Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

Interplay of Troponin- and Myosin-Based Pathways of Calcium Activation in Skeletal and Cardiac Muscle: The Use of W7 as an Inhibitor of Thin Filament Activation

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (K_I) were 70–80 μM for ATPase and 20–30 μM for tension, nearly >200-fold lower than BDM (20 mM and 5–8 mM, respectively). When W7 (50 μM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.     

BDM compared with P(i) and low Ca2+ in the cross-bridge reaction initiated by flash photolysis of caged ATP.

Using flash photolysis of caged ATP in skinned muscle fibers from rat psoas, we examined the inhibitory effects of 2,3-butanedione monoxime (BDM) on the contraction kinetics and the rate of ATP hydrolysis of the cross-bridges at approximately 10 degrees C. The hydrolysis rate was estimated from the stiffness records. The effects of BDM were compared with those of orthophosphate (P(i)) and of reduction in [Ca2+] (low Ca2+), and it was found that i) BDM and low Ca2+ inhibited ATPase activity to the same extent as they inhibited the steady tension, whereas P(i) inhibited ATPase activity much less than tension; ii) BDM and P(i) decreased tension per stiffness during the steady contraction more than did low Ca2+; iii) neither BDM nor low Ca2+ affected the initial relaxation of the fiber on release of ATP, but P(i) slightly slowed it; and iv) BDM hardly influenced the rate of contraction development after relaxation, although P(i) and low Ca2+ accelerated it. We concluded that BDM inhibits the Ca(2+)-regulated attachment of the cross-bridges and force-generation of the attached cross-bridges.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Induction of contractile activity by rigor complexes in myofibrils

The relation between ATPase rate and substrate concentration was investigated for myofibrils with varying amounts of added HMM. There was a biphasic, 3 to 5-fold increase in ATPase in the absence of Ca⁺⁺. In the absence of added HMM, the peak activity occurred at ≤ 0.1 mM MgATP. With increasing concentrations of HMM, the position and magnitude of the ATPase peak shifted to larger substrate concentrations and higher rates. The cofactor activity of regulated actin in myofibrils is activated to a similar degree by Ca⁺⁺ as by HMM (rigor links). SDS gel electrophoretic patterns of myofibrils mixed with HMM indicated the soluble HMM binds to myofibrils at 0.1 mM MgATP and is dissociated at higher MgATP concentrations. Thus, in well-regulated myofibrils in the absence of Ca⁺⁺ actin cofactor activity can be activated by rigor complexes.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Comparison of effects of smooth and skeletal muscle tropomyosins on interactions of actin and myosin subfragment 1

The ATPase activity of acto-myosin subfragment 1 (S-1) was measured in the presence of smooth and skeletal muscle tropomyosins over a wide range of ionic strengths (20-120 mM). In contrast to the 60% inhibitory effect caused by skeletal muscle tropomyosin at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on ionic strength. At low ionic strength (20 mM), smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM), it potentiates the ATPase activity 3-fold. All of these ATPase activities were measured at very low ratios of S-1 to actin, under conditions at which a 4-fold increase in S-1 concentration did not change the specific activity of the tropomyosin-acto.S-1 ATPase. Therefore, the potentiation of the ATPase activity by smooth muscle tropomyosin at high ionic strength cannot be explained by bound S-1 heads cooperatively turning on the tropomyosin-actin complex. To determine whether the fully potentiated rates are different in the presence of smooth muscle and skeletal muscle tropomyosins, S-1 which was extensively modified by N-ethylmaleimide was added to the ATPase assay to attain high ratios of S-1 to actin. The results showed that, under all conditions, the fully potentiated rates are the same for both tropomyosins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor.

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.     

Effects of BDM, [Ca2+]o, and temperature on the dynamic stiffness of quiescent cardiac trabeculae from rat

Studies of the passive mechanical properties of cardiac tissue have traditionally been conducted at subphysiological temperatures and various concentrations of extracellular Ca(2+) ([Ca(2+)](o)). More recently, the negative inotropic agent 2,3-butanedione monoxime (BDM) has been used. However, there remains a lack of data regarding the influence of temperature, Ca(2+), and BDM on the passive mechanical properties of cardiac tissue. We have used the dynamic stiffness technique, a sensitive measurement of cross-bridge activity, in which minute (approximately 0.2% of muscle length) sinusoidal perturbations are applied at various frequencies (0.2-100 Hz) to quiescent, viable right ventricular rat trabeculae at two temperatures (20 degrees C and 26 degrees C) and at two [Ca(2+)](o) (0.5 and 1.25 mM) in the presence and absence of BDM (20 mM). The stiffness spectra (amplitude and phase) were sensitive to temperature and [Ca(2+)](o) in the absence of BDM but insensitive in the presence of BDM. From the index of cross-bridge cycling (the ratio of high- to low-frequency stiffness amplitude), we infer that BDM inhibits a small degree of spontaneous sarcomere activity, thereby allowing the true passive properties of trabeculae to be determined. In the absence of BDM, the extent of spontaneous sarcomere activity decreases with increasing temperature. We caution that the measured mechanical properties of passive cardiac tissue are critically dependent on the experimental conditions under which they are measured. Experiments must be performed at sufficiently high temperatures (>25 degrees C) to ensure a low resting concentration of intracellular Ca(2+) or in the presence of an inhibitor of cross-bridge cycling.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

Oxygen exchange between Pi in the medium and water during ATP hydrolysis mediated by skinned fibers from rabbit skeletal muscle. Evidence for Pi binding to a force-generating state

Oxygen exchange between (18O4)Pi in the medium and water accompanies ATP hydrolysis catalyzed by the calcium-regulated MgATPase of vertebrate skeletal muscle. Exchange was observed in chemically skinned fibers from rabbit psoas muscle held isometrically and activated by 30 microM free Ca2+. The rate of exchange was approximately proportional to Pi concentration (up to 10 mM) and was characterized by an apparent second order rate constant greater than or equal to 475 M-1 S-1 (pH 7.1, ionic strength 0.2 M, 22 degrees C). Much less exchange occurred in the absence of Ca2+ or when ATP was replaced by ADP. It has been inferred from mechanical experiments that Pi can bind to a force-generating ADP-bound state of actomyosin with resultant suppression of force (Hibberd, M. G., Dantzig, J. A., Trentham, D. R., and Goldman, Y. E. (1985) Science 228, 1317-1319). The oxygen exchange results support this inference by providing direct evidence that Pi in the medium binds at the ATPase catalytic site in activated isometric fibers. The inter-relationship of these two effects involving Pi on mechanochemical coupling in muscle is discussed.     

Studies on Myofibrils from the Stored Muscle

Adenosine triphosphatase (ATPase) activity of myofibrils isolated from fresh muscle and the muscle stored at 4°C have been measured.

An increase in Mg-activated ATPase activity of myofibrils was caused by lengthened homogenization.

With the progress of aging of muscle, Mg-activated ATPase activity of myofibrils increased remarkably.

When myofibrils from pre-rigor and rigor muscle in 0.16 m KCl were treated with 0.6 m KCl-18 mm Tris-maleate solution (pH 7.0), Mg-activated ATPase activity of myofibrils at low ionic strength increased markedly. However, the Mg-activated ATPase activity of the myofibril isolated from the muscle stored at 4°C for 8 days (8-myofibril) increased slightly after the similar treatment.

The dependence of myofibrillar ATPase activity on KCl concentration became greater with the progress of aging of muscle.

These results may show that, as long as ATPase activity and the dependence of ATPase activity on KCl concentration are concerned, 8-myofibril is the most similar to the isolated actomyosin among myofibrils, although actomyosin in muscle may exist in a different form from that in solution. It is suggested that, with the progress of aging, the structural alteration of myofibril occurred and the myofibril became more susceptible to ATP-induced transformation.     

Isotonic contraction of skinned muscle fibers on a slow time base: effects of ionic strength and calcium

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1 degrees C as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCl to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCl concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCl concentration greater than or equal to 50 mM. In 0 and 20 mM KCl, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCl was about 0.6 and 0.9 times, respectively, that in 140 mM KCl. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCl. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.     

Thymol: a classical small-molecule compound that has a dual effect (potentiating and inhibitory) on myosin

The effect of thymol on the ATPase activity of myosin subfragment-1 (S1) and on the contractile properties of skinned skeletal muscle fibers was studied. At concentrations of 1.5-2 mM, thymol activated the S1 ATPase substantially and the actin-activated S1 ATPase modestly. At the same concentrations, the isometric force of skinned skeletal muscle fibers was modestly suppressed (11% at 2 mM). However, the kinetic parameters of contraction were suppressed more: the velocity of shortening and the rate of force redevelopment after shortening were suppressed by 43% and 31% at 2 mM, respectively. Thus, among other small-molecule inhibitors, thymol is unique in that it has opposite effects on the enzymatic activity and kinetic parameters of contraction. Thymol may serve as a potent tool for studying the mechanism of coupling between the ATPase reaction and contraction in muscle.     

Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures.

Tension responses to ramp stretches of 1-3% Lo (fiber length) in amplitude were examined in resting muscle fibers of the rat at temperatures ranging from 10 degrees C to 36 degrees C. Experiments were done using bundles of approximately 10 intact fibers isolated from the extensor digitorum longus (a fast muscle) and the soleus (a slow muscle). At low temperatures (below approximately 20 degrees C), the tension response consisted of an initial rise to a peak during the ramp followed by a complex tension decay to a plateau level; the tension decay occurred at approximately constant sarcomere length. The tension decay after a standard stretch at approximately 3-4.Lo/s contained a fast, an intermediate, and a (small amplitude) slow component, which at 10 degrees C (sarcomere length approximately 2.5 microns) were approximately 2000.s-1, approximately 150.s-1, and approximately 25.s-1 for fast fibers and approximately 2000.s-1, approximately 70.s-1 and approximately 8.s-1 for slow fibers, respectively. The fast component may represent the decay of interfilamentary viscous resistance, and the intermediate component may be due to viscoelasticity in the gap (titin, connectin) filament. The two- to threefold fast-slow muscle difference in the rate of passive tension relaxation (in the intermediate and the slow components) compares with previously reported differences in the speed of their active contractions; this suggests that "passive viscoelasticity" is appropriately matched to contraction speed in different muscle fiber types. At approximately 35 degrees C, the fast and intermediate components of tension relaxation were followed by a delayed tension rise at approximately 10.s-1 (fast fibers) and 2.5.s-1 (slow fibers); the delayed tension rise was accompanied by sarcomere shortening. BDM (5-10 mM) reduced the active twitch and tetanic tension responses and the delayed tension rise at 35 degrees C; the results indicate stretch sensitive activation in mammalian sarcomeres at physiological temperatures.     

Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].     

Role of protein kinase C in regulating smooth muscle electrical and contractile activity: the effect of phorbol ester

The effects of protein kinase C activation by 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) on the functions of guinea-pig smooth muscle taenia coli have been studied, using double-sucrose-gap method. A 15-20-min treatment of the muscle with 2 X 10(-8) M TPA caused a progressing inhibition of spontaneous electrical activity and mechanical tension, suppression of post-hyperpolarizing electrical and contractile "off-responses", a decrease in the number of action potentials during superthreshold membrane depolarization, depression of electrical and mechanical responses induced by acetylcholine, histamine, bradykinin mediators. The treatment of pre-depolarized (140 mM kappa+) muscle with 2.10(-8) TPA has led to a considerable reduction in contractile responses induced by the above mediators. The results obtained indicate that protein kinase C is capable of regulating both voltage-sensitive and receptor-operated ionic channels in smooth muscle cells.     

Effect of strain on actomyosin kinetics in isometric muscle fibers

Investigations were conducted into the biochemical and mechanical states of cross-bridges during isometric muscle contraction. Rapid length steps (3 or 6 nm hs(-1)) were applied to rabbit psoas fibers, permeabilized and isometric, at either 12 degrees C or 20 degrees C. Fibers were activated by photolysis of P(3)-1-(2-nitrophenyl)-ethyl ester of ATP infused into rigor fibers at saturating Ca(2+). Sarcomere length, tension, and phosphate release were recorded-the latter using the MDCC-PBP fluorescent probe. A reduction in strain, induced by a rapid release step, produced a short-lived acceleration of phosphate release. Rates of the phosphate transient and that of phases 3 and 4 of tension recovery were unaffected by step size but were elevated at higher temperatures. In contrast the amplitude of the phosphate transient was smaller at 20 degrees C than 12 degrees C. The presence of 0.5 or 1.0 mM added ADP during a release step reduced both the rate of tension recovery and the poststep isometric tension. A kinetic scheme is presented to simulate the observed data and to precisely determine the rate constants for the elementary steps of the ATPase cycle.