Phosphorylation of the Small Heat Shock-related Protein, HSP20, in Vascular Smooth Muscles Is Associated with Changes in the Macromolecular Associations of HSP20*

Colleen M. BrophyDagger §parallel **, Mary Dickinson§, and David Woodrum§parallel Dagger Dagger

From the Departments of Dagger  Surgery, § Medicine (Institute for Molecular Medicine and Genetics),  Cell Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912 and the parallel  Augusta Veterans Administration Medical Center, Augusta, Georgia 30910

    ABSTRACT
Top
Abstract
Introduction
References

Cyclic nucleotide-dependent vasorelaxation is associated with increases in the phosphorylation of a small heat shock-related protein, HSP20. We hypothesized that phosphorylation of HSP20 in vascular smooth muscles is associated with alterations in the macromolecular associations of HSP20. Treatment of bovine carotid artery smooth muscles with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and the adenylate cyclase activator, forskolin, led to increases in the phosphorylation of HSP20 and dissociation of macromolecular aggregates of HSP20. However, 3-isobutyl-1-methylxanthine and forskolin treatment of a muscle that is uniquely refractory to cyclic nucleotide-dependent vasorelaxation, human umbilical artery smooth muscle, did not result in increases in the phosphorylation of HSP20 or to dissociation of macromolecular aggregates. HSP20 can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (PKA) in both carotid and umbilical arteries and this phosphorylation of HSP20 is associated with dissociation of macromolecular aggregates of HSP20. Activation of cyclic nucleotide-dependent signaling pathways does not lead to changes in the macromolecular associations of another small heat shock protein, HSP27. Interestingly, the myosin light chains (MLC20) are in similar fractions as the HSP20, and phosphorylation of HSP20 is associated with changes in the macromolecular associations of MLC20. These data suggest that increases in the phosphorylation of HSP20 are associated with changes in the macromolecular associations of HSP20. HSP20 may regulate vasorelaxation through a direct interaction with specific contractile regulatory proteins.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    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). [gamma -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 beta -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 [gamma -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 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).

    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.

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.

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.

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.

To determine if the aggregates of the small heat shock proteins associated with specific elements of the contractile machinery, immunoblotting with antibodies against alpha -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 alpha -actin. Immunoreactive alpha -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.

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).


    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 alpha -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, alpha -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, alpha -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.

    ACKNOWLEDGEMENTS

We thank James Stull, Kanefusa Kato, and Mike Welsh for generously supplying antibodies, the labor and delivery nurses at the Medical College of Georgia, for umbilical cords, Shapiro's Meatpackers for bovine carotid arteries, and Shannon Lamb for technical assistance.

    FOOTNOTES

* This work was supported in part by an American Health Association Clinician Scientist Award (to C. M. B.), a Veterans Administration Merit Review Award, and National Institutes of Health Grant RO1 HL58027-01.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Institute for Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th St., Augusta, GA 30912. Tel.: 706-721-4761; Fax: 706-823-2269; E-mail: colleenb{at}mail.mcg.edu.

Dagger Dagger Supported by the M.D./Ph.D. program at the Medical College of Georgia.

    ABBREVIATIONS

The abbreviations used are: HSP, heat shock protein; IBMX, 3-isobutyl-1-methylxanthine; CHAPS, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonic acid; Temed, N,N,N',N'-tetramethylethylenediamine; MLD20, myosin light chain.

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Abstract
Introduction
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