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N L Stephens

Publications and source records attributed to N L Stephens.

At least 19 recordsLinked to original sources
PubMed2005

Regulation of pulmonary arterial myosin phosphatase activity in neonatal circulatory transition and in hypoxic pulmonary hypertension: a role for CPI-17.

Neonatal circulatory transition is dependent upon tightly regulated pulmonary circuit relaxation. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by pulmonary arterial myocyte relaxation failure. We examined the effect of short course (72 hour) in vivo normobaric hypoxia in newborn swine on smooth muscle contractile enzyme activity and regulatory phosphoprotein abundance, in tissue homogenates of 2nd to 4th generation pulmonary arteries. Myosin light chain kinase (MLCK) and phosphatase (MLCP) protein contents were unchanged in hypoxic pulmonary arteries compared to controls. MLCP activity increased in normoxic animals from birth to day 3. This was ablated by hypoxia; phosphatase activity, measured as in vitro myosin light chain dephosphorylation, was decreased significantly (P < 0.005) in the hypoxic group. Inhibitory site phosphorylations of MLCP myosin binding subunit at threonines 696 and 850 were similar in both hypoxic and normoxic subjects, suggesting that downregulation of MLCP in hypoxia does not involve this pathway. However, content of regulatory protein CPI-17 (protein kinase C-related phosphatase inhibitor) increased from birth in hypoxic subjects (P < 0.05); active (phosphorylated) CPI-17 protein abundance declined after birth in normals, but increased in hypoxic arteries (P < 0.05). This corresponded with the decrease in phosphatase activity. We speculate that CPI-17 may play a role in myosin phosphatase upregulation during neonatal circulatory transition, and in hypoxic inhibition of pulmonary phosphatase activity in PPHN.

Animals↗
PubMed2005

Isotonic relaxation of control and sensitized airway smooth muscle.

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 +/- 0.92 s vs. 7.78 +/- 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 +/- 0.01 s (control) vs. 12.74 +/- 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.

Animals↗
PubMed2001

Airway smooth muscle.

The greatest impetus to research in elucidating the fundamental biophysics and biochemistry of airway smooth muscle (ASM) has undoubtedly been provided by the need to understand how these are altered in asthma. Many of the biophysical and biochemical properties of this muscle have been reviewed before (Stephens, 1970; Stephens, 1977; Mulvaney, 1979; Souhrada and Loader, 1979; Stephens and Kroeger, 1980). They resemble those of striated muscle; however, even though mechanical properties are very similar, there are differences in biochemistry. For example, in smooth muscle, calcium-sensitive regulation of contraction is mediated by a calmodulin/myosin-light-chain kinase/phosphatase system, not by the familiar troponin-tropomyosin system (Gorecka et al., 1974; Mrwa and Ruegg, 1975; Dillon et al., 1981; Aksoy et al., 1982). Thus, the molecular mechanisms to be investigated in understanding disorders of increased smooth muscle contraction, which occur in allergic bronchospasm (Souhrada and Dickey, 1976), for example, may be quite different from those in striated muscle. Much of the following material is based on studies of canine tracheal smooth muscle (TSM) because there is evidence (Jenne et al., 1975) that it serves as a model for ASM-at least with respect to contractility down to the sixth generation of airways. Studies of isolated smooth muscle from smaller airways (Russell, 1978) are few and are based mainly on studies of lung strips (Lulich et al., 1976). Since then, we have developed a bronchial smooth muscle preparation (fifth generation) that allows precise study of those airways that are involved in allergic bronchospasm. Considerable work has been carried out on ASM from a variety of animal models of asthma. It should be pointed out that none of these reproduces the human disease exactly, and that they really should be identified as examples of nonspecific hyperreactivity. Be that as it may, the nonspecificity found in human patients in vivo and in animals (Peterson et al., 1971; Hargreave et al., 1980) suggests that the primary cause of asthma may reside at the muscle cell level. Whether it is the cell membrane, the excitation-contraction coupling apparatus, or the contractile machinery that is primarily involved, is not yet known with certainty.

Animals↗
PubMed2000

Different ontogeny of rate of force generation and shortening velocity in guinea pig trachealis.

Juveniles of many species, including humans, display greater airway responsiveness than do adults. This may involve changes in airway smooth muscle function. In the present work we studied force production and shortening velocity in trachealis from 1-wk-old (1 wk), 3-wk-old (3 wk), and 3-mo-old (adult) guinea pigs. Strips were electrically stimulated (60 Hz, 18 V) at their optimal length (l(o)) to obtain maximum active stress (P(o)) and rate of stress generation. Then, force-velocity curves were elicited at 2.5 s from the onset of the stimulus. By applying a recently developed modification of Hill's equation for airway smooth muscle, the maximum shortening velocity at zero load (V(o)) and the value alpha. gamma/beta, an index of internal resistance to shortening (Rsi), were calculated (alpha, beta, and gamma are the constants of the equation). P(o) increased little with maturation, whereas the rate of stress generation increased significantly (0.40 +/- 0.03, 0.45 +/- 0.03, 0. 51 +/- 0.03 P(o)/s for 1 wk, 3 wk, and adult animals). V(o) slightly increased early with maturation to decrease significantly later (1. 79 +/- 0.67, 2.45 +/- 0.92, and 0.55 +/- 0.09 l(o)/s for 1 wk, 3 wk, and adult animals), whereas the Rsi showed an opposite trend (14.98 +/- 5.19, 8.99 +/- 3.01, and 32.07 +/- 5.54 mN. mm(-2). l(o)(-1). s for 1 wk, 3 wk, and adult animals). This early increase of force generation in combination with late increase of Rsi may explain the changes of V(o) with age. An elevated V(o) may contribute to the incidence of airway hyperresponsiveness in healthy juveniles.

Aging↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Actins↗
PubMed1998

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.

Animals↗
PubMed1998

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

The properties of delayed rectifier K+ current [IK(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. IK(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 IK(dr) of myocytes of ragweed pollen-sensitized dogs were not different from age-matched control dogs. However, the sensitization reduced the availability of IK(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 IK(dr) amplitude, whereas an inactive diacylglycerol analog had no effect. The hyperpolarizing shift in voltage dependence of inactivation and/or modulation of IK(dr) by PKC may be two mechanisms that contribute to the enhanced reactivity of bronchial tissues from ragweed pollen-sensitized dogs.

Animals↗
PubMed1998

The contractile apparatus of airway smooth muscle. Biophysics and biochemistry.

Qualitatively the mechanical, structural, and biochemical properties of airway smooth muscles resemble those of all other smooth muscle. However, one important distinguishing feature of airway smooth muscle is that the major portion of isotonic shortening is completed within the first 3 s in a muscle whose contraction is 10 s. This indicates the importance of focusing on the changes that occur in these 3 s and also the limiting role of the maximum velocity of shortening in determining shortening data. There is evidence that the maximum capacity and velocity of shortening in human bronchial smooth muscle from patients with asthma are significantly greater than those obtained from healthy siblings. In the demonstration in which cells in culture are arrested by withdrawing all fetal calf serum, the cells alter their phenotype to cells that are very long (more than 200 micrometers) and shorten twice as much as cells freshly isolated when the tissue is new. Speculatively, if such cells developed in vivo they could account for the increased contractility of asthmatic airway smooth muscle. These cultured cells could also be excellent models for study of airway smooth muscle contractility.

Animals↗
PubMed1997

Quantitative densitometry of proteins stained with coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software.

In the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 micrograms, were scanned using reflectance densitometry with 127 microns step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 micrograms vs. volume were fit by a second-order regression: Volume = -0.58 x protein load2 + 16.82 x protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 micrograms and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS-PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.

Actins↗
PubMed1997

The cytoskeleton and the extracellular matrix in sensitized canine tracheal smooth muscle.

To investigate the mechanisms responsible for the increased shortening capacity (delta Lmax) of airway smooth muscle in ragweed pollen sensitized dogs, the alterations of biophysical and biochemical properties of cytoskeleton and extracellular collagen in tracheal smooth muscle (TSM) were studied. Smooth muscle passive elastic properties were not significantly altered by removal of cytoskeleton with guanidine HCI plus 2-mercaptoethnol; collagenase digestion reduced smooth muscle force development, but did not affect its delta Lmax and passive elastic properties in both sensitized and control dogs. There were no significant differences in the amount of cytoskeletal intermediate filament proteins, desmin and vimentin between sensitized and control TSM. The content of total collagen, collagen type I, and collagen cross-linking in sensitized TSM were significantly greater than in control. Collagen fibres in sensitized TSM was more resistant to collagenase attack. We conclude that increased delta Lmax in sensitized canine TSM is not the result of alterations in passive cytoskeletal and extracellular collagen structures.

Allergens↗
PubMed1997

Characterization of molecular determinants of smooth muscle cell heterogeneity.

Broad diversity in contractile and pharmacological properties of different smooth muscles is well recognized. Differences in proliferative capacity, electrophysiology, phenotypic marker protein content, matrix synthesis, and expression of cell-specific transcription factors between individual smooth muscle cells (SMCs) have also been reported. Precise developmental and molecular mechanisms underlying heterogeneity are not known; however, their elucidation is the thrust of much current research involving vascular smooth muscle. In contrast, limited studies of heterogeneity of subtypes of airway SMCs are available. In this report, we review molecular aspects of differentiation that may determine phenotypic heterogeneity of SMCs and also present data from our own studies characterizing heterogeneity in the proliferative capacity and marker protein content of airway SMCs. Using flow cytometry, cell cycle transit was monitored for cultured canine tracheal SMCs. Only 70% of arrested cells responded and traversed the cell cycle when stimulated with 10% fetal bovine serum. Furthermore, heparin inhibited 40% of serum-responsive cells from entering the cell cycle, suggesting that both serum- and heparin-sensitive and -insensitive airway SMCs exist. Flow cytometric analysis of contractile protein and DNA content in freshly dissociated canine tracheal SMCs revealed that diploid (approximately 87%) and tetraploid (approximately 13%) populations exist. Clusters of SMCs having "high" or "low" smooth muscle myosin or alpha-actin content were also discerned, indicating that distinct subtypes of SMCs exist in mature airways. Diversity of SMCs may be a critical factor determining specific responses of smooth muscles to a number of physiological or pathophysiological stimuli that may include, for example, inflammatory mediators in asthmatic airways.

Animals↗
PubMed1997

Airway responsiveness in two inbred strains of mouse disparate in IgE and IL-4 production.

The mouse provides an excellent model for genetic studies of asthma, which is characterized by airway hyperexcitability and hyperreactivity. The former is a function of the properties of the membrane of the airway smooth muscle (ASM), whereas the latter is a function, albeit indirectly, of the mechanical properties of the muscle contractile apparatus. The very small size of the muscle has in the past hampered its study. We report herein that contractile properties of tracheal smooth muscle (TSM) can be measured in mice. We examined TSM strips from two inbred strains of mouse, ASW and SJL, which are high and low IgE responders, respectively. Force-velocity relationships were measured in four groups of mice, two ASW (control and sensitized)/and two SJL (control and sensitized). Muscle strips from sensitized SJL mice exhibited shortening velocities (V0) and maximum shortening capacities (deltaLmax), that were significantly greater than those of the other groups. However, no difference was found between the two strains in maximal isometric force (P0). The two strains also showed differences in their potential to express cytokines such as interleukin-4 (IL-4) and IL-5 in ex vivo splenocyte cultures, as measured by the cytokines' messenger RNA (mRNA) and protein expression. The SJL strain, which exhibited TSM hyperreactivity, was found to produce significantly greater amounts of IL-4 than the ASW strain. We conclude that the altered contractile properties of TSM in sensitized SJL mice are independent of IgE response, but linked to increased amounts of IL-4.

Animals↗
PubMed1997

Heterogeneity of airway smooth muscle at tissue and cellular levels.

Heterogeneity of smooth muscle behavior is rapidly emerging as an important phenomenon. However, it is only recently that investigators have begun to explore its physiological and pathogenic importance. Stenmark's group recently reported the existence of four subpopulations of smooth muscle cells in pulmonary artery, and noted that only a specific group of cells was likely responsible for the hyperplasia of smooth muscle in the hypertensive artery. Our studies reported here were focused on determining whether heterogeneity of contractile properties existed in airway smooth muscle cells. Firstly, mechanical properties of muscle strips, free of cartilage and epithelium, were compared among the different generations of canine airway from trachea down to bronchial generation 6. Two distinct groups of smooth muscle were detected from these airways: (i) an extrapulmonary group, consisting of muscle strips from the trachea, and bronchial generation 1 and 2, characterized by higher maximum shortening capacity (delta Lmax) and zero-load velocity (V0) of shortening; (ii) an intrapulmonary group, containing bronchi from generation 3 to 6, with lower delta Lmax and V0. This led to the implication that smooth muscles from these two groups consisted of different types of cells. Secondly, single smooth muscle cells were isolated from trachea and bronchus (from generation 3 to 6) enzymatically, and the unloaded shortening capacity of fresh isolated cells was measured employing electrical field stimulation at room temperature. Two types of cells were identified from these preparations based on the length of muscle cells: type I, with a mean length of 110 +/- 3 microns (SE), predominated in trachea (up to 84%); type II, with a mean length of 200 +/- 9 microns (SE), accounted for 58% in bronchus. The mechanical properties of these two types of cells were also different. Type I cells shortened by 28% of their original lengths in response to maximal stimulation, while most type II cells (90% of total type II cells) shortened 16% (type IIA), and the remainder shortened 58% (type IIB). Because of the paucity of information about the orientation and arrangement of these different types of smooth muscle cells in the airway tree, it is difficult to apply these data directly for interpretation of differences in mechanical properties of smooth muscle cells obtained from strips from extrapulmonary and intrapulmonary airways. Existence of great numbers of cells with lower shortening capacity in intrapulmonary airways may be the major factor responsible for lower mechanical performance of these airway smooth muscle strips. The relatively lower mechanical performance of intrapulmonary airway smooth muscle may be of importance in preventing excessive narrowing of these airways during muscle contraction and optimizing airflow.

Animals↗
PubMed1997

Velocity of translation of single actin filaments (AF) by myosin heads from antigen-sensitized airway smooth muscle.

We have previously reported increased velocity of shortening (Vo) in the sensitized airway (0.36 1o/s, +/- SE) smooth muscle compared to the control (0.26 1o/s, +/- 0.017 SE) and subsequent experiments indicated this was due to increased phosphorylation of the 20 kDa myosin light chain resulting from increased total myosin light chain kinase activity. The motility assay technique described by Kron and Spudich was employed to determine whether additionally the molecular motor (actomyosin crossbridge) itself was altered in airway smooth muscle by ragweed pollen sensitization. The motility assay measures the velocity of actin filament translation by myosin molecules. The negative results of the motility assay were valuable in determining that the pathogenesis of allergic bronchospasm is not at contractile protein level but at regulatory enzyme level.

Actins↗
PubMed1996

Increased response to antigen and histamine release in smaller sensitized canine bronchi.

We studied the Schultz-Dale response in vitro in large and small size branches from 3rd to 6th generation bronchi from ragweed-sensitized dogs. The response to electric field stimulation (EFS) increased after antigen from 65.56 +/- 8.11 to 78.6 +/- 9.0 mN/mm2 of smooth muscle (P < 0.01), but no topographical difference was observed. The response to ragweed (% of the response to EFS) was 158.3 +/- 12 and 67.1 +/- 11.7 in strips from small and large branches respectively (P < 0.01), while no difference was observed between generations; when clustering bronchi according to dimension, it was 129.9 +/- 13.4 in small and 71.9 +/- 19.8 in large bronchi (P < 0.01). Histamine released from small and large branches was 2.90 +/- 1.01 and 0.76 +/- 0.20 (ng/mg of tissue) respectively (P < 0.05); no difference was found between generations. In conclusion, in sensitized dogs a greater response to antigen, which involves a higher histamine release, occurs in small compared to large bronchi. We suggest that control of distribution of ventilation occurs mainly at small bronchi level, which becomes the elective tissue to study the Schultz-Dale response. Finally, the classification of bronchi into generations is inadequate to study allergic bronchospasm.

Allergens↗