Changes of intracellular pH due to repetitive stimulation of single fibres from mouse skeletal muscle.

Abstract

SUMMARY1. The performance of skeletal muscle during repetitive stimulation may be limited by the development of an intracellular acidosis due to lactic acid accumulation. To study this, we have measured the intracellular pH (pHi) with the fluorescent indicator BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein) during fatigue produced by repeated, short tetani in intact, single fibres isolated from the mouse flexor brevis muscle.2. The pHi at rest was 7-33 + 0-02 (mean + S.E.M., n = 29, 22°C). During fatiguing stimulation pHi initially went alkaline by about 0 03 units (maximum alkalinization after about ten tetani). Thereafter pHi declined slowly and at the end of fatiguing stimulation (tetanic tension reduced to 30% of the original; 0-3 PO), pHi was only 0-063 + 0'011 units (n = 14) more acid than in control. 3. We considered three possible causes of acidosis being so small in fatigue: (i) a high oxidative capacity so that fatigue occurs without marked production of lactic acid; (ii) an effective transport of H+ or H+ equivalents out of the fibres; (iii) a high intracellular buffer power. 4. The oxidative metabolism was inhibited by 2 mM-cyanide in three fibres. After being exposed to cyanide for 5 min without stimulation, the tetanic tension was reduced to about 0-9IP. and pHi was alkaline by about 01 units. The fibres fatigued faster in cyanide and the pHi decline in fatigue was more than twice as large as that under control conditions. 5. Inhibition of Na+-H+ exchange with amiloride resulted in a slow acidification of rested fibres; resting pHi was not affected by either inhibition of HC03-Vlexchange with DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) or inhibition of the lactate transporter with cinnamate. 6. Fibres fatigued in cinnamate displayed a markedly larger acidification ( 0-4 pH units) and tension fell more rapidly than under control conditions; inhibition of Na+-H+ and HC03-Cl-exchange did not have any significant effect on fatigue. H. WESTERBLAD AND D. G. ALLEN lactate or butyrate frequently gave an infinite buffer power, which indicates that powerful pH-regulating mechanisms operate in these cases.8. The dependence of tetanic tension on pHi of rested fibres was studied by changing the Pco2of the bath solution. The tension was found to change with a slope of about 0 33PO (pH unit)-').9. The slowing of relaxation which occurs in fatigue is often attributed to acidosis. In agreement with this, acidification of rested fibres resulted in a marked slowing of relaxation.10. Relaxation was slower after about ten fatiguing tetani, i.e. at a time when pHi was increased. Thereafter relaxation slowed further and the greatest slowing was observed in the most acidified fibres. Thus, the slowing of relaxation in fatigue was due to both pH-independent and pH-dependent processes.11. In conclusion, the acidosis in fatigue was small and this was mainly because of an effective extrusion of H+ ions by the lactate transporter. Thus neither the tension decline nor the slowing of relaxation in fatigue ...

Ca2+-pumping impairment during repetitive fatiguing contractions in single myofibers: role of cross-bridge cycling

“…In the present study, the basal [Ca 2ϩ ] c in the last contraction with BTS was very similar to the basal [Ca 2ϩ ] c obtained at the fatigue time point in the control condition. This result is different from the results obtained in single mouse fibers (8); however, single mouse fibers do not develop significant acidosis during repetitive contractions (32), and experimental protocols between the two studies were quite different (8). Although we cannot speculate which metabolites were diminished during the BTS condition, the data obtained from the [Ca 2ϩ ] c decay periods (discussed below in details) suggest that accumulation of free Mg 2ϩ and ADP were likely reduced in the cytosol when the metabolic demand was diminished because of BTS.…”

“…2, pKA, the logarithmic dissociation constant, was set to 7.15 (32). Sb and Sa are the fluorescence signals from 440 nm excitation at pH 9.0 and pH 5.0, respectively, and the fluorescence ratio of Sb/Sa was determined as 0.73 Ϯ 0.06 (n ϭ 5 fibers).…”

Intracellular pH during sequential, fatiguing contractile periods in isolated singleXenopusskeletal muscle fibers

“…Fibers were incubated with 10 M of the membrane-permeant acetoxymethyl ester form of the [H ϩ ]cyt indicator 2Ј,7Ј-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes). Incubated fibers were illuminated with two rapidly alternating (20 Hz) excitation wavelengths of 440 and 490 nm, and the resulting fluorescence emission intensities at 535 nm were divided (490 nm/440 nm) to obtain the pH i-dependent signal (25). Absolute resting pHi was standardized by perfusing with 10 M of the K ϩ /H ϩ ionophore nigiricin in KCl-buffered (140 mM) Ringer and calibrating with three pH-standardized solutions in series: pH 6.5, 7.0, and 7.5 (25).…”

Skeletal Muscle Fatigue: Cellular Mechanisms

“…In other cases, however, pH i decreases only to ϳ6.8 or 6.9 at the point of exhaustion (30,215) (71,463). Furthermore, humans with myophosphorylase deficiency are unable to utilize muscle glycogen, and their muscles fatigue faster than normal individuals; this accelerated fatigue development occurs without any change in pH i (78 (310,391).…”