© 1996 American Heart Association, Inc.
Articles |
Endothelin-Dependent Actions in Cultured AT-1 Cardiac Myocytes
The Role of the 
Isoform of Protein Kinase C
From the Departments of Medicine (S.F.S.) and Pharmacology (T.J., E.P., H.Z., R.P.K., S.F.S.), Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Medicine and Pharmacology, Department of Medicine, College of Physicians and Surgeons, Columbia University, 630 W 168 St, New York, NY 10032.
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Abstract |
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Abstract The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC)-
immunoreactivity from the soluble to the
particulate cell fraction. The subcellular distributions of PKC
and
PKC
(also expressed by AT-1 cells) are not influenced by endothelin.
Using quantitative fluorescence microscopy with fura 2, we
examined the effects of endothelin on intracellular calcium. In
electrically driven myocytes, endothelin induces a rapid and transient
increase in the amplitude of the calcium transient. This is blocked by
both phorbol 12-myristate 13-acetate (PMA) pretreatment to
downregulate PKC and the PKC inhibitor chelerythrine,
arguing that PKC plays a critical role in endothelin
receptor–dependent increases in intracellular calcium. Endothelin
also stimulates mitogen-activated protein kinase (MAPK).
MAPK activation is markedly attenuated by pretreatment with PMA or
pertussis toxin (PTX, to inactivate susceptible G protein
subunits); it is completely prevented by combined pretreatment with
PMA and PTX. In contrast, it is not attenuated by chelation of
intracellular calcium with BAPTA. These findings indicate that the
pathway for endothelin receptor stimulation of MAPK involves PKC and
PTX-sensitive G protein(s). Thus, these studies identify a functional
role for PKC as a mediator of endothelin receptor–dependent
increases in cytosolic calcium and MAPK activity in AT-1 cells.
Accordingly, the AT-1 cell system should provide a uniquely useful
model to identify the intracellular targets for PKC and investigate
their function in the regulation of intracellular calcium homeostasis
and the induction of the growth response in cardiac myocytes.
Key Words: endothelin • protein kinase C • G proteins • mitogen-activated protein kinase • Ca2+
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Many hormones and neurotransmitters modulate myocyte electrical and contractile function and influence cardiac cell growth via specific interactions with cell surface receptors. Studies of the intracellular signaling mechanisms activated by these receptors have benefited from the availability of primary cultures of neonatal rat ventricular myocytes. In this system, it is evident that several agonists that produce positive chronotropic/inotropic responses and cardiomyocyte hypertrophy stimulate phosphoinositide hydrolysis.1 The phosphoinositide pathway has been linked to activation of PKC, stimulation of MAPK, and induction of early immediate genes. However, despite a large amount of correlative information, there still is substantial uncertainty regarding the role of these intracellular signaling molecules as modulators of cardiac myocyte contractile function and cellular growth. In part, these investigations have been hampered by the absence of a highly differentiated continuous cardiomyocyte cell line that can be produced in large amounts, maintained in culture for prolonged periods of time, and stably genetically manipulated.
The recently developed AT-1 cells may provide a model system suitable
for studying the regulation of intracellular signaling pathways and
their role in the control of cardiac gene expression and the modulation
of cardiac myocyte contractile function. The AT-1 cells are a
transplantable tumor lineage derived from transgenic mouse atrial
cardiomyocytes that express the simian virus 40 large-T
oncogene.2 These cells can be propagated in syngeneic
hosts and retain the capacity to divide in culture while maintaining a
highly differentiated cardiac phenotype. Specifically, cultured
AT-1 cardiomyocytes express adult cardiac-specific
proteins (-myosin heavy chain and -cardiac actin) and
connexin43 (the major protein of the cardiac gap
junction).3 The cells retain ultrastructural
features characteristic of cardiomyocytes, including
well-developed sarcomeres, transverse tubules, and intercalated
disks, as well as characteristic cardiac
electrophysiological properties; the cells
exhibit spontaneous electrical and contractile activity upon reaching
confluence in culture.2 3 4 5
There also is evidence that cultured AT-1 cardiomyocytes
express membrane receptors coupled to intracellular signaling
mechanisms.3 5 6 For example, activation
of muscarinic
cholinergic receptors leads to membrane
hyperpolarization via PTX-sensitive G
protein–dependent stimulation of a potassium
conductance.5 The observations that muscarinic agonists
stimulate phosphoinositide hydrolysis and inhibit
adenylyl cyclase activity in AT-1 cells constitute further evidence
that these cells express functionally active muscarinic cholinergic
receptors.6 Indeed, monoclonal antibodies specific for the
M2-mACh receptor subtype immunoprecipitate muscarinic
cholinergic receptors from these cells.6 AT-1 cells also
appear to express functional endothelin receptors, since endothelin
markedly stimulates secretion of ANF from AT-1 cells.3
However, the intracellular signaling mechanisms activated by
endothelin receptors in AT-1 cells have not been determined.
Accordingly, the goal of the present study was to define the
intracellular signaling pathway(s) activated by endothelin
receptors in AT-1 cells. The results identify a specific role for the
isoform of PKC in endothelin receptor–dependent activation of
MAPK and modulation of contractile function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Preparation of Cultured AT-1 Cells
The AT-1 cell transplantable tumor lineage was maintained by sequentially passaging the cells into syngeneic host mice (B6D2/F1, female, Jackson Labs, Bar Harbor, Me). The original AT-1 mouse colonies were established by Dr Loren Field (Krannert Institute, Indianapolis, Ind) and Dr William Claycomb (Louisiana State University, New Orleans, La). These experiments were performed on a lineage established at Columbia University derived from portions of a tumor cell slurry generously provided by Dr Dan Roden (Vanderbilt University, Nashville, Tenn). To isolate cells from tumor-bearing mice, the animals were anesthetized with isoflurane, and tumors were removed aseptically, minced well, and then further dissociated by gentle agitation for 90 minutes at 37°C in the presence of 150 U/mL Worthington type I collagenase in PBS. The cell suspension was mixed 1:1 with complete medium containing Excell 320 media (JRH Bioscience) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), 5% fetal bovine serum (Sigma Chemical Co), dexamethasone (1 nmol/L), and insulin (16 µg/mL, GIBCO), followed by gentle centrifugation. The cells were washed twice in complete medium, preplated for 30 minutes to decrease contamination by other cells with proliferative capability such as cardiac fibroblasts, and then plated at a density of 5x105 cells per milliliter onto circular fibronectin-coated glass coverslips (thickness, 0.1 mm; diameter, 31 mm; Biophysica Technologies, Inc; for fura 2 fluorescence experiments) or in fibronectin-coated culture dishes or multiwell plates. Cells were fed every 48 to 72 hours. Under these conditions, the primary cultures grow to confluence and usually form a synchronously beating monolayer. Although these cells can be passaged, this study was performed exclusively on primary cultures. Experiments were performed within 6 to 10 days of plating. Portions of minced tumor (0.1 to 0.3 mL) from each preparation also were subcutaneously injected into four separate mice to sustain the lineage. Tumors (5 to 10 g in size) developed within 6 weeks of injection.
Measurement of Inositol Phosphate Accumulation
Inositol
phosphate accumulation was measured according to
standard techniques as described previously.7 Briefly,
AT-1 cells were cultured in multiwell dishes in the presence of 3 µCi
[3H]myoinositol for 48 hours to label membrane
phosphoinositides. The monolayer culture was washed
extensively with HEPES-buffered saline to remove unincorporated
radioisotope. 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 0.7 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 (0.35 mL)
and H2O (0.5 mL) were added, and the mixture was vortexed
and then centrifuged at 1000g for 5 minutes to
separate the phases. The aqueous phase was transferred to Dowex
anion-exchange columns, and inositol phosphates were eluted
sequentially according to standard methods as described
previously.7
PTX-Dependent ADP-Ribosylation of G Proteins
A crude membrane
fraction was prepared by scraping AT-1 cells
into a buffer containing 0.25 mol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L
PMSF, and 50 mmol/L Trizma, pH 7.6, followed by
homogenization with a Polytron tissue
homogenizer and centrifugation at
43 000g for 45 minutes. Membranes were resuspended in 50
mmol/L Trizma buffer (pH 7.6) containing 2 mmol/L MgCl2 and
1 mmol/L EDTA at a concentration of 2 to 3 mg/mL. ADP-ribosylation
assays were performed as follows: membranes were incubated in 20 µL
of a 50 mmol/L Tris-chloride buffer (pH 8.0) containing 2 mmol/L
MgCl2, 1 mmol/L EDTA, 10 mmol/L dithiothreitol,
0.1% Lubrol PX, 10 mmol/L thymidine, 10 µmol/L
[32P]NAD (1.5 µCi per assay), and 20 µg/mL PTX
for 1
hour at 37°C. The reaction was terminated by addition of
SDS-polyacrylamide gel sample buffer and boiling for 5 minutes.
Electrophoresis was performed on vertical slab gels (resolving gel,
12% acrylamide; stacking gel, 3.9%
acrylamide) and was followed by
autoradiography.
Studies of PKC Activation
Soluble and particulate protein
fractions from AT-1 cells were
prepared essentially as described previously.8 Cells were
washed with PBS and then immediately transferred to ice-cold
homogenization buffer (20 mmol/L Tris-HCl, pH 7.5,
2 mmol/L EDTA, 2 mmol/L EGTA, 6 mmol/L ß-mercaptoethanol, 50
µg/mL aprotinin, 48 µg/mL leupeptin, 5 µmol/L pepstatin A, 1
mmol/L PMSF, 0.1 mmol/L sodium vanadate, and 50 mmol/L NaF), lysed by
sonication, and centrifuged at 100 000g for 1 hour.
The supernatant was removed (soluble fraction); 26.8±0.9% of the
total cell protein was recovered in this fraction (n=5). The pellet was
resuspended in homogenization buffer containing 1%
Triton X-100, incubated on ice for 10 minutes, and centrifuged
at 10 000g for 10 minutes at 4°C, and the supernatant was
saved (particulate fraction). Preliminary experiments established that
all PKC isoforms are completely extracted from the pellet by this
protocol. Samples were electrophoresed on an 8%
SDS-polyacrylamide gel and transferred to nitrocellulose.
Prestained molecular weight markers were electrophoresed in parallel.
After an incubation in 5% dry milk, 50 mmol/L Tris, pH 7.5, 200 mmol/L
NaCl, and 0.1% Triton X-100 (blocking buffer I) for 1 hour at room
temperature to block nonspecific binding, the nitrocellulose was probed
with a 1:500 dilution of primary PKC isoform–specific antisera in
3% BSA, 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 0.1% Triton X-100,
and 0.02% NaN3 overnight at 4°C. Five PKC
isoform–specific antisera were used. These antisera were generated
against synthetic peptides corresponding to amino acids 313 to 326 for
PKC, 313 to 329 for PKCß (a sequence common to both splice
variants of PKCß), or unique sequences in the carboxy-terminal
variable region of PKC
, PKC, and PKC. However, it should
be noted that additional atypical isoforms of PKC that are structurally
highly homologous to PKC in the carboxyl-terminal end of the
molecule (PKC
9 and PKC
10 ) also are
recognized by the anti–PKC antiserum.9 10
Thus, the
identification of the protein detected with this antibody as PKC is
tentative at this time. The nitrocellulose was then washed five times,
5 minutes each, with 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, and 2%
Triton X-100 and incubated in the same buffer containing 5% dry milk
(blocking buffer II) for 30 minutes at room temperature. To detect
bound primary antibody, blots were incubated for 1 hour at room
temperature with 125I-labeled goat anti-rabbit IgG
F(ab')2 fragment at a final dilution of 0.25 µCi/mL in
blocking buffer II. The nitrocellulose was washed seven times as
described above, dried, and autoradiographed with Kodak XAR film with
intensifying screens at -70°C.
Assay of MAPK Activity in SDS-Polyacrylamide Gels
Containing MBP
Cells were serum-starved for 2 days before 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 PMSF, and 0.3% [vol/vol]
ß-mercaptoethanol, pH 7.5). Homogenates were
centrifuged for 10 minutes at 10 000g, and then the
supernatants were diluted in SDS-PAGE sample buffer, boiled, and stored
at -70°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 20 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 30°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. Signals also were quantified with
a PhosphorImager 445SI (Molecular Dynamics). Kinases (42 and 44 kD)
that phosphorylate MBP were identified as MAPK on the basis
of their electrophoretic mobilities (immunoprecipitation studies were
not performed), and MAPK activity is reported as the combined
radioactivity in the 42- and 44-kD species.
Measurement of cAMP Accumulation
Intracellular cAMP was
measured essentially as described
previously.7 Briefly, AT-1 cells grown in 15.5-mm
multiwell dishes were preincubated for 60 minutes at room temperature
with 10 mmol/L theophylline. Assays were performed for 5 minutes at
room temperature and were terminated by removal of the incubation
buffer and addition of 0.5 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 -70°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
The method for
photometric measurement of cytosolic calcium in
fura 2–loaded cardiac cells has been published
previously.11 In brief, AT-1 cells cultured 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 37°C. AT-1 cells were rinsed with
fresh Tyrode's solution and maintained for at least 15 minutes at room
temperature before experimental protocols to allow for deesterification
of the dye.
The fluorescence of intracellular fura 2 was monitored with a device that alternately illuminates the cells with 340- and 380-nm light while measuring emission at 520 nm (Photon Technologies, Inc). Sampling rate for collection of ratio values was 100 Hz. Although cytosolic free calcium ion concentration theoretically can be calculated from the fura 2 fluorescence ratio at the two excitation wavelengths, 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.12 13 Because of these unavoidable uncertainties and because fura 2 fluorescence provides an extremely sensitive indicator of relative changes in intracellular calcium during experimental protocols (the primary focus of these studies), intracellular calcium is reported as the fura 2 fluorescence ratio.
Studies were performed using a 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 0.25 Hz using platinum wires embedded in the walls
of the superfusion chamber throughout the experimental protocol. A slow
stimulation rate was used in these studies for two reasons. First, our
preliminary experiments indicated that AT-1 cells maintained at room
temperature display rather slow endogenous spontaneous
beating rates (
10 beats per minute). Therefore, electrical
stimulation rates as slow as 0.25 Hz were sufficiently rapid to capture
and maintain constant beating rates during experimental protocols.
Second, diastolic calcium remains stable in AT-1 cells
stimulated at 0.25 Hz, whereas it tends to increase over time in some
AT-1 cells stimulated at more rapid rates (0.5 to 1 Hz). Accordingly,
the slower stimulation rate provided a more stable preparation to
examine the effects of endothelin on intracellular calcium.
Statistics
For measurements of the amplitude of calcium
transients before
and after drug, six successive transients were superimposed and
averaged. All data represent results of experiments on cells
from at least three separate cultures. Data are presented as
mean±SEM, and statistical comparisons were made using Student's
t test for paired observations with corrections by the
Bonferroni method as indicated. A value of P<.05 was
considered to be statistically significant.
Materials
Endothelin refers to the originally described
porcine/human
endothelin-1 species throughout the manuscript and was purchased from
Sigma. MBP also was purchased from Sigma. Antisera to PKC, PKCß,
PKC, PKC, and PKC were purchased from GIBCO-Life
Technologies,
Inc. TRAP (SFLLRN) was purchased from Bachem. Fura 2-AM and BAPTA-AM
were purchased from Molecular Probes. PMA and chelerythrine were
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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Stimulation of Phosphoinositide Hydrolysis
Stimulation of AT-1 cells with endothelin results in the hydrolysis of membrane phosphoinositides and the accumulation of IP1, IP2, and IP3. The effect of endothelin to stimulate IP1, IP2, and IP3 accumulation is concentration dependent, with an EC50 of
10 nmol/L and a maximum response obtained at 100 nmol/L of the
agonist (Fig 1
). IP3 and IP2
accumulation peak at 5 to 10 minutes (reaching 2.3±0.2- and
3.6±0.2-fold increases, respectively, over the corresponding
controls), whereas the accumulation of IP1 is progressive
for at least 45 minutes (Fig 2). Endothelin is the most
potent activator of phosphoinositide
hydrolysis identified in the present study.
Norepinephrine and the thrombin receptor agonist peptide
(SFLLRN), which are potent activators of
phosphoinositide hydrolysis in neonatal rat
ventricular myocytes,14 15 also stimulate
inositol phosphate accumulation in AT-1 cells. However, the responses
to these other agonists are exceedingly modest compared with the
response evoked by endothelin (Table 1).
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Activation of PKC
The hydrolysis of membrane
phosphoinositides would
be predicted to result in the formation of diacylglycerol, the
endogenous activator of PKC. Therefore, it was
of interest to determine whether endothelin activates PKC in
AT-1 cells. PKC is now recognized to comprise a family of structurally
related serine/threonine protein kinases that play a vital role in
cellular responses to hormones and drugs and in cell proliferation and
differentiation.16 PKCs can be subdivided into three
categories on the basis of their distinct cofactor requirements for
enzymatic activity.16 cPKCs (, ß, and ) are
activated by phosphatidylserine and
DAG/phorbol esters in a calcium-dependent fashion, suggesting a
role for increased cytosolic calcium in the in vivo regulation of cPKC.
In contrast, nPKCs (, , 
/L, and 
) are activated by
phosphatidylserine and DAG/phorbol esters but do
not require calcium for maximal enzymatic activation. Finally, aPKCs
( and /) exhibit distinct structural properties and are not
activated by DAG/phorbol esters; the mechanism for aPKC isoform
activation in vivo remains uncertain. Fig 3 illustrates
that AT-1 cells express conventional (), novel (), and atypical
() isoforms of PKC. PKCß and PKC were not detected in extracts
from three separate AT-1 cell preparations (although PKCß
immunoreactivity was detected in considerable abundance in extracts of
brain and PKC immunoreactivity was detected in considerable
abundance in extracts from neonatal rat heart included in these
experiments as positive controls; data not shown). In unstimulated AT-1
cells, PKC resides exclusively in the soluble fraction, whereas
PKC and PKC are distributed between the soluble and particulate
fractions.
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The effect of phorbol esters and hormones to translocate PKC
from the
soluble to the particulate fraction has been used as an indicator of
PKC activation. The phorbol ester PMA induces the rapid and complete
translocation of PKC and PKC from the soluble to the particulate
fraction of AT-1 cells. Prolonged treatment with PMA (24 hours) results
in the complete loss of PKC immunoreactivity from both the soluble
and particulate compartments and substantial downregulation of PKC.
However, a minor amount of PKC immunoreactivity (20% to 30% of
control levels) persists in the particulate fraction of AT-1 cells
pretreated with PMA for 24 hours. This result is consistent
with previous data in neonatal rat ventricular myocytes,
where PKC was found to be more resistant to PMA-induced
downregulation than was PKC.8 17 The abundance
or
subcellular distribution of PKC was not affected by PMA.
Endothelin, at concentrations that maximally activate
phosphoinositide hydrolysis, also causes the
subcellular redistribution of PKC (Fig 4). The
decrease in PKC immunoreactivity in the soluble fraction is rapid
(it is fully evident as early as 15 seconds after exposure to
endothelin) and sustained for >5 minutes. Endothelin induces a 54±9%
decrease in PKC in the soluble fraction (which contains 25% of
the total cell protein) and a coordinate 28±6% increase in PKC
in
the particulate fraction (which contains approximately twice as much
protein, n=3). Despite the continuous presence of endothelin, the
subcellular distribution of PKC tends to return toward baseline by
30 minutes. In contrast, the subcellular distribution of PKC is not
influenced by endothelin (even at the earliest time points, when
endothelin elevates intracellular calcium; see Fig 5 and
"Modulation of Intracellular Calcium" below). Endothelin also
does not affect the subcellular distribution of PKC (data not
shown). Although there are limited data to suggest that translocation
to the membrane may not be an essential prerequisite for activation of
PKC,18 translocation generally accompanies PKC activation.
Accordingly, these results suggest that endothelin selectively
activates PKC.
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Modulation of Intracellular Calcium
Endothelin
receptor–dependent stimulation of
phosphoinositide hydrolysis has been linked to changes
in intracellular calcium homeostasis in several cell
types.19 20 21 22 Therefore,
we examined whether endothelin
modulates intracellular calcium in AT-1 cells. Initial experiments were
performed in AT-1 cells, which were electrically driven at a basic
cycle length of 4000 milliseconds to maintain a constant beating rate
throughout the exposure to endothelin. Under these conditions,
endothelin induces a rapid and transient increase in
diastolic and peak systolic calcium (4.9±1.2% and
24.4±2.7% over basal, respectively; n=25, P<.05; Fig
5A
and Table 2). This results in an increase in the
amplitude of the calcium transient (43.4±5.7% over basal, n=25,
P<.05) and the appearance of spontaneous
diastolic calcium oscillations in 60% of the
cells studied (Fig 5A). The specificity of endothelin's
actions was
confirmed in control experiments demonstrating that neither
heat-denatured endothelin (100 nmol/L) nor BSA (100 nmol/L)
increases intracellular calcium in AT-1 cells. An effect of endothelin
to induce only a transient increase in intracellular calcium has been
described in other cell
types.19 23 24 25 Of note,
endothelin
also initiates periodic calcium oscillations in human
mesangial cells and vascular smooth muscle
cells.23 24 In this regard, AT-1 cells that displayed
diastolic calcium oscillations started with
significantly higher resting diastolic calcium ratios
(1.05±0.05, before exposure to endothelin) and achieved significantly
higher diastolic calcium ratios at the peak of the
endothelin response (1.12±0.07, n=15) when compared with AT-1
cells
that did not develop diastolic calcium
oscillations in the presence of endothelin (which had a
resting diastolic calcium ratio of 0.91±0.04 and a
diastolic calcium ratio after endothelin exposure of
0.93±0.04, n=10; these values are not significantly different
from
each other but are significantly lower than the values measured in
cells that display diastolic calcium
oscillations [P<.05]). In contrast, there was
no difference in systolic calcium ratios or calcium transient
amplitudes (before or after endothelin) between these groups. Although
this is consistent with the notion that diastolic
calcium oscillations arise as a result of spontaneous
release of calcium from the sarcoplasmic reticulum of
calcium-overloaded cells,26 this requires further
study. Endothelin also increases intracellular calcium in quiescent
AT-1 cells (Fig 5B), indicating that contractile activity is
not a
prerequisite for the endothelin-induced calcium response.
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Two
approaches were used to determine the role of PKC in the sequence
of events that leads to the increase in calcium with endothelin. First,
we took advantage of the actions of PMA, which acutely mimics the
effects of endogenously produced DAG to activate
PKC but, with chronic exposure, leads to the downregulation of PKC and
a concomitant loss of responsiveness to agonists that act through this
pathway. Pretreatment with PMA does not induce any changes in the
baseline characteristics of the calcium transients (Table 2).
However,
the effect of endothelin to increase the amplitude of the calcium
transient is markedly reduced (to 25.2±5.5% of the control response,
n=10), and endothelin never induces spontaneous diastolic
calcium oscillations in PMA-pretreated cells. In separate
experiments, we demonstrated that the PMA pretreatment does not
attenuate endothelin receptor–dependent stimulation of
phosphoinositide hydrolysis, indicating that the effect
of PMA to block the endothelin-dependent increase in cytosolic
calcium cannot be explained by a decrease in cell surface endothelin
receptor expression and/or activation of phospholipase C (data not
shown). As a second approach to determine whether PKC mediates the
endothelin-dependent rise in calcium, we used chelerythrine. This
compound is an inhibitor of the kinase domain of PKC with a
high degree of selectivity for PKC over other kinases.27
Chelerythrine alone does not influence the characteristics of the
calcium transients. However, it completely blocks the subsequent effect
of endothelin to raise intracellular calcium and induce
diastolic calcium oscillations. Taken together,
these experiments indicate that PKC plays a critical role in endothelin
receptor–dependent modulation of intracellular calcium. Given the
evidence that endothelin selectively activates the isoform
of PKC, these results argue that PKC mediates endothelin
receptor–dependent changes in cytosolic calcium.
Activation of MAPK
MAPK comprises a family of
serine/threonine kinases that become
activated in response to a variety of extracellular
stimuli.28 As a result of intense investigation in
numerous laboratories, it is now apparent that MAPKs integrate signals
from G protein–coupled receptors and tyrosine kinase
receptors.29 30 31 In their activated
form, MAPKs
phosphorylate a large array of downstream substrates,
including kinases (S6 kinase) and transcription factors (c-Jun and
c-Myc), thereby playing a pivotal role in signal transduction and
transcriptional regulation.31 32 To determine whether
endothelin activates MAPK in AT-1 cells, we assessed MBP
phosphorylation in cell lysates from
agonist-stimulated cells. By using an assay that measures
"in-the-gel" phosphorylation of MBP
following SDS-PAGE, we could characterize the MAPK species
activated in AT-1 cells. As depicted in Fig 6,
several high molecular weight renaturable proteins
phosphorylate MBP in this assay in a manner that is not
detectably changed by stimulation with endothelin. In contrast, bands
at both 42 and 44 kD, which correspond to the distinct isoforms of
MAPK, selectively increase in intensity upon stimulation with
endothelin. The phosphorylation is dependent on the
presence of MBP; no signal was detected in a control experiment in
which MBP is omitted from the gel (data not shown). Fig 6
illustrates
that endothelin rapidly increases MAPK activity; the increase is
detectable by 2 minutes and is maximal after 5 minutes of stimulation
with endothelin. The increase in MAPK activity is transient and
declines toward baseline values with continuous stimulation by
endothelin.
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Several mechanisms underlying heterotrimeric G
protein–coupled receptor activation of MAPK have been identified.
Gq-coupled receptors activate MAPK via a pathway
that involves the stimulation of phospholipase C and the activation of
PKC. The evidence that the calcium-sensitive PKC has Raf-1
kinase activity33 suggests a pathway involving the
sequential phosphorylation and activation of Raf-1,
MEK, and MAPK. Gi-coupled receptors (including the
receptors for lysophosphatidic acid, thrombin,
2-adrenergic agonists, and muscarinic
agonists34 35 36 37 38 )
also activate MAPK. Recent evidence
indicates that this results, at least in part, from the release of free
Gß subunits, which activate
MAPK39 40 ; whether this is a Ras-dependent or
Ras-independent mechanism is the subject of some
controversy.37 38 39 41
The next series of studies were designed to determine whether PKC plays
a role as a mediator of endothelin-dependent activation of MAPK.
The previous experiments established that prolonged pretreatment with
PMA (24 hours) completely downregulates PKC and results in a
substantial reduction in PKC immunoreactivity in AT-1 cells. Since a
minor amount of PKC persists in cells chronically treated with PMA,
we initially determined whether activation of MAPK through the phorbol
ester–sensitive isoforms of PKC could be completely prevented by
the PMA pretreatment protocol. Fig 7 demonstrates that
this is the case. PMA induces a 2.5±0.9-fold activation of MAPK over
the basal value in control AT-1 cells. The acute effect of PMA to
activate MAPK is abolished in lysates from cells exposed to PMA
for 24 hours, thereby validating the PMA pretreatment protocol as an
approach to investigate the actions of phorbol ester–sensitive
isoforms of PKC. Fig 7 shows that endothelin significantly
activates MAPK in cells pretreated with PMA to downregulate
PKC. However, the response is significantly attenuated. This suggests
that endothelin-dependent activation of MAPK is mediated, in part,
by a phorbol ester–sensitive PKC isoform. However, given the
evidence that the long-term effects of PMA may not be restricted to
downregulation of PKC, additional experiments were performed to
determine whether PMA exerts effects on elements in the signal
transduction cascade apart from PKC. As previously noted, control
experiments established that PMA pretreatment does not interfere with
endothelin receptor–dependent activation of phospholipase C (data
not shown). The decrease in endothelin 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 not significantly affected by the PMA pretreatment
protocol (2.45±0.3- and 2.27±0.3-fold stimulation over basal in
control and PMA-pretreated cultures, respectively; n=4). Taken
together, these results argue that endothelin acts, at least in part,
through a phorbol ester–sensitive PKC isoform to activate
MAPK. Given the evidence that endothelin selectively activates
PKC, these results identify a role for PKC in the signaling
pathway linking the endothelin receptor to activation of MAPK.
|
To
determine whether the pathway coupling the endothelin receptor to
activation of MAPK also involves a PTX-sensitive G protein, we
preincubated AT-1 cells with PTX. Fig 9 illustrates the
in vitro PTX-dependent [32P]ADP-ribose incorporation into
AT-1 cell G protein subunits in membranes from control cultures and
the absence of any [32P]ADP-ribose incorporation into
membranes from AT-1 cells pretreated with PTX. These results indicate
that the PTX pretreatment protocol completely ADP-ribosylates and
inactivates susceptible G protein subunits. Fig 7 shows
that the effect of endothelin to activate MAPK persists in
PTX-pretreated cells but that it is markedly attenuated. Endothelin
receptor–dependent stimulation of inositol phosphate accumulation
is not affected by the PTX pretreatment protocol (data not shown),
arguing that the effect of PTX to attenuate endothelin
receptor–dependent activation of MAPK cannot be explained by a
decreased receptor-dependent activation of phospholipase C.
Similarly, the effect of PMA to activate MAPK also is not
influenced by the PTX pretreatment, indicating that elements in the
signal transduction cascade from PKC to MAPK are intact in
PTX-pretreated cells. Finally, endothelin-dependent activation of
MAPK is totally prevented by the combined pretreatment with PMA and PTX
(Fig 7). Taken together, these results provide evidence that
endothelin
activates MAPK via a PKC-dependent pathway as well as a
PKC-independent signaling mechanism(s) that involves a PTX-sensitive G
protein.
|
There is recent evidence indicating that an increase in
intracellular
calcium, rather than PKC, is critical for receptor-dependent
activation of MAPK in cultured neonatal ventricular
myocytes.30 Since endothelin raises intracellular calcium
via a PKC-dependent mechanism, we considered the possibility that the
reduced MAPK activation by endothelin in PMA-pretreated cells is due to
loss of a calcium-dependent pathway rather than depletion of PKC.
To block the endothelin-dependent rise in intracellular calcium,
cells were incubated with the membrane-permeable acetoxymethyl
ester of the calcium-chelating compound BAPTA (50 µmol/L for 60
minutes). After chelation of intracellular calcium with BAPTA, calcium
transients are not detectable during electrical field stimulation (data
not shown). Chelation of intracellular calcium with BAPTA also
effectively prevents the endothelin-induced increase in
intracellular calcium (Fig 8A) but does not prevent the
endothelin-dependent activation of MAPK, either in control cells or
in cells pretreated with PTX to isolate the PKC-dependent pathway (Fig
8B and 8C). We cannot entirely exclude the
possibility that even in
BAPTA-AM–treated cells, endothelin induces small and localized
increases in intracellular calcium that critically mediate
receptor-dependent activation of MAPK. Nevertheless, these results
are most consistent with the conclusion that the PKC-dependent
pathway for endothelin activation of MAPK is not dependent on a rise in
intracellular calcium. It is noteworthy that chelation of intracellular
calcium with BAPTA also would be anticipated to prevent even a small
increase in the membrane association of PKC that might evade
detection in our assay system. Accordingly, the absence of any effect
of BAPTA to reduce endothelin receptor activation of MAPK provides
further support for the conclusion that the endothelin receptor acts
exclusively via PKC (and not PKC) to activate MAPK.
|
Modulation of cAMP Accumulation
Endothelin has been reported
to modulate intracellular cAMP
through actions at distinct endothelin receptor subtypes in many cell
types including cardiomyocytes.42 43 44
Therefore, we considered it necessary to determine whether the
endothelin receptor modulates intracellular cAMP in AT-1 cells. There
were two reasons for this concern. First, although the mechanism for
the endothelin receptor–dependent increase in intracellular
calcium appears to involve primarily endothelin
receptor-dependent stimulation of
phosphoinositide hydrolysis and activation of PKC, cAMP
plays a central role in the regulation of calcium homeostasis and
contractile function in myocytes. It is possible that a minor component
of the calcium response to endothelin receptor activation could be the
result of a separate pathway involving stimulation of adenylyl cyclase
and elevation of intracellular cAMP. Second, it was necessary to
consider the possibility that endothelin-dependent activation of
MAPK results, in part, from receptor-dependent changes in cAMP,
since cAMP has been reported to act as either a positive or negative
regulator of MAPK in different systems.40 45
Endothelin does not influence basal cAMP accumulation in control
or PTX-pretreated AT-1 cells (basal and endothelin-dependent cAMP
accumulation is 82±12 and 98±24 pmol per 15.5-mm dish,
respectively,
in control AT-1 cells and 102±17 and 125±46 pmol per 15.5-mm
dish,
respectively, in PTX-pretreated AT-1 cells; P=NS; n=5).
Fig 9 demonstrates that endothelin reduces cAMP accumulation in
response to the ß-adrenergic receptor agonist isoproterenol. The
effect of endothelin to reduce hormone-sensitive cAMP
accumulation is abolished by pretreatment with PTX according to a
protocol that completely ADP-ribosylates and inactivates
endogenous PTX-sensitive G-protein subunits. These
results establish that the endothelin receptor couples to inhibition of
cAMP accumulation via a PTX-sensitive G protein in AT-1 cells. A
similar effect of endothelin to inhibit isoproterenol-stimulated
cAMP formation has been observed in myocytes acutely isolated from the
adult rat ventricle44 (although not in myocytes cultured
from the neonatal rat atrium46 ). Of note, the absence of a
change in basal cAMP in response to endothelin argues that the
endothelin receptor elevates intracellular calcium and
activates MAPK via a cAMP-independent mechanism.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
The results of the present study establish that endothelin simultaneously activates several independently regulated signaling pathways in AT-1 cells. The major findings are as follows: (1) Endothelin stimulates phosphoinositide hydrolysis and selectively increases the membrane association of the calcium-insensitive
isoform of
PKC. (2) Endothelin elevates intracellular calcium via a PKC-dependent
pathway. It is important to note that because activation of the
endothelin receptor leads to the selective stimulation of PKC in
AT-1 cells, these studies implicate PKC as the mediator of the
endothelin receptor–dependent increase in cytosolic calcium. (3)
Endothelin activates MAPK via a PKC-dependent pathway as well
as a PKC-independent pathway that involves a PTX-sensitive G protein.
Further studies using BAPTA to chelate intracellular calcium provide
evidence that the PKC-dependent pathway for MAPK activation is directly
mediated by PKC rather than by the calcium-sensitive
PKC–dependent or the PKC–dependent rise in
intracellular
calcium. (4) Endothelin inhibits hormone-sensitive adenylyl cyclase
activity and activates MAPK via a PTX-sensitive G
protein. The effect of endothelin to reduce hormone-sensitive
adenylyl cyclase activity is not likely to contribute importantly
to endothelin receptor–dependent activation of MAPK and elevation
of intracellular calcium, since these responses occur in the absence of
prior hormonal stimulation.
AT-1 cells express both calcium-sensitive and
calcium-insensitive isoforms of PKC. Nevertheless, endothelin
induces the selective membrane association of only the
calcium-insensitive PKC; translocation of PKC cannot be
detected even at early time points, when endothelin markedly elevates
intracellular calcium. This observation contrasts with the recent
findings in noncardiac myocytes, where receptors coupled to
phosphoinositide hydrolysis increase the membrane
association of both cPKC () and nPKC () isoforms.47
In the previous study in other cell types, the rapid and transient
membrane association of PKC requires receptor-dependent
increases in both DAG and intracellular calcium, whereas DAG alone
induces the sustained membrane association of PKC.47
However, results in AT-1 cells are consistent with the recent
observation that endothelin and 1-adrenergic receptor
agonists specifically increase the membrane association of PKC and
PKC, but not PKC, in neonatal rat ventricular
myocytes.17 48 Assuming that translocation to the
membrane
is an essential prerequisite for cPKC activation (as noted, limited
evidence to the contrary has been presented18 ),
studies in cardiac myocytes provide very consistent evidence
that increases in intracellular calcium, associated with
receptor-dependent stimulation of phosphoinositide
hydrolysis, do not lead to activation of calcium-sensitive isoforms
of PKC in cardiac myocytes. The additional evidence that chelation of
intracellular calcium with BAPTA does not interfere with the
PKC-dependent pathway for endothelin receptor activation of MAPK
provides further support for the conclusion that calcium-sensitive
PKC does not play a role in the AT-1 cell response to endothelin.
Taken together, these studies suggest that there are fundamental
differences in the requirements for activation of calcium-sensitive
PKC isoforms between cardiac myocytes (which experience large
beat-to-beat oscillations in intracellular calcium
and therefore may be poorly suited to accommodate a regulatory function
for acute changes in intracellular calcium) and other cell types.
The endothelin receptor activates the 42- and 44-kD isoforms of
MAPK via two independently regulated pathways in AT-1 cells.
Endothelin-dependent activation of MAPK in AT-1 cells is partially
a PKC-dependent response. This is consistent with endothelin's
actions in cultured neonatal rat ventricular
myocytes.29 However, the isoform of PKC linked to the
activation of MAPK in cultured neonatal rat ventricular
myocytes could not be identified unambiguously, since endothelin
activates both PKC and PKC in those cells. In contrast,
AT-1 cells do not express PKC; activation by endothelin is confined
to the isoform of PKC. Thus, these studies in AT-1 cells identify a
link between activation of PKC and stimulation of MAPK. Further
studies suggest that the PKC–dependent pathway might involve the
direct activation of a phosphorylation cascade
involving Raf-1, MEK, and MAPK, since it is not the result of
PKC–dependent changes in intracellular calcium. In this regard,
PKC has been reported to have Raf-1 kinase activity.33
Recent studies in PKC–overproducing fibroblasts suggest that
PKC
might function in a similar fashion, since PKC was shown to function
as an oncogene by acting downstream of the Ras protein and immediately
upstream of the Raf-1 kinase.49 However, this and most
other studies probing the pathways involved in transducing signals from
G protein–coupled receptors to biochemical and functional
responses rely heavily on overexpression models. There is an element of
uncertainty in such models. For example, overexpression of normal or
mutated proteins may induce secondary changes in other elements in the
signaling pathway. Abnormally high levels of protein expression may
lead to promiscuous protein-protein interactions that do not
normally occur in the cell. Overexpression systems also can be
influenced by changes in the ratio of the protein of interest to other
proteins, cofactors, and substrates. In contrast, these studies of AT-1
cells have permitted us to identify PKC function in the context of
the normal signaling machinery of the cell. It is important to note
that these studies are the first to identify PKC function as it
relates to activation of MAPK in cardiac myocytes.
Endothelin receptors also activate MAPK in AT-1 cells through a
pathway that is sensitive to the inhibitory actions of PTX.
To our knowledge, this is the first example of PTX-sensitive G
protein–dependent activation of MAPK in
cardiomyocytes. Our findings are at odds with results in
other cell types where endothelin receptor–dependent activation of
MAPK has been reported to be insensitive to PTX.50 51
However, the accumulating evidence that the precise signaling pathway
leading to activation of MAPK can be influenced by the signaling
machinery available in a particular cell type may provide an
explanation for the discrepant results. At the present time, we
cannot identify the precise PTX-sensitive mechanism linking the
endothelin receptor to activation of MAPK. PTX-dependent release of an
inhibitory effect of endothelin on cAMP is unlikely, since
endothelin does not measurably alter basal cAMP levels in control and
PTX-treated cultures. Rather, a mechanism involving Gi
protein ß dimers, which recently have been shown to
activate MAPK,39 40 is more likely and requires
further study.
Results reported herein establish that endothelin elevates
intracellular calcium in AT-1 cells through a mechanism that involves
the activation of PKC. This conclusion is based on the observations
that endothelin selectively activates PKC and that two
approaches to interfere with signaling through the PKC limb of the
phosphoinositide signaling pathway abolish
(chelerythrine) or markedly attenuate (PMA pretreatment) the
endothelin-induced rise in intracellular calcium. The ability of
the highly selective PKC inhibitor chelerythrine to
completely block the response to endothelin suggests that the residual
component of the endothelin response that persists in PMA-pretreated
cells might be mediated by PKC and/or the minor fraction of PKC,
which is resistant to downregulation by PMA. Although
endothelin also induces a substantial rise in
IP3, there is no evidence that IP3
participates in endothelin-dependent modulation of intracellular
calcium. Absence of a role for IP3 is consistent
with recent evidence that IP3 receptors are expressed only
at very low levels in normal atrial and ventricular
myocardium52 and that the IP3 limb
of the phosphoinositide signaling pathway is not likely
to play a direct role in the physiological
receptor-dependent modulation of calcium homeostasis in
cardiomyocytes.
The PKC-dependent mechanism activated by endothelin, which
leads to a rise in intracellular calcium in AT-1 cells, remains to be
determined. Vigne et al22 have reported that in acutely
isolated rat atrial myocytes, endothelin mobilizes calcium from a
caffeine- and ryanodine-insensitive intracellular pool and promotes
calcium influx through the sarcolemma via a pathway that is not the
voltage-dependent L-type calcium channel. Consistent with
this result, Furukawa et al53 reported that endothelin
enhances calcium entry through T-type (but not L-type) calcium channels
in cultured neonatal rat ventricular myocytes. This
response was dependent on the activation of PKC and could constitute a
candidate mechanism for endothelin's actions in AT-1 cells. Endothelin
also has been reported to stimulate Na+-H+
exchange via a PKC-dependent pathway in acutely isolated adult rat
ventricular myocytes.54 55 This
endothelin-induced increase in Na+-H+
exchanger activity could secondarily elevate intracellular calcium as a
result of reduced calcium efflux or enhanced calcium influx via the
Na+-Ca2+ exchange. However, the effects of
endothelin to activate the Na+-H+
exchanger and enhance inotropy in adult ventricular
myocytes are not likely to be relevant to our observations in AT-1
cells, because their onset is delayed substantially relative to the
response reported herein (ie, they become detectable only after 4 to 6
minutes of continuous exposure to endothelin), they are long lasting,
and they occur without a detectable rise in intracellular
calcium.54 Clearly, the mechanism(s) linking the
endothelin receptor–dependent activation of PKC to the rise in
intracellular calcium in AT-1 cells requires further study. However,
these studies, which are the first to identify a specific role for
PKC in the regulation of cytosolic calcium in cardiac myocytes,
indicate that AT-1 cells constitute a unique model system to identify
the specific intracellular targets for PKC that mediate
receptor-dependent changes in intracellular calcium in cardiac
myocytes.
Our studies of the effect of endothelin on intracellular calcium were not routinely accompanied by simultaneous measurements of contractile function. However, preliminary experiments indicate that the endothelin-dependent elevation in intracellular calcium in AT-1 cells is associated with enhanced contractile motion (data not shown). The rapid and transient increases in intracellular calcium and contractile motion induced by endothelin in AT-1 cells should be considered in the context of previous evidence that endothelin's positive inotropic actions are slow in onset, persistent, and due to sensitization of the myofilaments to intracellular calcium, since they occur with little or no increase in intracellular calcium in ferret papillary muscles and rat ventricular myocytes, respectively.20 54 55 To our knowledge, there are no previous reports describing the effect of endothelin on intracellular calcium and contractile function in (murine) atrial myocytes. Thus, several factors that could contribute to the distinct mechanisms for endothelin-dependent modulation of contractile function observed in this and the previous studies must be considered. Apart from the obvious differences in species (ferret and rat versus mouse) and cell type (ventricle versus atrium), there are substantial differences in experimental protocols used in these studies that may be pertinent. Our experiments, which were confined to measurements of intracellular calcium within the first 3 minutes after the onset of superfusion with endothelin, would not detect an effect of endothelin to increase contractile motion that develops over 4 to 10 minutes and is independent of a rise in intracellular calcium. The possibility that endothelin also modulates contractile function in AT-1 cells via a mechanism that does not require a rise in intracellular calcium will be examined in future studies.
There is no evidence that cAMP plays a role in the endothelin receptor–mediated rise in intracellular calcium. This is not entirely surprising in view of the recent evidence that the SR of AT-1 cells contains a high level of Ca2+-ATPase (SERCA 2) but is strikingly deficient in phospholamban.56 In its dephosphorylated state, phospholamban is the inhibitor of SERCA 2. The phosphorylation of phospholamban by cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase releases the inhibitory effects of phospholamban on SERCA 2 and appears to constitute an important component of the cardiac contractile response to agents that elevate cAMP.57 In this regard, it is noteworthy that AT-1 cells, with reduced levels of phospholamban, display only a very modest increase in intracellular calcium in response to concentrations of isoproterenol, which induce large increases in intracellular cAMP (data not shown). Thus, on the basis of observations that endothelin does not influence basal cAMP accumulation in AT-1 cells and that the isoproterenol-dependent rise in cAMP induces only a modest change in intracellular calcium, we conclude that the endothelin receptor elevates intracellular calcium in AT-1 cells via a mechanism that does not involve cAMP.
To date, the absence of a highly differentiated
cardiomyocyte cell line has limited the scope of studies of
the molecular and biochemical mechanisms regulating signal transduction
process in cardiac myocytes. In this regard, AT-1 cells appear to be a
particularly useful model system, since their phenotype is
quite similar to that of normal mouse atrial cells. Specifically, AT-1
cells retain numerous structural features typical of atrial cardiac
myocytes, including intercalated disks, well-formed myofibrils,
transverse tubules, and atrial-specific cytoplasmic secretory
granules.2 The pattern of gene expression in AT-1 cells
also is quite similar to that observed in adult atrial
myocytes.3 58 In particular, AT-1 cells express only
adult
contractile protein isoforms (ie, -myosin heavy chain and
-cardiac actin). The proliferation of AT-1 cells without
reversion to an embryonic program of contractile protein expression is
noteworthy, since other processes that cause pathological cardiac
growth typically are associated with expression of fetal contractile
protein isoforms (ie, ß-myosin heavy chain and -skeletal
actin59 60 61 ). AT-1 cells also resemble
mature mouse atrial
myocytes in their expression of numerous cardiac muscle–specific
markers (desmin but not vimentin, troponin C, titan, the MM isoform of
creatine phosphate but not the BB isoform of creatine phosphate, and
connexin43), atrial-specific markers (ANF), SR proteins (SERCA 2
and calsequestrin but not phospholamban), and plasma membrane receptors
(-adrenergic, ß-adrenergic, and muscarinic receptors;
Reference 58 and L. Field, personal communication, 1995). However, the
assumption that gene expression in AT-1 cells in all respects resembles
mature atrial myocytes is not likely to be valid, since the pattern of
G-protein subunit expression in AT-1 cells is more similar to that of
neonatal than adult atria (ie, AT-1 cells express
Gs, Gi2,
Go, and Gß, but not
Gi1 or Gi36 ).
Electrophysiological studies indicate that the configuration of the AT-1 cell transmembrane action potential also resembles that of nontransgenic mouse atrial cardiomyocytes, although AT-1 cells are relatively depolarized and exhibit somewhat prolonged action potentials compared with nontransgenic atrial cells.2 5 The relatively limited information on membrane ion channel expression in AT-1 cells4 precludes any precise definition of the molecular basis for the subtle differences in electrical properties between AT-1 cells and nontransgenic atrial myocytes. However, pertinent to the present study, AT-1 cells hyperpolarize in a PTX-sensitive fashion in response to carbachol, providing evidence for potassium channel gating by activated muscarinic receptors typical of nontransgenic atrial myocytes.2 5 The additional observations that muscarinic receptor agonists stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase activity constitute further evidence that important intracellular signaling pathways are preserved in AT-1 cells.6 Thus, by most structural, biochemical, and electrophysiological criteria, AT-1 cells faithfully reproduce the phenotype of adult mouse atrial myocytes.
In summary, this is among the first studies to examine
receptor-activated signal transduction pathways in AT-1
cells. The results indicate that AT-1 cells will provide a useful model
system to investigate the cardiac actions of endothelin and that AT-1
cells also are uniquely suited to isolate and identify the cellular
actions of PKC. Finally, these propagating myocytes should be
amenable to stable genetic manipulations and thereby provide a valuable
resource for investigation of the signaling mechanisms involved in the
regulation of calcium homeostasis and the induction of
proliferative/hypertrophic cardiomyocyte growth.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by US Public Health Service–National Heart, Lung, and Blood Institute grants HL-49537 (Dr Steinberg) and HL-28958 (Dr Steinberg), a National Institutes of Health postdoctoral training grant in pharmacological sciences (grant 07271, Dr Jiang), and the Center for Heart Failure Research.
Received December 7, 1995; accepted February 20, 1996.
|
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