Mechanical strain has been implicated in
phenotypic changes, including alteration of gene expression in vascular
smooth muscle cells; however, the molecular basis for
mechanotransduction leading to nuclear gene expression is largely
unknown. We demonstrate in the present study that cyclic stretching of
vascular smooth muscle cells dramatically activates Jun N-terminal
kinase (JNK)/stress-activated protein kinase (SAPK) through an
autocrine mechanism. Stretch causes time- and
strength-dependent rise of the ATP concentration in media.
The stretch-induced activation JNK/SAPK is attenuated by the addition
of hexokinase or apyrase that scavenge ATP in media. Both the
P2 receptor antagonist and the A1
subtype-selective P1 receptor antagonist partially inhibit
stretch-induced activation of JNK/SAPK. The conditioned medium from
stretched cells contains an activity to stimulate JNK/SAPK. The
JNK-stimulating activity in the conditioned medium from stretched cells
is attenuated by the addition of apyrase or P1 and
P2 receptor antagonists. The addition of exogenous ATP or
adenosine induces dose-dependent activation of JNK/SAPK.
These results indicate that stretch activates JNK/SAPK in vascular
smooth muscle cells through mechanisms involving autocrine stimulation
of purinoceptors by ATP and its hydrolyzed product adenosine.
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INTRODUCTION |
The vascular wall is constantly exposed to mechanical forces of
hemodynamic origin. The tensile stress resulting from transmural pressure activates a contractile mechanism, which confers a mechanistic basis for autoregulation of blood flow (1, 2). Recent studies also
reveal that mechanical stretch exerts regulatory influences on gene
expression and thereby influences vascular tone and remodeling. We
recently demonstrated that cyclic stretching of vascular smooth muscle
dramatically increases the expression of the gene for the vasorelaxant
parathyroid hormone-related peptide (3-5). Others have reported that
stretch induces an increase in the gene expression of platelet-derived
growth factor-A chain in vascular smooth muscle cells (6). However,
little is known about the mechanisms by which mechanical force is
converted into intracellular signals coupled to nuclear gene expression
in vascular smooth muscle cells (7, 8).
Previous studies on cardiac myocytes demonstrate that stretching
activates the mitogen-activated protein kinase
(MAPK)1 cascade (9)
comprising Raf-1, MAPK/extracellular signal-regulated kinase (ERK)
kinase (MEK) and MAPK/ERK (10, 11). Additionally, static stretch causes
autocrine release of angiotensin II (AII), which mediates the major
part of stretch-induced activation of the MAPK cascade (11, 12).
Recently the novel serine/threonine kinase, Jun N-terminal protein
kinase (JNK)/stress-activated protein kinase (SAPK), has been shown to
be activated by a variety of extracellular stresses, including UV
irradiation, osmotic shock, protein synthesis inhibitors, and genotoxic
agents. JNK/SAPK can also be activated by growth factors, cytokines,
and G protein-coupled receptor agonists, including carbachol,
angiotensin II, and endothelin (13-18). JNK/SAPK is capable of
phosphorylating the transactivation domain of c-Jun (13, 14), ATF-2
(19), and Elk1 (20). The JNK-mediated phosphorylation of the
transcription factors brings about their increased activity leading to
induction of their target genes, including c-jun itself (20,
21). Recent studies reveal that, just like the MAPK cascade, JNK/SAPK
is activated through its phosphorylation by the upstream kinase
JNK/SAPK kinase (SEK1/MKK4/JNKK) (22-24), and that SEK1/MKK4/JNKK is,
in turn, activated by MEK kinase or related kinase (25-27). It is not
yet known how mechanical force affects the activity of JNK/SAPK cascade
in vascular smooth muscle.
While investigating the mechanisms by which stretch induces parathyroid
hormone-related peptide gene expression in vascular smooth muscle
cells, we noted that stretch induces a marked increase in the mRNA
level of c-jun. This prompted us to examine the possibility that stretching of vascular smooth muscle cells might activate JNK/SAPK. The results in the present study demonstrate that stretch induces potent activation of JNK/SAPK. Further, the present study provides evidence for the involvement of autocrine ATP and
purinoceptors in stretch-induced JNK/SAPK activation.
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MATERIALS AND METHODS |
Application of Mechanical Stretch to Cells--
Primary cultures
of rat aortic smooth muscle (RASM) cells were obtained from an
18-week-old Wistar rat by the explant method (3), and grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum (Commonwealth Serum Laboratory, Melbourne, Australia), 100 units/ml penicillin, and 100 µg/ml streptomycin (Wako Pure Chemicals,
Osaka, Japan) under an atmosphere of 95% air plus 5% CO2.
Before each experiment, confluent cells were serum-deprived by
incubation in serum-free Dulbecco's modified Eagle's medium
containing 0.2% bovine serum albumin (Fraction V, Sigma) for 24 h. RASM cells were subjected to repetitive cycles of mechanical stretch
and relaxation with a Flexercell strain unit (FX-2000, Flexcell Corp.,
McKeesport, PA) as described previously (3). The negative pressure to
the flexible bottoms was set to provide a maximum elongation of 3, 7, 15, or 20% of the silicone rubber and cells. All experiments were
carried out using alternation cycles of 0.5-s stretch and 0.5-s
relaxation at a rate of 60 cycles/min at 37 °C in 5%
CO2 in a humidified air.
Northern Blot Analysis--
Total RNA, isolated from RASM cells
by the acid guanidinium isothiocyanate/phenol/chloroform method, was
separated by formaldehyde, 1.0% agarose gel electrophoresis,
transferred onto a nylon membrane (Hybond N, Amersham Life Science) and
hybridized by cDNA probes with [
-32P]dCTP (NEN
Life Science Products) by the random priming method as described (3,
28). The radioactivity of corresponding bands was quantitated by Fuji
BAS 2000 bio-image analyzer (Fuji Film Co. Ltd., Tokyo, Japan). The rat
c-jun, human junB, and mouse junD
cDNAs were obtained from RIKEN Gene Bank (Tsukuba, Japan). The
human c-fos and c-myc cDNAs were obtained
from the Japanese Cancer Research Resources Bank (Tokyo, Japan).
JNK and ERK Assay--
The solid phase JNK assay was performed
as described in Ref. 13. JNK was recovered by incubation of cell lysate
with 10 µg of glutathione S-transferase
(GST)-c-Jun-(5-89) fusion protein bound to glutathione-Sepharose
beads. The pelleted beads were incubated with
[
-32P]ATP at 30 °C for 20 min, and the reaction was
terminated by adding 10 µl of 4 × Laemmli's SDS sample buffer
and boiling for 5 min. The samples were analyzed as described (30). The
bacterial expression plasmid of N-terminal amino acids 5-89 of c-Jun
fused to GST, pGEX-2T-c-Jun-(5-89) (29), was provided by Dr. A. S. Kraft (University of Alabama School of Medicine, Birmingham, AL). The immune complex JNK activity was determined as described (14, 30).
JNK was immunoprecipitated, using rabbit polyclonal anti-JNK1 C-terminal antibody (Santa Cruz, C-17). The immunoprecipitate was
incubated with 30 µl of the JNK assay buffer containing 3 µg of
GST-c-Jun-(5-89) at 30 °C for 30 min. The reaction was terminated and analyzed as described for the solid phase JNK assay.
The immune complex ERK assay was carried out as described (31). The
MAPKs ERK1 and ERK2 were immunoprecipitated using a mouse monoclonal
anti-MAPK antibody that recognizes both ERK1 and ERK2 (Zymed
Laboratories Inc., 03-6600).
Measurement of ATP Concentrations--
The ATP concentration in
culture media was measured with the Enlighten ATP assay system using
luciferase and luciferin (Promega). Briefly, culture media were
collected, mixed with trichloroacetic acid (a final concentration of
0.3%), and kept at 4 °C for 30 min. The media were then neutralized
by adding four volumes of 250 mM Tris acetate (pH 7.75),
and mixed with the ATP assay reagents. Luminescence was measured with a
Lumat LB9501 luminometer (Berthold, Bad Wildbad, Germany). The standard
curve was generated by using known concentrations of ATP.
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RESULTS |
We first determined whether cyclic stretching (60 cycle/min) of
vascular smooth muscle cells causes any change in the expression of
immediate-early genes. Northern blot analysis revealed that all of the
five immediate-early genes examined (c-jun, jun
B, jun D, c-fos, and c-myc) showed
increased expression in response to mechanical stretch (a maximal
elongation of 15%) (Fig. 1A). Among them, the induction of c-jun mRNA was most
prominent with a maximal 6-fold increase by 30 min. The induction of
c-jun mRNA increased with increasing strength of stretch
and became maximal at 15% stretch (Fig. 1B). In contrast,
stretch hardly changed the glyceraldehyde-3-phosphate dehydrogenase
mRNA level.

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Fig. 1.
Expression of immediate-early genes by
stretch. A, time course of c-jun,
junB, junD, c-fos, and
c-myc mRNA expression by stretch. B, stretch
dose-dependent expression of immediate-early genes. Cells
were subjected to stretch (a maximal elongation of 15%) for 0.5-4 h
(A), or to stretch of various magnitudes (a maximal elongation of 3-20%) for 0.5 h (B). Total RNA was
isolated from cells and analyzed for mRNAs of the immediate early
genes by Northern blotting. The same membrane was re-probed with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA as
an internal control.
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We next examined whether stretch activated JNK/SAPK in vascular smooth
muscle cells. Stretch caused a rapid and sustained activation of JNK.
The JNK activity, measured with a solid phase kinase assay (13), was
increased significantly over the basal value by 5 min, reached a
maximal value of 6-fold at 30 min, and then declined to a lower level
of 3-fold at 60 min (Fig. 2A). Measurement with a immune complex kinase assay using anti-JNK C-terminal antibody gave similar results (data not shown). The JNK
activation was dependent on the magnitude of stretch applied to cells
(Fig. 2B). The relationship between the extent of the stretch and the changes in JNK activity was quite similar to that observed for c-jun mRNA expression (Fig. 1B).
The vasoconstrictor AII, at a maximal concentration in terms of its
Ca2+ mobilizing activity, also activated JNK, but provided
a much weaker stimulus than stretch (Fig. 2A). We also
examined the effect of stretch on the activity of ERK, another MAPK
family member. As shown in Fig. 3, the
application of stretch stimulated the ERK activity with a maximal
stimulation of approximately 2-fold over the basal activity at 30 min.
AII induced activation of ERK to a comparable extent as stretch (Fig.
3).

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Fig. 2.
Activation of JNK/SAPK by stretch or
angiotensin II. A, time-dependent activation of
JNK/SAPK. B, stretch dose-dependent activation
of JNK/SAPK. Cells were subjected to either stretch (a maximal
elongation of 15%) or angiotensin II (A II)
(10 8 M) stimulation for 5-60 min
(A), or to stretch of various magnitudes (a maximal
elongation of 3-15%) for 30 min (B). The JNK/SAPK activity was measured by the solid phase kinase assay method using
GST-c-Jun-(5-89) as a substrate as described under "Materials and
Methods." Autoradiograms are shown in the upper panel in
A and B. The arrows indicate the position of GST-c-Jun-(5-89). Quantitation of JNK/SAPK activities are
shown in the lower panel in A and B,
where the JNK/SAPK activities (means of duplicate determinations) are
expressed relative to the activity in cells at time zero, which was
given an arbitrary value of 1. Two other experiments showed similar
results.
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Fig. 3.
Time-dependent activation of
MAPK/ERK by stretch or angiotensin II. Upper panel,
autoradiograms. The arrow indicates the position of myelin
basic protein (MBP). Lower panel, quantitation of
the MAPK/ERK activities. Cells were subjected to stretch (a maximal
elongation of 15%) or angiotensin II (A II)
(10 8 M) stimulation for 5 to 60 min. The
MAPK/ERK activities were measured as described under "Materials and
Methods." The MAPK/ERK activities (means of duplicate determinations)
were expressed relative to the activity in non-stretched cells, which
was given an arbitrary value of 1. Two other experiments showed similar results.
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We examined how vascular smooth muscle cells convert mechanical stimuli
into signals for activating JNK. Knowing that stretch causes activation
of stretch-activated (SA) cation channels in a variety of cell types
(32, 33), we sought to determine if SA cation channel activation is
involved in stretch-induced JNK/SAPK activation, by examining the
effect of Gd3+, a specific inhibitor of SA cation channel
function (34, 35). As shown in Fig. 4,
Gd3+ failed to suppress stretch-induced activation of
JNK/SAPK in vascular smooth muscle cells. Removal of extracellular
Ca2+ was also without any effect on stretch-induced
activation of JNK/SAPK (Fig. 4). Thus, SA cation channels or any other
Ca2+ channels are not likely to be involved in
stretch-induced JNK activation.

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Fig. 4.
Effects of the addition of Gd3+
and removal of extracellular Ca2+ on stretch-induced
JNK/SAPK activation. Upper panel, autoradiogram. Lower
panel, quantitation of the JNK/SAPK activities. Cells were subjected to stretch (a maximal elongation of 15%) for 30 min in the
presence of 50 µM Gd3+ or in the absence of
extracellular Ca2+ (0 mM Ca2+ plus
50 µM EGTA). The JNK/SAPK activities were measured with the solid phase kinase assay method and are expressed relative to the
activity in non-stretched cells, which was given an arbitrary value of
1. Another experiment showed similar results.
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The possible involvement of autocrine factors in stretch-induced JNK
activation was next examined. Conditioned media from cells subjected to
stretch for varied time periods were collected and applied to
non-stretched cells. The conditioned media from stretched cells did
cause the activation of JNK/SAPK (Fig.
5). In this assay, the JNK/SAPK
activating "factor" in conditioned media was detectable by 2 min,
and accumulated with time to reach a plateau value by 10 min.

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Fig. 5.
Activation of JNK/SAPK by stretch-conditioned
media. Upper panel, autoradiogram. Lower panel,
quantitation of the JNK/SAPK activities. Culture media conditioned by
non-stretched cells and cells subjected to stretch (maximal 15%
elongation) for 2, 5, 10, or 20 min were immediately applied to
non-stretched cells, and the incubation was carried out for 30 min.
Control cells received conditioned media from non-stretched cells. The
JNK/SAPK activity was measured with the solid phase kinase assay
method. The JNK/SAPK activities (means of duplicate determinations) are
expressed relative to the activity in cells that received conditioned
media from non-stretched cells, which was given an arbitrary unit of 1. The experiments were repeated twice with similar results. The
asterisk denotes a statistical significance
(p < 0.05) as compared with no stretch.
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Previous studies have shown that imposing flow on vascular endothelial
cells (36) and mechanical deformation of the cell membrane in mast
cells (37) and in mammary epithelial cells (38) induces release of ATP.
It is also known that vascular smooth muscle cells express cell surface
receptors for ATP and its hydrolyzed product, adenosine (39).
Therefore, we examined the possibility that ATP might be the autocrine
factor involved in stretch-induced JNK activation. The application of
stretch induced a rapid and sustained rise in the ATP concentration in the extracellular medium (Fig.
6A). The ATP concentration in
the medium reached a maximal level of 50-fold increase over the basal value by 5 min, and remained elevated over the basal value for at least
30 min. When the magnitude of stretch was increased, the ATP
concentration in the medium also increased (Fig. 6B), like
the activity of JNK and c-jun mRNA expression (Figs.
1B and 2B). To determine whether or not released
ATP is indeed involved in stretch-induced activation of JNK, we
examined the effects of agents that reduce the ATP concentration in
media. Apyrase is a well known ATP- and ADP-hydrolyzing enzyme.
Hexokinase catalyzes phosphorylation of glucose to generate glucose
6-phosphate at the expense of ATP (51). The addition of apyrase or
hexokinase nearly completely inhibited stretch-induced rise of the ATP
concentration in the medium (Fig.
7A). Both apyrase and
hexokinase also substantially, but not completely, inhibited
stretch-induced activation of JNK (Fig. 7B). Apyrase or
hexokinase were without inhibitory effects on basal JNK activity (data
not shown). The inhibitory effect of hexokinase on stretch-induced JNK
activation was likely not due to depletion of medium glucose, because
increasing glucose concentration up to 20 mM had no effect
on the ability of hexokinase to inhibit stretch-induced JNK activation
(data not shown). Furthermore, apyrase, when added to the conditioned
medium harvested from stretched cells, inhibited its JNK-stimulating
activity (Fig. 8). These observations
support the notion that ATP acts as a mediator of stretch-induced JNK
activation.

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Fig. 6.
Stimulation of ATP release by stretch.
A, time-dependent increase in the ATP
concentrations in culture media during stretching. B,
stretch dose-dependent increases in the ATP concentration in culture media. Cells were subjected to stretch (a maximal elongation of 15%) for 2-30 min (A), or to stretch of various
magnitudes (a maximal elongation of 3-15%) for 5 min (B).
The results are expressed as means ± S.E. of four to eight
determinations. The experiments were repeated three times
(A) and twice (B) with similar results.
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Fig. 7.
Inhibition of stretch-induced ATP release and
JNK/SAPK activation by ATP-trapping agents. A, inhibition of
ATP accumulation. B, inhibition of JNK/SAPK activation. Cell
were subjected to stretch (a maximal elongation of 15%) for 30 min in
the presence or absence of 5 units/ml hexokinase (Sigma) or 2 units/ml
apyrase (Sigma). The ATP concentration in culture media was measured by
using luciferin and luciferase as described under "Materials and
Methods." In B, the autoradiograms and quantitation of the
JNK/SAPK activities are shown. The JNK/SAPK activities were measured
with the solid phase kinase assay method. The results are expressed
relative to the activity in stretched cells without drugs, which was
given an value of 100%. The results represent means ± S.E. of
four to six (A) or three (B) determinations. The
experiments were repeated twice with similar results. The
asterisk denotes a statistical significance
(p < 0.05) as compared with stretch alone.
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Fig. 8.
Inhibition by apyrase of the JNK-stimulating
activity in the conditioned medium from stretched cells. The
conditioned medium (CM) with or without 2 units/ml apyrase
was rapidly harvested from cells subjected to stretch for 10 min or
non-stretched cells and were immediately applied to non-stretched
cells. The incubation was carried our for 30 min. The results are
means ± S.E. of three determinations. The asterisk
denotes a statistical significance (p < 0.05) as
compared with stretch conditioned medium.
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ATP and ADP act as agonists for P2 receptors, while the
hydrolyzed products of ATP, adenosine and AMP, act as agonists for P1 receptors (40, 41). We examined which of P1
and P2 receptors mediated the JNK activation in response to
stretch. The P2 receptor antagonist reactive blue-2 (RB-2)
(42, 43) partially inhibited stretch-induced JNK activation in a
dose-dependent manner (Fig. 9A). Suramin, another
P2 receptor antagonist with a different subtype selectivity
(44, 45), was without any inhibitory effect. The A1
subtype-selective P1 receptor antagonist,
8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (46, 47), but not the
A2 subtype-selective P1 receptor antagonist
8-(3-chlorostyryl)caffeine (47), also inhibited JNK activation (Fig.
9A). These results suggest that both RB-2-sensitive P2 and DPCPX-sensitive P1 receptors are
involved in stretch-induced JNK activation. By contrast, these
antagonists were without any effect on AII-induced JNK activation (data
not shown). Moreover, RB-2 and DPCPX both inhibited the JNK-stimulating
effect of the conditioned medium harvested from stretched cells (Fig.
9B). We examined the ability of exogenous purinoceptor
agonists to activate JNK in vascular smooth muscle cells. ATP, ADP,
adenosine, and UTP induced a time-dependent activation of
JNK (Fig. 9C). ATP, ADP, and adenosine each activated JNK
with similar dose-response relationships (Fig. 9D). UTP was
the most potent in activating JNK. Thus, ATP, adenosine, and presumably
ADP appear to mediate stretch-induced JNK activation.

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Fig. 9.
Effects of purinoceptor antagonists on
stretch-induced activation of JNK/SAPK and effects of exogenous
nucleotides and nucleoside on JNK/SAPK activity. A,
inhibition of stretch-induced JNK/SAPK activation by purinoceptor
antagonists. Cells were subjected to stretch (a maximal elongation of
15%) for 30 min in the presence of the P2 receptor
antagonists suramin (3~ × 10 4 M) or RB-2
(3~ × 10 5 to 3~ × 10 4 M),
the A1 receptor antagonist DPCPX (10 7 to
10 6 M), or the A2 receptor
antagonist 8-(3-chlorostyryl)caffeine (10 6
M). The autoradiogram and quantitation of the results are
shown. The JNK/SAPK activities were measured with the solid phase
kinase assay and are expressed relative to the activity in
non-stretched cells, which was given an arbitrary value of 1. The
asterisk denotes statistically significant difference
(p < 0.05) as compared with stretched cells.
B, inhibition by RB-2 and DPCPX of the JNK-stimulating activity in the conditioned medium (CM) harvested from
stretched cells. The conditioned medium was rapidly harvested from
cells subjected to stretch for 10 min or non-stretched cells and were immediately applied with or without a receptor antagonist to
non-stretched cells. The incubation was carried our for 30 min.
C, time-dependent activation of JNK/SAPK. Cells
were stimulated with 10 6 M amounts of ATP,
ADP, adenosine, or UTP for the indicated time periods. D,
activation of the JNK/SAPK activity by exogenous nucleotides. Cells
were stimulated with various doses of ATP, ADP, adenosine, or UTP for
20 min (for ATP and ADP) or 30 min (for adenosine and UTP). The
JNK/SAPK activity was measured with the solid phase kinase assay
method. The value represents -fold stimulation over the JNK/SAPK
activity in non-stimulated cells. The asterisk denotes a
statistical significance (p < 0.05) as compared with
stretch (A) or stretch conditioned medium
(B).
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DISCUSSION |
The present study demonstrates for the first time that mechanical
stress causes potent activation of JNK/SAPK in vascular smooth muscle
cells (Fig. 2). Stretch also induces modest activation of MAPK/ERK
(Fig. 3). Although stretch and the receptor agonist AII are similar in
that either stimulus can activate both JNK/SAPK and MAPK/ERK in
vascular smooth muscle cells, stretch activates JNK/SAPK much more
strongly than AII (Figs. 2 and 3). Stretch-induced JNK activation is
transient in the continued presence of stretch stimuli. This transient
nature might be explained by possible up-regulation by stretch of
JNK-inactivating phosphatase such as MKP-1, possible inactivation of
JNK kinase by phosphorylation, or other mechanisms. The JNK/SAPK
pathway is a recently identified downstream mediator linking
extracellular stimuli to nuclear events (27, 48). c-Jun, ATF-2, and
Elk1 are well characterized substrates for JNK/SAPK, and
JNK/SAPK-mediated phosphorylation activates these transcription factors
(13, 14, 16, 19, 20). These transcription factors appear to be
essential for transcriptionally activating a variety of genes in
response to external stimuli. Thus, JNK/SAPK likely acts as a mediator
for stretch-induced regulation of gene expression.
The activation of JNK/SAPK is induced by a variety of bioactive
substances including growth factors, cytokines, and G protein-coupled receptor agonists. Accordingly, we pursued the possibility that a
factor released from vascular smooth muscle cells upon stretching might
act in an autocrine/paracrine manner to activate JNK/SAPK. The present
study demonstrates that the conditioned medium harvested from stretched
cells contains a factor that activates JNK/SAPK (Fig. 5) and that ATP
is released in large quantities from cells upon stretching (Fig. 6).
The results shown in the present study imply that ATP is an
autocrine/paracrine factor to activate JNK/SAPK. First, when released
ATP was scavenged by the addition of the ATP-trapping agents, apyrase
and hexokinase, stretch-induced JNK/SAPK activation was considerably
suppressed (Fig. 7, A and B). Second, the
P2 receptor antagonist RB-2 inhibited stretch-induced
JNK/SAPK activation (Fig. 9A). The partial inhibition of
stretch-induced JNK activation by RB-2 might be explained by its weak
antagonistic potency and/or the partial contribution of P2
receptor (see below). Third, the JNK-stimulating effect of the
conditioned medium from stretched cells was suppressed by apyrase
treatment or the addition of RB-2 (Figs. 8 and 9B). Fourth,
the addition of exogenous ATP caused activation of JNK/SAPK in vascular
smooth muscle cells (Fig. 9C). However, the magnitude of
JNK/SAPK activation by exposure to the conditioned medium from
stretched cells was considerably smaller that induced by stretch
(compare Figs. 2 and 5). These differences might imply that stretch
activates JNK/SAPK through both autocrine-dependent and
autocrine-independent mechanisms. This notion is supported by the
findings showing that nearly complete inhibition of stretch-induced
increase in extracellular ATP by apyrase or hexokinase could not
totally abolish stretch-induced JNK activation (Fig. 7, A
and B). However, the incomplete inhibition of
stretch-induced JNK activation by hexokinase and apyrase could also be
because ADP, AMP, and adenosine in the presence of hexokinase, and AMP
and adenosine in the presence of apyrase still could activate purinoceptors in vascular smooth muscle cells (see below) (Fig. 9,
C and D).
Exogenous ATP at the concentration slightly higher than the maximal
medium concentration of ATP in stretched cells gives approximately 2-3-fold increase in the JNK activity (Fig. 9D), while
stretch usually causes more than 5-fold increase in the JNK activity. The difference may suggest the contribution of the hydrolyzed products
of ATP, i.e. ADP, AMP, and adenosine, besides ATP, as mentioned above. Second, it is known that extracellular nucleotides are
subjected to active hydrolysis by ectonucleotidases existing on the
plasma membrane (49, 50). Therefore, the concentration of ATP in the
medium might have been rapidly reduced by avid hydrolysis, although
much attention was paid for allowing for quick collection and transfer
of conditioned media. It is also possible that the ATP concentration in
the bulk media may be lower than that in the vicinity of ATP
transporters on the plasma membrane where purinoceptors also exist. In
airway epithelial cells, it was suggested that an ATP transporter and a
P2 receptor were present in close proximity on the plasma
membrane (51). It has been shown previously that medium concentrations
of autocrine peptides endothelin-1 and angiotensin II in stretched
cardiomyocyte cultures are lower than those of exogenous peptides
required for activation of MAPK (11, 12).
The A1 subtype-selective P1 receptor antagonist
DPCPX inhibited stretch-induced activation of JNK/SAPK (Fig.
9A). Further, the addition of exogenous adenosine caused
activation of JNK/SAPK (Fig. 9C). Consistent with this, the
addition of DPCPX to the conditioned medium inhibited its
JNK-stimulating effect (Fig. 9B), like the addition of
apyrase (Fig. 8). These findings, taken together, show that adenosine
as well as ATP are involved in stretch-induced activation of JNK/SAPK
through P1 and P2 receptors, respectively. Adenosine may be either released directly from the inside of cells, or
derived from hydrolysis of released ATP by ectonucleotidases, or both
(49).
It is of note that UTP was a more potent agonist to induce JNK
activation (Fig. 9D). Rat vascular smooth muscle cells
employed in the present study express at least two subtypes
(P2Y2 (=P2U) and P2Y6) (53) of the
G protein-coupled P2 receptors that show distinct agonist
and antagonist sensitivity profiles (42, 51). Both of them bind UTP
(43, 52). Previous studies (38, 54) demonstrated that mechanical
stimuli induced release of UTP as well as ATP from non-smooth muscle
cells. It is an interesting possibility that uracil nucleotides also
contribute to stretch-induced JNK activation. Cloned rat
P2Y6 receptor, but not rat P2Y2, is sensitive
to the P2 receptor antagonist RB-2, whereas both receptor subtypes are substantially resistant to suramin
(53).2 The results of the
antagonist sensitivity (Fig. 9A) and the relative nucleotide
potency (Fig. 9C) favor the view that P2Y6
largely mediates stretch-induced JNK activation.
We are grateful for Dr. H. Rasmussen for
critical reading of the manuscript and for Dr. K. Kurokawa for
encouragement. We thank R. Suzuki, F. Iwase, and W. Zhou for technical
and secretarial assistance.