| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
MS 0041 Received 1 September 1999; accepted after revision 30 January 2000.
 |
ABSTRACT |
|---|
 TopAbstract IntroductionMethodsResultsDiscussionReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Phosphorylation of the 20 kDa regulatory light chain of myosin (MLC) on Ser19 is the primary determinant of myosin's ATPase activity and contraction in arterial smooth muscle (Hai & Murphy, 1989). Most, but not all, stimuli induce a single steady-state relation between MLC phosphorylation and force (Ratz et al. 1989). One exception to regulation of force by MLC phosphorylation is the sustained phase of nitrovasodilator-induced relaxation, i.e. relaxation induced by cyclic 3':5'-guanosine monophosphate (cGMP). Initially, the nitrovasodilator nitroglycerine relaxed swine carotid artery by reducing both myoplasmic [Ca2+] ([Ca2+]i) (McDaniel et al. 1992) and the [Ca2+]i sensitivity of MLC phosphorylation (Van Riper et al. 1997). However, after 10 min of nitroglycerine application, [Ca2+]i and MLC phosphorylation increased back to levels similar to those observed without addition of nitroglycerine (McDaniel et al. 1992; Bárány & Bárány, 1993). These data suggest that the sustained phase of nitroglycerine-induced relaxation was no longer regulated by [Ca2+]i or MLC phosphorylation. In essence, nitroglycerine induces relaxation despite the presence of activated myosin, i.e. myosin with phosphorylated MLC.
We tested several hypotheses: (1) nitroglycerine-induced relaxation without proportional MLC dephosphorylation results from increases in [cGMP]; (2) nitroglycerine-induced relaxation without proportional MLC dephosphorylation results from inhibition of myosin attachment to thin filaments; and (3) nitroglycerine-induced relaxation without proportional MLC dephosphorylation results from phosphorylation of a specific protein that interacts with the thin filament.
In this study, we provide evidence in support of all three hypotheses. We also confirmed the finding of C. M. Brophy's laboratory (Beall et al. 1997) that nitroglycerine-induced relaxation was associated with phosphorylation of heat shock protein 20 (HSP20).
|
METHODS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Tissues, myosin phosphorylation assay and cGMP assay
Swine common carotid arteries were obtained from a slaughterhouse. Physiological saline solutions, dissection of medial strips, mounting and determination of the optimum length for stress development at 37°C were performed as described (Rembold & Murphy, 1988). The intimal surface was mechanically rubbed to remove the endothelium. Phosphorylation of the smooth muscle-specific 20 kDa myosin light chain was estimated in tissues mounted isometrically for force measurement. Tissues were frozen and homogenized as described (Rembold & Murphy, 1988) and the percentage MLC phosphorylation was determined by the acidic shift of the MLCs on two-dimensional electrophoretic separation (Rembold & Murphy, 1988; McDaniel et al. 1991). After Coomassie Blue staining and optical scanning, phosphorylation was estimated as moles inorganic phosphate (Pi) per mole total smooth muscle-specific MLC (mol Pi (mol MLC)-1). cGMP and cyclic 3':5'-adenosine monophosphate (cAMP) were assayed by radioimmunoassay after homogenization in 0·1 n HCl as described (McDaniel et al. 1991).
32PO4 loading
Isometrically mounted swine carotid medial tissues were loaded with 32PO4 for 5 h (Van Riper et al. 1995). After washout of 32PO4 for 1 h, the tissues were pharmacologically treated (e.g. histamine or histamine plus nitroglycerine). Then the tissues were frozen in an acetone-dry-ice slurry and homogenized in 0·5 ml of a solution comprising (mM): NaF, 100; sucrose, 80; EDTA, 10; Tes, 10; pH 7·4 (Rapoport et al. 1982, 1983; Draznin et al. 1986). The homogenate was centrifuged at 14000 g for 10 min and the proteins in the supernatant were separated by two-dimensional electrophoresis (isoelectric focusing (pI range, 5-7) followed by 12 % SDS polyacrylamide gel electrophoresis) (Driska et al. 1981; Aksoy et al. 1983). Proteins were transferred to nitrocellulose membranes. The nitrocellulose membranes were stained with colloidal gold, imaged with a Molecular Dynamics laser scanner for protein, and imaged with a Molecular Dynamics phosphoimager for 32P activity. Controls performed after these experiments revealed that the homogenization buffer used did not totally inactivate MLC phosphatase, so that MLC phosphorylation values were artificially high. Therefore, we could not estimate MLC phosphorylation levels in the 32P experiments.
Electrospray tandem mass spectroscopy
Coomassie Blue-stained spots containing the 20 kDa protein of interest were excised from five heavily loaded two-dimensional gels, minced, destained, alkylated and trypsinized as described (Henzel et al. 1993). Peptides were then run on a POROS 10 RC reverse-phase microcapillary HPLC and its output was directed into a Finnigan-MAT TSQ7000 electrospray tandem mass spectroscope (Henzel et al. 1993). Collisionally activated dissociation spectra were interpreted and the proposed peptide fragment sequences were then compared with published protein sequences (CAD spectral information on SEQUEST and partial peptide sequences on http://prospector.ucsf.edu/cgi-bin/msdigest.exe).
Measurement of oxygen consumption
Oxygen consumption and contractile stress were measured in swine carotid artery rings placed in a 1·2 ml airtight chamber containing a Clark-style oxygen electrode (Instech Labs) and connected to a force transducer (Grass FT03) as described (Wingard et al. 1994). Oxygen consumption was calculated from the rate of decline in PO2 divided by the wet weight of the tissue. Data reported are means ± 1 S.E.M. for the values obtained between 26 and 30 min after solution changes.
Centrifugation assays
Swine carotid arteries were pharmacologically treated and frozen in an acetone-dry-ice slurry (Rembold & Murphy, 1988). After air drying, the tissues were homogenized in 20 mM Tris pH 7·4 with 5 µl ml-1 proteinase inhibitory cocktail (Sigma) on ice, and then immediately centrifuged at 6000 g for 10 min at 2°C in a microfuge as described (Golenhofen et al. 1999). Samples of the whole homogenate, the pellet and supernatant were then separated by one-dimensional isoelectric focusing (isoelectric point (pI) range 5-8), blotted to nitrocellulose, immunostained with a rabbit commercially produced polyclonal anti-HSP20 antibody and detected with enhanced chemiluminescence.
Binding of HSP20 and peptides to thin filaments
Skeletal G-actin was extracted and purified from rabbit skeletal muscle acetone powder as described (Spudich & Watt, 1971), in accordance with national guidelines. The actin was polymerized by dialysis at 4°C against binding buffer consisting of 40 mM Tris, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0·01 % NaN3, pH 7·8 (Van Eyk et al. 1997). Smooth muscle tropomyosin (TM) was prepared as described (Bretscher, 1984) and dialysed at 4°C against several changes of binding buffer. The purified synthetic peptides were dissolved in actin-binding buffer and the pH checked. Protein concentrations were determined by the Lowry assay.
The actin centrifugation studies were performed as previously described (Van Eyk et al. 1997; Tripet et al. 1997). Briefly, each binding point was determined by mixing 5 nmol actin, 1·4 nmol tropomyosin (ratio of 7·1 mol actin per 2 mol tropomyosin - equivalent to 1 mol dimeric tropomyosin) and increasing quantities of peptide (0-23 nmol) in a total volume of 200 µl. The actin-TM- peptide mixture was incubated at room temperature for 10 min prior to being spun for 30 min at 100000 g in a TL-100 ultracentrifuge. Under these conditions, greater that 95 % of the actin was pelleted. The pellet was washed with binding buffer and dissolved in 90 µl 0·05 % aqueous trifluoroacetic acid. This was injected onto a Zorbax SB 300 reversed-phase column (250 mm × 4·6 mm i.d., 6·5 µm particle size, 300 Å (30 nm) pore size; Rockland Technologies) on a Varian HPLC system consisting of a 9012 solvent delivery system, 9100 autosampler and 9065 Polychrom. The various proteins and peptides were eluted using a 2 % eluent B per minute linear gradient, where eluent A was 0·05 % aqueous trifluoroacetic acid and eluent B was 0·05 % trifluoroacetic acid in acetonitrile, at a flow rate of 1 ml min-1. The peak areas were determined by integration at 210 nm for the peptide and TM and at 280 nm for actin. The peak areas were converted to nanomoles based on a standard curve obtained for each protein and peptide. The amount of protein and peptide pelleted in the absence of actin was subtracted from the amount pelleted in the presence of actin. Experiments were performed in triplicate. The Kd for HSP20 peptides was determined by fitting the binding data to the equilibrium binding equation with the best fit at n = 2 binding sites.
ATPase activity assay
Smooth muscle myosin S1 was prepared immediately prior to use (Greene et al. 1983). Reactions consisted of 25 µM F-actin, 8 µM tropomyosin, 0·6 µM myosin S1 and variable concentrations of synthetic HSP20 peptides. The buffer contained 40 mM Tris HCl, pH 7·5 at 37°C, 50 mM KCl and 1 mM DTT. Quantification of released Pi was determined as described (Pollard, 1982). Reactions were performed in a 96-well ELISA plate and were triggered by addition of 2 mM ATP. Reactions were terminated by addition of an equal volume (100 µl) of 6 % (w/v) SDS, 3 % ascorbic acid, 1 n HCl and 0·5 % ammonium molybdate. After 5 min incubation, the blue colour was stabilized with 2 % sodium citrate, 2 % sodium m-arsenite and 2 % acetic acid. The plates were read at 650 nm on an E-max precision microplate reader (Molecular Dynamics, Sunnyvale, CA, USA). Phosphate was determined by comparison with standards. Phosphate release was linear over the period of the experiment. Experiments were performed in triplicate.
Measurement of force in skinned smooth muscle
Swine carotid artery tissues were obtained as described above. Tissues were kept in a Hepes-buffered (pH 7·4), physiologically balanced salt solution prior to skinning. Tissues were demembranated (skinned) with 1 % Triton X-100 as described (Strauss et al. 1992). Small fibre bundles (5·0 mm in length and 100-200 mm in diameter) were dissected from the muscle strip and mounted for isometric force measurements as previously described (Strauss et al. 1992). Fibres mounted for mechanical measurements were immersed in a 250 µl bath of relaxing solution maintained at 20°C for a minimum of 10 min. Fibres generated 
2·5 mN of force within 2-10 min of stimulation (corresponding to a stress of 1·5 × 105 N m-2). The system had a usable sensitivity range down to 0·01 mN. All subsequently measured values were standardized to the values obtained in the control contraction cycle. We controlled for skinned fibre 'rundown' by performing parallel time experiments with both active and control peptides. 'Relaxing' solution was 10 mM MgCl2, 1 mM NaN3, 7·5 mM Na2ATP, 4 mM EGTA, 0·1 µM calmodulin (activity 6 × 105 U mg-1; Boehringer-Mannheim), 20 mM imidazole (pH 6·7), 10 mM phosphocreatine (Na2PCr) and 10 U ml-1 creatine phosphokinase as an ATP regenerating system. Relaxing solution contained Ca2+ at < 10 nM. The 'contracting' solutions were identical to relaxing solution except that they also contained either 2·0 or 3·6 mM CaCl2, resulting in a buffered free calcium concentration of 0·8 or 25 µM, respectively, 2·0 mM free Mg2+, 7·2 mM MgATP, and had an ionic strength of 110 mM, adjusted with potassium acetate.
Size-exclusion chromatography
Recombinant calmodulin was expressed in Escherichia coli strain BL-21 DE3 (Novy et al. 1993) and purified by hydrophobic interaction chromatography (Gopalakrishna & Anderson, 1992). Calmodulin was dialysed against actin-binding buffer with 1 mM CaCl2 at 4°C, with several changes. Mixtures of calmodulin and synthetic peptides at molar ratios of 1:4 were incubated at room temperature for 15 min. The peptide-protein mixtures were then run in actin-binding buffer plus 1 mM CaCl2 at 0·5 ml min-1 on a Superdex-75 column (Pharmacia) attached to a Varian HPLC system. The concentration of KCl in the buffer was adequate to eliminate non-specific ionic interactions between the protein, peptide and support. This maintains ideal size-exclusion behaviour. The effluent was monitored at 210 nm. Peaks were collected, lyophilized and identified by reversed-phase HPLC as described above for the actin-binding assay.
|
RESULTS |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Nitroglycerine-induced relaxation without proportional MLC dephosphorylation
Nitroglycerine-induced relaxation without proportional MLC dephosphorylation (i.e. uncoupling of force from MLC phosphorylation) is shown as a rightward shift in a steady-state MLC phosphorylation-force plot (Fig. 1). Only the higher nitroglycerine concentrations (1 and 10 µM) induced substantial relaxation and a rightward shift in the MLC phosphorylation-force relation. This suggests that relaxation without MLC dephosphorylation occurred only when nitroglycerine induced a substantial relaxation.
 |
View larger version [in this window] [in a new window] |
|
|
Relation between MLC phosphorylation and stress (force per cross-sectional area) in swine carotid arterial tissues stimulated with 0·5, 1 or 3 µM histamine (Hist) alone for 60 min (open symbols) or 3 µM histamine for 10 min followed by 0·01, 0·1, 1 or 10 µM nitroglycerine (NTG) for 50 min (filled symbols). The filled diamond shows the response after 3 µM histamine for 10 min followed by 300 µM 8-pCPT-cGMP for 50 min. Stress was normalized to the value produced by exposure to 109 mM [K+]o (K109) for 10 min. Data are presented as means ± 1 S.E.M. (n = 6-20). Where error bars are not visible, the S.E.M. is smaller than the size of the symbol. | ||
One possible explanation for relaxation without proportional MLC dephosphorylation is that the myosin light chains could be phosphorylated on amino acid residues (e.g. Ser1, Ser2, Thr9 or Thr18) that do not activate myosin's ATPase. Importantly, we previously found that addition of nitroglycerine to swine carotid artery induced a relaxation that was associated with MLC phosphorylation entirely on Ser19 (McDaniel et al. 1992). This result indicates that MLC phosphorylation at other, inactive sites cannot explain nitroglycerine-induced relaxation without proportional MLC dephosphorylation. Other vasodilators such as forskolin, which increase [cAMP], did not alter the relation between MLC phosphorylation and force in the swine carotid artery (McDaniel et al. 1991, 1994). These data suggest that relaxation without MLC dephosphorylation may be specific to increased levels of cGMP. However, we cannot rule out the possibility that this mechanism may be activated by larger increases in cAMP in the swine carotid artery or moderate increases in cAMP in other smooth muscles. Relaxation without MLC dephosphorylation has also been observed with some pharmacological treatments: Ca2+ depletion/repletion (Gerthoffer, 1987; Aburto et al. 1993), high extracellular [Mg2+] (D'Angelo et al. 1992) and okadaic acid (Tansey et al. 1990).
The role of cGMP in nitroglycerine-dependent relaxation without proportional MLC dephosphorylation
Fifty minutes after addition of 10 µM nitroglycerine to 3 µM histamine-stimulated tissues, [cGMP] was 0·35 ± 0·032 pmol (mg wet weight)-1. This was a significant (P < 0·001; Student's t test), 564 % increase in [cGMP] compared with the value observed in tissues only stimulated with histamine (0·052 ± 0·015 pmol (mg wet weight)-1, n = 4 experiments). Nitroglycerine also significantly increased measured [cAMP] by 36 % compared with histamine stimulation alone (0·73 ± 0·028 pmol (mg wet weight)-1 for histamine plus nitroglycerine vs. 0·54 ± 0·038 pmol (mg wet weight)-1, n = 4). This may represent a true increase in [cAMP] or an artifact secondary to the large increase in cGMP cross-reacting in the cAMP assay.
Relaxation without proportional MLC dephosphorylation was also observed after addition of 300 µM 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cGMP), a membrane-permeant cGMP analogue (Fig. 1, 
). These data support our first hypothesis that nitroglycerine-dependent relaxation without proportional MLC dephosphorylation was caused by increases in [cGMP] rather than by other effects of nitroglycerine.
Potential mechanisms: nitroglycerine reduces oxygen consumption
Nitroglycerine-dependent relaxation without proportional MLC dephosphorylation suggests the presence of another force regulatory system in smooth muscle. This second force regulatory system appears to be regulated by cGMP and important in relaxation rather than contraction. There are several candidate regulatory systems for force in smooth muscle (see reviews in Bárány, 1996). These include: (1) thin filament regulation by caldesmon or calponin, (2) thick filament regulation by Ca2+ binding to myosin or by heavy chain phosphorylation, (3) regulation via protein kinase C by an unknown cascade, and (4) non-cross-bridge-mediated stress in the form of postulated cytoskeletal domain cross-links. None of these appear to be specifically regulated by nitrovasodilators.
There are three potential mechanisms whereby nitroglycerine could induce relaxation without MLC dephosphorylation. (1) Nitroglycerine-induced relaxation could result from fragmentation or disassembly of thin filaments. (2) Nitroglycerine could also accelerate release of myosin from thin filaments so less force is generated per cross-bridge cycle (i.e. diminished power stroke without a change in the cycling rate of myosin). If either of these were the case, ATP consumption should increase from the futile cycling of phosphorylated myosin and fragmented actin filaments. (3) Conversely, if nitroglycerine were to induce relaxation by inhibiting the binding of phosphorylated myosin to thin filaments, ATP consumption should decrease. Steady-state oxygen consumption is an index of ATP utilization. In swine carotid artery, resting oxygen consumption was 42·3 ± 2·8 nmol min-1 g-1. Histamine, at a concentration of 3 µM, induced a contraction and significantly increased oxygen consumption to 69·9 ± 3·3 nmol min-1 g-1. Addition of nitroglycerine induced a 70 % relaxation and significantly decreased oxygen consumption to 45·2 ± 4·7 nmol min-1 g-1 (Fig. 2). These data suggest that nitroglycerine-induced relaxation did not occur by accelerating myosin release from actin or by inducing fragmentation or disassembly of actin filaments (mechanisms 1 and 2). Our data support the third mechanism: nitroglycerine-induced relaxation without proportional MLC dephosphorylation resulted from inhibition of phosphorylated myosin attachment to thin filaments. This inhibition could result from a nitroglycerine-sensitive regulatory system that is associated with either the thin filament or thick filaments.
 |
View larger version [in this window] [in a new window] |
|
|
Oxygen consumption and contractile force were measured in swine carotid tissues at rest and following sequential stimulation with 109 mM [K+]o (K109), after washout (Wash), 3 µM histamine (Hist), histamine plus 10 µM nitroglycerine (H + NTG), nitroglycerine alone (NTG) and after washout. Data are means ± 1 S.E.M. (n = 4-6). | ||
Identification of phosphorylated HSP20 in nitroglycerine-stimulated smooth muscle
Since okadaic acid is known to uncouple force from MLC phosphorylation (Tansey et al. 1990), we hypothesized that a phosphoprotein would be involved in nitroglycerine-induced relaxation. Draznin et al. (1986) reported that nitroglycerine treatment of intact rat aorta induced phosphorylation of several proteins. We repeated these studies in the swine carotid artery. Tissues were loaded with 32PO4 and cellular proteins separated by two-dimensional gel electrophoresis. In histamine-stimulated tissues, we found that addition of 10 µM nitroglycerine for 50 min primarily increased 32P labelling of a single protein with a molecular mass of approximately 20 kDa (labelled HSP20 in Fig. 3). Nitroglycerine also increased protein staining of the 20 kDa protein, consistent with a phosphorylation-dependent acidic shift in the pI of this protein (Fig. 3; the dephosphorylated form of HSP20 appears to comigrate with another protein). Histamine stimulation alone did not significantly alter 32PO4 labelling or protein staining of the 20 kDa protein. The 32P labelling and concentration of other proteins, such as MLC and HSP27, did not significantly change with addition of nitroglycerine (data not shown).
 |
View larger version [in this window] [in a new window] |
|
|
Representative gels resulting from homogenates of 32PO4-loaded swine carotid tissues that were unstimulated (top; Control), stimulated with 3 µM histamine alone for 60 min (middle), or stimulated with 3 µM histamine for 10 min followed by 10 µM nitroglycerine (NTG) for 50 min (bottom). Cellular proteins were separated in two dimensions: isoelectric focusing in the horizontal direction (approximately 6·0-5·5 pI, left to right) and SDS electrophoresis in the vertical direction (approximately 30-15 kDa, top to bottom). The left panels show protein staining with colloidal gold and the right panels 32P activity. The arrows indicate the 20 kDa protein (labelled HSP20) and a second phosphoprotein that was identified as heat shock protein 27 (HSP27) by electrospray tandem mass spectroscopy (data not shown). | ||
Figure 4 shows a correlation between 32P labelling of the 20 kDa protein and contractile force. Histamine stimulation (3 µM for 60 min) alone induced a prolonged contraction that was not associated with a significant increase in 32P labelling of the 20 kDa protein (upper tracing with bar showing 32P labelling). Addition of 10 µM nitroglycerine to histamine-stimulated tissues induced a rapid and prolonged relaxation that was associated with significantly increased 32P labelling of the 20 kDa protein (lower tracing). The increase in 32P labelling was 9·1 ± 2·6-fold 5 min after addition of nitroglycerine and 8·2 ± 2·4-fold at 50 min (bars on lower tracing, P < 0·05 for both compared with unstimulated tissues, n = 4-7).
 |
View larger version [in this window] [in a new window] |
|
|
Representative force tracings from the 32PO4-loaded swine carotid tissues that were stimulated with 3 µM histamine alone for 60 min (top trace), or stimulated with 3 µM histamine for 10 min followed by 10 µM nitroglycerine for 50 min (bottom trace). The bar graphs show means ± 1 S.E.M. of the 32P labelling (as a percentage of control) of the protein later identified as HSP20. *Significantly different from control (P < 0·05). | ||
We identified the 20 kDa protein as HSP20 (Swiss prot B53814) by electrospray tandem mass spectroscopy (Table 1). Trypsin digestion of the phosphorylated form of the protein resulted in peptides that produced collisionally activated dissociation spectra identical to those predicted from the published sequence of HSP20 (three representative peptides are shown in Table 1) (Kato et al. 1994). These data support our third hypothesis that nitroglycerine-induced relaxation without proportional MLC dephosphorylation results from phosphorylation of a specific protein. One of these mass spectroscopy-identified tryptic peptides from the 20 kDa protein was phosphorylated on a Ser residue. This phosphorylation corresponded to Ser16 of the HSP20 amino acid sequence. The amino acid sequence N-terminal to Ser16 (WLRRAS) resembles the consensus sequence for both cGMP-dependent protein kinase and cAMP-dependent protein kinase (Glass, 1990). Since we found above that nitroglycerine increased [cGMP] at the time point at which relaxation occurred without MLC dephosphorylation (Fig. 1) and when HSP20 was phosphorylated (Figs 3 and 4), our results are consistent with the hypothesis that cGMP mediates HSP20 phosphorylation.
Table 1. Identification of the 20 kDa protein as HSP20 by electrospray tandem mass spectroscopy
| Tryptic peptide | Amino acid sequence | MW |
| Mass spec. 1 | RAS(p)APXPGXSAPGR | 1430·4 |
| B53814 (14-27) | RAS(p)APLPGLSAPGR | 1430·5 |
| Mass spec. 2 | HFSPEEXAVK | 1157·8 |
| B53814 (82-91) | HFSPEEIAVK | 1157·3 |
| Mass spec. 3 | VVGEHVEVHAR | 1231·8 |
| B53814 (92-102) | VVGEHVEVHAR | 1232·4 |
Phosphorylated HSP20 preferentially sediments in the pellet with centrifugation
HSP20 is very similar to HSP27 and 
B-crystallin (Kato et al. 1994), proteins known to bind to and alter actin filament dynamics (see Discussion). We evaluated whether phosphorylated HSP20 could be a thin filament-binding protein with a centrifugation assay. We stimulated swine carotid artery with 10 µM histamine alone or 10 µM histamine plus 10 µM nitroglycerine. After freezing, the tissues were homogenized in a low salt buffer, separated on a one-dimensional isoelectric focusing gel, blotted to nitrocellulose, and immunostained for HSP20. Histamine stimulation alone induced a dark band of immunostaining and a lighter second band at a more acidic pI (Fig. 5). This lower band is consistent with phosphorylated HSP20. Histamine-induced HSP20 phosphorylation was calculated to be 0·04 ± 0·02 mol Pi (mol HSP20)-1 (mean ± 1 S.E.M., n = 4). Nitroglycerine-induced relaxation was associated with more intense staining of the phosphorylated HSP20 band. Nitroglycerine-induced HSP20 phosphorylation was calculated to be 0·19 ± 0·03 mol Pi (mol HSP20)-1 (mean ± 1 S.E.M., n = 7), a value significantly greater than that observed with histamine alone (P < 0·05).
 |
View larger version [in this window] [in a new window] |
|
|
Representative blot resulting from homogenates of swine carotid tissues that were stimulated with 10 µM histamine alone for 60 min (lanes 1-3) or stimulated with 10 µM histamine for 10 min followed by 10 µM nitroglycerine for 50 min (lanes 4-6). Cellular proteins were separated by isoelectric focusing in the vertical direction (approximately 6·0-5·5 pI), blotted to nitrocellulose, immunostained with an antibody to HSP20 and imaged with enhanced chemiluminescence. Total homogenates were loaded in lanes 1 and 4. After centrifugation at 6000 g for 10 min, the pellets were loaded in lanes 2 and 5, and the supernatants loaded in lanes 3 and 6. Phos HSP20, phosphorylated HSP20. | ||
Immediately after homogenization, some of the homogenate was centrifuged at 6000 g. Phosphorylated HSP20 preferentially sedimented in the pellet compared with the supernatant (this is most apparent in the samples from nitroglycerine-stimulated tissues; Fig. 5). When normalized to the amount of phosphorylated HSP20 observed in the homogenate, phosphorylated HSP20 was found in significantly greater amounts in the pellet (1·10 ± 0·16 times homogenate) compared with the supernatant (0·64 ± 0·16 times homogenate, P < 0·05 by paired t test). These data suggest that phosphorylation of HSP20 increased its affinity for a component of the pellet. Since HSP27 and B-crystallin are known to bind to thin filaments (Miron et al. 1991) and since Brophy et al. (1999) found that HSP20 cosediments with actin in vitro, the most likely candidate for HSP20 binding is the thin filament. While our in vitro centrifugation assay results were significant, the relative concentration of HSP20 in the pellet may not reflect that observed in vivo since dissociation may have occurred during homogenization and/or centrifugation.
Stoichiometry of HSP20 compared with actin
From protein staining of two-dimensional gels (Fig. 3), the ratio of the amount of phosphorylated HSP20 to total MLC was 0·57 ± 0·10 (n = 4). Murphy et al. (1974) reported that the swine carotid artery contained 9·7 mg g-1 of myosin and 30 mg g-1 of actin. Based on their estimate that 60 % of swine carotid artery is cellular, calculated concentrations of myosin and actin of 68 and 1100 µM, respectively, can be obtained. If we assume (1) that HSP20 and MLC bind Coomassie Blue stain equally and (2) that the homogenization buffer extracted HSP20 and MLC equally, we can estimate HSP20 stoichiometry. Given our HSP20/MLC ratio of 0·57 and the relative molecular masses, the estimated concentration of phosphorylated HSP20 is 46 ± 8 µM, corresponding to a phosphorylated HSP20/actin ratio of 1/24. If we assume that our estimate of HSP20 phosphorylation (0·19 ± 0·03 mol Pi (mol HSP20)-1 from Fig. 5) is reasonable, then the estimated concentration of total HSP20 is 236 ± 42 µM, which corresponds to a total HSP20/actin ratio of 1/5. Given the assumptions, this analysis suggests that HSP20 is present in a stoichiometry similar to that observed with troponin I (TnI) in skeletal muscle (1 TnI to 7 actin). It is therefore possible that HSP20 could be a thin filament regulatory protein.
Binding of HSP20 peptides to actin-TM
Interestingly, we noted that the amino acid residues 110-121 of HSP20 have a high degree of sequence homology with a region of cardiac and skeletal TnI termed the TnI inhibitory region (skeletal TnI104-115 and cardiac TnI136-147). Specifically, there is a conserved glycine (G), a phenylalanine (F) and four arginine (R) residues (Table 2). Therefore, we hypothesized that HSP20 may share some, but not all, of the thin filament regulatory properties of TnI. An important difference is that HSP20 appears to be regulated by a covalent modification (phosphorylation), while TnI is regulated by an allosteric interaction with troponin C (TnC).
Table 2. Sequence homology of a region of HSP20 (NCBI number: B53814) with other actin-binding proteins
B-crystallin, HSP27, HSP25 and the three peptides studied. Amino acids are shown as the single letter code. |, identical amino acid; 
, similar amino acid. Emboldened amino acid residues are those conserved between TnI and HSP20. The underlined glycine residues in the control peptide HSP20 sequence indicate those substituted for alanine. The sequence alignment was performed by ClustalW. (http://dot.imgen.bcm.tmc.edu:9331/cgi-bin/multi-align/multi-align.pl).
We characterized HSP20 amino acid residues 110-121 in a manner similar to that described for skeletal TnI104-115 (see Discussion). Two HSP20 synthetic peptides were synthesized: a short peptide equivalent to skeletal TnI104-115 (HSP20 residues 110-121, abbreviated HSP20110-121) and a longer peptide equivalent to skeletal TnI94-116 (HSP20 residues 100-121, abbreviated HSP20100-121; Table 2). A control HSP20 peptide was also synthesized in which two arginine residues were replaced with glycine ((R118G)(R121G) HSP20110-121; Table 2). These arginine residues were chosen based on the key role of their equivalent conserved residues in the TnI inhibitory region (TnI residues R115 and R112, Table 2; Van Eyk & Hodges, 1988).
The short peptide HSP20110-121 bound to actin-TM (Fig. 6A, 
). Binding increased further at high concentrations of HSP20110-121 (Fig. 6A, inset). The calculated Kd was 155 ± 82 µM if it is assumed that two peptide molecules bind per mole of actin (it would be higher if 3 bind per mole of actin). HSP20110-121 had a 2-fold lower affinity for actin-TM filaments than the published Kd for native TnI inhibitory peptide (TnI104-115) and a slightly higher affinity for actin-TM than the TnI synthetic peptide (F106G, R115G) TnI96-115 (Tripet et al. 1997).
 |
View larger version [in this window] [in a new window] |
|
|
A, the quantity of synthetic HSP20 peptide bound to actin-TM at various concentrations was measured, and the data plotted as molar ratio of peptide bound to actin-TM filaments (actin:TM, 7·1:2). The synthetic peptide HSP20110-121 ( | ||
Stoichiometric binding of HSP20110-121 to actin-TM filaments only occurred when the filaments contained tropomyosin (Fig. 6B, inset, ). Actin binding only in the presence of tropomyosin is a hallmark of TnI (Talbot & Hodges, 1979). This result suggests that HSP20 may share some of TnI's thin filament binding characteristics. The control peptide ((R118G)(R121G) HSP20110-121) bound actin 20 times more weakly than HSP20110-121 (Fig. 6A, 
). This demonstrates that the Arg118 and Arg121 amino acid residues are important in actin binding. These data support our third hypothesis that nitroglycerine-induced relaxation without proportional MLC dephosphorylation results from phosphorylation of a specific protein that interacts with the thin filament. HSP20 appears to contain an actin-binding sequence.
Compared with HSP20110-121, the longer synthetic peptide, HSP20100-121, bound to actin-TM with reduced affinity (Fig. 6A, 
). This is not uncommon since peptides with 'extra' amino acid residues (i.e. those not involved in binding) can reduce the affinity or biological activity. For example, the synthetic TnI peptide 96-131 is a poorer inhibitor when compared with TnI96-116, the peptide containing only the actin-TM-binding site (Tripet et al. 1997). Since there is only a single amino acid residue that is semi-conserved between TnI94-103 and HSP20100-109 it is not surprising that the longer peptide binds more poorly to actin. Interestingly, HSP20 residues 100-109 contain four negatively charged and two positively charged amino acids and two His residues that are 100 % conserved or semiconserved between HSP25, 27 and B-crystallin (Table 2). This suggests another role for this region of HSP20, a role shared by the other heat shock proteins. This region may be important in phosphorylation-dependent regulation of HSP20 and other heat shock proteins.
Effect of HSP20 peptides on actomyosin ATPase activity
If HSP20 functions as a TnI-like protein (i.e. preventing myosin attachment), then binding of HSP20 to actin should alter actin-activated myosin ATPase activity. The short peptide HSP20110-121 decreased actin-activated myosin ATPase activity, but only at high concentrations (Fig. 6B, ). The control peptide ((R118G)(R121G) HSP20110-121) did not alter actin-activated myosin ATPase activity (Fig. 6B, 
). This demonstrates that the Arg118 and Arg121 residues are important in the inhibition of ATPase by HSP20110-121. ATPase measurements were not performed with the long peptide, HSP20100-121.
Effect of HSP20 peptides on contractile force
If HSP20 functions as a TnI-like protein, then binding of HSP20 to actin should also relax smooth muscle. In skinned swine carotid artery, pretreatment with a high concentration of the short peptide HSP20110-121 (300 µM) attenuated the initial phase and abolished the sustained phase of a pCa 5·4 Ca2+-induced contraction (Fig. 7, third contraction - HSP20110-121 was present during the entire contraction). HSP20110-121 treatment inhibited Ca2+-induced sustained force by 81 ± 7 % (n = 6, P < 0·05, force measured 20 min after increasing Ca2+). Pretreatment with 300 µM of the control peptide ((R118G)(R121G) HSP20110-121) did not substantially alter the Ca2+-induced contraction (Fig. 7, second contraction).
 |
View larger version [in this window] [in a new window] |
|
|
Representative tracing of force generation by skinned swine carotid artery. The skinned tissue was contracted thrice by increasing pCa to 5·4. The first contraction was performed in the absence of any peptide. The second contraction was performed in the presence of 300 µM control peptide (R118G)(R121G) HSP20110-121. The final contraction was performed in the presence of 300 µM HSP20110-121. HSP20110-121, but not the control peptide, significantly attenuated the initial phase of contraction and abolished the sustained phase of contraction. Peptides were added 5 min before [Ca2+] was increased. | ||
Binding of HSP20 peptides to calmodulin
In addition to binding actin-TM, the TnI inhibitory peptide, TnI104-115, tightly binds TnC and weakly binds calmodulin (Malencik & Anderson, 1984; Cachia et al. 1986; Tobacman, 1996; Solaro & Van Eyk, 1996). To determine whether HSP20 binds calmodulin, high-performance size-exclusion chromatography was performed in the presence of calcium. None of the HSP20 peptides interacted with calmodulin under these conditions (Fig. 8). However, it is possible that the peptide could form a very weak calmodulin complex with extremely fast on and off rates.
 |
View larger version [in this window] [in a new window] |
|
|
A, size-exclusion chromatography (SEC) elution profile of a 4:1 mixture of HSP20 and calmodulin in the presence of calcium. Fractions were collected ( | ||
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Our results show that nitroglycerine-induced relaxation without MLC dephosphorylation was associated with increases in [cGMP]. A membrane-permeant cGMP analogue also induced relaxation without MLC dephosphorylation, suggesting that cGMP mediated the nitroglycerine response (Fig. 1). Nitroglycerine also decreased oxygen consumption, suggesting that nitroglycerine-induced relaxation is more likely to be caused by a mechanism that prevents myosin attachment to the thin filaments (Fig. 2). We found that nitroglycerine-induced relaxation was associated with phosphorylation of heat shock protein 20 on Ser16 (Figs 3 and 4). Finally, we found that a HSP20 peptide bound to thin filaments and inhibited force generation in skinned smooth muscle (Figs 5-7).
Characteristics of HSP20
HSP20 was originally discovered in extracts of human skeletal muscle because it coeluted with B-crystallin and HSP27 (Kato et al. 1994). HSP20 is expressed in skeletal muscle, cardiac muscle, stomach, intestine and bladder (Kato et al. 1994). The laboratory of Colleen Brophy was the first to study the role of HSP20 in smooth muscle relaxation. They found that cyclic nucleotide-dependent relaxation was associated with HSP20 phosphorylation in bovine carotid smooth muscle (Beall et al. 1997). The human umbilical artery is an 'experiment of nature' since it does not exhibit cyclic nucleotide-induced relaxation. While the umbilical artery contains HSP20, Brophy's group reported that forskolin did not induce HSP20 phosphorylation, suggesting a linkage between HSP20 phosphorylation and relaxation (Brophy et al. 1997). Jerius et al. (1999) reported that flow-dependent vasodilatation, a nitric oxide-mediated process, was associated with HSP20 phosphorylation. Brophy et al. (1999) also reported that HSP20 cosediments with actin in vitro. These data suggest that phosphorylation of HSP20 may be integral in cyclic nucleotide-induced relaxation.
HSP20 is highly homologous to HSP27 and B-crystallin (Kato et al. 1994), proteins known to affect actin filament dynamics. HSP25 (the rodent equivalent of HSP27) was first characterized as an inhibitor of actin polymerization (25 kDa actin inhibitory protein) (Miron et al. 1991). HSP25 is known to be present in vascular smooth muscle (Wilkinson & Pollard, 1993). Phosphorylation of HSP27 by p38 MAP (mitogen-activated protein) kinase increases the stability of actin filaments in cells exposed to cytochalasin D (Guay et al. 1997). Antibodies to HSP27 inhibited bombesin-induced contraction of rabbit anal smooth muscle (Bitar et al. 1991). B-crystallin is a heat shock protein expressed in mammalian lens, skeletal muscle and heart, but not in smooth muscle (Klemenz et al. 1990). Dephosphorylated B-crystallin, but not phosphorylated B-crystallin, was found to stabilize actin filaments and prevented cytochalasin-induced actin depolymerization (Wang & Spector, 1996).
Most importantly, HSP20 is the only one of these small heat shock proteins containing a cGMP/cAMP-dependent protein kinase consensus phosphorylation site (RRAS) (Kato et al. 1994). This is a potential link between increases in cGMP, HSP20 phosphorylation and relaxation. Based on the sequence similarity of HSP20 with other actin-binding heat shock proteins, we hypothesized that nitroglycerine-dependent phosphorylation of HSP20 causes HSP20 to interfere with or inhibit the normal function of the actin-containing thin filaments despite activation of myosin by MLC phosphorylation. We therefore tested whether HSP20 bound to thin filaments. We found that phosphorylated HSP20 cosedimented with thin filaments better than dephosphorylated HSP20 (Fig. 5). This is consistent with but does not prove that phosphorylated HSP20 binds to thin filaments. Our result differs from that observed by Brophy et al. (1999). In cosedimentation assays with actin alone, they found that unphosphorylated HSP20 sedimented in the pellet while phosphorylated HSP20 remained in the supernatant. This result suggests that the binding of HSP20 to thin filaments may depend on the constituents of the thin filament.
Could HSP20110-121 binding to thin filaments reduce force generation?
Interestingly, we noted that the amino acid residues 110-121 of HSP20 have a high degree of sequence homology with a region of cardiac and skeletal TnI termed the TnI inhibitory region (skeletal TnI104-115 and cardiac TnI136-147). Specifically, there is a conserved glycine, a phenylalanine and four arginine residues (Table 2). TnI is part of the troponin complex that is responsible for Ca2+-dependent regulation of striated and cardiac muscle contraction. Binding of the inhibitory region of TnI to actin-tropomyosin depends on Ca2+ binding to TnC (Tobacman, 1996; Solaro & Van Eyk, 1996). The skeletal TnI peptide TnI104-115 is the minimum TnI amino acid sequence required to inhibit the actin-TM-myosin (or myosin S1) ATPase activity (Talbot & Hodges, 1979). In TnI-extracted skinned cardiac muscle, addition of synthetic TnI104-115 alone is sufficient for muscle relaxation (Van Eyk et al. 1993). The majority of the conserved or semi-conserved amino acid residues shared by HSP20 and TnI are known to be crucial for the binding of TnI to actin-TM. Grand et al. (1982) showed that the proton magnetic resonance signals of one or more of the five arginine residues in skeletal TnI96-116 were perturbed by actin binding. Single glycine amino acid substitution throughout the synthetic skeletal TnI104-115 indicated that Arg115, Arg112, Lys107, Leu111 and Val114 are important for inhibition of the acto-TM-myosin S1 ATPase activity (Van Eyk & Hodges, 1988; Van Eyk et al. 1993; Van Eyk et al. 1997).
We therefore tested peptides from this region of HSP20 that is analogous to TnI. We found that the short peptide, HSP20110-121, abolished force in Ca2+-contracted swine carotid artery (Fig. 7) and partially inhibited the acto-S1-TM ATPase activity (Fig. 6). HSP20110-121 also displayed tropomyosin specificity, a hallmark of intact TnI and the peptide TnI104-115 (Campbell et al. 1992; Tripet et al. 1997). These data suggest that HSP20110-121 may regulate contractile force in a manner analogous to TnI104-115. Our data are most consistent with the hypothesis that binding of HSP20110-121 to the thin filament prevents attachment of phosphorylated cross-bridges to the thin filament (thereby reducing oxygen consumption).
However, HSP20110-121 is not a complete analogue of TnI104-115. High concentrations of HSP20110-121 were required to abolish Ca2+-activated force in skinned carotid artery (Fig. 7). HSP20110-121 also bound less tightly to actin-TM compared with the published values for TnI104-115 (Van Eyk & Hodges, 1988; Van Eyk et al. 1997; Tripet et al. 1997). Additionally, unlike intact TnI and TnI104-115 (Cachia et al. 1986), HSP110-121 did not bind calmodulin (Fig. 8), suggesting that HSP20110-121 cannot be regulated by a Ca2+-calmodulin-dependent mechanism (like TnI-TnC in striated muscle). These differences between HSP20 and TnI may represent differences in the regulation of these two proteins. The peptide HSP20110-121 does not contain the phosphorylation site (which is at Ser16). Since relaxation without myosin dephosphorylation was associated with phosphorylated HSP20, not dephosphorylated HSP20, we hypothesize that phosphorylation of HSP20 regulates binding of HSP20 to thin filaments. Our centrifugation data (Fig. 5) are consistent with the hypothesis that phosphorylation of HSP20 increases the affinity of HSP20 for the thin filament. The relatively low affinity of HSP20110-121 and its lack of calmodulin binding may be necessary to prevent dephosphorylated HSP20 from inhibiting thin filament function.
Intact HSP20 may have thin filament effects similar to those documented in other similar heat shock proteins (Miron et al. 1991; Wang & Spector, 1996; Guay et al. 1997). The actin-binding sequence in HSP20110-121 is conserved in other small heat shock proteins such as B-crystallin, HSP25 and HSP27 (Table 2). The fact that this region bound actin in a TM-specific manner but only bound weakly may reflect the initial function of these proteins. Early in evolution, weak interactions between heat shock proteins and actin-TM may have been important in the response to acute cellular stress, e.g. binding to protect the contractile proteins. This could explain the difference between these proteins and TnI. It is possible that the presence of a cGMP-dependent protein kinase phosphorylation site allowed HSP20 to evolve from a heat shock protein into a protein with additional functions.
Conclusion
Our working hypothesis is that cGMP-mediated phosphorylation of intact HSP20 increases the affinity of HSP20 for the thin filament. We hypothesize that this binding inhibits interaction of phosphorylated myosin with the thin filament, inducing a relaxation despite high levels of [Ca2+] and activated myosin. Data supporting these hypotheses are as follows. (1) Increases in [cGMP] and a membrane-permeant cGMP analogue induced relaxation without MLC dephosphorylation (Fig. 1). (2) cGMP-dependent relaxation was associated with phosphorylation of heat shock protein 20 on Ser16, a consensus site for cAMP/cGMP-dependent protein kinase (Figs 3 and 4). (3) Phosphorylated HSP20 cosedimented with thin filaments, consistent with phosphorylation-dependent regulation of the binding of HSP20 to thin filaments (Fig. 5). (4) A HSP20 peptide (HSP20110-121) bound to thin filaments in a manner similar to TnI and inhibited force generation in skinned smooth muscle (Figs 6 and 7). (5) Increases in cGMP also decreased oxygen consumption, suggesting that a mechanism that prevents myosin attachment to the thin filaments was responsible for the reduction in force (Fig. 2). Evaluation of this hypothesis will require evaluation of intact phosphorylated HSP20 in actin binding, ATPase and skinned fibre experiments.
These data have implications beyond the pharmacology of nitrovasodilators. Nitric oxide is released both by the endothelium and by non-adrenergic non-cholinergic (NANC) inhibitory nerves (Furchgott & Vanhoutte, 1989; Li & Rand, 1989; Bult et al. 1990). It is possible that nitric oxide-mediated HSP20 phosphorylation may be the mechanism responsible for endothelial- and NANC-mediated smooth muscle relaxation. As such, HSP20 could have a role in diseases such as hypertension, gastrointestinal motility disorders and erectile dysfunction. It is interesting that amino acid residues 110-121 of HSP20 are highly conserved among the other heat shock proteins. Thus, other small heat shock proteins may work in a similar manner with their actin affinities altered by phosphorylation by their respective kinases.
|
REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Aburto, T. K., Lajoie, C. & Morgan, K. G. (1993). Mechanisms of signal transduction during 2-adrenergic receptor-mediated contraction of vascular smooth muscle. Circulation Research 72, 778-785 |
[Abstract] |
| Aksoy, M. O., Mras, S., Kamm, K. E. & Murphy, R. A. (1983). Ca2+, cAMP, and changes in myosin phosphorylation during contraction of smooth muscle. American Journal of Physiology 245, C255-270 | [Medline] |
| Bárány, M. (1996). Protein phosphorylation during contraction and relaxation. In Biochemistry of Smooth Muscle Contraction, ed. Bárány, M., pp. 321-340. Academic Press, San Diego. | |
| Bárány, M. & Bárány, K. (1993). Dissociation of relaxation and myosin light chain dephosphorylation in porcine uterine muscle. Archives of Biochemistry and Biophysics 305, 202-204 | [Medline] |
| Beall, A. C., Kato, K., Goldenring, J. R., Rasmussen, H. & Brophy, C. M. (1997). Cyclic nucleotide-dependent vasorelaxation is associated with the phosphorylation of a small heat shock-related protein. Journal of Biological Chemistry 272, 11283-11287 | [Abstract/Full Text] |
| Bitar, K. N., Kaminski, M. S., Hailat, N., Cease, K. B. & Strahler, J. R. (1991). HSP27 is a mediator of sustained smooth muscle contraction in response to bombesin. Biochemical and Biophysical Research Communications 181, 1192-1200 | [Medline] |
| Bretscher, A. (1984). Smooth muscle caldesmon. Rapid purification and F-actin cross-linking properties. Journal of Biological Chemistry 259, 12873-12880 | [Abstract] |
| Brophy, C. M., Beall, A. C., Lamb, S., Dickinson, M. & Ware, D. J. (1997). Small heat shock proteins and vasospasm in human umbilical artery smooth muscle. Biology of Reproduction 57, 1354-1359 | [Abstract] |
| Brophy, C. M., Lamb, S. & Graham, A. (1999). The small heat shock protein-20 is an actin associated protein. Journal of Vascular Surgery 29, 326-333. | [Medline] |
| Bult, H., Boeckxstaens, G. E., Pelckmans, P. A., Jordaens, F. H., van Maercke, Y. M. & Herman, A. G. (1990). Nitric oxide as an inhibitory non-adrenergic non-cholinergic neurotransmitter. Nature 345, 346-347 | [Medline] |
| Cachia, P. J., Van Eyk, J. E., Ingraham, R. H., McCubbin, W. D., Kay, C. M. & Hodges, R. S. (1986). Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]. Biochemistry 25, 3553-3562 | [Medline] |
| Campbell, A. P., Van Eyk, J. E., Hodges, R. S. & Sykes, B. D. (1992). Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C. Biochimica et Biophysica Acta 1160, 35-54 | [Medline] |
| D'Angelo, E. K. G., Singer, H. A. & Rembold, C. M. (1992). Magnesium relaxes arterial smooth muscle by decreasing intracellular [Ca2+] without changing intracellular [Mg2+]. Journal of Clinical Investigation 89, 1988-1994 | [Medline] |
| Draznin, M. B., Rapoport, R. M. & Murad, F. (1986). Myosin light chain phosphorylation in contraction and relaxation of intact rat thoracic aorta. European Journal of Biochemistry 18, 917-928. | |
| Driska, S. P., Aksoy, M. O. & Murphy, R. A. (1981). Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. American Journal of Physiology 240, C222-233 | [Medline] |
| Furchgott, R. F. & Vanhoutte, P. M. (1989). Endothelium-derived relaxing and contracting factors. FASEB Journal 3, 2007-2018 | [Abstract] |
| Gerthoffer, W. T. (1987). Dissociation of myosin phosphorylation and active tension during muscarinic stimulation of tracheal smooth muscle. Journal of Pharmacology and Experimental Therapeutics 240, 8-15 | [Abstract] |
| Glass, D. B. (1990). Substrate specificity of the cyclic GMP-dependent protein kinase. In Peptides and Protein Phosphorylation, ed. Kemp, B. E., pp. 209-238. CRC Press, Boca Raton, FL, USA. | |
| Golenhofen, H., Ness, W., Koob, R., Htun, P., Schaper, W. & Drenckhahn, D. (1999). Ischemia-induced phosphorylation and translocation of stress protein B crystallin to Z lines of myocardium. American Journal of Physiology 274, H1457-1464. |
|
| Gopalakrishna, R. & Anderson, W. B. (1992). Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-sepharose affinity chromatography. Biochemical and Biophysical Research Communications 104, 830-836. | |
| Grand, R. J. A., Levine, B. A. & Perry, S. V. (1982). Proton-magnetic-resonance studies on the interaction of rabbit skeletal-muscle troponin I with troponin C and actin. Biochemical Journal 203, 61-68 | [Medline] |
| Greene, L. E., Sellers, J. R., Eisenberg, E. & Adelstein, R. S. (1983). Binding of gizzard smooth muscle myosin subfragment 1 to actin in the presence and absence of adenosine 5'-triphosphate. Biochemistry 22, 530-535 | [Medline] |
| Guay, J., Lambert, H., Gingras-Breton, G., Lavoie, J. N., Huot, J. & Landry, J. (1997). Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. Journal of Cell Science 110, 357-368 | [Abstract] |
| Hai, C.-M. & Murphy, R. A. (1989). Ca2+, crossbridge phosphorylation, and contraction. Annual Review of Physiology 51, 285-298 | [Medline] |
| Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C. & Watanabe, C. (1993). Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proceedings of the National Academy of Sciences of the USA 90, 5011-5015 | [Abstract] |
| Jerius, J. H., Karolyi, D. R., Mondy, J. S., Beall, A. C., Wootton, D., Ku, D., Cable, S. & Brophy, C. M. (1999). Endothelial-dependent vasodilation is associated with increases in the phosphorylation of a small heat shock protein (HSP20). Journal of Vascular Surgery 29, 678-684. | [Medline] |
| Kato, K., Goto, S., Inaguma, Y., Hasegawa, K., Morishita, R. & Asano, T. (1994). Purification and characterization of a 20-kDa protein that is highly homologous to -B-crystallin. Journal of Biological Chemistry 269, 15302-15309 |
[Abstract] |
| Klemenz, R., Frohli, E., Seiger, R. H., Schafer, R. & Aoyama, A. (1990). -B-Crystallin is a small heat shock protein. Proceedings of the National Academy of Sciences of the USA 88, 3652-3656. |
[Abstract] |
| Li, C. G. & Rand, M. J. (1989). Evidence for a role of nitric oxide in the neurotransmitter system mediating relaxation of the rat anococcygeus muscle. Clinical and Experimental Pharmacology and Physiology 16, 933-938 | [Medline] |
| McDaniel, N. L., Chen, X.-L., Singer, H. A., Murphy, R. A. & Rembold, C. M. (1992). Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i, [Ca2+]i sensitivity, and uncoupling stress from myosin phosphorylation. American Journal of Physiology 263, C461-467 | [Medline] |
| McDaniel, N. L., Rembold, C. M. & Murphy, R. A. (1994). Cyclic nucleotide dependent relaxation in vascular smooth muscle. Canadian The Journal of Physiology and Pharmacology 72, 1380-1385 | [Medline] |
| McDaniel, N. L., Rembold, C. M., Richard, H. L. & Murphy, R. A. (1991). cAMP relaxes arterial smooth muscle predominantly by decreasing cell [Ca2+]. The Journal of Physiology 439, 147-160 | [Abstract] |
| Malencik, D. A. & Anderson, S. R. (1984). Peptide binding by calmodulin and its proteolytic fragments and by troponin C. Biochemistry 23, 2420-2428 | [Medline] |
| Miron, T., Vancompernolle, K., Vandekerckhove, J., Wilchek, M. & Geiger, B. (1991). A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. Journal of Cell Biology 114, 255-261 | [Abstract] |
| Murphy, R. A., Herlihy, J. T. & Megerman, J. (1974). Force-generating capacity and contractile protein content of arterial smooth muscle. Journal of General Physiology 64, 691-705 | [Medline] |
| Novy, R. E., Sellers, J. R., Lin, J. J. & Lin, C.-N. (1993). In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants. Cell Motility and the Cytoskeleton 26, 248-261 | [Medline] |
| Pollard, T. D. (1982). Assays for myosin. Methods in Enzymology 85, 123-130 | [Medline] |
| Rapoport, R. M., Draznin, M. B. & Murad, F. (1982). Sodium nitroprusside-induced protein phosphorylation in intact rat aorta is mimicked by 8-bromo cyclic GMP. Proceedings of the National Academy of Sciences of the USA 79, 6470-6474 | [Medline] |
| Rapoport, R. M., Draznin, M. B. & Murad, F. (1983). Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 306, 174-176 | [Medline] |
| Ratz, P. H., Hai, C.-M. & Murphy, R. A. (1989). Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle. American Journal of Physiology 256, C96-100 | [Medline] |
| Rembold, C. M. & Murphy, R. A. (1988). Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circulation Research 63, 593-603 | [Abstract] |
| Solaro, R. J. & Van Eyk, J. E. (1996). Altered interactions among thin filament proteins modulate cardiac function. Journal of Molecular and Cellular Cardiology 28, 217-230 | [Medline] |
| Spudich, J. A. & Watt, S. (1971). The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. Journal of Biological Chemistry 246, 4866-4971 | [Medline] |
| Strauss, J. D., de Lanerolle, P. & Paul, R. J. (1992). Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle. American Journal of Physiology 262, C1437-1445 | [Medline] |
| Talbot, J. A. & Hodges, R. S. (1979). Synthesis and biological activity of an icosapeptide analog of the actomyosin ATPase inhibitory region of troponin I. Journal of Biological Chemistry 254, 3720-3723 | [Medline] |
| Tansey, M. G., Hori, M., Karaki, H., Kamm, K. E. & Stull, J. T. (1990). Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle. FEBS Letters 270, 219-221 | [Medline] |
| Tobacman, L. S. (1996). Thin filament-mediated regulation of cardiac contraction. Annual Review of Physiology 58, 447-481 | [Abstract] |
| Tripet, B., Van Eyk, J. E. & Hodges, R. S. (1997). Mapping of a second actin-tropomyosin and a second troponin C binding site within the C terminus of troponin I, and their importance in the Ca2+-dependent regulation of muscle contraction. Journal of Molecular Biology 271, 728-750 | [Medline] |
| Van Eyk, J. E. & Hodges, R. S. (1988). The biologic significance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin. Journal of Biological Chemistry 263, 1726-1732 | [Abstract] |
| Van Eyk, J. E., Strauss, J. D., Hodges, R. S. & Ruegg, J. C. (1993). A synthetic peptide mimics troponin I function in the calcium-dependent regulation of muscle contraction. FEBS Letters 323, 223-228 | [Medline] |
| Van Eyk, J. E., Thomas, L. T., Tripet, B., Wiesner, R. J., Pearlstone, J. R., Farah, C. S., Reinach, F. C. & Hodges, R. S. (1997). Distinct regions of troponin I regulate Ca2+-dependent activation and Ca2+ sensitivity of the acto-S1-TM ATPase activity of the thin filament. Journal of Biological Chemistry 272, 10529-10537 | [Abstract/Full Text] |
| Van Riper, D. A., McDaniel, N. L. & Rembold, C. M. (1997). Myosin light chain kinase phosphorylation in nitrovasodilator induced swine carotid artery relaxation. Biochimica et Biophysica Acta 1355, 323-330 | [Medline] |
| Van Riper, D. A., Weaver, B. A., Stull, J. T. & Rembold, C. M. (1995). Myosin light chain kinase phosphorylation in swine carotid artery contraction and relaxation. American Journal of Physiology 268, H2466-2475 | [Medline] |
| Wang, K. & Spector, A. (1996). -Crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner. European Journal of Biochemistry 242, 56-66 |
[Abstract] |
| Wilkinson, J. M. & Pollard, I. (1993). Immunohistochemical localization of the 25 kDa heat shock protein in unstressed rats: possible functional implications. Anatomical Record 237, 453-457 | [Medline] |
| Wingard, C. J., Paul, R. J. & Murphy, R. A. (1994). Dependence of ATP consumption on cross-bridge phosphorylation in swine carotid smooth muscle. The Journal of Physiology 481, 111-117 | [Abstract] |
The technical assistance of Barbara Weaver, Marcia Ripley, Elizabeth Chang, David Taylor and Mike Kinter is appreciated. Arteries were donated by Smithfield Co., Smithfield, VA, USA. C. M. Rembold is a Lucille P. Markey Scholar and J. E. Van Eyk is a Canadian Heart and Stroke Scholar. This work was partially supported by grants from the Lucille P. Markey Charitable Trust, the Jeffress Trust, the Virginia (C.M.R.) and New York (J.D.S.) Affiliates of the AHA, W. M. Keck foundation for the Center for Mass Spectroscopy, the Medical Research Council of Canada and the Ontario Heart and Stroke Foundation.
Corresponding author
C. M. Rembold: Box 146, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA.
Email: crembold{at}virginia.edu
This article has been cited by other articles:
|
|
 |
|
  S. J. Gunst and W. Zhang Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction Am J Physiol Cell Physiol, September 1, 2008; 295(3): C576 - C587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Komalavilas, R. B. Penn, C. R. Flynn, J. Thresher, L. B. Lopes, E. J. Furnish, M. Guo, M. A. Pallero, J. E. Murphy-Ullrich, and C. M. Brophy The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation Am J Physiol Lung Cell Mol Physiol, January 1, 2008; 294(1): L69 - L78. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Sandoval, E. R. Injeti, W. T. Gerthoffer, and W. J. Pearce Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2183 - H2192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Rembold Force suppression and the crossbridge cycle in swine carotid artery Am J Physiol Cell Physiol, September 1, 2007; 293(3): C1003 - C1009. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. F. Kelly and H. M. Snow Characteristics of the response of the iliac artery to wall shear stress in the anaesthetized pig J. Physiol., July 15, 2007; 582(2): 731 - 743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B E Cross, H M O'Dea, and D J MacPhee Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour Reproduction, April 1, 2007; 133(4): 807 - 817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Chen, K. Pavlish, H.-Y. Zhang, and J. N. Benoit Effects of chronic portal hypertension on agonist-induced actin polymerization in small mesenteric arteries Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1915 - H1921. [Abstract] [Full Text] [PDF]
|
|
|
|
