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This Article Full Text Full Text (PDF) All Versions of this Article: 555/2/423    most recent jphysiol.2003.058545v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ferenczi, E. A. Articles by Huang, C. L.-H. Search for Related Content PubMed PubMed Citation Articles by Ferenczi, E. A. Articles by Huang, C. L.-H.

¹ Physiological Laboratory, Department of Anatomy, ² Multi-Imaging Centre, Department of Anatomy and ³ Department of Pharmacology, University of Cambridge, Cambridge, UK

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 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 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 regulatory volume increases (RVIs). In situations where P_Cl/P_K is low, the same cotransporters would instead permit RVIs but at the expense of a capacity to stabilize E_m.

(Received 28 November 2003; accepted after revision 16 December 2003; first published online 23 December 2003)
Corresponding author C. L.-H. Huang: Physiological Laboratory, University of Cambridge, Cambridge, UK. Email: clh11{at}cam.ac.uk

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