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1 Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK
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Abstract |
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VR+ that fulfilled expectations of simple osmotic behaviour described in earlier reports. However, this was consistently followed by a slow increase in Vc (VS+) to 10–15% above osmotic predictions. Longer (>1 h) exposures to hypotonic solutions permitted a subsequent slow decrease in Vc (VS–), the eventual magnitude of which exceeded that of the preceding VS+. Restoration of isotonic conditions elicited a prompt recovery in Vc that matched simple osmotic predictions and thus left a net change in Vc. Such alterations in Vc attributable to VS+ then gradually reversed, while those due to VS– persisted. Both VS+ and VS– persisted under conditions of Cl– deprivation. The depolarization of Em that accompanied VR+ was consistent with dilution of intracellular [K+]. Em did not significantly alter during the subsequent VS transients. These empirical features of VS+ and VS– were analysed using the quantitative charge-difference model of Fraser and Huang, published in 2004. This attributed the VS+ to an electroneutral increase in the effective osmotic activity of normally membrane-impermeant intracellular anions. In contrast, the VS– could only be explained by an efflux of such anions and was accordingly comparable to organic anion-dependent regulatory volume decreases reported in other cell types.
(Received 9 December 2004;
accepted after revision 10 January 2005;
first published online 13 January 2005)
Corresponding author J. A Fraser: Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. Email: jaf21{at}cam.ac.uk
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionAppendixReferences |
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However, recent investigations of the response of skeletal muscle to extracellular hypertonicity in more physiological Cl–-containing Ringer solutions (Geukes Foppen et al. 2002; Ferenczi et al. 2004) demonstrated more complex relationships between Em and Vc. In contrast to the hyperpolarization of muscle fibres following fibre shrinkage, where K+ was the only significantly membrane-permeant ion (Adrian, 1956), similar osmotic reductions in Vc in such Cl–-containing solutions produced little change in Em. This stabilization of Em despite an increased [K+]i appeared to result from elevation (‘splinting’) of [Cl–]i above its electrochemical equilibrium concentration by the activity of cation–Cl– cotransport systems, including the Na+–K+–2Cl– cotransporter (NKCC; Geukes Foppen et al. 2002; Ferenczi et al. 2004). Thus, whereas the activity of such ion transporters is responsible for regulatory volume increase (RVI) in many other cell types (Lang et al. 1998a; Russell, 2000), it appears to regulate Em without significant influence upon Vc in skeletal muscle exposed to hypertonic solutions. Subsequent modelling showed that the high Cl– permeability (PCl) of skeletal muscle precluded a significant effect of cation–Cl– cotransport systems upon Vc without a far greater influence upon Em. In addition, any such changes to Vc or Em would reverse on cessation of the cation–Cl– cotransport activity, in contrast to the sustained changes to Vc and/or Em that could result from changes to intracellular impermeant anion content (Xi) and/or its effective mean valency (zX; Fraser & Huang, 2004).
The present experiments complement those recent studies of the effects of extracellular hypertonicity upon the relationship between Vc and Em (Ferenczi et al. 2004) by an examination of the effects of extracellular hypotonicity on this relationship, using measurements of Vc obtained using confocal xz-plane imaging and of Em using standard microelectrode techniques. The findings were then analysed in terms of a recent model (Fraser & Huang, 2004) that clarified the relationship between Vc and Em and described the possible mechanisms for their control in skeletal muscle.
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Methods |
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) measured using a standard, calibrated vapour pressure osmometer. All experiments were conducted at 20–22°C. The control, isotonic, solutions used were: (A) standard Cl–-containing Ringer solution (mM): 115 NaCl, 2.5 KCl, 1.8 CaCl2, 3 Hepes, 227 mosmol l–1; (B) standard SO42– Ringer solution: 75 Na2SO4, 1.25 K2SO4, 8 CaSO4, 3 Hepes, 225 mosmol l–1. The compositions of the hypotonic test solutions were identical to those of either solution A or B apart from a reduction in the concentration of the principal solute (NaCl or Na2SO4). The isotonic test solutions, which were used to control for the reduced extracellular ion concentrations in the hypotonic test solutions, contained extracellular ion concentrations that matched those in the corresponding hypotonic solutions, but additionally contained sufficient sucrose to render their tonicities equal to the isotonic control solutions. Sartorius and cutaneous pectoris muscles from cold-adapted Rana temporaria frogs (Blades Biological, Edenbridge, Kent, UK) previously killed by concussion followed by pithing (Schedule 1: Animals (Scientific Procedures) Act, Home Office, UK), were dissected in solution A. Several studies have reported comparable electrophysiological and osmotic properties in these two muscle types (Adrian, 1956; Ferenczi et al. 2004; Chin et al. 2004). For those experiments conducted under conditions of Cl– deprivation, the muscles were then gradually equilibrated with Cl–-free solutions by exposing them to a succession of Ringer solutions in which the Cl– content was progressively halved approximately every 5 min before a final transfer from 1 mM Cl– Ringer solution to Cl–-free Ringer solution. This protocol of gradual Cl– reduction avoided muscle twitching or contraction, in contrast to procedures that used more rapid removals of extracellular Cl–.
Cell volumes (Vc) were measured in groups of three to seven adjacent fibres in one- to three-fibre-thick cutaneous pectoris muscles mounted ventral side uppermost onto a coverslip that formed the base of a 0.5 ml microscope chamber. The chamber was sealed between solution changes to prevent evaporation. All solutions used to perfuse the muscle contained Sulphorhodamine B (Lissamine rhodamine B200: 75%; Aldrich-Sigma, UK) at a concentration of 62.5 µg ml–1 (Ferenczi et al. 2004). This membrane-impermeable dye stained the extracellular space and thereby highlighted muscle fibre edges, without influencing membrane electrophysiological properties (Gallagher & Huang, 1997). A Zeiss LSM-510 laser-scanning confocal microscope, incorporating an Axiovert 100M inverted microscope, was used to obtain images of fibres in the xz-plane using a 40x oil immersion objective. The Sulphorhodamine B was activated with a 543 nm wavelength laser and fluorescence emission captured at >560 nm. This generated images with a fluorescent extracellular space and dark fibre cross-sections.
Initial pilot scans in the xy-plane were obtained in order to ensure that the x-axes of the xz scans ran perpendicular to the fibre long axes. The definitive images were then obtained every 5–60 s in the xz-plane and in-house image analysis software was used to calculate the fibre cross-sectional areas. This software first corrected the fluorescence attenuation associated with scanning of the deeper fibre areas, according to calibration data obtained earlier and fully described by Ferenczi et al. (2004), then applied a noise filter and a threshold function. The cross-sectional area of each fibre in the field of view was then calculated. Since the muscle was secured at a constant length throughout each experiment, changes in the measured cross-sectional areas provided a direct indication of changes in cell volume. These cell volumes, Vc, were standardized relative to steady-state values obtained during an initial 10 min period in one of the control solutions, A or B. Volume recordings were not performed in muscles showing any evidence of reduced viability such as movement, contraction, T-system vacuolation or divergent volume changes between fibres within the field of view. Preliminary experiments showed that fibre lysis, indicated by the entry of dye into the cytoplasm, generally occurred within an hour of such changes.
These measurements of Vc were then compared with the corresponding predictions of cell volumes assuming that muscle fibres showed perfect osmometric behaviour. Thus, if Vp denotes such a predicted volume, Vc(a) the cell volume before the solution change, and e(a) and e(b) the extracellular osmolarity, measured using a vapour pressure osmometer, before (a) and after the solution change (b), respectively:
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| (1a) |
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| (1b) |
Membrane potentials (Em) were measured in sartorius muscles, known to have properties that closely resemble those of frog cutaneous pectoris muscles (Adrian, 1956), but providing considerably more fibres for electrophysiological study. They were mounted in a Perspex bath and pinned out stretched to 
1.5 times their in situ length in accordance with previous electrophysiological studies (Koutsis et al. 1995; Ferenczi et al. 2004), to give centre sarcomere lengths (2.4–2.5 µm), measured using the microscope eyepiece graticule, that were similar to the cutaneous pectoris fibres studied under confocal microscopy. The experiments were all carried out at room temperature (20–22°C) to enable results of the electrophysiological and volume studies to be compared. Test solutions were added to or withdrawn from the bath when necessary, with two washes between the additions of new solutions.
Significance of results was assessed using Student's paired two-tailed t test to a significance level of P < 0.05. Significance of volume changes was assessed by comparing mean Vc over 100 s periods (mean of at least 5 images, where n was defined as the number of fibres in each image) at predefined time points. Pilot experiments using both hypertonic and hypotonic solutions showed that passive volume changes took approximately 300 s and the slow volume increase that resulted from exposure to hypotonic solutions reached a maximum volume after approximately 60 min. Therefore, the time points at which volume changes were assessed for significance were the 100 s periods immediately prior to, 300 s after and 60 min after each solution change, or at the end of the experimental period. Mean Vc at each of these time periods was compared to mean Vc at the previous time period using Student's paired t test, or to the predicted volume (Vp) using Student's single-sample t test, as stated. In addition, in those experiments involving the return of fibres to isotonic Ringer solution following a period in hypotonic Ringer solution, Vc at the end of the experimental period was compared with that at the beginning.
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Results |
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VS+, follow the initial passive volume response, VR+, to exposures to hypotonic solutions
Figure 1 exemplifies the volume changes shown by groups of muscle fibres initially placed in a standard isotonic Ringer solution, then treated with a hypotonic Ringer solution. Application of the hypotonic extracellular solution initiated a pattern of volume changes (V) that differed significantly from the straightforward linear relationships between Vc and extracellular osmolarity reported previously (Blinks, 1965; Ferenczi et al. 2004).
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VR+, to a normalized of Vc to 1.32 ± 0.03 (n
= 4 fibres) at 5 min after the solution change, close to the predicted volume assuming simple osmotic behaviour and corresponding to a rate of volume increase of 5% of the initial volume per minute. The rate of this initial swelling was similar to the rate of passive osmotic volume changes following applications of hypertonic solutions (Ferenczi et al. 2004). However, Vc then showed a much slower increase (VS+) at a rate of 0.5% min–1 to reach a significantly (P < 0.01 on Student's paired t test versus the value of Vc at 5 min) larger Vc of 1.44 ± 0.03 at 60 min. Eleven muscles, in which Vc was recorded in 3–6 fibres of each, demonstrated similar rapid swelling to a mean value of Vc
= 1.30 ± 0.02 after 5 min exposures to hypotonic Ringer solution but an eventual Vc
= 1.42 ± 0.03 after 60 min, significantly exceeding the osmotic predictions (P
<< 0.01 on Student's single-sample t test). Figure 1B demonstrates that muscle fibre cross-sections in isotonic solutions were angular rather than circular in shape and that these volume changes largely involved changes in fibre shape to more circular profiles without predictable changes in fibre diameter.
Figure 2 shows typical confocal images in the xy-plane of single muscle fibres in standard Ringer solution (Fig. 2A) and after a 60 min exposure to hypotonic Ringer solution (Fig. 2B) through regions of interest that transected the xz-plane used for volume measurements. These show no significant differences in sarcomere length, as reflected in unchanged longitudinal distances between successive T-tubules. The VS+ was therefore not explicable in terms of an unexpected localized fibre shortening that could increase the local cross-sectional area whilst conserving total Vc. Figure 2 also shows that VS+ could not have resulted from an increase in T-tubular luminal as opposed to true cytosolic volume; to account for this 10% increase in Vc, the fractional tubular volume would have had to have increased >300-fold from its normal value of 0.003 (Peachey, 1965) to >0.10, contrasting with the similarity of the tubular appearances in Fig. 2A and B. Furthermore, although the T-tubules of amphibian muscle are too narrow (<20 nm) for accurate light microscopic measurements of their diameters (Martin et al. 2003), upper limits on the contribution of the T-system to these volume changes could be estimated using Delesse's principle, that the volume fraction, Vv, of an object embedded within a larger reference volume such as a muscle fibre is directly proportional to its area fraction in random sections. Vv was accordingly estimated by point counting (Weibel, 1979), using a transparent grid placed randomly over each of the digital images and containing a quadratic set of 0.5 µm spaced points generated by intersections on the grid. If Pi denotes the number of points falling on T-tubular lumina and Ptot the total number of points made upon the reference area, then the volume fraction is the dimensionless ratio Vv
=
Pi/Ptot. This gave statistically indistinguishable (P > 0.05) upper limits of tubular luminal volume fractions of 0.081 ± 0.011 (n
= 6) in standard Ringer solution and 0.065 ± 0.009 (n
= 6) in hypotonic Ringer solution.
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VS+ varies with the degree of extracellular hypotonicity
Figure 3 follows changes in Vc through a sequence of graded reductions in extracellular tonicity, followed by a final return of the fibres to standard isotonic Ringer solution. It illustrates a number of consistent features of the VR+ and VS+ phenomena. Fibres swelled rapidly on the introduction of 216 mosmol l–1 Ringer solution (a) to reach Vc values of 1.06 ± 0.01 (n
= 4 fibres) 5 min after the solution change, consistent with the simple osmotic prediction, Vp (horizontal bar). The subsequent VS+ changes increased Vc non-significantly (P > 0.05) to 1.07 ± 0.02 (n
= 4) over the next 20 min. However, the subsequent larger tonicity changes elicited progressively greater VS+ components. Thus, the transition from 216 to 200 mosmol l–1 Ringer solution (b) resulted in an initial VR+ that similarly matched osmotic expectations 5 min after the solution change. However, this was followed by a larger VS+ transient from 1.15 ± 0.02 at 5 min to 1.21 ± 0.02 (P < 0.05) prior to the next solution change 50 min later, a rate of volume increase of 0.11 ± 0.05% min–1. In contrast, during the VS+ following transfer to 171 mosmol l–1 Ringer solution (c) Vc increased from 1.43 ± 0.02 to 1.60 ± 0.04 (P < 0.01) in 40 min, a mean volume increase of 0.4 ± 0.1% min–1 that was significantly (P < 0.05) greater and more rapid than the increase that resulted from the transfer from 216 to 200 mosmol l–1 Ringer solution.
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VR+ followed by VS+ transients through each solution change finally resulted in an overall increase in Vc that significantly exceeded the simple osmotic expectations. Thus, the fibres in 171 mosmol l–1 Ringer solution eventually reached mean normalized volumes of 1.601 ± 0.037, against a Vp of 1.327 relative to the cell volume initially observed in 227 mosmol l–1 Ringer solution. The excess volume initially persisted on return to standard isotonic Ringer solution (d). Thus the return to isotonic 227 mosmol l–1 Ringer solution produced a rapid decrease in Vc to a value close to that predicted by assuming simple osmotic behaviour (Vp
=
Vc(a)
x (e(a)/e(b)) = 1.601 x (171/227) = 1.206). However, this passive osmotic shrinkage thus left a 20% residual elevation of Vc compared to its original value in standard Ringer solution that reflected the preceding, cumulative VS+ changes. This contrasted with the previously reported complete volume recovery in fibres returned to isotonic solutions following a sequential increase in extracellular tonicity (Ferenczi et al. 2004).
The adjustments in Vc that then followed were consistent with the VS+ being the consequence of an increased Vc under hypotonic, but not isotonic conditions. Thus, Fig. 3 indicates that fibres with similar Vc values showed contrasting slow volume transients, the directions of which depended on whether they were bathed in hypotonic Ringer solution (b) or demonstrated the residual swelling (d) that followed the exposures to extracellular hypotonicity. In the former case (b) fibres demonstrated the slow increase in volume, VS+, whereas in the latter case (d) there was a gradual recovery in volume to a significantly reduced Vc of 1.139 ± 0.013 (P < 0.01, n
= 4) at the end of the experimental period. The VS+ volume transients are thus not intrinsic consequences of increases in Vc above their resting values. Nor could the VS+ phenomenon be attributed to the reduction in the extracellular NaCl concentration ([NaCl]e); fibre volumes were not significantly altered by a 3 h exposure to isotonic, low [NaCl]e solutions (normalized Vc
=
–0.002 ± 0.003, n
= 8 fibres, 2 muscles after 3 h exposure) whose ionic composition equalled that of the 171 mosmol l–1 hypotonic Ringer solution used in Figs 1 and 3 (85 mM NaCl) but additionally contained sufficient sucrose to preserve normal tonicity (227 mosmol l–1).
VS+ persists under conditions of Cl– deprivation
Many cell types exhibit regulatory volume increase (RVI) or decrease (RVD) phenomena (Lang et al. 1998a,b; O'Neill, 1999) that result from cation–Cl– cotransporter activity mediated by the Na+–K+–2Cl– cotransporter (NKCC; Russell, 2000) or K+–Cl– cotransport (KCC; Lauf & Adragna, 2000), both of which have been identified in skeletal muscle (Hiki et al. 1999; Wong et al. 1999). Furthermore, many cell types increase their membrane Cl– permeabilities in response to swelling (Nilius et al. 1996; Okada et al. 2001). Experiments that exposed muscle fibres to hypotonic solutions in Cl–-free environments further excluded such a potential role of Cl–-dependent processes in these VS+ changes. Thus, Fig. 4 follows the mean relative volumes of a group of fibres that were exposed to hypotonic Cl–-free Ringer solution. The volume changes were similar to those in Cl–-containing Ringer solutions (Fig. 1) in that fibres showed an initial VR+ until Vc approximated Vp (horizontal bar) 5 min after the solution change. This was followed by a VS+ that gradually increased Vc (at a rate of 0.2% min–1) to 1.577 ± 0.025 after a 60 min exposure, significantly exceeding the simple osmotic expectations (Vp
= 1.36). VS+ thus persisted under conditions in which membrane-permeant anions were absent from the extracellular medium.
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VS+ is followed by a delayed slow volume decrease, VS–
The VS+ phenomenon was further investigated in fibres exposed to hypotonic solutions for longer periods. This revealed that the VS+ transients were followed after 60–70 min by a slow volume decrease, VS–. Figure 5 shows typical fibre volumes following 3 h exposures to hypotonic Cl–-containing Ringer solutions, demonstrating a slow and sustained volume decrease, VS–, beginning after the VS+ transient.
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VS–, persisted with accelerated kinetics under conditions of Cl– deprivation. These increased rates of VS+ and VS– in Cl–-free compared to Cl–-containing solutions made it possible to demonstrate several additional features of these slow volume transients within the 3–4 h period over which measurements of Vc in viable muscle fibres were possible, as follows.
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VR+, increasing Vc to values close to Vp (horizontal bar), as expected from a simple osmotic hypothesis. (2) A slow volume increase, VS+, that was nevertheless more rapid than its counterpart in Cl–-containing solutions then followed, increasing Vc to significantly above Vp (P < 0.01). (3) Exposures to extracellular hypotonicity of >1 h (Fig. 6B and C) then permitted an abrupt onset of a slow volume decrease, VS–, after 60–70 min, in common with the situation in the corresponding Cl–-containing solutions (Fig. 5). (4) This persisted at a constant rate that was faster than the VS– observed in Cl–-containing solutions (0.2% min–1 in Cl–-free versus 0.1% min–1 in Cl–-containing solutions) for at least 150 min (Fig. 6C). (5) The VS– process could eventually reduce Vc to significantly less than Vp (P < 0.01) for the prevailing extracellular tonicity (Fig. 6C), as opposed to simply reversing the preceding VS+ process.
Figure 6 also demonstrates the effects of (b) returning fibres to isotonic Cl–-free Ringer solution after long exposures to hypotonic Cl–-free Ringer solution, as follows. (1) In each case, the restoration to isotonic Ringer solution resulted in a rapid initial decrease in Vc, the magnitude of which matched the preceding VR+ and accordingly followed osmotic predictions (Vp, horizontal bar) that took into account the actual Vc immediately before the solution change. However, (2) Vc was then left with a residual difference from its baseline value, reflecting the volume changes attributable to the slow volume transients (VS+
+
VS–), corrected for the relative extracellular tonicities (e(hypo)/e(iso)). Thus, the shift in Vc from baseline values was close to (VS+
+
VS–) x (e(hypo)/e(iso)) in each case. (3) The initial passive, rapid decrease in Vc was then followed (Fig. 6A and B) by a much slower volume decrease that eventually equalled the magnitude of the VS+, corrected for the change in extracellular tonicity (thus, VS+
x (e(hypo)/e(iso))). It was not possible to demonstrate this correction for very long exposures to hypotonicity; although a downward trend was observed (Fig. 6C), it was non-significant (P > 0.05) at the limits of viability of the preparation. (4) In contrast to this apparent eventual correction of the volume gain during VS+, any volume loss attributable to a previous VS– persisted (Fig. 6B and C). Thus, fibres that showed a significant VS– remained residually shrunken on return to isotonic Cl–-free Ringer solution such that final Vc was significantly reduced from its initial baseline value (P < 0.01).
Resting membrane potentials, Em, during the volume changes
Adrian (1956) pointed out that osmotically induced changes in Vc would correspondingly concentrate or dilute any conserved intracellular ions and thereby potentially alter the resting membrane potential, Em. For example, increases in [K+]i/[K+]e resulting from osmotically induced reductions in Vc hyperpolarize the K+ Nernst potential, EK, and produce a corresponding shift in Em in fibres studied in Cl–-free hypertonic solutions (Adrian, 1956; Hodgkin & Horowicz, 1959). Such a relationship permits the influence of Vc upon Em to be predicted following a change in extracellular osmolarity. Thus, if the subscripts (iso) and (hypo) denote isotonic and hypotonic extracellular solutions, respectively, while the symbol denotes osmolarity:
if
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| (2) |
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| (3) |
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| (4) |
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| (5) |
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| (6) |
Equation (6) contains four implicit assumptions: (1) intracellular K+ content is conserved during changes in Vc; (2) Vc follows simple osmotic predictions (see Methods) over the range of extracellular osmolarities considered here; (3) Cl– is passively distributed; and (4) membrane Na+ permeability is low relative to that of K+. Any deviation of measured Em from these predictions might then reveal evidence of Em or Vc regulation.
The following control experiments therefore first confirmed that these assumptions were reasonable under steady-state conditions in isotonic solutions. First, Em was measured in fibres exposed to Ringer solutions in which sucrose partly but isosmotically replaced NaCl. This separated the effects upon Em of the reduced tonicity of hypotonic solutions from the effects of their reduced [NaCl]e and thus paralleled the investigation of Vc in solutions with similar isosmotic replacements of extracellular NaCl described above. Reductions in [NaCl]e from 120 to 85 mM then yielded no significant differences (P
>> 0.05 on Student's unpaired t test) between Em values as recorded over a 30 min period in standard isotonic Ringer solution (–90.4 ± 1.2 mV, n
= 13) and those obtained over 10 min periods immediately following (–90.1 ± 1.2 mV, n
= 9) and 60 min after the solution change (–89.3 ± 0.8 mV, n
= 34), respectively. Similar results were obtained after solution changes to 45 mM NaCl Ringer solution (–92.2 ± 1.3 mV, n
= 16 immediately following and –90.8 ± 1.0 mV, n
= 12 at 60 min; P > 0.05).
The second set of control experiments assessed the validity of assumption (3) above. Although early reports had suggested that Cl– is in electrochemical equilibrium across the membrane of resting skeletal muscle in isotonic solutions (Adrian, 1956; Hodgkin & Horowicz, 1959), more recent reports had suggested that Em in both mouse skeletal muscle in isotonic solutions and amphibian fibres in hypertonic solutions is slightly depolarized relative to EK owing to a combination of an elevated [Cl–]i/[Cl–]e due to cation–Cl– transporter activity and a high ratio of Cl– to K+ permeability, PCl/PK (Geukes Foppen et al. 2002; Ferenczi et al. 2004). The experiments accordingly examined Em in fibres in isotonic solutions under conditions of Cl– depletion, which would abolish any such cation–Cl– cotransport. Similar values of mean Em were obtained from fibres during an initial 30 min period in standard Ringer solution (–89.5 ± 1.3 mV, n = 16 fibres, 2 muscles) between 20 and 40 min (–92.3 ± 0.9 mV, n = 16 fibres, 2 muscles) and between 41 and 60 min after gradual equilibration with Cl–-free Ringer solution (–91.3 ± 0.5 mV, n = 24 fibres, 2 muscles). Therefore Cl– deprivation did not significantly affect values of Em under isotonic conditions (P > 0.05 in each case on Student's unpaired two-tailed t test versus values in standard Ringer solution).
Figure 7A then compares the influence of exposures to extracellular hypotonicity upon Em with the corresponding predicted values of Em (eqn (6)) over a wide range of tonicities, including those under which the Vc changes were examined. The muscle fibres were transferred from standard Cl–-containing Ringer solution to a hypotonic Cl–-containing Ringer solution and Em was measured soon after the solution change, to record Em changes due to VR+, and after 60 min, to record any additional changes due to VS+. Progressive reductions in tonicity produced increasing depolarizations of the Em measured between 5 and 15 min compared to values in isotonic Ringer solution (P
<< 0.01 in each case) that were close to predicted values of Em (dashed line) calculated from eqn (6) above. At tonicities of 192 and 119 mosmol l–1, Em remained stable after this initial depolarization. Only at the lowest tonicity (94 mosmol l–1) was there any significant depolarization (P < 0.01) between the early and late measurement periods, possibly reflecting the reduced fibre survival, loss of normal striations and inability to return to basal conditions that may follow such extreme reductions in extracellular tonicity (Paris et al. 1965). Even longer exposures to 171 mosmol l–1 solutions gave an Em of –87.1 ± 1.06 mV between 170 and 190 min (P > 0.05 against earlier time bins), demonstrating continued Em stability despite the VS– phenomenon. Figure 7B confirms similar relationships between extracellular tonicity and Em under Cl–-free conditions; decreases in extracellular tonicity depolarized Em as predicted by eqn (6) above (dashed line), giving stable Em values. Very long exposures to 164 mosmol l–1 solutions gave Em of –82.1 ± 1.06 mV between 170 and 190 min (P > 0.05 against earlier time bins), similarly demonstrating continued Em stability despite the marked VS– transient observed under similar conditions of Cl– deprivation.
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Em following the application of hypotonic solutions conformed to predictions that assumed perfect osmotic behaviour, conservation of intracellular K+ and dependence of Em upon EK. Furthermore, the subsequent slow volume transients produced no additional detectable Em in either Cl–-free or Cl–-containing Ringer solutions.
Mathematical modelling of the relationship between Em and Vc during VS+ and VS–
Skeletal muscle fibres exposed to hypotonic solutions thus showed: (1) slow volume transients, which followed an initial passive osmotic change (VR+), that first increased (VS+) and then decreased cell volume (VS–); and (2) accompanying alterations in Em that simply reflected a K+ Nernst equation in which osmotically induced increases in Vc directly altered [K+]i/[K+]e, in contrast to the stable values of Em observed in hypertonic solutions (Ferenczi et al. 2004). Both (1) and (2) persisted in Cl–-free solutions, which contain an insignificant concentration of membrane-permeable anions. This associated them with alterations in the intracellular membrane-impermeant solute (Xi) content, its osmotic activity, or its mean charge valency (zX), rather than Cl–-dependent transport processes (Fraser & Huang, 2004). However, the relationships between intracellular impermeant anion content (Xi), its mean charge valency per osmole (zX), Vc and Em are potentially complex. On the one hand, an efflux of charged X could alter its intracellular content, Xi, whilst preserving zX. On the other hand, the total intracellular charge carried by Xi would be unchanged following a flux of uncharged X or changes in the osmotic activity of X, and so such alterations in Xi would necessarily alter zX and in turn influence Em (Fraser & Huang, 2004). Nevertheless, it proved possible to model the effects of exposure of skeletal muscle to extracellular hypotonicity using the charge difference method of Fraser & Huang (2004) modified (see Appendix), to permit explicit modelling of changes in extracellular osmolarity.
Such modelling first assumed that the total charge carried by Xi was conserved within the cell unless there was a transmembrane flux of X. Secondly, [X]e was set to zero, as in the experimental solutions, and so no net inward fluxes of X were possible. Instead, Xi could be increased by simulating either an increase in its osmotic activity or cleavage of larger molecules. In this case, the total charge carried by Xi was conserved by keeping the product Vc x [X]i x zX constant by altering zX in inverse proportion to any change in [X]i x Vc. In contrast, decreases in total Xi content were modelled either by a similar charge-conserving mechanism or by the introduction of a small permeability term to X, permitting efflux of X and thus reducing the product Vc x [X]i x zX whilst preserving zX.
The model first demonstrated that an increase in Xi could result in a volume increase of comparable magnitude to VS+ without significant alteration of Em, despite the resultant decrease in zX. The simulation was started with extracellular solute concentrations similar to that of the isotonic low-[NaCl] Ringer solution, semiarbitrary intracellular concentrations, and with all other parameters, such as Na+–K+–ATPase density and ion permeabilities, as described by Fraser & Huang (2004) until all variables settled to stable values, at which point Vc was assigned a value of 1. Figure 8 illustrates results from the subsequent period of modelling, beginning with isotonic extracellular conditions. At point a extracellular tonicity was reduced and the system permitted to re-equilibrate. This reproduced the experimental findings of a rapid volume increase (VR+) and accompanying depolarization of Em. However, also note a rapid and small initial hyperpolarization on exposure to hypotonicity attributable to a transient Cl– influx secondary to the initial dilution of [Cl–]i.
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VS+ of similar magnitude to that demonstrated in Fig. 6B (Vc from 1.34 to 1.52), Em depolarized by only 2.9 mV, which is too close to the standard errors of the Em measurements to have been detected in the present study, and is thus compatible with the experimental data. The increase in Xi content was then stopped at point c.
At point d an efflux of X was modelled by assigning a small permeability term to Xi such that the permeability ratio PX:PK was 0.0001. This resulted in a volume decrease analogous to VS–, accompanied by a small depolarization, as would be expected for any process that increased overall anion efflux, but which was too small to be detected in the present study. However, Em stabilized at this slightly depolarized value, permitting the efflux of Xi to reduce Vc indefinitely, without further perturbation of Em or any intracellular ion concentrations (Fraser & Huang, 2004).
However, the efflux was stopped at point e after a VS– of similar magnitude to that demonstrated in Fig. 6B, and the extracellular conditions returned to those at the start of the modelling period. This increase in extracellular osmolarity resulted in a rapid Vc decrease. Vc then stabilized above its baseline value, due to the net increase in the total osmotic activity of Xi that took place during the VS+ and VS– processes. However, Em remained slightly depolarized (3 mV) compared to baseline values due to the reduction in zX and concomitant decrease in [K+]i.
Finally, at point f Xi content was gradually reduced in a reversal of the process started at point c, thus returning zX to its baseline value over the same period. This process was continued until both zX and [X]i reached their respective baseline values (g), at which point Vc was decreased from its original value, due to the loss of Xi content during the VS– process between points d and e. In contrast, Em, [K+]i, [Na+]i, [Cl–]i and [X]i were restored precisely to their original values.
A possible alternative mechanism for VS– was also modelled, in which the total intracellular charge carried by X (Vc
x
[X]i
x
zX) was conserved, as opposed to the net loss of charge during the VS– phase shown in Fig. 8. However, this did not permit reconstruction of the experimental results shown in Fig. 6C, where VS– reduced Vc from its maximum value to 1.15. A volume decrease of this magnitude with conservation of intracellular charge was shown to require approximately a doubling of zX, as the increase in cellular cation content resulting from the increase in zX magnitude would oppose the volume decrease caused by decreasing Xi content and additionally permit a significant increase in [K+]i, causing a hyperpolarization of 6 mV. Such a hyperpolarization was not detected (Fig. 7A and B). Further decreases in Vc from this value were shown to imply increasingly unrealistic values of zX, with a further doubling required to decrease Vc to 1, contrasting strongly with the indefinite Vc decreases possible with a simple efflux of X, which do not further perturb Em (Fraser & Huang, 2004).
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The experiments first confirmed that exposure of skeletal muscle fibres to hypotonic solutions elicited a rapid (>5% min–1) volume increase, VR+, that closely followed simple osmotic predictions as previously reported (Dydynska & Wilkie, 1963; Blinks, 1965). However, this swelling was followed by slower volume transients, not observed on earlier occasions, which may represent cellular volume regulatory phenomena. Their detection in the present study is likely to reflect the long exposure times used here (cf. Blinks, 1965) and the increased accuracy afforded by assessment of Vc using confocal xz-plane scanning rather than measurements of fibre diameter (cf. Reuber et al. 1963).
The slow volume transients showed two distinct phases. First, an initial slow (<0.5% min–1) volume increase, VS+, immediately followed VR+ and lasted approximately 50–60 min. VS+ produced increases in Vc to final values 10–15% greater than osmotic predictions, and was therefore suggestive of increases in total intracellular osmotic activity. It occurred in solutions of even slightly reduced relative tonicity (relative tonicity = 0.88). VS+ took place at a rate that gradually decreased until a maximum Vc was reached after approximately 50–60 min, contrasting sharply with the 2–5 min time courses of the passive VR+ volume changes and the passive volume changes previously reported in response to increases in extracellular tonicity (Ferenczi et al. 2004).
Second, a slow (<0.5% min–1) volume decrease, VS–, then began quite abruptly 60–70 min after the VR+. It was not possible to assess the maximum duration of the VS– phase because it continued beyond the limits of tissue viability in the isolated tissue preparation used here. However, it was observed to continue for at least 3 h, over which time the volume decrease could exceed the magnitude of the preceding VS+; fibre volumes then eventually decreased below the predicted values for the prevailing extracellular tonicity. This suggested that the VS– process did not simply represent a reversal of the preceding VS+ process.
Both the VS+ and VS– phenomena persisted, and indeed assumed more rapid kinetics, under conditions of Cl– deprivation. These features exclude possible mechanisms involving cation–Cl– cotransport systems or other Cl–-dependent processes, instead suggesting that Cl– redistribution (Hodgkin & Horowicz, 1959) otherwise limited the rates of both VS transients. Finally, fibres exposed to isotonic solutions with concentrations of NaCl that were reduced to levels identical to those of the hypotonic solutions used here showed stable volumes, suggesting that the slow volume transients were not a consequence of reduced extracellular ion concentrations.
Fibres that had been exposed to hypotonicity for various durations were then returned to the original isotonic solutions. This resulted in an initial cell shrinkage that was similar in magnitude and rate to the initial VR+ and therefore compatible with passive osmotic behaviour. However, the overall Vc changes during VS+ and VS– were then reflected in a residual Vc deviation from its baseline value. This permitted the identification of a second difference between the VS+ and VS– processes. After the initial rapid osmotic volume decrease on transfer from hypotonic to isotonic Ringer solution, fibres then showed a gradual volume decrease that was eventually equal in magnitude to the preceding VS+, allowing for the change in extracellular tonicity, at which point cell volumes stabilized. Cell volumes therefore returned precisely to their original resting values if the duration of exposure to hypotonic Ringer solution was insufficient to permit VS– to occur, demonstrating the full reversibility of VS+. However, if the exposures to hypotonicity were of sufficient duration to permit VS– the subsequent return to isotonic conditions continued to elicit a complete reversal of the VS+ process. This left final stable volumes that were less than the initial baseline volumes, suggesting that VS– produced a sustained loss of intracellular solute. Fibres that had undergone a period of VS+ and were then returned to isotonic Ringer solution additionally demonstrated that VS+ did not result from the fibre swelling per se. Thus, while swollen fibres in hypertonic Ringer solution demonstrated the VS+ phenomenon, fibres residually swollen to similar extents on return to isotonic Ringer solution due to a preceding VS+ phase instead showed volume recovery.
However, despite these slow volume transients, the changes in membrane potential (Em) during these exposures to hypotonic solutions remained close to predictions that assumed simple osmotic behaviour, conservation of intracellular K+, and a dependence of Em upon EK. Thus, exposures to hypotonic solutions with a range of relative tonicities from 0.75 to 0.42 caused rapid depolarizations compatible with a directly proportional reduction in the ratio [K+]i/[K+]e. Similar depolarizations were observed in Cl–-containing and Cl–-free solutions, thereby excluding any cation–Cl– cotransport-dependent stabilization of Em, in contrast to the Em stabilization reported in osmotically shrunken fibres (Ferenczi et al. 2004). Return to isotonic Ringer solution then permitted full recovery of Em to its resting value.
The present findings thus sharply contrast with the splinting of Em but a relatively simple dependence of Vc upon extracellular osmolarity in fibres studied in hypertonic solutions (Ferenczi et al. 2004). Nevertheless, they proved similarly amenable to a recent, charge-difference approach to quantitative modelling (Fraser & Huang, 2004) that invoked osmotic activity in charged intracellular impermeant anions and their possible transmembrane fluxes rather than the Cl–-dependent transport processes that had successfully accounted for the earlier observations in hypertonic solutions. Thus, such modelling suggested that the VS+ that immediately followed the hypotonic volume expansion, VR+, without detectable further depolarization of Em could reflect a reversible increase in osmotic activity in normally membrane-impermeant intracellular anions (Xi), whilst conserving the total intracellular charge carried by Xi (=
Vc
x
[X]i
x
zX) through a proportional decrease in zX. While such reductions in the magnitude of zX would reduce the maximum polarization of Em, the predicted depolarization was too small (<2–3 mV) for detection in the present study. VS+ may thus simply represent an unavoidable, even if inappropriate, consequence of the dilution of cellular contents, which might result in an increase in the osmotic pressure of Xi, for example due to the depolymerization of proteins (Lew & Bookchin, 1991), an alteration of cellular polymer water compartments (Bookchin et al. 1994), or a significant increase in the osmotic coefficient (
) of Xi with dilution. However, it is also conceivable that VS+ might represent a functional response to reduced Xi content that is inappropriately activated when Xi is diluted secondary to fibre swelling under hypotonic conditions. Under conditions of stable extracellular osmolarity, a reduction in [Xi] would tend to cause volume decrease (Fraser & Huang, 2004), rather than simply reflecting such a dilution of cellular contents, and therefore a mechanism analogous to VS+ that could increase Xi content or osmotic activity towards normal might then be an appropriate corrective response.
However, modelling showed that a converse decrease in Xi and conservation of the product Vc
x
[X]i
x
zX was not compatible with the magnitude of VS– observed experimentally, since such a mechanism would require an increase in the magnitude of zX to unreasonable values, thereby producing a significant hyperpolarization of Em, contrary to experimental observation. Rather, the delayed VS– could be readily explained in terms of a simple efflux of Xi, thereby reducing the total intracellular charge carried by Xi but conserving zX and hence Em while reducing the product Vc
x
[X]i
x
zX. This, together with the observed delay prior to its initiation, would be compatible with VS– representing an RVD phenomenon dependent on organic anion efflux that is activated by cell swelling, as has been described in other cell types (Lang et al. 1998a,b). Such a scheme is then compatible with the observed reversibility of VS+ and apparent irreversibility of VS–. If so, VS– would represent, to the best of our knowledge, the first demonstration of RVD in mature skeletal muscle.
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4 fibres in each case). Horizontal bars denote predicted cell volume, Vp, after each solution change.
) and after a much longer (50–70 min) exposure period (
). Where the S.E.M. is not shown, it is smaller than the data point. 