Effects of bicarbonate on resting potentials in mammalian skeletal muscle

In the absence of external HCO3, resting membrane potentials (Vm) in extensor digitorum longus muscle were depolarized as compared to the normal Vm in the presence of HCO3. Removal of Na or Cl form the HCO3-free media induced repolarization. In muscle in HCO3 buffer at 20 degrees C, internal K, Na, and Cl activities were analyzed with liquid ion selective microelectrodes. The averages were respectively, 119.7 +/- 2.1, 6.69 +/- 0.3, and 3.41 +/- 0.06 mM. In a high proportion of cells analyzed, the equilibrium potential for Cl was negative to Vm. Removing external HCO3, decreased internal K while internal Na and Cl increased. An increase in temperature and the application of HCO3 significantly lowered internal activities of both Na and Cl. Removal of HCO3 with temperature held constant caused a rapid depolarization, an increase in internal Na and Cl, and a decrease in internal K. Furosemide (10 microM) induced a repolarization of cells that were previously depolarized in the HCO3-free state, but the drug does not decrease internal Na.     

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.     

Ouabain stimulates unidirectional and net potassium efflux in resting mammalian skeletal muscle.

The present study compared ouabain-sensitive unidirectional K+ flux into (JinK) and out of (JoutK) perfused rat hindlimb skeletal muscle in situ and mouse flexor digitorum brevis (FDB) in vitro. In situ, 5 mM ouabain inhibited 54 +/- 4% of the total JinK in 28 +/- 1 min, and increased the net and unidirectional efflux of K+ within 4 min. In contrast, 1.8 mM ouabain inhibited 40 +/- 8% of the total JinK in 38 +/- 2 min, but did not significantly affect JoutK. In vitro, 1.8 and 0.2 mM ouabain decreased JinK to a greater extent (83 +/- 5%) than in situ, but did not significantly affect 42K loss rate compared with controls. The increase in unidirectional K+ efflux (JoutK) with 5 mM ouabain in situ was attributed to increased K+ efflux through cation channels, since addition of barium (1 mM) to ouabain-perfused muscles returned JoutK to baseline values within 12 min. Perfusion with 5 mM ouabain plus 2 mM tetracaine for 30 min decreased JinK 46 +/- 9% (0.30 +/- 0.03 to 0.16 +/- 0.02 micromol x min(-1) x g(-1)), however tetracaine was unable to abolish the ouabain-induced increase in unidirectional K+ efflux. In both rat hindlimb and mouse FDB, tetracaine had no effect on JoutK. Perfusion of hindlimb muscle with 0.1 mM tetrodotoxin (TTX, a Na+ channel blocker) decreased JinK by 15 +/- 1%, but had no effect on JoutK; subsequent addition of ouabain (5 mM) decreased JinK a further 32 +/- 2%. The ouabain-induced increase in unidirectional K+ efflux did not occur when TTX was perfused prior to and during perfusion with 5 mM ouabain. We conclude that 5 mM ouabain increases the unidirectional efflux of K+ from skeletal muscle through a barium and TTX-sensitive pathway, suggestive of voltage sensitive Na+ channels, in addition to inhibiting Na+/K+-ATPase activity.     

Internal citrate ions reduce the membrane potential for contraction threshold in mammalian skeletal muscle fibers.

An effect of internal citrate ions on excitation-contraction coupling in skeletal muscle is described. The threshold for contraction was measured in rat extensor digitorum longus, (EDL), and soleus muscle fibers using a two microelectrode voltage clamp technique with either KCl-filled or K3 citrate-filled current electrodes. Contraction thresholds were stable for many minutes with KCl current electrodes. In contrast, thresholds fell progressively towards the resting membrane potential, by as much as -15 mV over a period of 10 to 20 min of voltage-clamp with citrate current electrodes. In addition, prepulse inhibition was suppressed, subthreshold activation enhanced and steady-state inactivation shifted to more negative potentials. Fibers recovered slowly from these effects when the citrate electrode was withdrawn and replaced with a KCl electrode. The changes in contraction threshold suggest that citrate ions act on the muscle activation system at an intracellular site, since the citrate permeability of the surface membrane is probably very low. An internal citrate concentration of 5 mM was calculated to result from citrate diffusion out of the microelectrode into the recording area for 20 min. 5 mM citrate added to an artificial cell lowered the free calcium concentration from 240 to 31 microM. It is suggested that citrate modifies excitation-contraction coupling either by acting upon an anion-dependent step in activation or by reducing the free calcium and/or free magnesium concentration in the myoplasm.     

Effect of extracellular osmolality on cell volume and resting metabolism in mammalian skeletal muscle

The purpose of the present investigation was to establish an in vitro mammalian skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated, whole muscles [soleus and extensor digitorum longus (EDL)] were dissected from Long-Evans rats and incubated for 60 min in Sigma medium 199 (1 g of resting tension, bubbled with 95% O(2)-5% O(2), 30 +/- 2 degrees C, and pH 7.4). Medium osmolality was altered to simulate hyposmotic (190 +/- 10 mmol/kg) or hyperosmotic conditions (400 +/- 10 mmol/kg), whereas an isosmotic condition (290 +/- 10 mmol/kg) served as a control. After incubation, relative water content of the muscle decreased with hyperosmotic and increased with hyposmotic condition in both muscle types (P < 0.05). The cross-sectional area of soleus type I and type II fibers increased (P < 0.05) in hyposmotic, whereas hyperosmotic exposure led to no detectable changes. The EDL type II fiber area decreased in the hyperosmotic condition and increased after hyposmotic exposure, whereas no change was observed in EDL type I fibers. Furthermore, exposure to the hyperosmotic condition in both muscle types resulted in decreased muscle ATP and phosphocreatine (P < 0.05) contents and increased creatine and lactate contents (P < 0.05) compared with control and hyposmotic conditions. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acute alterations in muscle water content and resting muscle metabolism.     

Effects of membrane potential on mechanical activation in skeletal muscle

The effect of subthreshold depolarization on mechanical threshold was investigated in tetrodotoxin-poisoned mammalian and amphibian skeletal muscle fibers using a two-microelectrode voltage-clamp technique. Mechanical threshold was determined with a 2-ms test pulse. The immediate effect of depolarization was inhibition of the mechanical system. The consequent increase in the test pulse threshold was linearly related to the size of the depolarization and there was, on the average, a 10% increase in threshold for a 10-mV depolarization in mammalian fibers. The duration of the inhibitory period was also related to the size of the depolarization. Inhibition was interrupted by the onset of activation (seen as a reduction in the test pulse threshold), and in rat soleus fibers this occurred within 100 ms with a 20-mV depolarization, inhibition decayed within 10 ms. The decay of activation after brief conditioning pulses was initially rapid (on the average, the test pulse threshold recovered to 80% of its control value within 1 ms) and then slow (full recovery took 100-500 ms). After long conditioning pulses, activation often decayed into a period of inhibition. When depolarization (of 20 mV or more) was maintained for several seconds, the fibers became inactivated. Rat extensor digitorum longus and sternomastoid fibers were strongly inactivated by depolarization to -40 mV and the test pulse to +40 mV did not cause contraction.     

Three-dimensional structure of the Z band in a normal mammalian skeletal muscle

The three-dimensional structure of the vertebrate skeletal muscle Z band reflects its function as the muscle component essential for tension transmission between successive sarcomeres. We have investigated this structure as well as that of the nearby I band in a normal, unstimulated mammalian skeletal muscle by tomographic three- dimensional reconstruction from electron micrograph tilt series of sectioned tissue. The three-dimensional Z band structure consists of interdigitating axial filaments from opposite sarcomeres connected every 18 +/- 12 nm (mean +/- SD) to one to four cross-connecting Z- filaments are observed to meet the axial filaments in a fourfold symmetric arrangement. The substantial variation in the spacing between cross-connecting Z-filament to axial filament connection points suggests that the structure of the Z band is not determined solely by the arrangement of alpha-actinin to actin-binding sites along the axial filament. The cross-connecting filaments bind to or form a "relaxed interconnecting body" halfway between the axial filaments. This filamentous body is parallel to the Z band axial filaments and is observed to play an essential role in generating the small square lattice pattern seen in electron micrographs of unstimulated muscle cross sections. This structure is absent in cross section of the Z band from muscles fixed in rigor or in tetanus, suggesting that the Z band lattice must undergo dynamic rearrangement concomitant with crossbridge binding in the A band.     

Regulation of ATP supply in mammalian skeletal muscle during resting state-->intensive work transition

In the present debating paper, the problem how the rate of ATP supply by oxidative phosphorylation in mitochondria is adjusted to meet a greatly increased demand for ATP during intensive exercise of skeletal muscle is discussed. Different experimental results are collected from different positions of the literature and confronted with five conceptual models of the regulation of the oxidative phosphorylation system. The previously performed computer simulations using a dynamic model of oxidative phosphorylation are also discussed in this context. The possible regulatory mechanisms considered in the present article are: (A) output activation: an external effector activates directly only the output of the system (ATP turnover); (B) input/output activation: an external effector activates directly the output (ATP usage) and input (substrate dehydrogenation) of the system; (C) removal of substrate shortage: only ATP consumption and substrate supply by blood are directly activated; (D) removal of oxygen shortage: only ATP consumption and oxygen supply by blood are directly activated; (E) each step activation: an external effector activates both the ATP-consuming subsystem and all the steps in the ATP-producing subsystem (particular enzymes/carriers/blocks of oxidative phosphorylation, substrate supply, oxygen supply). The performed confrontation of the considered mechanisms with the presented results leads to the conclusion that only the each step activation model is quantitatively consistent with the whole set of experimental data discussed. It is therefore postulated that a universal effector/regulatory mechanism of a still unknown nature which activates all steps of oxidative phosphorylation should exist and be discovered. A possible nature of such an effector is shortly discussed.     

The effect of low ionic strength extracellular solutions on the resting potential in skeletal muscle fibers

Intracellular measurements of the resting potential were made in fibers of the frog sartorius muscle in solutions of varying salt composition and concentration to determine the effects of low ionic strength extracellular solutions on the resting potential. Changes in the glass microelectrode tip potential in low ionic strength solutions were minimized by adding ThCl₄ to the extracellular solution. These experimental conditions allowed measurement of the relationship of the resting potential to the concentration of the salt in the extracellular solution by replacing it with the nonionic substance, sucrose. Substitution of sucrose for the extracellular NaCl produced a stable depolarization which was logarithmically related to the NaCl concentration. Substitution of sucrose for choline Cl, instead of NaCl, produced the same degree of depolarization. When Na salts of anions less permeable than chloride (Br, I, NO₃) were used, the resting potentials in 116 mM solutions were close to those with chloride (±3mv). The depolarizations produced in low ionic strength solutions of these salts were significantly less than those with chloride.     

The dynamic properties of mammalian skeletal muscle

The dynamic characteristics of the rat gracilis anticus muscle at 17.5°C have been determined by isotonic and isometric loading. For a fixed initial length these characteristics were represented either as a family of length-velocity phase trajectories at various isotonic afterloads or as a series of force-velocity curves at different lengths. An alternate method of viewing these data, the length-external load-velocity phase space, was also generated. When the muscle was allowed to shorten from different initial lengths, the velocity of shortening achieved at a given length was lower for longer initial lengths. The amount of departure was also dependent upon the isotonic load, the greater the load the greater the departure. The departures were not caused by changes in the elastic elements of the muscle or fatigue in the ordinary sense. When the behavior of the muscle was investigated at different frequencies of stimulation, the shortening velocity was a function of the number of stimulating pulses received by the muscle at a given frequency. The shortening velocity of the rat gracilis anticus muscle is, therefore, not only a function of load and length, but also of an additional variable related to the time elapsed from onset of stimulation.     

Generation of resting membrane potential

This brief review is intended to serve as a refresher on the ideas associated with teaching students the physiological basis of the resting membrane potential. The presentation is targeted toward first-year medical students, first-year graduate students, or senior undergraduates. The emphasis is on general concepts associated with generation of the electrical potential difference that exists across the plasma membrane of every animal cell. The intention is to provide students a general view of the quantitative relationship that exists between 1) transmembrane gradients for K(+) and Na(+) and 2) the relative channel-mediated permeability of the membrane to these ions.     

Single chloride-selective channels active at resting membrane potentials in cultured rat skeletal muscle.

The patch-clamp technique was used to characterize channels that could contribute to the resting Cl-conductance in the surface membrane of cultured rat skeletal muscle. Two Cl- -selective channels, in addition to the Cl- -selective channel of large conductance described previously (Blatz and Magleby, 1983), were observed. One of these channels had fast kinetics and a conductance of 45 +/- 1.8 pS (SE) in symmetrical 100 mM KCl. The other had slow kinetics and a conductance of 61 +/- 2.4 pS. The channel with fast kinetics typically closed within 1 ms after opening and flickered between the open and shut states. The channel with slow kinetics typically closed within 10 ms after opening and displayed less flickering. Both channels were active in excised patches of membrane held at potentials similar to resting membrane potentials in intact cells, and both were open a greater percentage of time with depolarization. Under conditions of high ion concentrations, both channels exhibited nonideal selectivity for Cl- over K+ with the permeability ratio PK/PCl of 0.15-0.2. Additional experiments on the fast Cl- channel indicated that its activity decreased with lowered pHi and that SO2-4 and CH3SO-4 were ineffective charge carriers. These findings, plus the observation that the fast Cl- channel was also active in membrane patches on intact cells, suggest that the fast Cl- channel provides a molecular basis for at least some of the resting Cl- conductance. The extent to which the slow Cl- channel contributes is less clear as it was typically active only after excised patches of membrane had been exposed to high concentrations of KCl at the inner membrane surface.