Regulation of the contractile element of airway smooth muscle.

Regulation WILLIAM Department

of the contractile

element of airway smooth muscle

T. GERTHOFFER of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557

GERTHOFFER, WILLIAM T. Regulation of the contractile element of airway smooth muscle. Am. J. Physiol. 261 (Lung Cell Mol. Physiol. 5): Ll5-L28, 1991.-Smooth muscle of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, and calponin. There has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, identification of the sites phosphorylated on the regulatory myosin light chains, and the effect of myosin phosphorylation on stress development and crossbridge cycling rates have all been studied in some detail. We infer from these studies that besides myosin phosphorylation there is an important role for a thin filament Ca’+-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and may be phosphorylated during contraction. The use of intracellular Ca2’ indicators to estimate changes in intracellular Ca*+ ([Ca”];) and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca”’ is not fixed but might be modulated by undefined messengers or excitationcontraction pathways. This adds an additional challenge to the continuing effort to define the messengers and regulatory proteins that couple activation of membrane receptors to the contractile element in airway smooth muscle. caldesmon;

calponin

MUSCLE of the mammalian airways controls airway diameter and resistance to airflow. Smooth muscle tone is in turn controlled by a variety of external signals that are transduced to useful work by contractile proteins. A comprehensive view of airway regulation requires knowledge of each step in the flow of regulatory information. Studies of the source, metabolism, and receptors for extracellular messengers such as acetylcholine, histamine, and neuropeptides provide a view of the first steps in signal transduction (7, 13, 27, 76). Beyond the level of membrane receptors and ion channels, intracellular messengerssuch as diacylglycerol, inositol phosphates, and cyclic nucleotides participate in transmitting information to the contractile element (see Refs. 20 and 110 for recent reviews). Myoplasmic intracellular Ca2+ concentration ([Ca”‘]J is thought to be the final messenger controlling contractile proteins. The aim of this review is to address the question of how Ca2’ controls airway smooth muscle contraction at the level of the contractile element. Many studies of airway smooth muscle contraction at the cellular and biochemical level have employed tracheal smooth muscle from a variety of species as a model SMOOTH

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system; although it is clear that the major source of resistance and the site of the largest pressure drop is in the more central airways (102). There are differences in the responsiveness of tracheal and bronchiolar preparations to agonists, maximum stress development, and the mechanical organization of cartilage and muscle cells (reviewed by 76, 120). Nevertheless, the trachea is a convenient source of smooth muscle for biochemical and biophysical studies of the contractile element. Although there is a significant literature on contractile regulation of airway smooth muscle, many of the details of crossbridge regulation are not yet clear for either tracheal or bronchiolar smooth muscle. When the data set is incomplete we also will use data from studies of avian and mammalian vascular and visceral smooth muscles. CONTRACTILE ELEMENT COMPOSITION IN AIRWAY SMOOTH MUSCLE

The protein components of the contractile element of airway smooth muscle are similar to those found in other smooth muscles and include actin, myosin, tropomyosin, caldesmon, calponin, myosin light chain kinase (MLCK), and protein phosphatases. The interested reader is re-

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ferred to earlier reviews of smooth muscle biochemistry for a description of contractile protein composition and actomyosin adenosinetriphosphatase (ATPase) activity of other smooth muscles (4, 48, 126). I will review here the more recent studies of airway smooth muscle contractile proteins. Myosin

Smooth muscle myosin is a hexameric molecule composed of two heavy chains of 200-205 kDa, two 20-kDa light chains, and two 17-kDa light chains. Myosin heavy chains exist in three isoforms in airway smooth muscle termed MHCl and MHC2 and a “nonmuscle” isoform (25, 82). The two smooth muscle isoforms differ in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gels with MHC2 migrating faster than MHCl. The ratio of MHCl:MHC2 decreases during development of pigs but not humans (82). This shift of the myosin heavy chain from an apparently larger isoform (MHCl) to a smaller isoform (MHC2) is associated with a decrease in maximum stress development (normalized to myosin content) from birth to maturity (131). There also is a decrease in the Ca” sensitivity of chemically skinned tracheal muscle during maturation. Although these correlations are intriguing the functional significance of the two smooth muscle isoforms is uncertain. At this point there is no evidence for significant differences in actomyosin ATPase activity or tissue shortening velocity that corresponds to a particular isoform. Myosin heavy chains can be phosphorylated in vitro by protein kinase C and the multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II) (50, 71). Heavy chain phosphorylation occurs in cultured tracheal smooth muscle cells stimulated with ionomycin or phorbol dibutyrate (65). Ionomycin presumably increases [Ca”‘]i to activate CaM kinase II and phorbol dibutyrate activates protein kinase C. However, in intact tracheal smooth muscle, conditions that promote contraction (phorbol dibutyrate or carbachol) do not induce any change in heavy chain phosphorylation (65). Therefore, heavy chain phosphorylation may be important in proliferating cells, but may not be important in regulating contraction of differentiated airway smooth muscle. The 20-kDa regulatory myosin light chains are phosphoproteins critical for regulation of actomyosin ATPase and contraction. The role of 20-kDa light chain phosphorylation in contraction has been studied extensively and will be discussed in more detail below. Nonmuscle isoforms of the 20-kDa light chains have been identified in smooth muscles (33, 43), but whether these isoforms participate in contraction or in processes of cell proliferation or cell motility is unknown. There are acidic isoforms of tracheal 20-kDa light chain corresponding to the nonmuscle isoforms reported by Gaylinn et al. (33) comprising -6% of the total 20-kDa light chain (105). However, definitive sequence data are not available to identify these proteins as the nonmuscle isoform. The 17-kDa myosin light chains are present in two isoelectric variants and are essential for actomyosin ATPase activity (51).

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MLCK

MLCK of bovine tracheal muscle has been purified and studied in some detail by Stull and co-workers (133). The protein iS present in bovine tracheal smooth muscle at a molar concentration of 3.4 PM. Calmodulin is present in this tissue at 39 PM, and the Ca’+-calmodulin dissociation constant is 1 nM. These data and the kinetics of activation of MLCK by Ca2+-calmodulin suggest that neither the concentration of calmodulin nor MLCK are limiting for contraction and that initiation of contraction is limited by changes in [ Ca2+]i (reviewed by 133,147). This analysis is based on purified enzyme that is not phosphorylated. However, MLCK can be phosphorylated and its activity reduced in intact muscle (23, 134). Therefore, the notion that MLCK activity is controlled solely by [ Ca2+]; via calmodulin is probably incorrect. Mediators of airway constriction and dilation might modify the phosphorylation state of MLCK by regulating other protein kinases or phosphoprotein phosphatases, which could modify the K, for Ca2’-calmodulin. Phosphorylation of MLCK has important implications for physiological studies designed to test current theories of cross-bridge regulation by measuring [ Ca2+]i, myosin light chain phosphorylation, and mechanical responses. Assaying the MLCK activity ratio also may be required to test adequately any model of contractile regulation. Besides the normal Ca2+-regulated form of MLCK, there is indirect evidence in airway smooth muscle for a Ca2’- and calmodulin-independent kinase activity that phosphorylates the 20-kDa myosin light chains (95). The phosphatase inhibitor okadaic acid induces contraction of lamb tracheal smooth muscle in Ca2+-free medium that is not blocked by the calmodulin antagonist, W7, or the protein kinase C inhibitor H7. Another phosphatase inhibitor, calyculin-A, induces contraction of skinned smooth muscles at subthreshold Ca2+ concentration (59). Myosin phosphorylation induced by calyculin-A in Ca2’free solution is blocked by the nonspecific kinase inhibitors amiloride and K252a, but not by trifluoperazine and H7. These experiments are provocative but should be interpreted with caution because the inhibitors used are not very selective and no biochemical evidence for a Ca2+-independent MLCK activity has been reported. In contrast to MLCK there is very little information about myosin light chain phosphatase activities within airway smooth muscle. Paietta and Sands (99) characterized several phosphoprotein phosphatases purified from bovine tracheal smooth muscle, but there are no data identifying specific myosin light chain phosphatase(s). The maximum myosin light chain phosphatase activity has been estimated from rates of dephosphorylation of myosin in intact tissue (66), but there are no reports of the subtypes present or the regulation of the myosin light chain phosphatases in airway smooth muscle. This is a topic of increasing interest in part because of physiological studies that suggest myosin phosphatases might be regulated (96, 130). Actin and Thin Filament

Proteins

Actin exists in at least two smooth muscle isoforms and possibly a “nonmuscle” isoform (30). Several actin


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capping and severing proteins have been described that probably participate in assembly and remodeling of contractile and cytoskeletal actin filaments (54, 60,94, 116). There also are actin binding proteins such as filamin and desmin that maintain structure and mechanical integrity of actin filaments (reviewed in Ref. 126). There are no studies of the processes of actin filament assembly and remodeling in airway smooth muscle cells. Tropomyosin is a thin filament regulatory protein that exists in two isoforms (30). It appears to modulate activation of actomyosin ATPase along with myosin light chain phosphorylation and possibly in concert with caldesmon and calponin (16, 128). The specific roles of the two tropomyosin isoforms are not known. Caldesmon and calponin are proteins associated with thin filaments that may have important roles in smooth muscle contraction. Caldesmon purified from other smooth muscles is an elongated protein that binds actin, myosin, tropomyosin, calmodulin, and Ca2’ (reviewed recently by 17,78). Caldesmon inhibits actomyosin ATPase and the inhibition can be relieved by phosphorylation of caldesmon by CaM kinase II (2, 135). The potential role of caldesmon in regulating cross-bridge cycling is now a subject of lively debate. Both caldesmon and calponin are present in airway muscle (90, 106). Canine tracheal caldesmon is present only in the high-molecularweight form having an apparent molecular mass of 113 kDa determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (McCallum and Gerthoffer, unpublished observation). The sequence of the caldesmon gene from chicken gizzard yields a calculated molecular mass of 87 kDa (12), which is similar to the molecular mass determined by sedimentation velocity (41). The apparent molecular mass obtained by SDSPAGE is artifactually high because of anomalous migration of proteins in the gels. Tracheal caldesmon crossreacts with antibodies raised against chicken gizzard and bovine aorta caldesmon (106), but the amino acid sequence of airway smooth muscle caldesmon is unknown. Calponin is a smooth muscle-specific protein (40) associated with the thin filament; however, see Lehman (77) for a dissenting opinion. It has an apparent molecular mass of 32 kDa in canine tracheal smooth muscle based on SDS-PAGE (McCallum and Gerthoffer, unpublished observation). Aorta calponin shares sequence homology with the COOH-terminus of troponin T and binds to actin and tropomyosin. The actin binding domain has sequence homology to the putative actin binding domain of a-actinin (140). Calponin is a phosphoprotein that is a substrate for CaM kinase II and protein kinase C (89, 140, 149). Phosphorylation of calponin decreases binding to actin and relieves inhibition of actomyosin ATPase in vitro (149). Tropomyosin is not of acrequired for, but might participate in, inhibition tomyosin ATPase by calponin (149). Although airway smooth muscle caldesmon and calponin are of similar size and have antigenic epitopes in common with the chicken gizzard and bovine aorta proteins, there are many basic characteristics of the airway muscle proteins that remain undefined. Such characteristics include the amino acid sequences, the effects of these proteins on tracheal actomyosin ATPase activity and the pathways

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regulating caldesmon smooth muscle.

and calponin

CONTRACTILE

REGULATION

Regulation

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of Airway

in intact

airway

Smooth Muscle Tone In Vivo

Extracellular signals acting on airway smooth muscle cells are thought to be transduced to force by two general types of signaling pathways (reviewed by 20). Electromechanical coupling depends on ion channel function and ionic fluxes and is characterized by changes in membrane potential. Pharmacomechanical coupling is thought to be mediated by chemical (e.g., inositol phosphates, diacylglycerol) or ionic (e.g., Ca2+) messengers independent of changes in membrane potential. Myoplasmic Ca2+ is generally considered the final common messenger for any smooth muscle stimulant with [ Ca2+]i determining the active force produced. However, studies with intracellular Ca2+ indicators suggest that contractile element sensitivity to Ca2+ is variable and that there is not a simple relationship between [ Ca2+]i and force (reviewed in Ref. 69). The exact sequence of events between an increase in [Ca2+]i and development of force is an open question. Furthermore, the processes mediating a return to basal [Ca2+]i and relaxation (which are not necessarily a simple reversal of the activation processes) are also somewhat mysterious. Ca2+ Regulation of Contractile Element

Several regulatory schemes have been proposed including thick filament regulation by phosphorylation of myosin (reviewed by 67) and thin filament mechanisms involving caldesmon and calponin (91, 127, 137, 149). Both thick and thin filament mechanisms apparently require Ca2+ binding to a regulatory protein, calmodulin in the case of MLCK and possibly a calmodulin-like protein in the case of caldesmon (107). The Ca2’-binding protein that regulates calponin is unknown, but may be calmodulin if calponin is a substrate for CaM kinase II in vivo. Ca2+ -calmodulin-mediated phosphorylation of myosin is probably required for initiation of contraction and is a principal determinant of cross-bridge kinetics (46, 87). Many biochemical studies (reviewed in Ref. 47) demonstrate that phosphorylation of the 20-kDa myosin light chains increases actomyosin ATPase activity. Phosphorylation of the 20-kDa light chains increases the rate of product release (118) and increases maximum velocity ( Vmax). Reports of a linear relationship between myosin phosphorylation and actin-activated or actin/tropomyosin-activated myosin ATPase led to the initial models of smooth muscle regulation. In these early models, [Ca2+]i controlled MLCK, which phosphorylated myosin, thus increasing actomyosin ATPase causing cross-bridge cycling and force development. Relaxation occurred when myosin became dephosphorylated after cessation of the stimulus or in response to a smooth muscle relaxant. Unphosphorylated myosin was thought to be inactive. However, there are reports of significant actin-activated ATPase activity of unphosphorylated myosin purified from gizzard and aorta (144, 145). Activation of unphosphorylated myosin depends on conditions that favor


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filamentous myosin (which is most relevant to conditions in vivo). It was suggested that a second regulatory system inhibits actomyosin ATPase (144). Many studies of caldesmon regulation of gizzard and vascular smooth actomyosin ATPase are consistent with this hypothesis (80). It has been suggested that 1) caldesmon is phosphorylated during contraction, which relieves inhibition of actomyosin ATPase (3, 91, 111), or 2) Ca2+ binds to an unidentified protein, which binds to caldesmon and disinhibits actomyosin ATPase (80). A third regulatory system involving calponin also has been proposed (137). The role of calponin in vivo is unclear, but there is preliminary evidence that calponin is phosphorylated in intact airway smooth muscle (106). The existence of multiple regulatory mechanisms is inferred from several biochemical and physiological studies (reviewed by 67, 80), but the identities of the regulatory systems are unclear and the issue is controversial. I will review the evidence for each regulatory system and suggest a working model for regulation of tracheal smooth muscle contraction. Thick Filament

Regulation-The

Latch Hypothesis

Dillon et al. (26) showed that force was maintained in tonically contracting vascular smooth muscle by nonphosphorylated cross bridges termed “latch bridges.” A latch bridge is thought to form in an activated smooth muscle cell when an attached cross bridge is dephosphorylated, presumably by the same phosphatases that dephosphorylate myosin during relaxation. Dephosphorylation of the attached cross bridge induces a state of the cross bridge that supports tension but cycles very slowly or not at all (45,46,88). The mechanical result is reduced shortening velocity, which presumably reflects reduced actomyosin ATPase activity and reduced energy consumption. Recently, purified actin and myosin were used in an in vitro motility assay to show that phosphorylated myosin increased actin filament motility and that increasing the proportion of dephosphorylated myosin reduced actin mobility (148). When myosin was crosslinked with a sulfhydryl reagent to yield a weak actinbinding form, actin mobility was also reduced. It was suggested that cross bridges might exist in strongly bound (phosphorylated) and weakly bound (dephosphorylated) states and that the weakly bound state could in fact impose a significant load on the cycling cross bridges. If actin and myosin behave similarly in vivo some of the time-dependent changes in shortening velocity occurring in intact muscle might be caused by a shift to a dephosphorylated, attached state. Because force developed by muscles in the “latch” state still depended on Ca2’, a second Ca2’ regulatory mechanism was hypothesized, but no specific regulatory proteins were identified (5, 18, 37). This hypothesis stimulated much of the current interest in identifying new Ca” regulatory mechanism in smooth muscle. Recently the latch hypothesis was modified so that changes in MLCK and phosphatase activities could account for the dissociation of force and myosin phosphorylation (28, 46). The model is based on two important assumptions: that myosin light chain phosphatase activity is very high

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and is unregulated. By use of a kinetics approach to model contraction and myosin phosphorylation data from carotid artery, Murphy (87), suggested that a second Ca2’ regulatory system is not necessary to explain development of force and regulation of cross-bridge cycling. Experiments

Testing the Latch Hypothesis

The latch hypothesis is a rather fundamental departure from the regulatory mechanisms and the crossbridge behavior of striated muscles. For that reason and because smooth muscles are important effector cells in several systems the hypothesis has been thoroughly scrutinized. Two critical predictions of the hypothesis are 1) phosphorylation of myosin is necessary and sufficient for contraction; 2) myosin phosphorylation levels correlate with and control muscle shortening velocity. There are several preparations and conditions that fulfill both predictions (reviewed by 67 and 87), but there also are data inconsistent with both predictions. Some of the strongest evidence demonstrating the sufficiency of myosin phosphorylation in supporting contraction is Ca2’independent contraction of skinned fibers treated with ATPrS (72) and contraction of skinned gizzard smooth muscle induced by unregulated MLCK (146). MLCK was proteolyzed briefly to yield a Ca2+-independent enzyme that phosphorylated myosin in the skinned fiber and caused contraction at subthreshold Ca2’ concentration (pCa 8). Sufficiency of myosin phosphorylation is also supported by the study of Itoh et al. (61) in which shortening of isolated cells from toad stomach was blocked by microinjection of peptides that inhibit MLCK and calmodulin. There also are many correlative studies in which contraction or relaxation of intact and chemically skinned smooth muscle strips or isolated cells is proportional to the level of phosphorylation (reviewed by 68). Although these data are consistent with the sufficiency hypothesis, there are potential problems with each experiment. Smooth muscle skinned with Triton X-100, high concentrations of saponin or glycerol might lose critical regulatory proteins (74). The peptide inhibitors of MLCK and calmodulin injected into the living cell may not be selective at the concentrations achieved on injection. SMl augmented the [Ca”‘]i transient significantly while depressing cell shortening, suggesting multiple effects on regulatory proteins (62). A positive correlation of phosphorylation and force in intact cells could result from choosing stimuli that preferentially activate MLCK and not the other regulatory system, or the experimental manipulation might simply activate or inactivate all Ca2+-dependent regulatory systems to the same degree (121). Data Inconsistent with Myosin Phosphorylation as the Sole Regulatory Mechanism

In contrast to evidence suggesting a simple causal relationship between myosin phosphorylation and contraction there is evidence that myosin phosphorylation is not necessary (55,143) or sufficient (34,35) for smooth muscle contraction. In freshly skinned gizzard fibers


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Ca”-induced a contraction without a significant increase in myosin phosphorylation (143). Fibers stored for more than a week apparently “lost” the ability to contract independent of myosin phosphorylation, possibly because important regulatory proteins were lost from the preparation or were degraded during storage. Manganese ions induced contraction of skinned gizzard without a concomitant increase in myosin phosphorylation (55). The contractile effect of manganese is probably secondary to oxidation of contractile proteins because dithiothreitol reversed the effect. Moreland et al. (83) showed Ca*‘-calmodulin-dependent force maintenance in skinned carotid artery that is independent of myosin phosphorylation. They hypothesized a Ca*+-calmodulinregulated thin filament mechanism acting in parallel with myosin phosphorylation. The biochemical correlate to these studies is regulation of myosin ATPase by mechanisms independent of myosin phosphorylation (79,144). These data suggest that under some conditions myosin phosphorylation is not necessary for activation of actomyosin ATPase and contraction. Data from our laboratory address the issue of whether myosin phosphorylation is sufficient for contraction under all conditions. We dissociated myosin phosphorylation from force maintenance and force development with two experiments. When canine tracheal muscle was stimulated with carbachol and then treated with Ca*+-free solution containing ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) the muscles relaxed but myosin phosphorylation remained elevated at nearly steady-state levels of >0.4 mol Pi/mol light chain (34). When tracheal muscle was depleted of external Ca*’ and stimulated with carbachol myosin phosphorylation increased significantly but no significant force developed until CaC12 was added back to the bath solution (35). Phosphorylation greater than 0.3 mol Pi/ mol light chain was observed in Ca*+-depleted tissues stimulated with carbachol. In some preparations this level of myosin phosphorylation is associated with maximal contraction (39,113,121). Sparrow et al. (132) made a relevant observation using ATPrS to thiophosphorylate myosin in skinned fibers. Thiophosphorylation, which is presumed to be irreversible or slowly reversible, caused contraction in the presence of ATP. However, force was not maintained in low Ca*’ solution as would be expected if myosin remained phosphorylated and was sufficient to sustain force. The authors suggested that phosphatases slowly remove the thiophosphate from myosin. Another possibility is that Ca*’ acting at a second regulatory site is required for force maintenance. Recently a similar suggestion was made by Tansey et al. (138) based on the effect of okadaic acid, an inhibitor of types 1, 2A, and polycation modulatable phosphatases (10, 59). Bovine tracheal muscle previously stimulated with carbachol relaxed when treated with a low concentration of okadaic acid (3 PM). Fura- fluorescence decreased but myosin phosphorylation remained elevated at about 0.5 mol Pi/m01 light chain. Therefore, force and myosin have been dissociated in several airway smooth muscles (canine, guinea pig, and bovine) using several experimental protocols (Ca*’ depletion plus carbachol and okadaic acid plus carbachol in intact fibers and

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thiophosphorylation in skinned fibers). These results challenge the idea that myosin phosphorylation is sufficient for contraction and that it is the sole regulator of cross-bridge cycling. This issue can be analyzed further by reviewing the mechanical evidence that cross-bridge cycling rate is regulated in intact tracheal and bronchial smooth muscle. Mechanical Evidence for Latch Bridges in Airway Smooth Muscle

The hallmark of the latch state as first described (26) is an initial transient increase in shortening velocity on activation of a smooth muscle followed by a time-dependent decrease in velocity. Shortening velocity was measured by isotonic quick-release to a single low afterload of -12% of the force developed at the moment of release. Shortening velocity was thought to decrease as a function of time because myosin phosphorylation decreased as a function of time (5). Myosin phosphorylation decreased because the agonist (60 mM K’) causes a transient increase in [ Ca*+]i (112). Airway smooth muscle exhibits mechanical behavior similar to vascular smooth muscle (Fig. l), but there are important differences in the correlations of myosin phosphorylation, shortening velocity, and [ Ca*+] i. Shortening velocity transients occur in smooth muscle from the rabbit trachea (36), canine trachea (34,81), canine bronchi (64), and bovine trachea (66). Peiper and co-workers (104) reported a time-dependent decrease in the rate of postvibrational tension recovery of rat tracheal smooth muscle. The rate of postvibrational tension recovery depends in part on cross-bridge cycling rates (103). Shortening velocity transients occur during contraction of all airway muscle from all species tested to date. The issue of whether there is a coincident transient change in myosin light chain phosphorylation is less clear. Muscarinic activation of bovine tracheal muscle produces a transient increase in myosin phosphorylation that returns to basal values within 30 min (66, 123). K+depolarization produces a more modest myosin phosphorylation transient (123). Time-dependent changes in maximum shortening velocity were tightly correlated with time-dependent changes in myosin phosphorylation in neurally stimulated bovine tracheal muscle (66). In canine and rabbit tracheal muscle, muscarinic stimulation and K+-depolarization each produce only a modest transient increase in myosin phosphorylation (23,34,36, 81), but the time-dependent decrease in shortening velocity is greater than the decrease in myosin phosphorylation. This is consistent with data from uterine, colonic, and vascular smooth muscles in which disproportionate changes in tissue shortening velocity and myosin phosphorylation occur (14, 39, 44, 84, 122). Shortening velocity probably does not decrease only because of a [Ca*+]i transient, at least during muscarinic activation of canine tracheal muscle. Stimulation of canine tracheal muscle with carbachol causes a biphasic increase in [Ca*+]i with a significant sustained component (Fig. 1 and see ref. 97). Myosin phosphorylation increases rapidly with a modest initial transient followed by a significant sustained phase. This is similar to the


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Minutes FIG. 1. Time-dependent changes in regulatory processes during contraction of canine tracheal smooth muscle stimulated with 1 PM carbachol. A: active stress (0) increases monotonically, while lightly loaded shortening velocity (0) increases transiently. There is a modest myosin phosphorylation -(a) transient, but phosihorylation does not decrease as dramatically as shortening velocity from 30 s to 20 min [From Gerthoffer (34).] B: changes in the furafluorescence ratio (R340/380) and force were measured simultaneously using an isometric muscle bath with a Gould UC3 force transducer mounted on a JASCO CAF-100 fluorimeter. Muscle strips were loaded 7-9 h with 2.5 PM fura-2/AM, 37°C. One surface of muscle strip was illuminated alternately with excitation light at 340 and 380 nm (50 Hz). Fluorescence emission from the illuminated surface was measured at 500 nm. Signals from force transducer and fluorometer were digitized and stored on a microcomputer. Data are average of duplicate-strips from 1 animal. There was an initial [Ca2+]; transient followed by a-sustained increase in [ Ca2+] i.

temporal changes in [Ca2+]i. However, shortening velocity increases transiently and decays steadily even during the tonic phase of contraction (Fig. 1). Therefore, correlative experiments in airway smooth muscle do not all support the hypothesis that Ca2+ transients alone cause myosin phosphorylation transients, which in turn cause transient increases in tissue shortening velocity. The issue of what controls cross-bridge cycling rates in airway smooth muscle was considered by Seow and Stephens (119). They suggested that velocity might decrease by two mechanisms: a shift of myosin to the dephosphorylated, attached state (i.e., latch bridges), which imposes a load on the phosphorylated rapidly cycling cross bridges, or all active cross bridges might be slowed by some inhibitory mechanism. The hypothesis that cross bridges are dephosphorylated and then impose a load on phosphorylated cycling cross bridges has been challenged. In intact taenia coli, increasing external CaC12 from 1.9 to 4.5 mM increased Vmax without increasing myosin phosphorylation (122). In chemically skinned taenia coli, decreasing the ionic strength of the activating solution (pCa 4.4) from 75 to 18 mM decreased ATPase activity significantly without changing myosin phosphorylation (32). Therefore, a change in cross-bridge cycling rate as assayed by measuring tissue shortening velocity or actomyosin ATPase in skinned fibers does not require a concomitant change in myosin phosphorylation. Butler et al. (14) also found that slowing of the cross bridges in a latch state was not associated with

increased energy cost of external work which would be expected if there was an internal load on the cross bridges. These data and the poor correlation of myosin phosphorylation and shortening velocity in intact airway smooth muscle are more consistent with a general slowing of all cross bridges during contraction and support the possibility that other mechanisms in addition to myosin dephosphorylation regulate cross-bridge cycling. One criticism of this interpretation is that phosphorylation of a single light chain in the myosin molecule produces a cooperative effect such that full activation and maximum shortening velocity does not require stoichiometric phosphorylation of 2.0 mol Pi/m01 myosin (15, 129). Measuring myosin phosphorylation in tissues is a static measurement that shows the percentage of all 20-kDa myosin light chains phosphorylated. It does show the turnover rate of light chain phosphorylation, which might influence tissue shortening velocity (46). Therefore, tissue shortening velocity must be interpreted cautiously when testing hypotheses concerning cross-bridge regulation (86). There also is some uncertainty in correlating changes [Ca2+]i with mechanical and biochemical events in intact cells; see the analysis by Karaki in Ref. 69. Another important criticism of most correlative studies is that myosin might be phosphorylated at sites other than serine 19 and the poor correlation of shortening velocity and myosin phosphorylation is spurious. Muscarinic activation of airway muscle increases phosphatidyl inositol turnover, which could activate protein kinase C. If protein kinase C phosphorylates myosin in situ with no functional consequence there could arise an apparent dissociation of phosphorylation and velocity. This appears to be unlikely because stimulation of bovine tracheal smooth muscle with carbachol, serotonin, or high K+ increases phosphorylation only at serine 19, the primary regulatory site phosphorylated by MLCK (21, 65). There is no evidence that the agonists used in studies of intact muscle activate protein kinase C sufficiently to phosphorylate myosin light chains at the protein kinase C sites in vivo. This is a particularly important result because of the interest in the possible role of protein kinase C in phosphorylating smooth muscle myosin (109). It may be that the primary effect on the contractile system after activation of protein kinase C by agonists is to change [Ca”‘]i (65, 114) or to regulate other protein kinases (109) or perhaps to regulate phosphoprotein phosphatases. Although there are important limitations of studies of intact fibers, significant information has been obtained from such studies because the cellular regulatory mechanisms are intact. This is an important advantage over studies of skinned fibers and purified contractile proteins. Synthesis of information from all three types of experiments suggests an important role for thin filament regulation in smooth muscles (80, 83, 135). Thin Filament

Regulation

by Caldesmon

There are two effects of caldesmon that are relevant to cross-bridge regulation: 1) caldesmon inhibits actomyosin ATPase activity; the mechanism of inhibition may be by inhibiting myosin binding to actin (52, 141)


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or by reducing the rate of product release (79). 2) Caldesmon promotes “tight binding” or cross linking of actin to myosin at a site different from the ATPase site (57, 135). Inhibition of actomyosin ATPase is consistent with reduced shortening velocity and reduced energy consumption observed in tonic contractions (14, 101). The mechanical effects of actin-myosin cross linking by caldesmon are not at all clear. If such cross links occur in vivo and are analogous to the catch state of molluscan muscle then breaking the cross links should cause force to decay immediately and not redevelop (88, 115). This is not the mechanical behavior of carotid artery in the latch state. Step shortening causes a rapid transient relaxation followed by slow force redevelopment; behavior characteristic of a cross-bridge mechanism (37, 88). Unless the actin-caldesmon-myosin cross links are quite dynamic they are not the only mechanism for stress development after a quick-stretch. Caldesmon has some intriguing characteristics that suggest a role in thin filament regulation of the cross-bridge cycle, but there are several important questions to be answered. What is the physiologically important effect of caldesmon? Is it inhibition of myosin binding to actin, inhibition of product release, or cross linking of actin to myosin? How is caldesmon regulated in vivo? Does phosphorylation occur in intact muscle at a functional site at a rate consistent with a regulatory system? Does Ca2’-calmodulin have an important regulatory role? Thin Filament

Regulation

Intact

in force. The first observations of disproportionate changes in [Ca’+]i and force were reported by Morgan and Morgan (85). Several laboratories later reported dissociations of [ Ca’+]; and force in other vascular muscles (113, 114, 117), gastrointestinal (53, 96, 150), and airway smooth muscles (38, 97). There are a variety of treatments that can modify the [ Ca’+] i-force relationship in intact muscle (69). Stimulation of rat aortic strips with norepinephrine produces more tension for a given change in the furafluorescence ratio than does stimulation by K+ depolarization (117). Treatment of vascular muscle with phorbol esters produces higher force for a given change in [ Ca’+]i than does K+ depolarization (63, 97). Stimulation of tracheal smooth muscle with carbachol induces higher active stress and myosin phosphorylation at a given level of aequorin luminescence than does K+ depolarization (Fig. 2). Other treatments that modify the Ca2’ -force relationship in smooth muscle include chronic denervation of the guinea pig vas deferens (108) and smooth muscle relaxants such as adenosine, sodium nitroprusside, isoproterenol, and forskolin (1, 11, 70, 117). Skinned fibers prepared with staphylococcal a-toxin, ,B-escin, or low concentrations of saponin are being used in several laboratories to investigate this phenomenon. . 0 A Potassium Carbochol

IA

by Calponin

A regulatory role for calponin has been hypothesized based on its inhibitory effect on actomyosin ATPase and the effect of phosphorylation of calponin in vitro (137, 149). Several criteria used to establish a functional role for calponin phosphorylation have been satisfied. Kinase and phosphatase activities have been described (although not unequivocally identified), and there is a functional effect of phosphorylation (disinhibition of actomyosin ATPase). We recently addressed the criterion of correlation of calponin phosphorylation with function of intact airway smooth muscle. The rate of calponin phosphorylation was compared with the initial production and maintenance of isometric force (106). Calponin phosphorylation increased rapidly in response to carbachol, remained slightly elevated during a tonic contraction, and decreased during relaxation induced by chelation of external Ca2’ by EGTA. The time course of calponin phosphorylation is similar to the time course of changes in myosin phosphorylation and shortening velocity in tracheal smooth muscle (34, 35,81). Although the initial studies are intriguing there are many important questions to be answered before calponin can be assigned an important regulatory role in smooth muscle. EXCITATION-CONTRACTION CONTRACTILE ELEMENT

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COUPLING SENSITIVITY

AND TO CALCIUM

Smooth Muscle

Wider application of techniques for measuring changes in [Ca2+]i in living smooth muscles has shown that changes in [Ca2+]i are not always proportional to changes

=< G

n C -5.0

-4.9

-4.8 -4.7 log (L/L,,,)

-4.6

-4.5

FIG. 2. Stimulus-dependent differences in apparent Ca”’ sensitivity of active stress and myosin phosphorylation in intact canine tracheal smooth muscle. A: aequorin luminescence vs. active stress for muscles induced to contract by increasing concentrations of CaC12 in the presence of 1 PM carbachol (0) or 60 mM K+ (0). B: aequorin luminescence plotted vs. myosin phosphorylation for muscles activated with carbachol (A) or 60 mM K+ (A). Stimulation with carbachol-induced significantly higher active stress and myosin phosphorylation for a given change in aequorin luminescence (P < 0.05). [From Gerthoffer et al. (38).]


L22

INVITED

In these preparations the cell membranes are leaky to small molecules such as metal ions and nucleotides but appear to retain functional receptors and some coupling mechanisms. This was shown very clearly by the ability of norepinephrine plus GTP or GTPyS to shift the Ca2’force curve of mesenteric artery skinned with staphylococcal a-toxin to the left (93). Similar results have been reported for guinea pig ileum skinned with ,&escin (73) and bovine tracheal muscle skinned with saponin (75). Figure 3 illustrates muscarinic potentiation of Ca2+-induced contraction of a-toxin skinned canine tracheal smooth muscle. GTP (0.1 mM) produced a modest potentiation of the response to pCa 5.5. The addition of 1 PM carbachol then induced a biphasic contraction about equal to the maximum response to Ca”’ alone (pCa 4.5). Because [Ca”‘]i is clamped by extracellular EGTA buffer solutions, changes in force induced by agonists, antagonists, and guanine nucleotides are presumably not caused by changes in [ Ca2+]i. As a further precaution all solutions contained 10 PM ionomycin to release any stored Ca”+. Changes in the Ca2’ -force relationship are thought to reflect a change in contractile element sensitivity to Ca 2+ It ‘is not known to what extent agonists such as acetylcholine, histamine, or serotonin can modify contractile element sensitivity to Ca2’ in intact airway smooth muscle. Nor is it clear whether desensitization modifies the efficacy of relaxants such as ,&adrenoceptor agonists and methylxanthines. Some mediators of bronchomotor tone are known to interact postsynaptically to augment the responsiveness of airway muscle (reviewed by 76). Some of these drug interactions probably occur at the cell membrane and modify excitation-contraction coupling at steps before elevation of myoplasmic Ca2+. However, it is possible that potentiation or inhibition of the effect of Ca”’ at the contractile element might also occur. Possible Mechanisms Ca”’ Sensitivity

Regulating

the Apparent

The increase in force in response to agonists such as carbachol in Fig. 3 has been ascribed to a change in

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contractile element sensitivity to Ca2’. This terminology implies a change in the Ca2+-concentration requirement of Ca2+ binding proteins or Ca2+-dependent protein kinase activities such as MLCK or CaM kinase II. However, dissociation of force and [Ca2+]i also might be the result of increased contractile or regulatory protein phosphorylation secondary to activation of protein kinase C or inhibition of phosphoprotein phosphatase activities (75,92,96, 130). Each of these mechanisms is now being actively investigated. Stull and co-workers have shown that MLCK is phosphorylated and the catalytic activity of the kinase regulated in intact tracheal smooth muscle (134). The kinase activity ratio is much higher after muscarinic stimulation than after K+ depolarization; this is support for the notion that receptors are linked to mechanisms that change the actual Ca2+ sensitivity of the contractile element. Because K+ depolarization depresses the activity ratio during contraction the agoniststimulated state could be considered the normal sensitivity state of the contractile system and the K+-depolarized state to be one of “desensitization.” The critical question is what messenger(s) and regulatory proteins control the MLCK activity ratio? Based on skinned fiber studies G proteins are involved at some point, but the location, identity, and role of G proteins is unclear. There are several protein kinases activated by important mediators of airway muscle contraction: CAMP- and cGMP-dependent protein kinases, protein kinase C, and CaM kinase II. CAMP-dependent protein kinase, protein kinase C, and CaM kinase II all phosphorylate MLCK at a regulatory site called the A site (22, 49, 56), but only A-kinase and CaM kinase II do so at a rate and stoichiometry sufficient for a role in regulation (134). Protein kinase C may not have a significant effect; stimulation of intact muscle with phorbol dibutyrate induced phosphorylation of MLCK at the A site to only 3% (l34), although other sites were phosphorylated significantly. Direct phosphorylation of myosin light chains by protein kinase C is another pathway that might change the apparent Ca2’ sensitivity of the contractile element. The evidence supporting this idea is that phorbol esters under

250

1JJUCOfb

200

0.1

mU CTP

F 150 ; 100 2

0 LL

50 0

pea 4.5

9

-50 100

200 Minutes

3. Sensitization of chemically skinned tracheal smooth muscle by muscarinic stimulation. Canine tracheal smooth muscle was permeabilized with 20 pg/ml staphylococcal a-toxin for 20 min at room temperature using Ca” buffer solutions as described by Nishimura et al. (93). Ionomycin (10 PM) was included in relaxing (pCa 9) and activating (pCa 4.5 and 5.5) solutions to eliminate releasable Ca” stores. Fibers were stimulated with pCa 4.5 activating solution to elicit maximum Ca*+ -induced force followed by a control stimulation with pCa 5.5. Addition of 0.1 mM GTP to a second stimulation with pCa 5.5 solution induced transient contractions that were potentiated more than twofold by subsequent addition of 1 PM carbachol. A final stimulation with pCa 5.5 produced a contraction equal to the control response to pCa 5.5, suggesting that the potentiating effect of carbachol and GTP was readily reversible. FIG.


INVITED

some conditions induce contraction of some smooth muscles (19, 100). However, in other preparations phorbol esters relax contracting smooth muscle, inhibit neurogenie contractions, or have no effect (6, 35, 65). Furthermore, the evidence for an important functional effect of direct phosphorylation of myosin by protein kinase C in vivo is not very strong. Myosin light chains are phosphorylated in intact tracheal muscle stimulated by agonists only at the MLCK site (67). Uemoto et al. (139) used an in vitro motility assay to show that phosphorylation of the MLCK site of tracheal myosin light chains was required for motility. Phosphorylation of myosin by protein kinase C did not support motility, and phosphorylation of myosin by MLCK followed by phosphorylation by protein kinase C did not modify motility. Sutton and Haeberle (136) showed that phosphorylation of protein kinase C sites induced by treatment of skinned uterine smooth muscle with phorbol esters had no functional effect; phorbol ester treatment did not induce contraction and did not change tissue shortening velocity. Therefore, in studies where the phosphorylation sites are carefully defined, it appears agonists do not activate protein kinase C sufficiently to phosphorylate myosin light chains directly (65). In contrast to agonists, phorbol esters probably induce very prolonged activation of a high percentage of the total protein kinase C in the cell. Phorbol ester treatment can produce phosphorylation of myosin light chains in living smooth muscle at the protein kinase C sites (68, 125), but contraction is probably not caused by direct effects on the cross-bridge cycle mediated through myosin phosphorylation. Other possible mechanisms of action of phorbol esters include regulation of ion channels (31, 142) leading ultimately to changes in [Ca’+]i (98, 114), regulation of phosphatidyl inositol metabolism (9) which could ultimately influence [Ca”‘],, and phosphorylation of regulatory proteins such as caldesmon, calponin, other protein kinases and phosphoprotein phosphatases (109). Because treatment with phorbol esters might affect all these pathways it is important to identify the targets of protein kinase C and verify that functionally important sites are phosphorylated in response to physiological stimuli. Phorbol esters probably induce changes in protein phosphorylation in intact cells that do not occur in response to physiological or other pharmacological stimuli (67). In summary, the idea of variable sensitivity of the contractile element to Ca2+ in living smooth muscle cells is gaining increasing experimental support. It appears to occur in a variety of smooth muscles in response to many stimulants and in response to smooth muscle relaxants. From skinned fiber studies it seems that G proteins are important in the phenomenon, but the molecular details are unclear. Regulation of MLCK activity by phosphorylation and regulation of phosphoprotein phosphatases might contribute to the phenomenon, but again the details of the regulatory pathways remain to be determined. SUMMARY OF MECHANISMS CONTRACTILE ELEMENT SMOOTH MUSCLE

OF

REGULATING AIRWAY

Based on the evidence presented above there may be two Ca2+ regulatory systems acting on the cross-bridge

L23

REVIEW

cycle in airway smooth muscle: Ca2+-calmodulin-dependent phosphorylation of the 20-kDa myosin light chains and a Ca’+-dependent thin filament mechanism involving caldesmon, calponin, or both proteins acting together with tropomyosin (Fig. 4). Because we do not know many of the details of thin filament regulation it is not possible to construct an inclusive model of contractile regulation. What follows is a hypothesis based primarily on results of physiological and biochemical studies of airway smooth muscle contraction with liberal use of biochemical data from studies of gizzard and vascular smooth muscle. During the initial phase of carbachol-induced contraction, myosin light chain kinase (MLCK in Fig. 4) is activated by increased [Ca2+]i leading to rapid phosphorylation of myosin light chains (dotted line in Fig. 1A). If Ca2+ remains elevated above resting values, myosin phosphorylation remains elevated as well (24, 34, 36). If [Ca2+]i increases transiently, such as on stimulation with acetylcholine, myosin phosphorylation follows the transient. There is a rapid increase in the turnover rate of actomyosin ATPase and a rapid increase in crossbridge cycling rate as indicated by tissue shortening velocity (solid circles and dotted line in Fig. 1). The rapid increase in shortening velocity depends in part on increased myosin phosphorylation (open squares and dashed line in Fig. 1). Phosphorylation of myosin is probably rate limiting during the initiation of contraction (66, 129). Increased [Ca2+]i probably also activates CaM kinase II. Protein kinase C is activated by diacylglycerol released after agonist-induced hydrolysis of phosphatidylinositol bisphosphate (8, 29, 42). Rapid phosphorylation of calponin or caldesmon or both proteins might remove inhibition of actomyosin ATPase. Later (30 s-15 min) myosin phosphorylation, calponin, and caldesmon phosCa2+

2+

a MYOSIN

4. Schematic model of pathways regulating the contractile element of airway smooth muscle. Muscarinic stimulation by acetylcholine (ACh) increases [Ca”], by at least 2 mechanisms: voltagedependent influx of extracellular Ca2+ and release of stored Ca”. possibly in response to increased inositol trisphosphate (IP3). Ca2’ activates myosin light chain kinase (MLCK) which phosphorylates the 20-kDa myosin light chains, thus increasing actomyosin ATPase activity and rate of cross-bridge cycling. Ca”+ probably also activates multifunctional Ca*+/calmodulin-dependent protein kinase (CaM kinase II), which might phosphorylate other putative regulatory proteins such as caldesmon (CD) or calponin (CP); phosphorylation of these proteins might relieve inhibition of actomyosin ATPase, thus increasing crossbridge cycling rate and force production. FIG.


L24

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phorylation all decrease somewhat (34, 106). In some other smooth muscle preparations, depending on the stimulant used, contractile protein phosphorylation may well return to basal levels (26, 124). It may be that the respective protein kinase activities are inhibited during the course of contraction (109) or that protein phosphatases are activated to dephosphorylate contractile proteins (96, 130). If this is true there will be no simple relationship between myosin, caldesmon or calponin phosphorylation and [Ca2+]i. The concerted action of thick filament regulation and thin filament regulation could result in a complex but very flexible regulatory system in airway smooth muscle, particularly if the protein kinases and attendant phosphoprotein phosphatases are themselves regulated. An example would be protein kinase C (PKC) or CaM kinase II phosphorylating target proteins in a cascade mechanism as suggested by Rasmussen et al. (109). The question of what causes the time-dependent decrease in cross-bridge cycling rate remains unanswered. Phosphorylation of the 20-kDa myosin light chains plays a central role; it increases the Vmax of actomyosin ATPase, increases motility of actin filaments in in vitro motility assays, and is correlated under many conditions with increased tissue shortening velocity. However, it seems unlikely that myosin phosphorylation alone determines cross-bridge cycling rates because of reports of dissociation of the two events (14, 39, 44, 83, 121). Myosin phosphorylation might act in concert with the thin filament proteins, caldesmon and calponin. Unphosphorylated forms of these proteins inhibit actomyosin ATPase, the inhibition is relieved by phosphorylation and a few studies show there are increases in caldesmon and calponin phosphorylation in intact tissues (3, 100, 106). However, there are no studies defining the important phosphorylation sites or the protein kinases and phosphatases that control such phosphorylation. There is also a lack of information confirming the effects of these proteins on airway smooth muscle actomyosin ATPase or on actin motility in in vitro motility assays. This analysis considers only a single set of experimental conditions for which there is information on changes in [ Ca2+]i, changes in phosphatidylinositol turnover, contractile protein phosphorylation, tissue shortening velocity, and force. Because there is variability in the Ca2+force relationship, regulation of MLCK by phosphorylation and possibly regulation of phosphoprotein phosphatases, we cannot extend the analysis to other mediators of airway muscle tone or to agents used to produce bronchodilation. In summary, there has been significant recent progress in studies of contractile system regulation of airway smooth muscle. Regulation of myosin light chain kinase, myosin light chain phosphorylation, the phosphorylation sites and the functional effects of phosphorylation on stress development and cross-bridge cycling rates have been studied in some detail. We infer from these studies that there is an important role for a thin filament Ca2+-dependent regulatory mechanism. The potentially important thin filament proteins caldesmon and calponin are present in tracheal smooth muscle and preliminary studies indicate they are phosphorylated

REVIEW

during contraction. The use of intracellular Ca2+ indicators to estimate changes in [Ca2+]i and the development of several skinned fiber preparations have broadened the scope of physiological studies with airway smooth muscle and have suggested that the contractile element sensitivity to Ca2+ is not fixed but might be modulated by undefined messengers or excitation-contraction pathways. This adds an additional fascinating problem to the continuing effort to define the messengers and protein mediators that couple activation of membrane receptors to production of force by the contractile element. We gratefully acknowledge discussions of Jennifer Pohl, and Dr. Masatoshi Hori. This study was supported Blood Institute Grant HL-35805 Address for reprint requests: ogy, Howard Bldg., Reno, NV

the technical contributions and helpful Sandra McCallum, Dr. Hiroshi Ozaki, in part by National Heart, Lung, and and the Max Baer Heart Fund. W. T. Gerthoffer, Dept. of Pharmacol89557-0046.

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48. 49.

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56.

5% 58.

59.

60.

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