Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

This study investigated membrane transport mechanisms influencing relative changes in cell volume ( V ) and resting membrane potential ( E m ) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of E m despite cell shrinkage, which was attributable to elevation of intracellular [Cl − ] above electrochemical equilibrium through Na + –Cl − and Na + −K + −2Cl − cotransporter action following exposures to extracellular hypertonicity. Fibre volumes ( V ) determined by confocal microscope xz- scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl − -free, normal and Na + -free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials ( E m ). Fibres in isotonic Cl − -free, normal and Na + -free Ringer solutions showed similar E m values consistent with previously reported permeability ratios P Na / P K ( 0.03–0.05) and P Cl / P K (∼2.0) and intracellular [Na + ], [K + ] and [Cl − ]. Increased extracellular osmolarities produced hyperpolarizing shifts in E m in fibres studied in Cl − -free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such E m shifts, suggesting a Cl − -dependent stabilization of membrane potential. This stabilization of E m was abolished by withdrawing extracellular Na + or by the combined presence of the Na + –Cl − cotransporter (NCC) inhibitor chlorothiazide (10 μ m ) and the Na + −K + −2Cl − cotransporter (NKCC) inhibitor bumetanide (10 μ m ), or the Na + −K + -ATPase inhibitor ouabain (1 or 10 μ m ) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl − equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl − ] i above its electrochemical equilibrium. Such splinting of [Cl − ] i in combination with the high P Cl / P K of skeletal muscle stabilizes E m despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for reg