Porcine Airway Smooth Muscle Research Articles

Stiffness Mediated-Mechanosensation of Airway Smooth Muscle Cells on Linear Stiffness Gradient Hydrogels.

In obstructive airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), the extracellular matrix (ECM) protein amount and composition of the airway smooth muscle (ASM) is often remodelled, likely altering tissue stiffness. The underlying mechanism of how human ASM cell (hASMC) mechanosenses the aberrant microenvironment is not well understood. Physiological stiffnesses of the ASM were measured by uniaxial compression tester using porcine ASM layers under 0, 5 and 10% longitudinal stretch above in situ length. Linear stiffness gradient hydrogels (230 kPa range) were fabricated and functionalized with ECM proteins, collagen I (ColI), fibronectin (Fn) and laminin (Ln), to recapitulate the above-measured range of stiffnesses. Overall, hASMC mechanosensation exhibited a clear correlation with the underlying hydrogel stiffness. Cell size, nuclear size and contractile marker alpha-smooth muscle actin (αSMA) expression showed a strong correlation to substrate stiffness. Mechanosensation, assessed by Lamin-A intensity and nuc/cyto YAP, exhibited stiffness-mediated behaviour only on ColI and Fn-coated hydrogels. Inhibition studies using blebbistatin or Y27632 attenuated most mechanotransduction-derived cell morphological responses, αSMA and Lamin-A expression and nuc/cyto YAP (blebbistatin only). This study highlights the interplay and complexities between stiffness and ECM protein type on hASMC mechanosensation, relevant to airway remodelling in obstructive airway diseases.

Superoxide Anions Inhibit Intracellular Calcium Response in Porcine Airway Smooth Muscle Cells.

Background Superoxide anions (O 2 - ) have multiple effects on pulmonary parenchyma altering cell proliferation, cellular metabolism, and airway smooth muscle (ASM) contraction. Intracellular calcium ([Ca 2+ ] i ) concentration plays a significant role in the regulation of ASM contraction, relaxation, proliferation, and gene expression. Objective We investigated the effects of O 2 - on agonist-stimulated changes in [Ca 2+ ] i in ASM cells. Design/Methods Fura-2 AM-loaded, freshly isolated porcine ASM (PASM) cells were used to examine [Ca 2+ ] i release in response to acetylcholine (ACh), histamine, endothelin, caffeine, and thapsigargin (TPG) in the presence or absence of extracellular Ca 2+ . Results Exposure of PASM cells to xanthine and xanthine oxidase (X + XO) resulted in a time-dependent generation of O 2 - , inhibited by superoxide dismutase (SOD). Preincubating PASM cells with X + XO for 15- or 45-minute inhibited net [Ca 2+ ] i responses to ACh, histamine, caffeine, and TPG compared with control cells. Pretreating PASM cells with SOD for 30 minutes mitigated the inhibitory effect of X + XO treatment on ACh-induced Ca 2+ elevation suggesting role of O 2 - . X + XO treatment also inhibited caffeine- and TPG-induced Ca 2+ elevation suggesting effect of O 2 - on [Ca 2+ ] i release and reuptake mechanisms. Conclusion Superoxide attenuates [Ca 2+ ] i release, reuptake, and may interfere with physiological functions of ASM cells.

Inhibiting Actin Polymerization Increases Tension Cost in Porcine Airway Smooth Muscle

The polymerization of globular to filamentous actin in smooth muscle occurs upon activation and is associated with cytoskeletal remodeling. Remodeling of the cortical cytoskeleton in this manner allows for tethering of contractile proteins and force transmission to the extracellular matrix. Muscle contraction involves ATP hydrolysis and cross‐bridge cycling, which are both sensitive to internal load. During isometric activation of smooth muscle, ATP hydrolysis rate peaks initially but decreases with time, reaching a steady‐state, while maintaining maximum force. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle contraction. We hypothesized that the time‐dependent decrease in tension cost and ATP hydrolysis are associated with actin polymerization and a subsequent increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, ATP hydrolysis rate and isometric force were measured simultaneously after activation with maximum Ca2+ (pCa 4.0). Two strips were tested from each animal. One strip was treated with Cytochalasin‐D (Cyto‐D; 1 μM for 30 min), an inhibitor of actin polymerization, and one was an untreated, time‐matched control. We found that peak and steady‐state isometric force decreased by ~40%, and peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively, following Cyto‐D treatment. Thus, tension cost increased by ~100% and ~60% at peak and steady‐state, respectively. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate relates to an increase in internal load on cross‐bridges via actin polymerization. Inhibition of actin polymerization induced by Cyto‐D in smooth muscle impairs contractile protein tethering and therefore reduces internal loading, resulting in increases in tension cost, ATP hydrolysis rate and faster cross‐bridge cycling.

Cytoskeletal remodeling slows cross-bridge cycling and ATP hydrolysis rates in airway smooth muscle.

During isometric activation of airway smooth muscle (ASM), cross‐bridge cycling and ATP hydrolysis rates decline across time even though isometric force is sustained. Thus, tension cost (i.e., ATP hydrolysis rate per unit of force during activation) decreases with time. The “latch‐state” hypothesis attributes the dynamic change in cross‐bridge cycling and ATP hydrolysis rates to changes in phosphorylation of the regulatory myosin light chain (rMLC20). However, we previously showed that in ASM, the extent of rMLC20 phosphorylation remains unchanged during sustained isometric force. As an alternative, we hypothesized that cytoskeletal remodeling within ASM cells results in increased internal loading of contractile proteins that slows cross‐bridge cycling and ATP hydrolysis rates. To test this hypothesis, we simultaneously measured isometric force and ATP hydrolysis rate in permeabilized porcine ASM strips activated by Ca2+ (pCa 4.0). The extent of rMLC20 phosphorylation remained unchanged during isometric activation, even though ATP hydrolysis rate (tension cost) declined with time. The effect of cytoskeletal remodeling was assessed by inhibiting actin polymerization using Cytochalasin D (Cyto‐D). In Cyto‐D treated ASM, isometric force was reduced while ATP hydrolysis rate increased compared to untreated ASM strips. These results indicate that external transmission of force, cross‐bridge cycling and ATP hydrolysis rates are affected by internal loading of contractile proteins.

Mechanisms Underlying TNFα‐Induced Hyperreactivity in Airway Smooth Muscle

Inflammation and pro‐inflammatory cytokines (e.g., TNFα, IL‐13) induce airway smooth muscle (ASM) hyper‐reactivity and remodeling characteristic of asthma. We hypothesized that exposure to TNFα (and IL‐13) increases isometric force generation in porcine ASM via several underlying mechanisms: 1) increasing sensitivity to ACh stimulation; 2) enhancing the cytosolic Ca2+ ([Ca2+]cyt) response to ACh; 3) increasing the Ca2+ sensitivity of force generation; 4) enhancing the Ca2+‐dependent phosphorylation of myosin regulator light chain (rMLC); 5) promoting expression of non‐muscle rMLC; 6) increasing actin and myosin heavy chain (MyHC) content (i.e., the number of contractile units); and/or 7) promoting cytoskeletal remodeling (actin polymerization). Studies were performed on intact ASM strips (~1 mm wide × ~5 mm long) dissected from porcine tracheas obtained from a local abattoir. The ASM strips were placed in PSS with and without TNFα (100 ng/ml) for 24 h. The ACh concentration dependence of force generation ASM was determined (ACh range from 10 nM to 1mM), and the EC50 (i.e., the ACh concentration that produces 50% of maximal ASM force) was determined. In control, untreated ASM, the EC50 for ACh was 2.6 μM compared to 1.3 μM following TNFα treatment. We found that across all ACh concentrations, ASM force increased after TNFα treatment with an ~50% increase in maximum ASM force. The [Ca2+]cyt response induced by the EC50 [ACh] was similar in control and TNFα treated ASM. Thus, with an increase in force and no effect on [Ca2+]cyt, there was an apparent increase in Ca2+ sensitivity of force after TNFα treatment. However, in both control and TNFα treated ASM, the extent of rMLC phosphorylation induced by ACh stimulation was not affected by TNFα treatment. Importantly, the extent of rMLC phosphorylation at the ACh EC50 was comparable between control and TNFα treatment indicating no change in Ca2+ sensitivity. The expression of non‐muscle rMLC (determined by 2D gel electrophoresis) was also unaffected by TNFα treatment. Total actin and MyHC concentrations increased by ~45% following TNFα treatment; thus, the number of contractile units contributing to force increased. This was reflected by a marked increase in the Ca2+ activation of ATP consumption (cross‐bridge cycling) of ASM following TNFα treatment. Cytoskeletal remodeling was evaluated by ACh‐induced actin polymerization (F‐/G‐actin ratio) but was found to be unaffected by TNFa treatment. Together, our results show that two primary mechanisms underlie the marked increase in ASM force generation induced by 24‐h TNFα exposure: 1) an increase in ACh sensitivity, and 2) an increase in actin and MyHC concentrations (number of contractile units).Support or Funding InformationSupported by NIH grant HL126451 (GCS) and HL150890 (GCS)

Inhibiting Cytoskeletal Remodeling Increases Tension Cost in Airway Smooth Muscle

Cytoskeletal remodeling in smooth muscle occurs with activation and involves actin polymerization. Remodeling of the cortical cytoskeleton allows tethering of contractile proteins and force transmission to the extracellular matrix. Smooth muscle contraction involves cross‐bridge cycling and ATP hydrolysis, which are both sensitive to loading. During isometric activation, the rate of ATP hydrolysis is initially high but declines with time reaching a steady‐state, while peak force is maintained. Thus, tension cost, i.e., the rate of ATP hydrolysis per unit of force, decreases over time, reflecting increased efficiency of smooth muscle activation. We hypothesize that the time‐dependent decrease in ATP hydrolysis rate and tension cost is due to cytoskeletal remodeling and an increase in internal loading on cross‐bridges. In permeabilized porcine airway smooth muscle (ASM) strips, isometric force and ATP hydrolysis rate were simultaneously measured after maximum Ca2+ activation (pCa 4.0). From each animal, two ASM strips were tested: 1) Inhibition of actin polymerization and cytoskeletal remodeling using Cytochalasin‐D (Cyto‐D; treated (1 μM for 30 min); and 2) Untreated controls. Compared to untreated controls, Cyto‐D‐induced inhibition of actin polymerization reduced both peak and steady‐state isometric force by ~40%, while peak and steady‐state ATP hydrolysis rate increased by ~30% and ~20%, respectively. As a result, at peak and steady‐state, tension cost increased by ~100% and ~60%, respectively, following Cyto‐D treatment. These results support our hypothesis that the time‐dependent decrease in ATP hydrolysis rate reflects an increase in internal load on cross‐bridges via cytoskeletal remodeling. Cyto‐D inhibition of cytoskeletal remodeling impairs contractile protein tethering and thus reduces internal loading, resulting in faster cross‐bridge cycling and increased ATP hydrolysis rate.Support or Funding InformationSupported by NIH grant HL126451(GCS), HL150890 (GCS) and the Mayo Clinic

Mechanisms underlying TNFα-induced enhancement of force generation in airway smooth muscle.

Airway diseases such as asthma are triggered by inflammation and mediated by proinflammatory cytokines such as tumor necrosis factor alpha (TNFα). Our goal was to systematically examine the potential mechanisms underlying the effect of TNFα on airway smooth muscle (ASM) contractility. Porcine ASM strips were incubated for 24 h with and without TNFα. Exposure to TNFα increased maximum ASM force in response to acetylcholine (Ach), with an increase in ACh sensitivity (hyperreactivity), as reflected by a leftward shift in the dose–response curve (EC50). At the EC50, the [Ca2+]cyt response to ACh was similar between TNFα and control ASM, while force increased; thus, Ca2+ sensitivity appeared to increase. Exposure to TNFα increased the basal level of regulatory myosin light chain (rMLC) phosphorylation in ASM; however, the ACh‐dependent increase in rMLC phosphorylation was blunted by TNFα with no difference in the extent of rMLC phosphorylation at the EC50 ACh concentration. In TNFα‐treated ASM, total actin and myosin heavy chain concentrations increased. TNFα exposure also enhanced the ACh‐dependent polymerization of G‐ to F‐actin. The results of this study confirm TNFα‐induced hyperreactivity to ACh in porcine ASM. We conclude that the TNFα‐induced increase in ASM force, cannot be attributed to an enhanced [Ca2+]cyt response or to an increase in rMLC phosphorylation. Instead, TNFα increases Ca2+ sensitivity of ASM force generation due to increased contractile protein content (greater number of contractile units) and enhanced cytoskeletal remodeling (actin polymerization) resulting in increased tethering of contractile elements to the cortical cytoskeleton and force translation to the extracellular matrix.

TNFα enhances force generation in airway smooth muscle.

Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.

Prestretched airway smooth muscle response to length oscillation.

Airway smooth muscle (ASM) hyperconstriction is the cause of many respiratory diseases including asthma. In vitro testing has demonstrated that the active forces of ASM are reduced by length oscillation (LO) mimicking tidal breathing. In a previous study, we demonstrated that this force reduction can be further enhanced when superimposing oscillations (with certain frequencies and amplitudes) on this LO. In contrast, it has been reported that pressurizing the lung may help in relieving asthmatic airway constrictions. Ultimately, this pressurizing stretches the ASM and may disturb the acto‐myosin cross‐bridges in a manner similar to LO; however, it is of a static rather than dynamic nature. This research investigates the effect of combining both prestretch‐ and LO‐applications on contracted porcine ASM. Isolated porcine ASM relaxation was tested with a 0.56%, 2%, or 4% stretch of its reference length (L ref) in addition to LO. These oscillations are composed of a main wave mimicking the normal breathing (frequency of 0.33 Hz and amplitude of 4% L ref) and superimposed oscillations (frequencies of 20, 30, 40, 60 and 80 Hz and amplitude of 1% L ref). The oscillations were maintained for 10 min. The results demonstrate that a prestretch of 0.56% and 2% L ref does enhance the contracted ASM relaxation at certain superimposed length oscillations frequencies while of 4% L ref does not.

Heat Shock-Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β2-Adrenergic Receptor.

Severe bronchospasm refractory to β-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. β-agonist-induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of the small heat shock-related protein (HSP) 20. We hypothesized that a transducible phosphopeptide mimetic of HSP20 (P20 peptide) causes relaxation of human ASM (HASM) by interacting with target(s) downstream of the β2-adrenergic receptor (β2AR) pathway. The effect of the P20 peptide on ASM contractility was determined in human and porcine ASM using a muscle bath. The effect of the P20 peptide on filamentous actin dynamics and migration was examined in intact porcine ASM and cultured primary HASM cells. The efficacy of the P20 peptide in vivo on airway hyperresponsiveness (AHR) was determined in an ovalbumin (OVA) sensitization and challenge murine model of allergic airway inflammation. P20 peptide caused dose-dependent relaxation of carbachol-precontracted ASM and blocked carbachol-induced contraction. The β2AR inhibitor, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551), abrogated isoproterenol but not P20 peptide-mediated relaxation. The P20 peptide decreased filamentous actin levels in intact ASM, disrupted stress fibers, and inhibited platelet-derived growth factor-induced migration of HASM cells. The P20 peptide treatment reduced methacholine-induced AHR in OVA mice without affecting the inflammatory response. These results suggest that the P20 peptide decreased airway constriction and disrupted stress fibers through regulation of the actin cytoskeleton downstream of β2AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to β-agonists.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Nitric Oxide Donors Augment Interleukin-1β-Induced Vascular Endothelial Growth Factor in Airway Smooth Muscle Cells

Angiogenesis and microvascular leakage are features of chronic inflammatory diseases of which molecular mechanisms are poorly understood. We investigated the effects of interleukin-1β (IL-1β) on the expression and secretion of vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) in porcine airway smooth muscle cells (PASMC) in relation to a nitric oxide (NO) pathway. Serum-deprived (48h) PASMC were stimulated with IL-1β alone or with NO donor, L-arginine and/or NO synthase inhibitor L-NAME for 4 and 24h. IL-1β did not affect PlGF release, but augmented VEGF release (2.4-fold) after 24h. VEGF release was inhibited by L-NAME (531.8±52pg/ml), but restored and further elevated by L-arginine (1,529±287pg/ml). IL-1β up-regulated VEGF mRNA (1.8-fold) and this response was attenuated by L-NAME (1.1-fold) and augmented by L-arginine (3.8-fold) at 4h. Restoration of a NO pathway by L-arginine in L-NAME-treated cells resulted in elevated VEGF mRNA levels (2.2-fold). [(3)H]Thymidine incorporation assay revealed enhanced porcine pulmonary artery endothelial cell proliferation in response to IL-1β, VEGF and PlGF, and this mitogenic effect was not influenced via the NO pathway. Our results suggest that a NO pathway modulates VEGF synthesis during inflammation contributing to bronchial angiogenesis and vascular leakage.

Caveolin-1 and force regulation in porcine airway smooth muscle

Caveolae are specialized membrane microdomains expressing the scaffolding protein caveolin-1. We recently demonstrated the presence of caveolae in human airway smooth muscle (ASM) and the contribution of caveolin-1 to intracellular calcium ([Ca(2+)](i)) regulation. In the present study, we tested the hypothesis that caveolin-1 regulates ASM contractility. We examined the role of caveolins in force regulation of porcine ASM under control conditions as well as TNF-α-induced airway inflammation. In porcine ASM strips, exposure to 10 mM methyl-β-cyclodextrin (CD) or 5 μM of the caveolin-1 specific scaffolding domain inhibitor peptide (CSD) resulted in time-dependent decrease in force responses to 1 μM ACh. Overnight exposure to the cytokine TNF-α (50 ng/ml) accelerated and increased caveolin-1 expression and enhanced force responses to ACh. Suppression of caveolin-1 with small interfering RNA mimicked the effects of CD or CSD. Regarding mechanisms by which caveolae contribute to contractile changes, inhibition of MAP kinase with 10 μM PD98059 did not alter control or TNF-α-induced increases in force responses to ACh. However, inhibiting RhoA with 100 μM fasudil or 10 μM Y27632 resulted in significant decreases in force responses, with lesser effects in TNF-α exposed samples. Furthermore, Ca(2+) sensitivity for force generation was substantially reduced by fasudil or Y27632, an effect even more enhanced in the absence of caveolin-1 signaling. Overall, these results indicate that caveolin-1 is a critical player in enhanced ASM contractility with airway inflammation.

Logarithmic superposition of force response with rapid length changes in relaxed porcine airway smooth muscle

We present a systematic quantitative analysis of power-law force relaxation and investigate logarithmic superposition of force response in relaxed porcine airway smooth muscle (ASM) strips in vitro. The term logarithmic superposition describes linear superposition on a logarithmic scale, which is equivalent to multiplication on a linear scale. Additionally, we examine whether the dynamic response of contracted and relaxed muscles is dominated by cross-bridge cycling or passive dynamics. The study shows the following main findings. For relaxed ASM, the force response to length steps of varying amplitude (0.25-4% of reference length, both lengthening and shortening) are well-fitted with power-law functions over several decades of time (10⁻² to 10³ s), and the force response after consecutive length changes is more accurately fitted assuming logarithmic superposition rather than linear superposition. Furthermore, for sinusoidal length oscillations in contracted and relaxed muscles, increasing the oscillation amplitude induces greater hysteresivity and asymmetry of force-length relationships, whereas increasing the frequency dampens hysteresivity but increases asymmetry. We conclude that logarithmic superposition is an important feature of relaxed ASM, which may facilitate a more accurate prediction of force responses in the continuous dynamic environment of the respiratory system. In addition, the single power-function response to length changes shows that the dynamics of cross-bridge cycling can be ignored in relaxed muscle. The similarity in response between relaxed and contracted states implies that the investigated passive dynamics play an important role in both states and should be taken into account.

Regulation of sarcoplasmic reticulum Ca2+reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.

The extensive length-force relationship of porcine airway smooth muscle

The full functional length range of trachealis muscle was measured to identify a precise reference length and to assess the length changes that the myofilament lattice can accommodate. The initial reference length (L(10%)) was that where rest tension equaled 10% of total force (passive tension plus active force). Total force at this length served as a force reference (F(ref) = 219 +/- 12 kPa, N = 7). Muscles initially adapted at L(10%) for 30-60 min had no rest tension when shortened to <0.9 L(10%). Passive tension rose steeply and linearly with slope 11.2 F(ref)/L(10%) at lengths >1.04 L(10%). Rest tension at 1.1 L(10%) declined by <10% over 1 h. The steep slope and stability of rest tension at long lengths suggest that a parameter of the slope could serve as a precise, reproducible reference length. Active force was nearly constant at lengths 0.33-1.0 L(10%) and declined steeply at lengths between 0.1 and 0.2 L(10%), extrapolating to zero at 0.076 L(10%). Muscles visibly reextended during relaxation at lengths <0.25 L(10%). At long lengths, force extrapolated to zero at 1.175 L(10%). The >15-fold length range (0.076-1.175 L(10%)) for force generation and nearly constant force over a greater than threefold length range is likely produced by several structural accommodations, including filament sliding, an increased number of sliding filaments in series, and increased length of passive structures in series with the sliding filaments. Visible reextension during relaxation suggests that the lattice does not undergo plastic adaptations at lengths <25% L(10%) and that lattice plasticity is limited to a three- to fourfold length range.

Inhibition of myosin light‐chain phosphorylation inverts the birefringence response of porcine airway smooth muscle

Muscle birefringence, caused mainly by parallel thick filaments, increases in smooth muscle during stimulation, signalling thick filament formation upon activation. The reverse occurs in skeletal muscle, where a decrease in birefringence has been correlated with crossbridge movement away from the thick filaments. When force generation by trachealis muscle was inhibited with wortmannin, which inhibits myosin light-chain phosphorylation and thick-filament formation, but not the calcium increase caused by stimulation, the birefringence response inverted, suggesting crossbridge movement similar to that of skeletal muscle. Resistance to quick stretches was much greater in stimulated muscle than in unstimulated muscle before wortmannin treatment and no different in stimulated and unstimulated muscle after force inhibition by wortmannin. Before wortmannin treatment, stimulation reduced thick-filament cross-sectional areas in electron micrographs by 44%. After force inhibition by wortmannin, filament areas were not significantly different in stimulated and unstimulated muscle and not significantly different from those of relaxed muscle without wortmannin treatment. These results suggest that myofibrillar-space calcium causes crossbridges to move away from the thick filaments without firmly attaching to thin filaments.

Store-operated Ca2+Influx in Airway Smooth Muscle

Volatile anesthetics produce bronchodilation in part by depleting sarcoplasmic reticulum Ca stores in airway smooth muscle (ASM). Other bronchodilatory drugs are known to act via cyclic nucleotides (cyclic adenosine 3',5'-cyclic monophosphate, cyclic guanosine 3',5'-cyclic monophosphate). Intracellular Ca regulation in ASM involves plasma membrane Ca influx, including that triggered by sarcoplasmic reticulum Ca depletion (store-operated Ca entry [SOCE]). The authors hypothesized that anesthetics and bronchodilatory agents interact in inhibiting SOCE, thus enhancing ASM relaxation. In enzymatically dissociated porcine ASM cells imaged using fluorescence microscopy, sarcoplasmic reticulum Ca was depleted by 1 microm cyclopiazonic acid in 0 extracellular Ca, nifedipine, and potassium chloride (preventing Ca influx through L-type channels and SOCE). Extracellular Ca was rapidly reintroduced to selectively activate SOCE in the presence or absence of 1 minimum alveolar concentration (MAC) halothane, isoflurane, or sevoflurane. Anesthetic interference with SOCE regulation by cyclic nucleotides was examined by activating SOCE in the presence of (1) 1 microm acetylcholine, (2) 100 microm dibutryl cyclic adenosine 3',5'-cyclic monophosphate, or (3) 100 microm 8-bromo-cyclic guanosine 3',5'-cyclic monophosphate. SOCE was enhanced by acetylcholine, whereas volatile anesthetics and both cyclic nucleotides partially inhibited Ca influx. Preexposure to 1 or 2 MAC anesthetic (halothane > isoflurane > sevoflurane) inhibited SOCE. Only halothane and isoflurane inhibited acetylcholine-induced augmentation of Ca influx, and significantly potentiated cyclic nucleotide inhibition such that no influx was observed in the presence of anesthetics and cyclic nucleotides. These data indicate that volatile anesthetics prevent sarcoplasmic reticulum refilling by inhibiting SOCE and enhancing cyclic nucleotide blunting of Ca influx in ASM. Such interactions likely result in substantial airway relaxation in the presence of both anesthetics and bronchodilatory agents such as beta agonists or nitric oxide.

Agonist-induced cyclic ADP ribose production in airway smooth muscle

Cyclic ADP-ribose (cADPR) triggers sarcoplasmic reticulum (SR) Ca 2+ release in airway smooth muscle (ASM). SR Ca 2+ release is an important component of the intracellular Ca 2+ ([Ca 2+] i) response of ASM to agonists. Whether cADPR is endogenously produced in ASM during agonist stimulation has not been established. In this study, cADPR production was examined in acutely dissociated porcine ASM cells. ACh stimulation (⩾1 μM) significantly increased cADPR levels, peaking between 30 s and 1 min. This effect was inhibited by M 2 and M 3 muscarinic receptor antagonists. Histamine (⩾5 μM) increased cADPR levels to a greater extent than ACh, while diphenhydramine blocked histamine-induced cADPR elevation. Both bradykinin (100 nM) and endothelin-1 (100 nM) also increased cADPR levels to a greater extent than ACh or histamine. These results indicate that in porcine ASM, certain agonists acting via receptors increase cADPR levels. Furthermore, the extent of cADPR responses to agonist varies, possibly reflecting differences in G-protein coupling.

Halothane Does Not Inhibit the Functional Coupling between the β2-Adrenergic Receptor and the GαsHeterotrimeric G Protein

This study investigated whether halothane affects the functional coupling between the beta2 adrenergic receptor and the alpha subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (Galphas). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at Galphas and hence would not affect isoproterenol-induced relaxation of airway smooth muscle. Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. Galphas nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human beta1 or beta2 receptor each with human short Galphas. A radioactive, nonhydrolyzable analog of GTP, [S]GTPgammaS, was used as the reporter for nucleotide exchange at Galphas. Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in Galphas nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal Galphas nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted Galphas nucleotide exchange in both the beta1-Galphas and beta2-Galphas expressing membranes. However, the effect was significantly greater on beta1-Galphas coupling compared with beta2-Galphas coupling, with no effect on beta2-Galphas coupling at 2.8 MAC halothane. Halothane does not inhibit the biochemical coupling between the beta2 receptor and Galphas and hence does not affect the inhibition of calcium sensitivity induced by isoproterenol. Therefore, halothane should not affect the efficacy of beta2 agonists, as suggested by studies of in vivo animal models of asthma.