Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

Abstract

The effect of electrical stimulation on cell volume, V (c), and its relationship to membrane potential, E (m), was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V (c) of 19.8+/-0.62% (n=3) and 27.1+/-8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, DeltaE (m), of +23.6+/-0.98 mV (n=3). The extent to which this DeltaE (m) might contribute to the observed changes in V (c) was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K(+)](e). E (m) and V (c) varied linearly with log [K(+)](e) and [K(+)](e), respectively, in the range 2.5-15 mM (R (2)=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V (c) and E (m) (DeltaV (c)=0.85E (m)+68.53; R (2)=0.99) and hence derive the DeltaV (c) expected from the DeltaE (m) during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V (c) observed in active muscles could be reproduced by the corresponding [K(+)](e)-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl(-) deprivation abolished the relationship between E (m) and V (c), supporting a mechanism in which the depolarisation of E (m) drives a passive redistribution of Cl(-) and hence cellular entry of Cl(-) and K(+) and an accompanying, osmotically driven, increase in V (c).

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

“…The stimulation sequence produced an initial and significant positive shift in E m from −84±2 to −70±2.1 mV (n=10, 19; p<<0.05) in agreement with recent reports that used the same protocol [61]. Before stimulation all fibres tested fired action potentials on application of the standard 1.5 V shock.…”

“…In experiments where the muscle was first stimulated to fatigue, a constant-voltage isolated stimulator (Model DS2A-Mk.II) linked to a train/delay generator (Model DG2A, both Digitimer, Welwyn Garden City, UK) was used to expose the muscle to 80 sets of stimulation where each set consisted of a 2 s stimulus train with impulses 20 ms long of amplitude 20 V at a frequency of 10 Hz separated by rest intervals of 6 s before the introduction of the Vaseline bridge. This protocol of intermittent low-frequency tetanic electric-field stimulation was chosen because it has been used in previous studies in this muscle preparation [60,61], and it produces a greater force depression and a slower recovery than high-frequency continuous stimulation [64]. Solution changes were performed using a vacuum pump (BOC Edwards, Sussex, UK) to remove the solution in the bath and rinse the bath before adding the change solution.…”

Alterations in triad ultrastructure following repetitive stimulation and intracellular changes associated with exercise in amphibian skeletal muscle

“…The high permeability to Cl − relative to that of K + in amphibian skeletal muscle, however, then results in an accompanying Cl − entry which in turn increases the total intracellular solute content and drives water entry and an increase in cell volume (Boyle and Conway 1941; Usher-Smith et al. 2006b). This volume change can be prevented by replacing the extracellular Cl − with SO 4 2− to abolish Cl − fluxes.…”

“…The K + concentration of 15 mM was chosen as it produces a depolarisation similar to that seen after stimulation (35.3 ± 0.76 mV ( n  = 3) and 23.6 ± 0.98 mV ( n  = 3) respectively, Usher-Smith et al. 2006b) and that of 60 mM to maximise any possible effects on the triad ultrastructure. Figure 4a shows that both concentrations result in significant ( P  << 0.05, n  = ∼30) decreases in T-SR distance, with 60 mM having a significantly ( P  << 0.05, n  = ∼30) greater effect than 15 mM.…”

“…Muscles were exposed to 80 sets of stimulation where each set consisted of a 2 s stimulus given at intervals of 8 s with impulses 20 ms long at 20 V used at a frequency of 25 Hz (Usher-Smith et al. 2006a, b). Measurements of peak twitch tension recorded during individual 10 V (field strength ∼1 V/cm) stimuli 20 ms long using an isometric force transducer (Model 2 Number 724,485, Harvard Apparatus, USA) connected to a Model 1401 analogue to digital converter (Cambridge Electronic Design, Cambridge, UK) linked to a computer running Spike 2 version 3.19 for windows (Cambridge Electronic Design, UK) showed that this protocol reduces tetanic force to 25.7 ± 0.7% ( n  = 3) of its initial value.…”