Newman L. Stephens

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells

Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.Chronic asthma is characterized by hypertrophy and hyperplasia of airway smooth muscle cells (SMC) that limit airflow by a geometric effect. Whether contractility of airway SMC is altered is not clear. Cultured cells were used as a model of hyperplasia. Phenotypic changes seen indicated conversion to a synthetic, weakly contractile type. At confluence, although limited reversal of protein changes was seen, no restoration in contractility occurred. Phenotypic modulation of postconfluent cultured airway SMC under prolonged serum deprivation (arrested cells) is reported here. Two phenotypically distinct groups of cells were identified in primary airway SMC cultures: 1) elongated spindle-shaped cells, which expressed large amounts of smooth muscle contractile and regulatory proteins, and 2) flat and stellate cells, which expressed very little. The first group showed a surprising shortening capacity and a velocity that was even greater than that of the freshly isolated cells, whereas the second group became spherical and noncontractile. Even more surprising was that the myosin heavy chain (MHC) isoform (SM-B) generally said to be associated with the higher shortening velocity disappeared from the cell, while the content of the key rate-limiting regulating enzyme, myosin light chain kinase (MLCK), increased 30-fold. We conclude that a functional, contractile phenotype of airway SMC can be obtained by prolonged serum deprivation. We speculate that the increased contractility could be the result of increased phosphorylation of the 20-kDa myosin light chain resulting from increased content of smooth muscle MLCK rather than any increase in endogenous MHC ATPase activity. This model may be useful for study of SMC differentiation and contraction.

Sympathetic stimulation and hypertension in the pyridoxine-deficient adult rat.

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as gamma-aminobutyric acid, serotonin, dopamine, and norepinephrine. Adult male Sprague-Dawley rats placed on a pyridoxine-deficient diet for 8 weeks showed significant hypertension compared with pyridoxine-supplemented controls. Hypothalamic contents of pyridoxal phosphate, gamma-aminobutyric acid, and serotonin in the pyridoxine-deficient rats were significantly lower than those in pyridoxine-supplemented controls. Hypertension was associated with sympathetic stimulation. Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and gamma-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels. Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine-deficient rats. These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Calcium and smooth muscle contraction

The fact that smooth muscle exists in almost every hollow organ and is involved in a large number of disease states has led to a vast increase in smooth muscle research, covering areas from testing response to antagonists and agonists to measuring the molecular force generated by a single actin filament. Yet, the exact mechanisms regulating contractile response of smooth muscle remain unsolved. Calcium has been a central player in mediating smooth muscle contraction through binding with calmodulin, although there is evidence showing that under special circumstances smooth muscle can contract without change in intracellular Ca2+. In addition to the major regulatory pathway of Ca2+-calmodulin-mysoin light chain kinase, there are other thin filament linked regulatory mechanisms in which Ca2+-calmodulin dependent phosphorylation of calponin and caldesmon may be involved. Ca2+ sensitivity of smooth muscle contraction may vary under different situations and this has recently been recognized as an important regulatory mechanism. Examples are protein kinase C (PKC) dependent phosphorylation of myosin light chain kinase which results in partial inhibition of contraction, and activation of myosin light chain phosphatase. There is new evidence howing that not only does Ca2+ regulate contraction by regulating the interaction of contractile proteins in smooth muscle, but also that shortening of smooth muscle itself reduces intracellular Ca2+ concentration, via a negative feedback.

Increased Ca2+ and myosin phosphorylation, but not calmodulin activity in sensitized airway smooth muscles

The increased shortening velocity and capacity of airway smooth muscle (ASM) from ragweed pollen-sensitized dogs, which may be responsible for its in vivo airway hyperresponsiveness, have been shown to be associated with higher actomyosin adenosinetriphosphatase activity and greater level of phosphorylation of the 20-kDa myosin light-chain (MLC20) at rest and during contraction. Current studies show that the elevated level of phosphorylation may be the result of an increased myosin light-chain kinase (MLCK) activity due to excessive quantity of MLCK. There were no significant changes in total activity of calmodulin, a protein that binds and activates MLCK, in sensitized dog ASM (SASM) compared with control ASM (CASM). When normalized to the relative calmodulin content in the tissues, the specific calmodulin activities (means +/- SE) in sensitized tracheal smooth muscle (STSM) and sensitized bronchial smooth muscle (SBSM) and in their controls were not different (STSM 0.359 +/- 0.117, CTSM 0.339 +/- 0.136. SBSM 0.201 +/- 0.098, and control bronchial smooth muscle 0.213 +/- 0.056 nmol Pi.calmodulin content-1.min-1, respectively). Intracellular Ca2+ levels indicated by fura 2 fluorescent dye remained unaltered in SASM. We conclude that airway hyperresponsiveness may result from higher MLCK content in SASM rather than from changes in Ca(2+)-calmodulin activities, which is an example of alteration in Ca2- sensitivity of ASM.

The biophysics of asthmatic airway smooth muscle.

It is clear that significant advances have been made in the understanding of the physiology, biochemistry and molecular biology of airway smooth muscle (ASM) contraction and how the knowledge obtained from these approaches may be used to elucidate the pathogenesis of asthma. Not to belittle other theories of smooth muscle contraction extant in the field, perhaps the most outstanding development has been the formulation of plasticity theory. This may radically alter our understanding of smooth muscle contraction. Its message is that while shortening velocity and capacity are linear functions of length, active force is length independent. These changes are explained by the ability of thick filament protein to depolymerize at short lengths and to increase numbers of contractile units in series at lengths greater than optimal length or L(ref). Other advances are represented by the report that the major part of ASM shortening is complete within the initial first 20% of contraction time, that the nature and history of loading determine the extent of shortening and that these findings can be explained by the finding that the crossbridges are cycling four times faster than in the remaining time. Another unexpected finding is that late in the course of isotonic relaxation the muscle undergoes spontaneous activation which delays relaxation and smoothes it out; speculatively this could minimize turbulence of airflow. On the applied front evidence now shows the shortening ability of bronchial smooth muscle of human subjects of asthma is significantly increased. Measurements also indicate that increased smooth muscle myosin light chain kinase content, via increased actomyosin ATPase activity could be responsible for the changes in contractility.

Fetal ductus arteriosus ligation. Pulmonary vascular smooth muscle biochemical and mechanical changes.

To evaluate the smooth muscle mechanical and biochemical changes associated with persistent pulmonary hypertension syndrome of the newborn, we studied 31 fetal sheep in which the ductus arteriosus was ligated at 125 days of gestation. Sixty-one noninstrumented and six sham-operated fetuses served as controls. All animals were delivered by cesarean section at 137-140 days of gestation, and the experimental group had the ductus arteriosus ligated for 12 +/- 3 days. The ligated group demonstrated a higher mean (+/- SEM) pulmonary artery pressure (72.3 +/- 3.8 versus 54.1 +/- 2 mm Hg, p < 0.01) and right ventricular mean free wall weight (12.5 +/- 0.7 versus 6.8 +/- 0.3 g, p < 0.01) as compared with the sham-operated group. Significant changes in the pulmonary vascular smooth muscle of the ligated group were observed. The myosin content of vessels from the second through fifth generation demonstrated a significant increase in actin and myosin content (p < 0.01), but given their disproportional changes, the noninstrumented group demonstrated a lower actin/myosin ratio than the experimental group (p < 0.01). Changes in the myosin heavy chain isoform stoichiometry, characterized by an increase in both the mean high/low myosin heavy chain isoform ratio (1.8 +/- 0.3 versus 1.0 +/- 0.1, p < 0.05) and the nonmuscle isoform as a percentage of the total myosin heavy chain (12.4 +/- 0.7% versus 2.7 +/- 0.9%, p < 0.01), were also observed in the ligated as compared with the noninstrumented animals. In addition, the muscle Mg-ATPase activity was significantly (p < 0.05) reduced in the experimental group.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition

Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1 in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1 in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.

Airway smooth muscle contractile, regulatory and cytoskeletal protein expression in health and disease

The major part of research dealing with the biophysical and biochemical properties of airway smooth muscle is based on the assumption that the cells constituting the tissue are homogenous. For striated muscle this has been shown untenable. In recent years almost every property of vascular smooth muscle has been also demonstrated to be heterogeneous. This realization has been late in arriving on the airway smooth muscle research scene. Our own studies have shown that mechanical properties are, in quantitative terms, heterogeneously distributed down the airways and that contractility, for example, in extrapulmonary and intrapulmonary airways differs markedly. Another indication of heterogeneity is derived from studies of the biochemical properties of airway smooth muscle cells (ASMCs) in culture. Dramatic changes in phenotype expression were found with days in culture. Just after isolation from the tissue, the cells were of contractile type and contained mature isoforms of contractile, regulatory and cytoskeletal proteins. After the fourth day in culture the cellular phenotype changed such that contractile filaments diminished rapidly with smooth muscle isoforms being replaced by non-muscle isoforms. The cell assumed secretory or synthetic properties and commenced proliferating rapidly. It is possible that similar changes in phenotype could occur in vivo in cells undergoing hypertrophy or hyperplasia. Thus, a thickened medial layer of the type seen in the walls of airways from asthmatic airways is not necessarily one endowed with increased contractility and, in fact, the latter may be subnormal. Finally, using the so-called motility assay, we studied the velocity of translation of actin filaments by myosin molecules obtained from antigen-sensitized and control airway smooth muscle. We found no change in maximum velocity of actin translation. This was under conditions where the myosin light chain (MLC) was fully phosphorylated. However, in these tissues we found heterogeneity in myosin light chain kinase (MLCK) content which, we inferred, accounted for the difference in shortening velocity between control and sensitized muscle strips in vitro.

Delayed rectifier K+ current of dog bronchial myocytes: effect of pollen sensitization and PKC activation

The properties of delayed rectifier K+ current [ I K(dr)] of canine airway smooth muscle cells isolated from small bronchi and its modulation by protein kinase C (PKC) were studied by whole cell patch clamp. I K(dr) activated positive to -40 mV, with half-maximal activation at -16 ± 1.2 mV ( n = 15) and average current density of 31 ± 2.6 pA/pF ( n = 15) at +30 mV. The capacitive surface area, current density, and voltage dependence of activation of I K(dr) of myocytes of ragweed pollen-sensitized dogs were not different from age-matched control dogs. However, the sensitization reduced the availability of I K(dr) between -40 and -20 mV due to a hyperpolarizing shift in the voltage dependence of steady-state inactivation (-29.9 ± 1.2 in sensitized versus -26.0 ± 0.7 mV in control dogs, n = 9 and 11, respectively; P < 0.05). PKC activation with diacylglycerol analog or phorbol ester depressed I K(dr) amplitude, whereas an inactive diacylglycerol analog had no effect. The hyperpolarizing shift in voltage dependence of inactivation and/or modulation of I K(dr) by PKC may be two mechanisms that contribute to the enhanced reactivity of bronchial tissues from ragweed pollen-sensitized dogs.