Polylysine interaction
activates smooth muscle actin-myosin without LCzO phosphorylation
PAWEL T. SZYMANSKI, JOHN D. STRAUSS, GLENN DOERMAN, JOSEPH DISALVO, AND RICHARD J. PAUL Department of Physiology and Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576; and Department of Physiology, School of Medicine, University of Minnesota, Duluth, Minnesota 55812 Szymanski, Pawel T., John D. Strauss, Glenn Doerman, Joseph DiSalvo, and Richard J. Paul. Polylysine activates smooth muscle actin-myosin interaction without LC,,, phosphorylation. Am. J. Physiol. 262 (Cell Physiol. 31): Cl446-Cl455, 1992.-Phosphorylation/dephosphorylation of the 20-kDa light chain of smooth muscle myosin is a major regulator of actin-myosin interaction. Phosphatase inhibitors have thus been shown to enhance contraction in smooth muscle. The activity of type II phosphatase against phosphorylated myosin light chains is inhibited by polylysine (7). Thus we studied the effects of polylysine (lo-13 kDa) on actin-myosin interaction in permeabilized guinea pig taenia coli fibers and in bovine aortic actomyosin. Addition of polylysine (lo-20 PM) to Ca-ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid buffered solution ( [Ca2+] < 0.01 PM) elicited a contraction in fibers of 40 t 8% (n = 6) of maximally stimulated contractions ( [Ca2+] = 1.5 PM). Untreated fibers did not generate any significant force in parallel control experiments. Similarly, polylysine stimulated the ATPase activity both in fibers and actomyosin in a dose-dependent manner. This stimulation could be completely inhibited and abolished upon addition of heparin, a negatively charged heteropolysaccharide. In actomyosin previously phosphorylated with ATP+, polylysine in a concentration range of 2-13 PM did not further stimulate enzyme activity. These increases in activity were not connected with significant changes in the phosphorylation of 20-kDa myosin light chain nor could any incorporation of 32P associated with polylysine stimulation be detected in both skinned fibers and actomyosin by autoradiography of SDS gels. Our data indicate that polylysine increases actin-myosin interaction in both smooth muscle model systems by directly influencing contractile proteins. As such, polylysine may be a useful probe for the mechanism of activation of smooth muscle. contraction
of the mechanisms underlying the regulation of smooth muscle contractility is yet a subject of considerable controversy (1, 5, 11, 12, 16), it is generally accepted that the phosphorylation/dephosphorylation of the 20-kDa light chains of myosin (MLC; LC& is an important component. In its simplest form, phosphorylation of MLC allows the activation of the myosin Mg2+-adenosinetriphosphatase (ATPase) by actin, increasing the rate of cross-bridge cycling and consequent generation of force and/or shortening. Dephosphorylation would result in inactivation of myosin and relaxation. The more complex physiological responses of smooth muscle have led to modification of this simple “switch” hypothesis as well as to the proposal of several alternative modulatory mechanisms. However, several modeling studies (11, 24) have suggested that phosphorylation/dephosphorylation of MLC can be sufficient to explain the observed physiological responses. A phosphatase isolated from bovine aortic smooth WHILE
Cl446
THE NATURE
0363-6143/92
$2.00
Copyright
muscle was shown to dephosphorylate native myosin as well as isolated myosin light chains (6, 7). Its activity in vitro can be modulated by polycationic effecters such as lysine-rich histone H1, protamine, or polylysine, and hence it was called polycation-modulable phosphatase (7, 8). Subsequent studies revealed that polycationic modulation of phosphatase activity is a characteristic property of type II phosphatases (for review see Ref. 4). The activity of type II phosphatase against phosphorylated MLC is inhibited by polylysine in the concentration range of 5-10 PM (7). It was thus of interest to test whether polylysine could affect the contraction of smooth muscle simply by the inhibition of the dephosphorylation of MLC. In this study, we utilized a permeabilized or skinned fiber preparation to study the effects of polylysine in a structured model of smooth muscle. We report here that polylysine elicited a contraction and increased the ATPase activity in skinned fibers isolated from guinea pig taenia coli. Surprisingly, however, this contraction was not associated with any statistically significant changes in MLC phosphorylation. To provide further information on the mechanism underlying the polylysine-induced contraction, its effects on the ATPase activity of bovine aortic actomyosin were also studied. Our results indicate that polylysine increases actin-myosin interaction in both smooth muscle model systems independent of MLC or other protein phosphorylation/dephosphorylation. Thus polylysine may be a useful probe of the mechanism of regulation of actin-myosin interaction in smooth muscle. METHODS Skinned taenia coli fiber preparation. Taenia coli were dissected from guinea pigs and permeabilized or skinned as previously described (25). Briefly, strips of tissue were calcium depleted in a high potassium, ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)-buffered solution, pH 7.0. This was followed by treatment with a similar solution containing 1% Triton X-100 for 4 h to solubilize the cell membranes. The fiber bundles were stored at -30°C in 50% (vol/vol) glycerol and 20 mM imidazole, pH 6.7, 4 mM EGTA, 7.5 mM ATP, 10 mM MgC12, and 1 mM NaNs. Mechanical studies with skinned fibers. Skinned fiber bundles (5.0 mm long, 100-200 pm thick) were mounted horizontally with a nitrocellulose-based glue between an AME(SensoNor a.s) force transducer and rigid post. The position of this post and hence the length of the fiber were controlled by a micrometer. Changes in force were monitored with a linear chart recorder. The bundle was stretched by -5% from its unloaded length to establish a resting tension of 0.1-0.3 mN. Fibers were bathed in 300 ~1 of relaxing solution maintained at 25°C containing the following: 20 mM imidazole, pH 6.7,4 mM
0 1992 the American
Physiological
Society
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POLYLYSINE
ACTIVATES
SMOOTH
EGTA, 7.5 mM NaATP, 10 mM MgCIB, 1 mM NaN,, 0.1 PM calmodulin, and an ATP regenerating system consisting of IO mM phosphocreatine and IO U/ml creatine phosphokinase (solutions with 7.5 mM ATP are designated solution A). The calculated concentration of Ca2+ was CO.01 PM. Contraction was elicited by transferring the fiber to a solution similar to relaxing solution but also containing 2.0 mM CaCl,. This contracting solution has a calculated (IO) free calcium ion concentration of 1.54 PM, 1.94 mM free Mg2+, 7.2 mM MgATP, and an ionic strength of 110 mM. The concentrations of EGTA and CaCl, stock solutions as well as the free calcium in the above solutions were verified using an Orion calcium electrode (2). In addition to these standard solutions, solutions (designated as solution B) with reduced ATP (1 mM) were also studied to avoid the turbidity produced by the binding of ATP to polylysine. The compositions of these solutions were identical to those above (solution A) but with ATP reduced to 1 mM. The calculated concentrations of Ca2+ were ~0.01 and 1.66 PM and of Mg2+ were 6.49 and 6.65 mM for relaxing and contracting solutions, respectively. Upon transfer of the fibers to contracting solution, steady-state isometric forces of between 0.5 and 2.5 mN (m 150 mN/mm2) were generated. All fibers in each treatment group were first rinsed in relaxing solution for a minimum of IO min and then contracted in contracting solution to establish maximum isometric force (a control contraction). After the control contraction, fibers were transferred to relaxing solution until a stable base line had been reestablished. Then they were transferred to relaxing solutions, which contained either 10 or 20 PM polylysine. After a new level of isometric force was obtained, the concentration of polylysine was increased in 10 PM increments. After the changes in isometric force became stable, the fibers were transferred into fresh contracting solution (without polylysine) and the contractions were again monitored. To complete this protocol, fibers were transferred into either relaxing solution without polylysine or into relaxing solution plus heparin. In some fibers, the mechanical effects of a quick-step decrease in length of 10% of the initial fiber length were studied after a stable contraction in the presence of polylysine had been obtained. Tuenia coli skinned fiber homogenates. Freshly skinned fibers were homogenized at liquid N, temperatures using a dental amalgamator and stored at -80°C. Prior to an experiment, the powder obtained from this procedure was suspended in a solution containing 20 mM 2-(N-morpholino)propanesulfonic acid (MOPS), pH 6.7,lO mM MgC12, 4 mM EGTA, and 24 mM KCl. Preparation of bovine aortic actornyosin. Ca2+-sensitive actomyosin was prepared from bovine aortas according to the method described by Litten et al. (19). Briefly, the muscularis was extracted (4°C) at low ionic strength in a solution containing 80 mM KCl, 18 mM MOPS, pH 7.0, 4 mM MgC12, 4 mM EGTA, 4 mM ATP, 1 mM dithiothreitol (DTT), and 1 mM NaN,. The actomyosin was precipitated by dialysis against a lower ionic strength solution composed of 25 mM KCl, 4 mM MOPS, pH 7.0, 0.2 mM EGTA, 4 mM MgCl,, and 0.5 mM DTT, sedimented at 10,000 g for 15 min, washed four times in 8 vol of the same solution with 1% Triton X-100, and then washed five times with the identical solution but without Triton X-100. The final pellet was suspended in a solution containing 18 mM MOPS, pH 7.0,25 mM KCl, 1 mM MgC12, 1 mM DTT, and 60% (vol/vol) glycerol. Although not studied systematically, actomyosin could be stored at -80°C for several weeks without appreciable change in ATPase activity. ATPase activity measurements. The ATPase activity of both skinned fibers and actomyosin was studied under similar conditions in cuvettes of 600 ~1. Skinned fibers, =7 mm in length and 500-600 pm in diameter, were attached with a nitrocellulose-based glue between the arms of a stainless steel stirring rod.
MUSCLE
CONTRACTION
Cl447
Alternatively, actomyosin at a concentration of 100-200 pg/ml was placed in the cuvette. The reaction was carried out at 25°C in various solutions. The basic relaxing solution consisted of 20 mM MOPS, pH 6.7,lO mM MgCl,, 24 mM KCl, 4 mM EGTA, 0.5 mM DTT, 1 mM NaN,, and 1 mM ATP. Polylysine, lysine, heparin, type II phosphatase, or different concentrations of MgCl, were added as noted. Contracting solution was similar to relaxing solution but also contained 0.1 PM calmodulin and 3.6 mM CaC12 resulting in a calculated free calcium concentration of 14.9 PM, 8.9 mM free Mg2+, and an ionic strength not exceeding 110 mM. In some experiments, EGTA was added (with appropriate KOH to maintain constant pH) to achieve a final total concentration of 24 mM, which reduced the free calcium concentration to ~0.3 PM. All solutions contained an ATPregenerating system consisting of IO mM phospho(enol)pyruvate and 20 pg/ml pyruvate kinase. To measure ATPase activity in terms of the production of ADP, the pyruvate produced by the regenerating system was linked to the production of NAD by including 50 pg/ml of lactate dehydrogenase and 50 PM NADH (20). Rates of ATP hydrolysis were measured as a decrease of fluorescence of NADH in a Perkin-Elmer 650-10s spectrofluorimeter. The actomyosin ATPase activity of thiophosphorylated myosin was also measured. Thiophosphorylated myosin was prepared by incubation of actomyosin (2 mg/ml) in contracting solution containing 1 mM ATPyS for 45-60 min at 25°C in a total reaction volume of 800 ~1. The reaction was terminated by centrifugation at 10,000 g for 5 min and resuspension of the pellet in relaxing solution. After two further washes, the thiophosphorylated actomyosin was resuspended in the same relaxing buffer and used for ATPase measurements. Quantification of MLC phosphate content. Fibers isometrically mounted on wire frames or actomyosin were incubated at 25°C for 10 min in 200 ~1 of the various reaction mixtures described above. In all experiments, the reaction was started by adding ATP to achieve a final concentration of 1 mM. After the appropriate reaction time, the muscle fibers and actomyosin were denatured with 15% trichloroacetic acid for 10 min at 4°C. These were then rinsed (fiber bundles) or suspended (actomyosin) in a 20 mM imidazole, pH 6.7 buffer. MLC were separated on the basis of difference in their isoelectric point due to phosphorylation on urea-glycerol IEF-page gels with 2% Pharmalyte (pH 4.5 to pH 5.4; Ref. 13). Once separated, they were transferred into nitrocellulose paper. Proteins were identified using affinity-purified goat anti-myosin light chain antibodies and horseradish peroxidase-conjugated rabbit anti-goat antibodies (23). 4-Chloro-I-naphthol and peroxide were used to color the immunoreactive bands. Incorporation of 32P into taenia coli skinned fibers and bovine aortic actornyosin. Incorporation of 32P both into taenia coli skinned fibers and into bovine aortic actomyosin was measured following incubation at 25°C for 10 min in a reaction mixture of 75-100 ~1 containing 1 mg/ml of either fiber homogenate or actomyosin. The reaction solutions were identical to those previously described for ATPase measurements. In all experiments, the reaction was started by adding ATP to a final concentration 1 mM with [y-““P]ATP to yield a specific activity of 250 cpm/pmol ATP. After the appropriate reaction time, an equal volume of 2% sodium dodecyl sulfate (SDS) solution was added. The samples were then boiled for 5 min and subjected to SDS-slab gel electrophoresis on a continuous (5-20%) acrylamide gradient (32). Electrophoresis was run under constant current, first at 12.5 mA/gel (30 min) and then at 23 mA/gel (2-3 h). Gels were stained with Coomassie blue, destained in a methanol-acetic acid (40:20, vol/vol) solution and autoradiographed on X-ray film (Kodak XRP- 1). Phosphorylation levels were quantified by scanning densitometry of the Coomassie blue-stained gels, autoradiograms, and immunoblots
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Cl448
POLYLYSINE
ACTIVATES
SMOOTH
using a Zeineh soft laser scanning densitometry (Biomed Instruments) equipped with high-resolution optics and integrator. Phosphorylation values were calculated by integration of the spots corresponding to the phosphorylated MLC as a percent of the total integration of both the monophosphorylated and nonphosphorylated MLC. 32P incorporation into protein was calculated in the same manner, taking the level of MLC phosphorylation under Ca2+- calmodulin (CaM) as 100%; the amounts of protein in control, polylysine-treated, and Ca2+CaM-stimulated samples were identical. Each lane on the autoradiogram was scanned three times at various levels and a mean value was assigned to each 32P-labeled protein. Twelve fibers were run on a gel with four fibers per treatment condition, i.e., basal, basal + polylysine, and Ca2+-CaM. The densities assigned to each spot were averaged over four fibers for each
MUSCLE
CONTRACTION
condition. Thus each condition, including Ca2+-CaM, had a mean value with SE in arbitrary density units. Each mean was divided by the mean value in Ca2+-CaM (X 100%) to obtain a more relevant, normalized presentation. Thus all values had a SE. Preparation of type II phosphatase. Type II phosphatase was prepared from bovine lung by sequential steps involving ion exchange chromatography on DEAE-Sephacell, polylysine-agarose, and heparin-agarose according to method described by DiSalvo et al. (7, 8). Then, two cycles of chromatography on a MONO Q HR5/5 ion exchange column using the Pharmacia system for fast protein liquid chromatography were carried out with molecular filtration on LKB Ultra-gel AcA-34 between each cycle. The purified (-2,000-fold) enzyme was stored at -80°C in 20 mM tris(hydroxymethyl)aminomethane (Tris), pH
A
I
60
minutes
1
20 ,uM POLYLYSINE 1.5 PM
Caz+
I
30 minutes
(
0.01
pM
Ca2+
1.5 pM
Ca2+
1
2 mM free Mgz+
6.5
(
0.01
mM
pM
free
MgZ+
Caz+
Fig. 1. Record of isometric force in guinea pig taenia coli skinned fibers. At time = 0, tissues were transferred from relaxing solution ( [Ca2+] < 0.01 PM, [Mg2+] = 2 mM) to contracting solution ( [Ca2+] = 1.5 PM, [Mg2+] = 2 mM). After achieving a near steady state contraction, fibers were returned again to same relaxing solution (solution A, for details, see METHODS). Once near basal levels of force had been reattained, 2 protocols were performed. Protocol 1, A: polylysine (lo-20 PM) was added to relaxing solution, and, after achieving a steady state of force, fibers were returned again to contracting solution. Protocol 2, B: some fibers, at same point, were transferred to a high MgC12 relaxing solution (solution B, for details, see METHODS) consisting of [Ca”+] c 0.01 PM and [Mg2+] = 6.5 mM. After achieving a new steady-state contraction, polylysine (lo-20 PM) was added.
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POLYLYSINE
ACTIVATES
SMOOTH
7.4, buffer containing 0.5 mM DTT and 50% (vol/vol) glycerol. Protein determination. Protein concentration was determined by the Bradford procedure (3) using bovine plasma gamma globulin as a standard. Statistical analysis. Standard analysis of variance or the Student’s t test as appropriate were used for statistical analysis with P < 0.05 taken as an indication of a statistical significant difference. Chemicals. All chemicals were analytical grade or better. Polylysine refers to polylysine hydrobromide (lo-13 kDa) purchased from Sigma Chemical (P-6516). RESULTS
Mechanical studies with taenia coli skinned fibers. The addition of 20 PM polylysine (lo-13 kDa) to fibers in our standard relaxing solution ( [Ca2+] < 0.01 PM) elicited a contraction in skinned fibers (Fig. 1A). The magnitude of the developed force was equal to 40 t 8% (n = 6) of that measured in contracting solution ([ Ca2+] = 1.5 PM). In parallel control experiments in the absence of polylysine, no force was developed. The time course of the force development elicited by 20 PM polylysine had a half-time of 17.1 t 1.0 min (n = 6). This was similar to the value of 22.6 t 2.2 min (n = 6) obtained in the contracting solution; the difference for paired variates was not statistically significant. These times are somewhat longer than that previously observed (25) and are largely attributable to the lower temperature and calmodulin concentration used in the present study. Further addition of polylysine did not increase isometric force. Higher initial concentrations of polylysine were associated with a somewhat more rapid time course of force development; however, the maximal increase in force was similar to that observed at lower concentrations. Upon transfer of the polylysine-contracted fibers to contracting solution, isometric force increased. The final level of force achieved was variable ranging between 60 to 90% of that in the initial control contraction. Polylysine also affected the ability of the fibers to relax. Fibers contracted in the presence of Ca2+ relaxed within 15 min after transfer to Ca2+-free relaxing solution (the half-time of relaxation was 7.1 t 0.8 min, n = 3). In contrast, after exposure to polylysine the contracted fibers exhibited a slow and incomplete relaxation (after 2 h) upon return to the control solution. Relaxation was significantly accelerated by inclusion of heparin (190 pg/ml); basal values were reattained within 60-70 min. Heparin (190 pg/ml) added prior to polylysine (20 PM) inhibited the stimulatory effect on force generation in skinned fibers. A quick release of 10% of the length of the fiber, imposed after isometric force elicited by polylysine had become steady, was followed by a redevelopment of tension. This suggests that the contraction elicited by polylysine was not rigor. Our standard solutions for skinned fibers (solution A), contain 7.5 mM ATP and 10 mM MgCl,. In these solutions, polylysine induced a level of turbidity associated with the formation of an ATP-polylysine complex (27). This could be eliminated by increasing the MgC12-ATP ratio to lO:l, favoring Mg-ATP over polylysine-ATP. All subsequent experiments were thus repeated in these modified solutions (solution B). As shown in Fig. IB, the effects of polylysine on skinned fibers in these solutions
MUSCLE
Cl449
CONTRACTION
were qualitatively similar. However, as previously reported (14), 10 mM MgC12 under these conditions could elicit a contraction. The MgCl, contracture averaged -16.6 t 5.5%, and polylysine induced a further contraction of 14.8 t 3% (n = 4). If polylysine effectively removed all the ATP from the solution (a worse-case scenario), free Mg2+ would increase from 6.5 to 7.2 mM. We tested the effects of such a change by adding 1 mM MgC12 to our solution. A small increase, 1.7 k 0.5%, was seen in three fibers. Thus the increase in isometric force elicited by polylysine cannot be attributed in an increase in Mg2+. AWase activity studies. To further investigate the nature of the activation of contraction by polylysine, the ATPase activities of both fibers and actomyosin were studied. In the absence of Ca 2+, the ATPase activity of all the fibers studied averaged 2.54 t 0.17 nmol min-l mg dry weight-l (n = 29) and that of actomyosin averaged 2.91 t 0.10 nmolmin-l l mg protein-l (n = 36). In the presence of 14.9 PM Ca2+ and 0.1 PM calmodulin, the l
Eu 3 B 4 I3
l
160 140
@ I
120
vI
100 80
1200
1
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I
I
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__ 0
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..............*.............*. A I
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Fig. 2. ATPase activity as a function of either polylysine or lysine concentrations in relaxing solution. (Note that for polylysine used in these studies, its concentration can be expressed in terms of monomeric lysine units by multiplying by a factor of 80.) A: guinea pig taenia coli skinned fibers. B: bovine aortic actomyosin in presence of either polylysine (0) or lysine (a). Each value represents mean & SE of 3-9 different estimations. Basal ATPase activities, measured in relaxing conditions, were 2.54 t 0.17 nmolmin-l .mg dry weight-’ of fiber (n = 23) and 2.60 t 0.12 nmol~min-l~mg actomyosin protein-l (n = 17).
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Cl450
POLYLYSINE
ACTIVATES
SMOOTH
activities of fibers and actomyosin increased by 2.01- and 3. lo-fold, respectively. These controls were conducted in solutions (solution B) that contained a 1O:l Mg-ATP ratio for direct comparison to nonturbid, polylysine-containing solutions. To test whether high Mg2+ alone significantly increased the fiber ATPase in the absence of Ca2+, we compared ATPase activities in identical solutions save 10 mM vs. 3 mM MgC1, (-2 mM Mg2+). Fiber ATPase activities of 1.26 t 0.16 and 1.53 t 0.23 nmol emin-l mg dry weight-l (n = 9) were measured in 3 and 10 mM MgC12 solutions, respectively. The increase (21%) of ATPase activity in 10 mM MgC12-containing solutions was comparable with that of isometric force generation. When tested as paired variates, these differences in ATPase were statistically significant (P < 0.05). In the absence of Ca2+, polylysine elicits a dose-dependent increase in the ATPase activity of both fibers and actomyosin (Fig. 2). To achieve a level similar to that elicited by Ca2+-CaM,-8-12 PM polylysine were required for actomyosin. Fiber ATPase activity reached a plateau value of 160% at polylysine concentrations in the 80- to 160-PM range. Actomyosin ATPase activity appeared to saturate at polylysine concentrations of lOOhigher than control. In 600 PM and at a level -lo-fold subsequent studies, concentrations greater than 200 PM were not used as significant increases in ionic strength and could render interpretation of the data less straightforward. We also observed that 0.1 PM to 100 mM L-lysine did not alter basal actomyosin ATPase activity (Fig. 2). It should be noted that for the molecular weight of polylysine used, the concentration of polylysine is some 80-fold larger when expressed in terms of lysine monomeric units. For example, polylysine in the typical effector range of lo-20 PM would be on the order of millimolar lysine units. Thus we conclude that the effect of polylysine is not a simple charge effect. These effects of polylysine could be attributed to inhil
A
SKINNED FIBERS
B
MUSCLE
CONTRACTION
bition of phosphatase activity or alternatively stimulation of kinase(s). However, our assay conditions and protocol for preparation of bovine actomyosin produce nominally phosphatase-free actomyosin. We phosphorylated LC20 in our actomyosin by addition of ys2-ATP and Ca2+-CaM under conditions similar to that used for ATPase measurements. EGTA was added to reduce Ca2+ to < 0.3 PM, and LCzO dephosphorylation was monitored by gel autoradiography. Although some dephosphorylation could be measured, no significant dephosphorylation was measured over the initial lo-min period, a time over which our measurements of ATPase activity were made. Thus our observations on the activation of the actomyosin ATPase by polylysine suggest a mechanism other than inhibition of phosphatase. The following experiments, summarized in Fig. 3, A and B, provide functional evidence supporting this hypothesis. The addition of EGTA to chelate Ca2+ decreased the ATPase activity of the fibers to near basal levels. Subsequent addition of polylysine (40-160 PM) activated the fiber ATPase activity to the levels observed in Ca2+-CaM. In contrast, addition of EGTA did not markedly decrease the ATPase activity of actomyosin from the level seen in Ca2+ -CaM. This supports our previous data indicating that our bovine aortic actomyosin preparation is functionally free of phosphatase. Subsequent additions of polylysine in a range of 2.5 to 13 PM did not change the ATPase activity from those elicited by Ca2+-CaM. Actomyosin ATPase activity, however, could be further increased at higher concentrations of polylysine (176 PM). Thus, in a concentration range in which both contraction and fiber ATPase activity were stimulated by polylysine, the ATPase activity of actomyosin was not increased beyond that induced by Ca2+-CaM. This complementarity was also tested using thiophosphorylated actomyosin. As shown in Fig. 4, the ATPase activity of thiophosphorylated actomyosin was not increased by
ACTOMYOSIN
Fig. 3. ATPase activity in guinea pig taenia coli skinned fibers (A) and bovine aortic actomyosin (B). Assay was performed as described in METHODS in following solutions: I) relaxing, 2) contracting, 3) [Ca”+] = 0.3 PM, as a consequence of addition of 20 mM EGTA to contracting solution, and 4-6) identical to that of so&ion 3 but with added polylysine (4A, 40 ,uM; 5A, 160 PM; 4B, 2.5 PM; 5B, 12 PM; 6, 176 PM). Each value represents mean t SE of 3-6 fibers and 3-7 different estimations on actomyosin. All test values, with exception of solution 3 (A), were different from their respective controls (solution 1) at level of P < 0.05.
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POLYLYSINE
- THIOPHOSPHORYLAmD
ACTIVATES
ACTOMYOSIN
SMOOTH
-
T
1
Fig. 4. Effect of thiophosphorylation on ATPase activity in bovine aortic actomyosin. Assay was performed as described in METHODS in following reaction solutions: 0) before thiophosphorylation in relaxing solution and then posttreatment with ATPyS in 1) relaxing, 2) contracting, and 3 and 4) relaxing solution with either 2.8 or 13 PM polylysine, respectively. Each value represents mean t SE of 6 different estimations.
polylysine (2.8-13 PM) nor as expected by Ca2+-CaM. We also measured the effect of polylysine on the ATPase activity in partially dephosphorylated actomyosin to test our working hypothesis that the polylysine stimulation of actomyosin ATPase was not attributable to simple inhibition of phosphatase. These results are graphically summarized in Fig. 5. Actomyosin was phosphorylated in the presence of Ca2+-CaM and then EGTA was added to inhibit kinase activity. Type II phosphatase
MUSCLE
CONTRACTION
Cl451
(257 mu/ml) was then added to dephosphorylate myosin, and a significant decrease in ATPase activity was observed. Inhibition of phosphatase at this stage would be expected to result in a constant or slowly decreasing actomyosin ATPase activity as the kinase would be inhibited in the presence of EGTA. However, we found that the addition of polylysine (2 PM) increased the ATPase activity (Fig. 5). These results are also consistent with those shown in Figs. 3 and 4, in that the stimulatory effects of polylysine in low concentration ranges (2.543 PM) were complementary to that of phosphorylation, i.e., polylysine stimulation of the actomyosin ATPase did not exceed that of phosphorylation. Preincubation with heparin (25 pg/ml), a negatively charged heteropolysaccharide, had no effect on basal actomyosin activity but inhibited the stimulatory effects of 3.0 PM polylysine (Fig. 6). Furthermore, the addition of heparin (95 pg/ml) to actomyosin previously stimulated with polylysine (10 PM) decreased the ATPase activity to basal levels (Fig. 6). This is likely a consequence of the interaction between polylysine and heparin (29), which reduces the concentration of polymer charges available for modulation of polylysine-sensitive enzyme activity (9). This observation indicates that the effects are specific to polylysine and serves as a control for any effects of potential impurities or the counter ion (bromide) in the polylysine used in these studies. Phosphorylation studies. To directly test whether the stimulation by polylysine of force and ATPase activity was coupled to kinase stimulation and/or phosphatase inhibition, we measured the phosphorylation state of the 20-kDa MLC (MLC-Pi) both in fibers and actomyosin. MLC-Pi under either relaxed or contracted conditions was compared with that in the presence of polylysine in 14 [
-
I
. .
\
if 12
fl 40 % Yl 2
1
-
0 -
$6El 0 5
4-
G 4
2-
0
0
Fig. 5. ATPase activity of bovine aortic actomyosin measured as described in METHODS in following solutions: 1) relaxing, 2) contracting, 3) [Ca”+] = 0.3 PM, as a consequence of addition of 20 mM EGTA to contracting solution, 4) identical to soZution 3 but with type II phosphatase (257 mu/ml), and 5) identical to sohtion 4 but including 2 PM polylysine. Each value represents mean t SE of 4 different estimations. All test values were significantly different than basal (P < 0.05) and ATPase activity after addition of phosphatase significantly decreased as compared with that measured in the Ca2+-CaM and EGTA solution.
Fig. 6. described relaxing relaxing 3.0 FM pg/ml). Solutions 0.05.
ATPase activity of bovine aortic actomyosin measured as in METHODS in following solutions: 1) relaxing, 2 and 3) containing either with 2.8 or 10 PM polylysine, respectively, 4) solution with heparin (25 pg/ml), 5) same as sohtion 4 but with polylysine, and 6) same as solution 3 but with heparin (95 Each value represents mean t SE of 4 different estimations. 2 and 3 were different from control (solution I) at level of P
i -
j ,I *‘. ;
MLC----MLC-Pi
/
‘-
-
so-
-5.0 G,
G,
G,
1,
12
13
ACTOMYOSIN Fig. 7. Top: densitometric measurement of IEF gels of skinned fibers (left) and actomyosin (right). Bottom: IEF gels (G lanes) and immunoblots (I lanes) of skinned fibers (left) and actomyosin (75 pg, right). All symbols are identical for both panels. Samples were treated as described in METHODS under following conditions: I) relaxing solution, 2) in presence of 20 and 2.5 PM polylysine (fibers and actomyosin, respectively) in relaxing solution, and 3) contracting solution. Unphosphorylated MLC and MLC-P, are indicated. Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
POLYLYSINE
ACTIVATES
SMOOTH
Table 1. Myosin LC,, phosphorylation Basal
W+-CaM
Polylysine
Fibers 6.92&1.80 (5) 3.22kO.40 (5) 49.80+1.24 (3) Actomyosin 9.57k1.17 (14) 7.99k2.94 (5) 52.62k1.65 (15) Each value represents the mean + SE of (n) different estimations. Percent phosphorylation state of 20-kDa myosin light chains (MLC-P,) in fibers and actomyosin under following conditions: relaxing, relaxing plus polylysine, and contracting. Only values in Caa+/calmodulin (CaM) were different from control and polylysine at level of P < 0.05.
ATP into actomyosin or skinned fiber homogenates. Homogenates rather than whole skinned fibers were used to increase the amount of material for enhanced detectability and to ensure uniformity in solubilization and loading onto the gels. Similar to the previously described experiments, the effects of relaxing, contracting, and polylysine-containing solutions were studied. Under all conditions studied, again no significant changes in the protein pattern on SDS-page gradient gels were seen for either skinned fiber homogenates or actomyosin (Fig. 8). One predominant phosphoprotein with a molecular weight corresponding to the 20-kDa myosin light was observed in the autoradiograms with a 2.5-h exposure. This band was only lightly labeled under relaxing conditions or, importantly, in the presence of polylysine. A substantial increase in 32P incorporation for either preparation was seen in the presence of the Ca2+-CaM. To increase detectability of lesser phosphoproteins, longer exposures (9 h for fibers and 3 days for actomyosin) were examined (Fig. 8). Additional phosphoproteins can be
MUSCLE
CONTRACTION
Cl453
seen, but none were observed to change with the conditions studied; a statistical summary is given in Table 2. The effects of MgC1, and polylysine. Our data thus indicate that the contraction and stimulation of ATPase activity elicited by polylysine is independent of MLC-Pi. It has been reported that high concentrations of MgC12 can also stimulate smooth muscle contraction and ATPase activity in the absence of MLC phosphorylation (14). This MgC12 stimulation is of interest for it has been attributed to a shift in the 10s to 6s conformation of smooth muscle myosin. We thus studied the effects of polylysine in the presence of MgC12 ranging from 10 to 40 mM. From 3 to 10 mM, MgC12 did not significantly affect the basal actomyosin ATPase activity, whereas at concentrations greater than 10 mM, ATPase activity was increased as previously reported (14). However, subsequent addition of polylysine elicited a further increase in ATPase activity (Fig. 9). It is worth noting that the stimulation by MgClz (15-35 mM) was lower than that elicited by Ca 2+-CaM whereas, as seen in Fig. 3, polylysine could elicit an increase in ATPase similar to that of Ca2+-CaM Measure’ments of the effects of polylysine for MgC12 concentrations
activates smooth muscle actin-myosin without LCzO phosphorylation
PAWEL T. SZYMANSKI, JOHN D. STRAUSS, GLENN DOERMAN, JOSEPH DISALVO, AND RICHARD J. PAUL Department of Physiology and Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0576; and Department of Physiology, School of Medicine, University of Minnesota, Duluth, Minnesota 55812 Szymanski, Pawel T., John D. Strauss, Glenn Doerman, Joseph DiSalvo, and Richard J. Paul. Polylysine activates smooth muscle actin-myosin interaction without LC,,, phosphorylation. Am. J. Physiol. 262 (Cell Physiol. 31): Cl446-Cl455, 1992.-Phosphorylation/dephosphorylation of the 20-kDa light chain of smooth muscle myosin is a major regulator of actin-myosin interaction. Phosphatase inhibitors have thus been shown to enhance contraction in smooth muscle. The activity of type II phosphatase against phosphorylated myosin light chains is inhibited by polylysine (7). Thus we studied the effects of polylysine (lo-13 kDa) on actin-myosin interaction in permeabilized guinea pig taenia coli fibers and in bovine aortic actomyosin. Addition of polylysine (lo-20 PM) to Ca-ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid buffered solution ( [Ca2+] < 0.01 PM) elicited a contraction in fibers of 40 t 8% (n = 6) of maximally stimulated contractions ( [Ca2+] = 1.5 PM). Untreated fibers did not generate any significant force in parallel control experiments. Similarly, polylysine stimulated the ATPase activity both in fibers and actomyosin in a dose-dependent manner. This stimulation could be completely inhibited and abolished upon addition of heparin, a negatively charged heteropolysaccharide. In actomyosin previously phosphorylated with ATP+, polylysine in a concentration range of 2-13 PM did not further stimulate enzyme activity. These increases in activity were not connected with significant changes in the phosphorylation of 20-kDa myosin light chain nor could any incorporation of 32P associated with polylysine stimulation be detected in both skinned fibers and actomyosin by autoradiography of SDS gels. Our data indicate that polylysine increases actin-myosin interaction in both smooth muscle model systems by directly influencing contractile proteins. As such, polylysine may be a useful probe for the mechanism of activation of smooth muscle. contraction
of the mechanisms underlying the regulation of smooth muscle contractility is yet a subject of considerable controversy (1, 5, 11, 12, 16), it is generally accepted that the phosphorylation/dephosphorylation of the 20-kDa light chains of myosin (MLC; LC& is an important component. In its simplest form, phosphorylation of MLC allows the activation of the myosin Mg2+-adenosinetriphosphatase (ATPase) by actin, increasing the rate of cross-bridge cycling and consequent generation of force and/or shortening. Dephosphorylation would result in inactivation of myosin and relaxation. The more complex physiological responses of smooth muscle have led to modification of this simple “switch” hypothesis as well as to the proposal of several alternative modulatory mechanisms. However, several modeling studies (11, 24) have suggested that phosphorylation/dephosphorylation of MLC can be sufficient to explain the observed physiological responses. A phosphatase isolated from bovine aortic smooth WHILE
Cl446
THE NATURE
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$2.00
Copyright
muscle was shown to dephosphorylate native myosin as well as isolated myosin light chains (6, 7). Its activity in vitro can be modulated by polycationic effecters such as lysine-rich histone H1, protamine, or polylysine, and hence it was called polycation-modulable phosphatase (7, 8). Subsequent studies revealed that polycationic modulation of phosphatase activity is a characteristic property of type II phosphatases (for review see Ref. 4). The activity of type II phosphatase against phosphorylated MLC is inhibited by polylysine in the concentration range of 5-10 PM (7). It was thus of interest to test whether polylysine could affect the contraction of smooth muscle simply by the inhibition of the dephosphorylation of MLC. In this study, we utilized a permeabilized or skinned fiber preparation to study the effects of polylysine in a structured model of smooth muscle. We report here that polylysine elicited a contraction and increased the ATPase activity in skinned fibers isolated from guinea pig taenia coli. Surprisingly, however, this contraction was not associated with any statistically significant changes in MLC phosphorylation. To provide further information on the mechanism underlying the polylysine-induced contraction, its effects on the ATPase activity of bovine aortic actomyosin were also studied. Our results indicate that polylysine increases actin-myosin interaction in both smooth muscle model systems independent of MLC or other protein phosphorylation/dephosphorylation. Thus polylysine may be a useful probe of the mechanism of regulation of actin-myosin interaction in smooth muscle. METHODS Skinned taenia coli fiber preparation. Taenia coli were dissected from guinea pigs and permeabilized or skinned as previously described (25). Briefly, strips of tissue were calcium depleted in a high potassium, ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA)-buffered solution, pH 7.0. This was followed by treatment with a similar solution containing 1% Triton X-100 for 4 h to solubilize the cell membranes. The fiber bundles were stored at -30°C in 50% (vol/vol) glycerol and 20 mM imidazole, pH 6.7, 4 mM EGTA, 7.5 mM ATP, 10 mM MgC12, and 1 mM NaNs. Mechanical studies with skinned fibers. Skinned fiber bundles (5.0 mm long, 100-200 pm thick) were mounted horizontally with a nitrocellulose-based glue between an AME(SensoNor a.s) force transducer and rigid post. The position of this post and hence the length of the fiber were controlled by a micrometer. Changes in force were monitored with a linear chart recorder. The bundle was stretched by -5% from its unloaded length to establish a resting tension of 0.1-0.3 mN. Fibers were bathed in 300 ~1 of relaxing solution maintained at 25°C containing the following: 20 mM imidazole, pH 6.7,4 mM
0 1992 the American
Physiological
Society
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POLYLYSINE
ACTIVATES
SMOOTH
EGTA, 7.5 mM NaATP, 10 mM MgCIB, 1 mM NaN,, 0.1 PM calmodulin, and an ATP regenerating system consisting of IO mM phosphocreatine and IO U/ml creatine phosphokinase (solutions with 7.5 mM ATP are designated solution A). The calculated concentration of Ca2+ was CO.01 PM. Contraction was elicited by transferring the fiber to a solution similar to relaxing solution but also containing 2.0 mM CaCl,. This contracting solution has a calculated (IO) free calcium ion concentration of 1.54 PM, 1.94 mM free Mg2+, 7.2 mM MgATP, and an ionic strength of 110 mM. The concentrations of EGTA and CaCl, stock solutions as well as the free calcium in the above solutions were verified using an Orion calcium electrode (2). In addition to these standard solutions, solutions (designated as solution B) with reduced ATP (1 mM) were also studied to avoid the turbidity produced by the binding of ATP to polylysine. The compositions of these solutions were identical to those above (solution A) but with ATP reduced to 1 mM. The calculated concentrations of Ca2+ were ~0.01 and 1.66 PM and of Mg2+ were 6.49 and 6.65 mM for relaxing and contracting solutions, respectively. Upon transfer of the fibers to contracting solution, steady-state isometric forces of between 0.5 and 2.5 mN (m 150 mN/mm2) were generated. All fibers in each treatment group were first rinsed in relaxing solution for a minimum of IO min and then contracted in contracting solution to establish maximum isometric force (a control contraction). After the control contraction, fibers were transferred to relaxing solution until a stable base line had been reestablished. Then they were transferred to relaxing solutions, which contained either 10 or 20 PM polylysine. After a new level of isometric force was obtained, the concentration of polylysine was increased in 10 PM increments. After the changes in isometric force became stable, the fibers were transferred into fresh contracting solution (without polylysine) and the contractions were again monitored. To complete this protocol, fibers were transferred into either relaxing solution without polylysine or into relaxing solution plus heparin. In some fibers, the mechanical effects of a quick-step decrease in length of 10% of the initial fiber length were studied after a stable contraction in the presence of polylysine had been obtained. Tuenia coli skinned fiber homogenates. Freshly skinned fibers were homogenized at liquid N, temperatures using a dental amalgamator and stored at -80°C. Prior to an experiment, the powder obtained from this procedure was suspended in a solution containing 20 mM 2-(N-morpholino)propanesulfonic acid (MOPS), pH 6.7,lO mM MgC12, 4 mM EGTA, and 24 mM KCl. Preparation of bovine aortic actornyosin. Ca2+-sensitive actomyosin was prepared from bovine aortas according to the method described by Litten et al. (19). Briefly, the muscularis was extracted (4°C) at low ionic strength in a solution containing 80 mM KCl, 18 mM MOPS, pH 7.0, 4 mM MgC12, 4 mM EGTA, 4 mM ATP, 1 mM dithiothreitol (DTT), and 1 mM NaN,. The actomyosin was precipitated by dialysis against a lower ionic strength solution composed of 25 mM KCl, 4 mM MOPS, pH 7.0, 0.2 mM EGTA, 4 mM MgCl,, and 0.5 mM DTT, sedimented at 10,000 g for 15 min, washed four times in 8 vol of the same solution with 1% Triton X-100, and then washed five times with the identical solution but without Triton X-100. The final pellet was suspended in a solution containing 18 mM MOPS, pH 7.0,25 mM KCl, 1 mM MgC12, 1 mM DTT, and 60% (vol/vol) glycerol. Although not studied systematically, actomyosin could be stored at -80°C for several weeks without appreciable change in ATPase activity. ATPase activity measurements. The ATPase activity of both skinned fibers and actomyosin was studied under similar conditions in cuvettes of 600 ~1. Skinned fibers, =7 mm in length and 500-600 pm in diameter, were attached with a nitrocellulose-based glue between the arms of a stainless steel stirring rod.
MUSCLE
CONTRACTION
Cl447
Alternatively, actomyosin at a concentration of 100-200 pg/ml was placed in the cuvette. The reaction was carried out at 25°C in various solutions. The basic relaxing solution consisted of 20 mM MOPS, pH 6.7,lO mM MgCl,, 24 mM KCl, 4 mM EGTA, 0.5 mM DTT, 1 mM NaN,, and 1 mM ATP. Polylysine, lysine, heparin, type II phosphatase, or different concentrations of MgCl, were added as noted. Contracting solution was similar to relaxing solution but also contained 0.1 PM calmodulin and 3.6 mM CaC12 resulting in a calculated free calcium concentration of 14.9 PM, 8.9 mM free Mg2+, and an ionic strength not exceeding 110 mM. In some experiments, EGTA was added (with appropriate KOH to maintain constant pH) to achieve a final total concentration of 24 mM, which reduced the free calcium concentration to ~0.3 PM. All solutions contained an ATPregenerating system consisting of IO mM phospho(enol)pyruvate and 20 pg/ml pyruvate kinase. To measure ATPase activity in terms of the production of ADP, the pyruvate produced by the regenerating system was linked to the production of NAD by including 50 pg/ml of lactate dehydrogenase and 50 PM NADH (20). Rates of ATP hydrolysis were measured as a decrease of fluorescence of NADH in a Perkin-Elmer 650-10s spectrofluorimeter. The actomyosin ATPase activity of thiophosphorylated myosin was also measured. Thiophosphorylated myosin was prepared by incubation of actomyosin (2 mg/ml) in contracting solution containing 1 mM ATPyS for 45-60 min at 25°C in a total reaction volume of 800 ~1. The reaction was terminated by centrifugation at 10,000 g for 5 min and resuspension of the pellet in relaxing solution. After two further washes, the thiophosphorylated actomyosin was resuspended in the same relaxing buffer and used for ATPase measurements. Quantification of MLC phosphate content. Fibers isometrically mounted on wire frames or actomyosin were incubated at 25°C for 10 min in 200 ~1 of the various reaction mixtures described above. In all experiments, the reaction was started by adding ATP to achieve a final concentration of 1 mM. After the appropriate reaction time, the muscle fibers and actomyosin were denatured with 15% trichloroacetic acid for 10 min at 4°C. These were then rinsed (fiber bundles) or suspended (actomyosin) in a 20 mM imidazole, pH 6.7 buffer. MLC were separated on the basis of difference in their isoelectric point due to phosphorylation on urea-glycerol IEF-page gels with 2% Pharmalyte (pH 4.5 to pH 5.4; Ref. 13). Once separated, they were transferred into nitrocellulose paper. Proteins were identified using affinity-purified goat anti-myosin light chain antibodies and horseradish peroxidase-conjugated rabbit anti-goat antibodies (23). 4-Chloro-I-naphthol and peroxide were used to color the immunoreactive bands. Incorporation of 32P into taenia coli skinned fibers and bovine aortic actornyosin. Incorporation of 32P both into taenia coli skinned fibers and into bovine aortic actomyosin was measured following incubation at 25°C for 10 min in a reaction mixture of 75-100 ~1 containing 1 mg/ml of either fiber homogenate or actomyosin. The reaction solutions were identical to those previously described for ATPase measurements. In all experiments, the reaction was started by adding ATP to a final concentration 1 mM with [y-““P]ATP to yield a specific activity of 250 cpm/pmol ATP. After the appropriate reaction time, an equal volume of 2% sodium dodecyl sulfate (SDS) solution was added. The samples were then boiled for 5 min and subjected to SDS-slab gel electrophoresis on a continuous (5-20%) acrylamide gradient (32). Electrophoresis was run under constant current, first at 12.5 mA/gel (30 min) and then at 23 mA/gel (2-3 h). Gels were stained with Coomassie blue, destained in a methanol-acetic acid (40:20, vol/vol) solution and autoradiographed on X-ray film (Kodak XRP- 1). Phosphorylation levels were quantified by scanning densitometry of the Coomassie blue-stained gels, autoradiograms, and immunoblots
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Cl448
POLYLYSINE
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using a Zeineh soft laser scanning densitometry (Biomed Instruments) equipped with high-resolution optics and integrator. Phosphorylation values were calculated by integration of the spots corresponding to the phosphorylated MLC as a percent of the total integration of both the monophosphorylated and nonphosphorylated MLC. 32P incorporation into protein was calculated in the same manner, taking the level of MLC phosphorylation under Ca2+- calmodulin (CaM) as 100%; the amounts of protein in control, polylysine-treated, and Ca2+CaM-stimulated samples were identical. Each lane on the autoradiogram was scanned three times at various levels and a mean value was assigned to each 32P-labeled protein. Twelve fibers were run on a gel with four fibers per treatment condition, i.e., basal, basal + polylysine, and Ca2+-CaM. The densities assigned to each spot were averaged over four fibers for each
MUSCLE
CONTRACTION
condition. Thus each condition, including Ca2+-CaM, had a mean value with SE in arbitrary density units. Each mean was divided by the mean value in Ca2+-CaM (X 100%) to obtain a more relevant, normalized presentation. Thus all values had a SE. Preparation of type II phosphatase. Type II phosphatase was prepared from bovine lung by sequential steps involving ion exchange chromatography on DEAE-Sephacell, polylysine-agarose, and heparin-agarose according to method described by DiSalvo et al. (7, 8). Then, two cycles of chromatography on a MONO Q HR5/5 ion exchange column using the Pharmacia system for fast protein liquid chromatography were carried out with molecular filtration on LKB Ultra-gel AcA-34 between each cycle. The purified (-2,000-fold) enzyme was stored at -80°C in 20 mM tris(hydroxymethyl)aminomethane (Tris), pH
A
I
60
minutes
1
20 ,uM POLYLYSINE 1.5 PM
Caz+
I
30 minutes
(
0.01
pM
Ca2+
1.5 pM
Ca2+
1
2 mM free Mgz+
6.5
(
0.01
mM
pM
free
MgZ+
Caz+
Fig. 1. Record of isometric force in guinea pig taenia coli skinned fibers. At time = 0, tissues were transferred from relaxing solution ( [Ca2+] < 0.01 PM, [Mg2+] = 2 mM) to contracting solution ( [Ca2+] = 1.5 PM, [Mg2+] = 2 mM). After achieving a near steady state contraction, fibers were returned again to same relaxing solution (solution A, for details, see METHODS). Once near basal levels of force had been reattained, 2 protocols were performed. Protocol 1, A: polylysine (lo-20 PM) was added to relaxing solution, and, after achieving a steady state of force, fibers were returned again to contracting solution. Protocol 2, B: some fibers, at same point, were transferred to a high MgC12 relaxing solution (solution B, for details, see METHODS) consisting of [Ca”+] c 0.01 PM and [Mg2+] = 6.5 mM. After achieving a new steady-state contraction, polylysine (lo-20 PM) was added.
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POLYLYSINE
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7.4, buffer containing 0.5 mM DTT and 50% (vol/vol) glycerol. Protein determination. Protein concentration was determined by the Bradford procedure (3) using bovine plasma gamma globulin as a standard. Statistical analysis. Standard analysis of variance or the Student’s t test as appropriate were used for statistical analysis with P < 0.05 taken as an indication of a statistical significant difference. Chemicals. All chemicals were analytical grade or better. Polylysine refers to polylysine hydrobromide (lo-13 kDa) purchased from Sigma Chemical (P-6516). RESULTS
Mechanical studies with taenia coli skinned fibers. The addition of 20 PM polylysine (lo-13 kDa) to fibers in our standard relaxing solution ( [Ca2+] < 0.01 PM) elicited a contraction in skinned fibers (Fig. 1A). The magnitude of the developed force was equal to 40 t 8% (n = 6) of that measured in contracting solution ([ Ca2+] = 1.5 PM). In parallel control experiments in the absence of polylysine, no force was developed. The time course of the force development elicited by 20 PM polylysine had a half-time of 17.1 t 1.0 min (n = 6). This was similar to the value of 22.6 t 2.2 min (n = 6) obtained in the contracting solution; the difference for paired variates was not statistically significant. These times are somewhat longer than that previously observed (25) and are largely attributable to the lower temperature and calmodulin concentration used in the present study. Further addition of polylysine did not increase isometric force. Higher initial concentrations of polylysine were associated with a somewhat more rapid time course of force development; however, the maximal increase in force was similar to that observed at lower concentrations. Upon transfer of the polylysine-contracted fibers to contracting solution, isometric force increased. The final level of force achieved was variable ranging between 60 to 90% of that in the initial control contraction. Polylysine also affected the ability of the fibers to relax. Fibers contracted in the presence of Ca2+ relaxed within 15 min after transfer to Ca2+-free relaxing solution (the half-time of relaxation was 7.1 t 0.8 min, n = 3). In contrast, after exposure to polylysine the contracted fibers exhibited a slow and incomplete relaxation (after 2 h) upon return to the control solution. Relaxation was significantly accelerated by inclusion of heparin (190 pg/ml); basal values were reattained within 60-70 min. Heparin (190 pg/ml) added prior to polylysine (20 PM) inhibited the stimulatory effect on force generation in skinned fibers. A quick release of 10% of the length of the fiber, imposed after isometric force elicited by polylysine had become steady, was followed by a redevelopment of tension. This suggests that the contraction elicited by polylysine was not rigor. Our standard solutions for skinned fibers (solution A), contain 7.5 mM ATP and 10 mM MgCl,. In these solutions, polylysine induced a level of turbidity associated with the formation of an ATP-polylysine complex (27). This could be eliminated by increasing the MgC12-ATP ratio to lO:l, favoring Mg-ATP over polylysine-ATP. All subsequent experiments were thus repeated in these modified solutions (solution B). As shown in Fig. IB, the effects of polylysine on skinned fibers in these solutions
MUSCLE
Cl449
CONTRACTION
were qualitatively similar. However, as previously reported (14), 10 mM MgC12 under these conditions could elicit a contraction. The MgCl, contracture averaged -16.6 t 5.5%, and polylysine induced a further contraction of 14.8 t 3% (n = 4). If polylysine effectively removed all the ATP from the solution (a worse-case scenario), free Mg2+ would increase from 6.5 to 7.2 mM. We tested the effects of such a change by adding 1 mM MgC12 to our solution. A small increase, 1.7 k 0.5%, was seen in three fibers. Thus the increase in isometric force elicited by polylysine cannot be attributed in an increase in Mg2+. AWase activity studies. To further investigate the nature of the activation of contraction by polylysine, the ATPase activities of both fibers and actomyosin were studied. In the absence of Ca 2+, the ATPase activity of all the fibers studied averaged 2.54 t 0.17 nmol min-l mg dry weight-l (n = 29) and that of actomyosin averaged 2.91 t 0.10 nmolmin-l l mg protein-l (n = 36). In the presence of 14.9 PM Ca2+ and 0.1 PM calmodulin, the l
Eu 3 B 4 I3
l
160 140
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120
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100 80
1200
1
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I
I
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..............*.............*. A I
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Fig. 2. ATPase activity as a function of either polylysine or lysine concentrations in relaxing solution. (Note that for polylysine used in these studies, its concentration can be expressed in terms of monomeric lysine units by multiplying by a factor of 80.) A: guinea pig taenia coli skinned fibers. B: bovine aortic actomyosin in presence of either polylysine (0) or lysine (a). Each value represents mean & SE of 3-9 different estimations. Basal ATPase activities, measured in relaxing conditions, were 2.54 t 0.17 nmolmin-l .mg dry weight-’ of fiber (n = 23) and 2.60 t 0.12 nmol~min-l~mg actomyosin protein-l (n = 17).
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Cl450
POLYLYSINE
ACTIVATES
SMOOTH
activities of fibers and actomyosin increased by 2.01- and 3. lo-fold, respectively. These controls were conducted in solutions (solution B) that contained a 1O:l Mg-ATP ratio for direct comparison to nonturbid, polylysine-containing solutions. To test whether high Mg2+ alone significantly increased the fiber ATPase in the absence of Ca2+, we compared ATPase activities in identical solutions save 10 mM vs. 3 mM MgC1, (-2 mM Mg2+). Fiber ATPase activities of 1.26 t 0.16 and 1.53 t 0.23 nmol emin-l mg dry weight-l (n = 9) were measured in 3 and 10 mM MgC12 solutions, respectively. The increase (21%) of ATPase activity in 10 mM MgC12-containing solutions was comparable with that of isometric force generation. When tested as paired variates, these differences in ATPase were statistically significant (P < 0.05). In the absence of Ca2+, polylysine elicits a dose-dependent increase in the ATPase activity of both fibers and actomyosin (Fig. 2). To achieve a level similar to that elicited by Ca2+-CaM,-8-12 PM polylysine were required for actomyosin. Fiber ATPase activity reached a plateau value of 160% at polylysine concentrations in the 80- to 160-PM range. Actomyosin ATPase activity appeared to saturate at polylysine concentrations of lOOhigher than control. In 600 PM and at a level -lo-fold subsequent studies, concentrations greater than 200 PM were not used as significant increases in ionic strength and could render interpretation of the data less straightforward. We also observed that 0.1 PM to 100 mM L-lysine did not alter basal actomyosin ATPase activity (Fig. 2). It should be noted that for the molecular weight of polylysine used, the concentration of polylysine is some 80-fold larger when expressed in terms of lysine monomeric units. For example, polylysine in the typical effector range of lo-20 PM would be on the order of millimolar lysine units. Thus we conclude that the effect of polylysine is not a simple charge effect. These effects of polylysine could be attributed to inhil
A
SKINNED FIBERS
B
MUSCLE
CONTRACTION
bition of phosphatase activity or alternatively stimulation of kinase(s). However, our assay conditions and protocol for preparation of bovine actomyosin produce nominally phosphatase-free actomyosin. We phosphorylated LC20 in our actomyosin by addition of ys2-ATP and Ca2+-CaM under conditions similar to that used for ATPase measurements. EGTA was added to reduce Ca2+ to < 0.3 PM, and LCzO dephosphorylation was monitored by gel autoradiography. Although some dephosphorylation could be measured, no significant dephosphorylation was measured over the initial lo-min period, a time over which our measurements of ATPase activity were made. Thus our observations on the activation of the actomyosin ATPase by polylysine suggest a mechanism other than inhibition of phosphatase. The following experiments, summarized in Fig. 3, A and B, provide functional evidence supporting this hypothesis. The addition of EGTA to chelate Ca2+ decreased the ATPase activity of the fibers to near basal levels. Subsequent addition of polylysine (40-160 PM) activated the fiber ATPase activity to the levels observed in Ca2+-CaM. In contrast, addition of EGTA did not markedly decrease the ATPase activity of actomyosin from the level seen in Ca2+ -CaM. This supports our previous data indicating that our bovine aortic actomyosin preparation is functionally free of phosphatase. Subsequent additions of polylysine in a range of 2.5 to 13 PM did not change the ATPase activity from those elicited by Ca2+-CaM. Actomyosin ATPase activity, however, could be further increased at higher concentrations of polylysine (176 PM). Thus, in a concentration range in which both contraction and fiber ATPase activity were stimulated by polylysine, the ATPase activity of actomyosin was not increased beyond that induced by Ca2+-CaM. This complementarity was also tested using thiophosphorylated actomyosin. As shown in Fig. 4, the ATPase activity of thiophosphorylated actomyosin was not increased by
ACTOMYOSIN
Fig. 3. ATPase activity in guinea pig taenia coli skinned fibers (A) and bovine aortic actomyosin (B). Assay was performed as described in METHODS in following solutions: I) relaxing, 2) contracting, 3) [Ca”+] = 0.3 PM, as a consequence of addition of 20 mM EGTA to contracting solution, and 4-6) identical to that of so&ion 3 but with added polylysine (4A, 40 ,uM; 5A, 160 PM; 4B, 2.5 PM; 5B, 12 PM; 6, 176 PM). Each value represents mean t SE of 3-6 fibers and 3-7 different estimations on actomyosin. All test values, with exception of solution 3 (A), were different from their respective controls (solution 1) at level of P < 0.05.
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POLYLYSINE
- THIOPHOSPHORYLAmD
ACTIVATES
ACTOMYOSIN
SMOOTH
-
T
1
Fig. 4. Effect of thiophosphorylation on ATPase activity in bovine aortic actomyosin. Assay was performed as described in METHODS in following reaction solutions: 0) before thiophosphorylation in relaxing solution and then posttreatment with ATPyS in 1) relaxing, 2) contracting, and 3 and 4) relaxing solution with either 2.8 or 13 PM polylysine, respectively. Each value represents mean t SE of 6 different estimations.
polylysine (2.8-13 PM) nor as expected by Ca2+-CaM. We also measured the effect of polylysine on the ATPase activity in partially dephosphorylated actomyosin to test our working hypothesis that the polylysine stimulation of actomyosin ATPase was not attributable to simple inhibition of phosphatase. These results are graphically summarized in Fig. 5. Actomyosin was phosphorylated in the presence of Ca2+-CaM and then EGTA was added to inhibit kinase activity. Type II phosphatase
MUSCLE
CONTRACTION
Cl451
(257 mu/ml) was then added to dephosphorylate myosin, and a significant decrease in ATPase activity was observed. Inhibition of phosphatase at this stage would be expected to result in a constant or slowly decreasing actomyosin ATPase activity as the kinase would be inhibited in the presence of EGTA. However, we found that the addition of polylysine (2 PM) increased the ATPase activity (Fig. 5). These results are also consistent with those shown in Figs. 3 and 4, in that the stimulatory effects of polylysine in low concentration ranges (2.543 PM) were complementary to that of phosphorylation, i.e., polylysine stimulation of the actomyosin ATPase did not exceed that of phosphorylation. Preincubation with heparin (25 pg/ml), a negatively charged heteropolysaccharide, had no effect on basal actomyosin activity but inhibited the stimulatory effects of 3.0 PM polylysine (Fig. 6). Furthermore, the addition of heparin (95 pg/ml) to actomyosin previously stimulated with polylysine (10 PM) decreased the ATPase activity to basal levels (Fig. 6). This is likely a consequence of the interaction between polylysine and heparin (29), which reduces the concentration of polymer charges available for modulation of polylysine-sensitive enzyme activity (9). This observation indicates that the effects are specific to polylysine and serves as a control for any effects of potential impurities or the counter ion (bromide) in the polylysine used in these studies. Phosphorylation studies. To directly test whether the stimulation by polylysine of force and ATPase activity was coupled to kinase stimulation and/or phosphatase inhibition, we measured the phosphorylation state of the 20-kDa MLC (MLC-Pi) both in fibers and actomyosin. MLC-Pi under either relaxed or contracted conditions was compared with that in the presence of polylysine in 14 [
-
I
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Fig. 5. ATPase activity of bovine aortic actomyosin measured as described in METHODS in following solutions: 1) relaxing, 2) contracting, 3) [Ca”+] = 0.3 PM, as a consequence of addition of 20 mM EGTA to contracting solution, 4) identical to soZution 3 but with type II phosphatase (257 mu/ml), and 5) identical to sohtion 4 but including 2 PM polylysine. Each value represents mean t SE of 4 different estimations. All test values were significantly different than basal (P < 0.05) and ATPase activity after addition of phosphatase significantly decreased as compared with that measured in the Ca2+-CaM and EGTA solution.
Fig. 6. described relaxing relaxing 3.0 FM pg/ml). Solutions 0.05.
ATPase activity of bovine aortic actomyosin measured as in METHODS in following solutions: 1) relaxing, 2 and 3) containing either with 2.8 or 10 PM polylysine, respectively, 4) solution with heparin (25 pg/ml), 5) same as sohtion 4 but with polylysine, and 6) same as solution 3 but with heparin (95 Each value represents mean t SE of 4 different estimations. 2 and 3 were different from control (solution I) at level of P
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ACTOMYOSIN Fig. 7. Top: densitometric measurement of IEF gels of skinned fibers (left) and actomyosin (right). Bottom: IEF gels (G lanes) and immunoblots (I lanes) of skinned fibers (left) and actomyosin (75 pg, right). All symbols are identical for both panels. Samples were treated as described in METHODS under following conditions: I) relaxing solution, 2) in presence of 20 and 2.5 PM polylysine (fibers and actomyosin, respectively) in relaxing solution, and 3) contracting solution. Unphosphorylated MLC and MLC-P, are indicated. Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 10, 2019.
POLYLYSINE
ACTIVATES
SMOOTH
Table 1. Myosin LC,, phosphorylation Basal
W+-CaM
Polylysine
Fibers 6.92&1.80 (5) 3.22kO.40 (5) 49.80+1.24 (3) Actomyosin 9.57k1.17 (14) 7.99k2.94 (5) 52.62k1.65 (15) Each value represents the mean + SE of (n) different estimations. Percent phosphorylation state of 20-kDa myosin light chains (MLC-P,) in fibers and actomyosin under following conditions: relaxing, relaxing plus polylysine, and contracting. Only values in Caa+/calmodulin (CaM) were different from control and polylysine at level of P < 0.05.
ATP into actomyosin or skinned fiber homogenates. Homogenates rather than whole skinned fibers were used to increase the amount of material for enhanced detectability and to ensure uniformity in solubilization and loading onto the gels. Similar to the previously described experiments, the effects of relaxing, contracting, and polylysine-containing solutions were studied. Under all conditions studied, again no significant changes in the protein pattern on SDS-page gradient gels were seen for either skinned fiber homogenates or actomyosin (Fig. 8). One predominant phosphoprotein with a molecular weight corresponding to the 20-kDa myosin light was observed in the autoradiograms with a 2.5-h exposure. This band was only lightly labeled under relaxing conditions or, importantly, in the presence of polylysine. A substantial increase in 32P incorporation for either preparation was seen in the presence of the Ca2+-CaM. To increase detectability of lesser phosphoproteins, longer exposures (9 h for fibers and 3 days for actomyosin) were examined (Fig. 8). Additional phosphoproteins can be
MUSCLE
CONTRACTION
Cl453
seen, but none were observed to change with the conditions studied; a statistical summary is given in Table 2. The effects of MgC1, and polylysine. Our data thus indicate that the contraction and stimulation of ATPase activity elicited by polylysine is independent of MLC-Pi. It has been reported that high concentrations of MgC12 can also stimulate smooth muscle contraction and ATPase activity in the absence of MLC phosphorylation (14). This MgC12 stimulation is of interest for it has been attributed to a shift in the 10s to 6s conformation of smooth muscle myosin. We thus studied the effects of polylysine in the presence of MgC12 ranging from 10 to 40 mM. From 3 to 10 mM, MgC12 did not significantly affect the basal actomyosin ATPase activity, whereas at concentrations greater than 10 mM, ATPase activity was increased as previously reported (14). However, subsequent addition of polylysine elicited a further increase in ATPase activity (Fig. 9). It is worth noting that the stimulation by MgClz (15-35 mM) was lower than that elicited by Ca 2+-CaM whereas, as seen in Fig. 3, polylysine could elicit an increase in ATPase similar to that of Ca2+-CaM Measure’ments of the effects of polylysine for MgC12 concentrations
