Satellite and invasive cells in frog sartorius muscle

The occurrence and distribution of two cell types associated with normal and denervated frog skeletal muscle fibers are described. The first is the satellite cell. The general appearance and the number of satellite cells are not affected by long-term denervation. The second type of cell is the invasive cell. Invasive cells penetrate across the basal lamina and up to the core of the muscle fiber, without fusing with it. It is suggested that the origin of invasive cells is extramuscular, probably circulatory. Although invasive cells are more numerous in some denervated muscle, it is established that this is not a direct effect of denervation.     

Density and distribution of tetrodotoxin receptors in normal and detubulated frog sartorius muscle

Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the effects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in surface membrane is about 175/muM2 and that in the transverse tubular membrane is 41-52/mum2.     

Intracellular pH of frog sartorius muscle

A weak base, morpholine, has been labelled with ³H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20°C in a solution of the same of ionic strength as frog Ringer was found to be 8.45 ± 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indicator. Intracellular pH was then measured by distribution of morpholine (6.985 ± 0.08), nicotine (6.915 ± 0.03) and the weak acid 5,5′-dimethyl-2,4-oxazolidinedione (7.10 ± 0.05) and with pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H⁺ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Local movement in stimulated frog sartorius muscle

Local movement was recorded in tetanically contracting frog sartorius muscle to estimate the nonuniformity in the distribution of compliance in the muscle preparation and the compliance that resides in the attachments of the preparation to the measuring apparatus. The stimulated muscle was also subjected to rapid length changes, and the local movements and tension responses were recorded. The results indicate that during tension development at resting length the central region of the muscle shortens at the expense of the ends. After stimulation the "shoulder" in the tension, which divided the relaxation into a slow decline and a subsequent, rather exponential decay toward zero, was accompanied by an abrupt increase in local movement. We also examined the temperature sensitivity of the two phases of relaxation. The results are consistent with the view that the decrease in tension during relaxation depends on mechanical conditions. The local movement brought about by the imposed length changes indicates that the peak value of the relative length change of the uniformly acting part was approximately 20% less than the relative length change of the whole preparation. From these observations, corrections were obtained for the compliance data derived from the tension responses. These corrections allow a comparison with data in the literature obtained from single fiber preparations. The implications for the stiffness measured during the tension responses are discussed.     

Intracellular pH of frog sartorius muscle.

A weak base, morpholine, has been labelled with 3H and tested for its suitability as an indicator for intracellular pH, by distribution in the tissue water of frog sartorius muscle in the species Hyla litoria. Its pK'a at 20 degrees C in a solution of the same ionic strength as frog Ringer was found to be 8.45 +/- 0.02, which is in the range of maximal sensitivity. Morpholine equilibrated with the tissue in 17 h; it was shown that it was not bound to intracellular constituents, that it was not metabolised nor toxic in the concentrations used; it was therefore judged suitable as a pH indcator. Intracellular pH was then measured by distribution of morpholine (6.985 +/- 0.08), nicotine (6.915 +/- 0.03) and the weak acid 5,5'-dimethyl-2,4-oxazolidinedione (7.10 +/- 0.05) and the pH-sensitive microelectrodes (5.9, the equilibrium value). It was shown that the four significantly different values could not be reconciled in terms of experimental error, heterogeneity of intracellular pH, liquid junction potential differences, or binding of indicator molecules inside the fibre. They could, however, be reconciled if the fibre water had different structure and solvent properties from the extracellular water and all ions were distributed across the membrane as between two liquid phases containing different solvents. Then the H+ would be in equilibrium, as shown by the microelectrode measurement, but intracellular pH would be indeterminable and probably greater than 6.     

Ionic currents in nerve terminals of the frog sartorius muscle

Nerve terminal responses produced by stimulating the motor nerve were recorded extracellularly from the nerve endings of the frog sartorius muscle. A triphasic response occurred in the proximal areas of the nerve ending, beginning with a positive phase. Ionotophoretic application of tetrodotoxin, tetraethylammonium, and 4-aminopyridine indicated that the negative phase reflected inward sodium current and the third (positive) phase indicated outward potassium current. A late slow negative component was recorded using CaCl₂-filled electrodes during perfusion of erve-muscle preparations with a calcium-free solution containing tubocurarine. This component was dependent on the Ca²⁺ concentration present in the electrode, increasing when tetraethylammonium and 4-aminopyridine were added and disappearing under the effects of Co²⁺. Similar components were recorded using microelectrodes containing Sr²⁺ and Ba²⁺. It was deduced that the slow components in the response indicate currents passing through voltage-dependent calcium channels in the presynaptic membrane of the nerve ending. The time course of the calcium current is compared with that of transmitter release at the synapse.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. V. I. Ulanov-Lenin Kazan' State University. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 770–779, November–December, 1985.     

Diffusion and consumption of oxygen in the resting frog sartorius muscle

Adaptations of the method of Takahashi et al. (1966. J. Gen. Physiol. 50:317-333) were used to test the validity of the one-dimensional diffusion equation for O2 in the resting excised frog sartorius muscle. This equation is: (formula: see text) where x is the distance perpendicular to the muscle surface. t is time, P(x, t) is the partial pressure of O2,D and alpha are the diffusion coefficient and solubility for O2 in the tissue, and Q is the rate of O2 consumption. P(O, t), the time-course of PO2 at one muscle surface, was measured by a micro-oxygen electrode. Transients in the PO2 profile of the muscle were induced by two methods: (a) after an equilibration period, one surface was sealed off by a disc in which the O2 electrode was embedded; (b) when PO2 at this surface reached a steady state, a step change was made in the PO2 at the other surface. With either method, the agreement between the measured P(O, t) and that predicted by the diffusion equation was excellent, making possible the calculation of D and Q. These two methods yielded statistically indistinguishable results, with the following pooled means (+/- SEM): (formula: see text) At each temperature, D was independent of muscle thickness (range, 0.67-1.34 mm). The activation energy (EA) for diffusion of oxygen in muscle was -3.85 kcal/mol, which closely matches the corresponding value in water. Together with absolute values of D in water taken from the literature, the present data imply that (Dmuscle/DH2O) is in the range 0.59-0.69. This value, and that of EA, are in agreement with the theory of Wang (1954, J. Am. Chem. Soc. 76:4755-4763), suggesting that with respects to the diffusion of O2, to a useful approximation, frog skeletal muscle may be considered simply as a homogeneous protein solution.     

Visualization of satellite cells in living muscle fibres of the frog

Satellite cells were visualized in living muscle fibres of the frog. Single fibres or bundles consisting of a few fibres were isolated after treatment with collagenase, and viewed under the light microscope. Subsequent electron microscopy of identified cells confirmed that they were satellite muscle cells. Under the light microscope, satellite cells appear as fusiform cells, tapering into long fine processes usually orientated parallel to the muscle fibre axis. Horseradish peroxidase injected into the muscle fibre was not transferred to the satellite cells.     

K+ depolarization and phospholipid metabolism in frog sartorius muscle

K+ depolarization evokes phosphatidylinositol response, i.e. the increased 32P orthophosphate labelling of phosphatidylinositol in frog sartorii muscles. The phosphatidylinositol response seems to be closely related to K+ depolarization and not to the transient Ca2+ release at the beginning of depolarization. It ceases as soon as the muscles depolarized by 90 mmol/l KCl for a short period of time are repolarized, while it continues when the depolarization is maintained. When the muscles are depolarized with 20 mmol/l KCl, the phosphatidylinositol response is also observed. This response is not suppressed by drugs that block Ca2+ mobilization. Other agents like caffeine, azide or EGTA which induce some effects similar to that of K+ depolarization, do not evoke phosphatidylinositol response. Rather, they simply cause a decrease in the labelling of phospholipids, phosphatidylinositol being the least affected. In muscles derived from frogs maintained under healthy conditions Ca2+ release in the early phase of K+ depolarization does not cause significant changes in phospholipid labelling. However, in muscles from frogs starving for many months, a large decrease in the labelling of phospholipids is observed in the early phase of K+ depolarization. It is postulated that the changes in the physicochemical state of the membrane and not Ca2+ gating mechanism or free cell Ca2+ level are crucial in the phosphatidylinositol response in the frog sartorii muscles depolarized by high K+.     

Morphometric and histochemical characteristics of the frog sartorius muscle in ontogeny]

Morphometric and histochemical investigation of musculus sartorius was performed in ontogenesis in Rana ridibunda and Rana temporaria. Muscular composition was characterized according to the type of muscular fibres. Spectrum of lactatdehydrogenase isoenzymes was studied at different developmental stages. As the studies demonstrated, musculus sartorius underwent some essential changes in ontogenesis which manifested themselves in increasing number of muscular fibres and their areas, in changing LDG isoenzymic spectrum. Differentiation of the muscular fibres three types takes place at the 30th stage after P. V. Terentiev and depends on the nerve system maturation.     

Density and apparent location of the sodium pump in frog sartorius muscle

Summary The binding of the cardiosteroid³H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release.The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (B _max) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2×10^–7 m. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one.Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (15 hr). Increasing [K⁺]₀ from 2.5 to 10mm slows the time course of binding without any significant change in binding capacity of the muscle fibers.Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979)Biochim. Biophys. Acta 552:247] on the distribution of (Na⁺+K⁺)-ATPase among different cell membrane fractions from frog skeletal muscle.     

Series elasticity in frog sartorius muscle during release and stretch

When a stretch is applied to an isolated muscle during tetanic stimulation, the force developed is higher than the maximal isometric tension (Po). This force puts the series elastic component (SEC) under tension and in a domain which is not well defined in terms of tension-extension curve. In the present work, an attempt was made to determine the stiffness of the SEC for tensions greater than Po, using the sartorius muscle of the frog. For this purpose, rapid releases and stretches of different amplitudes were given during maximal isometric contractions. Plotting normalized tension (P/Po) against normalized length changes (negative or positive extensions, delta L/Lo.10(2] produced a tension-extension curve. The slopes of the linear part of each relationship on both sides of Po indicated an increase in SEC stiffness when the muscle was rapidly stretched. Furthermore, the transient character of the increase in stiffness was studied by measuring SEC stiffness during rapid releases applied at various time intervals after stretches: the muscle was found to be stiffer as the time interval was shorter. The results are discussed in terms of (i) non-linear behaviour of the passive and active parts of the SEC, (ii) enhancement of storage and release of potential energy.