SUMMARY1. The rate of efflux of lactate from isolated frog sartorius muscles is measured with a superfusion technique. Efflux curves are followed after raising the internal lactate level of the muscles by repetitive electrical stimulation over a 200 sec period.2. With an external pH of 7 0 or below the measured efflux rates following stimulation reach 100-150 n-mole/g. min. Increasing the pH of the superfusion fluid to 8-0 results in a two or threefold increase in the peak efflux rate. The effect is independent of the buffer system used and occurs fairly rapidly when the pH of the superfusion fluid is changed. This suggests that the effect of pH on lactate efflux is extracellular.3. The increase in efflux rate due to an increase in pH is dependent on buffer concentration. This fact together with measurements of surface pH changes in muscles following arrest of superfusion indicates that a pH gradient exists through the muscle thickness during lactate efflux.4. The low lactate efflux rate seen at a low buffer concentration (1 mM) is reduced to an even lower level by depolarization with potassium sulphate suggesting a membrane potential dependent component. At pH 8*0 with a high buffer concentration (25 mM) potassium sulphate only reduces efflux rate slightly.The observations are interpreted as indicating that a fraction of lactate lost is in the form of undissociated acid and that this fraction increases with increasing external pH.5. Conditions which favour loss of hydrogen ions and lactate from muscle are also associated with improved recovery of twitch tension.
H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20-25 mumol X g-1, resulting in a fall in intracellular pH of 0.3-0.5 units in mammalian muscle and 0.6-0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70-80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10-fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation-contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell.
The intracellular pH of frog sartorius muscles exposed to an extracellular pH 8.0 (25 mM HCO3-, 1% CO2) was 6.9-7.1. Following a fatiguing stimulation period (one tetanic contraction per second for 3 min), the intracellular pH was 6.5-6.7. When similar experiments were repeated with frog sartorius muscles exposed to pH 6.4 (2mM HCO3-, 1% CO2), the intracellular pH was 6.8-6.9 at rest and 6.3-6.4 following fatigue. So, in both experiments the intracellular pH decreased by 0.4-0.5 pH unit during fatigue. When the CO2 concentration of the bathing solution was increased from 1 to 30%, the intracellular pH of resting muscles decreased from 7.0 to 6.2-6.3. Although the effect of CO2 on the intracellular pH was greater than the fatigue effect, the decrease in tetanic force with CO2 was less than 40%, while during fatigue the tetanic force decreased by at least 70%. Therefore in frog sartorius muscle the decrease in tetanic force during fatigue exceeds the decrease that is expected from just a change in intracellular pH.
A model for oxygen transfer to cells from capillaries is considered in which mitochondria are either clustered at the cell periphery around capillaries or homogeneously distributed through the cytosol. The capillary Po2 required to supply cells utilizing oxygen at the same rate is much less when mitochondria cluster around capillaries. Two alternative mechanisms are considered for distributing energy from peripheral mitochondria to the rest of the cell; i.e., diffusion of ATP or creatine phosphate with enough creatine kinase to ensure equilibrium between the approximately P carriers. The latter has clear advantages and would appear to be adequate to supply a fairly large mitochondria-free cell core (e.g., 24-micrometer diameter) with very little change in ADP levels or in the free energy of ATP hydrolysis at maximum work rates. Thus, a viable alternative to the traditional Krogh model is presented which takes into account the inhomogeneity of the diffusion pathway as a result of mitochondrial clustering.
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