Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery

Melissa K. Meeks, Marcia L. Ripley, Zhicheng Jin, and Christopher M. Rembold

Cardiovascular Division, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia

Submitted 7 June 2004 ; accepted in final form 21 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca2+])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser16-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser16-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser16-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser157. Ser157-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser16-HSP20 phosphorylation. The relationship between Ser16-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level.

cAMP; cGMP; nitric oxide; vascular smooth muscle


INCREASES IN CYCLIC NUCLEOTIDE LEVELS induce smooth muscle relaxation via two general mechanisms. One mechanism is deactivation, in which reductions in myoplasmic Ca2+ concentration ([Ca2+]i) or increases in myosin phosphatase activity induce relaxation by reducing myosin regulatory light chain (MRLC) phosphorylation. The second mechanism is force suppression, in which there is a relaxation without a proportional reduction in [Ca2+]i or MRLC phosphorylation (13, 14, 17). Force suppression can be observed with increased cGMP concentration induced by nitric oxide (NO) (13), phosphodiesterase inhibitors (5), or increased cAMP concentration (20). Other treatments that induce force suppression include okadaic acid (22), some Ca2+-depletion protocols (8), Ca2+ channel blockers (12), high extracellular Mg2+ concentration ([Mg2+]o) (6), and other combinations of excitatory and inhibitory stimuli (2). Force suppression is not caused by phosphorylation of MRLC on amino acid residues other than Ser19 (13). NO-induced force suppression appears to involve increased cGMP concentration, given that a membrane-permeable cGMP analog also induces forced suppression (17).

Cyclic nucleotide-induced smooth muscle relaxation was found to correlate with phosphorylation of heat shock protein 20 (HSP20) on Ser16 (3, 4, 10, 26). Rembold and colleagues (17, 20) found that Ser16-HSP20 phosphorylation correlated with force suppression rather than the deactivation form of relaxation. They noted that HSP20 has a sequence homology with troponin I: a peptide containing this homology bound to thin filaments, reduced actin-activated myosin S1 ATPase activity, and relaxed skinned smooth muscle (17). We hypothesized that binding of Ser16-phosphorylated HSP20 to the thin filament turned "off" thin filaments so that phosphorylated myosin was unable to interact with the thin filament (i.e., a model similar to that for skeletal muscle troponin I). Such a model would explain reduced force despite MRLC phosphorylation.

If Ser16-HSP20 phosphorylation regulates force suppression, then the dependence of relaxation on increases in Ser16-HSP20 phosphorylation could suggest a mechanism(s) responsible for force suppression. There are three hypothetical mechanisms. The first hypothesis is that a threshold level of Ser16-HSP20 phosphorylation could inactivate a thin filament as a whole, i.e., there could be cooperative inactivation of whole thin filaments by HSP20. This would be the expected result if Ser16-HSP20 phosphorylation were to interfere with actin filament binding to cytoskeletal structures such as actinin, as proposed by Tessier et al. (23). It would also be the expected result if relaxation were to be associated with mechanically disrupted or depolymerized thin filaments. Contractile agonists have been reported to induce thin filament polymerization (16), suggesting the possibility that relaxation could be associated with reversal of thin filament polymerization. If this mechanism were operative, then increases in Ser16-HSP20 phosphorylation should not alter force until a threshold is obtained. Above the threshold, further increases in Ser16-HSP20 phosphorylation should significantly decrease force to near zero. A schema showing the predictions of this first hypothesis is shown in Fig. 1A.



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Fig. 1. Hypothetical schema of the proposed mechanisms for heat shock protein 20 (HSP20)-mediated force suppression. A: predictions for model 1 [a threshold level of HSP20 phosphorylation (HSP20p) inactivates a whole thick filament, i.e., cooperative inactivation]. B: predictions for models 2 and 3 (regional inactivation of thin filaments). For each model, 4 panels are shown from left to right representing 0, 20, 40, and 60% Ser16-HSP20 phosphorylation, respectively. Each panel shows 5 representative series of equal signs (=), which represent regions of thin filaments. An extra line ({equiv}) indicates that there is a phosphorylated HSP20 in that region. A heavy line replacing the equal sign indicates that the region is inactivated. A shows cooperative inactivation: 20% phosphorylation did not inactivate any region, but once over a threshold of slightly <40% phosphorylation, there was inactivation of entire filaments. B shows regional inactivation.

 
A second hypothesis is that a small region of a thin filament could be fully inactivated by phosphorylation of a single HSP20 on Ser16, i.e., HSP20 regulates myosin binding locally. If so, then there should be a linear reduction in force with increases in Ser16-HSP20 phosphorylation with a common intersection at zero force (i.e., x-intercept). This would be the expected result if Ser16-HSP20 phosphorylation were to act like troponin, as proposed by Rembold et al. (17).

A third hypothesis is that Ser16-HSP20 phosphorylation weakly inhibits myosin binding at either the thin- or thick-filament level. If so, then increases in Ser16-HSP20 phosphorylation should produce parallel reductions in force (i.e., similar slope of the relationship). A schema showing the predictions of the second and third hypotheses is shown in Fig. 1B.

The goal of this study was to evaluate these potential mechanisms. We measured the steady-state relationship between Ser16-HSP20 phosphorylation and force in both maximally and submaximally stimulated swine carotid arteries. As a measure of thin filament polymerization and depolymerization, we measured stimulus-induced changes in the relative amounts of F- and G-actin. Finally, we also determined the second phosphorylation site on swine HSP20, the "PKC" site, and evaluated its physiology.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Tissues. Swine common carotid arteries were obtained from a slaughterhouse and transported at 0°C in physiological salt solution (PSS). PSS contained (in mM) 140 NaCl, 4.7 KCl, 2 3-(N-morpholino)propanesulfonic acid, 1.2 Na2HPO4, 1.6 CaCl2, 1.2 MgSO4, 5.6 D-glucose, and 0.02 EDTA, with pH adjusted to 7.4 at 37°C. Dissection of the arteries into 2- to 3-mm rings was performed, and the intimal surface was mechanically rubbed to remove the endothelium (18).

Carotid artery rings were mounted, bathed in PSS at 37°C, and stretched to a force of 10 g. After 30 min, the rings were contracted with PSS containing an extracellular K+ concentration ([K+]o) of 109 mM (KCl substituted stoichiometrically for NaCl). After relaxation in PSS, the rings were repeatedly stretched to ~20 g. The tissues were then released to ~5 g and recontracted with 109 mM [K+]o PSS. This protocol results in rings set within 5% of their optimal length (18). The second 109 mM [K+]o PSS contraction was used for normalizing further forces. After relaxation in PSS, the length of each ring was measured. Rings were then 1) untreated (control); 2) activated with 1 or 10 µM histamine in PSS for 60 min; 3) activated with histamine in PSS for 30 min and then relaxed by the addition of 0.1, 0.3, 1, 3, or 10 µM forskolin for 30 min; 4) depolarized with 25, 30, or 40 mM [K+]o PSS for 90 min; or 5) depolarized with 25, 30, or 40 mM [K+]o PSS for 30 min and then relaxed by the addition of 0.1, 0.3, 1, 3, or 10 µM forskolin for 60 min. The longer times were required with [K+]o depolarization to attain stable steady-state force values. Rings were then frozen in an acetone-dry ice slurry. After air drying, the tissues were weighed and homogenized in a buffer containing 1% SDS, 10% glycerol, 20 mM dithiothreitol, and 0.05% bromphenol blue (20 mg wet wt/ml buffer).

Measurement of HSP20 and MRLC phosphorylation. Rabbit anti-HSP20 antibody was made commercially via repeated injection of gel-purified recombinant HSP20 (sequence confirmed by mass spectroscopy). After confirmation of an antigenic response, serum was collected and frozen for future use. Rabbit anti-MRLC antibodies were a gift from Subah Packer. Full-strength, half-strength, and quarter-strength dilutions of samples were then separated on one-dimensional isoelectric focusing gels (ampholytes were a 50:50 mixture of pI 5–8 and pI 4–6.5 for HSP20 and a 50:50 mixture of pI 4.5–5.4 and pI 4.0–6.5 for MRLC, where pI is the isoelectric point), blotted to nitrocellulose, immunostained with our rabbit polyclonal anti-HSP20 antibody (1:5,000) or rabbit polyclonal anti-MRLC antibody (1:4,000 in 1% bovine serum albumin and 0.01% sodium azide), and detected with enhanced chemiluminescence (ECL) (19). The dilutions ensured that the ECL detection system was in the linear range (19). Immunoblots were imaged with a digital camera and quantitated with UnScanIt software.

Cyclic nucleotide assays. Swine carotid rings were frozen as described in Tissues, homogenized in 0.1 M HCl, and analyzed for cAMP and cGMP by radioimmunoassay as described previously (15).

HSP20 phosphorylation sites. Unstimulated swine carotid rings were frozen as described in Tissues, homogenized, and centrifuged at 14,000 g for 10 min, and the supernatant proteins were separated first by isoelectric focusing (pI range 5–7) followed by 12% polyacrylamide gel electrophoresis (1, 7). Coomassie blue-stained spots containing HSP20 were excised from heavily loaded two-dimensional gels, minced, destained, alkylated, and trypsinized as described previously (9). Peptides were then run on a POROS 10 RC reversed-phase microcapillary HPLC, and its output was directed into a Finnigan-MAT TSQ7000 electrospray tandem mass spectroscope (9). Collisionally activated dissociation spectra were interpreted, and the proposed peptide fragment sequences were then compared with published protein sequences.

F/G-actin content. The relative amount of filamentous actin (F-actin) vs. globular-actin (G-actin) content in swine carotid arteries was determined with a commercial kit (BK037) from Cytoskeleton (Denver, CO). Lysis/F-actin stabilization buffer contained 50 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 50 mM NaCl, 5 mM MgCl2, 5 mM EGTA, 5% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% Tween 20, and 0.1% {beta}-mercaptoethanol, pH 6.9 at 30°C. Immediately before lysis/F-actin stabilization buffer was used, 100 mM ATP (10 µl/ml) and protease inhibitor cocktail (10 µl/ml: 1 µg/ml pepstatin, 1 µg/ml leupeptin, 10 µg/ml benzamidine, and 500 µg/ml tosylarginine methyl ester) were added.

Arteries were mounted, pharmacologically treated as described above, and then frozen with tongs cooled in liquid nitrogen. The frozen carotid was weighed and pulverized in a liquid nitrogen-chilled ceramic mortar and pestle. The resultant powder was divided equally between two chilled 1.5-ml Eppendorf tubes (the assay was performed in duplicate to increase accuracy). Lysis/F-actin stabilization buffer (50 µl/mg tissue wet) along with ATP and protease inhibitor cocktail (prewarmed at 30°C) were added to each tube. The tubes were then immediately vortexed and incubated at 30°C for 10 min. They were then immediately centrifuged at 16,000 g for 1 h at room temperature in an Eppendorf 5415C microcentrifuge. The supernatants were pipetted off the pellets, placed in empty 1.5-ml Eppendorf tubes, and placed on ice. The pellets were resuspended to the same volume as the supernatants by using ice-cold nanopure water plus 1 µM cytochalasin D. The pellet samples were left on ice (with vortexing every 15 min) for 1 h to dissociate F-actin. Half- and quarter-strength dilutions of each pellet and full-strength supernatant were then separated on 12% SDS-polyacrylamide gels, blotted to nitrocellulose, immunostained with a commercially produced rabbit polyclonal anti-actin antibody (AAN01; Cytoskeleton), and detected with ECL. Images were analyzed using UnScanIt software. Intensities were corrected for dilution, and the amount of F-actin as a percentage of total actin was calculated. The dilutions were corrected for offset and saturation errors as done previously with analyses of MRLC and HSP20 phosphorylation (20, 24).


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Identification of HSP20 phosphorylation sites. Figure 2 shows a two-dimensional gel from a swine carotid tissue that was not treated with agents that increase cAMP or cGMP concentration. The spot labeled S157 was excised and sequenced using mass spectroscopy; this spot was identified as HSP20 that was monophosphorylated on Ser157. Table 1 shows the predicted human, rat, and swine protein sequences based on published DNA sequences compared with the mass spectroscopy sequence labeled "protein." A mass spectroscopy sequence was obtained on all but 24 residues of HSP20 (the missing residues are shown in lowercase). The only difference between the sequenced protein and the predicted sequence from DNA sequencing was at residue 64: a valine in the protein sequence vs. a threonine in the DNA sequence. Swine HSP20 is similar to rat HSP20, which was previously shown to have three extra residues (compared with human) at locations 155–157, including a serine at 157 (25). These three extra residues, including Ser157, were also present in mouse and bovine HSP20. Figure 1 has other spots that were previously identified using mass spectroscopy (17). Of interest is the circle in Fig. 2 labeled S16 and S157 showing the location of the spot previously identified as Ser16-phosphorylated HSP20. This spot likely represents HSP20 diphosphorylation at both Ser16 and Ser157 (3, 20); however, prior sequencing did not include Ser157.



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Fig. 2. Two-dimensional gel subsequently used for mass spectroscopy. Six spots are labeled: S157, HSP20 phosphorylated on Ser157, sequenced with mass spectroscopy as described in text; S16 & S157, HSP20 phosphorylated on Ser16, sequenced with mass spectroscopy as described in Ref. 17 (this spot is also phosphorylated on Ser157, as indicated by mobility); HSP27, sequenced with mass spectroscopy as described in Ref. 17; and myosin regulatory light chain (MRLC), actin, and tropomyosin as identified in Ref. 7.

 

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Table 1. HSP20 sequences predicted from DNA and from mass spectroscopy sequencing including phosphorylation sites

 
Biochemical events during forskolin-induced relaxation. We measured the steady-state relationships among HSP20 phosphorylation, MRLC phosphorylation, and force in maximally and submaximally stimulated swine carotid artery relaxed using various concentrations of forskolin, a direct activator of adenylyl cyclase. Figure 3 shows all the biochemical measures and force data plotted against forskolin dose, and Fig. 4 shows the dependence of force on Ser19-MRLC phosphorylation.



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Fig. 3. Biochemical correlates of forskolin-induced relaxation in swine carotid artery contracted with histamine or a high extracellular K+ concentration ([K+]o). Tissues were unstimulated (open or filled diamonds); stimulated with 10 µM histamine and relaxed with 0.3, 1, 3, or 10 µM forskolin (filled circles at left); stimulated with 1 µM histamine and relaxed with 0.3, 0.6, 1, or 3 µM forskolin (filled squares at left); depolarized with 40 mM [K+]o and relaxed with 0.3, 1, 3, or 10 µM forskolin (open circles at right); depolarized with 30 mM [K+]o and relaxed with 0.3, 1, or 3 µM forskolin (open squares at right); or depolarized with 25 mM [K+]o and relaxed with 0.1, 0.3, or 1 µM forskolin (open triangles at right). Contraction times are given in MATERIALS AND METHODS. A: Ser16-HSP20 phosphorylation. B: Ser157-HSP20 phosphorylation. C: Ser19-MRLC phosphorylation. D: active stress as a fraction of a prior 109 mM [K+]o contraction (K109 force). The dotted line in A at right represents data for 10 µM histamine plus forskolin from A, left. Data are presented as means ± SE with n = 4–7. Some error bars are obscured by symbols.

 


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Fig. 4. Dependence of contractile stress on Ser19-MRLC phosphorylation in swine carotid artery. A: dependence of stress on Ser19-MRLC phosphorylation in tissues stimulated with 0.3, 1, 3, or 10 µM histamine (filled diamonds) or depolarized with 40, 30, or 25 mM [K+]o (open diamonds; data are from Fig. 3). These data were fit with a Hill plot, which is shown as a solid line [regression equation stress = 1.12 x Mp8.83/(0.208.83 + Mp8.83), where Mp is MRLC phosphorylation; r2 = 0.88]. B: dependence of stress on Ser19-MRLC phosphorylation in tissues stimulated with histamine and relaxed with forskolin (filled circles) or depolarized with [K+]o and relaxed with forskolin (open circles; data are from Fig. 3). The solid line represents the regression from data for histamine and [K+]o alone from A. Data are presented as means ± SE with n = 4–7. Some error bars are obscured by symbols.

 
Figure 4A shows the relationship between Ser19-MRLC phosphorylation and force induced by histamine or high [K+]o. Unstimulated (control) tissues had low force and Ser19-MRLC phosphorylation values of 0.14 ± 0.02 mol Pi/mol MRLC. Activation with histamine (Fig. 4A, filled diamonds) or high-[K+]o depolarization (open diamonds) increased both force and Ser19-MRLC phosphorylation such that a sigmoidal dependence of force on Ser19-MRLC phosphorylation was observed. A Hill curve fit revealed half-maximal force when Ser19-MRLC phosphorylation was 0.20 mol ± 0.01 Pi/mol MRLC. This relationship allows us to define forskolin-induced force suppression.

Increasing forskolin concentration caused dose-dependent reductions in force without significant changes in Ser19-MRLC phosphorylation (Fig. 3, C and D). Force suppression is demonstrated as a MRLC phosphorylation-force value significantly below (or to the right of) the sigmoidal dependence of force on Ser19-MRLC phosphorylation as shown in Fig. 4A. Addition of forskolin to histamine-stimulated tissues reduced force significantly below the regression line consistent with force suppression (Fig. 4B, filled circles). Addition of forskolin to high-[K+]o-depolarized tissues also reduced force so that the mean force values fell primarily below the line; however, most of the error bars included the regression line, so force suppression was not clearly demonstrable (Fig. 4B, open circles). Because high [K+]o alone induced MRLC phosphorylation values on the steep portion of the force-MRLC phosphorylation relationship, it was difficult to demonstrate force suppression as a downward shift in the relationship.

Ser157-HSP20 phosphorylation values were high in resting tissues and did not change with histamine stimulation or high-[K+]o depolarization (Fig. 3B). Addition of forskolin appeared to slightly decrease Ser157-HSP20 phosphorylation, although values remained quite high.

Ser16-HSP20 phosphorylation values were low in resting tissues and did not change with histamine stimulation or high-[K+]o depolarization (Fig. 3A). Increasing forskolin concentration increased Ser16-HSP20 phosphorylation and relaxed swine carotid artery in a dose-dependent manner. The concentration of histamine that activated the tissues did not affect forskolin-induced increases in Ser16-HSP20 phosphorylation. Similarly, the level of high-[K+]o depolarization did not affect forskolin-induced increases in Ser16-HSP20 phosphorylation. However, compared with high-[K+]o-depolarized tissues, the histamine-stimulated tissues had higher Ser16-HSP20 phosphorylation values when relaxed with 0.3 and 1 µM forskolin (dotted line in Fig. 3A, right).

The dependence of force on Ser16-HSP20 phosphorylation is detailed in Fig. 5 and Table 2. When tissues were activated by 1 or 10 µM histamine or 25 or 30 mM [K+]o, addition of forskolin produced a similar slope and x (HSP20 phosphorylation)-intercept in the dependence of force on Ser16-HSP20 (Table 2). When tissues were activated with 40 mM [K+]o, the dependence of force on Ser16-HSP20 phosphorylation had a significantly greater x (HSP20 phosphorylation)-intercept than during the other four stimulation protocols (slope appeared lower but did not reach statistical significance, P = 0.051).



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Fig. 5. Dependence of contractile stress on Ser16-HSP20 phosphorylation in swine carotid artery. Data are replotted from Fig. 3: filled circles represent 10 µM histamine plus forskolin, filled squares represent 1 µM histamine plus forskolin, open circles represent 40 mM [K+]o plus forskolin, open squares represent 30 mM [K+]o plus forskolin, and open triangles represent 25 mM [K+]o forskolin. Data are presented as means ± SE with n = 4–7. Some error bars are obscured by symbols.

 

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Table 2. Regression analysis of dependence of force on Ser16 HSP20 phosphorylation as determined by the form of contractile stimulation before addition of forskolin

 
F-actin changes during cyclic nucleotide-induced relaxation. Stimulation of swine carotid artery with 10 µM histamine significantly increased the relative amount of F-actin, decreased G-actin, increased Ser19-MRLC phosphorylation, increased stress, and did not change cAMP or cGMP concentration (Fig. 6). This increase in F-actin confirms prior studies in dog trachealis (16). Addition of 100 µM nitroglycerin or 1 µM forskolin to 10 µM histamine-stimulated tissues induced a significant relaxation that was not associated with a significant change in the relative amounts of F- or G-actin compared with histamine alone (Fig. 6, A and B). Addition of nitroglycerin to histamine-stimulated tissues significantly increased cGMP concentration without changing cAMP concentration (Fig 6C). Addition of forskolin to histamine-stimulated tissues significantly increased cAMP concentration without changing cGMP concentration. Addition of either forskolin or nitroglycerin to histamine-stimulated tissues was associated with force suppression (Fig. 6D, inset).



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Fig. 6. Stimulus-induced changes in actin polymerization. Six sets of swine carotid artery tissues were unstimulated (control), contracted with 10 µM histamine for 40 min, contracted with 10 µM histamine for 10 min followed by addition of 100 µM nitroglycerin for 30 min, or contracted with 10 µM histamine for 10 min followed by addition of 1 µM forskolin for 30 min. After freezing and pulverizing, tissues were analyzed for F-actin as a percentage of total actin (A). Data are also presented as percent change in G-actin (normalized to control contraction, B). When analyzed using a Kruskal-Wallis one-way ANOVA on ranks, the percent change in G-actin was significantly less for all 3 other treatments compared with control (*P = 0.015). A second set of swine carotid artery was treated identically and analyzed for cAMP (C, filled bars), cGMP (C, open bars), and MRLC phosphorylation (D). cAMP and cGMP are presented as pmol/mg wet wt on a log scale. E: contractile stress shown as a fraction of a prior 109 mM [K+]o contraction (forces from tissues analyzed in the F/G-actin assay are represented by filled bars, and forces from tissues analyzed for cAMP/cGMP/MRLC phosphorylation are represented by open bars). When analyzed using a one-way ANOVA, contractile stress for all 3 other treatments was significantly different from control (*), and contractile stress after histamine stimulation with the addition of nitroglycerin or forskolin was significantly different from that after histamine stimulation alone (@). D, inset: the dependence of contractile stress on Ser19-MRLC phosphorylation is shown using data replotted from left: filled circles represent 10 µM histamine plus forskolin or nitroglycerin, open circles represent unstimulated tissue or the histamine stimulation alone, and the solid line is the Hill plot from Fig. 4A.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our goal was to evaluate the mechanism whereby Ser16-HSP20 phosphorylation regulates smooth muscle force by analyzing the dependence of force on Ser16-HSP20 phosphorylation with partial activation of swine carotid media. This analysis was predicated on the general hypothesis that Ser16-HSP20 phosphorylation is the mediator of force suppression.

Our first hypothesis, that HSP20 mediates full inactivation of thin filaments, predicts a sigmoidal dependence of force on Ser16-HSP20 phosphorylation (see schema in Fig. 1). This was not observed (Fig. 5), and therefore, the first hypothesis (full inactivation of thin filaments) is not supported. These data therefore suggest that Ser16-HSP20 phosphorylation-induced relaxation is not likely to act via disruption of the linkage of thin filaments to cytoskeletal structures (23).

Prior studies suggested that contractile activation is associated with thin filament polymerization (16). We found that histamine stimulation of swine carotid artery increases the relative amount of F-actin compared with G-actin and is in agreement with this prior report (Fig. 6). Of interest, we employed a different method in a different tissue, suggesting that the increase in F-actin with activation is robust. We performed this analysis to test the hypothesis that force suppression was a reversal of this process, i.e., it was associated with F-actin depolymerization. This was not observed (Fig. 6). Relaxation induced by nitroglycerin or forskolin induced force suppression (Fig. 6D, inset) but did not alter the relative amount of F- or G-actin compared with histamine stimulation alone. Data for both the dependence of force on Ser16-HSP20 phosphorylation (Fig. 5) and the F/G-actin polymerization (Fig. 6) suggest that force suppression is not caused by a large depolymerization of thin filaments. It should be noted that the F/G-actin ratio will not detect changes in the structure of F-actin if changes do not alter the total amount of F-actin.

The second hypothesis, that HSP20 mediates regional inactivation of thin filaments, predicts that there should be a common x-intercept in the dependence of force on Ser16-HSP20 phosphorylation. We found similar x (HSP20 phosphorylation)-intercepts in tissues activated with 1 or 10 µM histamine or 25 or 30 mM [K+]o and then relaxed with forskolin. Tissues activated with 40 mM [K+]o had a significantly higher x (HSP20 phosphorylation)-intercept than tissues stimulated with the other four stimuli. It is possible that the higher [Ca2+]i induced by 40 mM [K+]o could be responsible for the increase in the x-intercept. This is the subject of further study.

The third hypothesis, that HSP20 mediates weak inhibition of myosin binding at either the thin- or thick-filament level, predicts that there should be a common slope of the dependence of force on Ser16-HSP20 phosphorylation. We did not find significantly different slopes with the five stimuli that were relaxed with forskolin, although it should be noted that the P value for the ANOVA was 0.051, suggesting the possibility of a type II error.

Our data did not clearly delineate between the second and third hypotheses. These data suggest that Ser16-HSP20 phosphorylation reduces force by either regional thin filament inhibition or weak inhibition of myosin binding at the thin- or thick-filament level. Our data do not allow us to determine whether this effect occurs at the level of cross-bridge attachment or maintenance of bound cross bridges. Further research is required to determine how such a mechanism could function.

We determined that the second phosphorylation site on swine carotid was Ser157. This is the site identified in rat platelets by Wang et al. (25) and is likely to be the same site of phosphorylation described by Beall et al. (3) in the COOH terminus of HSP20. In platelets, Ser157-HSP20 phosphorylation was mediated by insulin (25). In the swine carotid artery, Ser157-HSP20 phosphorylation was high and remained high with activation and with forskolin-induced relaxation. There was no clear relationship between Ser157-HSP20 phosphorylation and force (Fig. 3). Interestingly, Ser157 is not present in human HSP20 (11).

When tissues were activated with histamine, forskolin-induced relaxation was clearly associated with force suppression (Fig. 4). However, when tissues were depolarized with high [K+]o, forskolin-induced force suppression was more difficult to measure. This resulted from the steepness of the Ser19-MRLC phosphorylation-force relationship. Therefore, minor inaccuracies in measurement of Ser19-MRLC phosphorylation make it difficult to analyze the degree of force suppression in [K+]o-depolarized tissues. This result does not imply that force suppression does not occur when tissues are depolarized with high [K+]o; it suggests that it is difficult to measure. The steepness of the Ser19-MRLC phosphorylation-force relationship suggests the presence of cooperativity in the regulation of force by Ser19-MRLC phosphorylation: this is the subject of another investigation by investigators in our laboratory (21).

Our results confirm that forskolin induces force suppression in the histamine-stimulated swine carotid. If Ser16-HSP20 phosphorylation is found to be the mediator of force suppression, we propose that the mechanism for this force suppression is regional thin filament inhibition or weak inhibition of myosin binding at the thin or thick filament. We also found that the second phosphorylation site of swine HSP20 is Ser157.


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This research was supported by National Heart, Lung, and Blood Institute Grant HL-71191.


    ACKNOWLEDGMENTS
 
We thank Robin Woodson for technical support. Dr. Subah Packer graciously supplied the MRLC antibody. Smithfield Inc. (Smithfield, VA) donated the swine carotid arteries.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. M. Rembold, Box 801395, Cardiovascular Division, Univ. of Virginia Health System, Charlottesville, VA 22908-1395 (E-mail: crembold{at}virginia.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. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aksoy MO, Mras S, Kamm KE, and Murphy RA. Ca2+, cAMP, and changes in myosin phosphorylation during contraction of smooth muscle. Am J Physiol Cell Physiol 245: C255–C270, 1983.[Abstract]

2. Bárány M and Bárány K. Dissociation of relaxation and myosin light chain dephosphorylation in porcine uterine muscle. Arch Biochem Biophys 305: 202–204, 1993.[CrossRef][ISI][Medline]

3. Beall A, Bagwell D, Woodrum D, Stoming TA, Kato K, Suzuki A, Rasmussen H, and Brophy CM. The small 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]

4. Beall AC, Kato K, Goldenring JR, Rasmussen H, and Brophy CM. Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a small heat shock-related protein. J Biol Chem 272: 11283–11287, 1997.[Abstract/Free Full Text]

5. Chuang AT, Strauss JD, Steers WD, and Murphy RA. cGMP mediates corpus cavernosum smooth muscle relaxation with altered cross-bridge function. Life Sci 63: 185–194, 1998.[CrossRef][ISI][Medline]

6. D'Angelo EKG, Singer HA, and Rembold CM. Magnesium relaxes arterial smooth muscle by decreasing intracellular [Ca2+] without changing intracellular [Mg2+]. J Clin Invest 89: 1988–1994, 1992.[ISI][Medline]

7. Driska SP, Aksoy MO, and Murphy RA. Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am J Physiol Cell Physiol 240: C222–C233, 1981.[Abstract]

8. Gerthoffer WT. Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle. J Pharmacol Exp Ther 240: 8–15, 1987.[Abstract]

9. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, and Watanabe C. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90: 5011–5015, 1993.[Abstract/Free Full Text]

10. Jerius JH, Karolyi DR, Mondy JS, Beall AC, Wootton D, Ku D, Cable S, and Brophy CM. Endothelial-dependent vasodilation is associated with increases in the phosphorylation of a small heat shock protein (HSP20). J Vasc Surg 29: 678–684, 1999.[ISI][Medline]

11. Kato K, Goto S, Inaguma Y, Hasegawa K, Morishita R, and Asano T. Purification and characterization of a 20-kDa protein that is highly homologous to alpha-B-crystallin. J Biol Chem 269: 15302–15309, 1994.[Abstract/Free Full Text]

12. Katoch SS, Rüegg JC, and Pfitzer G. Differential effects of a K+ channel agonist and Ca2+ antagonists on myosin light chain phosphorylation in relaxation of endothelin-1-contracted tracheal smooth muscle. Pflügers Arch 433: 472–477, 1997.[CrossRef][ISI][Medline]

13. McDaniel NL, Chen XL, Singer HA, Murphy RA, and Rembold CM. Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i, [Ca2+]i sensitivity, and uncoupling stress from myosin phosphorylation. Am J Physiol Cell Physiol 263: C461–C467, 1992.[Abstract/Free Full Text]

14. McDaniel NL, Rembold CM, and Murphy RA. Cyclic nucleotide dependent relaxation in vascular smooth muscle. Can J Physiol Pharmacol 72: 1380–1385, 1994.[ISI][Medline]

15. McDaniel NL, Rembold CM, Richard HL, and Murphy RA. cAMP relaxes arterial smooth muscle predominantly by decreasing cell [Ca2+]. J Physiol 439: 147–160, 1991.[Abstract]

16. Mehta D and Gunst SJ. Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J Physiol 519: 829–840, 1999.[Abstract/Free Full Text]

17. Rembold CM, Foster B, Strauss JD, Wingard CJ, and Van Eyk JE. cGMP mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation. J Physiol 524: 865–878, 2000.[Abstract/Free Full Text]

18. Rembold CM and Murphy RA. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res 63: 593–603, 1988.[Abstract]

19. Rembold CM and O'Connor MJ. Caldesmon and heat shock protein 20 in nitroglycerin- and magnesium induced relaxation of swine carotid artery. Biochim Biophys Acta 1500: 257–264, 2000.[ISI][Medline]

20. Rembold CM, O'Connor MJ, Clarkson M, Wardle RL, and Murphy RA. HSP20 phosphorylation in nitroglycerin- and forskolin-induced sustained reductions in swine carotid media tone. J Appl Physiol 91: 1460–1466, 2001.[Abstract/Free Full Text]

21. Rembold CM, Wardle RL, Wingard CJ, Batts TW, Etter EF, and Murphy RA. Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation. Am J Physiol Cell Physiol 287: C594–C602, 2004.[Abstract/Free Full Text]

22. Tansey MG, Hori M, Karaki H, Kamm KE, and Stull JT. Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle. FEBS Lett 270: 219–221, 1990.[CrossRef][ISI][Medline]

23. Tessier D, Komalavilas P, Panitch A, Joshi D, and Brophy CM. The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin. J Surg Res 111: 152–157, 2003.[CrossRef][ISI][Medline]

24. Walker JS, Walker LA, Etter EF, and Murphy RA. A dilution immunoassay to measure myosin regulatory light chain phosphorylation. Anal Biochem 284: 173–182, 2000.[CrossRef][ISI][Medline]

25. Wang Y, Xu A, Pearson RB, and Cooper GJS. Insulin and insulin antagonists evoke phosphorylation of P20 at serine 157 and serine 16 respectively in rat skeletal muscle. FEBS Lett 462: 25–30, 1999.[CrossRef][ISI][Medline]

26. Woodrum DA, Brophy CM, Wingard CJ, Beall A, and Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol Heart Circ Physiol 277: H931–H939, 1999.[Abstract/Free Full Text]





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