Resting and action potentials under hypotonic conditions, unlike Na⁺ pump activity, depend only on the alteration of intracellular [Na⁺] and [K⁺] in frog skeletal muscle

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 (J_i^Na) 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 J_i^Na and is sufficient to fully account for the observed changes in resting potential (V_m) and AP.

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 (Π_0.5) that of Π₁ and the same ionic composition. In some experiments Cl^– was replaced by SO₄^2–. 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 Π_0.5 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 Π_0.5 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 (J_i^Na) 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.

In skeletal muscle, as in most excitable cells, the peak of the AP reaches a value not too far from that of E_Na because of the marked and transient increase of P_Na during its rising phase. It seems reasonable to assume that the ratio ([K⁺]_i in Π₁)/([K⁺]_i in Π_0.5) provides an estimate of the increment in fiber water content in Π_0.5. Taking the averaged values of [K⁺]_i from Eqns 1 and 2, i.e. 149 mmol l^–1 in Π_i and 92.8 mmol l^–1 in Π_0.5, 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 Π_0.5. This further indicates an increase in cell water of about 60% in Π_0.5 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 E_Na, would be 34.1 mV [=58 log(58.2/15)] in Π₁ and 46.2 mV [=58 log(58.2/9.3)] in Π_0.5. The mean peak AP (pAP) in Π₁ was 19.9 mV while in Π_0.5 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 E_Na, is close to that in fibers equilibrated in Π_0.5 (see Table 1). It is known that the maximum value of dV_m/dt (dV_m,max/dt) during the upstroke of the AP is an expression of the inward Na⁺ current (I_Na) during that period. As can be seen in Table 1, it amounted to 417 V s^–1 in NR while in Π₁ and Π_0.5 it was significantly lower. It can also be appreciated that the difference between the values of this parameter in Π₁ and Π_0.5 is not significant. This is not surprising because I_Na, and therefore dV_m,max/dt, is directly proportional to E_Na–V_m, the driving force (DF) on Na⁺. Although, as a result of intracellular Na⁺ dilution, E_Na is greater in Π_0.5 than in Π_i, it is also true that V_m, 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 Π_0.5.

The determination of the extra Na⁺ influx per AP (J_i^Na) 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 Π_0.5, which is in good agreement with the measurements of dV_m,max/dt.

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 P_Na/P_K ratio in Π_0.5 with respect to that in Π₁.

The increase in the activity of the Na⁺ pump, which is electrogenic, does not measurably affect V_m. The reason for this is as follows. The increase in active K⁺ influx produced by the Π₁ to Π_0.5 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 E_Na, increased in Π_0.5 with respect to its value in Π₁, in a predictable manner according to the fall in [Na⁺]_i in Π_0.5. On the other hand, the magnitude of dV_m,max/dt, an expression of the inward I_Na during the upstroke of the AP, in Π_0.5 was not different from that in Π₁, because the swelling in Π_0.5, 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 J_i^Na in Π_0.5 (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 J_i^Na 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 Π_0.5, 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 V_m 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.

 
• AP

  action potential

 
• DF

  driving force on ion x (E_x–V_m)

 
• E_x

  equilibrium potential of ion x

 
• J_i^Na

  extra Na⁺ influx per AP

 
• NR

  normal Ringer solution

 
• P_x

  permeability of ion x

 
• pAP

  peak AP

 
• V_m

  resting potential

 
• Π0.5

  hypotonic medium (Π₁/2)

 
• Π1

  isotonic medium