Cardiovascular Division, Department of Internal Medicine, University of Virginia Health System, Charlottesville, Virginia
Submitted 7 June 2004 ; accepted in final form 21 October 2004
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ABSTRACT |
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cAMP; cGMP; nitric oxide; vascular smooth muscle
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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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.
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MATERIALS AND METHODS |
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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 58 and pI 46.5 for HSP20 and a 50:50 mixture of pI 4.55.4 and pI 4.06.5 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 57) 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% -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).
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RESULTS |
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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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DISCUSSION |
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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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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