Warner, David O.

This study tested the hypothesis that the NO donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) in part by a cGMP-independent process that involves reversible oxidation of intracellular thiols. GSNO caused a concentration-dependent relaxation in ACh-contracted strips (EC 50 ∼1.2 μM) accompanied by a concentration-dependent increase in cytosolic cGMP concentration ([cGMP] i ). The soluble guanylate cyclase inhibitor methylene blue prevented the increase in [cGMP] i induced by 1 and 10 μM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of [cGMP] i to baseline, GSNO-induced relaxation persisted during continuous ACh stimulation. Dithiothreitol caused a rapid recovery of isometric force to values similar to those obtained with ACh alone in these strips. We conclude that GSNO relaxes CTSM contracted by ACh in part by oxidation of intracellular protein thiols.

This study determined whether the time-dependent decline in the rate of ATP hydrolysis by actomyosin ATPase during sustained isometric force can occur in the absence of a time-dependent decline in regulatory myosin light chain (rMLC) phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle. Maximal activation with 10 μM Ca2+induced sustained increases in isometric force, stiffness, and rMLC phosphorylation; however, the increase in the ATP hydrolysis rate was initially high but then declined to a steady-state level above that of the unstimulated muscle (basal 31.8 ± 5.8 nmol ⋅ cm−3⋅ s−1; peak 81.4 ± 11.3 nmol ⋅ cm−3⋅ s−1; steady-state 62.2 ± 9.1 nmol ⋅ cm−3⋅ s−1). Activation of strips in which the rMLC was irreversibly and maximally thiophosphorylated with adenosine 5′- O-(3-thiotriphosphate) also induced sustained increases in isometric force and stiffness but a nonsustained increase in ATP hydrolysis rate. There was no significant difference in the peak or steady-state isometric force, stiffness, or ATP hydrolysis rate or in the steady-state maximum unloaded shortening velocity between strips activated by 10 μM Ca2+or rMLC thiophosphorylation (0.058 ± 0.016 and 0.047 ± 0.011 muscle lengths/s, respectively). Mechanisms other than changes in rMLC phosphorylation contribute to the time-dependent decline in actomyosin ATPase activity during sustained activation of canine tracheal smooth muscle.

The purpose of this study was to determine the mechanism by which hydrogen peroxide (H2O2), an important inflammatory mediator, relaxes canine tracheal smooth muscle (CTSM). H2O2caused concentration-dependent relaxations of CTSM strips contracted with ACh or isotonic KCl [EC50of 0.24 ± 0.04 (SE) and 0.23 ± 0.04 mM, respectively]. Indomethacin (10 μM) decreased the sensitivity of both KCl- and ACh-contracted strips to H2O2. H2O2increased intracellular cAMP levels, an increase that was abolished by indomethacin. H2O2did not affect intracellular cGMP levels. In strips treated with indomethacin and contracted with ACh or isotonic KCl, H2O2-evoked relaxations were accompanied by increases in intracellular Ca2+concentration and decreases in regulatory myosin light chain phosphorylation. We conclude that H2O2decreases Ca2+sensitivity in CTSM by decreasing regulatory myosin light chain phosphorylation through inhibition of myosin light chain kinase and/or activation of smooth muscle protein phosphatases.

The effect of halothane on intracellular Ca2+concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+via increased Ca2+leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+release through ryanodine-receptor channels, and 3) inhibiting reuptake.

A β-escin-permeabilized canine tracheal smooth muscle preparation was used to test the hypothesis that cGMP decreases Ca2+sensitivity in airway smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that regulate Ca2+sensitivity and not by inhibiting Ca2+/calmodulin activation of the contractile proteins. 8-Bromo-cGMP (100 μM) had no effect on the free Ca2+concentration-response curves generated in the absence of muscarinic receptor stimulation. In the presence of 100 μM ACh plus 10 μM GTP, 8-bromo-cGMP (100 μM) caused a rightward shift of the free Ca2+concentration-response curve, significantly increasing the EC50for free Ca2+from 0.35 ± 0.03 to 0.75 ± 0.06 μM; this effect of 8-bromo-cGMP was concentration dependent from 1 to 100 μM. 8-Bromo-cGMP (100 μM) decreased the level of regulatory myosin light chain (rMLC) phosphorylation for a given cytosolic Ca2+concentration but had no effect on the amount of isometric force produced for a given level of rMLC phosphorylation. These findings suggest that cGMP decreases Ca2+sensitivity in canine tracheal smooth muscle primarily by inhibiting the membrane receptor-coupled mechanisms that modulate the relationship between cytosolic Ca2+concentration and rMLC phosphorylation.

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+concentration ([Ca2+]; i.e., the Ca2+sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in α-toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF[Formula: see text]; acute or 1-h exposure). Acute exposure to each compound increased Ca2+sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF[Formula: see text], but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF[Formula: see text] exposure required Ca2+during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca 2+ sensitivity [i.e., the amount of force for a given intracellular Ca 2+ concentration ([Ca 2+ ] i )]. We further investigated whether GSNO decreases Ca 2+ sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca 2+ ] i and the relationship between force and rMLC phosphorylation. GSNO (100 μM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca 2+ sensitivity in comparison to control strips without significantly decreasing [Ca 2+ ] i . GSNO reduced the amount of rMLC phosphorylation for a given [Ca 2+ ] i but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca 2+ sensitivity by affecting the level of rMLC phosphorylation for a given [Ca 2+ ] i , suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.

We tested the hypothesis that increases in force at a given cytosolic Ca2+concentration (i.e., Ca2+sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in α-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 μM Ca2+in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 μM) plus GTP (1 μM) significantly shifted the Ca2+-force relationship curve to the left (EC50: 0.39 ± 0.06 to 0.078 ± 0.006 μM Ca2+) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca2+-rMLC phosphorylation relationship curve was also shifted to the left (EC50: 1.26 ± 0.57 to 0.13 ± 0.04 μM Ca2+) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca2+concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca2+sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

Halothane is a potent bronchodilator that attenuates vagally mediated constriction of canine airways, in part by a direct action on the smooth muscle cell. In tracheal smooth muscle, acetylcholine (ACh) produces a transient peak increase in cytosolic ionized calcium concentration ([Ca2+]i), which declines to a sustained plateau concentration that is higher than that of the unstimulated muscle, and a sustained increase in force. The transient peak [Ca2+]i is caused primarily by Ca2+ release from the sarcoplasmic reticulum (SR), whereas the plateau [Ca2+]i is caused primarily by influx of extracellular Ca2+ across the plasma membrane. This study was conducted to investigate the effects of halothane on the 1) transient peak [Ca2+]i during force development (i.e., developed force) and 2) plateau [Ca2+]i during force maintenance (i.e., sustained force) induced by ACh in isolated strips of canine tracheal smooth muscle. Changes in [Ca2+]i were measured with the photoprotein, aequorin. In muscle strips contracted after incubation with 1.1 (n = 5) or 1.5 (n = 5) minimum alveolar concentration (MAC) halothane, halothane significantly attenuated the transient peak aequorin luminescence (AL) and developed force and significantly prolonged the time necessary to reach peak AL and developed force; these effects were not dose dependent. In muscle strips (n = 3) contracted with ACh, addition of halothane caused a reduction in sustained force but no decrease in plateau AL.(ABSTRACT TRUNCATED AT 250 WORDS)