Evaluation of proximity inhibition of DNA synthesis in 3T3 cells.

A microphotometric technique that displays rapid length changes of Spirostomum has been used to follow the variation with temperature of three kinetic parameters of myonemal contraction: contraction rate, relaxation rate and stimulus duration at threshold. In each case the exponential form of the relationship indicated that the gross rate constant might be equated with the limiting rate constant, k, of a driving chemical reaction, and from standard expressions of chemical kinetics the change in activation free energy appropriate to this reaction has been computed. The thermal dependence of contraction is described by an activation enthalpy (ΔLH?) of 21.7 kcal mol^?1, and the activation entropy (ΔLS?) of 26.8 e.u. is consistent with a model of contraction requiring neutralization of fixed myonemal charges by divalent cations. The analysis of thermal dependence of relaxation gives a negative activation entropy, a result predicted for a rate-limiting reaction involving dissociation of a neutral molecule. On the other hand, values of ΔLS? and ΔLH? for relaxation fall close to an isokinetic correlation drawn in the literature from analysis of the thermal dependence of ciliary beat frequency in different organisms, and for which breakdown of an ATP-ATPase complex could be the common rate-limiting reaction. ΔLS? for stimulus duration suggests that the rate-limiting step in excitation-contraction coupling is a reaction between ions of like charge, or ion pair formation from a neutral molecule.     

Energy-dependent contraction of swollen heart mitochondria--activation by butacaine.

Butacaine and certain other local anesthetics markedly stimulate the rate, extent, and efficiency of respiration-dependent contraction of heart mitochondria in nitrate salts at alkaline pH. The local anesthetics also induce respiratory control associated with contraction (i.e., the elevated rate of respiration during contraction declines to a State 4-like controlled rate when contraction is complete) so that the reaction at alkaline pH closely resembles the rapid and highly efficient process seen at neutral pH. Respiration-dependent contraction appears to be an osmotic response to cation extrusion on an endogenous cation/H⁺ exchanger (G. P. Brierley, M. Jurkowitz, E. Chavez, and D. W. Jung, 1977, J. Biol. Chem.252, 7932–7939). At alkaline pH, net ion extrusion is slow and inefficient due to the elevated permeability of the membrane to monovalent cations through a putative uniport pathway. Butacaine and other local anesthetics seem to decrease influx-efflux cycling of cations at alkaline pH by restricting cation influx through this uniport. Passive swelling at pH 8.3 in nitrate salts indicates that the uniport reaction is sensitive to Ca²⁺ and has a cation-selectivity of Na⁺ > K⁺ > Li⁺. Butacaine does not inhibit passive swelling under these conditions but produces effects identical to those of classical uncouplers and consistent with increased H⁺ conductance and accelerated influx of cations by cation/H⁺ exchange in nonrespiring mitochondria. However, since contraction in respiring mitochondria is inhibited by uncouplers but stimulated by butacaine, it is apparent that butacaine is not an effective proton conductor in energized mitochondria.     

Energy transfer and molecular switching II. Muscle contraction and enzymatic reactions

In Part I (Barrett, 1981), the concept of chemical parametric excitation was reviewed and applied to the process of nerve action potential excitation and regeneration. In the present paper, the chemical reactions involved in muscle contraction and the enzymatic reaction are examined and shown to be examples of chemical parametric excitation.It is demonstrated that in a model biochemical scheme for an enzymatic reaction, the enzyme is activated from a state, X, to a state, $\text{X}{}^1$, and in this activated state pumps the reaction parametrically. The concept of enzyme is identified with an excited state or state of disequilibrium permitting a release of energy during the dissipation, $\text{X}{}^1\text{→X}$, in the enzymatic reaction, which is powered by the release of energy in the return to the unexcited state X. The demonstration of parametric excitation relations for chemical systems indicates an explanation for the directionality of energy flow and designates an energy pumping role for an enzyme.In muscle contraction, the role of $\text{X}{}^1$ is played by actomyosin and Ca²⁺, and the enzymatic reaction is the hydrolysis of ATP. The release of energy caused by this hydrolysis reaction brings about the conformational changes underlying muscle contraction.     

In vivo electrophysiological responses of pedunculopontine neurons to static muscle contraction

The pedunculopontine nucleus (PPN) has previously been implicated in central command regulation of the cardiorespiratory adjustments that accompany exercise. The current study was executed to begin to address the potential role of the PPN in the regulation of cardiorespiratory adjustments evoked by muscle contraction. Extracellular single-unit recording was employed to document the responses of PPN neurons during static muscle contraction. Sixty-four percent (20/31) of neurons sampled from the PPN responded to static muscle contraction with increases in firing rate. Furthermore, muscle contraction-responsive neurons in the PPN were unresponsive to brief periods of hypotension but were markedly activated during chemical disinhibition of the caudal hypothalamus. A separate sample of PPN neurons was found to be moderately activated during systemic hypoxia. Chemical disinhibition of the PPN was found to markedly increase respiratory drive. These findings suggest that the PPN may be involved in modulating respiratory adjustments that accompany muscle contraction and that PPN neurons may have the capacity to synthesize muscle reflex and central command influences.     

Calcium efflux during the cold-induced contraction of mammalian striated muscle fibres

The efflux of ⁴⁵Ca from mammalian slow twitch muscle fibres has been studied to provide a measure of the concentration of free Ca²⁺ in the sarcoplasm. The kinetically complex early phases of washout of the isotope are succeeded by a prolonged slower phase which exhibits first-order kinetics. This later phase is accelerated by caffeine, by preventing oxidative phosphorylation and also during an isometric contraction, whether this contraction is produced by lowering the temperature or by electrical stimulation. The local anaesthetic tetracaine abolishes the contraction caused by cold and in this case the rate constant for efflux is progressively lowered as the temperature is reduced (Q₁₀ value of 2.3). The removal of external Na⁺ and Ca²⁺ reduces the efflux rate constant. Caffeine, sodium removal and the inhibition of oxidative phosphorylation, all potentiate the cold contraction and the associated extra ⁴⁵Ca efflux. Ca removal causes the cold contraction to become phasic. It appears that caffeine, sodium removal, the inhibition of oxidative phosphorylation and a decrease in temperature to below 10°C are all treatments which, like electrical stimulation, increase the sarcoplasmic free calcium concentration to varying degrees.     

Femoral artery inflow in relation to external and total work rate at different knee extensor contraction rates.

Whether limb blood flow is directly regulated to match the work rate, independent of the rate of contraction, remains elusive. This study therefore investigated the relationship between femoral arterial blood flow (FABF; Doppler ultrasound) and "external" (applied load) as well as "total" [external + "internal" (potential and kinetic energy changes of the moving lower leg)] work rate, during steady-state one-legged, dynamic, knee extensor exercise (1L-KEE) in the sitting position at different contraction rates. Ten subjects performed 1L-KEE at 30, 60, and 90 contractions/min (cpm) 1) at constant resistive loads of 0.2 and 0.5 kg inducing incremental external work rates (study I) and 2) at different relative resistive loads inducing constant external work rates of 9 and 18 W (study II). Moreover, 3) six subjects performed 1L-KEE at 60 and 100 cpm at incremental total work rates of 40, 50, 60, and 70 W (study III). In study I, FABF increased (P < 0.001) with increasing contraction frequency and external work rate, for each resistive load. In study II, FABF increased (P < 0.001) with increasing contraction frequency for each constant external work rate. Of major importance in study III, however, was that FABF, although increasing linearly with the total work rate, was not different (P = not significant) between contraction rates, at the total work rates of 40, 50, 60, and 70 W, respectively. Furthermore, FABF correlated linearly and positively with both the external and total work rate for each contraction frequency. In conclusion, the findings support the concept that leg blood flow during 1L-KEE in a normal knee extensor ergometer is matched directly in relation to the total work rate and metabolic activity, irrespective of the contraction frequency. The rate of contraction seems erroneously to influence the results only when it is related to the external work rate without taking into account the internal work component.     

Mechanically stimulated cytoskeleton rearrangement and cortical contraction in human neutrophils.

A mechanical test with micropipets is used to characterize cytoskeleton rearrangement and contraction induced by mechanical stresses in human neutrophils. The yield shear resultant of the cell cortex is on the order of 0.06 to 0.09 mN.m-1. The measured yield shear resultant suggests that the neutrophil cortex is a weakly cross-linked structure. When a tether is pulled out from the cell surface, a polymer structure starts to fill it and spreads out from the cell body. The rate of advancement of the polymerization front is almost constant and, therefore, is not diffusion limited. The measured rate is much smaller than the one of spontaneous actin polymerization, suggesting that the limiting process is either the dissociation of actin monomers from their dimers with the capping proteins or the rate of formation of new nucleation sites or both. Polymerization is also observed after applying sufficient mechanical stresses on a small portion of the cell surface. The polymerization is followed by mass transfer from the cell into the prestressed region and later on by contraction of the main cell body. The pressure generating the flow is located in the prestressed region and most probably is a result of its "swelling" and contraction. The contraction of the main cell body is very similar (in its time dependence and magnitude) to the contraction during phagocytosis. The measured maximum cortical tension is on the order of 0.5 mN.m-1, which for a 3.5-microns diameter pipet corresponds to a maximum contraction force of 11 nN.     

Dynamics of myosin-driven skeletal muscle contraction: I. Steady-state force generation

Skeletal muscle contraction is a canonical example of motor-driven force generation. Despite the long history of research in this topic, a mechanistic explanation of the collective myosin force generation is lacking. We present a theoretical model of muscle contraction based on the conformational movements of individual myosins and experimentally measured chemical rate constants. Detailed mechanics of the myosin motor and the geometry of the sarcomere are taken into account. Two possible scenarios of force generation are examined. We find only one of the scenarios can give rise to a plausible contraction mechanism. We propose that the synchrony in muscle contraction is due to a force-dependent ADP release step. Computational results of a half sarcomere with 150 myosin heads can explain the experimentally measured force-velocity relationship and efficiency data. We predict that the number of working myosin motors increases as the load force is increased, thus showing synchrony among myosin motors during muscle contraction. We also find that titin molecules anchoring the thick filament are passive force generators in assisting muscle contraction.     

Theory of muscle contraction II. Isotonic contraction

The theory of muscle contraction developed in Part I is extended to non-isometric cases. The basic feature of the approach is the strong viscous coupling of the movement of the counterionic (K⁺) layer with the movement of I-filaments. The surface conductance of the K⁺ layer governs the flux of H⁺ along the I-filaments which in turns regulates the rate of ATP hydrolysis. The energy output of the muscle becomes the function of its mechanical activity. By assuming linear dependence of the K⁺ layer's surface conductance on the velocity of shortening Hill's equation has been derived. With a set of reasonably chosen values of the basic parameters of the theory the values of Hill's constants have been computed. The theory has been also shown to provide the observed dependence of the isometric tension on the degree of the myofilamental overlap.     

Continuum rheology of muscle contraction and its application to cardiac contractility.

A set of constitutive equations is proposed to describe the mechanics of contraction of skeletal and heart muscle. Fiber tension is assumed to depend on the degree of chemical activation, the stretch ratio, and the rate of stretching of the fibers. The time rate of change of activation is governed by a differential equation. The proposed constitutive equations are used to model the time courses of isotonic and isometric twitches during contraction and relaxation phases of the muscle response to stimulation. Various contractility indices of the left ventricle are considered next by using the proposed constitutive equations. The present analysis introduces a new interpretation of the index of contractility (dP/dt)/P used in cardiac literature. It is shown that this index may not be related at all to the maximum speed of shortening and that it may be dependent on both preload and afterload. The development of pressure during isovolumetric contraction of the left ventricle is shown to be governed by a differential equation describing the time rate of change of tension during isometric contraction of myocardium fibers.     

Heat production and metabolism during the contraction of mammalian skeletal muscle

Methods are described whereby initial processes of muscular contraction may be investigated in a mammalian preparation, the soleus muscle of the rat. Conditions are chosen so that recovery is avoided. An isometric tetanus is investigated and an energy balance sheet is drawn up. It is found that there is more heat evolved than can be accounted for in terms of measured chemical reaction. This discrepancy is discussed with reference to the similar results that have been obtained using frog muscle.     

Energy-dependent contraction of swollen mitochondria: activation by nigericin.

The rate, extent, and efficiency of the energy-dependent contraction of heart mitochondria swollen in Na+ or K+ nitrate are all strongly activated by nigericin, an antibiotic which is known to support cation/H+ exchange in natural and model membranes. In the absence of nigericin, the cation selectivity sequence of energy-dependent contraction (Na+>Li+>K+>choline+) is identical to that of passive swelling in acetate salts, a reaction which is presumed to be dependent on an endogenous cation/H+ exchanger. These results strongly favor an osmotic mechanism for energy-dependent contraction which depends on electrogenic H+ ejection, H+/cation exchange, and electrophoretic anion efflux.     

High-speed video cinematographic demonstration of stalk and zooid contraction of Vorticella convallaria.

Stalk contraction and zooid contraction of living Vorticella convallaria were studied by high-speed video cinematography. Contraction was monitored at a speed of 9000 frames per second to study the contractile process in detail. Complete stalk contraction required approximately 9 ms. The maximal contraction velocity, 8.8 cm/s, was observed 2 ms after the start of contraction. We found that a twist appeared in the zooid during contraction. As this twist unwound, the zooid began to rotate like a right-handed screw. The subsequent stalk contraction steps, the behavior of which was similar to that of a damped harmonic oscillator, were analyzed by means of the equation of motion. From the beginning of stalk contraction, the Hookean force constant increased, and reached an upper limit of 2.23 x 10(-4) N/m 2-3 ms after the start of contraction. Thus, within 2 ms, the contraction signal spread to the entire stalk, allowing the stalk to generate the full force of contraction. The tension of an extended stalk was estimated to be 5.58 x 10(-8) N from the Hookean force constant of a stalk. This value coincides with that of the isometric tension of a glycerol-treated V. convallaria, confirming that the contractile system of V. convallaria is well preserved despite glycerol treatment.     

A kinetic model that explains the effect of inorganic phosphate on the mechanics and energetics of isometric contraction of fast skeletal muscle

A conventional five-step chemo-mechanical cycle of the myosin–actin ATPase reaction, which implies myosin detachment from actin upon release of hydrolysis products (ADP and phosphate, Pi) and binding of a new ATP molecule, is able to fit the [Pi] dependence of the force and number of myosin motors during isometric contraction of skeletal muscle. However, this scheme is not able to explain why the isometric ATPase rate of fast skeletal muscle is decreased by an increase in [Pi] much less than the number of motors. The question can be solved assuming the presence of a branch in the cycle: in isometric contraction, when the force generation process by the myosin motor is biased at the start of the working stroke, the motor can detach at an early stage of the ATPase cycle, with Pi still bound to its catalytic site, and then rapidly release the hydrolysis products and bind another ATP. In this way, the model predicts that in fast skeletal muscle the energetic cost of isometric contraction increases with [Pi]. The large dissociation constant of the product release in the branched pathway allows the isometric myosin–actin reaction to fit the equilibrium constant of the ATPase.     

Maintenance of the contraction time of the smooth muscle cell

It has been analyzed the speed of contraction (measured as Vmax) of guinea-pig intestine in vitro, after stimulation with carbachol. High doses of carbachol induce an high Vmax; but repeating the dose at short interval (4 min) the speed of contraction is reduced until it reaches values of Vmax 4,3. Low doses of carbachol determine a low Vmax that with repeating doses increases until Vmax 4,2. On the base of this tendency at the same Vmax, and from the anomalous behaviour of the intestines to one dose in front to different doses in carbachol. The excludes that the process could be due to a passive adaptation and hypothesizes a model of behaviour inside the cell. A model to which the myocells balance the stimulus in the way to make constant their velocity of contraction.     

Myonemal contraction of Spirostomum. I. Kinetics of contraction and relaxation

A microphotometric method is introduced that allows measurement of the contraction-relaxation kinetics of Spirostomum in response to electrical stimulation. The time course of contraction includes a rapidly contracting phase of some 4–5 mS during which cells shorten at a rate in excess of 100 cell lengths sec^?1. While a stimulus strength-duration curve determines the threshold of the response, the response to above threshold stimuli of different strengths and to trains of stimuli suggest that contraction of Spirostomum may not be an all-or-none event. The kinetics of relaxation following high stimulating voltages and repetitive after contractions also induced by high voltages are explained by excitation-contraction coupling through a stimulus-dependent intermediate effector, possibly the release of calcium ions. Changes in resting membrane potential detected by intracellular recording do not influence the initiation of contraction, while microinjection of calcium buffers above 10^?5 M Ca²⁺ invariably induces contraction.     

Nonuniform volume changes during muscle contraction.

We measured dynamic changes in volume during contraction of live, intact frog skeletal muscle fibers through a high-speed, intensified, digital-imaging microscope. Optical cross-sections along the axis of resting cells were scanned and compared with sections during the plateau of isometric tetanic contractions. Contraction caused an increase in volume of the central third of a cell when axial force was maximum and constant and the central segment was stationary or lengthened slightly. But changes were unequal along a cell and not predicted by a cell's resting area or shape (circularity). Rapid local adjustments in the cytoskeletal evidently keep forces in equilibrium during contraction of living skeletal muscle. These results also show that optical signals may be distorted by nonuniform volume changes during contraction.     

Protease induced novel ring contraction reaction of 1,3-oxazin-4-one derivatives

Protease induced ring contraction reaction of ethyl-4-oxo-3-phenyl-l-oxa-5-azaspiro[5,5]-undec-2-ene-2-car☐ylate (1) yielded 4-phenyl-3-hydroxy-1H-pyrrole-2,5-dione (5). This product and its derivatives have been characterized by comparing their total identity with authentic compounds. Involvement of basic amino acid residues for the initiation of the ring contraction reaction by abstracting the proton at position-3 of the oxazinone ring has been suggested. Chemical evidence for the base catalyzed reaction pathway of compound1 leading to the formation of compound5 is presented.     

Effect of bronchial smooth muscle contraction on lung compliance.

Lung compliance is generally considered to represent a blend of surface and tissue forces, and changes in compliance in vivo are commonly used to indicate changes in surface forces. There are, however, theoretical arguments that would allow contraction of airway smooth muscle to affect substantially the elasticity of the lung. In the present study we evaluated the role of conducting airway contraction on lung compliance in vivo by infusing methacholine (MCh) at a constant rate into the bronchial circulation. With a steady-state MCh infusion of 2.4 micrograms/min into the bronchial perfusate (perfusate concentration = 0.7 microM), there was an approximate doubling of lung resistance and a 50% fall in dynamic compliance. There were also significant decreases in chord compliance measured from the quasi-static pressure-volume curves and in total lung capacity and residual volume. When the same infusion rate was administered into the pulmonary artery, no changes in lung mechanics were observed. These results indicate that the conducting airways may have a major role in regulating lung elasticity. This linkage between airway contraction and lung compliance may account for the common observation that pharmacological challenges given to the lung usually result in similar changes in lung compliance and airway conductance. Our results also suggest the possibility that the lung tissue resistance, which dominates the measurement of lung resistance in many species, might in fact reflect the physical properties of conducting airways.     

Autonomic energy conversion. II. An approach to the energetics of muscular contraction

All discussions of muscle energetics concern themselves with the Hill force-velocity relation, which is also the general output relation of a class of self-regulated energy converters and as such contains only a single adjustable parameter —the degree of coupling. It is therefore important to see whether in principle muscle can be included in this class. One requirement is that the muscle should possess a working element characterized by a dissipation function of two terms: mechanical output and chemical input. This has been established by considering the initial steady phase of isotonic and isometric tetanic contraction to represent a stationary state of the fibrils (a considerable body of evidence supports this). Further requirements, which can be justified for the working element, are linearity and incomplete coupling. Thus the chemical input of the muscle may be expected to follow the inverse Hill equation (see Part I). The relatively large changes in activities of reactants which the equation demands could only be controlled by local operation of the regulator, and a scheme is outlined to show how such control may be achieved. Objections to this view recently raised by Wilkie and Woledge rest on at least two important assumptions, the validity of which is questioned: (a) that heat production by processes other than the immediate driving reaction is negligible, which disregards the regulatory mechanism (possibly this involves the calcium pump), and (b) that the affinity of the immediate driving reaction is determined by over-all concentrations. The division of heat production into “shortening heat” and “maintenance heat” or “activation heat” is found to be arbitrary.