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.     

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.     

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.     

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.     

A comparative study of the membrane structure in different types of muscle fibers in the frog

The muscle membrane of slow and fast fibers in cruralis and iliofibularis muscles and of intermediate fibers in submaxillaris muscle of the frog is studied in freeze-fracture replicas. A comparison of membrane folds, number, size and distribution of caveolae and of intramembrane particles (IMP) is given. In slow muscle fibers, the membrane folds are systematically present at the level of the I zone with a transversal continuity, whereas in fast and intermediate types the membrane folds are small and are randomly distributed. In slow muscle the caveolae are more numerous at the I zone than in the part corresponding to the center of the sarcomere. In fast muscle, small groups of caveolae form linear patterns, and in intermediate fibers the distribution is random. The number of caveolae in slow muscle fibers is two times more than in fast and intermediate fibers. The mean area of caveolae opening is largest in fast and smallest in slow muscle fibers. The number of IMP is significantly different in the three types of fibers, being highest in slow and lowest in intermediate fibers. The different pattern of folds in slow fibers may correspond to the different contractile properties of this fiber type. The presence of double the number of caveolae in slow fibers correlated to the less elaborate T system in this fiber type shows the possibility that slow fibers may be the result of an arrest during development for the performance of a different function. The difference in IMP density in the three muscle fiber types may be interpreted as the difference in their electrical properties.     

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.     

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.     

Buffer power and intracellular pH of frog sartorius muscle.

Intracellular pH (pHi) and buffer power of frog muscle were measured using pH-sensitive microelectrodes under conditions used previously in energy balance experiments because pH strongly influences the molar enthalpy change for phosphocreatine splitting, the major net reaction during brief contractions. The extracellular pH (pHe) of HEPES buffered Ringer's solution influenced pHi, but change in pHi developed slowly. Addition or removal of CO2 or NH3 from the extracellular solution caused a rapid change in pHi. The mean buffer power measured with CO2 was 38.4 mmol.l-1.pH unit-1 (+/- SEM 2.1, n = 49) and with NH3 was 36.2 (+/- SEM 5.5, n = 4) at 20-22 degrees C. At 5 degrees C, in experiments with CO2 the mean buffer power was 40.3 (+/- SEM 2.6, n = 3). For pHi values above approximately 7.0, the observed buffer power was greater than that expected from the values in the literature for the histidine content of intracellular proteins, carnosine and inorganic phosphate in the sarcoplasm. The measured pHi values were similar to those assumed in energy balance calculations, but the high measured buffer power suggests that other buffering reactions occur in addition to those included in energy balance calculations.     

High resolution scanning electron microscopy of frog sartorius muscle

A field emission-type scanning electron microscope (SEM) was used to study the three-dimensional ultrastructure of frog sartorius muscles. Various preparative procedures were tested to seeks better specimen preparation for high resolution SEM observation. Procedures should be chosen depending on the information desired. The cell surface and intracellular organization of muscle fibers were best visualized when the tissues were fixed with tannic acid-OsO4 and torn after critical point drying. The basal lamina appeared as a continuous felt-like layer, onto which fine filamentous materials adhered. The true outer surface of the sarcolemma was not seen, whereas the true inner surface was occasionally exposed and exhibited numerous caveolae, membraneous fragments and fine filaments attached to its surface. In freeze-fractured and dried tissues, the cleaved sarcolemma showed numerous apertures of caveolae and T-system tubules. Inside the cell, the myofibrils showed a typical branding pattern of the sarcomere. Thick myofilaments were regularly beaded except for the pseudo-H-zone. Around the myofibrils the sarcoplasmic reticulum and T-system were also clearly observed. The results are discussed with special reference to the usefulness and limitation of the high resolution SEM in studying the ultrastructure of cells and tissues.     

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.