Citations

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.

We hypothesized that decrements in maximum power output (W˙max) of the rat diaphragm (Dia) muscle with repetitive activation are due to a disproportionate reduction in force (force fatigue) compared with a slowing of shortening velocity (velocity fatigue). Segments of midcostal Dia muscle were mounted in vitro (26°C) and stimulated directly at 75 Hz in 400-ms-duration trains repeated each second (duty cycle = 0.4) for 120 s. A novel technique was used to monitor instantaneous reductions in maximum specific force (Po) andW˙maxduring fatigue. During each stimulus train, activation was isometric for the initial 360 ms during which Powas measured; the muscle was then allowed to shorten at a constant velocity (30% Vmax) for the final 40 ms, and W˙maxwas determined. Compared with initial values, after 120 s of repetitive activation, PoandW˙maxdecreased by 75 and 73%, respectively. Maximum shortening velocity was measured in two ways: by extrapolation of the force-velocity relationship ( Vmax) and using the slack test [maximum unloaded shortening velocity ( Vo)]. After 120 s of repetitive activation, Vmaxslowed by 44%, whereas Voslowed by 22%. Thus the decrease inW˙maxwith repetitive activation was dominated by force fatigue, with velocity fatigue playing a secondary role. On the basis of a greater slowing of Vmaxvs. Vo, we also conclude that force and power fatigue cannot be attributed simply to the total inactivation of the most fatigable fiber types.

There is a mediolateral gradient in activation of the parasternal intercostal (PI) muscle during inspiration. In the present study, we tested the hypotheses that serotonergic [5-hydroxytryptamine (5-HT)] input from descending central drive and/or intrinsic size-related properties of PI motoneurons leads to the differential activation of PI muscles. In dogs, PI motoneurons innervating the medial and lateral regions of the PI muscles at the T3–T5interspaces were retrogradely labeled by intramuscular injection of cholera toxin B subunit. After a 10-day survival period, PI motoneurons and 5-HT terminals were visualized by using immunohistochemistry and confocal imaging. There were no differences in motoneuron morphology among motoneurons innervating the medial and lateral regions of the PI muscle. However, the number of 5-HT terminals and the 5-HT terminal density (normalized for surface area) were greater in motoneurons innervating the medial region of the PI muscle compared with the lateral region. These results suggest that differences in distribution of 5-HT input may contribute to regional differences in PI muscle activation during inspiration and that differences in PI motoneuron recruitment do not relate to size.

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+concentration ([Ca2+]i) oscillations (5–30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]ito force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]ioscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]iand contraction in intact porcine ASM cells was found to be ∼450 ms. In β-escin-permeabilized ASM strips, photolytic release of caged Ca2+resulted in force generation after ∼800 ms. When calmodulin (CaM) was added, this delay was shortened to ∼500 ms. In the presence of exogenous CaM and 100 μM Ca2+, photolytic release of caged ATP led to force generation after ∼80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred ∼50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]iafter photolytic release of caged diazo-2 resulted in relaxation after a delay of ∼1.2 s and 50% reduction in force after ∼57 s. We conclude that in ASM cells agonist-induced [Ca2+]ioscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.

In the present study, we used real-time confocal microscopy to examine the effects of two nitric oxide (NO) donors on acetylcholine (ACh; 10 μM)- and caffeine (10 mM)-induced intracellular calcium concentration ([Ca2+]i) responses in C2C12mouse skeletal myotubes. We hypothesized that NO reduces [Ca2+]iin activated skeletal myotubes through oxidation of thiols associated with the sarcoplasmic reticulum Ca2+-release channel. Exposure to diethylamine NONOate (DEA-NO) reversibly increased resting [Ca2+]ilevel and resulted in a dose-dependent reduction in the amplitude of ACh-induced [Ca2+]iresponses (25 ± 7% reduction with 10 μM DEA-NO and 78 ± 14% reduction with 100 μM DEA-NO). These effects of DEA-NO were partly reversible after subsequent exposure to dithiothreitol (10 mM). Preexposure to DEA-NO (1, 10, and 50 μM) also reduced the amplitude of the caffeine-induced [Ca2+]iresponse. Similar data were obtained by using the chemically distinct NO donor S-nitroso- N-acetyl-penicillamine (100 μM). These results indicate that NO reduces sarcoplasmic reticulum Ca2+release in skeletal myotubes, probably by a modification of hyperreactive thiols present on the ryanodine receptor channel.

The multiplicity of mechanisms involved in regulation of intracellular Ca2+concentration ([Ca2+]i) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca2+]i. Heterogeneity in [Ca2+]iregulation is reflected by the presence of spontaneous, localized [Ca2+]itransients (Ca2+sparks) representing Ca2+release through ryanodine receptor (RyR) channels. Ca2+sparks display variable spatial Ca2+distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca2+]ithat occasionally trigger propagating [Ca2+]iwaves. Ca2+sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca2+]ioscillations that originate from foci with high spark incidence and also represent Ca2+release through RyR channels. With increasing agonist concentration, the peak of regional [Ca2+]ioscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i, representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca2+]ioscillations lies in the establishment of a global [Ca2+]ilevel for slower Ca2+-dependent physiological processes.

The effects of the β2-adrenoceptor agonist salbutamol (Slb) on isometric and isotonic contractile properties of the rat diaphragm muscle (Diamus) were examined. A loading dose of 25 μg/kg Slb was administered intracardially before Diamusexcision to ensure adequate diffusion. Studies were then performed with 0.05 μM Slb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch and maximum tetanic force were increased by ∼40 and ∼20%, respectively. Maximum shortening velocity increased by ∼15% after Slb treatment, and maximum power output increased by ∼25%. During repeated isotonic activation, the rate of fatigue was faster in the Slb-treated Diamus, but both Slb-treated and Ctl Diamusfatigued to the same maximum power output. Still, endurance time during repetitive isotonic contractions was ∼10% shorter in the Slb-treated Diamus. These results are consistent with the hypothesis that β-adrenoceptor stimulation by Slb enhances Diamuscontractility and that these effects of Slb are likely mediated, at least in part, by elevated cAMP.

Van Balkom, Roland H. H., Wen-Zhi Zhan, Y. S. Prakash, P. N. Richard Dekhuijzen, and Gary C. Sieck. Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle. J. Appl. Physiol. 83(4): 1062–1067, 1997.—The effects of corticosteroids (CS) on diaphragm muscle (Diam) fiber morphology and contractile properties were evaluated in three groups of rats: controls (Ctl), surgical sham and weight-matched controls (Sham), and CS-treated (6 mg ⋅ kg−1 ⋅ day−1prednisolone at 2.5 ml/h for 3 wk). In the CS-treated Diam, there was a selective atrophy of type IIx and IIb fibers, compared with a generalized atrophy of all fibers in the Sham group. Maximum isometric force was reduced by 20% in the CS group compared with both Ctl and Sham. Maximum shortening velocity in the CS Diamwas slowed by ∼20% compared with Ctl and Sham. Peak power output of the CS Diam was only 60% of Ctl and 70% of Sham. Endurance to repeated isotonic contractions improved in the CS-treated Diam compared with Ctl. We conclude that the atrophy of type IIx and IIb fibers in the Diam can only partially account for the CS-induced changes in isotonic contractile properties. Other factors such as reduced myofibrillar density or altered cross-bridge cycling kinetics are also likely to contribute to the effects of CS treatment.

The effects of corticosteroid (CS) treatment (prednisolone continuously administered subcutaneously at a flow rate of 2.5 μl/h, daily dose 5.6 mg/kg, for 3 wk) on neuromuscular junction (NMJ) morphology and neuromuscular transmission in rat diaphragm muscle (Dimus) were compared with weight-matched (Sham) and ad libitum fed control (Ctl) groups. Fibers were classified on the basis of myosin heavy chain (MHC) isoform expression. CS treatment caused significant atrophy of fibers expressing MHC2X(type IIx), either alone or with MHC2B(type IIx/b). Fibers expressing MHCslow(type I) and MHC2A(type IIa) were unaffected by CS. The planar areas of nerve terminals and motor endplates at type IIx/b fibers were smaller in CS-treated Dimuscompared with Sham and Ctl. However, CS-induced atrophy of type IIx/b fibers exceeded changes in NMJ morphology. Thus, when normalized for fiber diameter, NMJs were relatively larger in the CS-treated group compared with Ctl. Neuromuscular transmission failure, assessed in vitro by comparing force loss during repetitive (40 Hz) nerve vs. direct muscle stimulation, was less in CS-treated Dimus. These results indicate that alterations in NMJ morphology after CS treatment are dependent on fiber type and may contribute to improved neuromuscular transmission.

Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, and G. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl. Physiol. 82(6): 1836–1843, 1997.—Relaxation of airway smooth muscle (ASM) by β-adrenoceptor agonists involves reduction of intracellular Ca2+concentration ([Ca2+]i). In porcine ASM cells, acetylcholine induces [Ca2+]ioscillations that display frequency modulation by agonist concentration and basal [Ca2+]i. We used real-time confocal microscopy to examine the effect of salbutamol (1 nM to 1 μM), a β2-adrenoceptor agonist, on [Ca2+]ioscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of [Ca2+]ioscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 μM. These effects were mimicked by a cell-permeant analog of adenosine 3′,5′-cyclic monophosphate. The inhibitory effect of salbutamol was partially reversed by BAY K 8644. Salbutamol reduced [Ca2+]ieven when sarcoplasmic reticulum (SR) Ca2+reuptake and Ca2+influx were blocked. Lanthanum blockade of Ca2+efflux attenuated the inhibitory effect of salbutamol on [Ca2+]i. The [Ca2+]iresponse to caffeine was unaffected by salbutamol. On the basis of these results, we conclude that β2-adrenoceptor agonists have little effect on SR Ca2+release in ASM cells but reduce [Ca2+]iby inhibiting Ca2+influx through voltage-gated channels and by enhancing Ca2+efflux.