| Popis: |
This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (Em) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of Em 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 (Em). Fibres in isotonic Cl−-free, normal and Na+-free Ringer solutions showed similar Em values consistent with previously reported permeability ratios PNa/PK(0.03–0.05) and PCl/PK (∼2.0) and intracellular [Na+], [K+] and [Cl−]. Increased extracellular osmolarities produced hyperpolarizing shifts in Em 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 Em shifts, suggesting a Cl−-dependent stabilization of membrane potential. This stabilization of Em was abolished by withdrawing extracellular Na+ or by the combined presence of the Na+–Cl− cotransporter (NCC) inhibitor chlorothiazide (10 μm) and the Na+−K+−2Cl− cotransporter (NKCC) inhibitor bumetanide (10 μm), or the Na+−K+-ATPase inhibitor ouabain (1 or 10 μm) 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 PCl/PK of skeletal muscle stabilizes Em despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where PCl/PK is low, the same cotransporters would instead permit RVIs but at the expense of a capacity to stabilize Em. |
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