239

J. Physiol. (1977), 264, pp. 239-265 With 14 text-ftgure8 Printed in Great Britain

THE EFFECT OF LOWERING EXTERNAL SODIUM ON THE INTRACELLULAR SODIUM ACTIVITY OF CRAB MUSCLE FIBRES

BY R. D. VAUGHAN-JONES* From the Department of Physiology, University of Bristol, Bristol

(Received 9 June 1976) SUMMARY

1. Intracellular Na activity, aNa, was continuously measured in crab (Carcinus maenas) muscle fibres using a recessed-tip Na+-sensitive glass micro-electrode. Experiments could last up to several hours. a~a remained stable during prolonged experiments. The mean resting a0a was 8*4 + 0.02 mM (S.E. of mean for eighty-nine fibres) and the mean resting membrane potential was 65*3 mV + 0 3 (S.E. of mean for eighty-nine fibres). 2. Reducing [Na]0 to I normal (maintaining ionic strength with equivalent amounts of either Li or Tris) caused a large and rapid fall of aZa. There appeared to be two components of the effect, a fast and slow. The initial fast rate of decrease was about 3-5 m-mole/min decreasing to half this value in about 1 min. The rate of decrease of aya was not linearly related to aNa. The size of the fast change of aya was related to the magnitude of the Na gradient across the membrane. 3. High concentrations (2 x 10t4 M) of ouabain caused a very slow rise of apa by 1 or 2 mM/hr. This was equivalent to a net Na influx of between 1 and lO p-mole/cm2. sec, depending on whether or not a correction was applied to account for the increased surface area of the fibre caused by the invaginating cleft system. 4. The response to low Nao was virtually insensitive to the removal of Ko or to prolonged treatment with high concentrations of ouabain (2 x 10-4 M; 100 min) and so could -not readily be attributed to active Na/K pumping. 5. The response of aNa to low Nao was reversibly inhibited by high concentrations of Mn (50 mM) and by low concentrations of La (0.1 mM). La itself stimulated a rapid fall of a-a in normal Nao. *

Present address: Department of Pharmacology, South Parks Road, Oxford,

OX1 3QT.

240 R. D. VAUGHAN-JONES 6. Removal of Ca. and/or Mg, failed to reduce the response of aNa to low Na0. Removal of Ca. alone caused a slow rise of aNa whereas removal of Ca0 and Mg0 caused a rapid rise of aNa. 7. The response of ajla to low Nao was unaffected by the drugs D600 and Verapamil at concentrations known to inhibit divalent cation movements across membranes. 8. The response was unaffected by short-term pH changes from 6-3 to 8-3. 9. a?' and a0a were simultaneously measured in seven experiments, using recessed-tip Na+ and Cl--sensitive micro-electrode. Mean a?'= 30*8 mm + 2*5 (S.E. of mean) with a mean membrane potential of 66-8 + 0 7 mV (S.E. of mean). a?' on the basis of passive distribution for Cl- would be 28-9 mm, calculated from the mean membrane potential. aP? did not change in low Na0. 10. The two most reasonable interpretations of the results is that the changes of a;a are caused either by an active extrusion of Na+ on an ion-exchange carrier or by an intracellular sequestration of Nat, that occurs in low Na0, into a region where it is 'hidden' from the Na+sensitive electrode. This region could possibly be the sarcoplasmic reticulum. INTRODUCTION

Intracellular Na activity, aOa, can be measured directly with an ionsensitive electrode. This was first done by Hinke (1959) in crustacean muscle and by Lev (1964) in frog skeletal muscle. At first only brief measurements of apa were made, possibly because the early electrode designs had rather blunt tips and tended to cause extensive cell damage. However, the more recent recessed-tip design of electrode (Thomas, 1970; tip diameter 1 ,um) has been shown to be well suited for making continuous intracellular measurements of ion activities for periods of up to many hours (Thomas, 1972, 1974). The aim of the present experiments was to use the recessed-tip Na+sensitive electrode to determine the stable resting aOa of crustacean muscle and then to investigate how this changed in low Na0. By analogy with experiments on snail neurones (Thomas, 1972), it was thought that a.a would fall slowly under these conditions because of the activity of the Na/K pump and that the rate of decrease with time would provide information about the dependence of Na-pumping in muscle upon aba. Experiments carried out on frog skeletal muscle (Keynes & Swan, 1959; Mullins & Frumento, 1963; Harris, 1965) using radioactive tracers and Na-loaded muscles had already suggested that the efflux of Na, under the conditions of low Na0, was related to the cube of [Na], whereas, in

241 Na ACTIVITY OF CRAB MUSCLE nerve, a linear relationship had been found (Hodgkin & Keynes, 1956; Thomas, 1972). The results of the present study show that aNa is indeed very low in crustacean muscle, that it decreases very rapidly within a few minutes of reducing [Na]0 and that the rate of decrease is not linearly related to aNa. However, the effect is virtually insensitive either to high concentrations of ouabain or to the removal of K. and so cannot readily be attributed to the Na/K pump. The changes of aNa must be caused either by an active efflux of Na+ on an ion-exchange pump, or by a re-distribution of intracellular Na which occurs when [Na]o is low. A preliminary report of this work has already been published (Vaughan-Jones, 1976). METHODS Preparation The experiments were performed on the carpopodite extensor, or in a few experiments, flexor muscle of a walking leg of Carcinus maena8. The preparation itself was originally described by Fatt & Katz (1953). It consisted of the ventral half of the meropodite 'shell' to which was attached the carpopodite extensor muscle and its apodeme. The preparation was fixed by pressing the shell into a thin layer of silicone grease covering the bottom of the experimental chamber. The chamber, with mounted preparation, had a volume of 0 3 ml. and was superfused continuously with crab Ringer at a rate of about 2 ml./min. Solutions were led into the bath by means of a multi-way tap which could select between any one of ten different solutions without interrupting the flow. All experiments were carried out at room temperature, about 200 C. Solution The normal saline had the following composition: NaCl, 500 mM; KCl, 12 mM; CaCl2, 12 MM; MgCl2, 20 mM; Tris Cl, 10 mm. This is similar to the Fatt & Katz (1953) crab Ringer, except that no bicarbonate was present, and it is very similar to the composition of crab haemolymph (Cole, 1941). The osmolarity of all solutions was frequently measured using a freezing-point, depression osmometer to ensure that it was reasonably constant. The osmolarity of normal crab Ringer was about 1100 m-osmole/l. When a solution with a low or zero amount of a particular constituent was needed, it was made by substituting equivalent amounts of either LiCl or Tris Cl in order to maintain a constant ionic strength and osmolarity. Slightly more Tris Cl than LiCl had to be added when substituting, since Tris Cl is less dissociated than the other salts in the Ringer. High K solutions were initially made by raising K and removing equivalent amounts of Na but it was discovered that such Na removal affected a"a. So high K solutions were made by raising [K] from 12 to 120 mm and reducing [Na] from 500 to only 450 mm. This proved satisfactory and the slight increase in osmolarity of the solution had no visible effect on the muscle fibres. 1 mM-EGTA was added to zero Ca solutions; 1 mM-EDTA was added to zero Ca and Mg solutions. Solutions containing MnCl2, CoCl2 and LaCl3 were made by adding solid to normal crab Ringer. It was essential to prepare these particular solutions freshly before an experiment and to check and adjust their pH carefully. The pH of all solutions was 7-2-7-5.

242

R. D. VAUGHAN-JONES

Micro-electrodes Recessed-tip Na+-sensitive glass micro-electrodes were used throughout this study and were made as described by Thomas (1970, 1969) except that aluminosilicate glass (Corning code: 1720) rather than borosilicate glass was used for the outer insulating glass. After an experiment, electrodes were stored with their tips immersed in chromic acid and before using again were dipped and etched for about 20 min in strong alkali containing EDTA. Electrodes were calibrated before and after an experiment using solutions of known Na concentration (using Tris as a Na substitute). Since the ionic strength was constant a tenfold change in Na concentration represented a tenfold change in Na activity. Values for the activity coefficient of NaCl solutions were obtained from Parsons (1959). The Na+-sensitive glass is very selective for Na over all other cations in the experimental solutions at neutral pH except possibly for Li. But when Li was used as a Na substitute during 'low Na0 experiments', it was usually present at concentrations of 450 mM-Li with 50 mM-Na. At this concentration, Li had no measurable effect on the potential response of the Na+-sensitive electrode developed in the presence of 50 mM-Na. In a few experiments, concentrations of Na (substituted with Li) lower than 50 mm were used and so it is possible that readings of aea when the muscle was superfused with these solutions might have been slightly too high. Recessed-tip Cl--sensitive micro-electrodes have been described by Neild & Thomas (1972). These were calibrated in solutions of known Cl- activity. Acetate ions (to which the Cl--electrode was not sensitive) were used in place of Cl- to maintain constant ionic strength. Conventional micro-electrodes were used for recording the membrane potential. They were made from borosilicate glass tubing with a fine borosilicate glued inside. They were back-filled with 2-5 M-KCl buffered to pH 7-5 with Tris Cl and had resistances of 10-20 MD.

Electrical arrangements The method of continuous recording of intracellular Na+ has been described by Thomas (1972) for snail neurones. The same technique has been used in these experiments. A conventional KCl-filled glass micro-electrode and a recessed-tip Na+-sensitive micro-electrode are used simultaneously. Recordings are made by inserting both electrodes transversely into a single muscle fibre. The membrane potential was recorded as the difference between the conventional glass microelectrode inside the fibre and an indifferent agar bridge electrode in the bath. This was done using two unity-gain FET amplifiers and was displayed on an oscilloscope and one channel of a pen-recorder. Na activity was recorded as the potential difference between the Na+-sensitive electrode and the conventional glass microelectrode and was displayed on the second channel of the pen-recorder. The signal from the Na+-sensitive electrode was amplified by a Vibron electrometer (E.I.L. 62A). RESULTS

Measurement of normal aNa in crab muscle Fig. 1 shows the method of measuring the intracellular Na activity, aba, inside a crab muscle fibre. Both the Na+-sensitive electrode and the conventional micro-electrode were inserted into a single fibre. The electrodes were usually positioned close together (their tips about 30 ,um apart), although this was not critical. The Na+-sensitive electrode was

243 Na ACTIVITY OF CRAB MUSCLE calibrated both before and after the experiment and if its response to a tenfold change of aNa altered by more than 2 mV the measurement was discarded. This occurred only occasionally, the electrode's sensitivity usually remaining quite constant over a period of many hours. a;a was monitored for at least 10 min before its value was noted, only stable values being accepted. Sometimes there was an initial small increase in aNa which was probably associated with damage to the cell, but the level usually fell to a stable value within 15-20 min. If not, the electrodes were withdrawn and put into a fresh fibre. To check that both electrodes were properly sealed into the fibre, in the experiment shown in Fig. 1, the superfused Ringer was changed for about 2 min, to one containing 10 times the normal amount of K. This caused a large transient depolarization (over 25 mV in Fig. 1) which was measured on the membrane potential electrode. However, the reading of aNa remained constant showing that both electrodes were recording the same changes in membrane potential and must therefore have been completely intracellular. Only brief exposures to high Ko were used in case the raised Ko itself stimulated a change of ab;a. aTa was measured successfully by the above method in eighty-nine fibres. The mean value of aNa was 8-4 + 0-02 mm (S.E. of mean) with a range of 2-3-14-0 mm. Values of apa taken from different fibres of the same muscle within a preparation were very similar to each other, varying by 1 or 2 mm. Values from fibres in different preparations could vary widely. This mean value of apa is lower than that found by Hinke (1959) who used a Na+-sensitive electrode in the same preparation and obtained a mean value of 13-5 mm for twenty cells. However, Hinke's Na+-sensitive electrode was rather large and had to be inserted down the longitudinal axis of the muscle fibre and this probably caused damage to the fibre. The range of membrane potentials that he measured was 33-35 mV whereas the range for the present experiments was 54-74 mV with a mean of 65-3 mV. Fatt & Katz (1953) obtained a mean membrane potential for the same muscle of 70 mV. Values for aNa of 11-5-16-0 mm have also been reported for giant barnacle muscle fibres (Hinke, 1959; McLaughlin & Hinke, 1966; Allen & Hinke, 1971). However, a direct comparison with the present results is difficult since there may be a real difference between crab and barnacle muscle.

Effects of reducing extracellular [Na] Fig. 2 illustrates the effect of reducing [Na]o to -L normal, substituting either with Li or Tris. Since [Na]o in this solution was 50 mM, there

244

R. D. VAUGHAN-JONES

~1 350

10 min *0 10 E 20 -a

-r

30 40

50

20

20

10

10

3.5

0~ o c

2:

8-4 'El

---'

5.

._

0

70

35

8-4

C

kJ

5

z(U

3.5

Fig. 1. Pen recording of an experiment to measure the normal resting als and membrane potential of a crab muscle fibre. Before the experiment, the Na+-sensitive electrode (lower trace) was quickly tested by changing [Na]o in the bathing solution from 500 to 5-0 mm (Tris substituted). This represented a change of ala from 350 to 3-5 mm as indicated. Normal Nao was then restored and both electrodes were allowed to stabilize for a few minutes (indicated by dashed trace). At the first arrow, the Na+-sensitive electrode was inserted into the muscle fibre. It measured a potential which was the sum of the membrane potential plus a potential proportional to a~a. After a few minutes (at the second arrow) the voltage electrode was advanced into the same fibre, to measure the membrane potential which was displayed (upper trace). Since this was electronically subtracted from the Na+ electrode signal, the lower trace was now a true recording of aiN. The bar indicates the brief superfusion of a high K solution (120 mM). At the end of the experiment both electrodes were withdrawn from the fibre (voltage electrode first, then Na+ electrode) and the Na+ electrode was recalibrated. The two traces are slightly out of phase. This was to allow the pens on the recorder to move freely past each other.

Na ACTIVITY OF CRAB MUSCLE 245 was still a very small net passive Na influx (the experiments with ouabain suggest that this was almost negligible) and no net passive Na efflux. Low Na0 caused a remarkably rapid decrease of aj a, the level falling by about 3-5 mm within 4 min of changing the solution. The effect was essentially the same in either Li or Tris-substituted Ringer and in some experiments (not shown) it was virtually identical. There was 0

>

E

.0

10 min 20 20 40

-o E

60

65L

ftE

5i 3

_

', .-

Na

(Li)

'-

j Na(Tris)

^

u

10 K

Fig. 2. Pen recording of an experiment showing the effects on the membrane potential and a'a of reducing [Na]o to -j- normal (50 mM). Firstly a Li-substituted and secondly a Tris-substituted solution was perfused. That both electrodes were measuring the same potential changes was checked by finally and briefly superfusing a high K solution.

a concomitant small depolarization of the membrane potential by a few millivolts. Restoring Nao caused aNa to rise again, although occasionally it would not reach its original initial level, but would stabilize at a slightly lower aNa (see Fig. 5). In general, the effect could be repeated in the same fibre many times without any significant variation (see Fig. 13). Thirty-four successful experiments were performed, testing the response of apIa to i [Na]0 and there was also usually a brief superfusion of high Ko to check that both electrodes were completely intracellular. The response of different fibres from the same preparation was very similar although it varied greatly between preparations. The results are summarized in

Fig. 7B and will be examined more fully later.

R. D. VA UGHAN-JONES 246 Fig. 3A shows an experiment where a fibre was exposed to -k [Na]o (substituted with Li) for the longer period of 45 min. Part of the trace has been re-plotted in Fig. 3B on linear co-ordinates. ata decreased from 10 to about 6 mM in the first 4 min, and then fell more slowly over the rest of the period. In Fig. 3C, the rate of decrease of a0a has been plotted against ata for the whole 45 min period. The graph clearly shows that there is a non-linear relationship between the rate of decrease of a'a and the level of a'a in low Nao. This is in contrast with the findings of Thomas (1972) who conducted the same experiment on a snail neurone, using a Na+-electrode and found a linear relationship above a threshold level of aba. McLaughlin & Hinke (1966) reported a decrease of aia in barnacle muscle fibres, from 10-14 to 6-7 mm, when treated for 30 min in Na-free, sucrose Ringer. However, since they did not continuously measure a" a in their experiments, there are no details about the time course of the change. It is difficult to describe mathematically the relationship in crab muscle without a value for the final level of a"a eventually reached in low Na,. The graph in Fig. 3C yields a value for a"a at which the rate of decrease of a"a is zero. This value probably falls somewhere between 2-0 and 4 2 mm. A graph can then be plotted of the rate of decrease of a"a against a~a above 'threshold' on logarithmic axes as shown in Fig. 3D. The two extremes for the final level of a"a have been used: 4-2 mm (open circles) and 2-0 mm (filled circles). Neither value produced a simple straight-line relationship on logarithmic axes, and indeed, the curves shown in Fig. 3D suggest that there may be two components of the effect, a fast and a slow. The gradient of the 'fast component' fell between 2-25 (open circles) and 4-25 (filled circles), showing that during the initial, rapid changes of aja, the rate of change may be related to some power, possibly the cube, of aOs. It must be stressed here, however, that since the cause of the fast change of a's has not been fully established, an analysis such as this must be treated as merely descriptive. But it is interesting to note that in most of the experiments where [Na]O was reduced, there was a fast initial fall of ala followed by a slower decline (e.g. see Figs. 2, 5, 9 and 11).

The immediate question to arise from these experiments is: what does this change of aNa represent? One obvious suggestion is that it is an artifact caused either by an osmotic swelling of the fibre in low-Na solutions or caused by erroneous measurements from the Na+-sensitive electrode when it is not truly intracellular. An osmotic effect seems unlikely since the osmolarity of all solutions was frequently tested to ensure that it remained reasonably constant (see Methods). In a few accidental cases, where the osmolarity of a low-Na solution was significantly different from that of normal crab Ringer, then a change in the fibre diameter could easily be measured (using a graticule in the binocular microscope used to view the preparation) and the osmotic effect was accompanied by a general instability from both micro-electrodes

247 Na ACTIVITY OF CRAB MUSCLE which were usually dislodged. This did not occur with a low-Na solution that had the same osmolarity as normal Ringer and no shrinkage or swelling of fibres was visible. 10 A

z*co

61 6 I

0f' Na (Li) 3.5 -

-

C

__

0

0

10

I

20 -30 Time (min)

40

c 10 E

2-5 -

0

E E 1-0

Z- 2-0-

to

.M

0

0

01-5--

U

(U

0IV 001

0

0 0

0

co

opt I

C

I

6-

4-

I

Xi 3-0E

B

|

4I

.E

)o

E

101 8-

1-E

,

8-

20 min 0_1

"

0 u

0

-

ad0

00 4

5

6

7

8

9 10

0-1

1-0

10

aNa(m M ) aIa-threshold (mm ) Fig. 3. A, pen recording of an experiment showing the effect on aia of a prolonged superfusion of -91- [Na]0 solution (Li-substituted). B, graph of measurements of ail" taken from experiment shown in Fig. 3A plotted on a linear scale against time of exposure to j-- [Na]0. C, plot of the rate of decrease of a" against aNa for the period in -- [Na]0. D, log-log plot of rate of decrease of a" during exposure to -j- [Na]O against levels of a," above 'threshold' ('threshold' is defined as the final stable level of aNa achieved in this solution). Two values for 'threshold' have been used: 4-2 mm (open circles) and 2-0 mm (filled circles). Data taken from experiment shown in A.

Whilst there must always remain the possibility that the Na+-sensitive electrode was not truly intracellular throughout all the experiments performed, it does seem unlikely, especially since it could be clearly shown that the voltage electrode and the Na+ electrode were recording the same membrane potential changes suggesting that both electrodes

R. D. VA UGHAN-JONES 248 could efficiently seal into the same single fibre. Also, the ability to record stable levels of aija for many hours suggests that there was no local leakage of Na+ into the sarcoplasm at the point of insertion of the Na+ electrode. It would be difficult to conceive how a 'hole' or patch of membrane through which Na+ was leaking would neither enlarge nor, as is more likely, seal up after a few hours in a crab Ringer that contains such a high concentration (12 mM) of Ca, since it is known that Ca can very effectively help to re-seal membranes (de Mello, 1973). If a substantial local Na+ leak was occurring, it would be expected to greatly raise aria at regions just below the membrane, whereas regions deeper in the sarcoplasm should remain at a lower activity more representative of the 'real' value of a>a. This would imply that pushing a Na+-electrode deeper into the fibre would be expected to result in a lower reading of aNa. This was not found to be the case. Often the Na+ electrode could be gently advanced by 20 or 30,am, and providing the electrodes were not dislodged, the reading of a> remained unchanged. So it would seem that the change of a>a in low Nao was a real effect occurring in the sarcoplasm of the muscle fibre. This could be caused either by an active extrusion of Na+ across the cell membrane, or by an internal sequestration of a;ja. If the effect is because of an active outward pumping of Na+, it would be useful to know what the changes of aNa represent in terms of a Na+ efflux rate per unit area of membrane. This may be difficult to estimate accurately because the initial rate of change of ala may be approaching the maximum response time of the Na+ electrode. Bearing this in mind, however, the figures can be calculated. If one assumes that the activity coefficient for ionized Na is the same on both sides of the membrane, then measurements of aNa can be converted to [Na+], (the actual concentration of intracellular ionized Na) using the equation ala = a[Na+]i, where a is the activity coefficient for Na+ in the extracellular solution (obtained from Parsons, 1959). Changes in [Na+]i can then be related to an efflux of Na+ if one knows the surface area and diameter of the fibre. Since, in the case of barnacle giant fibres, the extensive cleft system can increase the surface area by up to 20 times (Selverston, 1967), it seems reasonable to assume that the surface area of Carcinus maenas fibres may be increased at least tenfold (large clefts certainly are visible). Taking this into account and making the approximation that the fibre is circular in cross-section (diameter of the fibre for experiment shown in Fig. 3A was measured at two different points using a graticule in the microscope and was 450 jum), then the fall of a la in Fig. 3A could represent an initial efflux of Na+ into low Na0 of about 70 p-mole/cm2 . sec declining within 30 sec to 45 p-mole/cm2see, within 1 min to 5 p-mole/cm2 see and finally after 17 min, to about 4 p-mole/cm2 . sec. \ There is very little available data concerning Na effluxes from crustacean muscle fibres into low-Na solution, which might offer a direct comparison with the present experiments. Brinley (1968) found that the resting efflux of radioactively labelled Na front barnacle fibres was reduced rather than raised in 0 Na solution. However,

Na ACTIVITY OF CRAB MUSCLE he noted that if

[Na]i had

249

been raised by an injection of Na, then the resting

24Na efflux increased when [Na]o was removed (using Li as a Na substitute), from 150 to nearly 1000 p-mole/cm2. sec within about 5 min and then declined over the next hour back to resting levels. If, once again, a correction is applied to Brinley's data to allow for the cleft system, the initial efflux into 0 Na solution is similar to that calculated from the present experiments.

Effects of zero external K and of ouabain Na could be actively extruded from the fibre in low Nao either by means of the ATP dependent Na/K pump or by means of an ion-exchange mechanism which is not directly ATP-dependent but which relies for its energy upon the potential energy stored in the existing gradients of >60 70_ E

80

90 Lgo 10 min E

_

|

^ RO

Na

__-_

_

_

_

~~ ANa

_

_

_

~ ¢Na

~~OK

'Na

Fig. 4. Pen recording of an experiment showing the effects on the membrane potential (upper trace) and a ' (lower trace) of the removal of external K. The response of a0', to -1- [Na]o was tested before, during and after the removal of external K. Li was used as a cation substitute.

certain ions across the cell membrane, for example, Ca. If the change of aNa is the result of active pumping Na/K, then it should be substantially reduced by either removing all the external K or by treatment with a cardiac glycoside, like ouabain. Three experiments were performed in which 0 K solution was superfused. Fig. 4 illustrates one such experiment. The response, during this period, of ajNa to low Nao was virtually unchanged from that seen both during and after treatment with 0 KO. However, it must also be noted that ai a did not rise significantly in any of the fibres and so possibly the interfibre spaces were still not completely K-free even though, in the experiment shown, the fibre was superfused with 0 KO for over 50 min, causing a hyperpolarization of the membrane potential of over 20 mV. Nevertheless, experiments on the Na pump in Maia and giant barnacle fibres, using radiotracers and Na-loaded muscles have indicated that 0 Ko can halve Na-pumping into normal Ringer within 10-20 min

250 R. D. VA UGHAN-JONES (Bittar, Caldwell & Lowe, 1967; Brinley, 1968; Bittar, Chen, Danielson, Hartmann & Tong, 1972). Fig. 5 shows an experiment where the superfusate was changed for 100 min to one containing 2 x 10-4 M ouabain. After 20 min aNa began to rise slowly (by about 1 0 mm in 30 min) but after 50 min of superfusion with ouabain, the response of a;a to low Na0 was only slightly reduced when compared with the control response.

f.Y@ 60E-

,

o6

E

--

E

,

t

20 min

80 6-

I4L Ouabai n (2 x10-4M) 510 Na

51s Na Fig. 5. Pen recording of an experiment showing the effects on membrane potential (upper trace) and a;a (lower trace) of prolonged treatment with ouabain (2 x 1O-4 M) in the superfusate. The response to low Na0 was tested before and during ouabain application. The low Na solutions used in this experiment were -51 normal (Li-substituted), i.e. [Na]0 was 10 mM.

In two other experiments, treatment with 2 x 104 M ouabain for about an hour caused aNa to rise by about 2 and 1 m-mole/hr, and in the fourth experiment 10-5M ouabain caused no rise whatsoever of apla over a 40 min period. In all experiments there was no substantial reduction in the response to low Nao. Since concentrations of ouabain higher than 0-2 mm were not used in these experiments, the possibility remains that maximal inhibition of Na pumping may not have occurred. However, it seems reasonable to assume that it was at least largely reduced. Ouabain in similar concentrations has been shown to inhibit substantially Na pumping in other crustacean muscle fibres (Bittar et al. 1967; Brinley, 1968) and Brinley reported that the optimal concentration of strophanthidin for inhibition of Na pumping in barnacle muscle was 10- M. So, if the change of aNa in low Nao represents an efflux of Na+ out of the fibre, then it seems more probable that it is achieved by a mechanism which is stimulated when external [Na] is reduced, rather than by the ATP-dependent Na/K pump. If, in Fig. 5, ouabain has indeed maximally blocked Na-pumping, then the slow rate of rise of a_,a will be caused by a passive Na+ influx, which

251 Na ACTIVITY OF CRAB MUSCLE under normal conditions would be balanced by active Na pumping at the same rate (assuming that there is minimal binding of Na+ ions as they enter the cell). This is equivalent to a net passive influx of 1-10 p-mole/ cm2.sec (fibre diameter = 450 #sm) depending on whether or not one corrects, as before, for the increase in fibre surface area owing to the cleft system. The real answer may well lie somewhere between these two values (also, if there was still some residual Na pumping with 0-2 mM ouabain, this value would be slightly larger). This suggests that the ouabain-sensitive Na pump in crab muscle extrudes Na+ at normal physiological levels of aNa at a rate similar to that seen in frog skeletal muscle (e.g. Horowicz & Gerber, 1965; 3-6 p-mole/cm2. sec). Values usually quoted for crustacean muscle (barnacle and spider crab) are up to twenty times higher (Bittar et al. 1967; Brinley, 1968) until one once again takes into account the effects of the extensive cleft system and it is likely that this can eliminate most of the discrepancy. It is of interest to compare the value of 1-10 p-mole/cm2. sec obtained for the passive Na influx in the presence of ouabain with the calculated Na influx that would have to occur to account for the rise of aa following the return from a low-Na to a normal-Na solution. From the experiment shown in Fig. 3A, it can be calculated that the average net Na influx during the 18 min period of recovery following low Nao treatment would be 84-84 p-mole/cm2. sec (allowing or not for the 'cleft system'). This is only an average value: the instantaneous rate at periods just after 15 min or so of recovery would be lower. But it is clear that the average influx rate under these recovery conditions would have to be at least eight times higher than the passive Na influx observed at rest with ouabain.

The present experiments show that if the ouabain-sensitive Na pump in crab muscle is the main mechanism for the net active extrusion of Na then it operates at much too slow a rate to account for such a fast decrease of aNa seen in low Nao, unless, of course, the Na pump is rapidly stimulated when [Na]o is reduced. Although an increase of Napumping in low Na0 certainly does occur in some tissues e.g. crab nerve (Baker & Connelly, 1966) and squid giant axon (Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt, 1969) a similar effect in the present experiments would have to be very much more pronounced to account for such a fast change of aNa and so seems to be an unlikely explanation especially since the effect persists virtually unchanged in rate and magnitude in the presence of 0-2 mM ouabain. In addition, Thomas (1972) has shown that the Na pump rate in a snail neurone is little affected by low Nao. The present experiments also show that the passive Na+ influx from normal Ringer is low, so that the Na+ influx from low Na solutions must have a negligible effect on the change of ai a seen in low Nao.

R. D. VAUGHAN-JONES

252

I~ ~

The relation between [Na]0 and a1 Fig. 6 shows an experiment where [Na]0 was reduced successively from 500 mm to 0, then 50, 100, 220 and back to 500 mm (substituting Na with Li). Removal of all Na0 produced as expected a large and rapid fall of a-a. Progressive restoration of Na0 produced a progressive rise of a:a. Fig. 6 shows that the effect was repeatable. The fibre was left for 10 min

500£

10 - If 2~~~~~~~~~~~~~~~~~~2 8_

60 2

wZ .0

|]3

E

>

J70 1 0

Fig. 6. Pen recording of an experiment showing the effects on membrane potential (lower trace) and aa (upper trace) of 0, 50, 100, 200 and 500 mm[Na]0 solutions. Solutions were Li-substituted. There is an interruption in the membrane potential record because the voltage electrode at this moment was pushed more firmly into the fibre. Therefore there is a simultaneous artefact appearing on the derived record of a"a.

about 4 min in each solution so that only the initial large changes of a:,a were seen. Fig. 7A shows a graph of aNa plotted against a Na for the second part of the experiment shown in Fig. 6. This was a stable experiment, where aNa recovered to its original resting level upon adding back all Na0. There is a near-linear relation between aNa and aNa, above a threshold value of about 2-25 mm a.a. In three other experiments, a similar picture emerged. This is to be compared with Fig. 7B, where the size of the fast changes of apa in 1- [Na]o (substituted with Li) are plotted against initial resting ala, for thirty-four individual experiments. It is clear that the higher the resting ala in a fibre, then the larger is the initial rapid change of aa in --L [Na]0. The results shown in Fig. 7A and

Na ACTIVITY OF CRAB MUSCLE 253 B indicate that the fast change of a a is largest when the concentration (or rather activity) gradient for Na across the muscle fibre membrane is reduced to the lowest level. This means that aNa in crab muscle is very sensitive to the transmembrane gradient for Na and that changes in this gradient are reflected in changes of aya. This would be the case, if the passive Na influx was normally very large and balanced by a very active Na pump, but the experiments so far seem to have removed this possibility. A

B 6

6

00V

S

5

0

Z-tv 1:

4 ~~

~

~

~

~

3

0

~

3 To~ ~~~~~~~~l Z.-

~ -

~

0

50 100 150 200 250 300 350 aNa (mM)

/0

0

06

0~~~~~~~~~~~~0 /O

°0

1

0/~K

U

0

2

4 6 8 agNa (mM)

10

12

Fig. 7. A, Values of as I taken from the second part of the experiment shown in Fig. 6 plotted against the activity of Na in the bathing solution, aOa (values of [Na]0 used in the experiment shown in Fig. 6 have been converted into activities). Values of a"' were measured after about 3 min in each new solution. The curve has been fitted by eye. Li was used as a Na substitute. B, Graph of the change of aea after 15 min exposure to -l- normal Na solution plotted against initial resting ala for thirty-four different muscle fibres (Li was used as a Na-substitute). The dashed line is the straight line of best fit (correlation coefficient = 0.9) intercepting the x-axis at 0ia= 2.42 mM.

A second possibility is that removal of some or all Na0, causing a decrease in the electrochemical Na gradient, allows Na to actively move out of the fibre in exchange for some other cation, moving passively down its electrochemical gradient. Na/Na exchange can be eliminated since the Na+ electrode only measures net changes of a Na and the effect occurs equally well in Li and Tris substituted solutions. An obvious candidate is a Na/Ca exchange pump which normally works by actively extruding Ca in exchange for a passive inward movement of Na. When [Na]O is lowered, the system operates 'backwards' and Na is extruded on an ion exchange carrier using the energy of the Ca gradient. Such a system has already been demonstrated in many excitable tissues including

254 R. D. VAUGHAN-JONES crustacean muscle (barnacle and spider crab; see Baker's review, 1972). To test the possibility of Ca or Mg involvement in the present experiments, substances were used which are known to block the movement of divalent cations across membranes, e.g. Mn, Co, La, D600 and Verapamil as well as testing the effect of removing Ca. and MgO.

Effects of external Mn and Co The effects of externally applied Mn were observed in five different experiments and Co was used in one experiment. At a concentration of 50 mm, both Mn and Co were found to substantially reduce the changes of at in low Na0. Fig. 8 shows one such experiment using 50 mM-MnCl2. Initially lowering [Na]o to one tenth (Li substituted) caused aNa to fall by about 2-5 mm in 7 min. After superfusion with 50 mM-MnCl2 in normal Ringer the change of ata in low Na0 was reduced to about 03 mm over the same test period. A second test after a further 5 min produced a change of a;a of about 0-5 mm. When the Mn was washed off and the fibre allowed to recover for 10 min, the response to low Na0 showed a recovery, a'a changing by just over 2 mm in 7 min. The effect of 50 mM-CoCl2 was very similar (not shown) in that the response of aNa to low Nao was very much reduced. Much lower concentrations of Mn produced variable results. In two experiments, 10 and 5 mm respectively blocked and substantially slowed down the rate of decrease of aba; in one experiment, 1 mm-Mn had no effect and in two more experiments, 10 mm-Mn appeared to stimulate a decrease of a.a without actually blocking the response to low Nao, although it was reduced in rate. Baker (1972) has reported that high concentrations of Mn (50 mm) inhibit the resting Ca influx in crab nerve and Chapman & Ochi (1972) have reported that Mn blocks low-Na contractures in frog heart muscle, the implication being that Na/Ca exchange is blocked under these conditions.

Effects of externally applied La Fourteen experiments were performed using La in concentrations ranging from 0*1 to 5 0 mm. Fig. 9 illustrates one such experiment. Superfusion of 3 mM-LaCl3 in Ringer immediately caused ala to fall rapidly. There was a slight depolarization of the membrane potential and any subsequent response to low Na0 was virtually abolished. The fibre was then washed for about 95 min in normal Ringer and a'a rose slowly back towards the initial resting level. The response to low Nao could then again be demonstrated and the whole experiment repeated.

255 Na ACTIVITY OF CRAB MUSCLE In all fourteen experiments La stimulated a fall of aPa and abolished the response of aya to low Nao. Not all the fibres were left long enough to see if the effect was reversible, but a recovery was seen in three of the fibres tested. La was effective even in concentrations of 0 I mm as shown in Fig. 10 and subsequently increasing the concentration of La did not produce any additional response. 0

c,, .M -

60

So~ ~5 mM-MnCI2

E O ~~~~~ 70

zEE .4.0 0 0

~ ~ ~

~

~

~

~

~

~~6

w

so mm-MnC10

57

toE~6

O Na

~

~

~

~

j0Na

t

14

10 mE

Fig. 8. Pen-recording of an experiment showing the effects on membrane potential (upper trace) and as a (lower trace) of -l'l, [Na]0, before, during and after the superfusion of a Ringer solution containing 50 mM-MnCl2 (Li was used as a Na-substitute). The bottom half of the Figure is continuous with the top half.

Effects of D600 and Verapamit Neither drug had any significant effect on resting a~a nor had any inhibitory effect on the response of ai a to low Na0. Three experiments were conducted using Verapamil at the concentration of 104 g/ml. (20 mmn), the dose reported by Baker (1972) to inhibit Ca influxes in crab nerve and two experiments were conducted using D600 at concentrations of 106 and 150M (for up to 40 mm). It was found however after long exposure to either drug that large contractures occurred whenever Na0 was reduced for more than 2-3 min. Ashley & Ellory (1972) and Ashley, Ellory & Hainut (1974) have reported that D600 has no effect on the 9

PHY

264

256 R. D. VA UGHAN-JONES membrane fluxes of radioactively labelled Ca and Mg in barnacle muscle and so it is perhaps not surprising that it has no apparent effect on aya in the present experiments. 20 min 'I

' " E

o

@0

50

- 70I

0.

a.

3 mM-LaCI3.

3

X

12 X

j1ONa

WONa

mm-LaCI3.

,o Na

WHONa

WHONa

Fig. 9. Pen-recording showing the effects on membrane potential (upper trace) and a~a (lower trace) of externally applied LaCl3 (3 mM). The response to -j1- [Na]0 was tested before, during and after application of LaCl3. The fibre was left for 1 hr and the experiment was then repeated. Li wvas used as a Na substitute. 20 min .0

fo

r- e"C)

-

a

70

-

1.0

E. E

,0 ~~~~~~~~~~~~~~ 0I

7 I-, 1: 5

~

~ ~ ~

ro'Na

~

~

~

~

~

~

~~~~~~.....

'Na

,'Na

Fig. 10. Pen-recording of an experiment showing the effects on membrane potential (upper trace) and ara (lower trace) of externally applied LaCl3 in concentrations of 0.1, 0*5 and l 0 mm. The response to -- [Na]0 (Lisubstituted) was tested before, during and after La application.

Effects of removing external Ca and Mg In a total of thirteen experiments, removal of Cao failed to either abolish or substantially reduce the change of ara seen in low Nao. Fig. 11 shows one experiment where a fibre was exposed to a 0 Ca solution (+ 1 mM-EGTA) for a total of 136 min, so that Ca trapped in the extracellular space had time to wash out and [Ca]o would be very low indeed.

257 Na ACTIVITY OF CRAB MUSCLE After the first hour in 0 Cao the fall of ara when [Na]o was reduced was still about the same as the control response, falling by about 3 mM in 8 min. In nine of the fibres, removal of Cao caused aNa to rise slowly by about 1 m-mole/hr. If external Ca was replaced, aNa would slowly return to the original resting level. In the experiment shown in Fig. 11, Mgo was also removed for 45 min and, in the absence of external Mg and Ca (+ 1 mM-EDTA) aI a rose very rapidly, in this case from 8-4 to 12-6 mm in 10 min, an average rate of increase of 0-42 m-mole/min. Once again, 30 min 0I_

-'r'

D MU E60 I E 410 _80

14 12

-

0 Ca

,'Na ,'Na Fig. 11. Pen-recording of an experiment showing the effects on membranepotential (upper trace) and a, a (lower trace) of removing external Ca and Mg, either individually or simultaneously. The response to --I- [Na]0 was tested, before, during and after divalent cation removal. Li was used as a cation substitute.

1,0Na

,O Na

however, the change of aNa in low Na0 was little affected. The rate and magnitude of the response in fact appeared larger. This is presumably related to the raised level of aNa induced in a zero Mg and Ca solution. Restoring Cao immediately stimulated a recovery of aNa which then appeared to be little affected by the re-addition of normal Mg0. This last effect was similar to one observed in a different experiment (not shown) where removal of either Cao or Cao plus Mgo caused aNa to rise, whereas removal of Mg0 alone had no effect and aNa remained stable. Six fibres were tested with 0 Cao plus 0 Mg0 (three using Li and three using Tris as a cation substitute) and in no case was there any significant inhibition of the change of aNa seen in low Na0. It is interesting to note in Fig. 11 that the rises of aNa seen in 0 Cao and Mg0 are not accompanied by any change of membrane potential. Li was used in this experiment as a cation substitute. When Tris was used instead, large depolarizations of 10-20 mV were seen which recovered, like aNa, on restoring the external Ca and Mg. 9-2

258 R. D. VA UGHAN-JONES Fig. 12 is a pen-recording of an experiment showing that low concentrations of LaCl.3 (3 mM) as well as blocking the response of ara to low Nao also completely blocked the effects of removing both Cao and MgO. Two such experiments were performed with identical results. >

TE50*

20 min

I

E.

70 0.

12 10 8

8E

06E

E Z-

12 10

3mm-LaC13

6

6

. 4

. ,Na

.

~I . 0Cag

I

L.*,.L

, Na 0CaOCa

.

co

4

Fig. 12. Pen-recording of an experiment showing the effects of externally applied La on both the response of aea to low Nao and to the removal of external divalent cations. Membrane potential (upper trace); a"a (lower trace).

In a few experiments Cao was raised but this usually caused a contracture of the fibre which dislodged the electrodes. High Cao also raised the osmolarity of the Ringer, so these experiments were not pursued. In some other experiments, however, Mg0 was raised to 225 mm and compensated for by lowering [Na]o to -L normal. In one experiment, the response of a.Na to a high-Mg, low-Na solution was very much reduced, but this effect was never found to be repeatable, even in the same fibre. More usually, the response was the same in the presence and absence of high Mgo.

Effect of changes in the external pH Since removal of Cao and Mgo had no inhibitory effect on the rapid fall of a'a in low Nao it was decided to test the possibility of other ions, like H+ or Cl-, being involved in a linked transport of Na+ across the cell membrane. In three experiments, short changes (7 or 8 min) in the pH of the superfusate were made, over the range of two pH units i.e. a hundredfold change of H+0. Fig. 13 shows one such experiment. The response of a;a to low Na0 was identical at three different pH levels in all three experiments.

Na ACTIVITY OF CRAB MUSCLE

259

Effect on a?' of low Na0 In seven experiments, both the intracellular Cl- activity, a?' and ajla were monitored simultaneously. For this experiment, three microelectrodes were inserted into the same fibre: the Na+-sensitive electrode, a recessed-tip Cl--sensitive electrode (Neild & Thomas, 1973) and a conventional membrane-potential electrode, which was used as the 10 min

E

( 5f

'Iu

3 co

C~vE

60

, Na,pH83 , Na, pH73 ,', Na, pH 6-3 Fig. 13. Pen-recording of an experiment showing the effects of three #Y- [Na]0 solutions of pH 6 3, 8-3 and 7 3 respectively. Membrane potential (lower trace); all (upper trace). Li was used as a Na-substitute.

reference electrode for both ion-sensitive electrodes. The average measured resting a?' of 30-8 mm (± 2-5 S.E. for seven fibres) was very close- to the value for a?' of 28-9 mm predicted from the average membrane potential (66.8 + 07 mV; S.E., seven fibres) on the basis of a passive distribution for Cl- across the cell membrane. Fig. 14 shows a pen-recording of one of the experiments. When [Na]0 was reduced (Li substituted), then aNa (middle trace), as usual, rapidly fell, but the reading of a?' (lower trace) remained unchanged. The effect of low [Na]o was tested in a total of five fibres, and in no case was there any apparent change of ac?, suggesting that a possible efflux of Na+ from the fibre into low Nao was not accompanied by a simultaneous active efflux of Cl-. The high-K solution used in the experiment shown in Fig. 14 caused 9-3

260 R. D. VA UGHAN-JONES a mild contracture of the fibre which disturbed the Na+-sensitive electrode at this point. The membrane potential and Cl--sensitive electrodes, however, were unaffected. As expected, a?' rose rapidly in this solution,

E (U

C

0

50+

0.

1 4 min J

C D

EU) 60

70-

K 7 . 13

;11

.9 .7

z

Z(

.5

40 .I--

E 30 . v.-

_p INa

U

10 K

20 1 Fig. 14. Pen-recording of an experiment showing the effects on ala, ac' and membrane potential (middle, lower and upper traces respectively) of -- [Na]o and a high K solution (120mM-K). The Na+-sensitive electrode became unstable after the exposure of the fibre to a 1O K solution and the recording of a~a after this point is therefore unreliable. Li was used as a Na-substitute.

most likely corresponding to a net passive influx of Cl- because of the large depolarization of the membrane potential. The slow repolarization of the membrane potential after high Ko is a common finding in these fibres (Fatt & Katz, 1953) and was accompanied by a slow fall of aFJ back to normal resting levels.

Na ACTI ViT Y OF CRAB MUSCLE

261

DISCUSSION

The level of a~la in crustacean muscle The present results confirm that there is a very low level of ionized Na in the sarcoplasm of crab muscle. If it is assumed that the activity coefficient of Na ions inside the fibre is the same as in the external superfusate, then the present mean value of a a of 8-4 mm would correspond to an intracellular concentration of ionized Na, [Na+]i of 12-0 mM. This is in dramatic contrast with measurements of the total fibre Na, [Na]i in crustacean muscle, using chemical analysis, where values of well over 50 mm are commonly quoted (Shaw, 1958; Hinke, 1959; McLaughlin & Hinke, 1966). It is clear, though, that much of the Na measured by chemical analysis is not sarcoplasmic in origin, but is either Na trapped in the surface invaginations of the cleft-system or Na compartmentalized within the sarcoplasmic reticulum. One possibility is that the intracellular activity coefficient for Na+ may be lower than it is extracellularly, which would mean that an aNa of 8-4 mm would correspond to a value for [Na+]1, much higher than 12*0 mm. However, measurements of the diffusion rates of Na+ in nerve (Hodgkin & Keynes, 1953) and of various electrolytes in skinned frog muscle fibres (Kushmerick & Podolsky, 1969) and crab muscle fibres (Bittar, 1973) show that most inorganic ions, except Ca, are fairly mobile in the cell cytoplasm, which suggests that the intracellular activity coefficient for Na+ may not be so very different on either side of the muscle fibre membrane. The effect on aNa of low Na0 The most striking findings of the present experiments are that quite rapid changes of [Na]0 are accompanied by fast changes of a-a. These changes are accompanied by a small depolarization of the membrane potential, are ouabain-resistant, and are blocked by the elements Mn, Co and La. They appear to be unaffected by removal of K or either Ca and/or Mg from the superfusate or by changes in the extracellular pH. The possibility that a reduction of the passive Na-influx in low Nao causes these changes seems to be excluded on the grounds that it is much too small to account for such large and rapid changes of aNa. This is because concentrations of ouabain which are known to inhibit substantially Na pumping in crustacean muscle caused only a slow rise of resting a-a over quite long periods of time. The fast changes of aNa could only be explained in terms of a reduction of the passive Na+ influx if the ATP-dependent Na pump in crab muscle was both extremely active and extremely ouabain-resistant.

R. D. VAUGHAN-JONES 262 The possibility that the changes of apa are an artifact because the Na+ electrode was not truly intracellular has already been fully considered (see Results) and it seems unlikely that this was consistently occurring in all the experiments especially when stable levels of aOa could be recorded for periods of up to many hours. If it was, then the real level of a3a in crab muscle would be the value at which no rapid change of aNa occurred in low Na0. From Fig. 7B this would be about 2-4 mM, an exceptionally low value which would represent a 150-fold change of aNa across the muscle fibre membrane. At present, explanations of the experimental results must be speculative-but the most reasonable interpretation is that the changes of a Na are real and are caused either by an active extrusion of Na+ on an ion-exchange carrier or by an intracellular sequestration of [Na+]i. Models have been constructed in favour of a 'bound fraction' of cellular Nai (Dick & Fry, 1973; McLaughlin & Hinke, 1966) but these only exchange slowly with the sarcoplasm and the extracellular environment and anyway, would be expected to be depleted rather than enriched in low-Na solution. However, the possibility exists that aNa which can itself exchange directly with the exterior might also be able to rapidly exchange indirectly via a second compartment like the sarcoplasmic reticulum which is extrasarcoplasmic in origin. Such multi-compartment hypotheses are not new (e.g. Keynes & Steinhardt, 1968; Brading, 1975) and would be supported by the observation that the changes of aya in low Nao seem to consist of two components. The mechanism of such a phenomenon here, however, is difficult to imagine, especially since it -appears to be relatively insensitive to changes in the concentrations of all external cations in the Ringer. Some of the evidence favours the view that the fall of aya in low Nao was because of a Na efflux in exchange for either Ca or Mg, a system similar to that found in other crustacean muscles (Baker, 1972; Ashley & Ellory, 1972; Ashley, Ellory & Hainut, 1974). Firstly, changes of aj a were very sensitive to the size of the Na gradient across the membrane; secondly, the effect was inhibited by the elements Co, Mn and La in concentrations which have been found to inhibit Na/Ca exchange in squid giant axon (see Baker, 1972) and thirdly, there was always very little change in membrane potential during such large changes of ai a suggesting that the Na/Ca (or Mg) exchange might be electroneutral. Since the resting membrane resistance of crab fibres is very low, however, any outward current across the membrane because of an uncoupled Na efflux would not be expected to hyperpolarize the membrane potential by more than a few mV so that a lack of effect on membrane potential need not necessarily indicate an electroneutral cation exchange (it is

263 Na ACTIVITY OF CRAB MUSCLE interesting to note, though, that the membrane potential consistently changed by a few millivolts in a depolarizing rather than a hyperpolarizing direction). The present experiments have also shown that the Na gradient in crab muscle is very high (more than a fortyfold change of a'"a on average, and in some cases, as in Fig. 10, as much as seventyfold) which means that the energy requirements for the normal operation of an electroneutral Na/Ca pump are nearly fulfilled (see Baker, 1972; 100 times the Na gradient would be adequate). Lastly, the time course of the fall of aNa in low Nao observed in the present experiments is very similar to the rise of [Ca2+], seen under the same conditions in spider-crab fibres (Baker, 1972) and in squid axon (Baker et al. 1971), using cells injected with aequorin. In view of these findings the lack of effect of 0 Cao and/or 0 Mg0 on the response of aNa to low Na0 was perhaps surprising unless one postulates that true Ca-free, Mg-free conditions had not been achieved. A whole muscle preparation was used during the present experiments and not single isolated fibres, so perhaps all the interfibre spaces were not completely Ca-free. Also, the presence of crab exoskeleton in the experimental chamber (see Methods) may well have provided a reservoir of extracellular Ca. However, since 1 mrik-EDTA was usually added to 0 Ca, 0 Mg solutions and 1 mM-EGTA to 0 Ca solutions (see MIethods), it is clear, that if Ca remained in the superfusion chamber it would have been at a very low concentration, and any Na/Ca exchange carrier in the membrane which worked under these conditions must have an extremely high affinity for [Ca2+]0. In the squid giant axon, where Na/Ca exchange has been more extensively investigated, this has not proved to be the case (Baker, Blaustein, Hodgkin & Steinhardt, 1969), since about 3 mM-[Ca]o are required to produce half-maximal activation of the Ca-sensitive Na efflux into 0 Nao (Li-substituted) Ringer solution. Therefore the possibility must still remain that the changes of aft in low Na0 are not caused by a Na efflux but represent Na+, moving into a region of the cell which is inaccessible to the Na+-sensitive electrode. The effects of La The fact that rises of ata seen in 0 Ca0 and Mgo are accompanied by a depolarization in Tris and not Li solutions suggests that they are caused by an increase in the resting membrane permeability producing an increased inward Na leak. Since La inhibits the rises of a:a seen in the absence of external divalent cations then it must inhibit the passive Na influx. This has been found by XViddicombe (1974) to occur in smooth

R. D. VA UGHAN-JONES muscle cells and Casteels, Van Breman & Wuytack (1972) reported that La, when externally applied to smooth muscle cells could prevent the decrease in membrane resistance caused by prolonged metabolic depletion. However, in the present experiments La also stimulated a rapid decrease of ara similar to, but not always the same as the response to low Na0 (see Fig. 10). It seems unlikely that La has acted in these experiments by just blocking a resting Na influx since the effect is so large and so rapid. It may be that La can affect aNa in other ways, possibly through the action of Ca since it is known that La can displace quite large quantities of membrane bound Ca (Widdicombe, 1974). The similarity between the response of apa to La and to low Nao certainly seems to suggest a common mechanism. 264

I would like to thank Dr R. C. Thomas, in whose laboratory these experiments were performed, for much advice and help throughout the course of this work, and Mrs Vicky Martin for technical assistance. Thank you also to R.C.T. and Dr T. B. Bolton for comments on the manuscript. This work was supported by a grant from the Science Research Council. REFERENCES

ALLEN, R. D. & HINKE, J. A. M. (1971). Sodium-lithium exchange in single muscle fibres of the giant barnacle. C(an. J. Physiol. Pharmacol. 49, 862-866. ASHLEY, C. C. & ELLORY, J. C. (1972). The efflux of Mg from single crustacean muscle fibres. J. Physiol. 226, 653-674. ASHLEY, C. C., ELLORY, J. C. & HAINUT, K. (1974). Calcium movements in single crustacean muscle fibres. J. Physiol. 242, 255-272. BARER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. molec. Biol. 24, 177-223. BAKER, P. F., BLAUSTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Physiol. 200, 431-458. BAKER, P. F., BLAUSTEIN, M. P., KEYNES, R. D., MANIL, J., SHAW, T. 1. & STEINHARDT, R. A. (1969) J. Physiol. 200, 459-496. BAKER, P. F. & CONNELLY, C. M. (1966). Some properties of the external activation site of the sodium pump in crab nerve. J. Physiol. 185, 270-297. BARER, P. F., HODGKIN, A. L. & RIDGWAY, E. B. (1971). Depolarization and calcium entry in squid giant axons. J. Phygiol. 218, 709-755. BITTAR, E. E. (1973). The diffusion coefficient of sodium in barnacle muscle fibres. Experientia 29, 553. BiTTAR, E. E., CALDWELL, P. C. & LOWE, A. G. (1967). The efflux of sodium from single crab muscle fibres. J. mar. biol. Ass. U.K. 47, 709-721. BITTAR, E. E., CHEN, S., DANIELSON, B. G., HARTMANN, H. A. & TONG, E. Y. (1972). An investigation of sodium transport in barnacle muscle fibres by means of the microsyringe technique. J. Physiol. 221, 389-414. BRADING, A. F. (1975). Sodium/sodium exchange in the smooth muscle of the guinea-pig taenia coli. J. Physiol. 251, 79-105. BRINLEY, F. J. (1968). Sodium and potassium fluxes in isolated barnacle muscle fibres. J. gen. Physiol. 51, 445-477. CASTEELS, E., VAN BREMAN, C. & WUYTACK, F. (1972). Effect of metabolic depletion on the membrane permeability of smooth muscle cells and its modification by La3+. Nature, New Biol. 239, 249-251.

Na ACTIVITY OF CRAB MUSCLE

265

CHAPMAN, R. A. & OCHI, R. (1972). The effects of manganese ions on the contractile responses of isolated frog atrial trabeculae. J. Physiol. 222, 56-58P. COLE, W. H. (1941). A perfusing solution for the lobster (Homaru8) heart and the effects of its constituent ions on the heart. J. gen. Physiol. 25, 1-6. DE MELLO, W. C. (1973). Membrane sealing in frog skeletal muscle fibres. Proc. natn. Acad. Sci. U.S.A. 70, 982-984. DICK, D. A. T. & FRY, D. J. (1973). Location of inexchangeable sodium in the nucleus and cytoplasm of oocytes of Bufo bufo exposed to sodium-free solutions. J. Physiol. 231, 19-29. FATT, P. & KATZ, B. (1953). The electrical properties of crustacean muscle fibres. J. Physiol. 120, 171-204. HARRIS, E. J. (1965). The dependence of efflux of sodium from frog muscle on internal sodium and external potassium. J. Physiol. 177, 355-376. HINKE, J. A. M. (1959). Glass micro-electrodes for measuring intracellular activities of sodium and potassium. Nature, Lond. 184, 1257-1258. HODGKIN, A. L. & KEYNES, R. D. (1953). The mobility and diffusion coefficients of K+ in giant axons from Sepia. J. Physiol. 119, 513-528. HODGKIN, A. L. & KEYNES, R. D. (1956). Experiments on the injection of substances into squid giant axons by means of a microsyringe. J. Phygiol. 131, 592-616. HOROWICZ, P. & GERBER, C. J. (1965). Effects of external potassium and Etrophanthidin on sodium fluxes in frog striated muscle. J. gen. Physiol. 48, 489-514. KEYNES, R. D. & SWAN, R. C. (1959). The effect of external sodium concentration on the sodium fluxes in frog sketal muscle. J. Physiol. 147, 591-625. KEYNES, R. D. & STEINHARDT, R. A. (1968). The components of the sodium efflux in frog muscle. J. Physiol. 198, 581-599. KUSHMERICK, M. J. & PODOLSKY, R. J. (1969). Ionic mobility in muscle cells. Science; N.Y. 166, 1297-1298. LEV, A. A. (1964). Determination of activity and activity coefficients of K and Na ions in frog muscle fibres. Nature, Lond. 201, 1132. McLAuGHLIN, S. G. A. & HINKE, J. A. M. (1966). Sodium and water binding in single striated muscle fibres of the giant barnacle. Can. J. Physiol. Pharmacol. '44, 837-848. MULLINS, L. F. & FRuMENTO, A. S. (1963). The concentration dependence of sodium efflux from muscle. J. gen. Physiol. 46, 629-654. NEILD, T. 0. & THOMAS, R. C. (1973). New design for a chloride-sensitive microelectrode. J. Physiol. 231, 7-8P. PARSONS, R. (1959). Handbook of Electrochemical Conctants. London: Butterworths. SELVERSTON, A. (1967). Structure and function of the transverse tubular system in crustacean muscle fibres. Am. Zool. 7, 515-525. SiAw, J. (1958). Further studies on ionic regulation in the muscle fibres of CarcinUs maena8. J. exp. Biol. 35, 902-919. THOMAS, R. C. (1970). New design for sodium-sensitive glass micro-electrode. J. Physiol. 210, 82-83P. THOMAS, R. C. (1972). Intracellular sodium activity and the sodium pump in snail neurones. J. Physiol. 220, 55-71. THOMAS, R. C. (1974). Intracellular pH of snail neurones measured with a new pH-sensitive glass microelectrode. J. Physiol. 238, 159-180. VAUGHAN-JONES, R. D. (1976). The effect of low-sodium solution and lanthanum on the sodium activity of crab muscle fibres. J. Physiol. 254, 40-41P. WIDDICOMBE, J. H. (1974). The effect of lanthanum on ion content and movement in the guinea pig taeniacoli. J. Physiol. 241, 106-107P.

The effect of lowering external sodium on the intracellular sodium activity of crab muscle fibres.

239 J. Physiol. (1977), 264, pp. 239-265 With 14 text-ftgure8 Printed in Great Britain THE EFFECT OF LOWERING EXTERNAL SODIUM ON THE INTRACELLULAR S...
3MB Sizes 0 Downloads 0 Views