the water balance of a serpulid polychaete … · sample (n = 8). sometime isn hypersaline the...

J. Exp. Biol. (1974), 60, 351-37° 351With 9 text-figures

Printed in Great Britain

THE WATER BALANCE OF A SERPULID POLYCHAETEMERCIERELLA ENIGMATICA (FAUVEL)

IV. THE EXCITABILITY OF THE LONGITUDINALMUSCLE CELLS

BY HELEN LE B. SKAERDepartment of Zoology, University of Cambridge*

{Received 17 December 1972)

INTRODUCTION

The body fluids of osmoconforming species living in media of fluctuating salinityare subject to dramatic changes in both osmotic and ionic concentration. This has beenshown to be true of the blood of Mercierella enigmatica (Skaer, 1974a, b). Evidence hasbeen presented which indicates that similar changes are common to all the body fluidsso that tissues bathed directly by extracellular fluid, such as the muscles, experienceconsiderable changes in concentration at the cell surface (Skaer, 1974c).

Studies on the ionic basis of electrical activity in nerve and muscle cells (reviewed byHodgkin, 1951) indicate that the preservation of excitability depends closely on themaintenance of ionic gradients across the cell membrane. Changes in the ionic com-position of the fluids bathing the cells alter these gradients, and abnormal electricalactivity in the cell results. However, M. enigmatica and other osmoconformers live andbehave normally despite large changes in the external medium. The influence offluctuations in the ionic composition of the bathing fluid on the electrical activity oftheir excitable cells is therefore of considerable interest. If the cells maintain steadyresting and action potentials despite such changes then it is possible that an activeprocess of ion transport could re-establish the normal ionic gradients across the cellmembrane even though the original absolute ion concentration probably could not berestored. Alternatively unconventional ionic mechanisms could compensate for theabnormal gradients and stable electrical activity could result. These possibilities havebeen investigated using the longitudinal muscle cells of M. enigmatica. Recordings fromthese cells fall into two classes which can be correlated with two types of longitudinalmuscle cell observed in the electron microscope (Skaer, 1974c).

METHODS

The electrophysiological apparatus and techniques for impaling the muscle cells andrecording electrical events from them have been described before (Skaer, 1974 c).Salines were made up to resemble the ionic composition of the blood of animals fromthe laboratory aquarium ('normal saline') and of animals equilibrated with glass-distilled water ('dilute saline') and 150% sea water ('hypersaline') (Table 1). The

• Present address: A.R.C. Unit of Invertebrate Chemistry and Physiology, Department of Zoology,University of Cambridge.

Sal

ine

Nor

mal

D

ilut

e D

ilut

e +

sucr

ose

Hyp

ersa

line

H

yper

osm

otic

co

ntro

l 94

0 m

~-

Na

+

50 m

~-

Na

+

Naf

fre

e 80

m~

-Ca

a+

3

m~

-Ca

2+

I0

30 ~

M-

CI

-

50 m

M-C

l-

C1-

fre

e 50

mM

C1-

3

mM

CaZ

+

Tab

le I

NaC

l (d

l)

18.7

56

-

-

35'0

76

I 8.

756

24.4

86

2.92

2 -

13'0

32

22.0

32

27.4

08

-

-

-

NaI

s (d

l)

22.0

50

7'45

0 7'

450

8.94

0 22

'050

77.6

29

-

-

36.6

54

13.7

08

-

70.0

30

70'0

30

70'0

30

Osm

otic

pr

essu

re

(mO

sm)

NaI

s =

sod

ium

iset

hion

ate.

KIs

= p

otas

sium

ise

thio

nate

. C

aAc =

cal

cium

ace

tate

.

Water balance of a serpulid polychaete M. enigmatica. IV 353

A tttfcb50 mV

200 msec

Fig. 1. Electrical activity recorded from the longitudinal muscle cells of animals dissected under:A, normal saline; B, dilute saline + sucrose, and C, hypersaline.

anion deficit was made up by including isethionate as the sodium salt in hypersalineand the sodium and potassium salts in dilute saline. For some experiments, dilutesaline was made isotonic with normal saline by adding sucrose.

The concentrations of sodium, calcium and chloride were varied individually insalines that contained the same concentration of other ions as normal saline. Thecompositions of these salines are given in Table 1.

Two poisons were used to inactivate the ion pumps: io~4 M ouabain (G-strophanthin,BDH; molecular weight 728-77) and 2X io~3 M cyanide as sodium cyanide (BDH;molecular weight 49-01). 2 x io~5 g/ml tetrodotoxin (TTX) was used to block sodiumcurrents. Its activity was tested on cockroach {Periplaneta) neurones, which have beenshown to depend on sodium for the inward current of the action potential (Yamasaki& Narahashi, 1958) (see Fig. 6B). 15 mM manganese chloride and 30 or 90 mM cobaltchloride were used to block calcium currents.

The pH of all unbuffered solutions was brought to 7-4 with o-1 N-HC1, o-1 N-H2SO4(chloride-deficient salines) or 5 % NaHCO3. The osmotic pressures of the salines weremeasured on an Osmette S osmometer (see Table 1). Since the osmotic pressures of theion-rich solutions are high, a control was made up by adding 253 g/1 of sucrose tonormal saline.

354 HELEN LE B. SKAER

Table 2. The influence of saline concentration on the action potentials ofthe longitudinal muscle cells of Mercierella enigmatica

V/sec

Saline

Normal(dissected)

Normal(intact)

Hypersaline(dissected)

Dilutesaline+ sucrose(dissected)

Cell type

Small

Large

Small

Small

Small

Large

R.P.(mV)

19-62±0-24(129)2984

±O'35(25)

20-50

±i-45(6)

20-63+ 056

(8)21-55

+ 0-41

( " )

28-25+ 0-63

(4)

Over-shoot(mV)

11-16+ 0-42(138)13-92

±1-05(26)

9-57+1-70

(7)663

±1-31(8)

17-36

±i'45( " )

25-25±3-i5

(4)

Under-shoot(mV)

25-67±0-38(116)3400

+ o-6o(22)

2586+ I-6I

(7)26-43

±0-57(7)

33-82+ 1-07

(11)

38-50+ 0-96

(4)

Rateof

rise

10-89+ 0-65(46)1294

±0-58(6)—

—

—

—

Rateoffall

24-81+1-24

(47)29-41

+ 1-26(6)—

—

—

—

Length(ms)

9'47±0-30(76)

9'75+ 0-96

(8)io-oo

±0-38(7)8-14

±0-63(7)1263

+ i-oo(11)

n-66±i-43

(4)

The figures are the means ± the standard error. The figures in brackets show the numbers in eachsample.

RESULTS

Changes in the total concentration of the external medium

Animals equilibrated in their tubes in 150 % sea water for 4 days show the normalwithdrawal response when stimulated by a moving shadow or by vibration; on beingremoved from their tubes, they move actively. If they are equilibrated in 150% seawater and dissected (as described, Skaer, 1974 c) under hypersaline, action potentialscan be recorded from the longitudinal muscle cells (Fig. 1C). These are comparedwith action potentials from control animals in Table 2. The activity in hypersaline doesnot differ significantly from recordings from cells of the small resting potential type(class I cells) in control animals. No cells of large resting potential (class II cells) havebeen impaled under hypersaline conditions but this is not surprising in such a smallsample (n = 8). Sometimes in hypersaline the action potential develops a plateau(Fig. 1C); the descending phase of the action potential levels out near the baseline intoa shoulder which may persist for 500 msec before the membrane potential is restored.

Animals equilibrated for 2 days in glass-distilled water show a completely normalbehaviour pattern, but when they are dissected under dilute saline attempts to impalethe longitudinal muscles are very rarely successful. Normal aquarium animals can besubjected to a sudden decrease in the concentration of the fluid bathing the musclesby dissecting them before changing the external medium. If animals from the aquariumare dissected and the bathing medium changed rapidly from normal saiine to dilutesaline, penetration of the muscle cells again becomes very difficult even though the

Water balance of a serpulidpolychaete M. enigmatica. IV 355

Normal 15 min 30 min

Normal 15 min 30 min l h 1+ h

L

I

Fig. 2. Tracings of electrical activity recorded from the longitudinal muscles of dissectedanimals at various times after the bathing medium had been changed from normal saline to:A, dilute saline; and B, dilute saline + sucrose. All the records are from class I cells.

animals continue to show normal movement. Activity has been recorded, however,from several different preparations (see Fig. 2 A). Action potentials persist for severalhours, but gradually the frequency of spontaneous activity decreases and more of thefibres have slow, flat action potentials with very little or no overshoot (see Fig. 2 A, 4 h).However, full action potentials have been recorded after as long as 18 h in dilute saline.If the external medium is replaced with normal saline, the frequency of normal typesof action potential increases again, and within an hour the preparation becomes indis-tinguishable from an animal taken directly from the marine aquarium. The slow, flataction potentials seen after several hours in dilute saline are similar to those recordedunder conditions of partial impalement in normal preparations. It is possible that cellsswell on being exposed suddenly to dilute saline. Swollen cells may be very much moredifficult to impale because they burst or fail to re-seal around the electrode.

A dilute saline was designed to test this possibility. Sucrose was added to dilutesaline so that its osmotic pressure was the same as normal saline. The cells of animalsexposed to this saline will not swell even though the ionic concentration of the mediumis rapidly reduced. The cells could be readily impaled, and electrical activity persistedfor many hours (see Fig. 2 B). The action potentials did not diminish in size but showeda tendency, rather, to increase. The results are summarized in Table 2. Although theresting potentials recorded in dilute saline with sucrose do not differ from those ofcontrol cells, the overshoot and undershoot figures for both types of cell are significantlylarger whether dissected or intact (see Fig. 2B, \ to \\ h). The lengths of the actionpotentials also tend to increase when the cells are bathed with dilute saline and sucrose.For comparison, action potentials from cells bathed in normal saline and dilute salinewith sucrose are shown in Fig. 1, as well as activity from cells bathed in hypersaline.

The action of poisonsOuabain

Animals that have been dissected and left in normal saline containing io"4 M ouabaincontinue to move for a period of up to 24 h. Action potentials can be recorded fromthe muscle cells for many hours (Fig. 3) but the frequency of records with a very small(i.e. < 10 mV) resting potential and small action potentials increases after about 4 h.


Normal l h 3 h 5 h 6h 7h

Normal 5h 6h 7h

L50 mV

200 msec

Fig. 3. Electrical activity recorded from the longitudinal muscle cells of Merderella enigtnaticaat various times after io~4 M ouabain had been introduced into the bathing medium. A, class Icells. B, class II cells. C, control. Animals were dissected and left in normal saline for a weekbefore the electrical activity was recorded.

Normal lOmin l h 3 h 6h 10 h

Normal 10 min

tttl h 3h

XXX * * = * =

6 h 10 h 24 h 33 h 45 h

L50 mV

200 msec

Fig. 4. Electrical activity recorded from the longitudinal muscle cells of Merderella enigtnaticaat various times after the bathing medium had been changed from normal saline to dilute saline+ sucrose containing io~4 M ouabain. A, class I cells. B, class II cells. C, control. Animals weredissected and left in dilute saline + sucrose for a week before the electrical activity was recorded.


Normal saline

50 HIM Na +

50 mV

200 msec

4-Na + free

MUIUUUU: r940 mM Na +

Fig. 5. Electrical activity recorded from the longitudinal muscle cells to show the effects ofaltered sodium concentration in the bathing medium. All the records are from class I cells.Normal saline contains 470 mM-Na+.

The interpretation of results is difficult, because the distinction between the two typesof electrical activity becomes blurred as the resting potentials decline. Nevertheless, itseems likely that the activity of class I cells (Fig. 3 A) declines faster than that of class IIcells (Fig. 3 B). The control animals were dissected and left in normal saline; the recordin Fig. 3 C was taken one week after dissection.

Animals that have been dissected and left in dilute saline + sucrose containing10-4 M ouabain continue to move for about 24 h, as was found for normal saline withadded ouabain. The pattern of action potentials recorded from the muscle cells is alsovery similar (see Fig. 4) except that in dilute saline with sucrose and ouabain the actionpotentials show an initial increase in size (Fig. 4 A and B; to 3 h). The control animalswere dissected and left in dilute saline + sucrose for a week before the record in Fig. 4 Cwas taken.

CyanideAnimals dissected and left in normal saline, with 2Xio"3M sodium cyanide added,

continue to move for four days after exposure to the cyanide solution. Action potentials


Table 3. The influence of external sodium concentration on the action potentials

of the longitudinal muscle cells of Mercierella enigmatica

Saline

Normal

940 mM-Na+

50 mM-Na+

Na+ free

2XIO"5 g/mlT T X

R.P.(mV)

19-62+ 0-24(129)

20-27±0-63

(J5)18-88

+ 0-46(33)1931

±o-S7(16)

19-50±068

(16)

Overshoot(mV)

11-16±0-42(138)

9 2 7

±1-13ds)10-14

+ 0-90(35)9-3S

±i-33(17)10-50

±1-09(20)

Undershoot(mV)

25-67±0-38(116)

2636+ I-OO

(14)

2429±065

(31)2636

+ 1-27(14)2645

+ 0-84(20)

Rateof

rise(V/s)

10-89±065(46)—

IO-IO+ o-6o(34)8-85

±0-79(17)—

Rateoffall

(V/s)

2481+1-24(47)—

23-80±1-18(34)2259

+ 1-82(17)—

The figures are the means ± the standard error. The figures in brackets show the numbers in eachsample.

tfc= 1U-1—t:Normal 3 min 6 min 1 h

L50 mV

200 msec

Saline

2mV L5 min

Fig. 6. A. Electrical activity recorded from the longitudinal muscle cells of Mercierella enig-matica to show the effect of 2 x io~5 g/ml of tetrodotoxin (TTX) in normal saline. B. Extra-cellular records of electrical activity of the ventral nerve cord of Periplaneta before and after2 x io~6 g/ml TTX had been introduced into the bathing medium.


16

14

12

10

§ 8

6 6 1-

4 -

2 -

10

[Ca]o(mM)

100

Fig. y. Graph to show the relationship between the height of the action-potential overshoot ofclass I cells and the concentration of calcium in the bathing medium. The insets show electricalactivity recorded from animals bathed in solutions of calcium concentration marked by the pointon the graph nearest each inset. Where the records show three traces, the lowest displays thedifferentiated signal derived from the action potential.

obtained from cells up to 11 h after exposure to cyanide appear to be undiminished insize. Because of technical difficulties, impalements after this time were not achieved.

Changes in sodium concentration

Electrical activity from muscle cells equilibrated in media of different sodium con-centration are shown in Fig. 5. The resting potential and the characteristics of theaction potential appear to be remarkably little affected by the concentration of sodiumin the extracellular medium (see Table 3). Moreover, tetrodotoxin, a poison whichspecifically blocks sodium conductance, has no significant effect on the action potentials(Fig. 6A).

Changes in calcium concentration

The characteristics of the action potentials do change, however, when the concen-tration of calcium in the medium is varied. The overshoot in 3 mM calcium is signifi-cantly smaller than in normal saline (31 mM calcium) and is significantly larger in80 mM calcium saline. The results are shown in Fig. 7 and Table 4. The fourth pointon the graph is for undissected aquarium animals in which the unbound calcium con-centration in the blood is 17-5 mM (Skaer, 1974ft). The slope of the line drawn is10-53 mV for a ten-fold change of external calcium concentration. The resting potentialsof cells in 80 mM and 3 mM calcium are not significantly different from those of cells innormal saline. The maximum rate of rise and fall of the action potential is substantiallyreduced when the external calcium concentration is lowered.


Table 4. The influence of external calcium concentration on the actionpotentials of the longitudinal muscle cells of Mercierella enigmatica

Saline

Normal

80 mM-Ca2+

3 mM-Ca2+

15 mM-Mn2+

30 mM-Co2+

90 mM-Co2+

R.P.(mV)

19-62±0-24(129)

18-32+ 061

(34)19-47

±0-30(101)

18-20+ 0-42

(69)1794

±0-56(34)14-75

+ 1-16(12)

Overshoot(mV)

I I - I 6+ 0-42(138)

16-03±0-94

(34)0-85

±0-52("4)

7-93+ 0-58

(70)

11-12±O-93

(34)5'42

±1-77(12)

Undershoot(mV)

25-67±0-38(116)

26-00+ 072

(32)

23-SS+ 0-44(106)

24-63±0-49

(64)24-06

±0-83(33)

20-08+ I-8I

(12)

Rateof

rise(V/s)

10-89+ 0-65

(46)—

2 2 6±0-15(109)

—

—

0-97+ 0-21

(II)

Rateoffall

(V/s)

2481±1-24

(47)—

8-85±0-54(109)

—

—

5-22±1-23

( I I )

Length(ms)

9 7 4±0-30(76)—

IO-I2±O-23(8l)

The figures are the means + the standard error. The figures in brackets are the numbers in eachsample.

Action potentials persist in dissected animals that are bathed with salines containing15 mM manganese but the height of the overshoot is significantly lower than in animalsdissected under normal saline. The addition of 30 mM cobalt to the normal salinebathing the muscle cells has no significant effect on the size of the overshoot but theaddition of 90 mM cobalt reduces it from 11-16 to 5-42 mV. In fact the effect of 90 mMcobalt is very similar to that of reducing the external calcium concentration; all para-meters of the action potential are reduced. 90 mM cobalt, however, also causes a de-crease in the level of the resting potential (Table 4), a characteristic that is shared to alesser degree by 30 mM cobalt and 15 HIM manganese solutions.

Changes in chloride concentration

The effects of altering the concentration of chloride ions in the medium are shown inFig. 8 and in Table 5. In salines containing twice the normal concentration of chloridethe size of the overshoot is significantly decreased. On the other hand a significantincrease in the height of the overshoot is obtained when the concentration of chlorideoutside the cell is decreased, but the graph in Fig. 8 clearly does not describe a singlestraight-line relation between overshoot height and external chloride concentration.The slope of the line joining points above the normal chloride concentration (515 mM)is 18-7 mV for a ten-fold concentration change. Below these concentrations the slopeabruptly decreases to 0-75 mV for a similar concentration change and finally there is aslight increase to 2-7 mV. There are no changes in the other parameters of the actionpotential that correlate completely with the alterations in the size of the overshoot

Water balance of a serpulidpolychaete M . enigmatica. IV 361

16 j

14

12

10

O 6

10 100

[Cl]0(mM)

1000

Fig. 8. Graph to show the relationship between the height of the action-potential overshoot ofclass I cells and the concentration of chloride in the bathing medium. The insets show electricalactivity recorded from animals bathed in solutions of chloride concentration marked by thepoint on the graph nearest each inset. Where the records show three traces, the lowest displaysthe differentiated signal derived from the action potential.

except for the changes in the length of the action potential (measured from the thresholdto the lowest point of the undershoot). Increasing the external chloride concentrationappears to decrease the length of the action potential, the reverse being found whenthe concentration is reduced (Table 5).

Changes in calcium and chloride concentrations

The size of the overshoot is very small (0-85 mV) when the concentration of calciumin the external medium is reduced to a tenth of the normal level. If the chloride con-centration is also reduced by a similar amount the overshoot of the action potential is7-40 mV, a significant increase over the size in reduced calcium.

Hypersaline control

The hypersaline control shows that there is a slight osmotic effect on the size of theovershoot (Table 5), i.e. there is a significant increase in height compared with the sizein normal saline. There is also a significant increase in the length of the action potential.


Table 5. The influence of external chloride concentration on the actionpotentials of the longitudinal muscle cells of Mercierella enigmatica

Saline

Normal

1030 mM-Cl~

50 mM-Cl-

Cl- free

5omM-Cl~;3 mM-Ca2+

Hypersalinecontrol

Restingpotential

(mV)

19-62+ 0-24

(129)

18-61+ 0-70

(18)

i8-35±0-32

(79)20-45

±o-43(22)

16-00+ 1-06

(13)

2O-OO

±0-56(II)

Overshoot(mV)

I I - I 6+ 0-42(138)

S-72±i-3S

(18)

11-85+ 0-49

(80)

16-28+ I-IO

(22)

7-40+ 0-91

(IS)16-18

+ 2-OO( » )

Undershoot(mV)

25-67±0-38(116)

21-59±0-79

(17)

25-27+ 0-46

(83)2801

+ 0-99(22)

19-57+ 1-18

(14)

25-56+ 1-07

(9)

Rateof

rise(V/s)

10-89+ 065(46)5-06

+ 0-58(18)

7-34±o-35(80)

—

0-92+ 0-22

(15)

996±069( I I )

Rateoffall

(V/s)

24-81+1-24

(47)1288

±1-30(18)

17-52±0-72(81)

—

4-72+ O-86(15)

20-53+ 1-26

(11)

Length(ms)

9-74±0-30(76)7-76

±0-50

(17)

12-90±0-25(7i)

1572±0-51(23)

12-92+ 0-92

(13)

II-OO+ O-I2(12)

The figures are the means ± the standard error. The figures in brackets are the number in eachsample.

DISCUSSION

Changes in the total concentration of the external medium

Comparison of the records in Fig. 1 and figures in Table 2 shows that the electricalactivity of the longitudinal muscles of Mercierella enigmatica does not diminish whenthe animals are equilibrated in media that are widely different from normal sea water.The resting potentials remain unaltered and the action potentials of muscle cells indilute saline (with or without sucrose) show if anything an increase in overshoot ratherthan the decrease that might be expected.

The effect on isolated muscle preparations of rapidly changing the ionic content ofthe bathing medium has been studied by Wells & Ledingham (1940). They found that arapid decrease in concentration caused inhibition of spontaneous contraction of isolatedmuscles of Arenicola marina and Nereis diversicolor. In contrast, the ability of Mer-cierella to move was unimpaired by rapid dilution of the external medium. It does,however, become more difficult to record from the muscle cells because they areswollen (Skaer, 1974a) and therefore more easily damaged.

The persistence of electrical and mechanical activity over such a wide range ofosmotic and ionic concentration of the bathing medium is clearly advantageous to anosmoconformer such as M. enigmatica, living in a wide range of salinities and often influctuating conditions. However, the ability of the longitudinal muscle fibres to supportaction potentials when the ionic concentration of the fluids bathing them varies sowidely presents the electrophysiologist with an intriguing problem. There are severaltheoretically possible ways of overcoming such a problem.


1. Compensating water movements

If the cells simply swell when the bathing medium is diluted and shrink in hyper-osmotic salines, the internal concentrations of ions in the cell will change so that thenormal ion gradients across the membrane tend to be preserved. Shaw (1955a, b;1958 a, b), working with Carcinus maenas, found that the ionic content of the musclecells changed quite substantially when the animals were exposed to diluted (40 %) seawater. Although the new concentrations of some ions (Ca2+ and Mg2+) could beexplained by assuming that the cells were swollen by 25 %, the concentrations of someothers (notably Na+ and K+) could not. Even where the new internal concentrationscould be explained by water influx, the dilution was insufficient to preserve the normalion gradient across the cell membrane. The maintenance of normal or near normalgradients could not therefore be the result simply of dilution of the cell contents.

In Mercierella enigmatica the extent to which the maintenance of normal ionicgradients across the cell membrane results from passive osmotic effects is probablyquite small. Measurements described previously (Skaer, 1974 a) indicate that the cellsare unlikely to swell by more than about 10% when the medium is diluted ten times.The possibility that the normal ionic gradients across the cell membrane are main-tained by active regulation must therefore be considered.

2. Active regulation

The normal ionic gradients across the excitable membrane could be maintained bythe activity of ion pumps. If this were the case the persistence of electrical activity afterchanging the composition of the bathing medium might be upset by poisons thatinhibit the pumps. Ouabain, a cardiac glycoside, specifically inhibits the sodium-potassium exchange pump (Schatzman, 1953). When dissected animals are exposed todilute saline with sucrose in the presence of ouabain, the action potentials continue andare enlarged in size, as they are in isotonic dilute saline alone. The electrical activitydoes eventually diminish after many hours, as it does in control preparations exposedto io"4 M ouabain in normal saline. It is clear that the adaptation of the muscle cells to adilute ionic environment does not depend on the activity of a sodium-potassiumexchange pump.

The normal ionic gradients across the muscle cell membranes might be maintainedafter changes in the composition of the bathing medium by ion pumps other than theouabain-sensitive sodium-potassium pump. However, the time course of cyanidepoisoning is very long, indicating that the muscle cells either contain large stores ofATP or can resynthesize ATP without recourse to respiration. In view of this, furtherexperiments using general metabolic poisons have not been pursued.

3. Low intracellular concentrations

If the intracellular concentration of the ion that carries the inward current wasextremely low, the gradient across the cell membrane might be sufficient to give afull action potential overshoot over the range of external concentration of that ionexperienced by the animal.

24 E X B 60


4. Variation in 'channel' density

If the number of' channels' allowing the ion carrying the inward current to cross themembrane was normally limiting and if the number were to increase as the concentra-tion of the ion in the external medium decreased, the action potential overshoot mightremain stable.

If any of these three possibilities (2-4) were true, one would not expect the overshootto be any more sensitive to the alteration of the external concentration of a single ionthan to the alteration of the concentrations of several or all the ions. In fact the sizes ofthe overshoot and undershoot would not be expected to change at all and the Nernstslopes for ions involved in carrying these currents would be horizontal.

5. Antagonistic ion effects

The action potential could be maintained by a series of antagonistic ion effects, suchthat the net inward and outward currents do not change as long as the relative concen-trations of the ions in the extracellular medium remains constant, no matter what theirabsolute concentrations may be. The ionic basis underlying such effects can be studiedby varying the concentration of one ion alone in the bathing medium. These experi-ments would also differentiate between the possibilities summarized in sections 2-4and antagonistic ion effects. In this study discussion will be restricted to the overshootof action potentials recorded from class I cells.

Sodium and calcium

The failure to alter the height of overshoot, the total rise or the rate of rise of theaction potential either by altering the sodium concentration of the bathing mediumover a very wide range or by introducing tetrodotoxin into the external solution sug-gests that sodium ions play no part in the passage of the inward pulse of current acrossthe cell membrane. On the other hand, altering the external calcium concentrationdoes produce an effect on the size of these parameters. If the height of the overshoot ofthe action potential is plotted against the log of the external calcium concentration(Fig. 7), a straight-line graph is obtained whose slope gives a figure of 10-53 m ^ changein height of the overshoot for a ten-fold change in external calcium concentration. If themembrane were permeable only to calcium ions, when the inward current of the actionpotential is flowing, the Nernst equation predicts that the slope for a ten-fold concen-tration change would be 29 mV. Thus, the passage of calcium ions into the cell does notaccount entirely for the inward current.

The influence of competitors for the calcium channels in the active membrane is notlarge unless they are present in rather high concentration. However, Geduldig &Junge (1968) working with Aplysia neurones found that a concentration of cobaltapproximately three times greater than the calcium concentration in the medium wasnecessary to block the calcium component of the overshoot. This is also found inMercierella enigmatica, though even a 90 mM concentration of cobalt in the mediumdoes not have such a large effect as reducing the calcium concentration ten times.

The results suggest then that sodium ions do not carry the inward current of theaction potential but that calcium ions probably do, though there is a component of the


inward current that must be carried by another ion. The occurrence of calcium-mediated action potentials is not uncommon in invertebrate muscle cells (crab muscle:Fatt & Katz, 1953; crayfish muscle: Fatt & Ginsborg, 1958; Abbott & Parnas, 1965;Takeda, 1967; barnacle muscle: Hagiwara & Naka, 1964; earthworm muscle: Hidaka,Ito & Kuriyama, 1969). Action potentials in which the inward current is carried bymore than one ion have also been recorded in other preparations (smooth muscle:Biilbring & Kuriyama, 1963; frog heart: Niedergerke & Orkand, 1966a, b; snailneurones: Kerkut & Gardner, 1967; Geduldig & Junge, 1968; Krishtal & Magura,1970; Jerelova, Krasts & Veprintsev, 1971; Sattelle, 1974) although in all of thesepreparations the inward current is carried partly by sodium ions.

Chloride

Alterations in the chloride concentration of the bathing medium have a profoundeffect on the size of the overshoot if the concentration of chloride is increased but onlya small effect if the concentration is reduced. The size of the overshoot is significantlyreduced when the chloride concentration in the bathing medium is doubled. Theosmotic pressure of the high-chloride saline is large (1800 mOsm) and it is conceivablethat the reduction is due to an osmotic effect on the cell. If the muscle cells shrink inhyperosmotic media, the intracellular concentration of ions would be increased andthus the gradient of calcium across the cell membrane would be reduced. This wouldtend to reduce the size of the overshoot. However, if the osmotic pressure of normalsaline is increased by adding sucrose to it (2020 mOsm) and animals are perfused withthis hypertonic saline, the size of the overshoot does not decrease, but rather shows atendency to increase slightly in size. It seems then that the reduction of the overshootin solutions of high chloride content is an ionic and not an osmotic effect.

Calcium and chloride together

When the concentration of both calcium and chloride are reduced to a tenth thenormal concentration, the size of the overshoot is larger by 6-55 mV (7'4o-o-85 mV)than when calcium alone is reduced. This figure is very much greater than the increasein overshoot produced by reducing chloride alone (0-69 mV). It is possible that theinfluence of external chloride concentration on the size of the overshoot is enhancedif the calcium concentration is also reduced. Such an interdependence of ion perme-abilities has been reported before (Krishtal & Magura, 1970; Jerelova et al. 1971;Sattelle, 1974).

The significant fact that emerges from the experiment of reducing both calcium andchloride in the external medium is that the height of the action potential is not markedlyaltered compared with the height in normal saline. The insensitivity of the overshootcan be inferred from the Nernst slopes for calcium and chloride (see Figs. 7 and 8).The maximum slopes are very similar, allowing for the valency difference, although inopposite senses. This means that the size of the overshoot is not very much altered ifthe ratio of calcium ions and chloride ions in the medium bathing the muscle cellsremains unchanged. Although the concentration of the body fluids of Mercierellaenigmatica varies over an enormously wide range, the ratios of the concentrations ofions in the blood do not change very much as is shown in Table 6.

24-2


Table 6

I

2

34

56

78

9

The

Equilibration medium

Glass-distilled waterAquarium sea water150% sea waterDilute saline

Table 7

in blood or salinebathing the tissues

0-07350-03380-04660-3039

Action-potential Action-potentiallength overshoot

Saline (ms) N (mV)

Normal1030 mM-Cl~50 mM-Cl-Cl- free5omM-Cl~;3 mM-Caa+

3 mM-Ca'+

Distilled water adapted(blood Cl- = 67-8 HIM)

Dilute saline + sucroseHypersaline adapted(blood Cl- = 843 HIM)

Hypersaline control

9-74+0-307-7610-50

12-90+0-25

12-92+0-92

10-12+0-2312-76

12-63 + 1-008-14+0-63

II-OO + O-I2

figures are the means ± the standard error.

76

17

2313

81

1

11

7

12

N is the number in

II-I6±O-42

572±i-3511 -85 ±0-4916-28! I-IO7-40 ±0-91

0-8510-5216

17-6311-456-6311-31

16-1812-00

> each sample.

When animals were dissected and perfused with a dilute saline (made up on the basisof the total concentration of ions in the blood and not the unbound concentrations),the overshoot increased significantly in size. Reference to Table 6 shows that the ratioof calcium to chloride in the bathing medium had also increased.

Duration of the action potential

The duration of the action potentials recorded from cells bathed in the differentsalines is shown in Table 7. These results can be interpreted in terms of the distributionof chloride across the membrane. If it is assumed that the external concentration ofchloride is normally greater than the internal concentration, then an increase in thelength of the action potential would be correlated with a decrease in the size of thegradient (salines 2, 3, 4, 6, 7; by reducing external chloride) and vice versa (salines 1and 8). Moreover, the magnitude of the change in duration is correlated with thedegree to which the chloride gradient is altered (cf. 2 and 3 or 1 and 8). The increase inthe duration of the action potential in the hypersaline control (9) (2020 mOsm) canalso be explained in terms of the chloride gradient. If the cells shrink, the internalchloride concentration might increase and the gradient might therefore be reduced.The chloride concentration in 3 mM-Ca2+ (5) is not different from that in normal salineand this is reflected in the unaltered length of the action potential. Similarly the dura-tions of the action potentials recorded from muscles bathed in salines 2 and 4 are notsignificantly different and these two salines both contain 50 mM-Cl~.

Water balance ofa serpulidpolychaete M. enigmatica. IV 367

Table 8

Tissue

Frog muscleCrab muscle(Carcinus maenas)

Squid nerve

Toad nerve

Human erythrocyteAlga (Nitella

translucens)

Intracellularchloride

concentration(mM)

1-552

39140

27

74I S '

Extracellularchloride

concentration(mM)

775 (plasma)524 (blood)

520 (sea water)520 (sea water)i n (interfibrillar

connective tissue)i n (plasma)1-3 (pond water)

Source

Conway, 1957Shaw, 19550

Steinbach, 1941Koechlin, 1954Shanes &Berman, 1955 a, b

Davson, 1964MacRobbie, 1962

It does seem then that there is quite a good correlation between the size of thechloride gradient and the duration of the action potential and, when the concentrationsof other ions are unaltered, between the duration of the action potential and the size ofthe overshoot (Table 7).

The chloride effect

Calcium and chloride ions appear to interact in the generation of the overshoot. Theinvolvement of chloride could either be indirect, for example, by influencing the calciumconductance, or direct, in that part of the membrane current is carried by chloride ions.For example, the overshoot could be maintained by the antagonistic effects of a calciuminflux and a chloride efflux. However, if an efflux of chloride leading to an overshoot ispostulated, it is necessary to assume that the concentration of chloride in the musclecells is greater than that in the extracellular fluids. This is not the case generally inanimal cells; the concentrations of chloride for some animal and plant cells are shownin Table 8. Only in the large algal plant cells (e.g. Nitella translucens) would an effluxof chloride be favoured (Gaffey & Mullins, 1958) where, since the cells lie naked inpond water, the chloride concentration of the external medium is very low. Hutter &Noble (i960) have suggested that a small component of the repolarizing current in frogmuscle fibres is carried by an influx of chloride ions. If such an influx carried the re-polarizing current in the muscle of Mercierella enigmatica, an alteration in the chloridegradient would have an effect both on the rate of fall of the action potential and on thesize of the undershoot. Neither of these parameters changes in a way that is consistentwith the hypothesis that the outward current is carried by chloride ions (Table 5).

A possible mechanism for the stability of the overshoot

If a chloride influx is involved in the development of the action potential then itwould occur before the undershoot is expressed. Such an influx of chloride couldantagonize the effect of a calcium influx and the peak of the overshoot would be reachedwhen the calcium and chloride currents were balanced so that the result of an increasein either the chloride conductance or the gradient of chloride across the membranewould be an increase in the chloride current and so a reduction in the size of the over-shoot (see Fig. 9). Thus an increase in the external chloride concentration would tendto reduce the height of the action potential and conversely a reduction in the chloride

368

Normalsaline

Overshoot

1116mV

HELEN LE B. SKAER

Normal C a "1/10 Cl-

11-85 mV

H-

1/10 Ca2+

Normal Cl"

0-85 mV

1/10 Ca2 +

1/10 Cl-

7-40 mV

| | | i I I 1 | i

nttttfctc

Outside Inside Outside Inside Outside Inside

Total i

Fig. 9. Postulated interaction between ion currents involved in the formation of the action-potential overshoot in different external media.

concentration would increase it. In fact this increase of the overshoot is only seen whenthe external calcium is also reduced and not when the external chloride is reduced inotherwise normal saline (Table 5). This effect could be explained by supposing that thechloride conductance is small compared with the calcium conductance so that theenhancing effects of reduced external chloride are normally swamped by the calciumcurrent. These ideas are summarized in Fig. 9.

It is not immediately clear how this hypothesis fits the data concerning the durationof the action potentials. Since the chloride current is not the principal inward or out-ward current, changes in its expression will probably have little effect on the maximumrates of rise and fall of the action potential. The present results certainly do not showany clear relationships between the chloride gradient and the signals from the dif-ferentiating circuit. But even if the rates of rise and fall are unaltered by the size of thechloride gradient, the duration of the action potential will be related to the height of theovershoot, and the greater the height of the action potential the longer the rise time will be.

Five hypotheses were put forward to account for the stability of the action potentialin M. enigmatica. The first hypothesis could contribute to but not account for itsstability. Hypotheses 2-4 all predict horizontal Nernst slopes for the ions involved incarrying the inward and outward currents of the action potential. This prediction is notborne out by experiment. Evidence has been presented to show that the height of theovershoot of the action potential results from the combined effects of calcium andchloride currents. These currents interact in such a way that the overshoot does notchange in height as long as the ratio of calcium and chloride in the medium bathing themuscle cells remains unaltered (Table 6, Fig. 9). Such interacting ion effects mightaccount for the stability of the whole of the resting and action potential of the longi-tudinal muscle cells of M. enigmatica. When the concentration of the external environ-

Water balance of a serpulidpolychaete M. enigmatica. IV 369

ment is changed the ratios of the concentrations of the ions in the blood are remarkablylittle altered despite large changes in their absolute concentrations (see Skaer, 19746).Thus the persistence of electrical activity in Merrierella, and possibly in other osmo-conforming species, could depend on the maintenance of the ratios of concentrations ofions in the body fluids rather than on the preservation of fixed concentrations of eachion species.

SUMMARY

1. The electrical activity of the two types of longitudinal muscles of an osmocon-forming polychaete worm, Merrier ella enigmatica, have been studied in media of widelyvarying osmotic and ionic composition. Activity persists practically unaltered in bothtypes of muscle cell.

2. The possible effects of osmotically induced changes in cell volume on the ionicgradients across the cell membranes are considered. It is concluded that the normalgradients are unlikely to be maintained as a result of such changes.

3. The involvement of ion pumps in the maintenance of the normal gradients acrossthe muscle cell membranes has been studied using specific and metabolic poisons. It isevident that the persistence of electrical activity in media of altered ionic content doesnot depend on the sodium-potassium exchange pump.

4. The ionic basis of the overshoot of action potentials recorded from cells of thesmall resting potential type has been studied. It is concluded that calcium ions but notsodium ions are responsible for the inward current although there is a component of theinward current carried by some other as yet unidentified ion.

5. Alterations in the external concentrations of chloride ions are found to alter boththe height of the overshoot and the length of the action potential.

6. Profound alterations in the overshoot height are produced only when the normalratio of calcium to chloride concentration in the external medium is altered. Possiblemechanisms to explain these effects are discussed.

7. It is suggested that the stability of the action potential in the muscle cells of M.enigmatica, despite large fluctuations in the salinity of the external medium, dependson the constancy of the ratios between the concentrations of the ions in the fluidsbathing the cells and not on the absolute concentrations of the ions.

This work was supported by a Science Research Council grant and a studentshipfrom Girton College, Cambridge. I am very grateful to my supervisor, Dr J. E. Tre-herne for his help and encouragement. I thank him, Dr R. Meech, Dr J. Oschman,Dr R. J. Skaer and Dr B. Wall for reading and criticising the manuscript. I am gratefulto Mr J. Rodford for help in drawing the figures.

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