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Am J Physiol Regul Integr Comp Physiol 279: R492-R498, 2000;
0363-6119/00 $5.00
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Vol. 279, Issue 2, R492-R498, August 2000

Stress causes decrease in vascular relaxation linked with altered phosphorylation of heat shock proteins

Leslie C. Fuchs1,2, Ararat D. Giulumian2, Louis Knoepp3, Walter Pipkin3, Mary Dickinson4, Chad Hayles3, and Colleen Brophy2,3,4,5,6

Departments of 1 Pharmacology and Toxicology, 3 Surgery, 4 Medicine (Institute for Molecular Medicine and Genetics), and 5 Cell Biology and Anatomy and the 2 Vascular Biology Center, Medical College of Georgia, Augusta 30912; and the 6 Augusta Veterans Administration Medical Center, Augusta, Georgia 30910


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cyclic nucleotide-dependent vascular relaxation is associated with increases in the phosphorylation of a small heat shock protein (HSP), HSP20. An increase in phosphorylation of another small HSP, HSP27, is associated with impaired cyclic nucleotide-dependent vascular relaxation. Expression of HSPs is altered by exposure to several types of cellular stress in vitro. To determine if behavioral stress in vivo alters vascular expression and phosphorylation of the small HSPs and cyclic nucleotide-dependent vascular relaxation, borderline hypertensive rats were stressed by restraint and exposure to air-jet stress 2 h/day for 10 days or remained in their home cage. Stress impaired relaxation of aorta to forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This was associated with an increase in the aortic expression and phosphorylation of HSP27, which was localized to the vascular smooth muscle, but a decrease in the amount of phosphorylated (P)-HSP20. To determine if P-HSP27 inhibits phosphorylation of HSP20, P-HSP27 was added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of cAMP-dependent protein kinase. P-HSP27 inhibited phosphorylation of HSP20 in a concentration-dependent manner. These data demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. The increase in P-HSP27 and decrease in P-HSP20 were associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress in vivo, an effect similar to that observed previously in response to cellular stress in vitro.

behavioral stress; vascular smooth muscle; hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXPOSURE OF ORGANISMS TO STRESS leads to activation of the hypothalamic-pituitary-adrenal axis with the production and release of catecholamines. On a cellular level, stress is associated with the induction of a group of proteins termed heat shock proteins (HSPs). The heat shock response was first described in Drosophila in which exposure to temperatures above normal led to an increase in the expression of HSPs (23). Subsequently, it has been determined that expression of HSPs can be elicited by a number of factors besides heat, such as circulating catecholamines, heavy metals, arsenite, hypoxia, free radicals, and osmotic stress (for review, see Ref. 3).

HSPs are divided into five major families on the basis of molecular weight and function. Although the precise functions of the HSPs are not known, many HSPs act as "molecular chaperones" and assist in the assembly, disassembly, stabilization, and internal transport of intracellular proteins. Cells stressed by a mild insult are more resistant to a subsequent stress, and this response termed stress tolerance is mediated by HSPs (3). Thus the induction of stress proteins protects the organism from repeated stresses and has been shown to protect the ischemic heart (21).

The small HSPs (molecular mass 20-30 kDa) may have a role in the regulation of smooth muscle tone. HSP27 and HSP20 are highly and constituitively expressed in muscle tissues (17). Increases in the phosphorylation of HSP27 have been associated with smooth muscle contraction (10, 19). Antibodies against HSP27 inhibit bombesin-induced contraction of rectal sphincter smooth muscle (5). Increases in the phosphorylation of HSP20 are associated with cyclic nucleotide-dependent relaxation of vascular smooth muscle and cyclic nucleotide-dependent inhibition of agonist-induced contraction (2, 30). Increases in phosphorylation of HSP20 are also associated with endothelium-dependent vasodilation (15). The addition of phosphopeptide analogs of HSP20 into transiently permeabilized vascular smooth muscle attenuates agonist-induced contraction (1). HSP20 was first identified as a by-product of the purification of HSP27, and the two small HSPs coexist in macromolecular aggregates (8, 17). Thus HSP27 and HSP20 may have interrelated roles in modulating the tone of smooth muscle, and the function of these proteins may be dependent on the phosphorylation state and macromolecular associations of the proteins.

Behavioral stress is associated with several vascular diseases including hypertension, atherosclerosis, and coronary artery disease (16, 24, 25). The borderline hypertensive rat (BHR), a first generation offspring of a female spontaneously hypertensive rat (SHR) and male Wistar Kyoto (WKY) rat, has been used extensively for studies on behavioral stress due to its sensitivity to several environmental challenges (26). Prolonged exposure of BHR to behavioral stress results in sustained hypertension that persists after removal of the stress (26). We have demonstrated that chronic behavioral stress (10 days) alters vascular contraction and relaxation in adult BHR (11, 12). These stress-induced alterations in vascular relaxation could contribute to pathogenesis of hypertension. Because HSPs are known modulators of vascular tone and are induced by several types of stress in vitro, the present study was designed to determine if exposure of BHR to behavioral stress in vivo alters vascular smooth muscle cyclic nucleotide-dependent relaxation and expression and phosphorylation of the small HSPs, HSP20 and HSP27.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Air-jet stress in BHR. Female SHR and male WKY rats were obtained from Taconic Farms (Germantown, NY) and bred at the Medical College of Georgia to obtain the first generation offspring BHR. Male BHR (13-14 wk) were randomly divided into two groups (control or stressed). The stressed group was restrained in tubular Plexiglas restrainers and exposed to air-jet stress for 2 h/day for 10 days, whereas the control group remained in their home cage for 10 days. Air-jet stress consisted of pulses of compressed air (15 lb./in.2) directed toward the face from an 1/8-in. opening at the front of the restrainer. Animals were subjected to a random duration of pulses (5 to 120 s) and interpulse intervals (5 to 120 s) 2 h/day for 10 days.

Hemodynamic measurements. On day 10, both stressed and unstressed rats were anesthetized with ketamine (50 mg/kg im) and acepromazine (16 mg/kg im). Under aseptic conditions, a cannula (PE-50 attached to PE-10) was placed in the femoral artery and exteriorized at the nape of the neck. The cannula was flushed with heparinized saline (100 U/ml). After a 1-day recovery period, arterial pressure and heart rate were monitored with a Grass recorder in unrestrained rats in their home cage. Heart rate was derived with a cardiotachometer that was triggered from the arterial pressure pulse.

Vascular reactivity. On day 11, rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The aorta was dissected and placed directly in cold HEPES buffer [(in mM) 140 NaCl, 4.7 KCl, 1.0 MgSO4, 1.0 NaH2PO4, 1.5 CaCl2, 10 glucose, and 10 HEPES, pH 7.4]. The endothelium was denuded mechanically by gently rubbing the luminal surface with forceps. This procedure results in complete removal of the endothelium, as verified previously with scanning electron microscopy and by the absence of relaxation to the endothelium-dependent vasodilator acetylcholine without affecting relaxation to the endothelium-independent vasodilator nitroprusside (15, 31). A portion of aorta was used for immunoblotting or immunofluorescence, whereas the remainder was used for vascular reactivity experiments.

Transverse rings, 1.0 mm in width, were cut, and each end was tied to a loop of 3-0 silk. The tissue was suspended in a muscle bath containing bicarbonate buffer [(in mM) 120 NaCl, 4.7 KCl, 1.0 MgSO4, 1.0 NaH2PO4, 10 glucose, 1.5 CaCl2, and 25 Na2HCO3, pH 7.4], equilibrated with 95% O2/5% CO2 at 37°C. The rings were fixed at one end to a stainless steel wire and attached to a Kent Scientific (Litchfield, CT) force transducer (TRN001) interfaced with a Data Translation A-D board, DT2801 (Data Translation, Marlboro, MA). Data was acquired with Daisylab software (Daisytec, Amherst, NH). Agonists were added directly to the bath, and the concentrations indicated represent the final concentration of the agonist. Vascular rings were progressively stretched, and isometric force generated in response to 110 mM KCl was monitored until optimal tension was produced (Lmax). After washing and equilibration for at least 30 min, vessels were precontracted with serotonin (10-6 M) for 15 min followed by the addition of either sodium nitroprusside (10-6 M) or forskolin (10-6 M). After each experiment, tissues were blotted and the wet weight was measured. Force was converted to stress (105 N/m2), calculated as force (g) × 0.0987/area, where area = wet weight (mg)/length (mm at Lmax)/1.055. The maximal relaxation response, which occurred within 10 min after sodium nitroprusside or forskolin treatment, was calculated as a percentage of the contractile response to serotonin.

Immunoblotting. Aortas were snap-frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle, and proteins were precipitated in 90% acetone, 10% trichloroacetic acid, and 10 mM dithiothreitol. The samples were centrifuged at 10,000 g, and the precipitated proteins were washed three times with acetone to remove the trichloroacetic acid. The proteins were then solublized in 6 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 10 mM dithiothreitol overnight at room temperature. The samples were centrifuged at 10,000 g, and the protein concentration of the supernatant was determined with a Pierce Coomassie Protein Assay Kit (Rockville, IL). Sample buffer (250 mM Tris, pH 6.8; 2% glycerol, 10 mM beta -mercaptoethanol, 2 mM EGTA, 4% SDS, 0.001% bromphenol blue) was added (1:1 dilution), and 30 µg of protein was separated on 15% SDS-PAGE gels and transferred to Immobilon for 210 V/h. The blots were air-dried and subsequently blocked with Tris-buffered saline (TBS; 10 mM Tris, 150 mM NaCl, pH 7.4)/5% milk for 1 h. The blots were then incubated with anti-HSP27 antibodies (1:10,000 dilution), phosphorylation state-specific HSP27 antibodies (1:500 dilution), anti-HSP20 antibodies (1:2,000 dilution), phosphorylation state-specific HSP20 antibodies (1:100 dilution), and anti-inducible HSP70 (1:1,000 dilution) in TBS/milk overnight at 4°C. The blots were washed three times (5 min each) in TBS/polyoxyethylenesorbitan monolaurate (Tween 20) (0.5%). Immunoreactive protein was determined with the use of 125I-protein A or enhanced chemiluminescence (DuPont NEN, Boston, MA). The blots were again washed six times (5 min each) in TBS/Tween 20 and exposed on PhosphorImager screens (Molecular Dynamics, Sunnyvale, CA) or X-ray film.

Immunofluorescence. Segments of rat aorta were fixed in 4% Formalin and embedded in paraffin. Six-micrometer cross sections were mounted on polylysine slides. The slides were deparaffinized with xylene and graded dilutions of ethanol. The sections were rinsed in PBS (10 mM phosphate, pH 7.5; 0.15 M NaCl) and incubated with anti-HSP27 antibodies (1:100 dilution) or phosphorylation state-specific HSP27 antibodies (1:100 dilution) for 12 h at 4°C. The sections were rinsed in PBS and incubated with Cy-3 conjugated donkey anti-mouse or goat anti-rabbit secondary antibodies (1:100 dilution) for 1 h at room temperature, rinsed again in PBS, and mounted with ProLong Antifade Kit (Molecular Probes, Eugene, OR). The slides were viewed with a Zeiss Axiophot microsocope interfaced with a digital Spot camera.

Cloning and expression of HSP20. For experiments determining the effect of phosphorylated (P)-HSP27 on phorphorylation of HSP20, recombinant HSP27 and recombinant HSP20 were used. Recombinant HSP27 was purchased from StressGen. The rat cDNA for HSP20 was PCR amplified with the use of sense (GAA TTC ATA TGG AGA TCC GGG TGC CTG TGC) and antisense (CGT ACT CGA GCT ACT TGG CAG CAG GTG GTG ACT) primers (synthesized by Gibco BRL, Grand Island, NY). The PCR products were ligated into PCR-script SK (+) cloning vector and ultimately transformed into BL21(DE3)pLysS cells (Promega, Madison, WI), as previously described (9). The HSP20 was affinity purified with the use of a HIS-bind resin column (Novagen, Madison, WI). Proteins from the fractions were separated on SDS-PAGE gels, and fractions containing a single band at 20 kDa were dialyzed against PBS/1% CHAPS.

In vitro phosphorylation of HSP20 and HSP27. Phosphorylation of HSP27 was accomplished by adding recombinant HSP27 (20 µg) to a reaction mixture with 0.25 U MAPKAP kinase II in 20 mM MOPS, pH 7.2, 25 mM beta -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol. The reaction was started with the addition of 200 µM ATP and stopped by separating the kinase from HSP27 with molecular sieving columns (Centricon 40, Amicon, Beverly, MA).

The effect of either nonphosphorylated HSP or P-HSP27 on phosphorylation of HSP20 was measured in a reaction mixture containing recombinant HSP20 (0.1 µg), 20 mM Tris (pH 7.4), 10 mM magnesium acetate, 40 nM of the catalytic subunit of cAMP-dependent protein kinase (PKA), and either P-HSP27 (0.01-1.0 µg) or nonphosphorylated HSP27 (0.01-1.0 µg). The reaction was initiated with the addition of 200 µM gamma -[32P]ATP (800 counts  ·  min-1  ·  pmol-1) followed by incubation for 30 min at 30°C. The reaction was stopped by the addition of 6.25 mM Tris (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, and 0.025% bromphenol blue and boiling for 5 min (1:1, vol/vol). The proteins were separated on 15% SDS-PAGE gels, and an autoradiogram was obtained.

Chemicals. HEPES and CHAPS were obtained from American Bioanalytical (Natick, MA). SDS, glycine, and Tris were from Research Organics (Cleveland, OH). 125I-protein A and 125I-protein were from Amersham (Arlington Heights, IL). Forskolin was from Calbiochem (La Jolla, CA). Piperazine diacrylamide and other electrophoresis reagents were from Bio-Rad (Hercules, CA). Sodium nitroprusside, serotonin, EGTA, EDTA, Tween 20, alpha -actin antibodies, and all other reagent grade chemicals were from Sigma (St. Louis, MO). Antibodies against HSP27 were from Dr. Michael Welsh (Ann Arbor, MI) (29) and those against HSP20 were from Dr. Kanefusa Kato (Aichi, Japan) (17). Phosphorylation state-specific anti-HSP20 and anti-HSP27 antibodies were produced and characterized as previously described (1, 18). Antibodies against the inducible form of HSP70 (iHSP70), gamma -[32P]ATP, 125I-protein A, 125I-IgG were from Amersham. Anti-mouse and anti-rabbit secondary antibodies were from Jackson Immunochemical (West Grove, PA). The catalytic subunit of cAMP-dependent PKA was purchased from Promega. MAPKAP kinase II was purchased from Upstate Biotechnology (Lake Placid, NY). Recombinant HSP27 was from StressGen (Victoria, BC, Canada).

Data analysis. Values are reported as means ± SE and n refers to the number of animals examined. The statistical difference between two groups was determined with Student's t-test and between multiple groups with one-way, repeated-measures ANOVA with the use of Sigma Stat software (Jandel Scientific, San Rafeal, CA). A P value <0.05 was considered significant. Densitometric analysis was performed on immunoblots in which 125I-protein A (for rabbit primary antibodies) or 125I-IgG (for mouse primary antibodies) was used as the secondary antibody with a PhosphorImager (Molecular Dynamics) and ImageQuant software (Molecular Dynamics).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hemodynamic measurements. Hemodynamic measurements were obtained from conscious, unrestrained BHR after 10 days of stress (stressed group, n = 10) or after 10 days in their home cage (control group, n = 9). Resting mean arterial pressure was 125 ± 3 mmHg in control BHR and 126 ± 4 mmHg in stressed BHR, and heart rate was 340 ± 15 beats/min in control BHR and 328 ± 12 beats/min in stressed BHR. Therefore, chronic stress did not significantly alter resting mean arterial pressure or heart rate in BHR.

Vascular reactivity. There were no significant differences in the magnitude of the contractile response to KCl (110 mM; Fig. 1A) or serotonin (10-6 M; Fig. 1B) in aortic smooth muscle from stressed compared with control BHR. To determine if air-jet stress led to alterations in smooth muscle relaxation responses, aortic smooth muscle was precontracted with serotonin (10-6 M), and the magnitude of relaxation in response to sodium nitroprusside (10-6 M) or to forskolin (10-6 M) was determined. These doses of sodium nitroprusside and forskolin led to complete relaxation of control aortic smooth muscle. However, in aortic smooth muscle from air-jet stressed BHR, the magnitude of the relaxation response to both sodium nitroprusside (Fig. 2A) and forskolin (Fig. 2B) was significantly less than from smooth muscle obtained from control animals.


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  		Fig. 1.   Contraction of isolated aorta harvested from control (C) and stressed (S) borderline hypertensive rat (BHR). The magnitude of the contractile responses to KCl (A; 110 mM) or serotonin (5-HT; B, 10-6 M) is shown (no significant differences were observed; n = 6-8 per group).



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  		Fig. 2.   Cyclic nucleotide-dependent relaxation of isolated aorta harvested from C and S BHR. Aorta were precontracted with serotonin (10-6 M), and the relaxation response to sodium nitroprusside (A; 10-6 M) or forskolin (B; 10-6 M) is shown (* P < 0.05 vs. C, n = 6-8 per group).

HSP expression and phosphorylation. Behavioral stress has been shown to increase expression of iHSP70, an indication of a stress response (28). To determine if stress led to induction of HSP70 in our model, immunoblotting was performed with the use of antibodies that recognize iHSP70. There were significant increases in the amount of immunoreactive iHSP70 in aorta from stressed compared with control BHR (Fig. 3).


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  		Fig. 3.   Expression of the inducible form of heat shock protein 70 (iHSP70) in aortic smooth muscle homogenates from C and S BHR. Homogenates were probed with antibodies against iHSP70. Top: representative immunoblots. The results of densitometric analysis of multiple blots are depicted graphically (* P < 0.05 vs. C, n = 6 per group).

The next set of experiments determined if stress led to alterations in the expression or phosphorylation of the small HSPs. Aortic smooth muscle tissue from control and air-jet treated BHR was homogenized, separated by SDS-PAGE, and transferred to Immobilon. The blots were probed with mouse monoclonal antibodies specific for HSP27 (29), affinity purified rabbit polyclonal antibodies that were produced with the use of phosphopeptides containing the serine 78 and 82 phosphorylation sites of HSP27 (18), affinity purified rabbit antibodies that were produced with the use of HSP20 purified from rat skeletal muscle (17), and affinity purified rabbit polyclonal antibodies that were produced with the use of phosphopeptides containing the serine 16 phosphorylation site of HSP20 (1). All of the antibodies produced a single band on blots from 15% SDS-PAGE gels with rat aortic homogenates, suggesting that the antibodies were specific.

In aortic smooth muscle homogenates from stressed animals, there was more immunoreactive HSP27 and immunoreactive P-HSP27 compared with control (Fig. 4). Immunoreactive P-HSP27 was very low in control homogenates. Immunoreactive HSP20 was increased in homogenates from stressed compared with control BHR. However, the expression of P-HSP20 was markedly decreased by exposure to stress (Fig. 5). Equal protein loading was confirmed by immunoblotting for alpha -actin. There were no differences in the amounts of immunoreactive alpha -actin between the aorta from stressed and control BHR (10.2 ± 0.9 vs. 11.4 ± 1.2 relative densitometric units, respectively, P > 0.05, n = 6).


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  		Fig. 4.   Expression and phosphorylation of HSP27 in aortic smooth muscle homogenates from C and S BHR. Homogenates were probed with antibodies against HSP27 or the phosphorylated isoforms of HSP27 (P-HSP27). Top: representative immunoblots. The results of densitometric analysis of multiple blots are depicted graphically (* P < 0.05 vs. C, n = 6-8 per group).



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  		Fig. 5.   Expression and phosphorylation of HSP20 in aortic smooth muscle homogenates from C and S BHR. Homogenates were probed with antibodies against HSP20 or P-HSP20. Top: representative immunoblots. The results of densitometric analysis of multiple blots are depicted graphically (* P < 0.05 vs. C, n = 6-8 per group).

Immunolocalization of HSP27. To determine the localization of HSP27, immunofluorescence studies were performed on Formalin-fixed, paraffin-embedded aortas from stressed and control BHR. Exposure to stress increased aortic immunofluorescent staining for HSP27. The staining pattern indicated that the immunoreactive HSP27 was diffusely cytoplasmic in the medial smooth muscle cells of the aortic wall (Fig. 6). Stress also increased immunofluorescent staining for P-HSP27. The immunoreactive P-HSP27 was localized to smooth muscle cells at the adventitial ( A) and lumenal ( L) aspect of the media. The only fluorescence detected when the primary antibody was ommitted or when mouse ascitic fluid or nonspecific rat serum was used as a primary antibody was the elastic lamella, which autofluoresced (data not shown). The antibodies used for HSP20 and for P-HSP20 did not produce consistent or specific staining in Formalin-fixed tissues.


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  		Fig. 6.   Immunofluorescence micrographs of rat aorta harvested from C or S BHR. Aorta were fixed in Formalin and probed with antibodies against HSP27 or P-HSP27 followed by Cy-3 conjugated secondary antibodies. The adventitia ( A) and lumen ( L) are denoted. All of the micrographs are in the same orientation. Immunoreactive HSP27 is denoted with an arrowhead, and the autofluorescing elastic lamella is denoted with an arrow. Magnification ×40.

Effect of P-HSP27 on phosphorylation of HSP20. Our findings demonstrate that stress increases P-HSP27 and decreases P-HSP20. Previous studies (7, 18, 30) suggest that stress-induced increases in P-HSP27 leads to impaired phosphorylation of HSP20. All of these conditions are associated with impaired cyclic nucleotide-dependent relaxation. The following experiment was designed to determine if P-HSP27 could inhibit HSP20 phosphorylation in vitro. Recombinant HSP27 was phosphorylated by MAPKAP kinase II, and the MAPKAP kinase II was removed with molecular sieving columns. Increasing concentrations of nonphosphorylated HSP27 or P-HSP27 were then added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of PKA. Nonphosphorylated HSP27 had no effect on phosphorylation of HSP20 (Fig. 7A). P-HSP27 inhibited the phosphorylation of HSP20 in a concentration-dependent and linear (r2 = 0.982) fashion (Fig. 7B). P-HSP27 did not inhibit the phosphorylation of another PKA substrate, KEMPtide (data not shown).


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  		Fig. 7.   Effect of P-HSP27 on phosphorylation of HSP20 in vitro. Increasing concentrations of nonphosphorylated HSP27 (A) and P-HSP27 (B) were added to a reaction mixture containing recombinant HSP20 and the catalytic subunit of protein kinase. The proteins were probed with antibodies against P-HSP20. The concentrations of HSP27 and P-HSP27 in the reaction mixture are depicted at bottom. This blot is representative of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BHR have been used extensively for studies on behavioral stress because they are sensitive to several environmental challenges (26). Prolonged exposure of BHR to behavioral stress results in sustained hypertension that persists after removal of the stress (26). However, as we observed previously (11, 12), exposure to only 10 days of air-jet stress did not alter the resting arterial pressure of conscious BHR in this study, allowing for determination of behavioral stress-induced changes in aortic vascular reactivity and HSP expression and phosphorylation before development of hypertension. In the present study, stress impaired relaxation of aortic smooth muscle to two substances that activate cyclic nucleotide-dependent signaling pathways, forskolin, which activates adenylyl cyclase, and sodium nitroprusside, which activates guanylyl cyclase. This decreased ability of the vascular smooth muscle to relax, which could contribute to development of stress-induced hypertension, was associated with an increase in expression of the phosphorylated isoform of HSP27 and a decrease in expression of the phosphorylated isoform of HSP20. These effects may be due to restraint, air-jet stress, or a combination of both stressors.

There is increasing evidence of a role for HSPs in vascular diseases such as hypertension. Hypertension induced by stress leads to increased expression of the stress-inducible form of HSP70 in vascular and adrenal tissue (28). Another large HSP, HSP90, modulates the function of nitric oxide synthase. Increased nitric oxide production in the mesenteric vasculature of portal hypertensive rats was mediated by HSP90 (27). Currently, the role of the small HSPs, HSP20 and HSP27, in hypertension is unknown. However, the small HSPs have been implicated in regulation of vasomotor tone.

Increases in the phosphorylation of HSP20 are associated with cyclic nucleotide- and endothelium-dependent vascular relaxation (2, 15). Phosphorylation of HSP27 is associated with impaired cyclic nucleotide-dependent relaxation (18). Additional data demonstrating an association between the phosphorylation of the small HSPs and smooth muscle tone include the finding that umbilical artery smooth muscle contains high levels of HSP27 and P-HSP27 and is uniquely refractory to cyclic nucleotide-dependent relaxation (4, 7, 20, 22). In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways with forskolin does not increase phosphorylation of HSP20 (30). Finally, the addition of phosphopeptide analogs of HSP20 into transiently permeabilized vascular smooth muscle inhibits agonist-induced contractions (1). The findings of the present study provide additional evidence that small HSPs modulate vascular tone. The stress-induced increase in phosphorylation of HSP27 and decrease in the amount of the phosphorylated isoform of HSP20 in aortic medial smooth muscle were associated with impaired cyclic nucleotide-dependent relaxation.

HSP20 and HSP27 share considerable sequence homology and appear to be biochemically associated in muscle tissues (8, 17). The decreased phosphorylation of HSP20 observed in aorta from stressed BHR in the present study, in umbilical smooth muscle (30), and in bovine carotid artery smooth muscle treated with arsenite (18) in previous studies may be due to a direct inhibition of the phosphorylation of HSP20 by P-HSP27. Our studies on the effect of phosphorylated recombinant HSP27 on the phosphorylation of recombinant HSP20 by PKA in vitro support this hypothesis. HSP27 that was phosphorylated in vitro by MAPKAP kinase II inhibited PKA-induced phosphorylation of HSP20 in a concentration-dependent manner. However, P-HSP27 did not inhibit the phosphorylation of another PKA substrate, KEMPtide, showing that P-HSP27 is not a direct inhibitor of PKA.

The mechanism by which HSP20 and HSP27 modulate vascular tone is unknown. Our studies suggest that the functions of HSP20 and HSP27 may be dependent on macromolecular associations with each other and the phosphorylation state of the HSPs. HSP20 and HSP27 can also interact with specific elements of the contractile machinery (8, 9, 17). HSP27 is an actin-binding protein and modulates actin filament dynamics (13). HSP27 has also been associated with thin filament regulatory proteins such as tropomyosin, caldesmon, and calponin (14). HSP20 binds to actin in vitro, and the association with actin is dependent on the phosphorylation state of HSP20 (9). Thus the small HSPs, HSP27 and HSP20, may modulate smooth muscle tone by interacting with specific elements of the contractile machinery, such as the thin filament actin and/or thin filament regulatory proteins, and by modulating the function of these proteins.

In conclusion, behavioral stress led to increases in the expression and phosphorylation of HSP27 and decreases in the amount of P-HSP20 in BHR aortic smooth muscle. These alterations in small HSP expression and phosphorylation were associated with impaired cyclic nucleotide-dependent relaxation of vascular smooth muscle. Finally, P-HSP27 inhibited the ability of PKA to phosphorylate HSP20 in vitro. These findings demonstrate that P-HSP27 can inhibit phosphorylation of HSP20. Additionally, the decrease in phosphorylation of HSP20 was associated with reduced cyclic nucleotide-dependent vascular smooth muscle relaxation in response to behavioral stress, an effect similar to that observed previously in response to cellular stress (18).

Perspectives

The data in this manuscript provide a molecular mechanism through which behavioral stress-induced increases in the amount and phosphorylation of HSP27 lead to impaired vascular relaxation. This mechanism involves inhibition of phosphorylation of HSP20 that appears to contribute to cyclic nucleotide-dependent relaxation. Although the current data are primarily correlative, it provides a potential molecular mechanism for impaired cyclic nucleotide-dependent relaxation. In addition, our data demonstrate that P-HSP27 can directly impair the phosphoylation of HSP20, supporting the hypothesis that a stochiometric relationship between the amounts of P-HSP20 and HSP27 may regulate the tone of vascular smooth muscle. These data are consistent with findings in human tissue (umbilical artery smooth muscle) and in another animal model (bovine carotid artery smooth muscle) ex vivo. Thus we believe these data provide a compelling molecular connection between stress and altered vascular smooth muscle responses.


    ACKNOWLEDGEMENTS

These experiments were supported in part by National Institutes of Health (NIH) Grant R29-HL-49924 to L. Fuchs and by a Veterans Administration Merit Review Award and NIH Grant RO1-HL-58027-01 to C. Brophy.


    FOOTNOTES

Address for reprint requests and other correspondence: L. C. Fuchs, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912 (E-mail: lfuchs{at}mail.mcg.edu).

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. §1734 solely to indicate this fact.

Received 11 January 2000; accepted in final form 7 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Beall, AC, Bagwell CA, Woodrum D, Stoming T, Kato K, Suzuki A, Rasmussen H, and Brophy CM. Heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation. J Biol Chem 274: 11344-11351, 1999[Abstract/Free Full Text].

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Am J Physiol Regul Integr Comp Physiol 279(2):R492-R498
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