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).

Figure 1. Calibration of xz-scanning distances as measured by confocal microscopy

A, measurements of maximum diameters of 25 μm reference microspheres within the xy-plane (▪; means of 5 readings; s.e.m.s smaller than the dimensions of the data points), apparent z-distances of a reference object (▴; means of 5 readings; s.e.m.s smaller than the dimensions of the data points). B, normalized measurements of cross-sectional areas of viable muscle fibres all plotted against z-distance away from the viewing coverslip.

Figure 1. Calibration of xz-scanning distances as measured by confocal microscopy

A, measurements of maximum diameters of 25 μm reference microspheres within the xy-plane (▪; means of 5 readings; s.e.m.s smaller than the dimensions of the data points), apparent z-distances of a reference object (▴; means of 5 readings; s.e.m.s smaller than the dimensions of the data points). B, normalized measurements of cross-sectional areas of viable muscle fibres all plotted against z-distance away from the viewing coverslip.

Figure 2. Confocal xz-scans of cross-sections of muscle fibres in Lissaminerhodamine-containing extracellular solution

The vertical cursors were drawn through the maximum dimensions of the fibre profiles, shown before the calibration and intensity corrections that preceded setting of intensity thresholds for the computation of cross-sectional area, for comparisons with changes in the fibre sectional areas before (A) and after (B) an alteration of extracellular osmolarity. The viewing coverslip forms the upper edge of each image.

Figure 3. Determinations of fibre cross-sectional areas following changes of extracellular osmolarity measured using confocal xz-scanning

A, changes in fibre cross-sectional area (μm²) over time (s) for six typical individual fibres of cutaneous pectoris muscle following perfusion of hypertonic normal Ringer solutions containing varying sucrose concentrations. Extracellular osmolarity was progressively increased and the ability of the muscle to recover was explored by returning to isotonic Ringer solution in two stages. The solutions were added in the following order (see arrows): 10 mm, 20 mm, 50 mm, 100 mm, wash-out with isotonic Ringer, second wash-out with isotonic Ringer and 200 mm sucrose-ringer. B, fibre cross-sectional areas for the individual fibres plotted against extracellular [1/osmolarity].

Figure 3. Determinations of fibre cross-sectional areas following changes of extracellular osmolarity measured using confocal xz-scanning

A, changes in fibre cross-sectional area (μm²) over time (s) for six typical individual fibres of cutaneous pectoris muscle following perfusion of hypertonic normal Ringer solutions containing varying sucrose concentrations. Extracellular osmolarity was progressively increased and the ability of the muscle to recover was explored by returning to isotonic Ringer solution in two stages. The solutions were added in the following order (see arrows): 10 mm, 20 mm, 50 mm, 100 mm, wash-out with isotonic Ringer, second wash-out with isotonic Ringer and 200 mm sucrose-ringer. B, fibre cross-sectional areas for the individual fibres plotted against extracellular [1/osmolarity].

Figure 4. Changes in normalized fibre volumes with the solution change procedures

A, changes in mean volume (±s.e.m.) normalized to values obtained in isotonic solutions over time (n= 6) through the solution change protocol (arrows) in Fig. 3. B, mean normalized volumes following changes from a 0 to a 10 mm (▪), 50–100 mm (♦) and a 0–200 mm (▴) external Ringer solution shown to a higher time resolution.

Figure 4. Changes in normalized fibre volumes with the solution change procedures

A, changes in mean volume (±s.e.m.) normalized to values obtained in isotonic solutions over time (n= 6) through the solution change protocol (arrows) in Fig. 3. B, mean normalized volumes following changes from a 0 to a 10 mm (▪), 50–100 mm (♦) and a 0–200 mm (▴) external Ringer solution shown to a higher time resolution.

Figure 5. Relationship between steady-state mean normalized volume and 1/osmolarity in different Ringer solutions

The open symbols show muscle volumes in normal (⋄ solution B), Na⁺-free (□ solution C) and Cl⁻-free (△ solution A) Ringer solutions to which different concentrations of sucrose had been added. •, recovery of volume on return to isotonic standard Ringer solution, confirming that the volume changes fully reversed on return to the isotonic solution. Data plotted as means ±s.e.m.; the s.e.m. is smaller than the dimensions of the data points where error bars are not visible. Data points from fibres studied in normal 100 mm sucrose Ringer solutions in the presence of 10 μm bumetanide, 10 μm bumetanide + 10 μm chlorothiazide and 10 μm ouabain superimposed upon each other. Therefore the mean ±s.e.m. of these values are listed in the text and are represented on the graph as the filled square (▪) that falls on the fitted line, indicating a persistence of simple osmotic behaviour in the presence of such agents.

Figure 6. The dependence of ΔE_m on extracellular osmolarity in Ringer solutions of different ionic composition

The measured ΔE_m (▪) are compared with predictions (▾ and dashed line; s.e.m.s are smaller than the dimensions of the data points) based upon the assumption that the muscle shows a simple osmotic behaviour in which intracellular K⁺ content is conserved and Cl⁻ is in electrochemical equilibrium to give a steady-state membrane potential defined by the K⁺ Nernst potential. A, Cl⁻-free Ringer solution (▪). There were no significant differences between measured ΔE_m in Cl⁻-free Ringer solution and the predicted values for a K⁺ electrode (two-tailed t test: P > 0.05 at each osmolarity, regression: r= 0.88, P value for a non-zero slope < 0.05). B, muscle in normal Ringer solution: the steady-state E_m values after 30 min at each osmolarity were significantly more depolarized than predicted (two-tailed t test significance levels: 10 mm: P < 0.01, 20 mm: P < 0.001, 50 mm: P < 0.001, 100 mm: P < 0.001, 200 mm sucrose Ringer solution: P < 0.01). C, muscle in Na⁺-free Ringer solution: there were no significant differences between the measured ΔE_m in choline Ringer solution and the predicted values (r= 0.94, P value for non-zero slope < 0.01). s.e.m.s are shown for both experimental data sets and the derived predictions: where not visible they are smaller than the dimensions of the data point.

Figure 6. The dependence of ΔE_m on extracellular osmolarity in Ringer solutions of different ionic composition

The measured ΔE_m (▪) are compared with predictions (▾ and dashed line; s.e.m.s are smaller than the dimensions of the data points) based upon the assumption that the muscle shows a simple osmotic behaviour in which intracellular K⁺ content is conserved and Cl⁻ is in electrochemical equilibrium to give a steady-state membrane potential defined by the K⁺ Nernst potential. A, Cl⁻-free Ringer solution (▪). There were no significant differences between measured ΔE_m in Cl⁻-free Ringer solution and the predicted values for a K⁺ electrode (two-tailed t test: P > 0.05 at each osmolarity, regression: r= 0.88, P value for a non-zero slope < 0.05). B, muscle in normal Ringer solution: the steady-state E_m values after 30 min at each osmolarity were significantly more depolarized than predicted (two-tailed t test significance levels: 10 mm: P < 0.01, 20 mm: P < 0.001, 50 mm: P < 0.001, 100 mm: P < 0.001, 200 mm sucrose Ringer solution: P < 0.01). C, muscle in Na⁺-free Ringer solution: there were no significant differences between the measured ΔE_m in choline Ringer solution and the predicted values (r= 0.94, P value for non-zero slope < 0.01). s.e.m.s are shown for both experimental data sets and the derived predictions: where not visible they are smaller than the dimensions of the data point.

Figure 6. The dependence of ΔE_m on extracellular osmolarity in Ringer solutions of different ionic composition

The measured ΔE_m (▪) are compared with predictions (▾ and dashed line; s.e.m.s are smaller than the dimensions of the data points) based upon the assumption that the muscle shows a simple osmotic behaviour in which intracellular K⁺ content is conserved and Cl⁻ is in electrochemical equilibrium to give a steady-state membrane potential defined by the K⁺ Nernst potential. A, Cl⁻-free Ringer solution (▪). There were no significant differences between measured ΔE_m in Cl⁻-free Ringer solution and the predicted values for a K⁺ electrode (two-tailed t test: P > 0.05 at each osmolarity, regression: r= 0.88, P value for a non-zero slope < 0.05). B, muscle in normal Ringer solution: the steady-state E_m values after 30 min at each osmolarity were significantly more depolarized than predicted (two-tailed t test significance levels: 10 mm: P < 0.01, 20 mm: P < 0.001, 50 mm: P < 0.001, 100 mm: P < 0.001, 200 mm sucrose Ringer solution: P < 0.01). C, muscle in Na⁺-free Ringer solution: there were no significant differences between the measured ΔE_m in choline Ringer solution and the predicted values (r= 0.94, P value for non-zero slope < 0.01). s.e.m.s are shown for both experimental data sets and the derived predictions: where not visible they are smaller than the dimensions of the data point.

Figure 7. Mapping of data points obtained from fibres in 200 mm sucrose Ringer solution onto corresponding predictions of a Goldman-Hodgkin-Katz (GHK) model assuming a conservation of intracellular Na⁺, K⁺ and Cl⁻ plotted over a range of relative membrane ionic permeabilities (P)

▪, expected ΔE_m following transfer of fibres from isotonic to 200 mm sucrose Ringer solution as a function of P_Cl/P_K. Values for P_Na/P_K, based on previously accepted ranges of values corroborated by experimental E_m values obtained in isotonic Cl⁻-free, normal and Na⁺-free Ringer solutions (▪ from top to bottom) of 0, 0.01, 0.02, 0.03, 0.05 and 0.1 are shown. Mapping of ΔE_m (boxes) obtained in Cl⁻-free (a) and normal (b) Ringer solutions onto the plots is consistent with previously reported values of P_Na/P_K and P_Cl/P_K and a conservation of intracellular Na⁺, K⁺ and Cl⁻. Those in Na⁺-free Ringer solutions (c) are consistent with a re-equilibration of Cl⁻.

Figure 8. Pharmacological studies of E_m

Mean (±s.e.m.) ΔE_m in the presence of the pharmacological agents: chlorothiazide (10 μm;agent 1), bumetanide (10 μm;agent 2), a combination of both chlorothiazide (10 μm) and bumetanide (10 μm) ( agent 3), and ouabain (10 μm; agent 4) when added to fibres studied in isotonic normal Ringer soution (A), in fibres treated with these agents both before and following transfer from isotonic to hypertonic (200 mm) sucrose Ringer solution (B), and in fibres first equilibrated in hypertonic (200 mm) sucrose Ringer solution for ∼30 min, then treated with these agents 1–4 (C): prior to recording of resting potentials. In panel A, only the histogram resulting from ouabain (agent 4) shows a significant membrane potential change (to a significance level of P < 0.001). In panel B, the histogram resulting from a combination of both chlorothiazide and bumetanide (agent 3) and from ouabain (agent 4) shows a significant membrane hyperpolarization (P < 0.001 and P < 0.01, respectively).

Figure 8. Pharmacological studies of E_m

Mean (±s.e.m.) ΔE_m in the presence of the pharmacological agents: chlorothiazide (10 μm;agent 1), bumetanide (10 μm;agent 2), a combination of both chlorothiazide (10 μm) and bumetanide (10 μm) ( agent 3), and ouabain (10 μm; agent 4) when added to fibres studied in isotonic normal Ringer soution (A), in fibres treated with these agents both before and following transfer from isotonic to hypertonic (200 mm) sucrose Ringer solution (B), and in fibres first equilibrated in hypertonic (200 mm) sucrose Ringer solution for ∼30 min, then treated with these agents 1–4 (C): prior to recording of resting potentials. In panel A, only the histogram resulting from ouabain (agent 4) shows a significant membrane potential change (to a significance level of P < 0.001). In panel B, the histogram resulting from a combination of both chlorothiazide and bumetanide (agent 3) and from ouabain (agent 4) shows a significant membrane hyperpolarization (P < 0.001 and P < 0.01, respectively).

Figure 8. Pharmacological studies of E_m

Mean (±s.e.m.) ΔE_m in the presence of the pharmacological agents: chlorothiazide (10 μm;agent 1), bumetanide (10 μm;agent 2), a combination of both chlorothiazide (10 μm) and bumetanide (10 μm) ( agent 3), and ouabain (10 μm; agent 4) when added to fibres studied in isotonic normal Ringer soution (A), in fibres treated with these agents both before and following transfer from isotonic to hypertonic (200 mm) sucrose Ringer solution (B), and in fibres first equilibrated in hypertonic (200 mm) sucrose Ringer solution for ∼30 min, then treated with these agents 1–4 (C): prior to recording of resting potentials. In panel A, only the histogram resulting from ouabain (agent 4) shows a significant membrane potential change (to a significance level of P < 0.001). In panel B, the histogram resulting from a combination of both chlorothiazide and bumetanide (agent 3) and from ouabain (agent 4) shows a significant membrane hyperpolarization (P < 0.001 and P < 0.01, respectively).

Figure 9. Simulation of the effects of NKCC transport following cell shrinkage induced by extracellular hypertonicity

Predicted corrections in volume (V,%) (□), membrane potential (E_m, mV) (△), [Na⁺]_i (♦), [K⁺]_i (▴) and [Cl⁻]_i (▪) (mm) mediated by NKCC activity following passive changes induced by a change in extracellular hypertonicity from isotonic (∼238 mosmol l⁻¹) to hypertonic (∼438 mosmol l⁻¹) Ringer solution. Each iteration of NKCC activity is then assumed to increment [Na⁺]_i and [K⁺]_i each by 10 μm, and thereby generate the abscissa, plotted in increments of 0.1 mm. In the case of a high P_Cl/P_K∼2 (A), full correction of E_m by 10–12 mV is only accompanied by a ∼2% volume increase, consistent with our experimental results. With a low P_Cl/P_K∼0.2 (B), the same increase in volume (∼2%) is accompanied by a profoundly smaller effect (<1 mV) on E_m, permitting large RVIs, even were E_m corrections to limit NKCC activity.

Figure 9. Simulation of the effects of NKCC transport following cell shrinkage induced by extracellular hypertonicity

Predicted corrections in volume (V,%) (□), membrane potential (E_m, mV) (△), [Na⁺]_i (♦), [K⁺]_i (▴) and [Cl⁻]_i (▪) (mm) mediated by NKCC activity following passive changes induced by a change in extracellular hypertonicity from isotonic (∼238 mosmol l⁻¹) to hypertonic (∼438 mosmol l⁻¹) Ringer solution. Each iteration of NKCC activity is then assumed to increment [Na⁺]_i and [K⁺]_i each by 10 μm, and thereby generate the abscissa, plotted in increments of 0.1 mm. In the case of a high P_Cl/P_K∼2 (A), full correction of E_m by 10–12 mV is only accompanied by a ∼2% volume increase, consistent with our experimental results. With a low P_Cl/P_K∼0.2 (B), the same increase in volume (∼2%) is accompanied by a profoundly smaller effect (<1 mV) on E_m, permitting large RVIs, even were E_m corrections to limit NKCC activity.