The effect of gramicidin A on the K+ conductance of the membrane of isolated frog skeletal muscle fibres.

Gramicidin A effects a drastic decrease of the membrane resistance of frog skeletal muscle fibres in isotonic K2SO4 solution. In detubulated fibres the effect is not so pronounced. The reduction of the membrane resistance is caused by an increase in the K+ conductance of the surface and T-system membranes of the muscle cell.     

Cross bridge slippage in skinned frog muscle fibres

Mechanically skinned single fibres of the semitendinosus muscles of Rana esculenta were investigated at ca. 4 C. The fibres were activated by a Ca²⁺ jump technique, which allowed the development of a steady isometric tension within several seconds of entering a calcium rich solution at 4 C. Sequences of length changes of different duration and amplitude were applied to the fibre. It could be demonstrated that the fibre behaved as a Hookean spring in the case of small amplitude length changes (up to 0.5% L₀, ramp duration 0.5 ms) and that a sequence of length changes induced reversible changes in fibre state. In contrast, large stretches (> 1% L₀) induced a muscle give if the stretch were not immediately preceded by a release. The data was interpreted on the basis of a strain induced detachment of cross bridges in combination with a rapid reattachment of presumably the same cross bridges in a discharged position. The rates of strain induced detachment and reattachment depended on the stretch amplitude. At amplitudes exceeding 2% L₀ the rates were estimated to be at least several thousands per second.     

Basolateral membrane potassium conductance is independent of sodium pump activity and membrane voltage in canine tracheal epithelium

Summary When secretagogues stimulate Cl secretion in canine tracheal epithelium, apical membrane Cl conductance (G _a ^Cl ) increases, and then basolateral membrane K conductance (G _b ^K ) increases. Conversely, inhibition ofG _a ^Cl results in a secondary decrease inG _b ^K . The coordination of the two membrane conductances and regulation ofG _b ^K is critical for maintaining constant intracellular ion concentrations and transepithelial Cl secretion. The purpose of this study was to test two hypotheses about the regulation ofG _b ^K . First, we asked whetherG _b ^K is directly linked to the activity of the Na,K-ATPase. We found that pump activity could be dissociated from K conductance. Inhibition of the Na pump with ouabain, in nonsecreting tissues led to an increase inG _b . Elevation of the bathing solution K concentration produced a similar effect. Addition of ouabain to secreting tissues did not appear to alterG _b . These results indicate thatG _b ^K does not directly parallel Na pump activity. Second, we asked whether changes inG _b ^K are voltage dependent. We prevented secretagogue-induced depolarization of the electrical potential difference across the basolateral membrane _b by clamping _b at its resting value during stimulation of Cl secretion with epinephrine. Despite maintaining _b constant, the typical changes in transepithelial resistance and the ratio of membrane resistances persisted. This observation indicates that depolarization is not required for the secretagogue-induced increase inG _b ^K . In addition we examined the effect of depolarizing and hyperpolarizing _b by passing transepithelial current in secreting and nonsecreting epithelia. Despite depolarizing and hyperpolarizing _b within the physiologic range, we observed no significant changes in transepithelial resistance or the ratio of membrane resistance that would suggest a change inG _b ^K . This observation indicates that changes in _b are not sufficient to alterG _b ^K . Thus,G _b ^K appears to be regulated by factors other than membrane voltage, or direct coupling to the Na pump.     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

Purinergic receptors expressed in human skeletal muscle fibres

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.     

Effect of high pH on the plasma membrane potential and conductance in Elodea densa

In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions. Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about -160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance. The application of pH 11, which caused a momentary hyper- or depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around -160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.     

Effect of temperature on the inward rectifier and gramicidin A-induced channels in the membrane of frog skeletal muscle fibres

The conductance of frog skeletal muscle fibres in isotonic K2SO4 solution has been measured. Experiments were carried out under current-clamp conditions using a double sucrose-gap technique. The potassium conductances of the inward rectifier and the gramicidin channel in the same muscle fibre were compared. Potassium conductance of the inward rectifier increased with the temperature, with a value of Q10 1.55 +/- 0.09 (n = 8) under hyperpolarization, and Q10 2.38 +/- 0.23 (n = 6) for the depolarizing stimulus, the difference between Q10 of potassium and gramicidin channels being statistically insignificant.     

Trichinella spiralis: skeletal muscle membrane potentials in infected and uninfected mice

Male albino mice were infected orally with 400 ± 10 excysted Trichinella spiralis larvae. Skeletal muscle resting membrane potentials were recorded from the tibialis anterior muscles of infected and uninfected mice on the following days postinfection (PI): 1–15, 18, 20, 24, 28, 30–60 (at 5-day intervals), 90, 120, 150, and 180. The membrane potentials were significantly (P < 0.05) lower in infected muscle (82 vs 85 mV) on Day 30 PI. On Days 60, 90, 120, 150, and 180 PI the mean membrane potential in infected muscle (62 mV) was about 23 mV lower than the mean for uninfected muscle (85 mV) and this difference was highly significant (P < 0.001). These findings are discussed in relationship to other physiological alterations known to occur in skeletal muscles infected with T. spiralis larvae.     

The number of satellite cells in slow and fast fibres from human vastus lateralis muscle

The aim of this investigation was to study the distribution of satellite cells in slow (type I fibres) and fast (type II fibres) fibres from human vastus lateralis muscle. This muscle is characterised by a mixed fibre type composition and is considered as the site of choice for biopsies in research work and for clinical diagnosis. Biopsy samples were obtained from five healthy young volunteers and a total of 1,747 type I fibres and 1,760 type II fibres were assessed. Satellite cells and fibre type composition were studied on serial muscle cross-sections stained with specific monoclonal antibodies. From a total of 218 satellite cells, 116 satellite cells were found in contact with type I fibres (53.6±8% of the satellite cells associated to type I fibres) and 102 satellite cells in contact with type II fibres (46.4±8% of the satellite cells associated to type II fibres). There was no significant difference (P=0.4) between the percentages of satellite cells in contact with type I and with type II fibres. Additionally, there was no relationship between the mean number of satellite cells per fibre and the mean cross-sectional area of muscle fibres. In conclusion, our results show that there is no fibre type-specific distribution of satellite cells in a human skeletal muscle with mixed fibre type composition.     

Caffeine and the myoplasmic calcium removal mechanisms in cut frog skeletal muscle fibres

Antipyrylazo III myoplasmic calcium transients were recorded in cut skeletal muscle fibres of the frog (Rana esculenta), using the double vaseline-gap voltage-clamp system. Intracellular calcium removal mechanisms were analysed, using a slightly modified model taken from the literature. Parameter values reported here are generally consistent with those obtained by the original model. Caffeine (0.5 mmol.l-1) moderately enhanced the overall myoplasmic calcium removal. In particular, the rate constant of the non-saturable uptake increased by 51% on the average, but there was a considerable fiber-to-fiber variation. The kinetic features of the binding sites representing the saturable uptake did not change significantly while the concentration of the available sites decreased by 36%. It is concluded that the caffeine-induced changes of the calcium removal components can be explained by supposing an increased resting myoplasmatic Ca2+ concentration in the presence of the drug.     

Urea loading enhances postfreeze performance of frog skeletal muscle

The wood frog (Rana sylvatica) is a terrestrial hibernator that can accumulate urea as an osmoprotectant in autumn and winter. This study tested the hypothesis that elevated urea can also function as a cryoprotectant in this freeze-tolerant species. Performance characteristics (threshold stimulus voltage, maximal isometric twitch and tetanic contraction forces, and (1/2) fatigue time) of isolated gastrocnemius muscles were measured before and after experimental freezing at -1.5 degrees C for 18 h, followed by thawing. Frozen/thawed muscles exhibited reduced function relative to baseline (prefreeze) levels; however, muscles preincubated in a saline solution containing urea (80 mmol l(-1)) performed substantially better in some tests than muscles incubated without urea. Concentrations of urea in these treated muscles, approximately 65 mmol l(-1), were within the physiological range in winter R. sylvatica. Reducing tissue urea levels to approximately 33 mmol l(-1) resulted in a similar pattern of response, although the differences between urea-incubated and saline-incubated muscles were not statistically significant. Tests of cryoprotective efficacy were also performed on gastrocnemius muscles from R. pipiens, a closely related, but freeze-intolerant species that hibernates aquatically and thus has little need to accumulate urea. Urea-treated muscles from this species performed no better than muscles incubated in saline, attesting that freeze tolerance cannot be conferred simply by augmenting cryoprotectant levels. Overall, these results bolster an earlier report that urea accumulated in response to low moisture availability can serve a cryoprotective role in freeze-tolerant ectotherms.     

Relationship of transepithelial electrical potential to membrane potentials and conductance ratios in frog skin

Summary Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4–6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiological events associated with voltage clamping (V _t =inside-outside potential). Increase and decrease ofV _t produced converse effects, related directly to the magnitude ofV _t .Hyperpolarization resulted in prompt decrease in inward transepithelial currentI _t and increase in fractional outer membrane resistancefR ₀ (as evaluated from small transient voltage perturbations) and in outer membrane potentialV ₀. Overshoot ofV ₀ was followed by relaxation to a quasi-steady state in minutes. Changes infR ₀ were progressive, with half times of some 1–5 sec. Changes in transepithelial slope conductanceg _t were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductancesg ₀ andg _i . Nevertheless, it was shown thatg ₀ is related inversely toV _t andV ₀. Presuming insensitivity ofV _i toV _t , the dependence ofg ₀ onV ₀ in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence ofg ₀ onV ₀ and/or cellular current. In contrast to previous findings in tight epithelia at open circuit, differences inV _t were associated with substantial differences infR ₀ and inner membrane potentialV _i . Hyperpolarization ofV _t over ranges commonly employed in studies of active transport and metabolism appears to increase significantly the electrochemical work per Na ion transported.     

Force generation and shift of mass between myosin and actin in skinned striated muscle fibres at low calcium concentrations

Skinned muscle fibres from the gracilis muscle of the rabbit were used to record small angle X-ray diffraction spectra under various contractile conditions. The intracellular calcium concentration, expressed as pCa, was varied between 8.0 and 5.74. Equatorial diffraction spectra were fitted by a function consisting of five Gaussian curves and a hyperbola to separate the (1.0), (1.1), (2.0), (2.1) and Z-line diffraction peaks. The hyperbola was used to correct for residual scattering in the preparation. The ratio between the intensities of the (1.1) and (1.0) peaks was defined as the relative transfer of mass between myosin and actin, due to crossbridge formation after activation by calcium. The relation between the ratio and the relative force of the fibre (normalized to the force at pCa 5.74 and sarcomere length 2.0 μm) was linear. At high pCa (from pCa 6.34 to 8.0) no active force was observed, while the ratio still decreased. Sarcomere length was recorded by laser diffraction. The laser diffraction patterns did not show changes in sarcomere length due to activation in the high pCa range (between 8.0 and 6.34). From these results the conclusion is drawn that crossbridge movement occurs even at subthreshold calcium concentrations in the cell, when no active force is exerted. Since no force is generated this movement may be related to crossbridges in the weakly bound state. Received: 20 June 1996 / Revised version: 12 January 1998 / Accepted: 18 March 1998     

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue. Received: 5 May 1998 / Revised version: 27 October 1998 / Accepted: 19 January 1999     

An integrative study of the temperature dependence of whole animal and muscle performance during jumping and swimming in the frog Rana temporaria

The aims of this study were: (1) to analyze individual variation in frog locomotor performance, (2) to compare the thermal sensitivity of jumping and swimming, and (3) to contrast whole animal versus muscle fiber performance at different temperatures. The jumping and swimming performance of Rana temporaria was analyzed at 5, 10, 15 and 20 °C. Muscle fiber bundles were isolated from lateral gastrocnemius and subjected to the length and activation patterns thought to occur in vivo. As temperature increased, locomotor performance in R. temporaria improved with a Q ₁₀ of 1.2 for both jump take-off velocity and mean swimming velocity. The slope of the relationship between performance and temperature (TE) was similar for both locomotor parameters and was described by the equation z-scores of locomotor performance = 0.127 × TE − 1.585. Although some frogs performed better than others relative performance was affected by locomotor type and temperature. Locomotor performance improved with temperature as the power required during take-off and the mean muscle power output increased with Q ₁₀ values of 1.7 and 1.6 respectively. The mean muscle power output during take-off was only 34% of the calculated requirements for the whole animal, suggesting the involvement of elastic strain energy storage mechanisms. Accepted: 2 September 1999     

Isolation and characterization of basement membrane from frog skeletal muscle

Isolation of basement membrane from frog skeletal muscle has been described. The membrane preparation contained 35 micrograms hexoses, 1.72 micrograms sialic acid, 6.8 micrograms phospholipids, 0.21 micrograms cholesterol/mg protein. Na + K-ATPase and 5'-nucleotidase could not be detected in the membrane preparation. Glycine accounted for about 20% of the total amino acids. On SDS-PAGE, the membrane resolved into 20-22 polypeptide bands.     

Factors in the inactivation of postjunctional membrane receptors of frog skeletal muscle

Several factors which influence the rate of inactivation of muscle postjunctional membrane (PJM) receptors during the sustained application of carbamylcholine (CARB) have been studied by two methods. The rate of inactivation was increased by elevating the tonicity of the bathing medium, by increasing the CARB concentration, by raising the calcium ion concentration, and by substituting SO₄ ⁼ for Cl^- ions in the extracellular fluid. The relative effectiveness of calcium and other divalent cations in receptor inactivation was compared. In the absence of calcium, other divalent cations such as magnesium, strontium, or manganese were not efficient substitutes for calcium. In the presence of calcium, the addition of strontium or manganese ions accelerated the rate of receptor inactivation, but the addition of magnesium (up to 12 mM) inhibited this process. The inactivation of the membrane receptors in denervated muscle fibers was found to be similar to that in innervated muscle fibers. Various factors in PJM receptor inactivation are discussed. It is suggested that PJM receptor inactivation is influenced by the binding of calcium ions to sites on the internal surface of the PJM.     

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.