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INTRODUCTION |
Agonist pre-contracted bovine carotid artery smooth muscle relaxes
with the addition of the guanylyl cyclase activator, sodium nitroprusside, or the adenylyl cyclase activator, forskolin. This relaxation is associated with increases in the phosphorylation of the
small heat shock-related protein,
HSP201 (1). In addition,
endothelial-dependent vasodilation of isolated segments of
bovine carotid arteries is also associated with increases in the
phosphorylation of HSP20 (2). However, HSP20 is not phosphorylated in a
muscle that is uniquely refractory to cyclic nucleotide-dependent relaxation, human umbilical artery
smooth muscle (3, 4). These data suggest that increases in the phosphorylation of HSP20 may mediate cellular signaling processes that
lead to vasorelaxation.
HSP20 was initially identified as a by-product of the purification of
another small heat shock protein, HSP27 (5). HSP27 has been shown to
modulate actin filament dynamics in cultured cells (7-9). Increases in
the phosphorylation of HSP27 have been associated with vascular smooth
muscle contraction (10-12). HSP20 has been shown to associate in
macromolecular aggregates with HSP27, in extracts from heart,
diaphragm, and soleus muscle cells (5). Both HSP20 and HSP27 dissociate
from the aggregated form in response to heat stress (5, 6). HSP20 and
HSP27 are predominant proteins in muscle tissues, which supports a
physiologic role for these proteins (5).
We hypothesized that the cellular functions of HSP20 and HSP27 are
modulated by both phosphorylation and macromolecular association with
specific proteins. Furthermore, we postulated that HSP20 and/or HSP27
are cellular signaling molecules that regulate the state of contraction
and relaxation of vascular smooth muscle.
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EXPERIMENTAL PROCEDURES |
Materials--
The catalytic subunit of
cAMP-dependent protein kinase was purchased from Promega
(Madison, WI). Hepes was obtained from American Bioanalytical (Natick,
MA). Urea, sodium dodecyl sulfate, glycine, and Tris were from Research
Organics (Cleveland, OH). Coomassie Brilliant Blue was from ICN
Biomedicals Inc. (Aurora, OH). [
-32P]ATP and
[32P]orthophosphate were from Amersham (Arlington
Heights, IL). 125I-Protein A from NEN Life Science Products
(Boston, MA). Forskolin and 3-isobutyl-1-methylanthine (IBMX) were from
Calbiochem (La Jolla, CA). Column calibration standards were obtained
from Pharmacia (Uppsala, Sweden). Piperazine diacrylamide and other
electrophoresis reagents were from Bio-Rad. CHAPS, EGTA, EDTA, Tween
20, HEPES, and all other reagent grade chemicals were from Sigma.
Rabbit polyclonal anti-MLC20 antibodies were from Dr. James
Stull (University of Texas, Dallas, TX), mouse anti-HSP27 antibodies
were from Dr. Michael Welsh (University of Michigan, Ann Arbor, MI)
(13), rabbit anti-HSP20 antibodies were from Dr. Kanefusa Kato (5). Goat anti-mouse and anti-rabbit secondary antibodies were from Jackson
Immunochemical (West Grove, PA). All other reagents were of analytical
grade. Protein concentrations were determined by the modified Bradford
assay (Pierce).
Preparation of Vascular Smooth Muscle Strips--
Intact bovine
carotid arteries were obtained from an abattoir (Shapiro's
meatpackers, Augusta, GA), and umbilical cords were from the labor and
delivery suite of the Medical college of Georgia and placed directly in
cold HEPES buffer (140 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM
NaH2PO4, 1.5 mM CaCl2,
10 mM glucose, and 10 mM Hepes, pH 7.4). The
adventitia was dissected from the carotid arteries and the endothelial
lining was denuded with a cotton-tipped applicator. Human umbilical
arteries were dissected free of Wharton's jelly. The arteries were
opened longitudinally, the endothelium denuded with a cotton-tipped
applicator, and thin (1 mM in diameter) transverse strips
were cut. Vessel viability was determined by concurrent muscle bath
experiments as described previously (3). The strips were equilibrated
for at least 60 min in Krebs bicarbonate buffer (KRB, 120 mM NaCl, 4.7 mM KCl, 1.0 mM
MgSO4, 1.0 mM NaH2PO4,
10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3) at 37 °C bubbled
with 95% O2, 5% CO2 to maintain a pH of 7.4. The strips were left in buffer alone (unstimulated) or treated with
IBMX (1 mM) and FSK (10 µM) for 10 min.
In Vitro Phosphorylation of Vascular Smooth Muscle
Homogenates--
Strips of bovine carotid artery smooth muscle were
homogenized in 10 mM Tris, 10 mM EDTA, 5 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 100 mM sodium fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, pH 7.4, and centrifuged
10,000 × g. The supernatants (1.2 mg of protein) were
phosphorylated in a reaction mixture containing 40 mM Tris,
pH 7.4, 20 mM magnesium acetate, 10 mM
K2HPO4, and 40 nM of the catalytic
subunit of cAMP-dependent protein kinase. The reaction was
initiated with the addition of 200 µM
[
-32P]ATP (800 cpm/pM) and incubated for
30 min at 30 °C. The reaction was stopped by the addition (1:1, v:v)
of 6.25 mM Tris, pH 6.8, 2% SDS, 5% 2-mercaptoethanol,
10% glycerol, 0.025% bromphenol blue and boiling for 5 min. The
proteins were acetone precipitated and re-suspended in 9 M
urea, 2% CHAPS, and 100 mM dithiothreitol for 24 h at
room temperature.
In Situ Phosphorylation--
Strips of bovine carotid artery
smooth muscle were incubated in low phosphate KRB containing 150 µCi/ml [32P]orthophosphate at 37 °C, for 4 h.
After stimulation, the vessels were snap frozen in liquid nitrogen,
ground to a fine powder with a mortar and pestle, and re-suspended in
90% acetone, 10% trichloroacetic acid, 10 mM
dithiothreitol. The suspension was quickly refrozen in liquid nitrogen
and then allowed to slowly return to room temperature. The suspension
was centrifuged (10,000 × g) and washed three times in
acetone. The pellets were dried under a stream of nitrogen and
re-solubilized in 9 M urea, 2% CHAPS, and 100 mM dithiothreitol for 24 h at room temperature. The
samples were then adjusted to 9 M urea, 2% CHAPS,
100 mM dithiothreitol, 15% glycerol, and 5% ampholines
(5 parts 6-8, 3 parts 5-7, and 2 parts 3-10).
Gel Electrophoresis--
Two-dimensional gel electrophoresis
using a modification of the method of O'Farrell and Hoschstrasser
et al. (14-16). In brief, 50 µg of protein was loaded
onto 12 × 15-cm slab isofocusing gels consisting of 4%
piperazine diacrylamide, 9 M urea, 5% ampholines (5 parts
6-8, 3 parts 5-7, and 2 parts 3-10), and 2% CHAPS. Temed (0.04%)
and ammonium persulfate (0.1%) were used to initiate polymerization. The cathode buffer consisted of 20 mM sodium hydroxide and
the anode buffer 10 mM phosphoric acid. The proteins were
focused for 10,000 V h. The gels were fixed in 10% trichloroacetic
acid and stained overnight with Neuhoff's Coomassie stain (17). The lanes were cut and applied to a 12% SDS-polyacrylamide gel. The gels
were dried and exposed to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Gel Filtration--
The strips of vascular smooth muscles
homogenized in 25 mM Hepes, 150 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, 2 mM
benzamidine, pH 7.4 (0.5 g tissue/1 ml of buffer), at 4 °C using a
Polytron homogenizer (Brinkman Instruments, Westbury, NY). The
homogenate was centrifuged 100,000 × g for 30 min at
4 °C. 200 µl of supernatant (containing 200 µg of total protein)
was applied to a Superose-6 HR 10/30 fast protein liquid chromatography
column (Pharmacia, Uppsala, Sweden). Fractions of 0.5 ml were
collected. For calibration, 100 µl of the following standards:
thyroglobulin (669 kDa, 5 mg/ml), catalase (232 kDa, 5 mg/ml), aldolase
(158 kDa, 5 mg/ml), and bovine serum albumin (67 kDa, 8 mg/ml) were
applied to the column.
Subcellular Fractionation--
The strips were homogenized in
homogenization buffer (25 mM Hepes, 150 mM
NaCl, 10 mM EDTA, 1 mM dithiothreitol, 2 mM benzamidine, pH 7.4 (0.5 g tissue/1 ml of buffer)) in a
Polytron homogenizer at 4 °C. The homogenate was centrifuged
1,000 × g to remove debris and the nuclear pellet. The
supernatants were diluted to 1 µg of protein/µl and 200 µl of
supernatant was centrifuged 10,000 × g. The pellet was
resuspended in 100 µl of homogenization buffer and 100 µl of 2 × sample buffer (6.25 mM Tris, pH 6.8, 2% SDS, 5%
2-mercaptoethanol, 10% glycerol, 0.025% bromphenol blue). To 100 µl
of the 10,000 × g supernatant, 100 µl of 2 × sample buffer was added. The remaining 100 µl of 10,000 × g supernatant was centrifuged 100,000 × g.
The 100,000 × g pellet was resuspended in 50 µl of
homogenization buffer and 50 µl of 2 × sample buffer. 50 µl
of 2 × sample buffer was added to the 100,000 × g supernatant. The samples were boiled for 8 min after the
addition of sample buffer.
Immunoblotting--
100 µl of each fraction from the column
was dot blotted onto nitrocellulose. The blots were fixed with 20%
methanol, dried, blocked with TBS (10 mM Tris, 150 mM NaCl, 0.5% Tween-20, pH 7.4), 5% milk, for 1 h,
washed 3 times with TBS, and then probed with anti-HSP27 antibodies
(1:4000 dilution in TBS, 5% milk) or anti-HSP20 antibodies (1:5000
dilution in TBS, 5% milk) for 1 h. The blots were washed 6 times
with TBS, 0.5% Tween and then with 125I-protein A,
125I-protein G, or enhanced chemiluminescence reagents (Pierce).
Data Analysis--
Values are reported as mean ± S.E. and
n refers to the number of experiments on tissues from
different animals. The statistical differences between two groups was
determined with Student's t test and between multiple
groups with one-way repeated measures analysis of variance (ANOVA)
using Sigma Stat software (Jandel Scientific, San Rafeal, CA). A
p value less than 0.05 was considered significant.
Densitometric analysis was performed with a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA).
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RESULTS |
cAMP-dependent Phosphorylation--
Treatment of
strips of bovine carotid artery smooth muscle with the adenylyl cyclase
activator, forskolin (10 µM), and the phosphodiesterase
inhibitor, IBMX (1 mM), for 10 min leads to increases in
the phosphorylation of HSP20 (Fig. 1 and
Table I). In a muscle that is uniquely
refractory to cyclic nucleotide-dependent relaxation, human
umbilical smooth muscle (3), treatment with forskolin (10 µM) and IBMX (1 mM) did not lead to
significant increases in the phosphorylation of HSP20 (Fig. 1, Table
I). HSP20 can be phosphorylated in vitro by the catalytic
subunit of cAMP-dependent protein kinase using homogenates
of either bovine carotid artery smooth muscle or human umbilical artery
smooth muscle (Fig. 1, Table I). While there are two phosphorylated isoforms of HSP20 in carotid artery smooth muscles with isoelectric points of 5.7 and 5.9, there is only one phosphorylated isoform of
HSP20 in umbilical artery smooth muscle, with a pI of 5.9. Other human
vascular tissues contain only the isoform with a pI of 5.9 (3). Thus,
the isoform with a pI of 5.9 is likely the most important isoform for
cyclic nucleotide-dependent vasorelaxation.

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Fig. 1.
Cyclic AMP-dependent
phosphorylation in bovine carotid artery smooth muscle. Whole cell
phosphorylation and two-dimensional gel electrophoresis was performed
on strips of carotid artery smooth muscle (Panels A and
B) and umbilical artery smooth muscle (Panels C
and D) that were unstimulated (Panels A and
C) or stimulated with forskolin and
3-isobutyl-1-methylxanthine (Panels B and D).
Homogenates of carotid (Panels E and F) and
umbilical (Panels G and H) were phosphorylated
in vitro in the absence (Panels E and
G) or presence of cAMP-dependent protein kinase
(Panels F and H). The arrowheads refer
to the relative mobility of HSP27 (12) and the arrows to the
relative mobility of HSP20 (1).
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Table I
Cyclic AMP-dependent phosphorylation in vascular smooth
muscle
Intact strips of carotid (BCA) and umbilical (HUA) artery smooth
muscles were phosphorylated in situ and left unstimulated (CONTROL) or
stimulated with IBMX and FSK. The proteins were separated by
two-dimensional electrophoresis and the extent of phosphorylation of
the isoform of HSP20 with a pI of 5.9 was determined with a
PhosphorImager and the results are reported as relative
densitometric units. The extent of phosphorylation of the three
isoforms of HSP27 were also determined and the total amount of
phosphorylated HSP27 is reported. A total of five experiments is
reported. Homogenates from carotid and umbilical arteries were also
phosphorylated in vitro in the absence (CONTROL) and presence of the
catalytic subunit of cAMP-dependent protein kinase (PKA).
The relative extent of the phosphorylation of HSP20 and HSP27 were
determined with a PhosphorImager and the results are reported as
relative densitometric units. A total of three experiments is reported.
The results are expressed as the mean ± S.E.; *,
p < 0.05 umbilical compared to carotid; #,
p < 0.05 stimulated compared to control.
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Activation of cyclic nucleotide-dependent signaling
pathways by treatment of carotid or umbilical artery smooth muscles
with IBMX and forskolin did not lead to increases in the
phosphorylation of HSP27 (Fig. 1, Table I). The in vitro
phosphorylation with PKA led to increases in the phosphorylation of
HSP27 in umbilical but not carotid arterial homogenates. Overall, there
was more phosphorylated HSP27 in umbilical compared with carotid artery smooth muscles.
Subcellular Fractionation--
To determine if HSP20 and HSP27
were predominantly in cytosolic fractions of vascular smooth muscles,
subcellular fractionation was performed on bovine carotid artery smooth
muscles. Both HSP20 and HSP27 were in the 10,000 and 100,000 × g supernatant fractions (Fig.
2). There was some immunoreactive HSP20
and HSP27 in the 10,000 × g pellet from the
unstimulated umbilical but not carotid artery homogenates (data not
shown). There was no immunoreactive HSP20 in the particulate fraction
from umbilical artery homogenates after IBMX/FSK stimulation (data not
shown). The distribution of HSP20 and HSP27 did not change with
IBMX/FSK treatment of carotid arteries.

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Fig. 2.
Subcellular fractionation of HSP20 and
HSP27. Strips of carotid artery smooth muscle were incubated in
bicarbonate buffer alone (lanes 1-4) or stimulated with
3-isobutyl-1-methylxanthine and forskolin for 10 min (lanes
5-8). The muscles were homogenized and the post-nuclear
supernatant was centrifuged 10,000 × g followed by
100,000 × g. The 10,000 × g
supernatant is shown in lanes 1 and 5;
10,000 × g pellet, lanes 2 and
6; 100,000 × g supernatant, lanes
3 and 7; and the 100,000 × g pellet,
lanes 4 and 8. The fractions were separated by
SDS-polyacrylamide gel electrophoresis (15% gels), transferred to
Immobilon, and the blots probed with antibodies against HSP20
(Panel A) or HSP27 (Panel B). The blots are
representative of three separate experiments.
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Macromolecular Aggregates of Heat Shock Proteins and Contractile
Elements--
Bovine carotid or human umbilical artery smooth muscles
were treated with buffer alone or with forskolin (10 µM)
and IBMX (1 mM), for 10 min. The strips were homogenized
and cytosolic supernatants were subjected to gel filtration as
described previously (18). Immunoblots of homogenates of carotid or
umbilical artery smooth muscles using specific polyclonal antibodies
against HSP20 (5) or specific monoclonal antibodies against HSP27 (13) produce single bands using either antibody. Thus, the fractions from
the gel filtration column were dot blotted and the amount of
immunoreactive protein was analyzed using a PhosphorImager. In
unstimulated carotid artery homogenates, the fraction containing the
peak amount of immunoreactive HSP20 was fraction 30 (Fig. 3, Panel A). Stimulation with
forskolin and IBMX led to a shift in the peak amount of immunoreactive
HSP20 to fraction 35 (Fig. 3, Panel A). In unstimulated
human umbilical artery homogenates, the fraction containing the peak
amount of immunoreactive HSP20 was again fraction 30 (Fig. 3,
Panel B). However, in homogenates from umbilical arteries
stimulated with forskolin and IBMX, which does not lead to relaxation
of these muscles (3) or to significant increases in the phosphorylation
of HSP20 (Fig. 3), the peak amount of immunoreactive HSP20 remained in
fraction 30 (Fig. 3, Panel C). The in vitro
phosphorylation of HSP20 using homogenates of bovine carotid arteries
also led to a shift of the peak amount of immunoreactive HSP20 from
fraction 30 to 35 (Fig. 3, Panel C). HSP20 can be
phosphorylated in vitro using homogenates of umbilical
artery smooth muscle (Fig. 1) and this was associated with a shift in
the peak amount of immunoreactivity from fraction 30 to 35 (Fig. 3,
Panel D).

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Fig. 3.
Dissociation as a result of
cAMP-dependent phosphorylation of HSP20. Strips of carotid
(Panels A and B) or umbilical (Panels
C and D) artery smooth muscle were stimulated with
forskolin and 3-isobutyl-1-methylxanthine (FSK/IBMX, Panels
A and C) or homogenates were phosphorylated in
vitro with the catalytic subunit of cAMP-dependent
protein kinase (PKA, Panels B and D). Cytosolic
supernatants were separated on a molecular sieving column and
immunoreactive HSP20 (relative densitometric units) was measured in
each of the column fractions (fraction number). The unstimulated
tissues are represented with a solid line and the stimulated
(FSK/IBMX or PKA) tissues with a dotted line. These results
are representative of five separate experiments.
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In unstimulated carotid artery homogenates, the fraction containing the
peak amount of immunoreactive HSP27 was fraction 35 (Fig.
4, Panel A). Stimulation with
forskolin and IBMX did not lead to a shift in the peak amount of
immunoreactive HSP27 (Fig. 4, Panel A). In unstimulated
human umbilical artery homogenates, there were two fractions with
immunoreactive HSP27, fractions 27 and 35 (Fig. 4, Panel B),
and stimulation with forskolin and IBMX did not lead to a shift in the
amount of immunoreactive HSP27 (Fig. 4, Panel C). The
in vitro phosphorylation of homogenates of either carotid or
umbilical arteries by PKA did not lead to a change in the fractions
containing immunoreactive HSP27 (Fig. 4, Panels B and
D).

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Fig. 4.
HSP27 exists in macromolecular aggregates
that do not dissociate with cAMP-dependent
phosphorylation. Strips of carotid (Panels A and
B) or umbilical (Panels C and D)
artery smooth muscle were not stimulated with
3-isobutyl-1-methylxanthine (Panels A and C) or
homogenates were phosphorylated in vitro with the catalytic
subunit of cAMP-dependent protein kinase (Panels
B and D). Cytosolic supernatants were separated on a
molecular sieving column and immunoreactive HSP27 (relative
densitometric units) was measured in each of the column fractions
(fraction number). The unstimulated tissues are represented with a
solid line and the stimulated tissues with a dotted
line. These results are representative of five separate
experiments.
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To determine if the aggregates of the small heat shock proteins
associated with specific elements of the contractile machinery, immunoblotting with antibodies against
-actin and the myosin light
chains (MLC20) was performed on column fractions. In
unstimulated bovine carotid artery homogenates, there was
immunoreactive MLC20 in fraction 30, but not in fraction 35 (Fig. 5A). After stimulation with forskolin and IBMX, immunoreactive MLC20 was in
fraction 35, but not in fraction 30. In the human umbilical
homogenates, the immunoreactive MLC20 was present in
fraction 35 in both unstimulated and IBMX/forskolin-treated muscles
(Fig. 5B). Immunoblots of samples from fractions 30 and 35 were also probed with antibodies against
-actin. Immunoreactive
-actin was present in fractions 30 and 35 from homogenates of both
carotid and umbilical arteries (data not shown).

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Fig. 5.
The regulatory myosin light chains.
Strips of carotid (Panel A) or umbilical (Panel
B) artery smooth muscle were not stimulated (solid
lines) or stimulated with forskolin and
3-isobutyl-1-methylxanthine (FSK/IBMX, dotted lines).
Cytosolic supernatants were separated on a molecular sieving column and
immunoreactive myosin light chains (relative densitometric units) was
measured in each of the column fractions (fraction number).
Representative immunoblots from column fractions 30 (lanes 1 and 3) and 35 (lanes 2 and 4) from
unstimulated muscles (lanes 1 and 2) or muscles
stimulated with FSK/IBMX (lanes 3 and 4) are
depicted in the upper right of each panel. The relative
mobility of molecular weight markers is noted on the left of
each immunoblot. These results are representative of three separate
experiments.
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To determine the apparent molecular weight of the macromolecular
aggregates, a calibration column was performed using proteins of known
molecular weight (Fig. 6). The catalase
standard (232 kDa) eluted from the column just after fraction 30 and
the aldolase standard (158 kDa) eluted just before fraction
35.

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Fig. 6.
Calibration of the Superose 6 column.
The following standards were applied to the Superose 6 column:
thyroglobulin (669 kDa, 5 mg/ml, peak 1), catalase (232 kDa,
5 mg/ml, peak 2), aldolase (158 kDa, 5 mg/ml, peak
3), and bovine serum albumin (67 kDa, 8 mg/ml, peak 4)
were applied to the column. The elution of each standard was confirmed
by the relative mobility of the proteins in each fraction on gel
electrophoresis (data not shown).
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DISCUSSION |
Cyclic nucleotide-dependent relaxation of vascular
smooth muscle is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20 (1). In this investigation, we
demonstrate that this increase in phosphorylation is associated with a
dissociation of macromolecular aggregates of HSP20 in a muscle that
displays normal cyclic nucleotide-dependent vasorelaxation, bovine carotid artery. The non-phosphorylated HSP20 eluted from the
column just before the catalase standard (232 kDa) and the phosphorylated HSP20 just before the aldolase standard (158 kDa). Thus,
the macromolecular aggregates of non-phosphorylated HSP20 are in an
aggregate with a molecular mass greater than 232 kDa and the
phosphorylated HSP20 in an aggregate with a molecular mass less than
158 kDa. Since the phosphorylated HSP20 was clearly in an aggregate
with a molecular mass greater than 67 kDa, it appears that the HSP20
aggregates do not dissociate completely into monomers after maximal
activation of cyclic nucleotide-dependent signaling
pathways with IBMX and forskolin.
In a muscle that is uniquely refractory to cyclic
nucleotide-dependent vasorelaxation, human umbilical artery
smooth muscle, HSP20 is not phosphorylated (3, 4). The macromolecular
aggregates of HSP20 from umbilical smooth muscle do not dissociate with
activation of cyclic nucleotide-dependent signaling
pathways. However, HSP20 is present in umbilical smooth muscle and can
be phosphorylated in vitro with the catalytic subunit of
cAMP-dependent protein kinase (PKA). The in
vitro phosphorylation of HSP20 is associated with disaggregation
of macromolecular aggregates.
Another small heat shock protein, HSP27, is a predominant protein in
vascular smooth muscle and is phosphorylated by receptor-initiated signaling cascades involving MAPKAP kinase-2 in smooth muscles (12).
Both HSP20 and HSP27 are predominantly cytosolic proteins in vascular
smooth muscles. HSP27 also exists in macromolecular aggregates in
bovine carotid artery smooth muscle, however, the aggregates do not
dissociate with activation of cyclic nucleotide-dependent signaling pathways. In carotid artery smooth muscle, HSP27 is in a
similar fraction as the HSP20 after stimulation with IBMX and
forskolin, suggesting that phosphorylated HSP20 may exist in a complex
with HSP27. In the umbilical artery smooth muscle immunoreactive
HSP27 was in two fractions and did not change after stimulation
of the tissues with IBMX and forskolin.
The initiation of vascular smooth muscle contraction is associated with
increases in the phosphorylation of the MLC20 (19). In this
investigation, we determined that the MLC20 were in the same fraction as non-phosphorylated HSP20 in cytosolic homogenates of
unstimulated bovine carotid artery smooth muscle. Stimulation of strips
of carotid artery smooth muscle with IBMX and forskolin led to a shift
of the MLC20 to fraction 35. This is the same fraction as
the HSP20 was in after IBMX and forskolin treatment. However, in both
unstimulated and IBMX/forskolin-treated umbilical artery smooth muscle,
the MLC20 were in fraction 35, suggesting that phosphorylated HSP20 may exist in an aggregate with the
MLC20. Immunoreactive
-actin was present in both
fractions 30 and 35 from both tissues. Using an actin co-sedimentation
assay, we have recently demonstrated that phosphorylated recombinant
HSP20 is associated with globular actin whereas non-phosphorylated
recombinant HSP20 is associated with filamentous actin (20). In
addition,
-actin co-immunoprecipitates with HSP20 (20). Thus, while
the precise mechanism by which HSP20 modulates vasorelaxation are not
known, these data suggest that HSP20 is associated with specific elements of the contractile machinery,
-actin and MLC20,
and may modulate vasorelaxation through a direct interaction with contractile elements.
While these studies examine vascular smooth muscles from different
arterial beds and different species, these two tissues display marked
differences in physiologic responses to activation of cyclic
nucleotide-dependent signaling pathways. Thus, these two
different tissues represent a useful model to compare the events
associated with cyclic nucleotide-dependent vasorelaxation. In addition, the doses of IBMX and forskolin used to activate cyclic
nucleotide-dependent cellular signaling pathways are
essentially maximal doses of each agent. These doses of IBMX and
forskolin lead to rapid and complete relaxation of agonist
pre-contracted carotid, but not umbilical artery smooth muscles (3).
Treatment of umbilical artery smooth muscles with IBMX and forskolin
does not lead to significant increases in the phosphorylation of HSP20 and in fact the amount of HSP20 phosphorylation after IBMX and forskolin treatment of umbilical artery smooth muscle did not exceed
basal levels of HSP20 phosphorylation in carotid arteries.
The data using these two muscles suggests that the small heat shock
proteins, HSP20 and HSP27, are predominantly cytosolic proteins in
vascular smooth muscles. Cyclic nucleotide-dependent increases in the phosphorylation of HSP20 leads to dissociation of
macromolecular aggregates of HSP20. In addition, activation of cyclic
nucleotide-dependent signaling pathways in vascular smooth
muscles leads to changes in the macromolecular associations of
MLC20. Thus, HSP20 may be an important downstream signaling mediator of cyclic nucleotide-dependent vasorelaxation and
may mediate relaxation through direct interaction with specific
elements of the contractile machinery.