OSMOTIC DETUBULATION IN FROG-MUSCLE ARISES FROM A REVERSIBLE VACUOLATION PROCESS

Isolated Rana temporaria sartorius muscle fibres were subject to intro duction and subsequent withdrawal of 400 mM extracellular glycerol, ex posures to high divalent ion concentrations and then fooling. Tubular detachment was then assessed through changes in the action potential a fterdepolarization. (1) The rapid (5-10 min) rather than slow fooling step (30 min) produced a gradual (30 min) development of detubulation arrested by the subsequent replacement of glycerol and reversed by add ition of 350 mM sucrose. Such osmotic agents influenced neither restin g potentials of intact or detubulated fibres nor action potentials in intact fibres. (2) Full tubular detachment was achieved by 40 min. Las er epifluorescence microscopy demonstrated an accompanying tubular vac uolation through its trapping of a Rhodamine dye. (3) Subsequent re-ad ditions (at 10-80 min) of glycerol restored the afterdepolarization in 30% of detubulated fibres and correspondingly reduced vacuolation. Su stained (> 60 min) exposures to 350 mM sucrose, applied between 30-60 min, both reversed tubular isolation in 70% of detubulated fibres and abolished tubular vacuolation. Finally, results from transient (10-30 min) sucrose exposures resembled the consequences of sustained applica tions of glycerol, suggesting that detubulation and its reversal resul t from an osmotic mechanism. (4) Nevertheless, irreversible changes de veloped after 70-80 min in 70% of detubulated fibres, a process hasten ed by slow cooling steps in the initial osmotic stress. The present st udy thus correlates morphological and electrophysiological consequence s of applying osmotic shock to skeletal muscle for the first time. It additionally differentiates reversible and irreversible components of detubulation. Finally, it suggests that detubulation results from the similarly reversible vacuolation observed under comparable osmotic con ditions, and that such vacuolation can eventually lead to irreversible detubulation.