containing 3 µmol/L of fura 2-AM and 1.5 µL/mL
of 25% (wt/vol in dimethyl sulfoxide) Pluronic F-127 for 20 minutes at
room temperature and placed in the superfusion chamber described above.
Voltage clamp was carried out using the perforated-patch
method25 to minimize disruption of cytosolic components.
The pore-forming antibiotic nystatin (Sigma Chemical Co) was
dissolved in dimethyl sulfoxide at 50 mg/mL, added to the pipette
solution (mmol/L: potassium glutamate 120, KCl 20, MgCl2 1,
Na2ATP 4, EGTA 0.5, mannitol 25, and HEPES 10, pH 7.2) at a
final concentration of 200 to 250 µg/mL, and dissolved by
ultrasonication. The entire pipette, including the tip, was filled with
the nystatin-containing solution.26 Pipette resistance
in external solution was 2 to 4 M
.
After forming a seal against
the cell, pores typically started to form
within 5 minutes. Series resistance during the experiment was typically
20 to 40 M. Voltage clamp was carried out using an Axopatch 200
amplifier, Digidata 1200 A/D interface, and Axotape 2.0 software (Axon
Instruments) running on a 486 computer. The current record was
simultaneously digitized on the second computer running the
PTI software and stored along with the fluorescence data.
Current records were normalized by cell capacitance, as measured by
the capacity compensation circuit of the amplifier.
The cell was
initially held at a potential of -70 mV. At the
start of data acquisition, a brief depolarization to 0 mV was imposed
to confirm that the particular cell being studied could generate a
voltage-dependent calcium transient. The cell was then maintained
at -70 mV for the duration of the experiment. Holding current and
fura 2 ratio were measured during superfusion with control Tyrode's
solution for 2 minutes, followed by solution containing 300 µmol/L
TRAP for 30 to 45 seconds, and then returned to control solution.
Statistics
For measurements of intracellular calcium and cell
motion, six
successive transients were superimposed and averaged. Amplitude and
width at half-maximal amplitude were measured for both the calcium
and motion transients. For studies of calcium and contractile function,
all data represent results of experiments on cells from at
least two separate cultures. For studies of IP and cAMP accumulation,
experiments were performed in triplicate. All biochemical studies were
replicated at least twice on different myocyte preparations. Data are
presented as mean±SEM, and statistical comparisons were made
using Student's t test for paired observations or ANOVA for
multiple comparisons as indicated. Significance was defined at the
P<.05 level.
Materials
TRAP (SFLLRN) was purchased from Bachem. Thrombin
was purchased
from Sigma. D609 was purchased from Kamiya Biomedical Co. Fura 2-AM was
purchased from Molecular Probes. PMA was purchased from LC Services.
All other chemicals were reagent grade and were obtained from standard
chemical suppliers.
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Results |
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Biochemical Signaling Mechanisms Activated by the Thrombin Receptor
Our previous studies established that thrombin stimulates a rapid and transient accumulation of IP3 and IP2, which is followed by a more gradual and sustained accumulation of IP1 in cultured neonatal rat ventricular myocytes.18 Fig 1
illustrates that this action is mimicked by TRAP.
IP1, IP2, and IP3
accumulation in response to TRAP is time dependent. The peaks in
IP2 and IP3 accumulation occur within the first
minute. Both in magnitude and kinetics, these responses are similar to
those induced by thrombin. Although the effects of thrombin and TRAP to
elevate IP2 and IP3 appear modest, the
agonist-induced increases in radioactivity in these fractions
represent twofold to fivefold increases over the very low
radioactivity recovered in these fractions in basal unstimulated cells.
At early time points (during the first 5 minutes), TRAP also elicits an
increase in IP1 accumulation that is similar in magnitude
to the response induced by thrombin. However, in contrast to the
response to thrombin, which subsequently wanes, prolonged exposure to
TRAP results in a large increase in IP1 accumulation, which
is progressive for at least 60 minutes. The effect of TRAP to stimulate
the formation of IP1, IP2, and
IP3 is concentration dependent, with an EC50 of
20 µmol/L (Fig 2).
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It recently has become
evident that a variety of extracellular stimuli
that activate the phosphoinositide signaling
pathway and induce hypertrophy in cardiac myocytes
activate MAPK,27 28 29 a family of
serine/threonine
kinases that play a pivotal role in signal transduction and
transcriptional regulation by phosphorylating a large array of
downstream substrates including kinases (S6 kinase) and transcription
factors (c-Jun,
c-Myc).29 30 31 To
determine whether thrombin and/or TRAP-dependent activation of
phosphoinositide hydrolysis also is associated with the
activation of MAPK in cardiac myocytes, we assessed
"in-the-gel" MBP phosphorylation in
lysates from agonist-stimulated cells. Fig 3
illustrates the effects of thrombin and TRAP to activate MAPK
at agonist concentrations that lead to equivalent initial stimulation
of phosphoinositide hydrolysis (1 U/mL and 300
µmol/L, respectively). In each case, MAPK species with molecular
masses of 42 and 44 kD are detectably activated by 1 minute and
maximally activated by 5 minutes. Other higher molecular weight
renaturable proteins also phosphorylate MBP in this assay,
but the intensity of MBP phosphorylation by these
proteins is not detectably changed by exposure to thrombin or TRAP. The
phosphorylation is dependent upon the presence of MBP;
no signal was detected in a control experiment in which MBP was omitted
from the gel (data not shown). Of note, the effect of TRAP to
activate MAPK is consistently larger and more
persistent than the response elicited by thrombin.
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Given the importance
of the PLC/PKC pathway as a mechanism for
heterotrimeric G protein–coupled receptor activation of MAPK, we
postulated that the incremental activation of MAPK by TRAP over that
induced by thrombin might be the consequence of the more robust and
persistent activation of PLC by TRAP than by thrombin. To investigate
the role of PKC as a mediator of thrombin- and TRAP-dependent
activation of MAPK, we took advantage of the actions of the
tumor-promoting phorbol ester PMA. This substance acutely mimics
the effects of endogenously produced diacylglycerol to
activate PKC but, with chronic exposure, leads to the
downregulation of phorbol ester–sensitive isoforms of PKC and a
concomitant loss of responsiveness to agonist activation of this
pathway. Previous studies from our laboratory established that cultured
neonatal rat ventricular myocytes express conventional
(
) and novel (
and 
) isoforms of PKC, which are completely
(PKC and PLC) or substantially (PKC, 80%) depleted from
the cell after prolonged treatment with PMA (24 hours).32
Fig 4 illustrates that PMA activates MAPK in
control cells, and this response is virtually abolished in cells
exposed to PMA for 24 hours, providing confirmation that activation
through the phorbol ester–sensitive isoforms of PKC is prevented
by the PMA pretreatment protocol. Pretreatment with PMA to downregulate
PKC also markedly attenuates both TRAP- and thrombin-dependent
activation of MAPK. The effect of PMA to block thrombin
receptor–dependent activation of MAPK in cardiac myocytes cannot
be explained by a decrease in cell surface thrombin receptor expression
(as has been shown to occur in other cell systems33 ),
since IP accumulations in response to TRAP and thrombin are not
significantly affected by the PMA pretreatment protocol (data not
shown). The decrease in thrombin receptor–dependent activation of
MAPK in cells chronically treated with PMA also is not due to loss of
functional MAPK molecules, since the effect of fibroblast growth factor
to activate MAPK through a receptor tyrosine kinase is
equivalent in control and PMA-pretreated cells (5.16±0.45- and
4.90±0.57-fold over basal, respectively). Finally, although
pretreatment with PMA for 24 hours virtually abolishes subsequent
PMA-dependent activation of MAPK, small but statistically significant
effects of TRAP and thrombin to activate MAPK persist in
PMA-pretreated cells; the magnitude of the residual activation of MAPK
by TRAP and thrombin in PMA-pretreated cells does not differ. Taken
together, these results provide evidence that TRAP and thrombin
activate MAPK via PKC-dependent and PKC-independent signaling
mechanisms and that the PKC-dependent pathway for activation of MAPK is
more effectively activated by TRAP than by thrombin.
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Cellular Actions of TRAP
The functional consequences of
myocyte stimulation with TRAP were
explored. TRAP profoundly increases the spontaneous beating rate of
cultured neonatal rat ventricular myocytes. Fig 5 illustrates a
typical experiment depicting the
increased automaticity in response to 300 µmol/L TRAP, and the
complete data are summarized in Fig 6. The effect of
TRAP to increase the spontaneous beating rate is concentration
dependent (EC50, 30 µmol/L; maximal response,
300 µmol/L) and more rapid as the concentration of agonist is
increased. Recovery from these effects of TRAP occurs within 3 to 4
minutes of washout. These effects of TRAP to modulate spontaneous
automaticity are equivalent to the actions previously reported for
thrombin.18
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), 30 µmol/L (
), and 300
µmol/L (
) (mean±SEM, n=4 for each).
In most cells (70%), the effect of TRAP to
modulate the contractile
rate precedes the rise in intracellular calcium ion concentration (see
Fig 5
). This difference in the kinetics of onset of these
responses
could indicate that the effect of TRAP to increase cytosolic calcium is
secondary to an increase in contractile rate. Alternatively, it is
possible that TRAP increases intracellular calcium and enhances
automaticity through independent mechanisms. To unambiguously determine
whether TRAP exerts a direct effect on cellular calcium
metabolism, independent of its effect to increase
contractile rate, these experiments were repeated in cells electrically
driven at a basic cycle length of 1000 milliseconds. This rate is
sufficiently rapid to maintain a constant beating rate during exposure
to TRAP. Fig 7 illustrates a typical experiment
demonstrating that TRAP induces a rapid and striking increase in both
diastolic and peak systolic calcium ion
concentration in electrically driven myocytes. TRAP-dependent increases
in intracellular calcium also are observed in cells driven at faster
and slower rates (basic cycle lengths, 500 and 2000 milliseconds; data
not shown). The increase in intracellular calcium is dose dependent, is
completely reversed during a 3- to 4-minute washout (as was previously
reported for thrombin18 ), and is not accompanied by any
significant change in the kinetics of the calcium transient (Table
1). Moreover, although TRAP markedly increases the
amplitude of the calcium transient, there are no associated changes in
the amplitude or the kinetics of the contraction (Table 1). In
fact,
the amplitude of cell shortening tends to decrease with more prolonged
exposure to TRAP (see Fig 7, bottom panel, tracing c), although
this
decrease is not statistically significant. This result is surprising,
since a rise in intracellular calcium generally results in increased
contractile function. Indeed, Table 2 demonstrates that
even modest increases in the amplitude of the intracellular calcium
transient induced by stepwise increments in extracellular calcium are
associated with substantial increases in the amplitude of the motion
transient. Taken together, the data presented in Tables 1 and
2
indicate that TRAP alters the relationship between the amplitude of the
calcium transient and the amplitude of the motion transient in neonatal
myocytes.
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Role of the Phosphoinositide Signaling Pathway in
the Contractile Response to TRAP
Receptor-dependent activation of
phosphoinositide hydrolysis is associated with changes
in cardiac contractile function. However, the precise role of second
messenger molecules generated by this pathway in the modulation of
contractile function has been very difficult to determine. Accordingly,
we used two approaches to investigate the role of the
phosphoinositide signaling cascade in the cellular
actions of TRAP. First, cells were pretreated for 24 hours with PMA to
downregulate PKC. Table 3 shows that pretreatment with
PMA does not attenuate the effect of TRAP to increase the spontaneous
contractile rate and to elevate cytosolic calcium. These results
suggest that the effects of TRAP to influence contractile function are
not mediated by phorbol ester–sensitive isoforms of PKC.
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As a
second strategy to investigate the role of the
phosphoinositide signaling pathway in the cellular
response to TRAP, we used the antiviral and antitumoral xanthate
compound D609, which has been shown to effectively inhibit PLC
activity.34 35 This alternative approach to block the
PKC
limb of the phosphoinositide signaling pathway was
required, since the long-term effects of PMA may not be limited to
downregulation of PKC. Moreover, by inhibiting PLC activity, D609
broadens the assessment to include the functional role of the
IP3 limb of the phosphoinositide signaling
pathway in the cellular response to thrombin receptor activation. Fig
8A verifies that D609 effectively inhibits
TRAP-dependent stimulation of IP accumulation. In separate experiments,
we demonstrated that ß-adrenergic receptor–dependent
stimulation of cAMP accumulation is similar in control and D609-treated
cells (207.6±25.5 and 240.4±30.7 pmol cAMP per well over the
corresponding controls, respectively; n=3). These results demonstrating
that the effect of the ß-adrenergic receptor agonist
isoproterenol to stimulate cAMP accumulation is completely spared by
the xanthate compound establish that the inhibitory effect
of D609 is specific for the phosphoinositide signaling
pathway. Fig 8B and 8C demonstrates that under
conditions in which
TRAP-dependent IP accumulation is profoundly inhibited by D609, the
effect of TRAP to increase automaticity in spontaneous myocytes and
increase cytosolic calcium in electrically driven myocytes persists.
These results provide evidence that TRAP enhances automaticity and
increases cytosolic calcium through a mechanism that is not dependent
upon the action of any phosphoinositide-derived
second messenger molecules.
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Role of Voltage-Sensitive Calcium Channels in the Calcium Response
to TRAP
Thrombin has been shown to stimulate calcium entry through
voltage-dependent calcium channels in frog myocytes,36
guinea pig myocytes,37 and rat portal vein smooth muscle
cells.38 To examine the possibility that the TRAP-induced
rise in cytosolic calcium is due to the activation of
voltage-sensitive calcium channels, myocytes were treated with the
calcium channel antagonist verapamil, and the
effect of TRAP was evaluated. Fig 9, top, illustrates
that verapamil markedly attenuates electrically stimulated
calcium transients but does not prevent the TRAP-induced rise in
cytosolic calcium. Electrically stimulated calcium transients were
completely abolished when cells were exposed to verapamil
in a superfusion buffer devoid of any added calcium, but the
TRAP-dependent rise in cytosolic calcium persisted (Fig 9,
middle). In
contrast, when caffeine was used to deplete intracellular calcium
stores, the TRAP-dependent increase in cytosolic calcium was markedly
attenuated (Fig 9, bottom). These results are most consistent
with the conclusion that the thrombin receptor–dependent rise in
calcium predominantly reflects the release of calcium from
intracellular stores.
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Perforated-patch voltage-clamp experiments were
carried out to
further probe the mechanism for thrombin receptor–dependent
modulation of calcium in cardiac myocytes. Fig 10
illustrates that even when the membrane potential is held at -70
mV to prevent activation of voltage-gated ion channels, TRAP still
induces a transient elevation of intracellular calcium. In three of
four cells, the elevation in calcium was associated with a measurable
increase in inward current (-5.22±1.11 pA/pF; mean capacitance,
27±4 pF). Of note, the one cell without a detectable change in current
in association with the rise in intracellular calcium had the smallest
increase in intracellular calcium. To determine whether the inward
current observed in these experiments is an initiating event or is the
response to the rise in calcium (eg, Na+-Ca2+
exchange current), two cells were treated with caffeine to deplete
intracellular calcium stores. Under these conditions, TRAP did not
induce any detectable changes in calcium or holding current,
consistent with the observed current being a secondary
event.
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Taken together, these results provide evidence that TRAP stimulates the release of calcium from intracellular stores. Although a critical role for transsarcolemmal influx of calcium to trigger intracellular calcium release cannot be excluded, the data demonstrating that the effect of TRAP to elevate intracellular calcium can occur in the presence of verapamil or when cells are held at -70 mV argue that the increase in calcium does not require activation of a voltage-gated calcium current.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Results of the present study establish that the thrombin receptor agonist peptide TRAP mimics thrombin's actions to stimulate phosphoinositide hydrolysis, activate MAPK, enhance automaticity, and elevate intracellular calcium in neonatal rat ventricular myocytes. These findings implicate the thrombin receptor, recently identified in cultured neonatal rat ventricular myocytes,19 in thrombin's cellular actions in these cells. These studies also provide insights into the role of second messenger molecules generated via the phosphoinositide pathway in thrombin receptor–dependent signaling in the heart. Results reported herein indicate that the thrombin receptor–dependent activation of MAPK is mediated, in part, by diacylglycerol activation of PKC. In contrast, phosphoinositide-derived second messenger molecules appear to play no role in thrombin receptor–dependent modulation of cardiac contractile function.
In our attempt to define the biochemical events regulated by the thrombin receptor, we observed that the nature of the thrombin receptor activation mechanism profoundly influences thrombin receptor coupling to downstream biochemical signaling cascades. Proteolytic activation of the thrombin receptor by thrombin results in the rapid and transient stimulation of phosphoinositide hydrolysis and activation of MAPK. In contrast, activation of the thrombin receptor by peptide agonist (TRAP) leads to a more robust and sustained stimulation of phosphoinositide hydrolysis and activation of MAPK. The similar kinetics for thrombin receptor–dependent stimulation of phosphoinositide hydrolysis and activation of MAPK suggests a relationship between these responses. This is supported by the observation that the incremental activation of MAPK by TRAP is abolished by pretreatment with PMA to downregulate phorbol ester–sensitive isoforms of PKC. Although the PKC-dependent pathway for MAPK activation appears to be more effectively activated by TRAP than by thrombin, both agonists have a similar (albeit smaller) effect to activate MAPK in PMA-pretreated cells. This suggests that an additional (more minor) PKC-independent pathway for MAPK stimulation is activated similarly by thrombin and TRAP. This is not surprising, given the evidence from several laboratories that the phosphoinositide signaling mechanism is one of several converging signal transduction pathways activated by thrombin. For example, in nonmyocytes thrombin stimulates tyrosine phosphorylation of various proteins,2 5 11 including the GAP protein, which activates Ras.2 Functional Ras, in turn, has been shown to be required for thrombin-induced mitogenesis.39 40 In cardiac myocytes, thrombin receptor activation induces hypertrophy and augments the expression of atrial natriuretic factor (one of a group of embryonic genes induced during myocyte hypertrophy) through a mechanism that appears to involve both PKC and protein tyrosine kinases.19 Thus, studies in diverse cell types are consistent with the notion that the integrated response to thrombin is dependent upon multiple converging signaling pathways.12 In this context, one might anticipate that the more robust and sustained activation of phosphoinositide hydrolysis and MAPK by TRAP than by thrombin would be associated with important functional consequences. However, both thrombin and TRAP have been reported to be equivalently effective inducers of atrial natriuretic factor expression in cultured cardiac myocytes.19 Nevertheless, the MAPK cascade has been postulated to play an important role in the hypertrophic response of cardiac myocytes, and the possibility that the dissimilar patterns of MAPK activation by TRAP and thrombin lead to distinct hypertrophic responses requires further study.
We also have attempted to elucidate the precise signaling mechanism(s) by which the thrombin receptor modulates cardiac contractile function. Our initial studies demonstrated that the kinetics of thrombin- and TRAP-dependent stimulation of phosphoinositide hydrolysis differ, whereas the kinetics of the automaticity and/or calcium responses to these agonists cannot be distinguished. Although this could suggest that the biochemical and functional responses are unrelated, this type of a comparison likely is not entirely valid; the biochemical experiments were performed on cells incubated in the presence of agonist for prolonged intervals, whereas measurements of calcium and contractile function were performed under conditions in which the cells were subjected to a rapid and transient bolus of agonist. Therefore, additional studies were required to determine whether the phosphoinositide signaling pathway mediates thrombin receptor–dependent changes in calcium and contractile function. There are several lines of evidence arguing that activation of the PLC/PKC pathway is neither necessary nor sufficient for thrombin-induced modulation of contractile function. First, prolonged treatment with PMA, which downregulates most isoforms of PKC, does not attenuate the contractile response to TRAP. Second, the effect of TRAP to stimulate the biochemical response and to modulate contractile function can be dissociated using D609. Third, norepinephrine is an even more robust and persistent activator of phosphoinositide hydrolysis than is thrombin but induces only a relatively minor increase in automaticity and does not increase intracellular calcium in these cells.41 Taken together, these results are the first to convincingly argue that second messenger molecules generated via phosphoinositide hydrolysis do not play an essential role in the rapid thrombin receptor–induced changes in cardiac contractile function. In view of previous evidence that thrombin increases calcium channel current in cardiac myocytes36 and smooth muscle cells38 and prolongs the duration of the action potential of Purkinje myocytes through a pathway that is sensitive to disruption by the calcium channel antagonist nisoldipine,18 we also considered the possibility that thrombin modulates contractile function by directly triggering the activation of a voltage-sensitive calcium channel. However, results reported herein establish that an influx of extracellular calcium through voltage-dependent calcium channels is not essential for the thrombin-dependent rise in cytosolic calcium (although we have not completely ruled out the possibility that such an action could contribute to the overall response to TRAP in actively beating myocytes). It is also noteworthy that when caffeine is used to prevent the TRAP-induced release of calcium from the sarcoplasmic reticulum, the effect of TRAP to increase the net inward current also is lost. This suggests that a net ion flux via a voltage-independent pathway (eg, a ligand-gated ion channel) does not initiate the TRAP-dependent rise in calcium. Thus, the thrombin receptor modulates contractile function through a mechanism that is distinct from the phosphoinositide signaling cascade and activation of a voltage-sensitive ion channel. The nature of the stimulus that links thrombin receptor activation to changes in contractile function remains elusive. It is possible that a signaling molecule such as phosphatidic acid, which has been reported to mobilize intracellular calcium42 and could be formed in D609-treated cells as a result of receptor-dependent hydrolysis of phosphatidylcholine by PLD,43 plays a role in this process. In this regard, cultured neonatal myocytes will provide a particularly useful model for future investigations designed to define the potential role of this and other signaling processes in the events that bridge the gap between thrombin receptor activation and changes in contractile function.
TRAP increases the amplitude of the calcium transient without increasing the amplitude of cell shortening in cultured neonatal rat ventricular myocytes. These results could suggest that TRAP activates a mechanism(s) that depresses myofibrillar calcium sensitivity. However, an alternative interpretation of the results is possible. Since cultured myocytes form a sheet of interconnecting cells that lack a single major axis of myofibrillar alignment, measurements of resting cell length are not possible. Therefore, the amplitude of cell shortening can be influenced by changes in contractile function during systole as well as diastolic contracture due to severe calcium overload. In contrast, the rod-shaped geometry of adult rat ventricular myocytes permits measurements of actual cell length at rest and during contraction and are well suited to distinguish these processes. Although we recognize that age-dependent differences in the contractile response to TRAP may exist and confound the interpretation of these studies, we have initiated studies on adult ventricular myocytes for this purpose. Recent experiments indicate that TRAP increases twitch amplitude in adult rat ventricular myocytes (authors' unpublished data, 1995). Assuming that similar mechanisms are operative in cultured neonatal myocytes, these data suggest that activation of the thrombin receptor leads to a positive inotropic response but that the magnitude of this response may be blunted in myocyte cultures due to concomitant changes in diastolic cell length.
It is difficult to envision a mechanism whereby thrombin would come into direct contact with myocardial cells and influence contractile function in the normal heart. We previously speculated that thrombin might assume importance as a humoral mediator of electrophysiological events in the setting of hemorrhagic infarction where the endothelial barrier is broken.18 However, Goldstein et al44 recently presented evidence that acute ischemia induced by thrombotic coronary occlusions results in a higher incidence of malignant ventricular arrhythmias than does nonthrombotic balloon occlusion, despite equivalent amounts of jeopardized myocardium. The implication of these studies is that factors derived from the thrombus or induced during thrombotic occlusion could be arrhythmogenic, even in the absence of direct hemorrhage into the myocardium. The notion that thrombin, produced during coronary thrombosis, directly influences myocytes in the ischemic regions of the heart is particularly intriguing in view of our recent finding that the actions of thrombin to stimulate phosphoinositide hydrolysis and enhance IP3 accumulation are exaggerated in hypoxic myocytes.45 Whether this translates into exaggerated contractile responses to thrombin in hypoxic myocytes remains to be determined. Experimental evidence that the direct and exaggerated effects of thrombin on the ischemic myocardium contribute importantly to the genesis of malignant arrhythmias would provide an important rationale for the development of therapeutic interventions designed to block thrombin's actions at its receptor on cardiac myocytes.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by US Public Health Service–National Heart, Lung, and Blood Institute grants HL-49537 and HL-43731.
Received October 25, 1995; accepted December 15, 1995.
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References |
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