- Email: [email protected]
Calcium Control of Smooth Muscle Contractility
Calcium Control of Smooth Muscle Contractility BY JAMES T. STULL, PHD,
KRISTINE E. KAMM, PHD,
ABSTRACT: Ca 2 + is a primary second messenger that binds to an intracellular receptor protein, calmodulin. Increases in cytosolic Ca 2 + concentration mediated by activation of cell surface receptors result in the formation of a Ca2 + calmodulin complex that regulates many Ca 2 + -dependent cellular processes. In smooth muscle, Ca 2 + /calmodulin activates myosin light chain kinase, which phosphorylates the regulatory light chain of myosin. This phosphorylation reaction increases the actin-activated MgATPase activity of myosin and is associated with increases in contractile properties, including force, stiffness, and maximal shortening velocity. These biochemical and biomechanical responses occur rapidly (seconds) in response to physiological stimulation involving neurotransmitter activation of smooth muscle cells. Thus, the Ca2 + -dependent phosphorylation of the myosin light chain is a primary event in activation of smooth muscle contraction. KEY INDEXING TERMS: Calcium; Calmodulin; Myosin; Phosphorylation; Smooth Muscle; Second Messengers; Contraction; Myosin Light Chain Kinase. [Am J Med Sci 1988; 296(4):241-245.]
I
t is well established that a number of hormones and neurotransmitters exert their effects on cellular functions by regulating cytosolic Ca 2 + concentrations. Ca 2 + may flow through Ca 2 + channels in the plasma membrane and/or may be mobilized from intracellular storage sites due to the stimulation of specific cell surface membrane receptors. The actions of Ca 2 + are, in turn, mediated by specific cytosolic Ca 2 + receptor proteins such as calmodulin. In a simplified scheme, stimulation of receptors results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two
From the Department of Physiology and Moss Heart Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. The authors acknowledge the National Institutes of Health for partial support of the research described in this article. Reprint requests: James T. Stull, PhD, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
DORIS A. TAYLOR, PHD
second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (Figure 1). Diacylglycerol activates protein kinase C, which may catalyze the phosphorylation of cellular proteins as an integral component of signal transduction. Inositol 1,4,5-trisphosphate stimulates the release of Ca 2 + from intracellular storage sites. The other presentations in this symposium focus on specific aspects of signal transduction mechanisms, including formation of diacylglycerol and activation of protein kinase C. We will discuss the other branch of the second messenger system, namely the Ca 2 + /calmodulin pathway in relation to regulation of smooth muscle contractility. The sliding filament theory of muscle contraction proposes that shortening is the result of cyclic attachment and detachment of the globular heads of myosin in thick filaments to actin in thin filaments. 1 This cycling process, associated with hydrolysis of A TP, allows for sliding of thin and thick filaments past each other. In striated muscles, Ca 2 + binding to thin filament regulatory proteins, ie, the troponin complex, allows for the cyclic interaction of myosin with actin. However, troponin is absent from smooth muscle, and it is thought that a primary event in smooth muscle activation is the Ca 2 + -dependent phosphorylation of the 20 kDa regulatory light chain subunit of myosin by Ca 2 + /calmodulin stimulation of myosin light chain kinase (Figure 1). 2 Myosin is a highly asymmetric molecule, composed of two globular heads and an ahelical rod-like tail. The tail is made up of two high molecular weight (200 kDa), heavy chain subunits that comprise the backbone of the thick filament. Each heavy chain forms a globular head that projects from the surface of the thick filament and contains the actin-binding domain and the catalytic site for ATP hydrolysis. In addition, each head contains a 20 kDa regulatory light chain subunit that is phosphorylated. Phosphorylation of myosin light chain in skeletal muscle results in potentiation of isometric twitch tension, 3 whereas phosphorylation in smooth muscle leads to contraction. 2 Biochemical Properties of Contraction
Calmodulin, a major intracellular receptor protein for Ca 2 +, mediates activation of a number of target enzymes and thereby is implicated in Ca 2 + regulation of a variety of cellular events. 4 Calmodulin is ubiquitous. It is a low molecular weight (16. 7 kDa) protein 241
Smooth Muscle Contractility
~3 ccf-+ -----~ Co 2 ++ Calmodulin
0.5 sec
Myosin Light Chain
S Kinase Co~+· Calmodulin~ Co~+ · CM • MLCK ~
., / 1 Myo:~ l_jg-h~ ~;ain
Myosin
ATP ADP
Myosin-®
Phosphatase Contraction~
tActin-Activated MgATPose
Figure 1. A general scheme for regulation of tracheal smooth muscle contraction. Release of acetylcholine (Ach) from parasympathetic nerves results in the stimulation of cholinergic muscarinic receptors, leading to the hydrolysis of phosphoinositides and formation of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). These events result in an increase in cytosolic Co 2 + concentrations, activation of the calmodulin-dependent myosin light chain kinase and phosphorylation of myosin light chain.
that is composed of four Ca 2 + binding sites with affinities for Ca 2 + in the micromolar range. A simplified me-chanism for the Ca 2 + -dependent activation of myosin light chain kinase by calmodulin involves two sequential reactions (Figure 1). The first step is Ca 2 + binding to calmodulin, which induces a conformational change in calmodulin. 4 As reviewed by Cox, 5 the binding of Ca 2+ to calmodulin is complex. Two general models have been proposed. The first model proposes that the four binding sites are independent and have similar affinities for Ca 2 +. The second model proposes that there are two pairs of sites with positive cooperativity of binding within each pair and a marked difference between the two pairs in affinity for Ca 2 +. The second step of the Ca 2 + -dependent activation involves Ca 2 + /calmodulin binding to myosin light chain kinase. This complex then catalyzes the phosphorylation of the 20 kDa regulatory light chain of myosin (Figure 1). The Ca2 + -dependent activation by calmodulin requires Ca 2 + occupancy of most, if not all, Ca 2 + binding domains. 3 •5 Because three cr four Ca2 + binding sites have to be filled with Ca 2 +, activation of myosin light chain kinase is positively cooperative in regard to Ca 2 + concentrations, whereas activation by calmodulin follows simple hyperbolic kinetics (Figure 2). Activation results from binding of one calmodulin to one kinase catalytic subunit, and the concentration of calmodulin required for half-maximal activation of myosin light chain kinase is only 1 nM. 6 For myosin light chain phosphorylation to be a physiologically important reaction, there has to be an enzyme(s) that catalyzes dephosphorylation (Figure 1). Pato and coworkers 7•8 isolated a number of protein
242
phosphatases from smooth muscle that have unique biochemical properties. One of these has been purified to homogeneity and consists of two types of protein subunits (58 kDa and 40 kDa). Onishi et al 9 isolated another smooth muscle phosphatase with three types of protein subunits (67 kDa, 54 kDa and 34 kDa). At this time it is not clear if there are one or more protein phosphatases that catalyze the dephosphorylation of myosin in smooth muscle cells. There is no evidence that these protein phosphatases are regulated by second messenger systems . Phosphorylation of myosin results in an increase in the actin -activated MgA TPase activity of myosin and thus initiates myosin crossbridge cycling on actin filaments. Sellers 10 found that phosphorylation increases the maximal velocity of the myosin MgA TPase activity, with little effect on the ability of myosin to bind to actin. Nonphosphorylated myosin was not activated by actin. Other investigators found that phosphorylation of smooth muscle myosin significantly increases its ability to bind to actin.U At high actin concentrations, the MgATPase activity of nonphosphorylated myosin could be increased. Additional investigations will be necessary to elucidate the specific mechanism(s) by which phosphorylation affects myosin MgATPase activity. Cellular Indices of Contraction
Permeable smooth muscle fibers provide a model system for studying factors that regulate contractilityY The permeabilization, or skinning procedure, which may be performed by chemical means, removes the sarcolemma of smooth muscle cells so that proteins and other chemicals may be added to the prep aration under precisely controlled conditions. Numerous investigations performed with skinned fibers have provided evidence that the Ca 2 + dependence of force is conferred by calmodulin activation of myosin light
%
Ma• Rote
Figure 2. Myosin light chain kinase activity surface. Activity of the purified kinase is plotted on the vertical axis as a function of Co2+ and calmodulin concentrations (as indicated on the horizontal axis). The solid lines represent the experimental data, whereas the dotted lines connect corresponding points calculated from a mathematical model. 29 Reprinted with permission. 29 October 1988 Volume 296 Number 4
Stull, Kamm, and Taylor
chain kinase, with resulting phosphorylation of myosin light chains. 2 Addition of Ca 2 + to skinned smooth muscle fibers results in an increase in the phosphate content of the myosin light chain and contraction. The sensitivity of the contractile force to activation by Ca 2 + can be increased by the addition of exogenous calmodulin, as predicted from the mechanism of myosin light chain kinase activation (Figure 2). Agents that bind to calmodulin inhibit myosin light chain kinase activity and contraction. Exposure of skinned smooth muscle fibers to A TP')'S results in thiophosphorylation of the light chains. Thiophosphorylated light chains are poor substrates for protein phosphatases, so activation is irreversible upon removal of Ca 2 +. The addition of a calmodulin-independent, proteolytic fragment of myosin light chain kinase produces a Ca 2 +-independent contraction. These observations demonstrate a primary role for phosphorylation of myosin light chain in smooth muscle contraction. In intact smooth muscles, typical values for phosphorylation are 0.1 mol phosphate per mol light chain in muscles that do not exhibit spontaneous contractile activity or active tone. 2 Stimulation with agonists or KCl increases the myosin light chain phosphorylation that precedes changes in isometric force. Addition of pharmacologic agents that interfere with the Ca 2 +-dependent activation of myosin light chain kinase results in a decrease in the extent of phosphate incorporation into the light chain and proportional decreases in isometric force. Such pharmacologic agents include calmodulin antagonists and Ca 2 + channel antagonists. 13 It is important to understand the cellular process involved in myosin light chain kinase activation in relation to smooth muscle contractility. Intracellular determinants dictate a positively cooperative activation of myosin light chain kinase at Ca 2 + concentration less than 1 J.LM. These determinants include: (1) the requirement for Ca 2 + occupancy of four binding sites in calmodulin for activation; (2) a high molar ratio of calmodulin to kinase; (3) a high cellular content of calmodulin (10-40 J.LM) and kinase (1 J.LM), which are 10,000- to 1,000-fold greater than the dissociation constant of Ca~+. calmodulin for myosin light chain kinase; and (4) an increase in the affinity for Ca 2 + when calmodulin binds to the kinase. In resting tracheal smooth muscle cells in culture, the cytosolic free Ca 2 + concentration is about 150 nM. 14 Agonist (carbachol, serotonin, or histamine) or Ca 2 + ionophore (ionomycin) stimulation resulted in an increase in cytosolic free Ca 2 + concentration and in the extent oflight chain phosphorylation. Maximal values of cytosolic Ca 2 + concentration were greater with ionophore (34 J.LM) than with pharmacologic agonists (500 nM). However, the quantitative relationships between cytosolic Ca 2 + concentrations and light chain phosphorylation were similar, ie, a single curve described the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
relationship obtained under all conditions of stimulation. Unstimulated levels of light chain phosphorylation were low (0.09 mol phosphate per mol light chain), as expected for a Ca 2 +-dependent reaction. When cytosolic Ca 2 + concentrations increased above 200 nM, light chain phosphorylation increased, with half-maximal phosphorylation (0.30 mol phosphate per mol light chain) occurring at 240 nM. The Hill coefficient for the relationship between light chain phosphorylation and cytosolic free Ca 2 + concentration was approximately 3, indicating an apparent positively cooperative relationship. Thus, small increases in cytosolic free Ca 2 + concentration lead to increased myosin light chain phosphorylation. Physiologic States of Contraction
Studies with smooth muscles by many investigators have demonstrated a dependency of force generation on myosin phosphorylation. 2 In general, these studies have been conducted on tissues activated by addition of agonists to the bathing medium. This approach allows for asynchronous times of arrival and different rates of increase in the concentration of an agonist at each cell as the agonist diffuses into the muscle strip. These diffusional delays have effects on the rate and maximal extent of biochemical responses. 15 Therefore, we developed a physiologic preparation for the rapid and synchronous activation of the smooth muscle cells via release ofneurotransmitter. 16•17 After field stimulation of bovine trachealis muscle, acetylcholine release from cholinergic nerve fibers activates muscarinic receptors on smooth muscle cells. At various times after field stimulation, muscle strips were frozen with a rapid-release electronic freezing device. The frozen muscle samples were then analyzed for myosin light chain phosphorylation. Upon stimulation, there was a 500 millisecond latency before the onset of increases in force, stiffness, and myosin light chain phosphorylation (Figure 3). After the latency period, myosin light chain was phosphorylated from 0.04 to 0.80 mol phosphate per mol light chain by 3.5 seconds. This rapid phosphorylation had a pseudofirst-order rate constant of 1.1 per second. The pseudo-first-order rate constant provided evidence that there was no negatively cooperative or ordered process in the phosphorylation reaction. These results were interesting, considering the controversy regarding the biochemical observations on the nonequivalence of myosin heads in phosphorylation reactions, ie, whether the first head of myosin was phosphorylated much more rapidly than the second head. 18- 20 Therefore, we developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin (not myosin light chain) in smooth muscle. There was no detectable myosin phosphorylation in unstimulated muscle when the extent of light chain phosphorylation measured directly was only 0.02 mol phosphate
243
Smooth Muscle Contractility
.
Field Stimulation
I
I
~
f/$#/$#/$#/#//#/##/$/#//#ai-7~
1.0 -
.C::: 0
.<::.
~
08-
.<::.
o>
::::i d._ 0.60
~
0.4
~ 0
.<::.
5t
0 .<::.
0.2
u.
0
E
0 0
2
3
4
5
Time (sec)
Figure 3. Temporal development of active stillness, active force, and light chain phosphorylation during the onset of contraction. Stillness and force are expressed as a fraction of the maximal change observed above values measured at rest. The insert shows the time course on an expanded scale up to 1 second. Reprinted with permission. 17
per mol of light chain. After 2.5 seconds of neural stimulation, myosin light chain phosphorylation increased to a value of 0.63 mol of phosphate per mol of light chain, which resulted in 40% diphosphorylated myosin. The relationship between the extent of light chain phosphorylation and the relative amount of diphosphorylated myosin was consistent with a random phosphorylation process in smooth muscle cells. Following the period of latency, myosin light chain is rapidly phosphorylated and force and stiffness increase rapidly (Figure 3). Stiffness increases coincident with phosphorylation, and both increase more rapidly than isometric force. The coincident increase in stiffness and myosin light chain phosphorylation during smooth muscle activation suggests that each myosin head attaches independently to actin thin filaments upon phosphorylation. With continuous neural stimulation of tracheal smooth muscle for prolonged periods of time, after maximal responses are obtained, there is an apparent decrease in cytosolic Ca 2 + concentration with reductions in the extent of myosin light chain phosphorylation and maximal shortening velocity (Figure 4). Similar studies in tracheal smooth muscle have demonstrated a decrease in Ca 2 + -dependent phosphorylase a formation. The developed force is maintained during a sustained period of stimulation (Figure 4). This phase of contraction is characterized by relatively low levels of free Ca 2 + and myosin light chain phosphorylation, associated with low maximal velocity of shortening with force maintenance. 2•21 This physiologic state originally was proposed to be caused by the formation of a "latch bridge," ie, force is maintained because of a different state of the myosin crossbridge whereby it remains attached longer and cycles more
244
slowly. 22 •23 The maintenance of force is Ca 2 + -dependent, so a second Ca 2 + -dependent regulatory mechanism appears to be required. The latter Ca 2 + regulatory system presumably has a higher sensitivity to activation by Ca 2 + than myosin light chain kinase. 24 There is considerable interest in the cellular mechanisms for force maintenance with low values of light chain phosphorylation. Recent theories include: 1. Force maintenance by attached crossbridges for which detachment rates are greatly slowed after dephosphorylation. 25 2. Unregulated crossbridges,u for which Ca 2 + regulation is attributable to additional proteins in thin filaments. 26 One such protein could be caldesmon, a calmodulin-binding protein found in thin filaments isolated from smooth muscle. Caldesmon binding to actin thin filaments inhibits actin-activated myosin MgATPase activity. The inhibition is reversed by Ca 2 + /calmodulin. 3. Changes in the intermediate filament domain of smooth muscle cells. Filaments are distributed in two domains represented by (a) intermediate filaments free of myosin, but containing filamin, actin, a-actinin, and desmon and (b) contractile protein filaments containing actin, myosin, tropomysin, and caldesmon. 27 Further, intermediate filament proteins may be phosphorylated by protein kinases and, in particular, protein kinase C. 28 These phosphorylation reactions may affect the dynamic regulation of assembly and turnover of intermediate filaments. Thus, it has been hypothesized that tonic force maintenance may be associated with phosphorylation-mediated structural rearrangements of intermediate til-
1.0
0.40
0.8
0 32
_g <.)
0.6
-
~
0 24
~
~
0.4
.
'
~
016
0
>----(sec 1------l
a.
~
~
>'
~
0.2
l
0.08
0
r----------- ( min )------1
Figure 4. Time course of force, myosin light chain phosphorylation, and maximal shortening velocity (V 0 ) in neurally stimulated strips of tracheal muscle. For measurement of light chain phosphorylation, strips were frozen during rest or at indicated limes between 2 seconds and 30 minutes of stimulation. Force at time of freezing is expressed as a fraction of force obtained with a maximal neural precontraction (P < .01). Reprinted with permission. 16 October 1988 Volume 296 Number 4
Stull, Kamm, and Taylor
ament proteins following protein kinase C activation. Identification of the precise mechanism(s) that may account for the physiologic properties of tonic contraction will require additional investigation. Conclusions
Neurotransmitters and hormones activate cell surface receptors and affect cellular processes via different second messenger signals. Ca 2+ was first recognized as a second messenger when its central role in skeletal muscle contraction and glycogen metabolism was identified. We now appreciate the importance of Ca 2 + in mediating many types of cellular responses via the intracellular receptor protein calmodulin. The control of smooth muscle contractility by Ca 2 + /calmodulin has provided a model system for studies of Ca 2 + -dependent cellular processes. Integrated biochemical, cellular, and physiologic investigations have defined a general scheme in which Ca 2 + binds to calmodulin and the Ca 2 + /calmodulin complex activates myosin light chain kinase. Catalysis of myosin light chain phosphorylation then leads to contraction of smooth muscle. Although these aspects of smooth muscle contractility are established, important questions remain concerning the mechanisms, including other second messenger systems, that modify the control of smooth muscle contractility by Ca 2 +. References 1. Goldman YE , Brenner B: Special topic: molecular mechanism of muscle contraction. Annu Rev Physiol49:629- 636, 1987. 2. Kamm KE, Stull JT: The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacal Toxicol25:593-620, 1985. 3. Stull JT, Nunnally MH, Moore RL, Blumenthal DK: Myosin light chain kinases and myosin phosphorylation in skeletal muscle, in WeberG (ed): Advances in Enzyme Regulation. New York , Pergamon Press, 1985, ed 23, pp 123- 140. 4. Klee CB, Vanaman TC: Calmodulin. Adv Protein Chem 35: 213- 321, 1982. 5. Cox JA: Interactive properties of calmodulin. Biochem J 249: 621-629, 1988. 6. Blumenthal DK, Stull JT: Activation of skeletal muscle myosin light chain kinase by Ca 2+ and calmodulin. Biochemistry 19:5608- 5614, 1980. 7. Sellers JR, Pato MD: The binding of smooth muscle myosin light chain kinase and phosphatases to actin and myosin. J Bioi Chem 259:7740- 7746, 1984. 8. Pato MD, Kerc E: Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. J Bioi Chem 260:12359- 12366, 1985. 9. Onishi H, Umeda J, Uchiwa H, Watanabe S: Purification of gizzard myosin light-chain phosphatase, and reversible changes in the ATPase and superprecipitation activities of actomyosin in the presence of purified preparations of myosin
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
10.
11.
12.
13.
14. 15.
16.
17.
18.
19.
20. 21.
22.
23.
24.
25.
26. 27. 28.
29.
light-chain phosphatase and kinase. J Bioi Chem 91:265-271, 1982. Sellers JR: Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin. J Bioi Chem 260: 15815- 15819, 1985. Wagner PD, Vu N-D: Regulation of the actin-activated ATPase of aorta smooth muscle myosin. J Bioi Chem 261 : 7778- 7783, 1986. Meisheri KD , Ruegg JC, Paul RJ: Studies on skinned fiber prepa rations, in Grover AK , Daniel EE (eds): Calcium and Contractility. Clifton, New Jersey, Humana Press, 1985, pp 191- 224. Asano M, Stull JT: Effects of calmodulin antagonists on smooth muscle contraction and myosin phosphorylation, in Hidaka H , Hartshorne DJ (eds) : Calmodulin Antagonists and Cellular Physiology. Orlando, Florida, Academic Press, 1985, pp 225- 260. Taylor DA, Stull JT: Ca 2 + dependence of myosin phosphorylation in tracheal smooth muscle cells. Biophys J 53:598a, 1988. Kamm KE, Murphy RA: Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion. Experientia 41 :1010- 1017, 1985. Kamm KE, Stull JT: Myosin phosphorylation, force, and maximal shortening velocity in neurally stimulated tracheal smooth muscle. Am J Physioi249:C238-C247, 1985. Kamm KE, Stull JT: Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness. Science 232:80- 82, 1986. Persechini A, Hartshorne DJ: Ordered phosphorylation of the two 20,000 molecular weight light chains of smooth muscle myosin. Biochemistry 22:4 70-4 76, 1983. Sellers JR, Chock PB, Adelstein RS: The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state. J Bioi Chem 258:14181- 14188, 1983. Trybus KM , Lowey S : Mechanism of smooth muscle myosin phosphorylation. J Bioi Chem 260:15988- 15995, 1985. Rembold CM, Murphy RA: Myoplasmic calcium, myosin phosphorylation, and regulation of the crossbridge cycle in swine arterial smooth muscle. Circ Res 58:803-815, 1986. Dillon PF, Aksoy MO, Driska SP, Murphy RA: Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211:495-497, 1981. Driska SP, Aksoy MO and Murphy RA: Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am J Physioi240:C222-C233, 1981. Chatterjee M , Murphy RA: Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle. Science 221 :464-466, 1983. Hai C-M, Murphy RA: Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am J Physiol254:C99C106, 1988. Marston SB, Smith CW J: The thin filaments of smooth muscles. J Muscle Res Cell Motil6:669- 708, 1985. Small JV, Furst DO, DeMay J: Localization of filamin in smooth muscle. J Cell Biol102:210-220, 1986. Rasmussen H , Takuwa Y, ParkS: Protein kinase C in the regulation of smooth muscle contraction. Federation of American Societies for Experimental Biology Journal1:177- 185, 1987. Blumenthal DK, Stull JT: Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin. Biochemistry 19:5608- 5614, 1980.
245
KRISTINE E. KAMM, PHD,
ABSTRACT: Ca 2 + is a primary second messenger that binds to an intracellular receptor protein, calmodulin. Increases in cytosolic Ca 2 + concentration mediated by activation of cell surface receptors result in the formation of a Ca2 + calmodulin complex that regulates many Ca 2 + -dependent cellular processes. In smooth muscle, Ca 2 + /calmodulin activates myosin light chain kinase, which phosphorylates the regulatory light chain of myosin. This phosphorylation reaction increases the actin-activated MgATPase activity of myosin and is associated with increases in contractile properties, including force, stiffness, and maximal shortening velocity. These biochemical and biomechanical responses occur rapidly (seconds) in response to physiological stimulation involving neurotransmitter activation of smooth muscle cells. Thus, the Ca2 + -dependent phosphorylation of the myosin light chain is a primary event in activation of smooth muscle contraction. KEY INDEXING TERMS: Calcium; Calmodulin; Myosin; Phosphorylation; Smooth Muscle; Second Messengers; Contraction; Myosin Light Chain Kinase. [Am J Med Sci 1988; 296(4):241-245.]
I
t is well established that a number of hormones and neurotransmitters exert their effects on cellular functions by regulating cytosolic Ca 2 + concentrations. Ca 2 + may flow through Ca 2 + channels in the plasma membrane and/or may be mobilized from intracellular storage sites due to the stimulation of specific cell surface membrane receptors. The actions of Ca 2 + are, in turn, mediated by specific cytosolic Ca 2 + receptor proteins such as calmodulin. In a simplified scheme, stimulation of receptors results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two
From the Department of Physiology and Moss Heart Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas. The authors acknowledge the National Institutes of Health for partial support of the research described in this article. Reprint requests: James T. Stull, PhD, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
DORIS A. TAYLOR, PHD
second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (Figure 1). Diacylglycerol activates protein kinase C, which may catalyze the phosphorylation of cellular proteins as an integral component of signal transduction. Inositol 1,4,5-trisphosphate stimulates the release of Ca 2 + from intracellular storage sites. The other presentations in this symposium focus on specific aspects of signal transduction mechanisms, including formation of diacylglycerol and activation of protein kinase C. We will discuss the other branch of the second messenger system, namely the Ca 2 + /calmodulin pathway in relation to regulation of smooth muscle contractility. The sliding filament theory of muscle contraction proposes that shortening is the result of cyclic attachment and detachment of the globular heads of myosin in thick filaments to actin in thin filaments. 1 This cycling process, associated with hydrolysis of A TP, allows for sliding of thin and thick filaments past each other. In striated muscles, Ca 2 + binding to thin filament regulatory proteins, ie, the troponin complex, allows for the cyclic interaction of myosin with actin. However, troponin is absent from smooth muscle, and it is thought that a primary event in smooth muscle activation is the Ca 2 + -dependent phosphorylation of the 20 kDa regulatory light chain subunit of myosin by Ca 2 + /calmodulin stimulation of myosin light chain kinase (Figure 1). 2 Myosin is a highly asymmetric molecule, composed of two globular heads and an ahelical rod-like tail. The tail is made up of two high molecular weight (200 kDa), heavy chain subunits that comprise the backbone of the thick filament. Each heavy chain forms a globular head that projects from the surface of the thick filament and contains the actin-binding domain and the catalytic site for ATP hydrolysis. In addition, each head contains a 20 kDa regulatory light chain subunit that is phosphorylated. Phosphorylation of myosin light chain in skeletal muscle results in potentiation of isometric twitch tension, 3 whereas phosphorylation in smooth muscle leads to contraction. 2 Biochemical Properties of Contraction
Calmodulin, a major intracellular receptor protein for Ca 2 +, mediates activation of a number of target enzymes and thereby is implicated in Ca 2 + regulation of a variety of cellular events. 4 Calmodulin is ubiquitous. It is a low molecular weight (16. 7 kDa) protein 241
Smooth Muscle Contractility
~3 ccf-+ -----~ Co 2 ++ Calmodulin
0.5 sec
Myosin Light Chain
S Kinase Co~+· Calmodulin~ Co~+ · CM • MLCK ~
., / 1 Myo:~ l_jg-h~ ~;ain
Myosin
ATP ADP
Myosin-®
Phosphatase Contraction~
tActin-Activated MgATPose
Figure 1. A general scheme for regulation of tracheal smooth muscle contraction. Release of acetylcholine (Ach) from parasympathetic nerves results in the stimulation of cholinergic muscarinic receptors, leading to the hydrolysis of phosphoinositides and formation of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ). These events result in an increase in cytosolic Co 2 + concentrations, activation of the calmodulin-dependent myosin light chain kinase and phosphorylation of myosin light chain.
that is composed of four Ca 2 + binding sites with affinities for Ca 2 + in the micromolar range. A simplified me-chanism for the Ca 2 + -dependent activation of myosin light chain kinase by calmodulin involves two sequential reactions (Figure 1). The first step is Ca 2 + binding to calmodulin, which induces a conformational change in calmodulin. 4 As reviewed by Cox, 5 the binding of Ca 2+ to calmodulin is complex. Two general models have been proposed. The first model proposes that the four binding sites are independent and have similar affinities for Ca 2 +. The second model proposes that there are two pairs of sites with positive cooperativity of binding within each pair and a marked difference between the two pairs in affinity for Ca 2 +. The second step of the Ca 2 + -dependent activation involves Ca 2 + /calmodulin binding to myosin light chain kinase. This complex then catalyzes the phosphorylation of the 20 kDa regulatory light chain of myosin (Figure 1). The Ca2 + -dependent activation by calmodulin requires Ca 2 + occupancy of most, if not all, Ca 2 + binding domains. 3 •5 Because three cr four Ca2 + binding sites have to be filled with Ca 2 +, activation of myosin light chain kinase is positively cooperative in regard to Ca 2 + concentrations, whereas activation by calmodulin follows simple hyperbolic kinetics (Figure 2). Activation results from binding of one calmodulin to one kinase catalytic subunit, and the concentration of calmodulin required for half-maximal activation of myosin light chain kinase is only 1 nM. 6 For myosin light chain phosphorylation to be a physiologically important reaction, there has to be an enzyme(s) that catalyzes dephosphorylation (Figure 1). Pato and coworkers 7•8 isolated a number of protein
242
phosphatases from smooth muscle that have unique biochemical properties. One of these has been purified to homogeneity and consists of two types of protein subunits (58 kDa and 40 kDa). Onishi et al 9 isolated another smooth muscle phosphatase with three types of protein subunits (67 kDa, 54 kDa and 34 kDa). At this time it is not clear if there are one or more protein phosphatases that catalyze the dephosphorylation of myosin in smooth muscle cells. There is no evidence that these protein phosphatases are regulated by second messenger systems . Phosphorylation of myosin results in an increase in the actin -activated MgA TPase activity of myosin and thus initiates myosin crossbridge cycling on actin filaments. Sellers 10 found that phosphorylation increases the maximal velocity of the myosin MgA TPase activity, with little effect on the ability of myosin to bind to actin. Nonphosphorylated myosin was not activated by actin. Other investigators found that phosphorylation of smooth muscle myosin significantly increases its ability to bind to actin.U At high actin concentrations, the MgATPase activity of nonphosphorylated myosin could be increased. Additional investigations will be necessary to elucidate the specific mechanism(s) by which phosphorylation affects myosin MgATPase activity. Cellular Indices of Contraction
Permeable smooth muscle fibers provide a model system for studying factors that regulate contractilityY The permeabilization, or skinning procedure, which may be performed by chemical means, removes the sarcolemma of smooth muscle cells so that proteins and other chemicals may be added to the prep aration under precisely controlled conditions. Numerous investigations performed with skinned fibers have provided evidence that the Ca 2 + dependence of force is conferred by calmodulin activation of myosin light
%
Ma• Rote
Figure 2. Myosin light chain kinase activity surface. Activity of the purified kinase is plotted on the vertical axis as a function of Co2+ and calmodulin concentrations (as indicated on the horizontal axis). The solid lines represent the experimental data, whereas the dotted lines connect corresponding points calculated from a mathematical model. 29 Reprinted with permission. 29 October 1988 Volume 296 Number 4
Stull, Kamm, and Taylor
chain kinase, with resulting phosphorylation of myosin light chains. 2 Addition of Ca 2 + to skinned smooth muscle fibers results in an increase in the phosphate content of the myosin light chain and contraction. The sensitivity of the contractile force to activation by Ca 2 + can be increased by the addition of exogenous calmodulin, as predicted from the mechanism of myosin light chain kinase activation (Figure 2). Agents that bind to calmodulin inhibit myosin light chain kinase activity and contraction. Exposure of skinned smooth muscle fibers to A TP')'S results in thiophosphorylation of the light chains. Thiophosphorylated light chains are poor substrates for protein phosphatases, so activation is irreversible upon removal of Ca 2 +. The addition of a calmodulin-independent, proteolytic fragment of myosin light chain kinase produces a Ca 2 +-independent contraction. These observations demonstrate a primary role for phosphorylation of myosin light chain in smooth muscle contraction. In intact smooth muscles, typical values for phosphorylation are 0.1 mol phosphate per mol light chain in muscles that do not exhibit spontaneous contractile activity or active tone. 2 Stimulation with agonists or KCl increases the myosin light chain phosphorylation that precedes changes in isometric force. Addition of pharmacologic agents that interfere with the Ca 2 +-dependent activation of myosin light chain kinase results in a decrease in the extent of phosphate incorporation into the light chain and proportional decreases in isometric force. Such pharmacologic agents include calmodulin antagonists and Ca 2 + channel antagonists. 13 It is important to understand the cellular process involved in myosin light chain kinase activation in relation to smooth muscle contractility. Intracellular determinants dictate a positively cooperative activation of myosin light chain kinase at Ca 2 + concentration less than 1 J.LM. These determinants include: (1) the requirement for Ca 2 + occupancy of four binding sites in calmodulin for activation; (2) a high molar ratio of calmodulin to kinase; (3) a high cellular content of calmodulin (10-40 J.LM) and kinase (1 J.LM), which are 10,000- to 1,000-fold greater than the dissociation constant of Ca~+. calmodulin for myosin light chain kinase; and (4) an increase in the affinity for Ca 2 + when calmodulin binds to the kinase. In resting tracheal smooth muscle cells in culture, the cytosolic free Ca 2 + concentration is about 150 nM. 14 Agonist (carbachol, serotonin, or histamine) or Ca 2 + ionophore (ionomycin) stimulation resulted in an increase in cytosolic free Ca 2 + concentration and in the extent oflight chain phosphorylation. Maximal values of cytosolic Ca 2 + concentration were greater with ionophore (34 J.LM) than with pharmacologic agonists (500 nM). However, the quantitative relationships between cytosolic Ca 2 + concentrations and light chain phosphorylation were similar, ie, a single curve described the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
relationship obtained under all conditions of stimulation. Unstimulated levels of light chain phosphorylation were low (0.09 mol phosphate per mol light chain), as expected for a Ca 2 +-dependent reaction. When cytosolic Ca 2 + concentrations increased above 200 nM, light chain phosphorylation increased, with half-maximal phosphorylation (0.30 mol phosphate per mol light chain) occurring at 240 nM. The Hill coefficient for the relationship between light chain phosphorylation and cytosolic free Ca 2 + concentration was approximately 3, indicating an apparent positively cooperative relationship. Thus, small increases in cytosolic free Ca 2 + concentration lead to increased myosin light chain phosphorylation. Physiologic States of Contraction
Studies with smooth muscles by many investigators have demonstrated a dependency of force generation on myosin phosphorylation. 2 In general, these studies have been conducted on tissues activated by addition of agonists to the bathing medium. This approach allows for asynchronous times of arrival and different rates of increase in the concentration of an agonist at each cell as the agonist diffuses into the muscle strip. These diffusional delays have effects on the rate and maximal extent of biochemical responses. 15 Therefore, we developed a physiologic preparation for the rapid and synchronous activation of the smooth muscle cells via release ofneurotransmitter. 16•17 After field stimulation of bovine trachealis muscle, acetylcholine release from cholinergic nerve fibers activates muscarinic receptors on smooth muscle cells. At various times after field stimulation, muscle strips were frozen with a rapid-release electronic freezing device. The frozen muscle samples were then analyzed for myosin light chain phosphorylation. Upon stimulation, there was a 500 millisecond latency before the onset of increases in force, stiffness, and myosin light chain phosphorylation (Figure 3). After the latency period, myosin light chain was phosphorylated from 0.04 to 0.80 mol phosphate per mol light chain by 3.5 seconds. This rapid phosphorylation had a pseudofirst-order rate constant of 1.1 per second. The pseudo-first-order rate constant provided evidence that there was no negatively cooperative or ordered process in the phosphorylation reaction. These results were interesting, considering the controversy regarding the biochemical observations on the nonequivalence of myosin heads in phosphorylation reactions, ie, whether the first head of myosin was phosphorylated much more rapidly than the second head. 18- 20 Therefore, we developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin (not myosin light chain) in smooth muscle. There was no detectable myosin phosphorylation in unstimulated muscle when the extent of light chain phosphorylation measured directly was only 0.02 mol phosphate
243
Smooth Muscle Contractility
.
Field Stimulation
I
I
~
f/$#/$#/$#/#//#/##/$/#//#ai-7~
1.0 -
.C::: 0
.<::.
~
08-
.<::.
o>
::::i d._ 0.60
~
0.4
~ 0
.<::.
5t
0 .<::.
0.2
u.
0
E
0 0
2
3
4
5
Time (sec)
Figure 3. Temporal development of active stillness, active force, and light chain phosphorylation during the onset of contraction. Stillness and force are expressed as a fraction of the maximal change observed above values measured at rest. The insert shows the time course on an expanded scale up to 1 second. Reprinted with permission. 17
per mol of light chain. After 2.5 seconds of neural stimulation, myosin light chain phosphorylation increased to a value of 0.63 mol of phosphate per mol of light chain, which resulted in 40% diphosphorylated myosin. The relationship between the extent of light chain phosphorylation and the relative amount of diphosphorylated myosin was consistent with a random phosphorylation process in smooth muscle cells. Following the period of latency, myosin light chain is rapidly phosphorylated and force and stiffness increase rapidly (Figure 3). Stiffness increases coincident with phosphorylation, and both increase more rapidly than isometric force. The coincident increase in stiffness and myosin light chain phosphorylation during smooth muscle activation suggests that each myosin head attaches independently to actin thin filaments upon phosphorylation. With continuous neural stimulation of tracheal smooth muscle for prolonged periods of time, after maximal responses are obtained, there is an apparent decrease in cytosolic Ca 2 + concentration with reductions in the extent of myosin light chain phosphorylation and maximal shortening velocity (Figure 4). Similar studies in tracheal smooth muscle have demonstrated a decrease in Ca 2 + -dependent phosphorylase a formation. The developed force is maintained during a sustained period of stimulation (Figure 4). This phase of contraction is characterized by relatively low levels of free Ca 2 + and myosin light chain phosphorylation, associated with low maximal velocity of shortening with force maintenance. 2•21 This physiologic state originally was proposed to be caused by the formation of a "latch bridge," ie, force is maintained because of a different state of the myosin crossbridge whereby it remains attached longer and cycles more
244
slowly. 22 •23 The maintenance of force is Ca 2 + -dependent, so a second Ca 2 + -dependent regulatory mechanism appears to be required. The latter Ca 2 + regulatory system presumably has a higher sensitivity to activation by Ca 2 + than myosin light chain kinase. 24 There is considerable interest in the cellular mechanisms for force maintenance with low values of light chain phosphorylation. Recent theories include: 1. Force maintenance by attached crossbridges for which detachment rates are greatly slowed after dephosphorylation. 25 2. Unregulated crossbridges,u for which Ca 2 + regulation is attributable to additional proteins in thin filaments. 26 One such protein could be caldesmon, a calmodulin-binding protein found in thin filaments isolated from smooth muscle. Caldesmon binding to actin thin filaments inhibits actin-activated myosin MgATPase activity. The inhibition is reversed by Ca 2 + /calmodulin. 3. Changes in the intermediate filament domain of smooth muscle cells. Filaments are distributed in two domains represented by (a) intermediate filaments free of myosin, but containing filamin, actin, a-actinin, and desmon and (b) contractile protein filaments containing actin, myosin, tropomysin, and caldesmon. 27 Further, intermediate filament proteins may be phosphorylated by protein kinases and, in particular, protein kinase C. 28 These phosphorylation reactions may affect the dynamic regulation of assembly and turnover of intermediate filaments. Thus, it has been hypothesized that tonic force maintenance may be associated with phosphorylation-mediated structural rearrangements of intermediate til-
1.0
0.40
0.8
0 32
_g <.)
0.6
-
~
0 24
~
~
0.4
.
'
~
016
0
>----(sec 1------l
a.
~
~
>'
~
0.2
l
0.08
0
r----------- ( min )------1
Figure 4. Time course of force, myosin light chain phosphorylation, and maximal shortening velocity (V 0 ) in neurally stimulated strips of tracheal muscle. For measurement of light chain phosphorylation, strips were frozen during rest or at indicated limes between 2 seconds and 30 minutes of stimulation. Force at time of freezing is expressed as a fraction of force obtained with a maximal neural precontraction (P < .01). Reprinted with permission. 16 October 1988 Volume 296 Number 4
Stull, Kamm, and Taylor
ament proteins following protein kinase C activation. Identification of the precise mechanism(s) that may account for the physiologic properties of tonic contraction will require additional investigation. Conclusions
Neurotransmitters and hormones activate cell surface receptors and affect cellular processes via different second messenger signals. Ca 2+ was first recognized as a second messenger when its central role in skeletal muscle contraction and glycogen metabolism was identified. We now appreciate the importance of Ca 2 + in mediating many types of cellular responses via the intracellular receptor protein calmodulin. The control of smooth muscle contractility by Ca 2 + /calmodulin has provided a model system for studies of Ca 2 + -dependent cellular processes. Integrated biochemical, cellular, and physiologic investigations have defined a general scheme in which Ca 2 + binds to calmodulin and the Ca 2 + /calmodulin complex activates myosin light chain kinase. Catalysis of myosin light chain phosphorylation then leads to contraction of smooth muscle. Although these aspects of smooth muscle contractility are established, important questions remain concerning the mechanisms, including other second messenger systems, that modify the control of smooth muscle contractility by Ca 2 +. References 1. Goldman YE , Brenner B: Special topic: molecular mechanism of muscle contraction. Annu Rev Physiol49:629- 636, 1987. 2. Kamm KE, Stull JT: The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacal Toxicol25:593-620, 1985. 3. Stull JT, Nunnally MH, Moore RL, Blumenthal DK: Myosin light chain kinases and myosin phosphorylation in skeletal muscle, in WeberG (ed): Advances in Enzyme Regulation. New York , Pergamon Press, 1985, ed 23, pp 123- 140. 4. Klee CB, Vanaman TC: Calmodulin. Adv Protein Chem 35: 213- 321, 1982. 5. Cox JA: Interactive properties of calmodulin. Biochem J 249: 621-629, 1988. 6. Blumenthal DK, Stull JT: Activation of skeletal muscle myosin light chain kinase by Ca 2+ and calmodulin. Biochemistry 19:5608- 5614, 1980. 7. Sellers JR, Pato MD: The binding of smooth muscle myosin light chain kinase and phosphatases to actin and myosin. J Bioi Chem 259:7740- 7746, 1984. 8. Pato MD, Kerc E: Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. J Bioi Chem 260:12359- 12366, 1985. 9. Onishi H, Umeda J, Uchiwa H, Watanabe S: Purification of gizzard myosin light-chain phosphatase, and reversible changes in the ATPase and superprecipitation activities of actomyosin in the presence of purified preparations of myosin
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
10.
11.
12.
13.
14. 15.
16.
17.
18.
19.
20. 21.
22.
23.
24.
25.
26. 27. 28.
29.
light-chain phosphatase and kinase. J Bioi Chem 91:265-271, 1982. Sellers JR: Mechanism of the phosphorylation-dependent regulation of smooth muscle heavy meromyosin. J Bioi Chem 260: 15815- 15819, 1985. Wagner PD, Vu N-D: Regulation of the actin-activated ATPase of aorta smooth muscle myosin. J Bioi Chem 261 : 7778- 7783, 1986. Meisheri KD , Ruegg JC, Paul RJ: Studies on skinned fiber prepa rations, in Grover AK , Daniel EE (eds): Calcium and Contractility. Clifton, New Jersey, Humana Press, 1985, pp 191- 224. Asano M, Stull JT: Effects of calmodulin antagonists on smooth muscle contraction and myosin phosphorylation, in Hidaka H , Hartshorne DJ (eds) : Calmodulin Antagonists and Cellular Physiology. Orlando, Florida, Academic Press, 1985, pp 225- 260. Taylor DA, Stull JT: Ca 2 + dependence of myosin phosphorylation in tracheal smooth muscle cells. Biophys J 53:598a, 1988. Kamm KE, Murphy RA: Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion. Experientia 41 :1010- 1017, 1985. Kamm KE, Stull JT: Myosin phosphorylation, force, and maximal shortening velocity in neurally stimulated tracheal smooth muscle. Am J Physioi249:C238-C247, 1985. Kamm KE, Stull JT: Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness. Science 232:80- 82, 1986. Persechini A, Hartshorne DJ: Ordered phosphorylation of the two 20,000 molecular weight light chains of smooth muscle myosin. Biochemistry 22:4 70-4 76, 1983. Sellers JR, Chock PB, Adelstein RS: The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state. J Bioi Chem 258:14181- 14188, 1983. Trybus KM , Lowey S : Mechanism of smooth muscle myosin phosphorylation. J Bioi Chem 260:15988- 15995, 1985. Rembold CM, Murphy RA: Myoplasmic calcium, myosin phosphorylation, and regulation of the crossbridge cycle in swine arterial smooth muscle. Circ Res 58:803-815, 1986. Dillon PF, Aksoy MO, Driska SP, Murphy RA: Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211:495-497, 1981. Driska SP, Aksoy MO and Murphy RA: Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am J Physioi240:C222-C233, 1981. Chatterjee M , Murphy RA: Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle. Science 221 :464-466, 1983. Hai C-M, Murphy RA: Cross-bridge phosphorylation and regulation of latch state in smooth muscle. Am J Physiol254:C99C106, 1988. Marston SB, Smith CW J: The thin filaments of smooth muscles. J Muscle Res Cell Motil6:669- 708, 1985. Small JV, Furst DO, DeMay J: Localization of filamin in smooth muscle. J Cell Biol102:210-220, 1986. Rasmussen H , Takuwa Y, ParkS: Protein kinase C in the regulation of smooth muscle contraction. Federation of American Societies for Experimental Biology Journal1:177- 185, 1987. Blumenthal DK, Stull JT: Activation of skeletal muscle myosin light chain kinase by calcium(2+) and calmodulin. Biochemistry 19:5608- 5614, 1980.
245