THE AFTERDEPOLARIZATION IN RANA-TEMPORARIA MUSCLE-FIBERS FOLLOWING OSMOTIC SHOCK

Rana temporaria sartorius muscle fibres were exposed to varied sequenc es of solution and temperature changes that have been employed hithert o in procedures that sought to decouple the transverse tubules from th e surface membrane. The incidence of such detubulation was assessed in large numbers of fibres through demonstrating a loss or otherwise of the after-depolarization that normally reflects successful tubular pro pagation of the surface action potential. This criterion yielded asses sments of the existing detubulation techniques in agreement with earli er results. The experiments then developed an improved detubulation pr ocedure that required only brief (15 min) exposures to glycerol, its r eplacement in a single step by a Ca2+/Mg2+-Ringer solution for 30 min, and rapid cooling from room temperature (19-21 degrees C) to 6-10 deg rees C prior to final restoration of the normal Ringer solution. This sequence of steps yielded an optimal incidence (98%) of detubulation i n viable surface fibres that were amenable to electrophysiological stu dies. Studies that systematically modified the detubulation procedure demonstrated that the omission of any one step in the protocol signifi cantly reduced the incidence of detubulation with or without accompany ing deteriorations in fibre resting potentials. Successful detubulatio n accordingly required an initial exposure to an optimal glycerol conc entration that lasted for a minimal duration and for its abrupt withdr awal. Inclusion of a cooling step within 30 min after glycerol withdra wal was coincident with, and critical to, optimal tubular isolation. T hus, cooling steps that either preceded, or that followed the glycerol withdrawal step by more than 60 min, resulted in a sharp reduction in the incidence of detubulation. Similarly, a critical period of exposu re to Ca2+/Mg2+ Ringer solution also promoted detubulation without com promising the recovery of stable and satisfactory resting potentials. The findings reported here remain consistent with a primarily osmotic mechanism for detubulation. However, they demonstrated additional and important influences of temperature and of divalent cation concentrati on on the extent of tubular detachment when such factors were modified during the time course of the expected volume changes that followed e ach adjustment in osmotic condition.