Noradrenaline effects on cultured rat sensory neurones exposed to morphine in utero or in vitro.

Dorsal root ganglion (DRG) neurones cultured from newborn rats were chronically exposed to 1 microM morphine. Untreated cells and in vitro morphine treated cells were cultured for 1 or 2 weeks from control offspring while in utero morphine treated cells were cultured for 1 week from offspring of morphine dependent rats. Noradrenaline (1 microM) was applied by pressure ejection. Intracellular recordings measured the change in action potential duration (APD). Noradrenaline significantly decreased the APD of 1-week-old untreated cells while the 2-week-old untreated cells and the groups of morphine treated cells showed no significant change of APD following the drug application. Thus, newborn rat DRG neurones demonstrated a different sensitivity to noradrenaline with the culture duration or with morphine exposure.

Muscle performance, voluntary activation and perceived effort in normal subjects and patients with prior poliomyelitis.

Many people previously affected by polio complain of increased fatigue, weakness and pain many years after the initial illness. Although electromyographic abnormalities have been found in these patients, the cause of their increased weakness is not well understood. Previous studies have shown decreased strength and impaired exercise performance in those with prior polio, but the level of voluntary drive to the muscle has not been investigated. The present study investigated maximal voluntary activation without fatigue and both peripheral and central components of muscle fatigue during exercise in 21 subjects with poliomyelitis 20-40 years previously, and 20 healthy, age-matched control subjects. Voluntary activation and strength of the elbow flexors were quantified using twitch interpolation during maximal isometric voluntary contractions both at rest, and during fatigue induced by 45 min of repeated isometric contractions. Compared with the control subjects, patients with prior polio had impaired voluntary activation both when the elbow flexors were not fatigued and during fatiguing submaximal exercise. During exercise, polio subjects also had lower twitch amplitudes and increased subjective fatigue. Central and peripheral fatigue were more marked in those with the post-polio syndrome. The impaired voluntary activation with unfatigued muscles in polio subjects indicates that defective central or reflex drive may contribute to their new weakness.

Effect of caffeine and high potassium on normal and dystrophic mouse EDL muscles at various developmental stages.

EDL muscles from normal and dystrophic (dy2j) mice of various ages were examined. Muscles were divided into three groups according to age: 7 to 14 days postnatal, 16 to 21 days postnatal, and 6 months old, to assess age and/or phenotype related differences in the muscle response to caffeine or high K+. The response of normal muscles to caffeine decreased with age and reached adult characteristics between the second and third week of postnatal life. Their response to high K+ also changed during postnatal development; specifically, the time taken to recover to 50% pretest twitch tension decreased with age, probably reflecting developmental changes in Cl- conductance. Up to 21 days of age, the sensitivity of dystrophic muscles to both caffeine and high K+ was essentially similar to normal, while marked differences were observed in the adult. Taken altogether, our results suggest that while the maturation of a number of systems might be delayed in dystrophic muscles at preclinical stages of the disease, their e-c coupling and SR function (Ca2+ release and reuptake) appear to be quite normal. Our results further suggest that the "abnormal" responses of dystrophic muscles at more advanced stages of the disease, when challenged by drugs acting on either of these systems, may be explained in terms of changes in muscle fiber type proportions.

Volume changes during contraction of isolated frog muscle fibers.

A microscope objective and electronic imaging system were used to determine how isolated frog skeletal muscle fibers adjust their volume during an isometric tetanus. Cross-sectional area and volume of the middle third of a fiber increased rapidly with the development of active tension, which indicates that contraction produced components of force perpendicular to the long axis. The extreme ends are known to shorten whether or not the middle of a fiber is isometric or stretched. Shortening of the ends may shift water towards the middle, which could account for the volume changes we observed. The cytoskeletal matrices of muscle evidently adjust rapidly during contraction to maintain a dynamic equilibrium between the axial and radial forces that stabilize the whole cell. The Z disks have been shown to expand during active, but not passive, tension development. Z disks might be the elastic elements of the muscle cytoskeleton primarily involved in rapid balancing of the radial components of active force.

Effect of low Ca2+ solution on muscle contraction of developing, preclinical dystrophic (dy2j) mice.

EDL muscles from normal and dystrophic (dy2j) mice aged 7 to 21 days of postnatal life were examined. Muscles were divided into 2 groups according to age, 7 to 14 days and 16 to 21 days postnatal, so as to assess age- and/or phenotype-related differences in the muscle response to low Ca2+ solution. Tension production was already much impaired in "predystrophic" muscles. At this stage, however, there was essentially no difference in twitch kinetics between normal and dystrophic muscles. Upon exposure to low Ca2+ solution, twitch responses of both normal and dystrophic muscles declined in a similar manner. In the youngest animals studied (7 to 14 days), the tetanic responses of both normal and dystrophic muscles to low Ca2+ solution were also similar. In animals 15 to 21 days old, however, the tetanic tension developed in low Ca2+ solution by dystrophic muscles, was significantly less than that of normal. Moreover, under these conditions (i.e., in low Ca2+ solution), and following tetanic stimulation, the membrane potential of dystrophic muscles in this age group was significantly more depolarized than that of normal muscles. Our results suggest that the ability of the cell to deal with extracellular Ca2+ is normal in predystrophic muscles up to 21 days of postnatal life. The results also clearly point to the fragility of the membrane in these muscles.

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.

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.

Actions of caffeine on fast- and slow-twitch muscles of the rat.

1. The effects of caffeine (0.2-20 mmol l-1) have been examined on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat. 2. At 25 degrees C, threshold caffeine concentration for an observable increase in resting [Ca2+]i was 0.2 and 1.0 mmol l-1 for soleus and EDL muscles respectively. Increases in resting force were first detectable at about 0.5 mmol l-1 caffeine for soleus muscles and 5.0 mmol l-1 caffeine for EDL muscles and occurred in the range 0.2-0.4 mumol l-1 [Ca2+]i for soleus and 0.7-0.9 mumol l-1 for EDL. 3. Caffeine potentiated the twitch responses of soleus and EDL in a dose-related manner. The soleus was more sensitive in this respect, with 50% potentiation occurring at 1 mmol l-1 caffeine compared with 3.5 mmol l-1 for the EDL. Concentrations of caffeine below 2 mmol l-1 potentiated Ca2+ transients associated with twitches in both soleus and EDL muscles with no apparent change in the decay rate constant. 4. High concentrations of caffeine (greater than 2 mmol l-1) further potentiated peak Ca2+ in the EDL but depressed it in the soleus. The rate of decay of the Ca2+ transient in high caffeine was significantly prolonged in the soleus but remained unaffected in the EDL. 5. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) had little effect on force or [Ca2+]i at concentrations known to significantly increase intracellular cyclic AMP levels. 6. The Ca2+ transient during fused tetani was characterized by an initial peak, a decline to a plateau level and sometimes a gradual rise towards the end of the stimulus train. Peak [Ca2+]i during normal tetani ranged between 1.1 and 2.4 mumol l-1 in the soleus and 1.9 and 4.0 mumol l-1 in the EDL. 7. Caffeine potentiated both force and [Ca2+]i during tetanus. Since the increase of the Ca2+ transient was significantly greater than potentiation of force, it is likely that saturation of myofilaments occurs. The primary effect of caffeine on the Ca2+ transient was an elevation of the plateau phase. 8. Caffeine concentrations below 5 mmol l-1 potentiate twitch and tetanic force in both fast- and slow-twitch mammalian skeletal muscles primarily by increasing both the basal and stimulus-evoked release of Ca2+ from the sarcoplasmic reticulum.

The action of ryanodine on rat fast and slow intact skeletal muscles.

1. The action of ryanodine on force development of bundles dissected from rat extensor digitorum longus (EDL) and soleus muscles has been examined. 2. Ryanodine (100-5000 nM) irreversibly depressed twitch and tetanic tension of both muscle types in a dose-related manner. 3. At concentrations above 250 nM, ryanodine induced a slowly developing, dose-dependent contracture which could not be blocked by 5 mM-Co2+. Increasing the stimulation rate or decreasing the oxygenation of the preparation accelerated the rate of contracture development while the total removal of extracellular Ca2+ was required to prevent it. 4. Following the relaxation of the initial contracture (IC) in Ca2+-free solution, a second type of contracture (SC) could be induced by the readdition of Ca2+. This contracture differed from IC in that it was dependent on Ca2+ in the millimolar range and was prevented by 5 mM-Co2+. Both IC and SC were relaxed by perfusion with Ca2+-free, EGTA-containing solution. 5. Subcontracture doses of ryanodine (100 nM) markedly potentiated caffeine contractures of both muscle types. 6. Asymmetric charge movement in EDL fibres was recorded with the Vaseline-gap technique. The amount of charge moved near threshold was virtually unaffected by the presence of 10 microM-ryanodine over the time examined. 7. The results are consistent with the suggestion that ryanodine locks the calcium release channels of the sarcoplasmic reticulum (SR) in an open subconductance state with reduced conductance. It appears that lowering the external calcium concentration might still inactivate the release channels after they have been blocked open by ryanodine, possibly by an effect on the T-tubular voltage sensor.

Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibres.

1. The effects of 2,3-butanedione monoxime (BDM, 0.2-10 mmol/l) have been examined at different temperatures on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat and on Ca2+-activated isometric force of mechanically skinned soleus and EDL fibres. Ca2+ release and uptake by the sarcoplasmic reticulum has also been investigated directly on skinned EDL fibres at 1 mmol/l BDM at 23 degrees C. 2. BDM bound calcium with low affinity (KCaBDMapp = 55.9 +/- 4.3 M-1 at 22 degrees C, pH 7.10, ionic strength approximately 155 mmol/l) and did not affect the kinetics and quantum yield of the Ca2+-induced aequorin luminescence. 3. BDM reversibly reduced both force- and Ca2+-dependent aequorin light during the twitch and tetanus of all intact-muscle preparations in a dose-related manner. Twitch responses of soleus fibres were more sensitive to BDM than were EDL fibres. Higher concentrations of BDM were needed to abolish the tetanic force response than the twitch response in both muscle types. 4. The initial rate of rise and the decay rate constants of twitches in both soleus and EDL muscles were increased in the presence of BDM. Accordingly, a higher frequency of stimulation was required to produce fusion of the tetanus. This phenomenon could be explained by the reduced magnitude of the Ca2+ transient. 5. BDM reduced maximal Ca2+-activated force development in mechanically skinned soleus and EDL muscle fibres in a dose-dependent fashion. This depression of force was less sensitive to BDM than were the light transients in intact fibres. Furthermore, BDM affected EDL skinned fibres to a greater extent than soleus skinned fibres, in contrast with its effects on intact fibres. 6. At concentrations of BDM greater than 2.5 mmol/l, BDM significantly decreased the sensitivity of the contractile apparatus to calcium. The relative force-pCa (= -log [Ca2+]) curves for both soleus and EDL skinned fibres were shifted to the right (i.e. to higher calcium concentrations) in a dose-related manner. 7. BDM (10 mmol/l) slowed maximal Ca2+-activated force development of skinned preparations of both fibre types at all temperatures investigated (4-24 degrees C). 8. BDM reduced stiffness in skinned preparations maximally activated by calcium in proportion to the reduction in the isometric force response. BDM also inhibited rigor force of all fibres but only if it was present before the onset of rigor.(ABSTRACT TRUNCATED AT 400 WORDS)

Paralysis of skeletal muscle by butanedione monoxime, a chemical phosphatase.

The chemical phosphatase butanedione monoxime (BDM) reversibly inhibited twitches and tetanic contractions in bundles of rat soleus fibres in a dose-dependent manner (2-20 mM) but had no effect on the amplitude or time course of action potentials. In addition, BDM reversibly reduced the amplitude of potassium contractures demonstrating a depressant effect on contraction not mediated by action potentials. BDM had no effect on asymmetric charge movement but depressed calcium currents across the surface membrane in voltage-clamped fibres. The most significant effect of BDM on excitation-contraction coupling was a reduction in the amplitude of the calcium transient associated with contraction in aequorin-injected fibres. While these experiments do not eliminate the possibility of a direct effect of BDM on contractile filaments, reduction of calcium release from the sarcoplasmic reticulum, at least at low concentrations of BDM (below 2 mM), would seem to be the main mechanism for the inhibition of contractions in rat skeletal muscle.

The effect of alcohols on aequorin luminescence.

The effect of alcohols from ethanol to octanol on aequorin luminescence was biphasic; enhancement of both peak light and total photon yield at low concentrations, and inhibition of these parameters at high concentrations. The potency of alcohols to exert these effects was in the same order as the oil/water partition coefficients of the alcohols. It is argued that neither of these effects is related to calcium binding by aequorin, since alcohols enhanced calcium-independent luminescence, and the inhibition of responses is not associated with a reduction in the rate of aequorin 'consumption' on binding of calcium.

Relationship between intracellular calcium concentration and relaxation of rat fast and slow muscles.

Following injection of aequorin into small bundles of rat soleus and extensor digitorum longus muscles, simultaneous measurements of light (proportional to the intracellular free calcium ion concentration ([Ca2+]i)) and force were recorded during muscle twitches over a range of temperatures from 10 to 37 degrees C. The Arrhenius plots for rate of decline of [Ca2+]i following a twitch were linear and yielded the same activation enthalpy (Ea) for both muscle types, indicating that the same process controls Ca2+ uptake in both fast and slow muscles. Arrhenius plots of force decay rate constants showed a break around 20 to 25 degrees C with the Ea's above the break being similar to the Ea's for decline of [Ca2+]i and indicating a close relationship between rate of relaxation and decline of [CA2+]i above this temperature.

The measurement of changes in intracellular free calcium during action potentials in mammalian neurones.

This report describes the techniques we have developed to record changes in intracellular calcium concentration which take place during action potentials in the cell body of rat dorsal root ganglion neurones in vitro. The photoprotein, aequorin, was microinjected into the cell body of individual sensory neurones and light output from Ca2+-activated aequorin molecules was recorded with a photomultiplier tube attached to a modified inverted microscope. Aspects of the technology outlined here include: cell culture methods; a flow chamber for electrophysiological experiments on cell culture preparations; modifications to our inverted microscope; use of 150 mM KCl-filled microelectrodes; and an electronic device for processing the photomultiplier output. Some preliminary results are presented.

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.

Endurance properties of respiratory and limb muscles.

Endurance properties of the inspiratory and expiratory muscles were compared with those of the flexors and extensors of the elbow in healthy volunteers. During a series of sustained contractions separated by rest intervals of one minute there was a progressive decline in the force produced by both muscle groups acting at the elbow and by the expiratory muscles. By contrast, the ability of the inspiratory muscles to generate force recovered completely within the one minute intervals. The decline in force during the first sustained contraction was similar for the inspiratory muscles and those acting at the elbow. During series of repeated brief maximal contractions (duty cycle 50%) the inspiratory muscles fatigued less than the other muscle groups. This apparent resistance to fatigue shown by the inspiratory muscles may be due to many factors including the central organization of their motoneurones and to local neuromuscular properties. Evidence supporting a contribution from intrinsic muscle properties is provided by studies of isolated curarized muscles.

Use of aequorin to study excitation--contraction coupling in mammalian smooth muscle.

Considerable information is available for intact skeletal and cardiac muscle cells on the role of changes in intracellular Ca2+ (Cai2+) levels during excitation-contraction coupling. Cai2+ levels seem to be similarly important in smooth muscle. However, only two published reports describe the use of indicators to directly measure [Cai2+] in intact smooth muscle cells. One described the use of murexide in molluscan smooth muscle, the other the use of aequorin in single amphibian cells. Their results differed: very long calcium transients were described for the molluscan muscle, compared with very brief ones for the amphibian cells. We present here the first description of the relationship between calcium transients and contraction of whole, mammalian smooth muscle injected with aequorin. The Ca2+ transient we observed has a slow rise, a long duration, and there was no delay between the peak of the Ca2+ transient and the onset of force. This contrasts with the amphibian findings but resembles the molluscan data. We observed a delay between the onset of the increase in [Cai2+] and the onset of the rise in tension which might be explained, at least in part, by the presence of a threshold [Cai2+] for concentration.

The role of sulfhydryl groups in contraction of vascular smooth muscle.

While high concentrations (50 micronmol/l) of merurial sulfhydryl (SH) reagents p-chloromercuribenzoate (PCMB) and p-chloromercuriphenyl sulfate (PCMPS) caused a nonspecific inhibition of constrictor responses in isolated rabbit ear arteries, lower concentrations had selective effects. At a concentration of 10 micronmol/l both agents abolished responses to epinephrine and reduced responses to barium and calcium, but responses to caffeine were unaffected by PCMB and potentiated by PCMPS. Responses to histamine were little affected by PCMPS, but with PCMB, histamine responses were first reduced but later potentiated. In contrast, ethacrynic acid, a nonmercurial SH inhibitor which gains access to the intracellular space, failed to show any specificity of action and inhibited contractile responses to all these agonists. The results suggest that SH inhibitors whose actions are limited to the cell membrane exert both specific and nonspecific effects. The specific effect may be at SH groups associated with the alpha adrenoceptor while nonspecific effects may involve changes in membrane permeability.