Latrunculin B increases force fluctuation-induced relengthening of ACh-contracted, isotonically shortened canine tracheal smooth muscle

Abstract

-We hypothesized that differences in actin filament length could influence force fluctuationinduced relengthening (FFIR) of contracted airway smooth muscle and tested this hypothesis as follows. One-hundred micromolar AChstimulated canine tracheal smooth muscle (TSM) strips set at optimal reference length (L ref) were allowed to shorten against 32% maximal isometric force (F max) steady preload, after which force oscillations of Ϯ16% F max were superimposed. Strips relengthened during force oscillations. We measured hysteresivity and calculated FFIR as the difference between muscle length before and after 20-min imposed force oscillations. Strips were relaxed by ACh removal and treated for 1 h with 30 nM latrunculin B (sequesters G-actin and promotes depolymerization) or 500 nM jasplakinolide (stabilizes actin filaments and opposes depolymerization). A second isotonic contraction protocol was then performed; FFIR and hysteresivity were again measured. Latrunculin B increased FFIR by 92.2 Ϯ 27.6% L ref and hysteresivity by 31.8 Ϯ 13.5% vs. pretreatment values. In contrast, jasplakinolide had little influence on relengthening by itself; neither FFIR nor hysteresivity was significantly affected. However, when jasplakinolide-treated tissues were then incubated with latrunculin B in the continued presence of jasplakinolide for 1 more h and a third contraction protocol performed, latrunculin B no longer substantially enhanced TSM relengthening. In TSM treated with latrunculin B ϩ jasplakinolide, FFIR increased by only 3.03 Ϯ 5.2% L ref and hysteresivity by 4.14 Ϯ 4.9% compared with its first (pre-jasplakinolide or latrunculin B) value. These results suggest that actin filament length, in part, determines the relengthening of contracted airway smooth muscle.actin filament dynamics; force oscillations; isotonic contractions; hysteresivity A NUMBER OF PREVIOUS STUDIES in animals (22,24,25,27) and humans (7,29) have demonstrated that tidal breathing per se reduces airway constriction during contractile stimulation, and deep breathing does so more effectively. Even a single deep inspiration can substantially reverse experimentally induced bronchoconstriction in normal individuals (1, 4, 11). However, the ability of deep breathing to dilate the airways is absent in asthmatic subjects (4, 11). Understanding why this protective mechanism fails in asthma is the focus of ongoing investigation in several laboratories and is the ultimate goal of the present study.Fredberg and coworkers (13, 14) studied the influence on airway smooth muscle shortening of the load fluctuations imposed by breathing. They stimulated bovine trachealis strips with ACh and allowed them to shorten isotonically against a constant load to a steady-state length and then superimposed sinusoidal force oscillations of increasing amplitudes (to simulate tidal breathing) on the constant mean load (5). These increasing force oscillations caused substantial smooth muscle relengthening, despite continued contractile stimulation. They showed that relengthening coul...

Force Fluctuation induced Relengthening of Acetylcholine-contracted Airway Smooth Muscle

“…dynamics (i.e., cycling rate) likely plays a role. Fredbergs's laboratory and our work (Figures 2 and 3) have demonstrated increased hysteresivity with larger force fluctuations (1)(2)(3). Hysteresivity is a reflection of actomyosin crossbridge cycling.…”

“…We also found that the phosphorylation level of the regulatory myosin light chain 20 (rMLC 20 ) was not different between control and latrunculin B-treated tissues that had been flash-frozen near the end of the 20-minute oscillation protocol (1). These data indicated that the increased FFIR in the presence of latrunculin B could not be accounted for by a reduction of crossbridge activation in these TSM preparations.…”

“…We have presented published data that strongly implicate FFIR as a key component of this breathing effect (1)(2)(3)(4). Furthermore, we have suggested, based on our previous work, that actin filament length is a major determinant of FFIR (1). Actin filament length is regulated by many pathways, including those that may be activated by inflammatory mediators known to be present in asthmatic airways.…”

“…We suspect that the augmentation of FFIR with p38 MAPK inhibition may be due in part to inhibition of HSP27 phosphorylation, preventing its uncapping from actin and thus favoring shorter actin filaments. Shorter actin filaments allow for a greater degree of relengthening with force fluctuations (1). Of note, p38 MAPK is activated by a number of inflammatory mediators and cytokines (23,24).…”

“…That is, it seems that the airways (airway smooth muscle) of healthy individuals reacted differently to the stretch from a DI than did those of individuals with asthma. Therefore, we designed in vitro experiments (1,4) to assess whether oscillating (to simulate breathing) contracted and shortened airway smooth muscle (to simulate narrowed bronchi) could be relengthened (airway opening) and to determine whether the relengthening could be physiologically regulated.…”