Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

Abstract

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,suggestingaCl − -dependentstabilizationofmembranepotential.ThisstabilizationofE 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 . This study investigated the relationship between changes in cell volume (V ) and resting membrane potential (E m ) in amphibian skeletal muscle fibres exposed to osmotic stress brought about by increased Emily A. Ferenczi and James A. Fraser were equal contributors to this paper. extracellular osmolarity. It was prompted by classic reports that the osmotic and electrophysiological properties of Cl − -depleted frog skeletal muscle followed predictions for a freely distensible, semipermeable sac containing a fixed quantity of solute in which cell volume decreased linearly with extracellular osmotic pressure and that this volume change resulted in membrane hyperpolarization reflecting the increased (Adrian, 1956;Blinks, 1965). Significant, osmotically induced cell volume changes in skeletal muscle similarly occur in vivo during normal activity (Sjojaard et al. 1985;Kowalchuk et al. 1998).The present experiments extended the above reports. First, they introduced an assessment of cell volume (V ) using confocal microscope scanning of amphibian skeletal muscle fibres in the xz-plane over time. This offered significant advantages over earlier measurements that only assessed steady-state fibre diameter in response to extracellular osmotic change (cf. Dydynska & Wilkie, 1963;Blinks, 1965). The cylindrical geometry of skeletal muscle fibres made it possible to simplify earlier confoca...

Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

“…Previous studies have characterised the relationship between cell volume, V c , and membrane potential, E m , in quiescent muscle [9,13,14], and others separately demonstrated that activity alters both V c and E m (e.g. [25,28,30,36]).…”

“…Relative muscle fibre volumes, V c , were recorded continuously in the superficial fibres of the very thin cutaneous pectoris muscle preparation throughout the experimental protocols using confocal microscope xz-scanning to allow a consistent detection of even small (2-5%) changes in cell volume given established calibration and correction techniques [9]. Measurements were made in the course of and following intermittent low-frequency electric-field stimulation which was chosen as it produces a greater force depression and a slower recovery than high-frequency continuous stimulation [45].…”

“…The muscles were dissected and pinned out in normal Ringer solution and then mounted, ventral side uppermost, in a 0.5-ml volume chamber on a coverslip that formed the floor of the chamber. As described previously [9], this arrangement permitted free flow of fluid around the ventral aspect of this very thin muscle, whilst its dorsal aspect remained in contact with the coverslip. The muscles were imaged using a Zeiss LSM-510 laser scanning confocal microscope system incorporating an Axiovert 100 M inverted microscope (Zeiss, Jena, Germany) using the ×40 oil immersion objective in the presence of the fluorescent membrane-impermeant dye, Sulforhodamine B (Lissamine rhodamine B200 75%; SigmaAldrich, Poole, UK) added to the bathing solution at a concentration of 62.5 μg ml -1 [12].…”

“…First, they characterised changes in V c during and after stimulation using laser confocal microscope xz-plane scanning [9] and plastic sections of muscles fixed in glutaraldehyde. The extent to which these observations in stimulated fibres could be replicated by manipulations of E m , measured by conventional microelectrode techniques, in quiescent fibres was then explored using extracellular solutions containing increased concentrations of potassium.…”

Optogenetic activation of mechanically insensitive afferents in mouse colorectum reveals chemosensitivity

“…Rather than encoding hyperosmotic challenge, most channels are activated by hypotonic stimuli and presumed cell swelling, including TrpV4 that is widely expressed in visceral sensory neurons (6,22,32). In contrast, hypertonic stimuli cause hyperpolarization in some cells (16,33) and presumably result in cell membrane shrinkage, contributing to an increased activation threshold for stretch-activated ion channels, consistent with a previous finding that AHS significantly reduced colorectal afferent response frequency and increased response threshold to mechanical stretch (21). The underlying mechanism(s) for short-latency, long-lasting responses of tonic-spiking MIAs and most probing-responsive afferents to AHS remain unresolved and await further study.…”