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.     

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.     

Variability in the shapes of satellite cells in normal and injured frog sartorius muscle

Surface topography of satellite cells is examined in the scanning electron microscope after application of a new method to eliminate connective tissue components. We describe the overall shape of satellite cells in normal frog muscle and show the range of variation in normal structure. Small increases in the width of tails and changes in the cell outline occur after muscle injury in vivo and after incubation in vitro. These changes may be signs of early mobilization of satellite cells related to their eventual role in muscle regeneration.     

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.     

Effects of caffeine on the intracellular distribution of calcium in frog sartorius muscle

The influence of caffeine on the intracellular distribution of calcium in the frog sartorius muscle was studied by differential centrifugation in an attempt to identify the locus of action of this alkaloid. The problem was approached in two ways. In the first, the locus of action was sought by relating the kinetic functions of ⁴⁵Ca washout curves of muscles to changes in the distribution of ⁴⁵Ca in the isolated fractions from the same muscles. It was not possible to make any correlation of the ⁴⁵Ca-washout curves to the activity in the fractions; the relative distribution of this nuclide remained essentially unchanged at 1-, 2-, and 3-hour intervals along the curve. The washout curves appear to be the net effect of a complex interaction of the calcium in pools containing both readily exchangeable calcium and calcium which has a slow exchange or turnover rate. The second approach centered upon the examination of the effect of caffeine on the intracellular distribution of ⁴⁵Ca and of calcium among the cellular fractions. Caffeine treatment resulted in a distinct increase in the calcium content of the mitochondrial fraction and a decrease in the calcium of the microsomal fraction. Electron micrographic studies revealed significant morphological changes in the whole muscle and in the isolated mitochondrial fraction after the muscle had been exposed to caffeine in a concentration producing irreversible contracture or rigor (10 mM). The increase in calcium content of the mitochondrial fraction after caffeine treatment may be due to an actual accumulation of calcium by the mitochondria or may be the consequence of the appearance of granular vesicles in the fraction.     

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.     

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+.     

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.     

The distribution of orthogonal assemblies and other intercalated particles in frog sartorius and rabbit sacrospinalis muscle.

Freeze-fracture replicas have been prepared from two fast-acting vertebrate muscles (frog sartorius and rabbit sacrospinalis) and are described with particular reference to the distribution of intercalated particles in the plasma membrane. T-tubule and SR cisternae. Orthogonal assemblies of small particles are present on the A face plasma membrane in each instance, and their distribution (in sartorius) is found to be random with respect to the underlying myofibrillar sarcomere repeat. Such assemblies are not present on A or B faces of T-tubule or SR cisternae. Asymmetric particle distribution is described for fracture faces of the T-tubules and SR: the profuse particle packing of the SR A face is uniform from terminal cisternae to medial fenestrated collar. Intercalated particles are present on A and B faces of T-tubule fractures; more commonly on the latter. These results are compared with studies on insect muscles, and a comparative approach to further studies on the correlation between membrane structure and function is discussed.     

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.