The effect of extracellular tonicity on the anatomy of triad complexes in amphibian skeletal muscle.
Martin CA., Petousi N., Chawla S., Hockaday AR., Burgess AJ., Fraser JA., Huang CL., Skepper JN.
Ultrastructural features of tubular-sarcoplasmic (T-SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca2+ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T-SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca(2+)-release events ('sparks', in 100 mM-sucrose-Ringer solution), sustained Ca2+ elevations and propagated Ca2+ waves (> or = 350-500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (approximately 50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T-SR junctional anatomy. It increased the tubular diameters and T-SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca2+ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T-SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity.