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. 1999 Sep 1;519 Pt 2(Pt 2):527-38.
doi: 10.1111/j.1469-7793.1999.0527m.x.

F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs

K A Jones  1 , W J Perkins, R R Lorenz, Y S Prakash, G C Sieck, D O Warner
Affiliations

F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs

K A Jones et al. J Physiol. .

Abstract

1. Dynamic actin reorganization involving actin polymerization and depolymerization may play an important functional role in smooth muscle. 2. This study tested the hypothesis that F-actin stabilization by phalloidin increases tension cost (i.e. ATP hydrolysis rate per unit of isometric force) during Ca2+-induced activation of Triton X-100-permeabilized canine tracheal smooth muscle. 3. Adenosine 5'-triphosphate (ATP) hydrolysis rate was quantified using an enzyme-coupled NADH fluorometric technique, regulatory myosin light chain (rMLC) phosphorylation was measured by Western blot analysis, and maximum unloaded shortening velocity (Vmax) was estimated by interpolation of the force-velocity relationship to zero load during isotonic loading. 4. Maximal activation with 10 microM free Ca2+ induced sustained increases in isometric force, stiffness, and rMLC phosphorylation. However, the increase in ATP hydrolysis rate initially reached peak values, but then declined to steady-state levels above that of the unstimulated muscle. Thus, tension cost decreased throughout steady-state isometric force. 5. Following incubation of permeabilized strips with 50 microM phalloidin for 1 h, the increases in isometric force and stiffness were not sustained despite a sustained increase in rMLC phosphorylation. Also, after an initial decline, tension cost increased throughout activation. Phalloidin had no effect on Vmax during steady-state isometric force or on rMLC phosphorylation. 6. These findings suggest that dynamic reorganization of actin is necessary for optimal energy utilization during contraction of permeabilized airway smooth muscle.

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Figures

Figure 1
Figure 1. Representative trace showing the effect of free Ca2+ concentration on NADH fluorescence in a Triton X-100-permeabilized canine tracheal smooth muscle strip
The rate of decline in NADH fluorescence during a 4.9 s period was measured and used to quantify the ATP hydrolysis rate based on a prior calibration of the system with known NADH concentrations. ATP hydrolysis rates were normalized for tissue volume and expressed as nanomoles per centimetre cubed per second.
Figure 2
Figure 2. Effect of phalloidin on the time course of changes in isometric force, stiffness and ATP hydrolysis rate in Triton X-100-permeabilized canine tracheal smooth muscle strips maximally activated by 10 μm free Ca2+
Permeabilized strips were superfused with relaxing solution (see Methods for composition) containing 1 % methanol or 50 μm phalloidin in 1 % methanol for 1 h prior to activation. In preliminary studies, 1 % methanol had no effect on the increases in isometric force (upper panel), stiffness (middle panel) or ATP hydrolysis rate (lower panel) induced by 10 μm free Ca2+. Values are means ±s.e.m. using data obtained from 7 animals.
Figure 3
Figure 3. Effect of phalloidin on the time course of changes in the relationship between isometric force and stiffness in Triton X-100-permeabilized canine tracheal smooth muscle strips maximally activated by 10 μm free Ca2+
Permeabilized strips were superfused with relaxing solution (see Methods for composition) containing 1 % methanol or 50 μm phalloidin in 1 % methanol for 1 h prior to activation. These relationships were constructed from the data shown in Fig. 2, upper and middle panels. Values are means using data obtained from 7 animals. Error bars have been omitted for the purpose of clarity.
Figure 4
Figure 4. Effect of phalloidin on the time course of changes in tension cost (isometric force/ATP hydrolysis rate) in Triton X-100-permeabilized canine tracheal smooth muscle strips maximally activated by 10 μm free Ca2+
Permeabilized strips were superfused with relaxing solution (see Methods for composition) containing 1 % methanol or 50 μm phalloidin in 1 % methanol for 1 h prior to activation. Tension cost was calculated as ATP hydrolysis rate (nmol cm−3 s−1) divided by isometric force (N cm−2). Values are means ±s.e.m. using data obtained from 7 animals
Figure 5
Figure 5. Effect of phalloidin on the time course of changes in rMLC phosphorylation in Triton X-100-permeabilized canine tracheal smooth muscle strips maximally activated by 10 μm free Ca2+
Permeabilized strips were incubated in relaxing solution (see Methods for composition) containing 1 % methanol or 50 μm phalloidin in 1 % methanol for 1 h prior to activation. Values are means and s.e.m. using data obtained from 5 animals.
Figure 6
Figure 6. Effect of phalloidin on Vmax in Triton X-100-permeabilized canine tracheal smooth muscle strips maximally activated by 10 μm free Ca2+
Permeabilized strips were incubated in relaxing solution (see Methods for composition) containing 1 % methanol (upper panel) or 50 μm phalloidin in 1 % methanol (lower panel) for 1 h prior to activation. The data were fitted with the hyperbolic Hill equation (P+a)(V+b) = (Po+a)b, where a and b are the asymptotes of the hyperbola. Values are means ±s.e.m. using data obtained from 5 animals.
Figure 7
Figure 7. Representative images showing simultaneous fluorescence labelling of G-actin in rat diaphragm (upper panels) and canine tracheal smooth muscle (lower panels)
The G-actin was labelled with rhodamine-DNase I and imaged using confocal fluorescence microscopy. The image intensity gain was determined using an unlabelled tissue section to eliminate tissue autofluorescence and was kept constant throughout the experiment.
Figure 8
Figure 8. Representative images showing simultaneous fluorescence labelling of G-actin (left panel) and F-actin (right panel) in intact (upper panel) and permeabilized (lower panel) canine tracheal smooth muscle
The G- and F-actin were labelled with rhodamine-DNase I and Alexa-488-phalloidin, respectively, and visualized using confocal fluorescence microscopy. The image intensity gain was determined for each channel using an unlabelled tissue section to eliminate tissue autofluorescence and was kept constant throughout the experiment.
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