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. 1999 Sep 15;519 Pt 3(Pt 3):829-40.
doi: 10.1111/j.1469-7793.1999.0829n.x.

Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle

D Mehta  1 , S J Gunst
Affiliations

Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle

D Mehta et al. J Physiol. .

Abstract

1. The role of actin polymerization in the regulation of smooth muscle contractility was investigated in canine trachealis muscle strips. The effect of contractile activation on the content of monomeric globular (G)-actin was estimated by the method of DNase I inhibition. The G-actin content was 30 % lower in extracts of muscle strips activated with 10-4 M acetylcholine (ACh) than in extracts from unstimulated muscle strips. The decrease in G-actin in response to contractile stimulation was prevented by latrunculin-A, an agent that prevents actin polymerization by binding to G-actin monomers. 2. The inhibition of actin polymerization by latrunculin-A markedly depressed force development in response to ACh but had no effect on ACh-induced myosin light chain (MLC) phosphorylation. Latrunculin also suppressed the length sensitivity of force during ACh-induced isometric contractions. The actin-capping agent cytochalasin-D also markedly inhibited force and caused only a slight decrease in MLC phosphorylation. Cytochalasin-D also inhibited force in alpha-toxin-permeabilized muscle strips that were activated either by Ca2+ or by ACh at constant pCa. No disorganization of smooth muscle cell ultrastructure was detected by electron microscopy or by immunofluorescence microscopy of muscles treated with either agent. 3. The results suggest that the polymerization of actin is stimulated by the contractile activation of tracheal smooth muscle and that this actin polymerization contributes directly to force development. In addition, actin filament remodelling contributes to the length sensitivity of tracheal smooth muscle contractility.

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Figures

Figure 1
Figure 1. Effects of latrunculin (A and B) and cytochalasin-D (C and D) on force and myosin light chain (MLC) phosphorylation in tracheal muscle strips stimulated with 100 μm ACh under isometric conditions
Latrunculin (0.1, 0.5, or 1 μm) significantly reduced force development; however it had no significant effect on MLC phosphorylation (n = 5). Cytochalasin (0.5, 1 or 10 μm) significantly reduced both force development and MLC phosphorylation (n = 5). Neither cytochalasin-D nor latrunculin affected MLC phosphorylation in unstimulated muscles. Arrows represent the mean value of MLC phosphorylation in unstimulated muscle strips. Force is normalized to the maximal force obtained in response to 100 μm ACh in the absence of cytochalasin or latrunculin (untreated strips). Data are means ±s.e.m.* Values significantly different from untreated muscle strips (P < 0.05).
Figure 2
Figure 2. Effect of cytochalasin-D on contractions induced by increasing [Ca2+] to pCa 5 (A) or by adding 10−4 M ACh at pCa 7 (B) in α-toxin-permeabilized muscle strips
Cytochalasin-D (1 or 10 μm) caused significant inhibition of force in muscle strips stimulated with 10 μm Ca2+or with ACh (n = 3). Force was normalized to the maximal force obtained in response to Ca2+ or ACh in muscle strips not treated with cytochalasin. Data are means ±s.e.m.* Values significantly different from untreated muscle strips (P < 0.05).
Figure 3
Figure 3. Effect of latrunculin (A and B) or cytochalasin (C and D) on the length sensitivity of force and MLC phosphorylation in muscle strips contracted isometrically at 0.6Lo, 0.8Lo or Lo with 10 μm ACh
○, □, muscle strips not treated with cytochalasin or latrunculin; •, ▪, strips treated with cytochalasin or latrunculin. In untreated muscle strips, force was significantly different at lengths of 0.6Lo, 0.8Lo and Lo (A and C) (n = 8-18). In the presence of latrunculin, no significant differences in muscle force were observed at these muscle lengths (n = 8-18), whereas in the presence of cytochalasin force was significantly different at Lo and at 0.6Lo (n = 8). Differences in MLC phosphorylation at 0.6Lo and Lo were statistically significant in untreated muscle strips as well as in strips treated with cytochalasin or latrunculin (n = 8-10). Forces at 0.6Lo and at 0.8Lo were normalized to the force obtained at Lo in untreated muscle strips.
Figure 4
Figure 4. Comparison of the effects of differences in active tension (A and B) or differences in muscle length (C and D) on the sensitivity of contraction to latrunculin
In strips contracted to different tensions at the same muscle length (A), the inhibitory effect of latrunculin (Lat-A) on force was similar (B) (n = 6). In strips contracted to similar tensions at different lengths, Lo and 0.6Lo (C), latrunculin caused significantly more force inhibition at Lo (D) (n = 6). ▪, contraction with 10 μm ACh; formula image, contraction with 0.1 μm ACh. Force was normalized to the force obtained in response to 10 μm ACh at Lo in the absence of latrunculin. Percentage inhibition of force by latrunculin was calculated as the percentage reduction in force after treatment. * Values significantly different from each other (P < 0.05).
Figure 5
Figure 5. Effect of muscle length (A) and cytochalasin-D or latrunculin (B) on the G-actin content of muscle strips after 5 min stimulation with 100 μm ACh
A, at muscle lengths of either Lo or 0.6Lo, stimulation with ACh caused a significant decrease in G-actin content. Differences in G-actin content in muscles at 0.6Lo or Lo were not significant in stimulated or in unstimulated muscles (n = 8). B, the G-actin content in muscles stimulated with ACh in the presence of 1 μm latrunculin (Lat-A) was not significantly different from that in unstimulated muscles, whereas the G-actin content of muscles stimulated with ACh in the presence of 10 μm cytochalasin (CCD) was significantly lower than that in unstimulated muscles. Values of G-actin content are normalized to values at Lo in unstimulated strips (n = 8). * Values significantly different from values at Lo in unstimulated tissues.
Figure 6
Figure 6
Representative electron micrographs of 60 nm thick longitudinal sections of unstimulated tracheal muscle strips. A, untreated muscle strips; B, strips treated with 10 μm cytochalasin-D; C, strips treated with 1 μm latrunculin. Scale bar, 0.5 μm.
Figure 7
Figure 7
Representative confocal images of single dissociated unstimulated tracheal smooth muscle cells stained with rhodamine-phalloidin. Top, untreated; middle, treated with 10 μm cytochalasin-D; bottom, treated with 1 μm latrunculin-A.
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