Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries SUZANNE MORELAND, JUNJI NISHIMURA, CORNELIS VAN BREEMEN, HEE YUL AHN, AND ROBERT S. MORELAND Department of Pharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543; Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146; and Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101 Moreland, Suzanne, Junji Nishimura, Cornelis van Breemen, Hee Yul Ahn, and Robert S. Moreland. Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries. Am. J. PhysioZ. 263 (Cell FhysioL. 32): C540-C544, 1992.~-Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in a-toxin-permeabilized smooth muscle. We used a-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca”‘]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca 2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G proteindependent increase in myofilament Ca2 + sensitivity. myosin light chain phosphorylation; muscle mechanics; shortening velocity; stiffness; a-toxin; mesenteric artery; sensitization; G proteins PRIMARY EVENT involved in the initiation of smooth muscle contraction following an increase in cytosolic free Ca2+ is phosphorylation of the ZO-kDa myosin light chain (MLC) catalyzed by the calmodulindependent MLC kinase (for review see Ref. 3). The sensitivity of the myofilaments to the cytosolic [Ca”‘] can be modulated as was first demonstrated by Morgan and Morgan (13). Nishimura et al. (14), using arteries permeabilized with a-toxin from Staphylococcus aureus, demonstrated that the agonist-induced increase in myofilament Ca2+ sensitivity is dependent on G proteins. This finding has since been corroborated by several groups using a-toxin (58), saponin (2, 9), or ,B-escin (8) to permeabilize the smooth muscle preparations. The dependence of this phenomenon on G proteins is based on the evidence that guanosine 5’-O-(3-thiotriphosphate) (GTP$S) produces an irreversible increase in myofilament Ca2+ sensitivity and guanosine 5’-0-(2thiodiphosphate) (GDPpS) inhibits the agonist plus
THE
c540
0363-6143/92
$2.00
Copyright
GTP increase in Ca2+ sensitivity. Whether the G proteins involved in this mechanism are the membraneassociated heterotrimeric G proteins (for review seeRef. 1) or the small cytoplasmic G proteins such as rho ~21 as recently suggested by Hirata et al. (4) is not known. Although the phenomenon of an agonist-induced increase in myofilament Ca2+ sensitivity is well accepted, the mechanism(s) involved in this G protein-dependent system has not been established. Fujiwara et al. (2) first demonstrated that the enhanced Ca2+ sensitivity of saponin-permeabilized vascular fibers in response to stimulation in the presence of GTPyS was accompanied by an increase in MLC phosphorylation. This finding was reproduced in saponin-permeabilized tracheal tissue (9) and in a-toxin-permeabilized fibers from both vascular and nonvascular sources (5-8). Kubota et al. (9) recently demonstrated that the increase in MLC phosphorylation following GTPyS stimulation is located in Serl”, the site catalyzed by MLC kinase. To account for the agonist and G protein-dependent increase in both Ca2+ sensitivity and MLC phosphorylation levels, Somlyo et al. (16) proposed G protein-dependent inhibition of a MLC phosphatase. We have also shown that agonist plus GTP increases force in cu-toxin-permeabilized vascular tissue (14, 15). However, in contrast to the studies described above, steady-state levels of MLC phosphorylation are not increased compared with stimulation with Ca”+ alone (15). This apparent discrepancy in whether or not MLC phosphorylation levels are increased by stimulation with an agonist plus GTP needed to be addressed. Therefore, the goal of this study was to determine the temporal relationship between force and MLC phosphorylation levels during stimulation by either Ca2+ alone or Ca2+ in combination with an agonist plus GTP. We also estimated shortening velocity and stiffness to obtain a more complete understanding of the mechanism(s) underlying the modulation of myofilament Ca”+ sensitivity. MATERIALS
AND METHODS
Adult male New Zealand White rabbits (2-2.5 kg) were killed by 100% CO, inhalation. Second- and third-order branches of the mesenteric artery (~250 pm outer diameter) were isolated and cleaned of fat and connective tissue. Small helical strips -5
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
TRANSIENT
MLC
PHOSPHORYLATION
mm in length were cut as close to perpendicular to the long axis of the vessel as possible. The strips used for isometric and isotonic force measurements were rnounted between two tweezers, one attached to a force transducer and the other to a motor in a Muscle Research System (Wissenschaftliche Gerate, Heidelberg), and perfused with a physiological salt solution (PSS) previously described (12). The strips used for the determination of MLC phosphorylation levels were equilibrated in a small beaker containing PSS. All experiments were performed at room temperature. The vascular strips were permeabilized with Staphylococcus aureus a-toxin as described by Nishimura et al. (14). After equilibration, the strips were stimulated with 10 mM ATP for 30 min, which produced a phasic contraction and desensitization of purinergic receptors. All subsequent solutions contained a minimum of 1 mM ATP to maintain purinergic receptor desensitization. The strips were then contracted with 10 PM norepinephrine (NE) and I mM ATP in a calcium-free PSS containing 2 mM ethylene glycol-his@-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) for 45 min to deplete cellular calcium. After this calcium depletion step, the smooth muscle cells were permeabilized by the addition of 2,500 hemolytic units/ml of a-toxin (GIBCO-BRL Life Sciences) for 60 min in a solution composed of (in mM) 2 EGTA, 100 K-methanesulfonate, 5.38 MgC&, 5.35 Na,ATP, 10 creatine phosphate, and 20 tris(hydroxymethyl)aminomethane (Tris) maleate (pH 7.0). All Ca2+-contracting solutions were composed of (in mM) 10 EGTA, 20 Tris maleate (pH 7.0), 10 creatine phosphate, 4 MgATP, 1 free Mg2+, appropriate CaCl, to achieve the desired free [ Ca2+], and appropriate K-methanesulfonate to maintain ionic strength at 0.12 M. The compositions of these solutions were calculated by a computer program that solves the appropriate multiequilibrium association equations (see Ref. 11 for a full description of the program). All permeabilized strips were perfused with fresh solution every 10 s providing a complete change of bath volume. Estimates of shortening velocity were performed by subjecting the strips to isotonic quick releases to afterloads ranging from 0.12 to 0.45 times the force at the instant of release. The force step was stable within 5-25 ms following release. The change in length from 200 to 300 ms following the release was used to calculate shortening velocity at a single afterload. Maximal shortening velocity was calculated by a linear transformation of the hyperbolic force-velocity relationship constructed from at least four releases to different afterloads. Estimates of tissue stiffness were performed by imposing a 500-Hz sine wave length change (0.05% L,) and measuring the resultant change in force with respect to length. All estimates of stiffness were normalized to those determined in the same tissues in response to 10 PM Ca2+. The motor was interfaced to a microcomputer for the initiation of experimental protocols, collection of data, and data analysis (software written by Dr. Konrad Guth and modified by Dr. Steven Petrou and Alex Fielding). MLC phosphorylation levels were determined on long (~10 mm) strips of the mesenteric arteries. After equilibration and permeabilization, the tissues were exposed to a Ca2+-containing solution in the presence of 10 ,uM ionomycin for varying times and then rapidly frozen in a dry ice-acetone slurry containing 6% trichloroacetic acid. The tissues were slowly thawed to room temperature and then transferred to a vial containing 50 ~1 of a homogenization solution previously frozen in liquid nitrogen, composed of 1% sodium dodecyl sulfate, 10% glycerol, and 20 mM dithiothreitol. The vial containing tissue, solution, and a small stainless steel ball was cooled in liquid nitrogen and placed in a dental amalgamator (Wig-A-Bug, Crescent Dental) for homogenization of the tissue. This procedure was repeated and then the vial was allowed to reach 4°C and the contents were transferred to a storage tube. The vial was rinsed with 50 ~1 of
IN PERMEABILIZED
c543.
ARTERIES
homogenization solution, which was added to the storage tube, and then stored at -80°C. Within 1 wk of homogenization, the samples were thawed and subjected to two-dimensional electrophoresis as described previously (12). After electrophoresis, the separated proteins were subjected to high-field-intensity Western blotting to nitrocellulose membranes. Visualization of the blotted proteins was performed using AuroDye forte gold protein stain (Amersham) as described by Kitazawa et al. (5). Quantitation of the stained blots was performed by scanning densitometry of the nitrocellulose paper made transparent by immersion in decalin as described by Maruyama et al. (10). Values are reported as moles Pi per mole MLC by integration of the spot corresponding to the phosphorylated MLC as a percent of the total of both the phosphorylated and unphosphorylated MLC. Group mean values were compared using unpaired two-tailed Student’s t test. A P value of ~0.05 was taken as significant. RESULTS
a-Toxin-permeabilized mesenteric tissues were contracted with 0.3 PM Ca2+ in the presence or absence of 10 PM NE plus 10 PM GTP. Force developed to significantly higher levels in the presence of NE plus GTP (Fig. 1A). The forces shown in this figure were normalized as a percent of a maximal contraction in the same tissue in response to 10 PM Ca2+. In control experiments, tissues were contracted three consecutive times with 10 PM Ca2+ or were contracted with 10 PM Ca2+ prior to the addition of NE plus GTP and again following relaxation from the NE plus GTP contraction. In all cases, the 10 PM Ca2+induced contractions were reproducible to within 5%. The addition of 10 PM ionomycin or A23187 did not affect either the rate of force development or the maximal level of force attained, indicating that organellar Ca2+ was not involved in the contractile response. Figure 1B shows the corresponding time course of the change in MLC phosphorylation levels under similar stimulation A
80
1
01 0
1 10
Minutes
J 20
0.01
I
I
1
0
10
20
Minutes
Fig. 1. Time course of force (A) and myosin light chain (MLC) phosphorylation (B) in response to 0.3 PM Ca2+ alone (0) or 0.3 ,uM Ca2+ plus 10 PM norepinephrine (NE) plus 10 PM GTP (D) in cu-toxinpermeabilized mesenteric arteries. All contractions were performed in presence of 10 mM EGTA to maintain intracellular [Ca2+] constant. Force data were normalized as a percent of maximal response obtained to 10 PM Ca2+ alone. In selected experiments, time course of force and MLC phosphorylation was extended to 60 min. Force and MLC phosphorylation levels were maintained for entire 60-min period: 48.2 + 7.2% maximal force (n = 4); 0.22 -t 0.07 mol Pi/m01 MLC (n = 2) with 0.3PM Ca2+ alone and 78.9 k 5.1% maximal force (n = 4); 0.27 + 0.06 mol Pi/m01 MLC (n = 2) with 0.3 PM Ca”+ plus 10 PM NE plus 10 PM GTP. Values shown are means + SE for 3-5 determinations. * P < 0.05 compared with Ca2+ alone. ** P < 0.05 compared with MLC phosphorylation levels at 2 min of stimulation with Ca2+ alone.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
c542
TRANSIENT
MLC
PHOSPHORYLATION
IN PERMEABILIZED
conditions. All determinations of MLC phosphorylation levels shown in Fig. 1B were performed in the presence of 10 PM ionomycin. During stimulation with 0.3 PM Ca2+ alone, MLC phosphorylation levels increased more or less monotonically, although the level of MLC phosphorylation at 20 min of stimulation with 0.3 PM Ca”+ alone was significantly lower than those at 1 and 2 min of stimulation. In contrast, during stimulation with 0.3 PM Ca2+ plus 10 PM NE plus 10 PM GTP, MLC phosphorylation levels initially increased to significantly higher levels than those obtained in response to Ca2+. However, with continued agonist stimulation, MLC phosphorylation levels fell to levels similar to those in 0.3 PM Ca2+ alone. Therefore, agonist stimulation of the a-toxin-permeabilized arterial strip resulted in a large transient in MLC phosphorylation, although the tissue was incubated in 10 PM ionomycin and the Ca2+ was buffered by 10 mM EGTA. These conditions should maintain the [Ca”‘] constant. Cross-bridge characteristics were further examined by estimating isotonic shortening velocities during contractions in response to Ca 2+ alone and in the presence of NE plus GTP. Figure 2A shows the time courses of isotonic shortening velocity during stimulation with 0.25 ,uM Ca2+ in the presence and absence of 10 PM NE plus 10 PM GTP. Although the addition of NE plus GTP transiently increased MLC phosphorylation levels, it had no effect on shortening velocities. To determine if the measurements of shortening velocity were Ca2+ dependent, the experiments were repeated during stimulation with a higher [Ca2+]. In 10 PM Ca2+, shortening velocities were significantly higher than during stimulation with 0.25 PM Ca2+; however, the addition of agonist plus GTP still had no effect on cross-bridge cycling rates (Fig. 2B). The data in Fig. 2 show the time course of shortening velocity at a single afterload of ~0.13 times the force at the instant of release. Estimates of the maximal velocity of shortening (V,,) were performed during steady-state conditions to more precisely examine cross-bridge cycling rates (Table 1). V, at steady state (20 min of stimulation) was Ca2+ dependent but was not affected at either [Ca2+] by the addition of NE plus GTP. This table also demonstrates that steady-state MLC phosphorylation levels were significantly increased in response to 10 ,uM Ca2+ compared with 0.3 PM Ca2+. The addition of NE plus GTP did not
ARTERIES
Table 1. Maximal velocity of shortening and MLC phosphorylation during steady-state contractions of a- toxin-permeabilized mesenteric arteries Condition
MIX
vo
0.25 pM Ca”+ 0.25 pM Ca”+ +lOhMNE + 10 pM GTP 10 ,uM Ca”+ 10 PM Ca2+ + 10 pM NE + 10 pM GTP
0.047+0.005 0.051t0.003
(6) (8)
0.23t0.04 0.25kO.01
(4) (4)
0.070+0.006* 0.072+0.0083(
(7) 10)
0.68*0.05* 0.67+0.04t
(4) (4)
Values are means t SE for number of determinations in parentheses. Vo, maximal velocity of shortening in Lo/s. Myosin light chain (MLC) phosphorylation levels in mol Pi/m01 MLC. cu-Toxin-permeabilized smooth muscle preparations were activated under appropriate conditions for 20 min to achieve steady-state force maintenance. Elevation of [Ca”+] significantly increased V,, and steady-state MLC phosphorylation levels whereas addition of NE plus guanosine triphosphate (GTP) had no effect. * P < 0.05 compared with 0.25 PM Ca”‘. t P < 0.05 compared with 0.25 FM Ca2+ plus NE plus GTP.
increase steady-state MLC phosphorylation levels at either [Ca2+]. Estimates of stiffness were performed to determine if the NE plus GTP-dependent increase in force resulted from a recruitment of additional cross bridges or a change in the force generation per cross bridge. The relationship between stress and stiffness was similar during stimulation with 0.25 and 10 PM Ca2+ alone and in the presence of 10 PM NE plus 10 PM GTP (Fig. 3). Therefore, the agonist and G protein-dependent force enhancement was apparently due to recruitment of cross bridges. DISCUSSION
The results of this study clearly demonstrate that, in the presence of Ca2+, agonist- and GTP-dependent stimulation of a-toxin-permeabilized vascular smooth muscle
125
_
100
-
6 G 2 IL
7 .,
r 0
/
/
;o.04v
+
: 0.03
ki z
-
.-if?
.- F gj
0.03
-
4
0.02
E
.-
;
I 0.01
s o,oo
i
g 0
IO
20
t 0.00
./‘,. ,’ // /’ / ,’ i , / ./,. / ‘-
0.25 pM Ca*+ + NE + GTP
t
r
g
/
i 0
Minutes
Fig. 2. Time course of isotonic shortening velocity PM Ca2+ alone (0) or 0.25 PM Ca”’ plus 10 PM NE 10 PM Ca2+ alone (0) or 10 PM Ca2+ plus 10 PM (II) (B). Data are from single isotonic releases to time of release. Values shown are means ,+ SE for
10
20
Minutes
in response to 0.25 plus GTP (m) (A) and NE plus 10 PM GTP -0.13 times force at 6-10 determinations.
‘/ .’
,/’ / ,
~
C lj
/
’ /’ / ,’
-
,
). / I / , , .’ /
,’ , , .’ / ./ ,
5
0.04
‘/
75-
ii
>;
, ‘/
0
-
A0 .-c
Phosphorylation
,
./ .‘/
/
,’
// / /i
,
i’ 1
.*’
10 pM Ca*+ + NE + GTP
Fig. 3. Steady-state force (solid bars) and stiffness (hatched bars) determinations in response to 0.25 and 10 FM Ca”+ alone and in presence of 10 PM NE plus 10 PM GTP. Data were normalized as a percent of response obtained to 10 PM Ca 2+ alone. Absolute values for force are as follows (in mg): 0.25 FM Ca fL+ alone = 24.6 + 3.5; 0.25 FM Ca”+ plus NE plus GTP = 87.8 + 6.7; 10 PM Ca’+ alone = 129.9 + 9.8; 10 PM Ca”+ plus NE plus GTP = 140.6 +: 7.3. Values shown are means + SE for 4-6 determinations.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
TRANSIENT
MLC
PHOSPHORYLATION
induces a large transient increase in the levels of MLC phosphorylation. This transient increase in MLC phosphorylation levels is observed under conditions of constant intracellular [Ca”+] and is accompanied by a monotonic increase in force. In contrast, stimulation of a-toxin-permeabilized vascular smooth muscle by Ca2+ alone induces a smaller monotonic increase in force and a very blunted transient, or even monotonic, increase in MLC phosphorylation. NE plus GTP enhanced both force and MLC phosphorylation early in the contractions in agreement with previous reports by Kitazawa et al. (5, 7, 8), who showed increased MLC phosphorylation levels at the peak of force development in response to an agonist plus GTP. After the initial increase, MLC phosphorylation decreased to a value similar to that obtained in response to Ca2+ alone in spite of the fact that NE plus GTP maintained force at an elevated level. A transient in MLC phosphorylation was previously reported in permeabilized phasic smooth muscle, but in this case force fell concomitantly with the decline in MLC phosphorylation (8). The observation that ionomycin did not affect force or MLC phosphorylation in response to NE plus GTP indicates that Ca2+ release from the sarcoplasmic reticulum was not involved in the agonist-induced contraction. This is in agreement with the finding of Somlyo et al. (18) that, in fluo-3-loaded a-toxin-permeabilized smooth muscle, flash photolysis of caged GTPyS increased force with no change in the [Ca”+]. Moreover, Somlyo and colleagues (6, 18) have shown by X-ray microanalysis that Ca-EGTA enters the cytosol of all cells in the cu-toxinpermeabilized tissue. Taken together, these results strongly suggest that the [Ca2+] remained constant in our preparation during stimulation with NE plus GTP. Stimulation with GTPyS alone or in combination with an agonist results in a maintained G protein-dependent increase in MLC phosphorylation during the steady-state phase of force maintenance (2, 9). We have also demonstrated that activation of the a-toxin-permeabilized mesenteric artery with 10 ,uM GTPyS results in a maintained elevation in MLC phosphorylation levels (15). The difference in results obtained with GTP$S, an irreversible activator of G proteins, and GTP, the presumed physiological mediator of contraction in vivo, suggests that GTPyS may mask the normal course of events that occur during agonist stimulation of vascular tissue as GTP is hydrolyzed. In response to either an increase in [Ca2+] or the addition of NE plus GTP, there was a concomitant increase in stiffness and force. The finding that the relationship between stiffness and force was not altered by the addition of NE plus GTP indicates that the enhancement in force development occurs by an increase in the number of force-generating cross bridges. Yamakawa and Brozovich (20) have shown in a-toxin-permeabilized single swine carotid artery cells that the increase in force is greater than the increase in stiffness following agonist plus GTP stimulation compared with stimulation by Ca2+ alone. The discrepancy in results may be due to speciesor tissue differences. Alternatively, small but significant agonistinduced effects on stiffness or force that are demonstrable
IN PERMEABILIZED
ARTERIES
c543
in the single cell might be obscured in the tissue. Cross-bridge cycling rate, as estimated by isotonic shortening velocity, was increased by Ca2+ but not by NE plus GTP. Fujiwara et al. (2) have shown that stimulation of saponin-permeabilized rabbit mesenteric artery by GTPrS significantly increases shortening velocity as measured by the slack test. Presumably this difference is the result of stimulation with GTPyS compared with NE plus GTP. It is also possible that the estimates of shortening velocity determined by Fujiwara et al. (2) using the slack test were more sensitive to small G protein-mediated changes than the single lightly loaded releasesshown in Fig. 2. This should not, however, account for the absence of a NE plus GTP-dependent increase in V,, during steady state. Our results would suggest that steady-state V,, depends solely on the [Ca2+] or possibly on the Ca2+dependent steady-state MLC phosphorylation. Our results are not consistent with a simple and direct relationship between force and MLC phosphorylation. Somlyo et al. (16) suggested that the increase in myofilament Ca2+ sensitivity is mediated by inhibition of a regulated MLC phosphatase. More recently, Kubota et al. (9) showed in saponin-permeabilized bovine trachea that the GTPyS-dependent increase in total MLC phosphorylation is due solely to increased phosphate incorporation at Serl!‘, the site phosphorylated by MLC kinase. Both groups have presented evidence demonstrating depressed MLC phosphatase activity following G protein activation (7, 9). Our results during the development phase of contraction are consistent with the hypothesis of G proteinmediated downregulation of a MLC phosphatase. However, the events that occur during force maintenance cannot be explained by this hypothesis. A recent study by Kubota et al. (9) indicated that regulation of MLC kinase activity is not involved. Therefore, a mechanism distinct from the MLC kinase-phosphatase system is required to account for maintenance of the receptor and G proteindependent enhanced force with MLC phosphorylation levels similar to those in response to Ca2+ alone. The mechanism(s) involved are beyond the scope of this study, but it will be interesting to investigate potential regulatory roles for thin filament-based proteins (19) or cooperative cross-bridge interactions (17). In summary, we have clearly demonstrated that the agonist plus GTP-dependent increase in force in the a-toxin-permeabilized mesenteric arteries is accompanied by a transient increase in MLC phosphorylation levels. Although the initial peak levels of MLC phosphorylation were significantly elevated compared with the effect of increasing Ca2+ alone steady-state levels of MLC phosphorylation were similar’ in the presence and absence of the agonist plus GTP. The receptor and G proteindependent increase in force was associated with an increased number of attached cross bridges; however, the cross-bridge cycling rate was unchanged and regulated solely by the [Ca”‘]. These results are consistent with the hypothesis that the enhancement during force development involves downregulation of a MLC phosphatase. However, the maintenance phase of agonist-enhanced force cannot be explained by this hypothesis and requires additional and as yet unidentified components.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
c544
TRANSIENT
MLC
PHOSPHORYLATION
This study was supported, in part, by National Heart, Lung, and Blood Institute Grants HL-37956 (to R. S. Moreland) and HL-3565’7 (to C. van Breemen), American Heart Association Southeastern Pennsylvania Affiliate Grant-in-Aid (to R. S. Moreland), and postdoctoral fellowships from the American Heart Association, Florida Affiliate (to J. Nishimura) and Southeastern Pennsylvania Affiliate (to H. Y. Ahn). Address for reprint requests: R. S. Moreland, Bockus Research Institute, Graduate Hospital, 415 S. 19th St., Philadelphia, PA 19146. Received
24 March
1992; accepted
in final
form
2 June
1992.
REFERENCES 1. Freissmuth, M., P. J. Casey, and A. G. Gilman. G proteins control diverse pathways of transrnembrane signaling. FASEB J. 3: 2125-2131, 1989. 2. Fujiwara, T., T. Itoh, Y. Kubota, and H. Kuriyama. Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J. Physiol. Lond. 408: 535-547, 1989. 3. Hartshorne, D. J. Biochemistry of the contractile process in smooth muscle. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, p. 423-482. 4. Hirata, K., A. Kikuchi, T. Sasaki, S. Kuroda, K. Haibuchi, Y. Matsuura, H. Seki, K. Saida, and Y. Takai. Involvement of rho ~21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J. Biol. Chem. 267: 7819-7822, 1992. 5. Kitazawa, T., B. D. Gaylinn, G. H. Denney, and A. P. Somlyo. G-protein mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J. BioZ. Chem. 266: 1708-1715, 1991. 6. Kitazawa, T., S. Kobayashi, K. Horiuch, A. V. Somlyo, and A. P. Somlyo. Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J. Biol. Chem. 264: 5339-5342, 1989. Kitazawa, T., M. Masuo, and A. P. Somlyo. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc. NlatZ. Acad. Sci. USA 88: 9307-9310, 1991. Kitazawa, T., and A. P. Somlyo. Desensitization and muscarinic resensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle. Biochem. Biophys. Res. Commun. 172: 1291-1297, 1990. Kubota, Y., M. Nomura, K. E. Kamm, M. C. Mumby, and J. T. Stull. GTPyS-dependent regulation of smooth muscle contractile elements. Am. J. Physiol. 262 (CelZ Physiol. 31): C405c410, 1992.
IN PERMEABILIZED
ARTERIES
T., T. Mikawa, and S. Ebashi. Detection of cal10. Maruyama, cium binding proteins by 4Wa autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J. Biothem. 95: 511-519, 1984. l1 Moreland, R. S., and R. A. Murphy. Determinants of Ca2+’ dependent stress maintenance in skinned swine carotid media. Am. J. Physiol. 251 (Cell Physiol. 20): C892-C903, 1986. 12. Moreland, S., and R. S. Moreland. Effects of dihydropyridines on stress, myosin phosphorylation, and V,, in smooth muscle. Am. J. PhysioZ. 252 (Heart Circ. Physiol. 21): H1049-H1058, 1987. 13. Morgan, J. P., and K. G. Morgan. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J. Physiol. Lond. 351: 155-167, 1984. J., M. Kolber, and C. van Breemen. Norepi14. Nishimura, nephrine and GTPyS increase myofilament Ca2+ sensitivity in a-toxin permeabilized arterial smooth muscle. Biochem. Biophys. Res. Commun. 163: 929-935, 1988. 15. Nishimura, J., S. Moreland, R. S. Moreland, and C. van Breemen. Regulation of the Ca 2+-force relationship in permeabilized arterial smooth muscle. Adv. Exp. Med. BioL. 304: 11 l-127, 1991. 16 . Somlyo, A. P., T. Kitazawa, B. Himpens, G. Matthijs, K. Horiuti, S. Kobayashi, Y. E. Goldman, and A. V. Somlyo. Modulation of Ca2+ -sensitivity and of the time course of contraction in smooth muscle: a major role of protein phosphatases? Adv. Protein Phosphatases 5: 181-195, 1989. A. V., Y. E. Goldman, T. Fujimori, M. Bond, D. R. 17. Somlyo, Trentham, and A. P. Somlyo. Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. J. Gen. PhysioZ. 91: 165-192, 1988. A. V., T. Kitazawa, K. Horiuti, S. Kobayashi, D. 18. Somlyo, Trentham, and A. P. Somlyo. Heparin-sensitive inositol trisphosphate signaling and the role of G-proteins in Ca”+-release and contractile regulation in smooth muscle. Prog. Clin. BioZ. Res. 327: 167-182, 1990. 19. Winder, S. J., C. Sutherland, and M. P. Walsh. Biochemical and functional characterization of smooth muscle calponin. Adv. Exp. Med. BioZ. 304: 37-51, 1991. M., and F. V. Brozovich. Agonist stimulation 20. Yamakawa, increases force in a-toxin permeabilized single vascular smooth muscle cells (Abstract). Biophys. J. 61: A164, 1992.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
THE
c540
0363-6143/92
$2.00
Copyright
GTP increase in Ca2+ sensitivity. Whether the G proteins involved in this mechanism are the membraneassociated heterotrimeric G proteins (for review seeRef. 1) or the small cytoplasmic G proteins such as rho ~21 as recently suggested by Hirata et al. (4) is not known. Although the phenomenon of an agonist-induced increase in myofilament Ca2+ sensitivity is well accepted, the mechanism(s) involved in this G protein-dependent system has not been established. Fujiwara et al. (2) first demonstrated that the enhanced Ca2+ sensitivity of saponin-permeabilized vascular fibers in response to stimulation in the presence of GTPyS was accompanied by an increase in MLC phosphorylation. This finding was reproduced in saponin-permeabilized tracheal tissue (9) and in a-toxin-permeabilized fibers from both vascular and nonvascular sources (5-8). Kubota et al. (9) recently demonstrated that the increase in MLC phosphorylation following GTPyS stimulation is located in Serl”, the site catalyzed by MLC kinase. To account for the agonist and G protein-dependent increase in both Ca2+ sensitivity and MLC phosphorylation levels, Somlyo et al. (16) proposed G protein-dependent inhibition of a MLC phosphatase. We have also shown that agonist plus GTP increases force in cu-toxin-permeabilized vascular tissue (14, 15). However, in contrast to the studies described above, steady-state levels of MLC phosphorylation are not increased compared with stimulation with Ca”+ alone (15). This apparent discrepancy in whether or not MLC phosphorylation levels are increased by stimulation with an agonist plus GTP needed to be addressed. Therefore, the goal of this study was to determine the temporal relationship between force and MLC phosphorylation levels during stimulation by either Ca2+ alone or Ca2+ in combination with an agonist plus GTP. We also estimated shortening velocity and stiffness to obtain a more complete understanding of the mechanism(s) underlying the modulation of myofilament Ca”+ sensitivity. MATERIALS
AND METHODS
Adult male New Zealand White rabbits (2-2.5 kg) were killed by 100% CO, inhalation. Second- and third-order branches of the mesenteric artery (~250 pm outer diameter) were isolated and cleaned of fat and connective tissue. Small helical strips -5
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
TRANSIENT
MLC
PHOSPHORYLATION
mm in length were cut as close to perpendicular to the long axis of the vessel as possible. The strips used for isometric and isotonic force measurements were rnounted between two tweezers, one attached to a force transducer and the other to a motor in a Muscle Research System (Wissenschaftliche Gerate, Heidelberg), and perfused with a physiological salt solution (PSS) previously described (12). The strips used for the determination of MLC phosphorylation levels were equilibrated in a small beaker containing PSS. All experiments were performed at room temperature. The vascular strips were permeabilized with Staphylococcus aureus a-toxin as described by Nishimura et al. (14). After equilibration, the strips were stimulated with 10 mM ATP for 30 min, which produced a phasic contraction and desensitization of purinergic receptors. All subsequent solutions contained a minimum of 1 mM ATP to maintain purinergic receptor desensitization. The strips were then contracted with 10 PM norepinephrine (NE) and I mM ATP in a calcium-free PSS containing 2 mM ethylene glycol-his@-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA) for 45 min to deplete cellular calcium. After this calcium depletion step, the smooth muscle cells were permeabilized by the addition of 2,500 hemolytic units/ml of a-toxin (GIBCO-BRL Life Sciences) for 60 min in a solution composed of (in mM) 2 EGTA, 100 K-methanesulfonate, 5.38 MgC&, 5.35 Na,ATP, 10 creatine phosphate, and 20 tris(hydroxymethyl)aminomethane (Tris) maleate (pH 7.0). All Ca2+-contracting solutions were composed of (in mM) 10 EGTA, 20 Tris maleate (pH 7.0), 10 creatine phosphate, 4 MgATP, 1 free Mg2+, appropriate CaCl, to achieve the desired free [ Ca2+], and appropriate K-methanesulfonate to maintain ionic strength at 0.12 M. The compositions of these solutions were calculated by a computer program that solves the appropriate multiequilibrium association equations (see Ref. 11 for a full description of the program). All permeabilized strips were perfused with fresh solution every 10 s providing a complete change of bath volume. Estimates of shortening velocity were performed by subjecting the strips to isotonic quick releases to afterloads ranging from 0.12 to 0.45 times the force at the instant of release. The force step was stable within 5-25 ms following release. The change in length from 200 to 300 ms following the release was used to calculate shortening velocity at a single afterload. Maximal shortening velocity was calculated by a linear transformation of the hyperbolic force-velocity relationship constructed from at least four releases to different afterloads. Estimates of tissue stiffness were performed by imposing a 500-Hz sine wave length change (0.05% L,) and measuring the resultant change in force with respect to length. All estimates of stiffness were normalized to those determined in the same tissues in response to 10 PM Ca2+. The motor was interfaced to a microcomputer for the initiation of experimental protocols, collection of data, and data analysis (software written by Dr. Konrad Guth and modified by Dr. Steven Petrou and Alex Fielding). MLC phosphorylation levels were determined on long (~10 mm) strips of the mesenteric arteries. After equilibration and permeabilization, the tissues were exposed to a Ca2+-containing solution in the presence of 10 ,uM ionomycin for varying times and then rapidly frozen in a dry ice-acetone slurry containing 6% trichloroacetic acid. The tissues were slowly thawed to room temperature and then transferred to a vial containing 50 ~1 of a homogenization solution previously frozen in liquid nitrogen, composed of 1% sodium dodecyl sulfate, 10% glycerol, and 20 mM dithiothreitol. The vial containing tissue, solution, and a small stainless steel ball was cooled in liquid nitrogen and placed in a dental amalgamator (Wig-A-Bug, Crescent Dental) for homogenization of the tissue. This procedure was repeated and then the vial was allowed to reach 4°C and the contents were transferred to a storage tube. The vial was rinsed with 50 ~1 of
IN PERMEABILIZED
c543.
ARTERIES
homogenization solution, which was added to the storage tube, and then stored at -80°C. Within 1 wk of homogenization, the samples were thawed and subjected to two-dimensional electrophoresis as described previously (12). After electrophoresis, the separated proteins were subjected to high-field-intensity Western blotting to nitrocellulose membranes. Visualization of the blotted proteins was performed using AuroDye forte gold protein stain (Amersham) as described by Kitazawa et al. (5). Quantitation of the stained blots was performed by scanning densitometry of the nitrocellulose paper made transparent by immersion in decalin as described by Maruyama et al. (10). Values are reported as moles Pi per mole MLC by integration of the spot corresponding to the phosphorylated MLC as a percent of the total of both the phosphorylated and unphosphorylated MLC. Group mean values were compared using unpaired two-tailed Student’s t test. A P value of ~0.05 was taken as significant. RESULTS
a-Toxin-permeabilized mesenteric tissues were contracted with 0.3 PM Ca2+ in the presence or absence of 10 PM NE plus 10 PM GTP. Force developed to significantly higher levels in the presence of NE plus GTP (Fig. 1A). The forces shown in this figure were normalized as a percent of a maximal contraction in the same tissue in response to 10 PM Ca2+. In control experiments, tissues were contracted three consecutive times with 10 PM Ca2+ or were contracted with 10 PM Ca2+ prior to the addition of NE plus GTP and again following relaxation from the NE plus GTP contraction. In all cases, the 10 PM Ca2+induced contractions were reproducible to within 5%. The addition of 10 PM ionomycin or A23187 did not affect either the rate of force development or the maximal level of force attained, indicating that organellar Ca2+ was not involved in the contractile response. Figure 1B shows the corresponding time course of the change in MLC phosphorylation levels under similar stimulation A
80
1
01 0
1 10
Minutes
J 20
0.01
I
I
1
0
10
20
Minutes
Fig. 1. Time course of force (A) and myosin light chain (MLC) phosphorylation (B) in response to 0.3 PM Ca2+ alone (0) or 0.3 ,uM Ca2+ plus 10 PM norepinephrine (NE) plus 10 PM GTP (D) in cu-toxinpermeabilized mesenteric arteries. All contractions were performed in presence of 10 mM EGTA to maintain intracellular [Ca2+] constant. Force data were normalized as a percent of maximal response obtained to 10 PM Ca2+ alone. In selected experiments, time course of force and MLC phosphorylation was extended to 60 min. Force and MLC phosphorylation levels were maintained for entire 60-min period: 48.2 + 7.2% maximal force (n = 4); 0.22 -t 0.07 mol Pi/m01 MLC (n = 2) with 0.3PM Ca2+ alone and 78.9 k 5.1% maximal force (n = 4); 0.27 + 0.06 mol Pi/m01 MLC (n = 2) with 0.3 PM Ca”+ plus 10 PM NE plus 10 PM GTP. Values shown are means + SE for 3-5 determinations. * P < 0.05 compared with Ca2+ alone. ** P < 0.05 compared with MLC phosphorylation levels at 2 min of stimulation with Ca2+ alone.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
c542
TRANSIENT
MLC
PHOSPHORYLATION
IN PERMEABILIZED
conditions. All determinations of MLC phosphorylation levels shown in Fig. 1B were performed in the presence of 10 PM ionomycin. During stimulation with 0.3 PM Ca2+ alone, MLC phosphorylation levels increased more or less monotonically, although the level of MLC phosphorylation at 20 min of stimulation with 0.3 PM Ca”+ alone was significantly lower than those at 1 and 2 min of stimulation. In contrast, during stimulation with 0.3 PM Ca2+ plus 10 PM NE plus 10 PM GTP, MLC phosphorylation levels initially increased to significantly higher levels than those obtained in response to Ca2+. However, with continued agonist stimulation, MLC phosphorylation levels fell to levels similar to those in 0.3 PM Ca2+ alone. Therefore, agonist stimulation of the a-toxin-permeabilized arterial strip resulted in a large transient in MLC phosphorylation, although the tissue was incubated in 10 PM ionomycin and the Ca2+ was buffered by 10 mM EGTA. These conditions should maintain the [Ca”‘] constant. Cross-bridge characteristics were further examined by estimating isotonic shortening velocities during contractions in response to Ca 2+ alone and in the presence of NE plus GTP. Figure 2A shows the time courses of isotonic shortening velocity during stimulation with 0.25 ,uM Ca2+ in the presence and absence of 10 PM NE plus 10 PM GTP. Although the addition of NE plus GTP transiently increased MLC phosphorylation levels, it had no effect on shortening velocities. To determine if the measurements of shortening velocity were Ca2+ dependent, the experiments were repeated during stimulation with a higher [Ca2+]. In 10 PM Ca2+, shortening velocities were significantly higher than during stimulation with 0.25 PM Ca2+; however, the addition of agonist plus GTP still had no effect on cross-bridge cycling rates (Fig. 2B). The data in Fig. 2 show the time course of shortening velocity at a single afterload of ~0.13 times the force at the instant of release. Estimates of the maximal velocity of shortening (V,,) were performed during steady-state conditions to more precisely examine cross-bridge cycling rates (Table 1). V, at steady state (20 min of stimulation) was Ca2+ dependent but was not affected at either [Ca2+] by the addition of NE plus GTP. This table also demonstrates that steady-state MLC phosphorylation levels were significantly increased in response to 10 ,uM Ca2+ compared with 0.3 PM Ca2+. The addition of NE plus GTP did not
ARTERIES
Table 1. Maximal velocity of shortening and MLC phosphorylation during steady-state contractions of a- toxin-permeabilized mesenteric arteries Condition
MIX
vo
0.25 pM Ca”+ 0.25 pM Ca”+ +lOhMNE + 10 pM GTP 10 ,uM Ca”+ 10 PM Ca2+ + 10 pM NE + 10 pM GTP
0.047+0.005 0.051t0.003
(6) (8)
0.23t0.04 0.25kO.01
(4) (4)
0.070+0.006* 0.072+0.0083(
(7) 10)
0.68*0.05* 0.67+0.04t
(4) (4)
Values are means t SE for number of determinations in parentheses. Vo, maximal velocity of shortening in Lo/s. Myosin light chain (MLC) phosphorylation levels in mol Pi/m01 MLC. cu-Toxin-permeabilized smooth muscle preparations were activated under appropriate conditions for 20 min to achieve steady-state force maintenance. Elevation of [Ca”+] significantly increased V,, and steady-state MLC phosphorylation levels whereas addition of NE plus guanosine triphosphate (GTP) had no effect. * P < 0.05 compared with 0.25 PM Ca”‘. t P < 0.05 compared with 0.25 FM Ca2+ plus NE plus GTP.
increase steady-state MLC phosphorylation levels at either [Ca2+]. Estimates of stiffness were performed to determine if the NE plus GTP-dependent increase in force resulted from a recruitment of additional cross bridges or a change in the force generation per cross bridge. The relationship between stress and stiffness was similar during stimulation with 0.25 and 10 PM Ca2+ alone and in the presence of 10 PM NE plus 10 PM GTP (Fig. 3). Therefore, the agonist and G protein-dependent force enhancement was apparently due to recruitment of cross bridges. DISCUSSION
The results of this study clearly demonstrate that, in the presence of Ca2+, agonist- and GTP-dependent stimulation of a-toxin-permeabilized vascular smooth muscle
125
_
100
-
6 G 2 IL
7 .,
r 0
/
/
;o.04v
+
: 0.03
ki z
-
.-if?
.- F gj
0.03
-
4
0.02
E
.-
;
I 0.01
s o,oo
i
g 0
IO
20
t 0.00
./‘,. ,’ // /’ / ,’ i , / ./,. / ‘-
0.25 pM Ca*+ + NE + GTP
t
r
g
/
i 0
Minutes
Fig. 2. Time course of isotonic shortening velocity PM Ca2+ alone (0) or 0.25 PM Ca”’ plus 10 PM NE 10 PM Ca2+ alone (0) or 10 PM Ca2+ plus 10 PM (II) (B). Data are from single isotonic releases to time of release. Values shown are means ,+ SE for
10
20
Minutes
in response to 0.25 plus GTP (m) (A) and NE plus 10 PM GTP -0.13 times force at 6-10 determinations.
‘/ .’
,/’ / ,
~
C lj
/
’ /’ / ,’
-
,
). / I / , , .’ /
,’ , , .’ / ./ ,
5
0.04
‘/
75-
ii
>;
, ‘/
0
-
A0 .-c
Phosphorylation
,
./ .‘/
/
,’
// / /i
,
i’ 1
.*’
10 pM Ca*+ + NE + GTP
Fig. 3. Steady-state force (solid bars) and stiffness (hatched bars) determinations in response to 0.25 and 10 FM Ca”+ alone and in presence of 10 PM NE plus 10 PM GTP. Data were normalized as a percent of response obtained to 10 PM Ca 2+ alone. Absolute values for force are as follows (in mg): 0.25 FM Ca fL+ alone = 24.6 + 3.5; 0.25 FM Ca”+ plus NE plus GTP = 87.8 + 6.7; 10 PM Ca’+ alone = 129.9 + 9.8; 10 PM Ca”+ plus NE plus GTP = 140.6 +: 7.3. Values shown are means + SE for 4-6 determinations.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
TRANSIENT
MLC
PHOSPHORYLATION
induces a large transient increase in the levels of MLC phosphorylation. This transient increase in MLC phosphorylation levels is observed under conditions of constant intracellular [Ca”+] and is accompanied by a monotonic increase in force. In contrast, stimulation of a-toxin-permeabilized vascular smooth muscle by Ca2+ alone induces a smaller monotonic increase in force and a very blunted transient, or even monotonic, increase in MLC phosphorylation. NE plus GTP enhanced both force and MLC phosphorylation early in the contractions in agreement with previous reports by Kitazawa et al. (5, 7, 8), who showed increased MLC phosphorylation levels at the peak of force development in response to an agonist plus GTP. After the initial increase, MLC phosphorylation decreased to a value similar to that obtained in response to Ca2+ alone in spite of the fact that NE plus GTP maintained force at an elevated level. A transient in MLC phosphorylation was previously reported in permeabilized phasic smooth muscle, but in this case force fell concomitantly with the decline in MLC phosphorylation (8). The observation that ionomycin did not affect force or MLC phosphorylation in response to NE plus GTP indicates that Ca2+ release from the sarcoplasmic reticulum was not involved in the agonist-induced contraction. This is in agreement with the finding of Somlyo et al. (18) that, in fluo-3-loaded a-toxin-permeabilized smooth muscle, flash photolysis of caged GTPyS increased force with no change in the [Ca”+]. Moreover, Somlyo and colleagues (6, 18) have shown by X-ray microanalysis that Ca-EGTA enters the cytosol of all cells in the cu-toxinpermeabilized tissue. Taken together, these results strongly suggest that the [Ca2+] remained constant in our preparation during stimulation with NE plus GTP. Stimulation with GTPyS alone or in combination with an agonist results in a maintained G protein-dependent increase in MLC phosphorylation during the steady-state phase of force maintenance (2, 9). We have also demonstrated that activation of the a-toxin-permeabilized mesenteric artery with 10 ,uM GTPyS results in a maintained elevation in MLC phosphorylation levels (15). The difference in results obtained with GTP$S, an irreversible activator of G proteins, and GTP, the presumed physiological mediator of contraction in vivo, suggests that GTPyS may mask the normal course of events that occur during agonist stimulation of vascular tissue as GTP is hydrolyzed. In response to either an increase in [Ca2+] or the addition of NE plus GTP, there was a concomitant increase in stiffness and force. The finding that the relationship between stiffness and force was not altered by the addition of NE plus GTP indicates that the enhancement in force development occurs by an increase in the number of force-generating cross bridges. Yamakawa and Brozovich (20) have shown in a-toxin-permeabilized single swine carotid artery cells that the increase in force is greater than the increase in stiffness following agonist plus GTP stimulation compared with stimulation by Ca2+ alone. The discrepancy in results may be due to speciesor tissue differences. Alternatively, small but significant agonistinduced effects on stiffness or force that are demonstrable
IN PERMEABILIZED
ARTERIES
c543
in the single cell might be obscured in the tissue. Cross-bridge cycling rate, as estimated by isotonic shortening velocity, was increased by Ca2+ but not by NE plus GTP. Fujiwara et al. (2) have shown that stimulation of saponin-permeabilized rabbit mesenteric artery by GTPrS significantly increases shortening velocity as measured by the slack test. Presumably this difference is the result of stimulation with GTPyS compared with NE plus GTP. It is also possible that the estimates of shortening velocity determined by Fujiwara et al. (2) using the slack test were more sensitive to small G protein-mediated changes than the single lightly loaded releasesshown in Fig. 2. This should not, however, account for the absence of a NE plus GTP-dependent increase in V,, during steady state. Our results would suggest that steady-state V,, depends solely on the [Ca2+] or possibly on the Ca2+dependent steady-state MLC phosphorylation. Our results are not consistent with a simple and direct relationship between force and MLC phosphorylation. Somlyo et al. (16) suggested that the increase in myofilament Ca2+ sensitivity is mediated by inhibition of a regulated MLC phosphatase. More recently, Kubota et al. (9) showed in saponin-permeabilized bovine trachea that the GTPyS-dependent increase in total MLC phosphorylation is due solely to increased phosphate incorporation at Serl!‘, the site phosphorylated by MLC kinase. Both groups have presented evidence demonstrating depressed MLC phosphatase activity following G protein activation (7, 9). Our results during the development phase of contraction are consistent with the hypothesis of G proteinmediated downregulation of a MLC phosphatase. However, the events that occur during force maintenance cannot be explained by this hypothesis. A recent study by Kubota et al. (9) indicated that regulation of MLC kinase activity is not involved. Therefore, a mechanism distinct from the MLC kinase-phosphatase system is required to account for maintenance of the receptor and G proteindependent enhanced force with MLC phosphorylation levels similar to those in response to Ca2+ alone. The mechanism(s) involved are beyond the scope of this study, but it will be interesting to investigate potential regulatory roles for thin filament-based proteins (19) or cooperative cross-bridge interactions (17). In summary, we have clearly demonstrated that the agonist plus GTP-dependent increase in force in the a-toxin-permeabilized mesenteric arteries is accompanied by a transient increase in MLC phosphorylation levels. Although the initial peak levels of MLC phosphorylation were significantly elevated compared with the effect of increasing Ca2+ alone steady-state levels of MLC phosphorylation were similar’ in the presence and absence of the agonist plus GTP. The receptor and G proteindependent increase in force was associated with an increased number of attached cross bridges; however, the cross-bridge cycling rate was unchanged and regulated solely by the [Ca”‘]. These results are consistent with the hypothesis that the enhancement during force development involves downregulation of a MLC phosphatase. However, the maintenance phase of agonist-enhanced force cannot be explained by this hypothesis and requires additional and as yet unidentified components.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
c544
TRANSIENT
MLC
PHOSPHORYLATION
This study was supported, in part, by National Heart, Lung, and Blood Institute Grants HL-37956 (to R. S. Moreland) and HL-3565’7 (to C. van Breemen), American Heart Association Southeastern Pennsylvania Affiliate Grant-in-Aid (to R. S. Moreland), and postdoctoral fellowships from the American Heart Association, Florida Affiliate (to J. Nishimura) and Southeastern Pennsylvania Affiliate (to H. Y. Ahn). Address for reprint requests: R. S. Moreland, Bockus Research Institute, Graduate Hospital, 415 S. 19th St., Philadelphia, PA 19146. Received
24 March
1992; accepted
in final
form
2 June
1992.
REFERENCES 1. Freissmuth, M., P. J. Casey, and A. G. Gilman. G proteins control diverse pathways of transrnembrane signaling. FASEB J. 3: 2125-2131, 1989. 2. Fujiwara, T., T. Itoh, Y. Kubota, and H. Kuriyama. Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J. Physiol. Lond. 408: 535-547, 1989. 3. Hartshorne, D. J. Biochemistry of the contractile process in smooth muscle. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, p. 423-482. 4. Hirata, K., A. Kikuchi, T. Sasaki, S. Kuroda, K. Haibuchi, Y. Matsuura, H. Seki, K. Saida, and Y. Takai. Involvement of rho ~21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J. Biol. Chem. 267: 7819-7822, 1992. 5. Kitazawa, T., B. D. Gaylinn, G. H. Denney, and A. P. Somlyo. G-protein mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J. BioZ. Chem. 266: 1708-1715, 1991. 6. Kitazawa, T., S. Kobayashi, K. Horiuch, A. V. Somlyo, and A. P. Somlyo. Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J. Biol. Chem. 264: 5339-5342, 1989. Kitazawa, T., M. Masuo, and A. P. Somlyo. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc. NlatZ. Acad. Sci. USA 88: 9307-9310, 1991. Kitazawa, T., and A. P. Somlyo. Desensitization and muscarinic resensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle. Biochem. Biophys. Res. Commun. 172: 1291-1297, 1990. Kubota, Y., M. Nomura, K. E. Kamm, M. C. Mumby, and J. T. Stull. GTPyS-dependent regulation of smooth muscle contractile elements. Am. J. Physiol. 262 (CelZ Physiol. 31): C405c410, 1992.
IN PERMEABILIZED
ARTERIES
T., T. Mikawa, and S. Ebashi. Detection of cal10. Maruyama, cium binding proteins by 4Wa autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J. Biothem. 95: 511-519, 1984. l1 Moreland, R. S., and R. A. Murphy. Determinants of Ca2+’ dependent stress maintenance in skinned swine carotid media. Am. J. Physiol. 251 (Cell Physiol. 20): C892-C903, 1986. 12. Moreland, S., and R. S. Moreland. Effects of dihydropyridines on stress, myosin phosphorylation, and V,, in smooth muscle. Am. J. PhysioZ. 252 (Heart Circ. Physiol. 21): H1049-H1058, 1987. 13. Morgan, J. P., and K. G. Morgan. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J. Physiol. Lond. 351: 155-167, 1984. J., M. Kolber, and C. van Breemen. Norepi14. Nishimura, nephrine and GTPyS increase myofilament Ca2+ sensitivity in a-toxin permeabilized arterial smooth muscle. Biochem. Biophys. Res. Commun. 163: 929-935, 1988. 15. Nishimura, J., S. Moreland, R. S. Moreland, and C. van Breemen. Regulation of the Ca 2+-force relationship in permeabilized arterial smooth muscle. Adv. Exp. Med. BioL. 304: 11 l-127, 1991. 16 . Somlyo, A. P., T. Kitazawa, B. Himpens, G. Matthijs, K. Horiuti, S. Kobayashi, Y. E. Goldman, and A. V. Somlyo. Modulation of Ca2+ -sensitivity and of the time course of contraction in smooth muscle: a major role of protein phosphatases? Adv. Protein Phosphatases 5: 181-195, 1989. A. V., Y. E. Goldman, T. Fujimori, M. Bond, D. R. 17. Somlyo, Trentham, and A. P. Somlyo. Cross-bridge kinetics, cooperativity, and negatively strained cross-bridges in vertebrate smooth muscle. J. Gen. PhysioZ. 91: 165-192, 1988. A. V., T. Kitazawa, K. Horiuti, S. Kobayashi, D. 18. Somlyo, Trentham, and A. P. Somlyo. Heparin-sensitive inositol trisphosphate signaling and the role of G-proteins in Ca”+-release and contractile regulation in smooth muscle. Prog. Clin. BioZ. Res. 327: 167-182, 1990. 19. Winder, S. J., C. Sutherland, and M. P. Walsh. Biochemical and functional characterization of smooth muscle calponin. Adv. Exp. Med. BioZ. 304: 37-51, 1991. M., and F. V. Brozovich. Agonist stimulation 20. Yamakawa, increases force in a-toxin permeabilized single vascular smooth muscle cells (Abstract). Biophys. J. 61: A164, 1992.
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 12, 2019.
