Exogenous caldesmon promotes relaxation of guinea-pig skinned taenia coli smooth muscles: inhibition of cooperative reattachment of latch bridges?

 In smooth muscle, the state of prolonged contraction (latch state) is associated with very slow energy turnover and cycling of crossbridges that are dephosphorylated. A similar state may be reproduced in skinned fibres when the calcium-induced contraction is terminated by calcium removal with ethylenebis(oxonitrilo)tetraacetate (EGTA) and, during the slow relaxation that follows, force is maintained by dephosphorylated crossbridges that cycle slowly or not at all and may cooperatively reattach after detachment (Khromov et al. 1995, Biophys J 69:2611–2622). In guinea-pig skinned taenia coli that has been pretreated by prolonged incubation with caldesmon (5 μM), the rate of relaxation is approximately 1.6 times greater than in untreated controls, with half-times of relaxation being 1.3 and 2.1 min, respectively. In contrast, preloading the fibres with calponin does not accelerate relaxation. Preloading the fibres with caldesmon also accelerates the relaxation of skinned fibres from the state of rigor contraction when the latter is terminated by immersion into an ATP-containing relaxing solution or, in the presence of inorganic phosphate (Pi), also by flash-photolytic release of ATP from caged-ATP. Even in the latter case, relaxation is comparatively slow, possibly because of cooperative reattachment of dephosphorylated crossbridges which delays net crossbridge detachment and hence relaxation. We propose that by inhibition of cooperative reattachment caldesmon accelerates relaxation, even in the presence of Pi, and that the latch-like state of skinned fibres is supported by dephosphorylated cooperatively attaching crossbridges and may be regulated by the activity of caldesmon in the smooth muscle cell. Received: 25 February 1997 / Received after revision: 29 May 1997 / Accepted: 2 June 1997     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.     

Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle

The relation between the isoform distribution of the myosin 17 kDa essential light chain (LC₁₇) and the mechanical properties of smooth muscle was investigated. The relative content of the basic (LC_17b) and acidic (LC_17a) isoelectric variants of the 17 kDa myosin light chain was determined in different mammalian smooth muscle tissues. The relative content of LC_17b varied between muscles: rabbit rectococcygeus 0%, rabbit trachea 5%, guinea-pig taenia coli 21%, rat uterus 38%, rabbit aorta 56% and rat aorta 60%. The rate of tension development was determined following photolysis of cagedadenosine triphosphate (ATP) in skinned fibres activated with thiophosphorylation of the regulatory light chains. The half-time for force development was 0.67 s in rabbit rectococcygeus, 1.6 s in rabbit trachea, 1.13 s in guineapig taenia coli and 1.38 s in rabbit aorta. The maximal shortening velocity (v _max) was determined with the isotonic quick release technique in skinned fibre preparations activated with thiophosphorylation. v _max was 0.25 muscle lengths per second (ML/s) in rabbit rectococcygeus, 0.24 ML/s in rabbit trachea, 0.17 ML/s in guinea-pig taenia coli, 0.11 ML/s in rat uterus and 0.03 ML/s in rabbit aorta. The range of variation in v _max between muscles was larger than in the half-time for force development. The inverse relationship between v _max and the relative content of LC_17b in the investigated muscles suggests that the type of essential myosin light chain influences the v _max in smooth muscle.     

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca²⁺. The ionic strength was adjusted with either KMeSO₃ or ethylene glycol bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid, dipotassium salt (K₂EGTA) in the range of /2=65–215 mM, or I.E. 49–194 mM, where I.E. denotes ionic equivalent. Following rigor development at a/2 of 165–215 mM (I.E. 144–194 mM), the liberation of approximately 0.5 mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10–20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a /2 of 65 mM and 110 mM (I.E. 60–100 mM), the initial detachment phase was shortened and force decreased by only approximately 5–10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20–80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65 mM and 215 mM.     

A low cost high intensity flash device for photolysis experiments

Novel techniques of flash photolysis experiments require high intensity light sources in the near UV. We describe here a simple and inexpensive flash device which may compete with bulky and expensive laser systems if the experiments do not necessitate very short light pulses. Using a particular optical arrangement and stored electrical energy, a variation of the parameters voltage and capacitance led to a difference in light output by a factor of more than two. The system is used to relax both skeletal and smooth muscle fibres in the rigor state by releasing up to 2 mM ATP from 12.5 mM caged-ATP.