Sieck, Gary C.

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 relaxes airway smooth muscle in part by a direct effect on the smooth muscle cell. The purpose of this study was to investigate the possible role of cyclic nucleotides in this direct effect. Strips of canine tracheal smooth muscle in vitro were contracted with acetylcholine (ACh) and then exposed to 0.7-2.6% halothane. Isometric force and the intracellular concentrations of adenosine cyclic 3',5'-monophosphate ([cAMP]i) guanosine cyclic 3',5'-monophosphate ([cGMP]i), and free calcium ([Ca2+]i, using the fluorescent Ca(2+)-sensitive dye fura 2) were measured. ACh caused significant increases in force, [cAMP]i, [cGMP]i, and [Ca2+]i. Subsequent exposure of the strips to halothane caused an additional increase in [cAMP]i, decreases in force and [Ca2+]i, and no effect on [cGMP]i. The additional increase in [cAMP]i was similar to that produced by a concentration of isoproterenol (0.03 microM) that caused equipotent relaxation. Indomethacin abolished the increase in [cAMP]i produced by ACh and abolished the additional increase in [cAMP]i produced by halothane. In contrast, indomethacin had no effect on the decreases in force and [Ca2+]i. These findings suggest that in canine tracheal smooth muscle contracted with ACh 1) halothane increases [cAMP]i by a cyclooxygenase-dependent mechanism and 2) the increase in [cAMP]i produced by halothane is not responsible for the relaxation or the decrease in [Ca2+]i.

The mechanism of the direct relaxing effect of halothane on airway smooth muscle may involve a decrease in 1) cytosolic calcium concentration ([Ca2+]i) and/or 2) the force produced for a given [Ca2+]i (i.e., the “sensitivity” of the myofibrillar contractile system to Ca2+). This study was conducted to test the hypothesis that halothane reduces the sensitivity of the myofibrillar contractile system to Ca2+ during muscarinic receptor stimulation of canine tracheal smooth muscle. Isolated smooth muscle strips were mounted in a photometric superfusion system, stretched to their optimal length for force development, and loaded with the fluorescent Ca2+ indicator, fura 2, for simultaneous recording of fura 2 fluorescence and isometric force. Emission fluorescence intensities due to excitation at 340 (F340)- and 380 (F380)-nm wavelengths were measured and F340/F380 was used as an index of [Ca2+]i. After superfusion with Ca(2+)-free physiological salt solution (PSS) containing 1 or 100 microM acetylcholine (ACh), two consecutive cumulative concentration-response curves to CaCl2 (0.01–2.4 mM) were generated for each strip; one curve was generated in the presence of halothane. In strips stimulated with 1 (n = 6) or 100 (n = 6) microM ACh, the cumulative addition of CaCl2 to the Ca(2+)-free PSS caused concentration-dependent increases in both F340/F380 and force. In strips stimulated with 1 microM ACh, 2.4 +/- 0.3% halothane proportionally attenuated increases in both F340/F380 and force.(ABSTRACT TRUNCATED AT 250 WORDS)

Maximum velocity of the actomyosin ATPase reaction ( VmaxATPase) and ATP consumption rate during maximum isometric activation (ATPiso) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 − (ratio of ATPisoto VmaxATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21–66 yr). A quantitative histochemical procedure was used to measure VmaxATPase. In permeabilized fibers, ATPisowas measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC2Xand MHC2Acompared with fibers singularly expressing MHC2Aand MHCslow(39 vs. 52 and 56%, respectively). Tension cost (ratio of ATPisoto generated force) also varied with fiber type, being highest in fibers coexpressing MHC2Xand MHC2A. We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.

The effect of chronic exogenous testosterone (T) treatment on neuromuscular transmission in the diaphragm (Dia) muscle of adult male rats was determined. The contribution of neuromuscular transmission failure (NTF) to Dia fatigue was evaluated by superimposing intermittent direct muscle stimulation on repetitive nerve stimulation of isometric contraction in vitro. T treatment significantly reduced the contribution of NTF to Dia fatigue by ∼20% ( P < 0.001). Fiber type-specific effects on NTF were determined by measuring Dia fiber glycogen levels subsequent to repetitive nerve or muscle stimulation. T treatment had no effect on glycogen depletion in Dia type I and IIa fibers regardless of stimulation route. In the control group, type IIx fibers demonstrated significantly less glycogen depletion after nerve stimulation compared with direct muscle stimulation ( P < 0.05), suggesting the presence of NTF. In contrast, T treatment increased glycogen depletion of type IIx fibers during nerve stimulation to levels similar to those after direct muscle stimulation. These data indicate that testosterone treatment substantially improves neuromuscular transmission in the Dia.

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.

It has been found that maximum specific force (Fmax; force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in Fmax reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHCslow and MHCneo, respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHCslow and coexpressing MHCneoproduced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHCslow and MHCneo contributes to the lower Fmax seen in early postnatal development.

In the present study, myosin heavy chain (MHC) content per half sarcomere, an estimate of the number of cross bridges available for force generation, was determined in rat diaphragm muscle (Diam) fibers expressing different MHC isoforms. We hypothesize that fiber-type differences in maximum specific force [force per cross-sectional area (CSA)] reflect the number of cross bridges present per CSA. Studies were performed on single, Triton X-100-permeabilized rat Diamfibers. Maximum specific force was determined by activation of single Diamfibers in the presence of a high-calcium solution (pCa, −log Ca2+concentration of 4.0). SDS-PAGE and Western blot analyses were used to determine MHC isoform composition and MHC content per half sarcomere. Differences in maximum specific force across fast MHC isoforms were eliminated when controlled for half-sarcomere MHC content. However, the force produced by slow fibers remained below that of fast fibers when normalized for the number of cross bridges available. On the basis of these results, the lower force produced by slow fibers may be due to less force per cross bridge compared with fast fibers.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Diam) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (Po) and maximum unloaded shortening velocity ( Vo). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHCneoin the Diampersisted, and emergence of MHC2Xand MHC2Bwas delayed. By D-21 and D-28, relative expression of MHC2Aand MHC2Bwas reduced in DNV compared with control (CTL) animals. Expression of MHCneoalso reappeared in adult Diamby 2–3 wk after DNV, and relative expression of MHC2Bwas reduced. At each age, Powas reduced and Vowas slowed by DNV, compared with CTL. In CTL Diam, postnatal changes in Poand Vowere associated with an increase in fast MHC isoform expression. In DNV Diam, no such association existed. We conclude that, in the Diam, DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.

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.