Cardiac glycosides inhibit detubulation in amphibian skeletal muscle fibres exposed to osmotic shock

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.