Slow volume transients in amphibian skeletal muscle fibres studied in hypotonic solutions

The influence of extracellular hypotonicity on the relationship between cell volume (V(c)) and resting membrane potential (E(m)) was investigated in Rana temporaria skeletal muscle. V(c) was measured by confocal microscope imaging of fibres through their transverse (xz) planes, and E(m) was determined using standard microelectrode techniques. Hypotonic solutions first elicited a rapid increase in fibre volume, DeltaV(R+) that fulfilled expectations of simple osmotic behaviour described in earlier reports. However, this was consistently followed by a slow increase in V(c) (DeltaV(S+)) to 10-15% above osmotic predictions. Longer (>1 h) exposures to hypotonic solutions permitted a subsequent slow decrease in V(c) (DeltaV(S-)), the eventual magnitude of which exceeded that of the preceding DeltaV(S+). Restoration of isotonic conditions elicited a prompt recovery in V(c) that matched simple osmotic predictions and thus left a net change in V(c). Such alterations in V(c) attributable to DeltaV(S+) then gradually reversed, while those due to DeltaV(S-) persisted. Both DeltaV(S+) and DeltaV(S-) persisted under conditions of Cl- deprivation. The depolarization of E(m) that accompanied DeltaV(R+) was consistent with dilution of intracellular [K(+)]. E(m) did not significantly alter during the subsequent DeltaV(S) transients. These empirical features of DeltaV(S+) and DeltaV(S-) were analysed using the quantitative charge-difference model of Fraser and Huang, published in 2004. This attributed the DeltaV(S+) to an electroneutral increase in the effective osmotic activity of normally membrane-impermeant intracellular anions. In contrast, the DeltaV(S-) could only be explained by an efflux of such anions and was accordingly comparable to organic anion-dependent regulatory volume decreases reported in other cell types.