Muscle temperature, contractile speed, and motoneuron firing rates during human voluntary contractions.

A study was made of motoneuron firing rates and mechanical contractile parameters during maximum voluntary contraction of human hand muscles. A comparison of muscles that had been fatigued after a 60-s maximum voluntary contraction (MVC) with muscles that were cooled by approximately 5 degrees C showed that the contractile properties, in particular the rates of contraction and relaxation, were similarly affected in both conditions. In contrast, the motoneuron firing rate was affected differently by the two treatments. In the case of the fatigued muscles the motoneuron firing rate was reduced by 36%, as was expected from previous studies, but in the case of the cooled muscles, there was no significant change in the motoneuron firing rate. We conclude that the reflex reduction in the motoneuron firing rate seen in the fatigued muscle is not triggered directly by a change in the mechanical properties of the muscle.

A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase.

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.

Variation in the normalized tetanic force of single frog muscle fibres.

1. The forces produced in maximal fixed-end tetani of single fibres isolated from the anterior tibialis muscle of the frog Rana temporaria have been measured at sarcomere lengths of 2.2 microns and temperatures near 0 and 10 degrees C. 2. When normalized by either cross-sectional area or dry weight per unit length at a sarcomere length of 2.2 microns, the forces vary over a twofold range. 3. The normalized force is not significantly correlated with the velocity of unloaded shortening or the twitch characteristics of the fibres. Lack of variability of these two quantities (together with histochemical evidence) suggest that only one fibre type is present in the experimental sample. 4. The steady rate of energy liberation (stable, heart rate) of the fibres during isometric tetani is positively correlated with the normalized force, indicating that extra ATP splitting is required to produce higher forces. 5. Fibres with a higher ratio of dry weight per unit length to cross-sectional area ('dry density') show a higher force when normalized by area, but not when normalized by dry weight per unit length. 6. Fibres with a more circular cross-sectional profile produce more force when normalized by either cross-sectional area or dry weight per unit length. The significance of this correlation is unclear. 7. The contribution of various sources to the total overall variation in normalized force is assessed. It is suggested that a diffusible substance or substances may be involved in modulating fibre force.

The energetics of work and heat production by single muscle fibres from the frog.

During active shortening the heat rate in isolated muscle fibres is greater than during isometric contraction, and increases with velocity of shortening (V), but at a decreasing rate as the maximum velocity (V0) is approached. For shortening at V less than 0.25 V0 the amount of extra heat produced during a period of shortening is proportional to the distance shortened, but for rapid shortening (V greater than 0.5 V0) the extra heat increases less than proportionally with the distance shortened. After a period of shortening the higher heat rate returns to the isometric level over a period of several hundred milliseconds. A similar period of increased heat rate is seen after a quick release. After shortening 10% of muscles slack length at 0.1 V0 the amount of heat produced during tension redevelopment is similar to that after a quick release. But after more rapid shortening there is less heat production than after a quick release.

A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP.

1. The enthalpy changes during individual reaction steps of the myosin subfragment 1 ATPase were studied with the use of a new stopped-flow calorimeter [Howarth, Millar & Gutfreund (1987) Biochem. J. 248, 677-682]. 2. At 5 degrees C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (delta H = +64 kJ.mol-1). 3. ADP binding is accompanied by a biphasic enthalpy change. 4. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization. 5. Protons are involved in all four principal steps of the myosin subfragment 1 ATPase.

A stopped-flow calorimeter for biochemical applications.

A rapid-response stopped-flow calorimeter for small samples of reagents is described. The construction, performance characteristics and operational limitations are described, along with an example of its ability to resolve the kinetics of an enzyme-catalysed hydrolysis. It is thought likely that the method would find useful application in a variety of chemical and biochemical investigations.

Absolute values of myothermic measurements on single muscle fibres from frog.

The heat and force produced in tetanic contraction of single fibres from anterior tibialis muscle of the frog Rana temporaria have been observed at measured temperatures close to 1 and 10 degrees C. Heat was measured using a Hill-Downing type thermopile. In control experiments with a resistor of known heat capacity comparable to a single muscle fibre, it was found that Peltier and Joule heating produced identical thermopile outputs. The Peltier method was used to introduce a known amount of heat into the system in each experiment with a muscle fibre. From the response to this heating the heat capacity of each preparation was obtained and used to calculate the absolute amount of heat production from the thermopile output. The heat produced during tetanic contraction (H) could be described by Aubert's equation [H = Ha (1 - e-t/tau) + hbt]. In some fibres there was no labile heat (Ha), whereas in others it was clearly present. The stable heat rate (hb) was strongly temperature dependent (Q10 = 4.06). At 0 degree C the stable heat rate (normalized by dry weight) in the single fibres was significantly greater than that in whole anterior tibialis muscle.

Simultaneous heat and tension measurements from single muscle cells.

Simultaneous force and heat measurements were made in single cells from skeletal muscle of the frog during isometric twitches and tetani at 10 and 0 degree C. A Hill- Downing type thermopile of low heat capacity was used. In twitches, peak force development was found to be well correlated with heat production at both temperatures, during posttetanic twitch potentiation (at 10 degrees C) and during posttetanic twitch depression (at 0 degree C). In a twitch at 0 degree C, heat production started less than 14 msec after the stimulus had begun, before force development. As in whole muscle, the heat during a tetanus could be separated into two components: an early component produced at an exponentially decreasing rate, labile heat, and a steady rate, stable maintenance heat rate. Increasing temperature from 0 to 10 degrees C doubled the stable maintenance heat rate. At the higher temperature the time constant of labile heat production was halved and the quantity of labile heat decreased. When two tetani were given at 10 degrees C, a 5 min rest interval was required before the second tetanus produced the same force and heat as the first. At 0 degree C this interval was at least 10 min. With shorter intervals, both heat and force were depressed. At 10 degrees C both were depressed equally but at 0 degree C the effect on heat was greater than on force. At both temperatures labile heat was depressed to a greater extent than the stable maintenance heat rate. Results are interpreted in terms of possible calcium-parvalbumin interaction during a tetanus.

Heat production by single fibres of frog muscle.

The heat produced during contractions of preparations consisting of one or a few muscle fibres was measured for the first time. Fibres were dissected from the anterior tibialis muscles of the frog, Rana temporaria. Measurements were made with thermopiles of a design based on that described by Howarth et al. (1975). Although the fibre preparations were small, measurable signals could be recorded because the heat capacity of the thermopiles was also small. The output of the thermopile was amplified by a galvanometer circuit. In all the experiments the ends of the preparation were held in a fixed position during stimulation ("isometric'). Observations were made of heat production during twitches and tetanic contractions. The heat produced in a twitch of a single fibre depended on the stimulus strength in an all-or-nothing way. The results show that the amount of heat produced in individual twitches is fairly constant at different temperatures in the range 3-15 degrees C. In contrast, the heat produced in tetanic contractions is considerably greater at higher temperatures. The time course of heat production in a tetanus was influenced by temperature such that the early rapid phase of heat production was less obvious at the higher temperature. The quantities of heat produced by fibre preparations were in reasonable agreement with those produced by whole muscles when the comparison was made on the basis of heat produced per g wet weight of tissue.

The initial heat production in garfish olfactory nerve fibres.

A study has been made of the temperature changes associated with the passage of a single impulse in the non-myelinated fibres of the garfish olfactory nerve: and the time course of these temperature changes has been compared with the time course of the electrical events during the action potential. As in other non-myelinated nerves studied the observed temperature changes result from a biphasic initial heat production consisting of a transient evolution of heat (the positive heat) followed by a rapid heat reabsorption (referred to as the negative heat). There is no evidence of any additional phases of initial heat production. At 0 degrees C the measured positive initial heat is 224 mucal/g impulse (937 muJ/g impulse); and the corresponding negative initial heat is 230 mucal/g impulse (962 muJ/g impulse). The residual initial heat is very small, being about -6 mucal/g impulse (-25 muJ/g impulse). In the range 0-10 degrees C there is no significant effect of temperature on the magnitude of either the positive or the negative phases of heat production. The experimental thermal records were analysed to determine the true time course of the temperature changes in the nerve undistorted by the recording system. The time course of the temperature changes does not fit with that of the transmembrane voltage change as represented by the monophasic compound action potential recorded externally from the same point on the nerve. A better fit is obtained if the temperature changes are compared with the square of the voltage change in accordance with the view that the heat derives almost wholly from free energy changes and entropy changes in the membrane capacity. The best fit is obtained if it is assumed that the membrane potential does not discharge to zero during the action potential but that at the peak of the action potential the charge (and hence the p.d.) across the membrane capacity retains about 24% of its resting value.

The recovery heat production in non-myelinated garfish olfactory nerve fibres.

1. The recovery heat production of the non-myelinated fibres of garfish olfactory nerve has been measured. 2. At about 20 degrees C the total recovery heat was 381 +/- 26 microcal g-1 impulse-1 at a stimulation frequency of 2 sec-1. 3. The time constant of decay of the recovery heat production after a brief period of stimulation was 78.7 +/- 3.1 sec at about 20 degrees C. 4. Changing the temperature (by +/- 5 degress C) had little effect on the total recovery heat produced. 5. However, lowering the temperature reduced both the rate of rise, and the maximum rate of recovery heat production whereas the time constant of decay was increased. Raising the temperature produced corresponding changes in the opposite direction. 6. the recovery heat production measured in the present experiments is consistent with the previously measured oxygen consumption in the same preparation.

The optical spike. Structure of the olfactory nerve of pike and rapid birefringence changes during excitation.

1. Electron microscope studies on the olfactory nerve of the pike revealed a population of 4.2 million, densely packed unmyelinated nerve fibres; 95% are small fibres (average diameter 0.19 mum, narrow modal class), 5% are larger (average diameter 0.6 mum). Each fibre is bounded by an axonal membrane with a bilayer structure (80 A thickness). 2. The olfactory nerve is birefringent (negative with respect to fibre axis) and shows at 20 degrees C an average retardation R = 23 mn. The birefringence becomes more negative on lowering the temperature. 3. With the passage of an action potential a rapid, transient increase of retardation--the optical spike-- occurs; deltaR = 0.04 nm. The optical spike corresponds to the time course of structural changes in the axon membrane during excitation; it begins later, peaks earlier and decays more quickly than the voltage changes as recorded externally in the present study.

The heat production associated with the passage of a single impulse in pike olfactory nerve fibres.

1. A study has been made of the temperature changes associated with the passage of a single impulse in the non-myelinated fibres of the pike olfactory nerve. 2. The initial heat occurs in two phases: a burst of positive heat, followed by an evolution of negative heat. The positive and negative heats, and the net initial heat, are temperature-dependent. 3. At 0 degrees C the measured positive initial heat is 44.2 mucal/g.impulse; and the corresponding negative initial heat is 48.9 mucal/g.impulse. There is thus a net initial heat that is negative, of about 4.7 mucal/g.impulse. 4. The positive heat has a positive temperature coefficient, being increased by a factor of 1.86 when the temperature is rasied from 0 degrees C to 10 degrees C. 5. The negative initial heat also increases when the temperature is raised, but less than the positive initial heat. As a result, the net initial heat tends to become positive at higher temperatures. 6. Because of temporal dispersion of the action potential over the face of the thermopile, the observed temperature changes are smaller than those that occur at a single point in the nerve close to the stimulating cathode. The value of the positive heat at 0 degrees C corrected for temporal dispersion is estimated to be about 62 mucal/g.impulse: the corresponding value for the negative heat is about 67 mucal/g.impulse. 7. All records were analysed in terms of only two phases of initial heat (one positive, one negative). No analysis required four phases; but it is unclear whether this finding reflects a true absence of four phases, or merely the inability of the recording equipment to resolve them. 8. The positive heat seems to be derived from two sources. First, there is a dissipation of the free energy stored in the membrane capacity. Secondly, there is an evolution of heat corresponding with a decrease in entropy of the membrane dielectric with depolarization.

Heat production non-myelinated nerves.

Experiments with the C fibres of the rabbit vagus nerve have established that heat is evolved during the depolarizing phase of the action potential and is absorbed during the repolarizing phase. Subsequent studies using the pike olfactory nerve indicate that the heat production begins at a high rate very early in the depolarizing phase and is completed in advance of the peak of the spike. This would be expected if the heat arised from the energy released by the discharge of the membrane capacitance which varies as the square of the membrane potential; but estimates of the stored energy fall short of the observed heat production by a factor of two or three times. The prominent cooling phase suggests that a substantial part of the heat may arise from an entropy change. Such an entropy change would be expected to result from the change in the electrical stress in the dielectric of the membrane capacitance, and may thus be manifestation of reversible changes in the molecular architecture of the insulating matrix of the membrane.

The origin of the initial heat associated with a single impulse in mammalian non-myelinated nerve fibres.

1. A study has been made of the temperature changes associated with the passage of a single impulse in rabbit desheated vagus nerves.2. The initial changes consist of an evolution of positive heat followed by a reabsorption of most of it; i.e. there is a phase of positive and a phase of negative heat production.3. The size of the positive heat, its time of onset, and the time of onset of the negative heat have been measured by an analogue method of analysis. In addition, these parameters, together with the size of the negative heat and the duration of both phases of initial heat, have been studied with the aid of a computer, and also by conventional heat block analysis.4. At about 5 degrees C the measured positive heat is 7.2 mucal/g. impulse. It starts as soon as the compound action potential reaches the thermopile and lasts for about 107 msec.5. This positive heat decreases with increasing temperature, the ratio of heat at 4 degrees C to that at 14 degrees C being 1.86.6. The measured negative heat at about 5 degrees C is 4.9 mucal/g. impulse. It starts 102 msec after the onset of positive heat, and lasts for about 240 msec.7. When the sodium of Locke solution is replaced by lithium the positive heat is reduced by 19%, but the negative heat is increased by 22%.8. In potassium-free solutions the positive heat is hardly affected (increase of 5%), but the negative heat is more than doubled. As a result the nerve may become briefly colder than its initial temperature by about 2 mu degrees C.9. The effect of sodium-deficient solutions on the positive heat is somewhat variable, but the negative heat is consistently diminished.10. Replacement of the chloride of Locke solution by sulphate or nitrate has little effect on the positive heat. The negative heat is reduced in size by 26% and in duration by 22%.11. Replacement of most of the sodium of Locke solution by barium reduces or abolishes the negative heat, and increases the measured size of the positive heat nearly threefold.12. Veratrine (10(-5) g/ml.) produces a nearly tenfold increase in the net positive heat.13. Ouabain (1 mM) and antimycin A (1 mug/ml.) applied for 30-60 min have little effect on the initial heat production.14. Over the temperature range 5-15 degrees C, and for the ionic solution changes described above, there is close agreement in timing between the positive heat and the rising phase of the action potential, and between the negative heat and the falling phase.15. Because of the inevitable temporal dispersion of the action potential over the face of the thermopile, the observed temperature changes are smaller than those which occur at a single point in the nerve close to a stimulating electrode. The corrected value of the positive heat at 5 degrees C is 24.5 mucal/g. impulse, while that of the negative heat is 22.2 mucal/g. impulse.16. The heats of mixing of the ions in solution that interchange during the action potential are much too small to account for the observed initial heats, but an exchange of ions associated with fixed charges might make a significant contribution to the heats.17. The condenser theory, according to which the positive heat represents the dissipation of electrical energy stored in the membrane capacity, while the negative heat results from the recharging of the capacity, appears unable to account for more than half of the observed temperature changes.18. It seems probable that the greater part of the initial heat results from changes in the entropy of the nerve membrane when it is depolarized and repolarized.