Perspective Airway Smooth Muscle and Asthma Primal de Lanerolle Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois
"Asthma morbidity and mortality are on the rise. From 1980 to 1987, the prevalence rate of asthma in the United States increased 29 percent, and death rates for asthma as the firstlisted diagnosis increased 31 percent. In 1988, asthmarelated health care expenditures exceeded $4 billion in the United States" (1). Nevertheless, apart from understanding that bronchodilators are effective in treating asthmatic symptoms, we know little about the underlying mechanisms at the molecular level in asthma. One reason for this is that asthma is a syndrome that involves multiple tissues (e.g., mast cells, neutrophils, epithelial cells, smooth muscles) and the interactions between these tissues. In addition, a number of hormones, nerves, and neurotransmitters can affect these cells and their interactions. In light of the central role of airway smooth muscle in asthma, a great deal of research has focused on understanding the molecular basis of the regulation of smooth muscle contraction and whether there are alterations in these regulatory mechanisms in asthma. Intense research efforts over the past 15 years have clearly demonstrated that the regulation of smooth muscle contraction involves the phosphorylation of the 20 kD light chain subunit of smooth muscle myosin (MLC 20 ) . Biochemical experiments 'have demonstrated that the transfer of the terminal phosphate group from ATP to ser 19 on MLC 20 , with the subsequent formation of a phosphoester bond, results in the stimulation of the actin-activated ATPase activity of smooth muscle myosin (2). Other studies have shown that the enzyme catalyzing this phosphorylation reaction, myosin light chain kinase (MLCK), is a calciumcalmodulin-dependent enzyme (2). Based on these studies, the following sequence of events was postulated to occur during smooth muscle contraction: (1) an increase in intracellular calcium results in the binding of calcium to calmodulin; (2) the calcium-calmodulin complex then binds to and activates MLCK; (3) the active enzyme phosphorylates MLC 20 ; (4) the phosphorylated myosin interacts with actin to hydrolyze ATP; (5) the energy released following the hydrolysis of ATP is used to move
(Received for publication October 15, 1992) Address correspondence to: Primal de Lanerolle, Ph.D., Department of Physiology and Biophysics, University of Illinois (M/C 901), P.o. Box 6998, Chicago, IL 60680. Abbreviations: cyclic adenosine monophosphate, cAMP; myosin light chain kinase, MLCK; 20 leD light chain subunit of smooth muscle myosin, MLC 20 • Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 565-566, 1992
thick and thin filaments past each other, which results in contraction as described by the sliding filament model; (6) relaxation results from dephosphorylation of the MLC 20 by phosphoprotein phosphatases following a decrease in intracellular calcium and the inactivation ofMLCK. Many physiologic experiments using airway and other smooth muscles have been performed to test this hypothesis and, by and large, these experiments have validated the central points of this hypothesis (see reference 3 for a more complete discussion of this subject). The heart of this hypothesis is that changes in MLCK activity result in changes in the contractile properties of smooth muscles and this is where muscle biochemistry and asthma research intersect. If an increase in MLCK activity results in smooth muscle contraction, is it possible that a chronic increase in MLCK activity and/or improper activation of MLCK and/or changes in the enzymatic characteristics of the myosin molecule could be responsible for the airways hyperreactivity seen in asthma? The article by Jiang and colleagues in this issue of the journal (4) addresses the first possibility. These investigators have developed a canine ragweed pollen-sensitized model of airways hyperresponsiveness. A previous paper from this laboratory reported an increase in actomyosin ATPase activity in airway muscle from sensitized dogs compared with control (nonsensitized) dogs (5). In the present study, they have investigated the mechanism for this increase. They now report a significant increase in MLC 20 phosphorylation in the bronchial smooth muscle in sensitized dogs compared with control both at rest and during electrical stimulation. Since this increase in MLC 20 phosphorylation could be due to an increase in MLCK activity or a decrease in phosphoprotein phosphatase activity, they also investigated whether there are changes in the levels of the activities of these enzymes. Their conclusion from these studies is that there is a significant increase in MLCK activity with no apparent change in MLC20 dephosphorylating activity. Thus, these studies for the first time present data suggesting that an increase in MLCK activity and MLC 20 phosphorylation are directly involved in airways hyperresponsiveness. MLCK could also be indirectly involved in airway hyperresponsiveness. It has been known for many years that increases in cyclic adenosine monophosphate (cAMP) are correlated with airway muscle relaxation (3) and it has been suggested that the airway manifestations of asthma are related to a lack of responsiveness to cAMP (6). There are two ways in which cAMP could affect MLCK activity. First, cAMP has been postulated to decrease intracellular calcium
566
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
levels (7), which would result in a net reduction in MLCK activity, lower levels of MLC 20 phosphorylation, and less force. The second possibility is based on the observation that phosphorylation by cAMP-dependent protein kinase decreases MLCK activity (8). Phosphorylation ofMLCK at two sites decreases the affinity of MLCK for the activating calcium/calmodulin complex, thereby decreasing the number of active MLCK molecules at a given submaximal intracellular calcium concentration (3). Thus, prior stimulation with agents that increase cAMP levels would result in slower force generation and less total force following stimulation with a contractile agonist (3). In order to test this hypothesis, we have immunoprecipitated MLCK from tracheal smooth muscle and found an increase in MLCK phosphorylation only in the presence of an increase in intracellular cAMP levels (9). In addition, we have demonstrated that pretreatment of tracheal muscle with isoproterenol results in a slower rate of MLC 20 phosphorylation and force generation following the addition of carbachol than in muscles not treated with isoproterenol (10). Although these studies are highly suggestive that MLCK phosphorylation could playa role in modulating the contractile properties of smooth muscles, many studies are required to establish this relationship. These include establishing that intracellular MLCK activity is decreased following an increase in cAMP levels and verifying that the proper sites on MLCK are phosphorylated following an increase in cAMP levels. In conclusion, studies from a number of different groups have suggested plausible mechanisms by which changes in the contractile apparatus in airway smooth muscle could result in the types of changes seen in asthma. Interestingly, changes in MLCK activity, either through an increase in MLCK protein or changes in MLCK activity due to phosphorylation, are at the heart of both mechanisms. Perhaps the most pressing need at the moment is to follow up these studies with others on tissues from asthmatic animals and humans. For instance, it would be very interesting to know whether airway muscle from asthmatics expresses more MLCK mRNA or protein than from nonasthmatics using Northern and Western blot analyses. It is also important to determine whether there are differences in the primary structures of MLCK and myosin subunits in asthmatics
versus nonasthmatics. It has been shown recently that hypertrophic cardiac myopathy results from mutations in the myosin heavy chain (11). Although it is too much to hope that a single mutation will explain airway muscle hyperreactivity or hypertrophy, we should not dismiss the possibility of a genetic basis for asthma out of hand. Moreover, PCR technology makes it possible to study small pieces of tissue, such as those obtained at surgery. Ultimately, extending the types of studies described by Jiang and colleagues (4) to human tissue and combining them with molecular biology techniques should greatly increase our understanding of the molecular basis of asthma. Acknowledgments: This work was supported in part by Grants HL35808 and HL024 I I from the National Institutes of Health. Dr. de Lanerolle is the recipient of the Florence and Arthur Brock Career Investigator Award from the Chicago Lung Association.
References 1. Lenfant, C. 1991. Foreword. In Guidelines for the Diagnosis and Management of Asthma. U.S. Public Health Service Publication #91-3042. 2. Hartshorne, D. J. 1987. Biochemistry of the contractile process in smooth muscle. In Physiology of the Gastrointestinal Tract. 2nd ed. L. R. Johnson, editor. Raven Press, New York. 423-482. 3. de Lanerolle, P., and R. J. Paul. 1991. Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am. J. Physiol. 261(Lung Cell. Mol. Physiol. 5):Ll-Ll4. 4. Jiang, H., K. Rao, A. J. Halayko, X. Liu, and N. L. Stephens. 1992. Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle. Am. J. Respire Cell Mol. Bioi. 7: 567-573. 5. Kong, S. K., R. P. C. Shiu, and N. L. Stephens. 1986. Studies of myofibrillar ATPase in ragweed sensitized canine pulmonary smooth muscle. J. Appl. Physiol. 60:92-96. 6. Szentivanyi, A. 1968. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. Allergy 42:203-232. 7. Van Breeman, c., and K. Saida. 1989. Cellular mechanisms regulating [Ca 2 +] , smooth muscle. Annu. Rev. Physiol. 51:315-329. 8. Adelstein, R. S., M. A. Conti, D. R. Hathaway, and C. B. Klee. 1978. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase. J. Bioi. Chem. 253:8347-8350. 9. de Lanerolle, P., M. Nishikawa, D. A. Yost, and R. S. Adelstein. 1984. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 223:1415-1417. 10. Obara, K., and P. de Lanerolle. 1989. Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle. J. Appl. Physiol. 66: 2017-2022. II. Tanigawa, G., 1. A. Jarcho, S. Kass et al. 1990. A molecular basis for familial hypertrophic cardiomyopathy: an od{3 cardiac myosin heavy chain hybrid gene. Cell 62:991-998.
"Asthma morbidity and mortality are on the rise. From 1980 to 1987, the prevalence rate of asthma in the United States increased 29 percent, and death rates for asthma as the firstlisted diagnosis increased 31 percent. In 1988, asthmarelated health care expenditures exceeded $4 billion in the United States" (1). Nevertheless, apart from understanding that bronchodilators are effective in treating asthmatic symptoms, we know little about the underlying mechanisms at the molecular level in asthma. One reason for this is that asthma is a syndrome that involves multiple tissues (e.g., mast cells, neutrophils, epithelial cells, smooth muscles) and the interactions between these tissues. In addition, a number of hormones, nerves, and neurotransmitters can affect these cells and their interactions. In light of the central role of airway smooth muscle in asthma, a great deal of research has focused on understanding the molecular basis of the regulation of smooth muscle contraction and whether there are alterations in these regulatory mechanisms in asthma. Intense research efforts over the past 15 years have clearly demonstrated that the regulation of smooth muscle contraction involves the phosphorylation of the 20 kD light chain subunit of smooth muscle myosin (MLC 20 ) . Biochemical experiments 'have demonstrated that the transfer of the terminal phosphate group from ATP to ser 19 on MLC 20 , with the subsequent formation of a phosphoester bond, results in the stimulation of the actin-activated ATPase activity of smooth muscle myosin (2). Other studies have shown that the enzyme catalyzing this phosphorylation reaction, myosin light chain kinase (MLCK), is a calciumcalmodulin-dependent enzyme (2). Based on these studies, the following sequence of events was postulated to occur during smooth muscle contraction: (1) an increase in intracellular calcium results in the binding of calcium to calmodulin; (2) the calcium-calmodulin complex then binds to and activates MLCK; (3) the active enzyme phosphorylates MLC 20 ; (4) the phosphorylated myosin interacts with actin to hydrolyze ATP; (5) the energy released following the hydrolysis of ATP is used to move
(Received for publication October 15, 1992) Address correspondence to: Primal de Lanerolle, Ph.D., Department of Physiology and Biophysics, University of Illinois (M/C 901), P.o. Box 6998, Chicago, IL 60680. Abbreviations: cyclic adenosine monophosphate, cAMP; myosin light chain kinase, MLCK; 20 leD light chain subunit of smooth muscle myosin, MLC 20 • Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 565-566, 1992
thick and thin filaments past each other, which results in contraction as described by the sliding filament model; (6) relaxation results from dephosphorylation of the MLC 20 by phosphoprotein phosphatases following a decrease in intracellular calcium and the inactivation ofMLCK. Many physiologic experiments using airway and other smooth muscles have been performed to test this hypothesis and, by and large, these experiments have validated the central points of this hypothesis (see reference 3 for a more complete discussion of this subject). The heart of this hypothesis is that changes in MLCK activity result in changes in the contractile properties of smooth muscles and this is where muscle biochemistry and asthma research intersect. If an increase in MLCK activity results in smooth muscle contraction, is it possible that a chronic increase in MLCK activity and/or improper activation of MLCK and/or changes in the enzymatic characteristics of the myosin molecule could be responsible for the airways hyperreactivity seen in asthma? The article by Jiang and colleagues in this issue of the journal (4) addresses the first possibility. These investigators have developed a canine ragweed pollen-sensitized model of airways hyperresponsiveness. A previous paper from this laboratory reported an increase in actomyosin ATPase activity in airway muscle from sensitized dogs compared with control (nonsensitized) dogs (5). In the present study, they have investigated the mechanism for this increase. They now report a significant increase in MLC 20 phosphorylation in the bronchial smooth muscle in sensitized dogs compared with control both at rest and during electrical stimulation. Since this increase in MLC 20 phosphorylation could be due to an increase in MLCK activity or a decrease in phosphoprotein phosphatase activity, they also investigated whether there are changes in the levels of the activities of these enzymes. Their conclusion from these studies is that there is a significant increase in MLCK activity with no apparent change in MLC20 dephosphorylating activity. Thus, these studies for the first time present data suggesting that an increase in MLCK activity and MLC 20 phosphorylation are directly involved in airways hyperresponsiveness. MLCK could also be indirectly involved in airway hyperresponsiveness. It has been known for many years that increases in cyclic adenosine monophosphate (cAMP) are correlated with airway muscle relaxation (3) and it has been suggested that the airway manifestations of asthma are related to a lack of responsiveness to cAMP (6). There are two ways in which cAMP could affect MLCK activity. First, cAMP has been postulated to decrease intracellular calcium
566
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
levels (7), which would result in a net reduction in MLCK activity, lower levels of MLC 20 phosphorylation, and less force. The second possibility is based on the observation that phosphorylation by cAMP-dependent protein kinase decreases MLCK activity (8). Phosphorylation ofMLCK at two sites decreases the affinity of MLCK for the activating calcium/calmodulin complex, thereby decreasing the number of active MLCK molecules at a given submaximal intracellular calcium concentration (3). Thus, prior stimulation with agents that increase cAMP levels would result in slower force generation and less total force following stimulation with a contractile agonist (3). In order to test this hypothesis, we have immunoprecipitated MLCK from tracheal smooth muscle and found an increase in MLCK phosphorylation only in the presence of an increase in intracellular cAMP levels (9). In addition, we have demonstrated that pretreatment of tracheal muscle with isoproterenol results in a slower rate of MLC 20 phosphorylation and force generation following the addition of carbachol than in muscles not treated with isoproterenol (10). Although these studies are highly suggestive that MLCK phosphorylation could playa role in modulating the contractile properties of smooth muscles, many studies are required to establish this relationship. These include establishing that intracellular MLCK activity is decreased following an increase in cAMP levels and verifying that the proper sites on MLCK are phosphorylated following an increase in cAMP levels. In conclusion, studies from a number of different groups have suggested plausible mechanisms by which changes in the contractile apparatus in airway smooth muscle could result in the types of changes seen in asthma. Interestingly, changes in MLCK activity, either through an increase in MLCK protein or changes in MLCK activity due to phosphorylation, are at the heart of both mechanisms. Perhaps the most pressing need at the moment is to follow up these studies with others on tissues from asthmatic animals and humans. For instance, it would be very interesting to know whether airway muscle from asthmatics expresses more MLCK mRNA or protein than from nonasthmatics using Northern and Western blot analyses. It is also important to determine whether there are differences in the primary structures of MLCK and myosin subunits in asthmatics
versus nonasthmatics. It has been shown recently that hypertrophic cardiac myopathy results from mutations in the myosin heavy chain (11). Although it is too much to hope that a single mutation will explain airway muscle hyperreactivity or hypertrophy, we should not dismiss the possibility of a genetic basis for asthma out of hand. Moreover, PCR technology makes it possible to study small pieces of tissue, such as those obtained at surgery. Ultimately, extending the types of studies described by Jiang and colleagues (4) to human tissue and combining them with molecular biology techniques should greatly increase our understanding of the molecular basis of asthma. Acknowledgments: This work was supported in part by Grants HL35808 and HL024 I I from the National Institutes of Health. Dr. de Lanerolle is the recipient of the Florence and Arthur Brock Career Investigator Award from the Chicago Lung Association.
References 1. Lenfant, C. 1991. Foreword. In Guidelines for the Diagnosis and Management of Asthma. U.S. Public Health Service Publication #91-3042. 2. Hartshorne, D. J. 1987. Biochemistry of the contractile process in smooth muscle. In Physiology of the Gastrointestinal Tract. 2nd ed. L. R. Johnson, editor. Raven Press, New York. 423-482. 3. de Lanerolle, P., and R. J. Paul. 1991. Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am. J. Physiol. 261(Lung Cell. Mol. Physiol. 5):Ll-Ll4. 4. Jiang, H., K. Rao, A. J. Halayko, X. Liu, and N. L. Stephens. 1992. Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle. Am. J. Respire Cell Mol. Bioi. 7: 567-573. 5. Kong, S. K., R. P. C. Shiu, and N. L. Stephens. 1986. Studies of myofibrillar ATPase in ragweed sensitized canine pulmonary smooth muscle. J. Appl. Physiol. 60:92-96. 6. Szentivanyi, A. 1968. The beta adrenergic theory of the atopic abnormality in bronchial asthma. J. Allergy 42:203-232. 7. Van Breeman, c., and K. Saida. 1989. Cellular mechanisms regulating [Ca 2 +] , smooth muscle. Annu. Rev. Physiol. 51:315-329. 8. Adelstein, R. S., M. A. Conti, D. R. Hathaway, and C. B. Klee. 1978. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase. J. Bioi. Chem. 253:8347-8350. 9. de Lanerolle, P., M. Nishikawa, D. A. Yost, and R. S. Adelstein. 1984. Increased phosphorylation of myosin light chain kinase after an increase in cyclic AMP in intact smooth muscle. Science 223:1415-1417. 10. Obara, K., and P. de Lanerolle. 1989. Isoproterenol attenuates myosin phosphorylation and contraction of tracheal muscle. J. Appl. Physiol. 66: 2017-2022. II. Tanigawa, G., 1. A. Jarcho, S. Kass et al. 1990. A molecular basis for familial hypertrophic cardiomyopathy: an od{3 cardiac myosin heavy chain hybrid gene. Cell 62:991-998.
