Loop diuretics inhibit detubulation and vacuolation in amphibian muscle fibres exposed to osmotic shock.

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detubulation following osmotic shock, was investigated in amphibian skeletal muscles. These were exposed to a glycerol-Ringer solution (18 min), an isotonic Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 microM) to these solutions sharply reduced the incidence of detubulation, assessed by abolition or otherwise of action potential after-depolarisations, from 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 microM bumetanide). It dramatically reduced the number and fraction of muscle volume occupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horseradish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, similarly was reduced with 2 microM bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulation protocol. Thus, it exerted its maximum effect when added to the glycerol-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas fibre diameters measured using scanning electron microscopy returned to normal during glycerol treatment relative to those of control fibres left in isotonic Ringer, addition of 2.0 microM bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics. furosemide and ethacrynic acid similarly influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introduction of extracellular glycerol. This might then enable the subsequent Na-K-ATPase dependent water extrusion that produces the tubular distension (vacuolation) and detachment (detubulation) following glycerol withdrawal, phenomena also observed in muscular dystrophy.