title: Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions.
abstract: 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 x z - 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) ( approximately 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 microM) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor bumetanide (10 microM), or the Na(+)-K(+)-ATPase inhibitor ouabain (1 or 10 microM) 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 co-transporters would instead permit RVIs but at the expense of a capacity to stabilize E(m).
authors.affiliation: Physiological Laboratory, University of Cambridge, Cambridge, UK.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Ferenczi
authors.forename: James A
authors.initials: JA
authors.lastname: Fraser
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Chawla
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
authors.forename: Christof J
authors.initials: CJ
authors.lastname: Schwiening
authors.forename: Christopher L-H
authors.initials: CL
authors.lastname: Huang
mesh_terms: D000818:Animals; D002138:Calibration; D048429:Cell Size / Q000502:physiology; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006982:Hypertonic Solutions / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011896:Rana temporaria; D012964:Sodium / Q000502:physiology; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors
chemical_list: D004791:Enzyme Inhibitors; D006982:Hypertonic Solutions; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase