From the Department of Pharmacology, Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854
Protein synthesis in H9c2 ventricular myocytes
was subject to rapid inhibition by agents that release
Ca2+ from the sarcoplasmic/endoplasmic reticulum,
including thapsigargin, ionomycin, caffeine, and arginine vasopressin.
Inhibitions were attributable to the suppression of translational
initiation and were coupled to the mobilization of cell-associated
Ca2+ and the phosphorylation of eIF2
. Ionomycin and
thapsigargin produced relatively stringent degrees of Ca2+
mobilization that produced an endoplasmic reticulum (ER) stress response. Translational recovery was associated with the induction of
ER chaperones and resistance to translational inhibition by Ca2+-mobilizing agents. Vasopressin at physiologic
concentrations mobilized 60% of cell-associated Ca2+ and
decreased protein synthesis by 50% within 20-30 min. The inhibition
of protein synthesis was exerted through an interaction at the V1
vascular receptor, was imposed at physiologic extracellular Ca2+ concentrations, and became refractory to hormonal
washout within 10 min of treatment. Inhibition was found to attenuate
after 30 min, with full recovery occurring in 2 h. Translational
recovery did not involve an ER stress response but rather was derived
from the partial repletion of intracellular Ca2+ stores.
Longer exposures to vasopressin were invariably accompanied by
increased rates of protein synthesis. Translational inhibition by
vasopressin, but not by Ca2+-mobilizing drugs, was both
preventable and reversible by treatment with phorbol ester, which
reduced the extent of Ca2+ mobilization occurring in
response to the hormone. Larger and more prolonged translational
inhibitions occurred after down-regulation of protein kinase C. This
report provides the first compelling evidence that hormonally induced
mobilization of sarcoplasmic/endoplasmic reticulum Ca2+
stores is regulatory upon mRNA translation.
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INTRODUCTION |
Ca2+ sequestered by the endoplasmic reticulum
(ER)1 is essential for
optimal rates of protein synthesis occurring in nucleated mammalian
cells (reviewed in Ref. 1). This control involves coupling of the rates
of mRNA translation to those of protein translocation into the
organelle for subsequent processing or folding. Depletion of ER
sequestered Ca2+ to slow glycoprotein processing and
transport competence, or introduction of a reducing environment to
suppress ER processing of proteins with disulfide cross-links, results
in the activation of the double-stranded RNA-activated protein kinase,
the phosphorylation of eIF2
, the inhibition of eIF2B, and the
slowing of translational initiation (2-6). An adaptive response,
characterized by translational accommodation to continued depletion of
ER Ca2+ stores by drugs such as ionomycin or thapsigargin
or to the continued presence of a thiol-reducing agent, occurs over
several hours. This adaptive response is dependent on increased
expression of the ER resident chaperone, GRP78/BiP (1, 4, 7, 8). Inductions of this chaperone and of recovery from translational inhibition depend on activation of grp78 transcription and,
in some cell types, a growth-promoting factor. The acute suppression of
translational initiation by agents that inhibit ER protein processing,
the induction of the ER chaperones GRP78 and GRP94, and the recovery of
translational activity are characteristic of the "ER stress
response" (1, 9, 10). Translational suppression, however, is not
required for expression of the later events. Both GRP78 and
translational tolerance can be induced by Ca2+-mobilizing
or thiol-reducing agents at concentrations that do not suppress protein
synthesis (7).
The physiologic significance of translational suppression by conditions
provoking the ER stress response is unclear. However, Ca2+-mobilizing hormones, including epinephrine,
angiotensin, and vasopressin, have been found to slow protein synthesis
over several minutes in isolated hepatocytes (11-13). Inhibitions were
reduced during incubations in Ca2+-depleted media and
overturned at supraphysiologic Ca2+ concentrations (11).
Corresponding reductions were observed in the polysomal contents of
excised portions of perfused rat liver in response to hormones and
manipulations of the Ca2+ content of the perfusing medium
(14). It was proposed that hormonally induced changes in intracellular
Ca2+ homeostasis provide a mechanism for regulating the
rate of protein synthesis in normal hepatocytes. Given the transient
viability and low synthetic rates of dispersed hepatocytes and the
technical limitations associated with perfused rat liver, it has not
been possible to correlate translational rates with changes in
cell-associated Ca2+ or eIF2
phosphorylation, to examine
the reversibility of hormonally imposed inhibitions, or to ascertain
whether an adaptive response occurs during continuous hormonal
treatments. Although cultured cells are better suited to such studies,
cell lines exhibiting translational suppression in response to hormones
that mobilize Ca2+ from the ER to the cytosol have not been
identified.
We now report that H9c2 ventricular myocytes respond to drugs that
deplete the sarcoplasmic/endoplasmic reticulum (S(E)R) of
Ca2+ and to physiologic concentrations of arginine
vasopressin with a rapid inhibition of mRNA translation at
initiation. Inhibitions are coupled to the mobilization of
cell-associated Ca2+ and to the phosphorylation of eIF2
.
Ionomycin and thapsigargin produced relatively stringent degrees of
Ca2+ mobilization in which translational recovery was
associated with the induction of ER chaperones. Translational recovery
from inhibition by vasopressin did not involve an ER stress response
but derived instead from the partial repletion of intracellular
Ca2+ stores. Inhibition of protein synthesis by
vasopressin became refractory to hormonal washout within 10 min
of treatment. These findings support the validity of hormonally
mediated regulation of mRNA translation involving mobilization of
Ca2+ sequestered within the S(E)R in ventricular
myocytes.
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EXPERIMENTAL PROCEDURES |
Materials--
H9c2(2-1) rat embryonic ventricular myocytes
were obtained from the American Type Culture Collection at passage
number 12. Arginine vasopressin, lysine vasopressin, desmopressin,
endothelin-1, angiotensin II, phenylephrine, caffeine, and phorbol
12-myristate 13-acetate (PMA) were purchased from Sigma. Ionomycin and
thapsigargin were from Calbiochem. Monoclonal antibody to eIF2
was
the gift of Dr. Lynn O'Brien, University of Rochester.
Ampholines were purchased from Galliard-Schlesinger (pH 4-8) and from
Oxford Glycosystems (pH 3.5-10).
L-[4,5-3H]Leucine was purchased from Amersham
Life Science, Inc., and [45Ca+2]CaCl2 was purchased from
NEN Life Science Products.
General Methodology--
Stock H9c2 cells were propagated in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and subcultured before confluence. For experiments,
monolayers were allowed to reach confluence after which cultures were
maintained for 1 day in Dulbecco's modified Eagle's medium
supplemented with 5% horse serum to promote differentiation of
myoblasts into myotubes (15). Before treatments, cells were
equilibrated for 5-15 min with serum-free Ham's F-10 medium modified
to contain 25 mM leucine and 0.2 mM Ca2+. Leucine pulse incorporation was measured as described
previously (16) for monolayers in multiwell trays (2.4 cm2/well) and for 15-min incubations. Incubations were
conducted in triplicate and results are presented as the mean ± S.E. of values obtained. Findings were reproduced on at least two
separate occasions. [35S]Methionine labeling,
one-dimensional 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) of
detergent-solubilized extracts of methionine-labeled cells, and
autoradiography were conducted as described previously (17). Ribosomal
and polyribosomal size distributions were measured by density gradient
centrifugation as described previously (18).
Measurement of Cell-associated
45Ca2+--
Cells in 12-well (4.8 cm2/well) plates were pre-equilibrated for 2 h in 1 ml
of modified F-10 medium containing 0.2 mM Ca2+
and 45CaCl2 (0.02 Ci/mmol) before treatments.
After treatments, monolayers were washed twice with 2 ml of ice-cold
buffered saline containing 2.5 mM LaCl3,
dissolved in 500 µl of 1% sodium dodecyl sulfate, and analyzed for
radioactivity. 45Ca2+ in combination with 5 mM EGTA was added to non-equilibrated samples and the
preparations washed and solubilized immediately to assess the
contribution of extracellular 45Ca2+. These
values (3% of experimental values) were subtracted from experimental
values. Results, presented in nanomoles of cell-associated Ca2+/mg of protein, are expressed as the mean ± S.E.
of values obtained for triplicate incubation samples. Findings were
reproduced on at least two separate occasions.
Determination of the Phosphorylation State of
eIF2
--
Duplicate monolayers (2.4 cm2) of multiwell
trays were lysed at 40 °C in 200 µl of sample buffer containing
3% ampholines (4 parts pH 4-8 and 1 part pH 3-10), 0.4% Tween 20, 2%
-mercaptoethanol, and 9.5 M urea. Lysates were then
subjected to slab gel isoelectric focusing in the presence of 9.5 M urea as described (19) to separate the phosphorylated and
non-phosphorylated forms of the
-subunit of eIF2. Gels were treated
with 1 M Tris, pH 8.8, and blotted onto polyvinylidene
difluoride membranes under basic conditions. Phosphorylated and
non-phosphorylated eIF2
were immunodetected with monoclonal antibody
to eIF2 and chemiluminescence as described previously (5). Films were
scanned by UMAX Magic Scan version 1.3.3. Analysis of the
relative amounts of phosphorylated and non-phosphorylated subunit was
performed on a Macintosh Quadra 700 computer using the public domain
NIH Image program.2
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RESULTS |
Inhibition of Protein Synthesis in H9c2 Myocytes by Vasopressin:
the Roles of S(E)R Ca2+ Stores and Protein Kinase
C--
Agents that deplete ER Ca2+ stores inhibit protein
synthesis in non-muscle cells in approximately 6-12 min (1-7, 17).
Similar inhibitions were observed in H9c2 myocytes exposed for 30 min to agents that release Ca2+ from the sarcoplasmic reticulum
or ER (Table I). The divalent cation
ionophore, ionomycin, inhibited leucine incorporation and mobilized
cell-associated Ca2+, with larger effects observed at 25 as
compared with 5 nM drug. Thapsigargin (1 µM),
an irreversible inhibitor of Ca2+ uptake by the S(E)R,
mobilized 75% of cell-associated Ca2+ while inhibiting
protein synthesis by 93%. Caffeine (5 mM), which releases
Ca2+ from the sarcoplasmic reticulum of cardiac muscle
(20), mobilized 45% of H9c2 cell-associated Ca2+ and
inhibited leucine incorporation by 40%. Hormones that have been
reported to increase cytosolic free Ca2+
([Ca2+]i) in normal neonatal or H9c2 ventricular
myocytes (21-25) were also examined for the ability to suppress
protein synthesis and mobilize Ca2+ stores. Phenylephrine
(10 µM) and epinephrine (data not shown) elicited
marginal responses, whereas angiotensin II and endothelin-1 were
ineffective. Arginine vasopressin (1 µM), however,
mobilized approximately two thirds of the total cell-associated
Ca2+ and suppressed protein synthesis by 45%. Inhibitions
of protein synthesis in the range of 35-40% occurred at quite low
arginine vasopressin concentrations (10 nM), maximized to
slightly higher values (
50%) at 0.1-1 µM hormone,
and were attributable to an interaction at the V1 vascular receptor
(Fig. 1). Lysine vasopressin, a slightly
less potent agonist selective for the V1 vascular receptor, elicited
similar degrees of inhibition at 0.01-10 µM, whereas desmopressin, a long acting synthetic agonist selective for the V2
renal receptor, at identical concentrations did not affect leucine
incorporation.
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Table I
Effects of various Ca2+-mobilizing agents on leucine
incorporation and cellular Ca2+ contents
H9c2 cells were pre-equilibrated for 2 h in F-10 medium containing
0.2 mM CaCl2 either with or without 4.5 µCi/ml
45CaCl2. Agents were then added at the indicated
concentrations, and the incubation was continued for 30 min.
Cell-associated Ca2+ was determined for prelabeled
preparations, and pulse incorporation of [3H]leucine into
proteins was determined for non-prelabeled preparations.
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Fig. 1.
Inhibition of leucine incorporation by
vasopressin analogs at varying concentrations. H9c2 cells were
treated for 30 min in modified Ham's F-10 medium containing the
indicated concentrations of arginine vasopressin ( ), desmopressin
( ), or lysine vasopressin ( ). [3H]Leucine was then
added, and incorporation of labeled amino acid into protein was
determined after 15 min.
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The inhibition of leucine incorporation in H9c2 cells that developed in
response to the mobilization of sequestered Ca2+ was
readily reversed by restoration of the cation to the extracellular medium (Fig. 2). To generate
Ca2+-depleted preparations, H9c2 cells were pretreated for
15 min in medium lacking Ca2+ and containing 0.1 µM ionomycin and 1 mM EGTA. Preparations were then washed with albumin-containing medium to remove ionophore (26) and
equilibrated in medium containing 1 mM EGTA and graded increases in Ca2+. At Ca2+ concentrations in
the physiological range (1 mM in excess of EGTA), leucine
incorporation was stimulated approximately 8-10-fold. Activation of
the V1 vascular receptor has been established to signal an increase in
[Ca2+]i, which is dependent, in part, on the
release of ER sequestered cation to the cytosol by inositol
trisphosphate (27). The Ca2+ dependence of the vasopressin
inhibition of protein synthesis was examined as a function of
increasing extracellular concentrations of the cation (Fig. 2).
Vasopressin had no effect on leucine incorporation at low extracellular
Ca2+ concentrations but reduced incorporation at 1 mM free cation by half. Supraphysiologic (2-3
mM) extracellular Ca2+ concentrations did not
reverse the inhibition attributable to vasopressin.

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Fig. 2.
Extracellular Ca2+ concentration
dependence of leucine incorporation in the absence and presence of
arginine vasopressin. H9c2 cells were pretreated for 15 min in
F-10 medium lacking Ca2+ and containing 1 mM
EGTA and 100 nM ionomycin to deplete Ca2+
stores. Cells were washed twice with low Ca2+ medium
lacking ionomycin and containing 2 mg/ml fatty acid free bovine serum
albumin to bind ionophore. After addition of albumin-free medium
containing 1 mM EGTA, preparations were adjusted with the indicated concentrations of Ca2+ and were incubated for 30 min. Incubations were continued for an additional 30 min in the absence
( ) or presence ( ) of 10 µM arginine vasopressin.
[3H]Leucine was then added, and pulse incorporation into
protein was determined.
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The time dependence of the inhibition of protein synthesis by
vasopressin was determined for cells incubated for 2 h with 0.2 mM Ca2+, which is sufficient to maintain rates
of leucine incorporation in untreated cells (Fig.
3). Within 20-30 min of hormone
addition, maximal inhibition of leucine incorporation was observed.
Thereafter, pulse incorporation rates were found to rise, with full
recovery of activity observed at 2 h. The addition of EGTA in
excess of Ca2+ produced declining pulse incorporation rates
that plateaued at 50% of control values from 1-2 h. Vasopressin added
in combination with the chelator provoked larger inhibitions of leucine
incorporation (67% at 30 min), which were sustained through 2 h
of incubation.

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Fig. 3.
Time dependence of vasopressin inhibition of
protein synthesis at low and high extracellular Ca2+
concentrations. H9c2 cells in F-10 medium containing 0.2 mM Ca2+ were treated for the indicated times
with 10 µM arginine vasopressin ( ), 1 mM
EGTA ( ), or 10 µM arginine vasopressin and 1 mM EGTA ( ). [3H]Leucine was added, and
pulse incorporation into protein was determined.
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Activation of phospholipase C in response to occupation of V1 vascular
receptors is associated with the intracellular generation of both
inositol trisphosphate and diacylglycerol (27). The potential
involvement of protein kinase C in translational regulation by
vasopressin was therefore explored utilizing PMA as an activator of the
enzyme. In control experiments, exposure of H9c2 cells to PMA for up to
30 min did not affect either leucine incorporation or cell-associated
Ca2+ (data not shown). By 45 min, however, leucine
incorporation tended to rise modestly. The effects of vasopressin on
leucine incorporation were examined in cells that had been either
pretreated for 15 min with 1 µM PMA or carried as
untreated controls (Table II). PMA
pretreatment was found to abolish the inhibition of leucine incorporation that occurred in untreated cells in response to the
subsequent addition of vasopressin. PMA also fully reversed (within 5 min) pre-existing inhibitions of leucine incorporation attributable to
vasopressin (data not shown). In contrast, suppression of leucine
incorporation occurring in H9c2 cells treated with ionomycin,
thapsigargin, sodium arsenite, or dithiothreitol or with heat shock
(43 °C for 20 min), was neither prevented nor reversed by PMA (data
not shown). Ca2+ mobilization in response to vasopressin
was reduced, but not abolished, by PMA (Table II). Vasopressin
mobilized approximately 37% of cell-associated Ca2+ in
PMA-pretreated cells as compared with 50% release in non-treated cells; this degree of mobilization was completed within 10 min.
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Table II
Leucine incorporation and cell-associated Ca2+ at various
times of treatment with arginine vasopressin in the absence and
presence of phorbol myristate acetate
H9c2 cells were pre-equilibrated for 2 h in F-10 medium containing
0.2 mM CaCl2 and in the absence or presence of 4.5 µCi/ml 45CaCl2. Cells were then pretreated for 15 min
with either solvent (0.05% Me2SO) or phorbol myristate acetate
(PMA, 1 µM). After incubation for the indicated times in
the presence of vasopressin (AVP, 10 µM), cell-associated
Ca2+ and pulse incorporation of [3H]leucine were
determined.
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Chronic exposure to phorbol ester was also employed to investigate the
effects of protein kinase C down-regulation on vasopressin signaling.
Conditions were chosen that are reported to produce down-regulation of
the kinase in neonatal cardiomyocytes (28). H9c2 cells were pretreated
for 16 h in complete growth medium with or without the addition of
PMA. The time dependence of vasopressin inhibition of protein synthesis
was then examined (Fig. 4).
Non-pretreated preparations responded to hormone with a rapid
suppression of protein synthesis that maximized at 20 min. Thereafter,
pulse incorporation rates increased such that the 2-h values exceeded those of untreated controls. In preparations exposed chronically to
PMA, vasopressin evoked larger (80%) inhibitions of leucine incorporation that were sustained for 40 min. Protein synthesis in
these preparations recovered from inhibition by the hormone slowly,
with only partial restoration of incorporation rates observed at 2 h. The protein kinase C inhibitors, calphostin C and chelerythrine, were tested in H9c2 cells at concentrations reported to inhibit the
enzyme in vivo (29, 30). Each of these agents produced inhibition of leucine incorporation and the release of cell-associated Ca2+ within 30 min by indeterminant mechanisms (data not
shown).

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Fig. 4.
Effect of chronic phorbol ester treatment on
the recovery of protein synthesis from inhibition by arginine
vasopressin. H9c2 cells were pretreated for 16 h in complete
growth medium containing either 0.05% Me2SO ( ) or 1 µM phorbol myristate acetate ( ). Pretreated
preparations were incubated in serum-free F-10 medium containing 10 µM arginine vasopressin for the indicated times, and
pulse incorporation of [3H]leucine into proteins was then
determined.
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Inhibition of Translational Initiation and Phosphorylation of
eIF2
by Vasopressin--
The polyribosomal contents of H9c2 cells
were examined after treatments with thapsigargin, vasopressin, PMA, and
PMA in combination with vasopressin (Fig.
5). As anticipated, polysomes almost
completely disappeared in response to thapsigargin, which slows
initiation relative to peptide chain elongation in non-muscle cells
(17). Vasopressin also reduced polysomal content, but not as
dramatically as thapsigargin. Polyribosomes were preserved in
incubations with PMA alone, and polysome disaggregation in response to
vasopressin was reduced after brief pretreatment with the phorbol
ester. To correct for potential differences in loading of gradients,
the amounts of 80 S monosomes, small polysomes, and large polysomes were quantitated by absorbance at 254 nm and related to each other. For
each treatment condition, the ratio of 80 S monosome:small polysome:large polysome was determined to be: control preparation, 30:10:10; thapsigargin, 67:3:0; vasopressin, 45:11:9; PMA, 24:10:10; PMA + vasopressin, 35:12:12. By this analysis, vasopressin caused 80 S
monosomes to increase and polysomes to decrease, typical of a slowing
of initiation. Large polysomes were reduced preferentially. By
contrast, PMA, which stimulates amino acid incorporation, caused polysomes to accumulate at the expense of 80 S monosomes, consistent with increased rates of initiation. Effects of vasopressin were largely
overturned by PMA, such that ribosomal profiles under this condition
were similar to those of controls. Thapsigargin produced almost
complete initiation blockade.

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Fig. 5.
Polysome contents of H9c2 cells treated with
thapsigargin, arginine vasopressin, phorbol myristate acetate, or both
phorbol myristate acetate and arginine vasopressin. H9c2 cells in serum-free F-10 medium were treated for 30 min without drug
(a), with 100 nM thapsigargin (b),
with 10 µM arginine vasopressin (c), with 1 µM phorbol myristate acetate (d), or with both
phorbol myristate acetate and arginine vasopressin (e).
Lysates of variously treated preparations were then subjected to
sucrose density gradient centrifugation for analysis of ribosomal size
distributions. The arrow indicates the migration position of
the 80 S monosome.
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Suppression of translational initiation in non-muscle cells in response
to drugs that deplete ER Ca2+ stores is attributable to the
phosphorylation of the
-subunit of eIF2 by double-stranded
RNA-activated protein kinase (1-4). The phosphorylated form of eIF2
is known to inhibit the GTP/GDP exchange factor, eIF2B, thereby
preventing eIF2 recycling (19). Because eIF2B is usually present at
much lower concentrations than eIF2, phosphorylations of 20-30% of
the eIF-2
pool cause substantial inhibitions of eIF2B activity in
most cell types (5, 6, 13, 31, 32). Addition of
Ca2+-mobilizing drugs or vasopressin to H9c2 cells resulted
in phosphorylation of eIF2
corresponding closely with the degree of
inhibition of leucine incorporation (Fig.
6). In untreated preparations, 94-100% of eIF2
was present in the non-phosphorylated form (A,
lanes 1 and 5; B, lanes 1 and 8; C, lanes 1 and 6).
The strongest phosphorylations of eIF2
and concomitant suppressions
of leucine incorporation occurred in response to 1 µM
thapsigargin (A, lane 2). Caffeine at 5 mM (A, lane 3) and vasopressin at 10 µM (A, lane 4) were each less
effective than thapsigargin in promoting eIF2
phosphorylation and
inhibiting leucine incorporation. Increasing degrees of eIF2
phosphorylation and accompanying inhibitions of protein synthesis were
observed in response to increasing concentrations of ionomycin (B, lanes 2-6). Within the same cell sampling,
eIF2
was phosphorylated and protein synthesis was inhibited to
comparable extents by 30 nM ionomycin (B,
lane 4) and by 10 µM vasopressin
(B, lane 7). Vasopressin produced eIF2
phosphorylation and inhibition of leucine incorporation at 15 and 30 min (C, lanes 2 and 3) that dissipated by 120 min (C, lane 4) of treatment. Brief
exposure to PMA abolished eIF2
phosphorylation and translational
suppression in response to 30 min of treatment with vasopressin
(C, lane 5 as compared with lane
3).

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Fig. 6.
Effects of arginine vasopressin and other
Ca2+-mobilizing agents on the phosphorylation state of
eIF2 in H9c2 cells. Cells in serum-free F-10 medium were
treated as described below. Preparations were denatured, subjected to
slab gel isoelectric focusing, and immunoblotted for eIF2 . The
migration positions of the authentic phosphorylated and
non-phosphorylated subunits are indicated. The percent of eIF2 in
the phosphorylated state was determined as described under
"Experimental Procedures." Additional samples were taken for
measurements of [3H]leucine pulse incorporation, with
results provided as percent of the untreated control value.
A, effects of brief treatments with thapsigargin, caffeine,
or arginine vasopressin. Cells were incubated without drug (lanes
1 and 5), with 100 nM thapsigargin (Tg, lane 2), with 10 mM caffeine
(CFN, lane 3), or with 10 µM arginine vasopressin (AVP, lane 4) for 30 min.
B, effects of ionomycin at varying concentrations and of 10 µM vasopressin. Cells were treated without drug (lanes 1 and 8), with the indicated concentration of ionomycin (IM,
lanes 2-6), or with 10 µM vasopressin
(AVP, lane 7) for 30 min. C, effects
of time of treatment with vasopressin and of treatment with phorbol
myristate acetate before challenge with vasopressin. Cells were
incubated without drug (lanes 1 and 6) or with 10 µM vasopressin for 15, 30, or 120 min (AVP,
lanes 2, 3, and 4,
respectively). An additional sample was pretreated with 1 µM PMA for 15 min and then challenged for 30 min with 10 µM vasopressin (lane 5).
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Translational Recovery from Inhibition by Vasopressin--
Neither
the acute inhibition of protein synthesis by hormone nor recovery from
this inhibition was altered by actinomycin D at concentrations
inhibitory to gene transcription, by rapamycin at concentrations that
inhibit the ribosomal p70 S6 kinase (33), or by cAMP-elevating agents
and analogs at concentrations that inhibit signal transduction
dependent on Ras (34, 35) (data not shown). Earlier findings (see Table
II and Figs. 3 and 4) were consistent with roles for protein kinase C
activation and Ca2+ in overturning the inhibition of
leucine incorporation by vasopressin. The degree to which H9c2 cells
replenish their Ca2+ stores during recovery from
translational inhibition by vasopressin was therefore examined. Leucine
incorporation and cell-associated Ca2+ were measured after
varying times of treatment with vasopressin (Fig.
7). Inhibition of incorporation
paralleled the decline in cell-associated Ca2+ during the
first 30 min of hormonal treatment. At 20-30 min, 60% of
Ca2+ stores were mobilized and protein synthesis was
inhibited 50%. Protein synthesis steadily recovered from inhibition
thereafter, with full recovery being observed by 100 min. At 2 h
of treatment, pulse incorporation rates were 20% higher than those in
untreated controls. During the recovery period (30-100 min)
cell-associated Ca2+ also increased but at slower rates
than observed for leucine incorporation. Full recovery of pulse
incorporation was associated with the restoration of approximately
one-fourth of the cation initially mobilized.

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Fig. 7.
Leucine incorporation and cell-associated
Ca2+ at varying treatment times with arginine
vasopressin. H9c2 cells were pre-equilibrated for 2 h in F-10
medium containing 0.2 mM CaCl2 and in the
absence or presence of 4.5 µCi/ml 45CaCl2.
Vasopressin (10 µM) was then added and the incubation
continued for the indicated times. Cell-associated Ca2+ was
determined for prelabeled preparations ( ), and pulse incorporation of [3H]leucine into proteins was determined for
non-prelabeled preparations ( ).
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A variety of cell types respond to protracted ER Ca2+
mobilization with an ER stress response that includes the induction of GRP78 and the development of translational tolerance to
Ca2+-mobilizing agents. H9c2 cells that had recovered from
translational suppression by vasopressin over 2 h and
corresponding untreated controls were examined for the development of
such tolerance upon challenge with various concentrations of ionomycin,
with thapsigargin at 1 µM, or with vasopressin at 10 µM (Table III). Comparable
rates of leucine incorporation were observed for the hormonally
pretreated and non-treated cells before challenge. Leucine
incorporation in both preparations was reduced in a
dose-dependent manner in response to increasing
concentrations of ionomycin and was inhibited extensively in response
to thapsigargin. A brief challenge with vasopressin, however,
suppressed leucine incorporation in the non-treated controls by 68%
but had no effect on incorporation in the vasopressin-pretreated
preparations. Cell-associated Ca2+ was also determined for
non-treated and vasopressin-pretreated preparations before and after
challenge with Ca2+-depleting drugs (Table III). The
Ca2+ contents of vasopressin-pretreated cells were 45% of
those of non-treated controls. Ionomycin mobilized Ca2+ in
a dose-dependent fashion from both the non-treated and the vasopressin-pretreated preparations. The Ca2+ contents of
both preparations were reduced to comparably low values by 150 nM ionomycin and by 1 µM thapsigargin. After
brief challenge with vasopressin, 65% of cell-associated
Ca2+ was mobilized from the non-treated controls but no
Ca2+ was released from the hormone-pretreated
preparations.
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Table III
Effects of Ca2+-mobilizing agents on leucine incorporation
before and after recovery of protein synthesis from inhibition by arginine vasopressin
H9c2 cells were pre-equilibrated for 2 h in F-10 medium containing
0.2 mM CaCl2 and in the absence or presence of 4.5 µCi/ml 45CaCl2. Vasopressin (AVP, 10 µM) was added where indicated, and the incubation was
continued for 2 h. Agents were then added at the indicated
concentrations. After 30 min of treatments, cell-associated Ca2+ and pulse incorporation of [3H]leucine were
determined.
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To verify that H9c2 cells are capable of expressing the ER stress
response, myocytes were incubated for 8 h at varying
concentrations of ionomycin or with 0.5 µM thapsigargin.
Preparations were washed to remove ionophore, and proteins were
pulse-labeled with [35S]methionine and sampled for
SDS-PAGE (7.5%) and autoradiography (Fig.
8). Preferential labeling of GRP78 and
GRP94 was observed in response to treatments with 10-100
nM ionophore, with optimal labeling occurring at 30 nM (lanes c-f as compared with lane
a). A modest increase in labeling of other proteins was also apparent in the treated preparations. Incubation with 5 nM ionomycin
did not promote increased expression of the GRPs or stimulation of overall protein synthesis (lane b). The GRPs were also
labeled preferentially in thapsigargin-pretreated preparations
(lane g). Thapsigargin was not removed by the washing
procedure, however, as evidenced by the decrease in overall protein
labeling. Additional samples were pretreated for 2 h in the
absence and presence of vasopressin before incubations in the absence
or presence of ionomycin (30 nM). Preparations were washed
and proteins were pulse-labeled and analyzed by SDS-PAGE and
autoradiography as above (Fig. 8). Although labeling of most cellular
proteins was modestly increased in the vasopressin-treated sample,
expression of the GRPs was not selectively increased by the hormone
(lane k as compared with lane i). No selective
labeling of the GRPs was observed during shorter incubations with
vasopressin (data not shown). However, pretreatment with vasopressin
followed by incubation with ionomycin resulted in greater
pulse-labeling of GRP78 and GRP94 than was observed in samples
incubated with ionophore alone (lane l as compared with
lane j).

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Fig. 8.
Induction of the Glucose Regulated Proteins
in H9c2 cells by Ca2+-mobilizing agents. H9c2 cells in
F-10 medium were incubated without drug (lanes a and
h), with 5, 10, 15, 30, or 100 nM ionomycin (lanes b-f, respectively), or with 1 µM
thapsigargin (lane g) for 8 h. In a separate
experiment, cells were incubated for 2 h in F-10 medium with
(lanes k and l) or without (lanes i
and j) 1 µM vasopressin followed by 8 h
in the absence (lanes i and k) or presence
(lanes j and l) of 30 nM ionomycin.
Cells were washed with medium lacking drugs and containing 2 mg/ml
fatty acid free bovine serum albumin. Proteins were then pulse-labeled by incubation for 30 min in medium containing 5 µM
methionine and 20 µC/ml [35S]methionine, and lysates
were analyzed by SDS-PAGE (7.5%) and autoradiography. The migration
position of molecular weight markers is indicated on the
ordinate in kDa.
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To ascertain whether translational tolerance to inhibition by
vasopressin accompanies induction of the GRPs, H9c2 cells were pretreated for 8 h in the absence or presence of ionomycin or thapsigargin (irreversible). Preparations were washed and then challenged with ionomycin at increasing concentrations, with
thapsigargin, or with vasopressin. After 30 min of treatments, samples
were analyzed by measurement of leucine pulse incorporation (Table IV). Incorporation in the control
preparations was inhibited in the typical fashion by ionophore,
thapsigargin, and vasopressin. By contrast, protein synthesis in
ionomycin-pretreated preparations was markedly resistant to suppression
by these agents. No inhibition by ionophore was observed except at the
highest (150 nM) concentration tested, and 10 µM vasopressin was ineffective. Although thapsigargin suppressed incorporation in ionomycin-pretreated preparations, the
extent of this suppression was not as great as in controls. Samples
pretreated with thapsigargin had somewhat lower synthetic rates as
compared with untreated controls, but protein synthesis was completely
unaffected after challenge with ionomycin at all concentration tested,
with thapsigargin, or with vasopressin.
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Table IV
Tolerance to translational inhibition by vasopressin and other
Ca2+-mobilizing agents after expression of the GRPs
H9c2 cells were incubated for 8 h in serum-free F-10 medium in the
absence of drug or with 300 nM ionomycin or 1 µM thapsigargin. Cells were washed twice with medium
lacking drugs and containing 2 mg/ml fatty acid-free bovine serum
albumin. Agents were then added at the concentrations indicated below.
After 30 min of treatments, pulse incorporation of
[3H]leucine into proteins was determined.
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Reversibility of Translational Inhibition by
Vasopressin--
Physiologic responses to vasopressin are known to
persist despite receptor internalization, which, depending on cell
type, at 37 °C occurs within 3-20 min of receptor occupation
(36-38). It was nonetheless unclear whether the translational
inhibition imposed by vasopressin in H9c2 cells was reversible or
persisted upon removal of the hormone. Cells were therefore treated
with vasopressin for 0, 2, 5, 10, 15, or 20 min, and the preparations were washed three times with medium lacking hormone. Washed
preparations were then incubated for 30 min in fresh medium lacking or
containing vasopressin, followed by measurements of leucine pulse
incorporation (Table V). Inhibitions of
protein synthesis attributable to vasopressin ranged from 55% at 2 min
to 69% at 20 min of treatment. These inhibitions were reversible by
removal of hormone at 2 and 5 min of treatment, but thereafter were
irreversible.
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Table V
Reversibility of translational inhibition by vasopressin
H9c2 cells were treated with vasopressin (100 nM) for the
indicated times. Medium was then removed, and the cells were washed three times with medium lacking vasopressin. Medium lacking or containing hormone was then added as indicated. After 30 min, medium
was again replaced with fresh medium lacking or containing vasopressin,
and pulse incorporation of [3H]leucine into protein was
determined.
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DISCUSSION |
Although Ca2+ sequestration by the ER has been
previously established to maintain optimal rates of translational
initiation in mammalian cells, the drugs utilized to mobilize the
cation invariably led to the development of an ER stress response. This report provides the first compelling evidence that hormonally induced
mobilization of S(E)R Ca2+ stores is regulatory upon
translation in that it is receptor-mediated, observable at
physiological extracellular Ca2+ concentrations, and
reversible without the generation of a stress response. Translational
inhibitions coinciding with mobilization of S(E)R Ca2+
stores of H9c2 ventricular myocytes were imposed by arginine vasopressin at concentrations that raise [Ca2+]i
in normal cardiomyocytes (39, 40). Inhibitions were exerted as a
consequence of a hormonal interaction at the V1 vascular receptor (39,
40), the receptor expressed in normal cardiac tissue (27). Vasopressin
exerted a persistent, albeit decreasing, inhibition of translation for
periods of 1-2 h. This result was not surprising in that vasopressin
receptors are known to undergo internalization within 3-20 min in
various tissues after hormonal binding. The physiologic effects of the
hormone persist after internalization (27). The ability of the hormone to signal translational inhibition in H9c2 cells was reversible by
washing for only several minutes. Circulating vasopressin has a
relatively long half-life (approximates 20 min) and undergoes degradation in the liver and kidney but not in the pulmonary
circulation (41).
Inhibition of the translational process in H9c2 cells by treatment with
vasopressin involved imposition of the same mechanisms as those exerted
by ionomycin, which destroys intracellular Ca2+ gradients,
and thapsigargin, which blocks the Ca2+-ATPase of the
S(E)R. Vasopressin, like ionomycin and thapsigargin (1-7, 17), slowed
translation at the initiation step and caused eIF2
to become
phosphorylated. Phosphorylation of this initiation factor correlated
closely with hormonal inhibitions of protein synthesis under all
experimental conditions tested. As has been observed invariably with
agents that inhibit translational initiation by mobilizing ER
Ca2+ stores (1, 4, 5, 7, 8, 17), protein synthesis in cells
expressing increased concentrations of ER chaperones was tolerant to
inhibition by vasopressin. The mechanism through which vasopressin
caused Ca2+ stores of H9c2 cells to be mobilized was not
explored. Unlike depletion of Ca2+ stores by thapsigargin
or ionophores, Ca2+ depletion in response to vasopressin
began to attenuate after 30 min. Attenuation presumably involved either
decreased production of, enhanced degradation of, or declining
responsiveness to intracellular messenger(s) that mobilize
Ca2+ from the S(E)R to the cytosol. Vasopressin acting at
V1 vascular receptors of various tissues is established to signal the
activation of phospholipase C and the generation of inositol
trisphosphate during the first few min of treatment (27). Inositol
trisphosphate is widely documented as an intracellular mediator of
hormone-dependent Ca2+ mobilization, although
cyclic ADP-ribose has also been advanced as a putative physiologic
regulator of ryanodine-sensitive Ca2+-dependent
release processes in intact mammalian systems, including heart (20, 42,
43). Mediation of the Ca2+-mobilizing effects of
vasopressin by cyclic ADP-ribose in H9c2 cells would be consistent with
the actions of caffeine, a pharmacologic activator of the cardiac
ryanodine receptor (20), in promoting Ca2+ release,
translational suppression, and eIF2
phosphorylation. Caffeine
affects these parameters in a quantitatively similar manner to
vasopressin. Eicosanoids have also been proposed to mediate the
Ca2+-mobilizing actions of vasopressin in smooth muscle
cells (27).
As in various non-muscle cell types (1, 4, 5, 7, 8, 17), recovery of
H9c2 cells from translational inhibition by Ca2+-mobilizing
drugs was contingent upon induction of expression of ER chaperones. In
contrast, recovery from translational inhibition by vasopressin
depended on partial re-accumulation of Ca2+ and, most
probably, the activation of protein kinase C. Indirect evidence,
including abolition of vasopressin inhibition in the presence of a
phorbol ester and prolonged inhibition of translation under conditions
wherein the enzyme is known to be down-regulated, supports a role for
protein kinase C in this event. Both calphostin C and chelerythrine at
concentrations used widely to investigate the involvement of protein
kinase C in various processes, produced the mobilization of
Ca2+ and the phosphorylation of eIF2
. It was not
possible to determine whether the effects of these inhibitors derived
from inhibition of protein kinase C activity or from actions at other
sites. Any action through protein kinase C would imply that the enzyme
is partially coupled to translational rates in H9c2 cells in the absence of hormonal influences. No other requirements for recovery of
translational activity could be identified. Other protein kinases that
are signaled in response to vasopressin, such as ribosomal S6 kinases
(44) and mitogen-activated protein kinase (45, 46), could not be
implicated. Although activation of V1 vascular receptors is known to
signal increased transcription of the immediate early genes (27),
transcriptional events were not required for translational
recovery.
Recovery from translational inhibition by vasopressin was associated
with partial, rather than full, restoration of cell-associated Ca2+, presumably at the S(E)R. Information is lacking
regarding whether H9c2 or other cell types possess critical ER or S(E)R
"pools" or subcompartments of Ca2+ supporting
translation. Translational inhibition in GH3 and HepG2 cells occurs at somewhat greater degrees of ER Ca2+
depletion than are required to impede protein processing within the
organelle (1, 7, 26). Inhibition of protein processing invariably
appears to trigger the phosphorylation of eIF2
in response to
Ca2+ depletion. Ca2+ re-accumulation in H9c2
cells after 2 h of vasopressin treatment, therefore, may restore
ER function sufficiently that protein synthesis can resume. PMA,
presumably by activating protein kinase C, reduced the degree to which
vasopressin lowered cell-associated Ca2+.
Although ionomycin (30 nM) and vasopressin (10 µM) generated comparable degrees of cation mobilization
and eIF2
phosphorylation over 30 min, only ionomycin produced a
subsequent ER stress response. The ionophore at 30 nM
clearly signaled comparable degrees of GRP78 induction and
translational tolerance in H9c2 cells to those observed in other cell
types (1, 4, 5, 7-10, 17). Continued exposures to either ionomycin or
thapsigargin elicited strong inductions of GRP78 and GRP94 and
development of translational tolerance to ER stressors in H9c2 cells
without additional requirements for auxiliary promoters (serum, PMA,
growth factors, cAMP analogs) observed in GH3, NIH-3T3, and
myeloma cells (1, 4, 5, 7, 17). Vasopressin treatment of H9c2 cells
differs from other Ca2+-mobilizing drugs by permitting
partial recovery of cell-associated Ca2+ after 30 min. This
recovery, which is accompanied by increasing rates of protein
synthesis, may sufficiently restore S(E)R function as to ablate the
development of an ER stress response.
Vasopressin exerts inotropic actions on cardiovascular performance that
was until recently, attributed to hormonal effects at the vasculature
and kidney. The hormone is now appreciated to stimulate lipid
metabolism in perfused hearts (47) and H9c2 cells (24), to increase
[Ca2+]i in cardiomyocytes (39), to potentiate
ventricular L-type currents via V1 vascular receptor
stimulation (40), and to cause atrial natriuretic factor to be secreted
from cardiomyocytes (48). The translational suppression observed in
H9c2 cells in response to vasopressin provides additional evidence that
the heart is directly targeted by this hormone. The functional
significance of the short-term suppression of ventricular protein
synthesis accompanying mobilization of cell-associated Ca2+
by the hormone remains to be clarified. Both muscle contraction, which
is supported by [Ca2+]i, and mRNA
translation, which is supported by S(E)R sequestered Ca2+,
are energy-intensive processes. Reduced translation accompanying release of S(E)R Ca2+ to the cytosol may therefore function
to divert ATP production toward supporting increased contractile
activity. In addition to its antidiuretic actions, vasopressin
serves broadly as a growth factor and, depending on cell type, can
promote either hypertrophy or hyperplasia (27). Induction of specific
gene expression and enhanced rates of protein synthesis are both
required for growth responses to vasopressin. In support of a
growth-promoting effect of vasopressin on H9c2 cells, prolonged
exposure to the hormone was invariably accompanied by increased rates
of amino acid incorporation into protein. It is anticipated that H9c2
cells should provide a highly useful model system for investigating
both the acute and the chronic effects of vasopressin on the
biochemistry and physiology of ventricular myocytes.