SUMMARY

It is well established that hypotonicity generates a marked and unexpected increase in active Na+ efflux in frog muscle fibers as well as in other cells like cardiac myocytes, astrocytes, brain synaptosomes and renal cells. The effect of hypotonicity on the electrical activity of skeletal muscle related to Na+ and K+ voltage-gated channels, however, has not been specifically addressed. The results of the present investigation show that the changes in resting and action potentials produced by hypotonicity can be fully explained by the reduction of intracellular [Na+] and [K+] due to the increase in cellular water content.

INTRODUCTION

In frog skeletal muscles exposed to hypotonic media the active extrusion of Na+, at variance with the expected reduction due to the fall in its intracellular concentration ([Na+]i), increases (Venosa, 1978). In this tissue, the effect is produced, apparently, by the incorporation of spare Na+/K+-ATPase units into the sarcolemma (Venosa, 1991; Venosa, 2003). The hypotonic stimulation of the sodium pump has also been observed in brain synaptosomes (Mongin et al., 1992; Aksensev, 1994), astrocytes (Mongin et al., 1994), cardiomyocytes (Walley et al., 1993; Bewick et al., 1999) and renal cells (Coutry et al., 1994). In skeletal muscle, at least, the effect is triggered by the stretching of the cell membrane caused by the swelling resulting from exposure to hypotonic media.

It was thought that if, in frog muscle, hypotonicity produces changes in the voltage-gated Na+ and K+ channels involved in the excitation process, akin to those elicited in Na+/K+ active transport, then the action potential (AP) and the corresponding extra Na+ influx (JiNa) would also be affected under hypotonic conditions. The results of the present study show that the fall in [Na+]i and intracellular K+ concentration ([K+]i), produced by the rise in cell water content when in hypotonic media, does not alter JiNa and is sufficient to fully account for the observed changes in resting potential (Vm) and AP.

MATERIALS AND METHODS

Experiments were performed on isolated frog (Leptodactylus ocellatus L.) paired sartorius muscles. Animals were maintained and the experiments were conducted in accordance with the guidelines of the local Ethics Committee, which are similar to those of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996). Before dissection, the animals were chilled in an ice–water mixture until fully immobile and then double pithed.

The normal Ringer solution had the following composition (mmol l–1): NaCl 115; KCl 2.5; CaCl2 1.8; Na2HPO4 2.15; and NaH2PO4 0.85 (pH 7.18). The reference isotonic medium (Π1) was similar to the normal saline except that 62 mmol l–1 NaCl, one-half of the total osmolarity, was replaced by an osmotically equivalent concentration of sucrose as determined using a vapor pressure osmometer (Wescor model 5100C, Wescor Inc., Logan, UT, USA). The hypotonic media (no sucrose added) had an osmotic pressure one-half (Π0.5) that of Π1 and the same ionic composition. In some experiments Cl was replaced by SO42–. The electrical measurements were made using conventional glass microelectrodes filled with 3 mol l–1 KCl with a resistance of 10–15 MΩ and coupled to a high imput impedance electrometer (WPI, New Haven, CT, USA), whose output was recorded online by a data acquisition system (Power Lab/410, AD Instruments, Sydney, Australia) connected to a personal computer. The effects of hypotonicity were studied in muscles exposed to Π0.5 for 1.5 h. During that period, no regulatory volume decrease (RVD) was observed. In fact, in our experience, RVD was not observed during much longer exposures to Π0.5 either. In some experiments the AP and its time derivative (obtained with a function module; Frederic Haer & Co., Brunswick, ME, USA) were directly recorded in a digitizing oscilloscope with screen memory (Tektronix model 5223, Beaverton, OR, USA). To diminish the twitch movement and the subsequent dislodgment of the microelectrode, muscles with the inner face up were stretched (20%) and a small bundle of fibers were stimulated externally using a thin tungsten electrode. APs were elicited with rectangular pulses lasting 1 ms, delivered by a stimulator (Grass model S48, Quincy, MA, USA) through a Grass isolation unit (model SIU 5).

Determination of the extra Na+ influx per impulse (JiNa) with 22Na+ (New England Nuclear, Boston, MA, USA) was done using a technique previously described (Venosa, 1974; Kotsias and Venosa, 2001).

Student's t-test was used to estimate the statistical significance of differences. Values are expressed as means ± s.e.m.

RESULTS

The resting potential

In hypotonic medium the equilibrium potential of Na+ and K+ (ENa, EK) as well as Vm are altered because of the decrease of [K+]i and [Na+]i. Vm is also strongly dependent on the extracellular K+ concentration ([K+]o), particularly at values greater than 10 mmol l–1. In frog muscle fibers, the relationship between Vm and the concentration of Na+ and K+ is expressed by Eqn 1 (Hodgkin and Horowicz, 1959):
formula
(1)
where α represents the ratio of Na+ and K+ permeabilities (α=PNa/PK), which under normal conditions is of the order of 0.01–0.02. R, T and F have their usual meanings. As [K+]o increases (>10 mmol l–1), α[Na+]o becomes negligible compared with [K+]o and can be suppressed in Eqn 1. Moreover, at 20°C, and using log instead of ln, Eqn 1 becomes:
formula
(2)
which expresses a linear relationship between Vm and log[K+]o where 58 mV is the slope (S) of the line for an ideal K+ electrode. The measurement of Vm at different [K+]o, under isotonic and hypotonic conditions was done in Cl-free media (Cl replaced by SO42–) to avoid Cl transients upon solution changes (Hodgkin and Horowicz, 1959) and assuming [K+]i is constant. To keep the osmolarity constant, the increments in [K+]o were made by equimolar reductions of extracellular Na+ concentration ([Na+]o). Fig. 1 shows the plot of Vm as a function of log[K+]o in both isotonic (Π1) and hypotonic medium (Π0.5). The linear fitting of the data corresponds to a relationship of the form Vm=S log[K+]oS log[K+]i (r=0.999 and 1.000 for Π1 and Π0.5, respectively). Under both conditions the fibers behave similar to K+ electrodes with S values of 55.2 and 55.7 mV for Π1 and Π0.5, respectively. On the other hand, the extrapolation to Vm of 0 mV, which should occur when [K+]o=[K+]i, yielded values of 151.7 mmol l–1 for Π1 and 93.0 mmol l–1 for Π0.5.
Eqn 1 can be rearranged so that:
formula
(3)
where [K+]o is the independent variable, 1/[K+]i is the slope and α[Na+]o/[K+]i is a constant given by eVmF/RT when [K]o=0. Fig. 2 shows the plot of the data for [K+]o between 1.25 and 58 mmol l–1 for both Π1 (r=0.999) and Π0.5 (r=0.0994). The linear fitting of the data yielded [K+]i values of 146.4 mmol l–11) and 92.6 mmol l–10.5), which are not significantly different from those calculated from the plot in Fig. 1. On the other hand, for [K+]o=0 the first term on the right-hand side of Eqn 3 vanishes and the magnitude of α can be easily calculated. Thus for Π1, α=0.0063 and for Π0.5 α=0.0113. These values are not too far from the value of 0.01 reported by Hodgkin and Horowicz (Hodgkin and Horowicz, 1959).
Fig. 1.

Resting membrane potential (Vm, in mV) as a function of log[K+]o in muscles equilibrated in reference isotonic medium (Π1) and hypotonic medium with an osmotic pressure one-half that of Π10.5). Each experimental point represents the mean (±s.e.m.) of between 34 and 39 fibers. The linear fitting yielded slopes of 55.2 mV (r=0.999) for Π1 and 55.7 mV (r=1.000) for Π0.5, values which are close to the theoretical 58 mV for a K+ electrode. The extrapolation to Vm=0 mV, where [K+]o=[K+]i, indicates [K+]i=151.7 mmol l–1 for Π1 and [K+]i=93 mmol l–1 for Π0.5.

Fig. 1.

Resting membrane potential (Vm, in mV) as a function of log[K+]o in muscles equilibrated in reference isotonic medium (Π1) and hypotonic medium with an osmotic pressure one-half that of Π10.5). Each experimental point represents the mean (±s.e.m.) of between 34 and 39 fibers. The linear fitting yielded slopes of 55.2 mV (r=0.999) for Π1 and 55.7 mV (r=1.000) for Π0.5, values which are close to the theoretical 58 mV for a K+ electrode. The extrapolation to Vm=0 mV, where [K+]o=[K+]i, indicates [K+]i=151.7 mmol l–1 for Π1 and [K+]i=93 mmol l–1 for Π0.5.

Fig. 2.

eVmF/RT as a function of [K+]o in the presence of both Π1 and Π0.5. It can be seen that the relationship is fairly linear in accordance with Eqn 3 (see text) in the [K+]o range 1.25–58 mmol l–1. The slope of the lines (1/[K+]i) provides a measure of [K+]i: 146.4 mmol l–1 for Π1 and 92.6 mmol l–1 for Π1. Each experimental point represents the mean (±s.e.m.) of between 12 and 39 fibers.

Fig. 2.

eVmF/RT as a function of [K+]o in the presence of both Π1 and Π0.5. It can be seen that the relationship is fairly linear in accordance with Eqn 3 (see text) in the [K+]o range 1.25–58 mmol l–1. The slope of the lines (1/[K+]i) provides a measure of [K+]i: 146.4 mmol l–1 for Π1 and 92.6 mmol l–1 for Π1. Each experimental point represents the mean (±s.e.m.) of between 12 and 39 fibers.

The AP

In skeletal muscle, as in most excitable cells, the peak of the AP reaches a value not too far from that of ENa because of the marked and transient increase of PNa during its rising phase. It seems reasonable to assume that the ratio ([K+]i in Π1)/([K+]i in Π0.5) provides an estimate of the increment in fiber water content in Π0.5. Taking the averaged values of [K+]i from Eqns 1 and 2, i.e. 149 mmol l–1 in Πi and 92.8 mmol l–1 in Π0.5, and assuming [Na+]i=15 mmol l–1 in Π1, similar to that in NR (Venosa and Horowicz, 1973), a [Na+]i of 9.3 mmol l–1 [=15(92.8/149)] can be estimated for fibers equilibrated in Π0.5. This further indicates an increase in cell water of about 60% in Π0.5 relative to Π1 (149/92.8=1.61), which is similar to that found previously in the same preparation (Venosa, 2003). With these values of [Na+]i, the calculated ENa, would be 34.1 mV [=58 log(58.2/15)] in Π1 and 46.2 mV [=58 log(58.2/9.3)] in Π0.5. The mean peak AP (pAP) in Π1 was 19.9 mV while in Π0.5 it was 27.9 mV, close to the value of 29.4 mV measured in the presence of NR where the ratio [Na+]o/[Na+]i, and therefore ENa, is close to that in fibers equilibrated in Π0.5 (see Table 1). It is known that the maximum value of dVm/dt (dVm,max/dt) during the upstroke of the AP is an expression of the inward Na+ current (INa) during that period. As can be seen in Table 1, it amounted to 417 V s–1 in NR while in Π1 and Π0.5 it was significantly lower. It can also be appreciated that the difference between the values of this parameter in Π1 and Π0.5 is not significant. This is not surprising because INa, and therefore dVm,max/dt, is directly proportional to ENaVm, the driving force (DF) on Na+. Although, as a result of intracellular Na+ dilution, ENa is greater in Π0.5 than in Πi, it is also true that Vm, because of intracellular K+ dilution, is less negative, so that the DF is virtually the same under the two conditions (Table 1). Fig. 3 shows representative APs and their time derivative of fibers equilibrated in NR, Π1 and Π0.5.

Table 1.

Action potential data

Action potential data
Action potential data

The determination of the extra Na+ influx per AP (JiNa) yielded mean values of 3.31±0.88 nmol g–1 AP–1 (N=8) in Π1 and 3.47±0.69 nmol g–1 AP–1 (N=6) in Π0.5, which is in good agreement with the measurements of dVm,max/dt.

Fig. 3.

Representative records of action potentials (APs) and their time derivatives in three different fibers equilibrated in normal Ringer solution (NR) (A), Π1 (B) and Π0.5 (C). In each record the lower curve corresponds to the AP and the upper one to its time derivative. The base line of the derivative record (dVm/dt=0) coincides with the 0 mV of the membrane potential record (Vm=0). The upper calibration bar corresponds to dVm/dt and the lower one to Vm.

Fig. 3.

Representative records of action potentials (APs) and their time derivatives in three different fibers equilibrated in normal Ringer solution (NR) (A), Π1 (B) and Π0.5 (C). In each record the lower curve corresponds to the AP and the upper one to its time derivative. The base line of the derivative record (dVm/dt=0) coincides with the 0 mV of the membrane potential record (Vm=0). The upper calibration bar corresponds to dVm/dt and the lower one to Vm.

DISCUSSION

In frog muscle (Venosa, 1978; Venosa, 1991; Venosa, 2003) as well as in several other cell types, hypotonicity produces a marked and unexpected increase in the active extrusion of Na+. The aim of the present experiments was to find out how the basic electrical properties of muscle fibers are affected under similar conditions. As described above, hypotonicity depolarizes the resting potential in a predictable fashion, mainly due to the fall in [K+]i and practically no change in the value of the PNa/PK ratio in Π0.5 with respect to that in Π1.

The increase in the activity of the Na+ pump, which is electrogenic, does not measurably affect Vm. The reason for this is as follows. The increase in active K+ influx produced by the Π1 to Π0.5 transfer is of the order of 0.7 pmol cm–2 s–1 (Venosa, 1991). Given the stoichiometry of the pump is 3 Na+/2 K+, the corresponding increase in active Na+ transport would be 0.7×3/2=1.05 pmol cm–2 s–1; that is, a net outward transfer of 1.05–0.70=0.35 pmol cm–2 s–1 or a current density of 0.35×10–12 mol cm–2 s–1×96500 C mol–1=3.4×10–8 A cm–2. Assuming a membrane resistance of 4000Ωcm2 (Katz, 1966), this current density would produce a hyperpolarization of only 3.6× 10–8 A cm–2×4000Ωcm2=0.14 mV.

With regard to the AP parameters, the magnitude of pAP, which strongly depends on ENa, increased in Π0.5 with respect to its value in Π1, in a predictable manner according to the fall in [Na+]i in Π0.5. On the other hand, the magnitude of dVm,max/dt, an expression of the inward INa during the upstroke of the AP, in Π0.5 was not different from that in Π1, because the swelling in Π0.5, and the consequent fall of both [Na+]i and [K+]i, produced virtually no change in the DF. This is supported by the fact that JiNa in Π0.5 (3.47±0.69 nmol g–1 AP–1) was not different from that in Π1 (3.31±0.88 nmol g–1 AP–1). In this regard it is worth mentioning that when JiNa is expressed in terms of the superficial sarcolemma [430 cm2 g–1 (Venosa, 1991)], we have 7.70 and 8.07 pmol cm–2 AP–1 for Π1 and Π0.5, respectively. These values, in what might be the result of a species difference, are about one-third of those previously determined in sartorii from Rana pipiens (552 cm2 g–1) in the presence of 60 mmol l–1 [Na+]i (Venosa, 1974).

In conclusion, it is interesting to note that, in frog muscle, while hypotonicity generates a series of changes in active Na+ transport, involving an increase in the membrane density of Na+ pumps, apparently through the insertion of spare pumps in the sarcolemma mediated by actin filaments of the cytoskeleton (Venosa, 2003), no changes of that sort seem to occur to the voltage-gated Na+ channels. Instead, the observed changes in Vm and AP promoted by hypotonicity can be fully explained by the reduction of [Na+]i and [K+]i due to the increase in cell water content.

LIST OF SYMBOLS AND ABBREVIATIONS

     
  • AP

    action potential

  •  
  • DF

    driving force on ion x (ExVm)

  •  
  • Ex

    equilibrium potential of ion x

  •  
  • JiNa

    extra Na+ influx per AP

  •  
  • NR

    normal Ringer solution

  •  
  • Px

    permeability of ion x

  •  
  • pAP

    peak AP

  •  
  • Vm

    resting potential

  •  
  • Π0.5

    hypotonic medium (Π1/2)

  •  
  • Π1

    isotonic medium

This work was supported by CONICET (the National Research Council of Argentina).

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