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Aging: effects on the prostaglandin production by skeletal muscle of male rhesus monkeys (Macaca mulatta).

Prostaglandin (PG) production from [1-14C] arachidonic acid was measured in homogenates of sartorius muscle of aged (more than 20 years) and young adult (7 to 11 years) rhesus monkeys. Total production by the aged series (3.21 +/- 0.31 (S.E.) nmol PG per g N per min) was about two times that of the young series (1.76 +/- 0.14 (S.E.) nmol per g N per min). Epinephrine-stimulated PG production was also twice as great in the aged muscle series as in the young adult series (7.22 +/- 0.74 (S.E.) and 3.54 +/- 0.52 (S.E.) nmol per g N per min). This difference was mainly due to greater production of 6-keto-PGF1 alpha and PGF2 alpha. In both series, the addition of 500 microM epinephrine to the assay media significantly increased production of all types of PGs measured (6-keto-PGF1 alpha, PGF2 alpha, PGE2 plus thromboxane B2 [TXB2], the stable breakdown product of TXA2, and PGD2). The absolute increases in all types of PGs studied in the presence of epinephrine were significantly greater in the aged muscle, with the exception of PGF2 alpha. The distribution patterns of the various types of PGs studied were similar in all series, except for 6-keto-PGF1 alpha; this PG accounted for a greater percentage of the total production by aged muscle than by young adult muscle (p less than 0.05).

6-Ketoprostaglandin F1 alpha↗

Changes of thick filament structure during contraction of frog striated muscle.

The strongest myosin-related features in the low-angle axial x-ray diffraction pattern of resting frog sartorius muscle are the meridional reflections corresponding to axial spacings of 21.4 and 14.3 nm, and the first layer line, at a spacing 42.9 nm. During tetanus the intensities of the first layer line and the 21.4-nm meridional decrease by 62 and 80% respectively, but, when the muscle is fresh, the 14.3-nm meridional intensity rises by 13%, although it shows a decrease when the muscle is fatigued. The large change in the intensity of the 21.4-nm meridional reflection suggests that the projected myosin cross-bridge density onto the thick filament axis changes during contraction. The model proposed by Bennett (Ph.D. Thesis, University of London, 1977) in which successive cross-bridge levels are at 0,3/8, and 5/8 of the 42.9-nm axial repeat in the resting muscle, passing to 0, 1/3, and 2/3 in the contracting state, can explain why the 21.4-nm reflection decreases in intensity while the 14.3-nm increases when the muscle is activated. The model predicts a rather larger increase of the 14.3-nm reflection intensity during contraction than that observed, but the discrepancy may be removed if a small change of shape or tilt of the cross-bridges relative to the thick filament axis is introduced. The decrease of the intensity of the first layer line indicates that the cross-bridges become disordered in the plane perpendicular to the filament axis.

Animals↗

Initial heat production in isometric from muscle at 15 degrees C.

An infrared radiation-detecting system was used to measure initial heat production in bull frog sartorius muscle at 15 degrees C. Numerous tests with the system showed that thermal artifacts were not noticeable. Many previous measurements with myothermic thermopiles were corroborated with this method. In addition, a cooling phase as large as 0.39 of peak exothermicity was found during and after relaxation. Cooling diminished with both increasing sarcomere length and increasing duration of mechanical activity. No large rapid increase in heat rate accompanied a 0.6 reactivation at the peak of twitch tension. Above rest length, initial heat rate and the heat produced up to the peak of tension decreased nearly proportionally with overlap of myofilaments, while the total twitch initial heat decreased slightly.

Animals↗

Effects of drugs on partially and completely denervated skeletal muscle of the frog.

The sensitivity of completely denervated and the innervated end-plate regions of partially denervated frog's sartorius muscles to stimulation by drugs has been investigated. The drugs were tested on muscles isolated 12 to 39 days following denervation. On the basis of the results obtained most of the drugs could be put into one of two main groups. The first group included choline, tetramethylammonium, carbachol and decamethonium; these drugs readily stimulated non-denervated muscles, and chronic denervation, either partial or complete, greatly increased the sensitivity of the muscles. The second group included pilocarpine and carbamate of (2-hydroxypropyl) trimethyl ammonium chloride (Bethanechol, U.S.P.). The muscles were relatively insensitive to stimulation by these two drugs. They could stimulate the muscles if applied in high enough concentrations, but the responses thus obtained could not be distinguished from responses produced by similar concentrations of sucrose and chronic denervation did not increase the sensitivity of the muscles to stimulation by these two drugs. Methacholine, like the drugs in the second group, was unable to stimulate the muscles in low concentrations, but chronic denervation rendered the muscles sensitive to stimulation by this drug. Tetraethylammonium presented a completely different pattern of activity which was consistent with a hypothesis that in low doses this drug stimulates skeletal muscle by causing a release of acetylcholine from nerve endings in the muscle.

Acetylcholine↗

Further evidence for a potassium-like action of lithium ions on sodium efflux in frog skeletal muscle.

1. The efflux of labelled sodium as well as net sodium and lithium changes were studied in aged high sodium sartorius muscles of the South American frog Leptodactilus ocelatus.2. In the presence of 2.5 mM potassium in the media, the replacement of external sodium with lithium or magnesium resulted in an increase in sodium efflux. The magnitude of such increase was always larger in lithium.3. With the absence of potassium in the media, the response of sodium efflux to replacement of external sodium varied with the cation used as a substitute. In lithium Ringer there was always a noticeable increase, whereas in magnesium there was always a marked reduction. The same results were observed when calcium was substituted for magnesium.4. The replacement of 60 mM external sodium with sucrose did not prevent the stimulating effect of 5 mM potassium on sodium efflux, nor the inhibitory action of 10(-4)M ouabain. This indicates that neither sucrose by itself, nor the lowering of the ionic strength, modified to an appreciable extent the function of the sodium pump.5. Net sodium extrusion took place against an electrochemical gradient in potassium-free - 50 mM sodium - mM lithium Ringer. About 75% of this efflux was ouabain sensitive.6. Muscles made both sodium and lithium rich and incubated in potassium-free - 60 mM sodium - 50 mM lithium Ringer also showed net sodium extrusion against an electrochemical gradient, which was 85% ouabain sensitive. This extrusion took place even under conditions where the changes in free energy favouring lithium entry were always lower than the changes in free energy opposing sodium going out. This indicates that a sodium-lithium exchange by a counter-transport process is unlikely.7. External potassium reduced the ouabain sensitive lithium influx in muscles incubated in lithium Ringer. The values found were 5.90 +/- 0.39 mu-mole/g.hr and 2.66 +/- 0.43 mumole/g.hr in potassium-free and 15 mM potassium respectively. At the same time potassium had no effect on the ouabain-insensitive lithium uptake.8. Muscles incubated in potassium-free-magnesium Ringer had a residual sodium efflux which could not be accounted for by passive movement. About 40% of it was abolished by 10(-4)M ouabain. This ouabain-sensitive part could be a consequence of some stimulation of the sodium pump by potassium leaking out of the cells. If this is correct it should be inhibited by external sodium and should not contribute to the total sodium efflux in potassium-free sodium media.9. Magnesium was used as the reference cation to study the sodium-stimulated sodium efflux under potassium-free conditions. The total sodium efflux amounted to 0.668 hr(-1) (rate constant) and was 71% ouabain sensitive.10. The present experiments demonstrated that lithium ions have a direct stimulating effect on sodium efflux in high sodium skeletal muscle, and strongly support the notion that this effect is produced by an activation of the sodium pump through a potassium-like action.

Animals↗

Inward calcium current in twitch muscle fibres of the frog.

1. Voltage clamp experiments using the three micro-electrode voltage clamp technique were performed on sartorius muscles of the frog. 2. By blocking potassium currents with tetraethylammonium and replacing chloride ions with sulphate a slow inward current was detected. 3. The slow inward current is mainly carried by calcium, since it is abolished by cobalt and D-600, it depends on external calcium, and is not affected by removing external sodium or by tetrodotoxin (TTX). 4. The slow inward current has a mean threshold of -40 mV, reaches a mean maximum value at ca. 0 mV of 81 microamperemetercm-2 and has a mean reversal potential of +38 mV. 5. The calcium current is inactivated by the application of 2 sec conditioning prepulses according to a sigmoid curve with V(h) = -42 mV and k = 6.2 mV. 6. The slow time course of this calcium current makes it rather unlikely that it participates in contraction during a twitch, but it might be activated during long depolarizations as potassium contractures.

Action Potentials↗

The energy liberation of frog skeletal muscle in tetanic contractions containing two periods of shortening.

1. Heat and work production have been measured in pairs of frog sartorius muscles undergoing two periods of rapid isovelocity shortening at 0 degrees C. The first (conditioning) shortening occurred in the sarcomere length range 2.70--2.25 micrometer (as measured in resting muscles) and the second (test) shortening in the range 2.25--2.10 micrometer. The shortening heat associated with the test shortening was obtained as the difference in heat production between pairs of tetani which were identical except for the presence of the test shortening. 2. The shortening heat associated with the test shortening was reduced when it was preceded by the conditioning shortening; with no interval between shortenings its value was 52 +/- 3% (mean +/- S.E. of mean, n = 6) of that without the conditioning shortening. As the interval between shortenings was increased the shortening heat became larger; its recovery was more than half complete with an interval of 0.3 sec. 3. The work produced in the test shortening was also reduced in tetani which contained the conditioning shortening; its dependence on the interval between shortenings was similar to that of the shortening heat.

Animals↗

Succinylcholine and muscle excitability.

Succinylcholine lowers the resting membrane potential taken with microelectrodes similarly in nerve-scarce and innervated portions of frog sartorius muscle. Twitches to electrical excitation of the nerve-scarce pelvic end of the muscle are also rapidly reduced. The results indicate that succinylcholine probably acts generally on the muscle membrance to diminish excitability.

Animals↗

Nitrate ions: Potentiation of increased permeability to sugar associated with muscle contraction.

Nitrate ions potentiate twitch tension and enhance the increase in permeability to sugar which occurs in electrically stimulated frog sartorius muscles. However, the potentiating effect of nitrate ions on permeability is not dependent upon an increase in twitch tension. The possible relation of changes in permeability to alterations of the concentration of calcium ions in the cell is discussed.

Animals↗

Dependence of cell pH and buffer capacity on the extracellular acid-base change in the skeletal muscle of bullfrog.

Intracellular pH was measured with single- or double-barreled liquid ion-exchanger microelectrodes in the bullfrog sartorius muscle perfused in vitro. A neutral carrier ligand was used for pH sensor of microelectrodes. Average slopes of the single-barreled microelectrodes were -56.4 +/- 1.34 mV/pH (n = 30) and the double-barreled -52.6 +/- 1.34 mV/pH (n = 65). While changing acid-base parameters of bathing media (pHe from 6.7 to 8.4, PCO2 from 3.7 to 37 mmHg, and HCO3- concentrations from 5 to 75 mM), paired muscle cell pH (pHi) and membrane potential (EM) values were determined at 23 degrees C. In control conditions (pHe = 7.6, HCO3- = 15 mM, PCO2 = 11 mmHg), pHi and EM (n = 20) averaged 6.99 +/- 0.04 (S.E.) and -69.2 +/- 2.2 mV, respectively. A negative correlation was observed between pHi and EM (correlation coefficient r = -0.564, p less than 0.002). The change in EM per unit pH change was approximately -30mV, indicating that the H+ distribution across the cell membrane only incompletely obeys the Donnan rule. The pHi varied more or less with pHe. Namely, changes in pHe and PCO2 at constant HCO3- produced relatively large changes in pHi, but elevation of pHe and HCO3- at constant PCO2 produced relatively minor rise in pHi. The stability of pHi or the size of buffer capacity were proportional to external HCO3- concentrations. These data suggested that a transmembrane distribution of buffer pairs depends largely on non-ionic diffusion of CO2-HCO3- buffer system and partly on ion fluxes of HCO3- or H+.

Acid-Base Equilibrium↗

[Study of the mechanics and small-angle equatorial x-ray pattern of the frog skeletal muscle during transition and rigor at different temperatures].

Mechanical characteristics and low-angle equatorial X-ray patterns from frog sartorius muscle passing into iodoacetate rigor under isometric conditions at temperatures 2 degrees-25 degrees C were studied. It is ascertained that during the rigor tension development at all the temperatures Z-reflection intensity increases and those of the (10), (11), (20), (21) and (30) reflections decrease. The last three reflections disappear then still in the phase of the rigor tension development. It is found that the sarcomere lengths remain not always invariable, especially at high temperatures, when the muscle passes into rigor, and can both decrease and increase in the sample place which is investigated by means of X-ray diffraction method. It is shown that the decrease of the I10/I11 relation in some experiments at high temperatures is only due to the sarcomere length decrease. The merging time of the Z and (11) reflections depends both on the temperature and on the sarcomere length change. Thus essential changes correlated with the rigor tension development, and resulted in the Z-reflection intensity increase take place in tetragonal lattice of Z-band and in the I-band region located near Z-band. In A-band the hexagonal lattice order change for the worse is marked only. It is proposed that the mechanism of the rigor tension development differs from that of tension development in ordinary contraction of the skeletal muscle.

Animals↗

Membrane lipid layers vs. polarized water dominated by fixed ions: a comparative study of the effects of three macrocyclic ionophores on the K+ permeability of frog skeletal muscle, frog ovarian eggs, and human erythrocytes.

The effects of 10(-7) M valinomycin, nonactin, and monactin on human erythrocytes, frog sartorius muscle, and frog ovarian oocytes in the presence of varying external K+ concentration were studied. The results showed essentially a consistent but relatively modest increase of the K+ permeability constant in cm/sec with all three antibiotics on human erythrocytes. No change in response to any one of the antibiotics was observed in frog muscles or in frog ovarian eggs. These results and reports of similar failure to demonstrate ionophore-mediated increase of K+ permeability in squid axon and inner membrane of the liver mitochondria led to the conclusion that lipid membrane barrier to ionic traffic may be significant in the human erythrocytes but even here one must regard the evidence as tentative. In contrast, for the majority of other cell types studied, the data indicate the primary, if not exclusive route of ion traffic, is via the nonlipid component of the cell membrane. The evidence that these nonlipid paths are the fixed charge-polarized water layer complex and that they cover much of the cell surface of many types of living cells was discussed.

Animals↗

Effect of phlorizin on the action potential and voltage-dependent ionic conductances in frog skeletal muscle.

The effects of phlorizin on the action potential and voltage-dependent ionic conductances were investigated in frog (Rana esculenta) sartorius muscles. In order to study the ionic currents, phase plane trajectories of the action potentials were analysed. As a consequence of lowering the overshoot by 19%, the amplitude of the action potential was decreased on the effect of phlorizin applied at a concentration of 2 mmol/l. The maximum rate of rise of the action potential (V +max) was reduced on the average by 23%. The maximum rate of fall of the spike potential (V -max) was also diminished by about 25% and the repolarization was prolonged consequently. Due to phlorizin treatment the early negative after-potential was significantly lowered or was not noticeable at all. Analysing the phase plane trajectories of the action potentials the peak inward (Na) current was found to be decreased by about 16% despite the unchanged maximum Na conductance. At the Na equilibrium potential a 9.2 mV shift towards negative direction was observed. Both the peak outward (K) current and the maximum outward (K) conductance were reduced by 25 and 29%, resp. tau K, the time constant reflecting the kinetic properties of K conductance was increased by 36%. Phlorizin treatment prevented the repetitive activity induced by cevadine. The inhibiting actions of phlorizin on fast ionic currents developed also in cevadine-treated muscles. All of the effects of phlorizin were reversible. The presented data show that phlorizin, the well-known inhibitor of phosphorylase and a competitive antagonist of sugar uptake, is also capable of modifying the electrical properties of the frog skeletal muscle membrane.

Action Potentials↗

Effects of organophosphate insecticides on the cholinergic receptors of frog skeletal muscle.

The organophosphates octamethyl pyrophosphoramide, Bidrin, and phosphoric acid 2,2-dichlorovinyl dimethyl ester inhibit the membrane voltage response of frog sartorius muscles to carbamylcholine in a manner expected of either a slowly reversing competitive inhibitor or a noncompetitive inhibitor. The inhibition reverses with a time course of minutes, depending upon the temperature. The inhibition by these compounds is reduced by d-tubocurarine but is unaffected by hexamethonium. This may indicate that the organophosphate binding sites are near the cholinergic binding site since both d-tubocurarine and hexamethonium are competitive inhibitors of cholinergic agonists and d-tubocurarine is the larger compound.

Acetylcholine↗

Membrane potential dependence of potential oscillation induced by cevadine in striated muscle.

The dependence of the membrane potential oscillation induced by cevadine on the actual transmembrane potential was studied in the frog sartorius muscle. 1. If the membrane potential oscillation is recorded for hours, its amplitude is seen to decrease slowly and smoothly and the membrane potential measured during the resting period among the waves of oscillation also decreases simultaneously. This means that the depolarization increases. 2. The increase of depolarization results not only in a decrease in the amplitude of the oscillation but the oscillation ceases between -40 and -55 mV as well. 3. The phenomenon reappears if the membrane is partially repolarized on a cevadine treated muscle fibre on which the membrane potential oscillation has already ceased as a consequence of the relatively marked depolarization. 4. Changing the membrane potential either to a value more positive than -40 mV, or to one more negative than -90 mV the developed oscillation activity may reversibly be suspended. 5. According to the above results, the amplitude of the membrane potential oscillation depends on the actual membrane potential. If other factors which may influence the oscillation parameters are unchanged, the relationship between the oscillation amplitude and the membrane potential can be characterized by a linear equation. In these cases there is a close correlation between the measured values and the calculated ones.

Animals↗

Increased responsiveness to dietary lysine deficiency of pectoralis major muscle protein turnover in broilers selected on breast development.

It has been previously established that growth and carcass qualities of chicks are modified by genotype and dietary amino acid supply. In this study, we evaluated the effects of lysine deficiency and genetic selection on muscle protein metabolism. Chicks originating from an experimental line selected for breast development (QL) and its control line (CL) were provided ad libitum access to isoenergetic diets containing 20% crude protein but differing in their lysine content (0.75 or 1.01%). Protein fractional synthesis rates (FSR) were measured in vivo in the pectoralis major and sartorius muscles of 3-wk-old chickens (flooding dose of [3H]phenylalanine). Fractional breakdown rates (FBR) were estimated as the difference between synthesis and deposition. Lysine deficiency reduced (P < 0.001) growth performance and muscle weights and increased (P < 0.05) muscle FSR, capacity for protein synthesis (muscle RNA:Protein, Cs) and FBR. Although QL birds grew faster and had heavier pectoralis major muscles than CL birds (P < 0.05), there was no line difference in sartorius weight (P = 0.15). No difference between the lines was observed in sartorius protein metabolism (P > 0.14). In the pectoralis major muscle, chicks of both lines receiving an adequate lysine intake also exhibited similar protein turnover rates. However, in chicks fed the lysine-deficient (0.75% lysine) diet, FSR and Cs were higher in QL than in CL chicks (P < 0.05), and FBR tended (P = 0.07) to be higher in QL chicks. This increased protein turnover in the QL birds on the lysine-deficient diet suggests that the responsiveness of muscle protein metabolism to amino acid supply is modified by genetic selection for breast development.

Animals↗

Effects of low calcium and calcium antagonists on skeletal muscle staircase and fatigue.

The purpose of this investigation was to determine the effects of low extracellular calcium and calcium antagonists on skeletal muscle staircase and fatigue. Initial experiments revealed that, brief exposure (10 minutes) of single frog sartorius muscle to diltiazem, D-600 (5 and 30 microM) and low calcium Ringer's solution (LCR, calcium replaced by magnesium and EGTA) had little effect on isometric twitches evoked every 30 seconds. However, when stimulated at 1 per second for 15 minutes, the calcium antagonists significantly decreased the magnitude and time course of the staircase, whereas LCR decreased only the time course. Each experimental condition significantly increased the rate of fatigue while diltiazem and D-600 both increased the magnitude of fatigue. Following the stimulation period, caffeine (10 mM) elicited contractures from all muscles whereas high potassium (180 mM) elicited contractures from control muscles only. These results indicate that calcium channel antagonists depress the skeletal muscle staircase response. They also indicate that these compounds as well as LCR enhance the fatigue process. Extracellular calcium influx may therefore have some influence on skeletal muscle twitches during prolonged repetitive activity.

Animals↗

Protein kinases A and C stimulate the Na+ active transport in frog skeletal muscle without an appreciable change in the number of sarcolemmal Na+ pumps.

AIM: The activation of both protein kinases A (PKA) and protein kinases C (PKC) in some cell types increases and in others reduces active Na+ efflux. These effects have been ascribed to either a change in the rate of ionic translocation by a fixed number of Na+ pumps or, a change in the number of plasma membrane pumps. The purpose of the present experiments was to study the effect of activating PKA and PKC on the Na+ extrusion by the Na+ pump in frog skeletal muscle. METHODS: Na+ (22Na+) fluxes and ouabain (3H-ouabain) binding were measured in frog sartorius muscles. RESULTS: Both activation of PKA and PKC increased the active Na+ extrusion by a factor of two; these effects were not additive. Ouabain binding experiments indicated that the pump stimulation by activation of these kinases is not associated with any significant increase in the number of plasma membrane pumps. Stimulation of the active Na+ efflux by protein kinase activation (no change in the number of sarcolemmal pumps) and by hypotonicity (increase in the number of pumps) could be elicited in the same preparation and they were additive. CONCLUSION: It is concluded that in frog skeletal muscle fibres, (1) activation of both PKA and PKC stimulate the Na+ pump by increasing its rate of ionic translocation; and (2) two modes of Na+ active transport (with and without an increase in the number of pumps) are operative, and can be at work simultaneously, a phenomenon to be reckoned with.

1-Methyl-3-isobutylxanthine↗