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PubMed1987

Qualitative measurements of the entry of L-lactate into single surface fibres of frog skeletal muscle using a lactate-sensitive microelectrode.

The present results demonstrate the sensitivity of the Corning chloride liquid ion exchanger 477913 to L-lactate. Microelectrodes filled with this exchanger showed responses to changes in L-lactate concentration in chloride-free solutions. In these experiments L-lactate replaced gluconate in equimolar amounts. Microelectrodes filled with this exchanger were used to qualitatively detect changes in intracellular anion in chloride-depleted frog sartorius muscle fibres during exposure to extracellular concentrations of L-lactate. The increase in intracellular anion concentration is consistent with the movement of L-lactate into the cell. This microelectrode enables one to qualitatively monitor changes in intracellular L-lactate in chloride-free experiments without incorporating selectivity coefficients.

Animals↗
PubMed1985

Ionic conductances in frog short skeletal muscle fibres with slow delayed rectifier currents.

Short (0.8-1.6 mm) lumbricalis fibres of Rana pipiens were voltage clamped by a two-micro-electrode technique at 5 degrees C in sucrose hypertonic Ringer solution (SHR). Terminated linear cable analysis suggests that if the current electrode is placed near the centre of the fibre length and the voltage-sensing electrode is placed 0.19 times the fibre length from the current electrode, the fibre can be adequately voltage clamped and the conductance may be simply calculated as I/V for fibre length constants from 1.0 to 0.15 mm. In SHR solution lumbricalis fibres have action potentials with peak amplitudes of only +2 to 7 mV and a slow, gradual repolarization, distinct from the action potentials observed in sartorius muscle. In 60 mM-Na+ SHR the inward Na current could be adequately controlled over the fibre length, providing an estimated Na conductance (GNa) of 8.9 mS/cm2. The magnitude of GNa and GK (delayed rectifier) in lumbricalis fibres was approximately 20% of that reported for sartorius and semitendinosus, although the resting conductances were similar. Fibres demonstrated delayed rectifier currents with complex patterns of activation suggesting two components of conductance (fast, GK,f and slow, GK,s) which were combined together in varied amounts: (a) GK,f activated rapidly to a maximum within 80 ms at 0 mV as previously described (Adrian, Chandler & Hodgkin, 1970a); (b) GK,s activated gradually with depolarizations below -50 mV and achieving peak currents at about 400 ms at 0 mV. In about 10% of lumbricalis fibres studied, GK,s occurred in isolation with a peak magnitude of 1.4 +/- 0.4 mS/cm2 (+/- S.D.). GK,s activation kinetics and tail currents are described by a squared two-state (l2) Hodgkin-Huxley model and have a Q10 of 2.8. These currents inactivated with a time constant of 5-7 s at 0 mV. Isolated GK,s with identical kinetics was also observed in certain sartorius fibres studied with the three-electrode voltage clamp. The fractional amount of GK,s in the combined delayed rectifier (GK,s + GK,f) currents could be estimated from analysis of the late activation phase with depolarization. Combined delayed currents were described by summing GK,f currents using a n4 model with GK,s currents defined by the l2 model.

Action Potentials↗
PubMed1998

The tubular vacuolation process in amphibian skeletal muscle.

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes 'vacuolation' in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface ('detubulation'), particular if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for he first time two types of vacuole, 'open' and 'closed', whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed closed to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. 'Open' vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. 'Closed' vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca/+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by 'open' vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole 'closure' took place well after formation of 'open' vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of 'open' vacuole formation. These findings identify electrophysiological 'detubulation' of striated muscle with 'closure' of initially 'open' vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.

Animals↗
PubMed1988

Evidence for increased perfusion heterogeneity in skeletal muscle during reduced flow.

Recently, it has been demonstrated (D. Cousineau, C. P. Rose, D. Lamoureux, and C. A. Goresky, 1983, Cir. Res., 53, 719-730; K. Tyml, 1986, Microvasc. Res., 32, 84-98) that heterogeneity of microvascular flow depends on tissue metabolism. The objective of this study was to examine the possibility that, independent of metabolism, heterogeneity is also a function of flow. Using an intravital video-microscopic approach, we evaluated heterogeneity in the frog sartorius muscle at different flow rates while maintaining the muscle in the same exercised state. The flow was altered via partial aortal clamping. Exercised state was achieved by direct electrical stimulation. Heterogeneity of flow was evaluated in terms of the coefficient of variation (CV = SD/mean) computed from simultaneous measurements of red cell velocities in a capillary network. In addition to velocity analysis, the number of perfused capillaries crossing a 1-mm test line on the video monitor was counted. Among 10 networks from eight muscles, the overall preocclusion hyperemic velocity and CV were 0.42 +/- 0.15 SD mm/sec and 40 +/- 12%, respectively. The overall capillary count was 16.4 +/- 3.0 cap/mm. In all networks, increasing clamping reduced the mean hyperemic velocity and increased CV. For reductions to less than 50% of the preocclusion velocity, CV increased significantly (P less than 0.05), up to 88%. In 6 networks only, increasing clamping reduced capillary count, down to 10.2 cap/mm. This reduction was due to flow stoppages in capillaries situated randomly throughout the network. The data demonstrate for the first time that, for the same exercised state, heterogeneity of velocity depends on the mean velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Neuromuscular blocking action of cadmium and manganese in isolated frog striated muscles.

In isolated frog sciatic nerve--gastrocnemius muscle preparations, exposure for 60 min to Cd2+ (0.1-2 mM) caused a marked attenuation of the twitch tension developed by stimulation of nerves but no or only a slight inhibition of the tension produced by direct stimulation at maximum twitch height. The inhibitory effect was partially reversed by 1 mM cysteine. In frog sartorius muscles, the addition of Cd2+ (0.1 mM) alos abolished the end-plate potential evoked by nerve stimulation but did not suppress potential changes induced by iontophoretically applied acetylcholine. The addition of Mn2+ (2 and 5 mM) attenuated the response of muscles to both direct and indirect stimulation; a greater attenuation of the latter was observed. The inhibition was not reserved by cysteine but was partially reversed by excess Ca2+. Contractile responses of frog rectus abdominis muscles to acetylcholine were not significantly affected by Cd2+ and Mn2+ but were attenuated by d-tubocurarine in a dose-dependent manner. Impulse conduction along sciatic nerves was not impaired by Cd2+ and Mn2+ but was by procaine. It appears that Cd2+ interferes with the release of acetylcholine from motor nerve terminals by reducing the transmembrane influxes of Ca2+, the influx possibly relating to SH groups of membrane constituents.

Acetylcholine↗
PubMed1999

Cardiac glycosides inhibit detubulation in amphibian skeletal muscle fibres exposed to osmotic shock.

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.

Action Potentials↗
PubMed1979

Quantitation of chronic microwave radiation effects on muscle cell electrical excitable properties: a temperature dependence analysis of the H-H cable and membrane current parameters of irradiated cells.

Frogs Rana pipiens maintained under constant laboratory environmental conditions were subjected daily to chronic microwave exposure with pulsed microwave (2.88 GHZ) at a power density of 10 mW/cm2, during 0.1 hour, for periods up to 100 days. The whole body Specific Absorption Rate (SAR) was of 1.5 mW/g per 1 mW/cm2. The passive and dynamic electrical parameters of sartorius muscle cells from control and irradiated frogs as a function of temperature in the range from 10 C to 30 C, have been analyzed. This temperature dependence analysis, presented in another work, was planned to be used in this paper for detecting those possible "masked" chronic microwave effects in complex membrane mechanisms highly temperature sensitive, and tightly related to the excitation and propagation of bioelectrical responses. The temperature dependence of the passive (Vr, Gm, Cm, and tau) and the active cell electrical parameters (theta, V*, Vs, VNa, Vn, V+, V-, kr, kNa, kK, tau Na, tau K, -gNa, gNa, gK, INa, and IK) was not altered by chronic microwave exposure. The striking observation reported in another work, about the presence of two groups of cell electrical parameters, characterized by their dependence with temperature, was reproduced on the irradiated muscle cells. Results have indicated that there was not muscle cell cumulative bioeffects resulting from microwave exposure to 10 mW/cm2, over a 0.1 hour period.

Action Potentials↗
PubMed1972

The effect of shortening on the time-course of active state decay.

The active state describes the force developed in a muscle when the contractile elements are neither lengthening nor shortening. Recently it was suggested that perturbations used to measure the active state also alter the time-course of the active state. The present research was undertaken to assess quantitatively the effect of two such perturbations, isotonic shortening and quick release, on the active state in frog sartorius muscle. Methods were developed which allowed the determination of active state points following periods of controlled isotonic shortening or quick release early in the contraction cycle. All experiments were carried out within the plateau region of the length-tension curve. Both isotonic shortening and quick release altered the active state decay. The active state force decreased as the extent of shortening or release was increased. For each 0.1 mm of isotonic shortening there was a 2% decrease in active state force. Quick release produced a larger decrement. From this data we conclude that the time-course of active state can be measured only in relative terms because it is altered by the motion which takes place in the contractile machine while the active state is being measured. This finding helps to resolve paradoxes in the literature relating to the time-course of the active state, calculated and experimentally determined isometric tetanic myograms, and the heat of shortening.

Animals↗
PubMed1982

Increased permeability to sugar following muscle contraction. Inhibitors of protein synthesis prevent reversal of the increase in 3-methylglucose transport rate.

Contractile activity is followed by an increase in the permeability of muscle to sugar. The increase in sugar transport rate persists unchanged for hours before gradually returning to base-line (Holloszy, J. O., and Narahara, H. T. (1965) J. Biol. Chem. 240, 3493-3500). This prolonged delay suggested that the recovery process might involve protein synthesis. We have investigated this possibility. A large increase in permeability to [3-3H]methylglucose occurred in frog sartorius muscles stimulated to contract 60 times/min for 30 min. Cycloheximide or puromycin at concentrations that inhibited protein synthesis completely blocked reversal of the increase in permeability to 3-methylglucose. The recovery process was not irreversibly impaired, as permeability returned to base-line in muscles that were washed and incubated without protein synthesis inhibitors for a second 24-h period. Reversal of the increase in permeability to 3-methylglucose induced by a high concentration of epinephrine was also prevented by cycloheximide. In contrast, cycloheximide did not prevent reversal of the increases in sugar transport rate induced by either insulin or trypsin. These results provide evidence that exercise and catecholamines activate sugar transport by a process that requires protein synthesis for its reversal, while the increases in permeability induced by insulin and trypsin involve a different mechanism.

3-O-Methylglucose↗
PubMed1989

Sartorius transposition to protect vascular grafts in the groin.

Fifty-two patients who required reexploration through the groin incision within seven days of an arterial procedure were studied to assess the value of sartorius transposition. Twenty-four group I patients had the sartorius muscle used to cover the vascular graft at reoperation while 28 group II patients had a standard closure. The groups were similar with respect to age, initial arterial operation, incidence of diabetes, and use of antibiotics and postoperative anticoagulation. Wound complications were frequent in both groups (62.5% vs. 57.1%, respectively) but outcome of these wound problems was quite different. Graft infection and anastomotic disruption were significantly less frequent in group I patients. Because fewer infected prostheses had to be removed group I patients were less likely to require high level amputations. Sartorius transposition is an effective method of protecting vascular grafts jeopardized by the high frequency of wound problems associated with early reoperation.

Aged↗
PubMed1984

Time-resolved X-ray diffraction studies of cross-bridge movement and their interpretation.

The purpose of these studies has been to obtain information about the structural behaviour of the cross-bridges during contraction. Since there are so few reflections still present in the part of the X-ray diagram produced by cross-bridges in a contracting muscle they cannot on their own give a detailed picture. However, they can give information of a more general nature - much in the same way as measurements of tension may do, for example - and the patterns can also tell us what structural regularities are no longer present during contraction. The experiments which I will describe have been carried out in nearly all cases on frog sartorius muscles using synchrotron radiation as an intense X-ray source. The necessary facilities were provided by the European Molecular Biology Laboratory Outstation on the storage ring DORIS at DESY Hamburg. The results to which I will refer have in many cases already been described in papers published or in press (Huxley, 1979; Huxley, Faruqi , Bordas , Koch and Milch , 1980; Huxley, Simmons, Faruqi , Kress , Bordas and Koch, 1981; Huxley, Faruqi , Kress , Bordas and Koch, 1982), to which reference may also be made for experimental details.

Animals↗
PubMed1992

Capillary as a communicating medium in the microvasculature.

The preceding study (Dietrich and Tyml, 1992. Microvasc. Res. 43) demonstrated that a local application of norepinephrine (NE) on a capillary in a skeletal muscle produces a temporary reduction in blood flow within this capillary. The reduction is mediated via constriction of the supplying arteriole. The objective of the present study was to address the mechanism by which the local NE stimulus is propagated from the capillary to the arteriole. Using intravital video microscopy we measured red blood cell velocity in capillaries, and diameter of supplying arterioles, in the sartorius muscle in anesthetized frogs. Velocity responses were measured following iontophoretic application of NE (3 mM in the pipette) on the capillary, with or without pretreatment with 0.9 mM tetrodotoxin (nerve-specific sodium channel blocker), 30 mM lidocaine (nonspecific sodium channel blocker), and 30 mM yohimbine (alpha 2-receptor blocker). Diameter responses were measured before and after capillary damage introduced by microcautery. Tetrodotoxin did not block the NE-induced velocity reduction (i.e., from 0.2 to 0.07 mm/sec), while lidocaine attenuated it. Yohimbine blocked it only when applied on the same site as NE. Capillary damage abolished the NE-induced arteriolar constriction (i.e., from 27.8 to 21.5 microns). We conclude that the observed responses were not due to (1) direct diffusion of NE from the capillary to the arteriole, (2) conduction along adrenergic nerves, or (3) venous-arteriolar diffusional cross-talk. We interpret our data to indicate that the capillary itself could function as a communicating medium.

Animals↗
PubMed1966

Studies on ion accumulation in muscle cells.

A comparison is made between the quantitative predictions of equilibrium ionic distribution in living cells according to the membrane theory (Donnan equilibrium) and according to the association-induction hypothesis. This comparison shows that both theories predict competitive effects of one permeant ion on the equilibrium concentration of another permeant ion; but within the limit of experimental accuracy only the association-induction model predicts quantitatively significant specific competition of one specified ion with the accumulation of another specified ion. The equilibrium distributions of K(+), Rb(+), and Cs(+) ions in frog sartorius muscle were studied and quantitatively significant specific competition was demonstrated; these results favor the association-induction hypothesis (adsorption on cell proteins and protein complexes and partial exclusion from cell water). Based on this model we estimated that at 257deg;C, the apparent association constants for K(+), Rb(+), and Cs(+) ion are 665, 756, and 488 (mole/liter)(-1). We found that the total concentration of adsorption sites (no less than 240 mmole/kg of fresh cells) agrees with the analytically determined concentrations of beta- and gamma-carboxyl groups of muscle cell proteins (260 to 288 mmole/kg).

Animals↗
PubMed1985

Isolated hindlimb perfusion in dogs: the effect of perfusion pressures on the oxygen supply (ptO2 histogram) to the skeletal muscle.

During regional isolated perfusion, neoplasms in extremities are treated with high doses of chemotherapeutic drugs by means of an extracorporeal circuit. The question is whether optimal tissue perfusion, which is essential for the therapy, is obtained by regulation of the extracorporeal circuit on an adequate perfusion flow or on an adequate perfusion pressure. To determine which perfusion pressure is needed to maintain adequate tissue perfusion, hindlimbs of six dogs were perfused at perfusion pressures 0, 15, 25, and 50 mm Hg below systemic mean arterial pressure. A multiwire polarographic oxygen electrode placed on the sartorius muscle permitted quantitative evaluation of tissue oxygenation by means of ptO2 histograms. Our results indicated that the perfusion pressure must be equal to or within 15 mm Hg below systemic mean arterial pressure to obtain optimal tissue perfusion. To maintain this perfusion pressure, high perfusion flows of about five to 10 times control femoral flows were needed. At a perfusion pressure 50 mm Hg below systemic mean arterial pressure, perfusion flow was normal, but tissue perfusion was severely impaired.

Animals↗
PubMed1985

Are endothelial cells rich in filaments involved in the phenomenon of electrically induced stoppages of flow in frog capillaries?

The aim of this study was to determine the frequency of occurrence of the phenomenon in skin and muscle capillaries in both young and mature frogs and to examine the ultrastructure of endothelial cells found in these capillaries. Using microelectrode techniques, trains of pulses of durations up to 90 s, amplitudes -0.2 to -2.0 V, frequencies 10 to 64 Hz and pulse widths 5 to 50 msec were applied to microscopically visualized capillaries situated either in skin (knee region) or at the surface of a sartorius muscle in metamorphosed, intermediately aged or mature frogs. Reproducible flow stoppages induced by these trains of pulses occurred in 2.3%, 1.4% and 6.7% of all tested muscle capillaries and in 11.1%, 82.5% and 68.6% of all skin capillaries in the three age groups, respectively. Examinations of electron micrographs of very thin sections of both skin and muscle capillaries revealed that only 13 out of 47 (27.7%) and 4 out of 45 (8.9%) endothelial cells in these two tissues contained filament bundles. In light of these ultrastructural findings, the dramatically high occurrence of the phenomenon of electrically induced stoppages of flow in skin capillaries (in both intermediately aged and mature frogs) cannot be explained in terms of the hypothesis involving endothelial cells rich in filaments. It is concluded that caution must be exercised when interpreting this phenomenon in terms of the several microvascular responses which the electrical stimulation can elicit.

Age Factors↗
PubMed1984

The influence of velocity of length change on tension development in skeletal muscle: model calculations and experimental results.

Force responses obtained during constant velocity length changes on skeletal muscle tissue are simulated by means of two cross-bridge models proposed by Huxley and Simmons (1971, Nature 233, 533-538) and by Julian et al. (1974, Biophys. J. 14, 546-562). An implicit method was used for the numerical approximation in the simulations. The simulated force transients due to constant velocity length changes are found to be in qualitative agreement with re-investigated experimental results obtained from the whole sartorius muscle of the frog. A non-linear tension transient is observed, dependent both on amplitude and on velocity of release revealing an inflexion which gives the transient a shoulder shape. When velocity is increased the inflexion occurs earlier and at a lower tension value. A non-linear transient is observed during stretches performed at moderate velocities. Force responses are found to deviate concavely downwards from a linear time course. Simulations, however, predict a rather linear tension transient for comparable velocities. Implications of the experimental findings are discussed for both models.

Animals↗
PubMed1979

Inhibition of aminopyridine-induced contractile activity in skeletal muscle by tetrodotoxin and by magnesium.

The effects of tetrodotoxin and magnesium have been studied on aminopyridine-induced contractile activity seen in the absence of nerve stimulation. In the chick biventer cervicis muscle both tetrodotoxin and magnesium pretreatment prevented the development of fasciculations and contractures in the presence of 4-aminopyridine and 3,4-diaminopyridine. Both tetrodotoxin and magnesium abolished aminopyridine induced fasciculations and contractures. Tetrodotoxin did not reduce postjunctional sensitivity to the agonists acetylcholine and carbachol whereas magnesium produced some reduction of postjunctional sensitivity. It is concluded that conducted action potentials must be involved in the aminopyridine-induced contractile activity. In the frog sartorius muscle aminopyridines occasionally induced repetitive endplate potentials suggesting that the compounds induce repetitive nerve activity. In both tetrodotoxin and magnesium treated preparations 4-aminopyridine produced only a moderate increase in miniature endplate potential frequency. It is concluded that aminopyridines increase nerve membrane excitability resulting in the generation of repetitive action potentials in the absence of nerve stimulation.

Aminopyridines↗
PubMed1982

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres.

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.

Action Potentials↗