THE WATER BALANCE OF A SERPULID POLYCHAETE MERCIERELLA ENIGMATICA (FAUVEL)

Transcription

1 J. Exp. Biol. (1974), 60, With 9 text-figures Printed in Great Britain THE WATER BALANCE OF A SERPULID POLYCHAETE MERCIERELLA ENIGMATICA (FAUVEL) IV. THE EXCITABILITY OF THE LONGITUDINAL MUSCLE CELLS BY HELEN LE B. SKAER Department of Zoology, University of Cambridge* {Received 17 December 1972) INTRODUCTION The body fluids of osmoconforming species living in media of fluctuating salinity are subject to dramatic changes in both osmotic and ionic concentration. This has been shown to be true of the blood of Mercierella enigmatica (Skaer, 1974a, b). Evidence has been presented which indicates that similar changes are common to all the body fluids so that tissues bathed directly by extracellular fluid, such as the muscles, experience considerable changes in concentration at the cell surface (Skaer, 1974c). Studies on the ionic basis of electrical activity in nerve and muscle cells (reviewed by Hodgkin, 1951) indicate that the preservation of excitability depends closely on the maintenance of ionic gradients across the cell membrane. Changes in the ionic composition of the fluids bathing the cells alter these gradients, and abnormal electrical activity in the cell results. However, M. enigmatica and other osmoconformers live and behave normally despite large changes in the external medium. The influence of fluctuations in the ionic composition of the bathing fluid on the electrical activity of their excitable cells is therefore of considerable interest. If the cells maintain steady resting and action potentials despite such changes then it is possible that an active process of ion transport could re-establish the normal ionic gradients across the cell membrane even though the original absolute ion concentration probably could not be restored. Alternatively unconventional ionic mechanisms could compensate for the abnormal gradients and stable electrical activity could result. These possibilities have been investigated using the longitudinal muscle cells of M. enigmatica. Recordings from these cells fall into two classes which can be correlated with two types of longitudinal muscle cell observed in the electron microscope (Skaer, 1974c). METHODS The electrophysiological apparatus and techniques for impaling the muscle cells and recording 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 from the laboratory aquarium ('normal saline') and of animals equilibrated with glassdistilled 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.

2 Osmotic pressure (mosm) Table I Saline Normal Dilute Dilute + sucrose Hypersaline Hyperosmotic control 940 m~-na+ 50 m~-na+ Naf free 80 m~-caa+ 3 m~-ca2+ I030 ~M-CI- 50 mm-cl- C1- free 50 mm C1-3 mm CaZ+ NaCl (dl) '076 I ' NaIs (dl) '450 7' ' '030 70'030 NaIs = sodium isethionate. KIs = potassium isethionate. CaAc = calcium acetate.

3 Water balance of a serpulid polychaete M. enigmatica. IV 353 A tttfcb 50 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 hypersaline and the sodium and potassium salts in dilute saline. For some experiments, dilute saline was made isotonic with normal saline by adding sucrose. The concentrations of sodium, calcium and chloride were varied individually in salines that contained the same concentration of other ions as normal saline. The compositions 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 ) 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 sodium currents. Its activity was tested on cockroach {Periplaneta) neurones, which have been shown 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 cobalt chloride 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 % NaHCO 3. The osmotic pressures of the salines were measured on an Osmette S osmometer (see Table 1). Since the osmotic pressures of the ion-rich solutions are high, a control was made up by adding 253 g/1 of sucrose to normal saline.

4 354 HELEN LE B. SKAER Saline Normal (dissected) Normal (intact) Hypersaline (dissected) Dilute saline + sucrose (dissected) Table 2. The influence of saline concentration on the action potentials of the longitudinal muscle cells of Mercierella enigmatica Cell type Small Large Small Small Small Large R.P ±0-24 (129) 2984 ±O'35 (25) ±i-45 (6) (8) (") (4) Overshoot (138) ±1-05 (26) (7) 663 ±1-31 (8) ±i'45 (") ±3-i5 (4) Undershoot ±0-38 (116) o-6o (22) I-6I (7) ±0-57 (7) (11) (4) Rate of rise (46) 1294 ±0-58 (6) V/sec Rate of fall (47) (6) Length (ms) 9'47 ±0-30 (76) 9' (8) io-oo ±0-38 (7) 8-14 ±0-63 (7) i-oo (11) n-66 ±i-43 (4) The figures are the means ± the standard error. The figures in brackets show the numbers in each sample. RESULTS Changes in the total concentration of the external medium Animals equilibrated in their tubes in 150 % sea water for 4 days show the normal withdrawal response when stimulated by a moving shadow or by vibration; on being removed from their tubes, they move actively. If they are equilibrated in 150% sea water and dissected (as described, Skaer, 1974 c) under hypersaline, action potentials can be recorded from the longitudinal muscle cells (Fig. 1C). These are compared with action potentials from control animals in Table 2. The activity in hypersaline does not 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) have been impaled under hypersaline conditions but this is not surprising in such a small sample (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 into a 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 normal behaviour pattern, but when they are dissected under dilute saline attempts to impale the longitudinal muscles are very rarely successful. Normal aquarium animals can be subjected to a sudden decrease in the concentration of the fluid bathing the muscles by dissecting them before changing the external medium. If animals from the aquarium are dissected and the bathing medium changed rapidly from normal saiine to dilute saline, penetration of the muscle cells again becomes very difficult even though the

5 Water balance of a serpulidpolychaete M. enigmatica. IV 355 Normal 15 min 30 min L Normal 15 min 30 min lh 1+ h Fig. 2. Tracings of electrical activity recorded from the longitudinal muscles of dissected animals 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 several hours, but gradually the frequency of spontaneous activity decreases and more of the fibres 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 types of action potential increases again, and within an hour the preparation becomes indistinguishable from an animal taken directly from the marine aquarium. The slow, flat action potentials seen after several hours in dilute saline are similar to those recorded under conditions of partial impalement in normal preparations. It is possible that cells swell on being exposed suddenly to dilute saline. Swollen cells may be very much more difficult 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 dilute saline so that its osmotic pressure was the same as normal saline. The cells of animals exposed to this saline will not swell even though the ionic concentration of the medium is rapidly reduced. The cells could be readily impaled, and electrical activity persisted for many hours (see Fig. 2 B). The action potentials did not diminish in size but showed a tendency, rather, to increase. The results are summarized in Table 2. Although the resting potentials recorded in dilute saline with sucrose do not differ from those of control cells, the overshoot and undershoot figures for both types of cell are significantly larger whether dissected or intact (see Fig. 2B, \ to \\ h). The lengths of the action potentials 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 saline with sucrose are shown in Fig. 1, as well as activity from cells bathed in hypersaline. The action of poisons Ouabain Animals that have been dissected and left in normal saline containing io" 4 M ouabain continue to move for a period of up to 24 h. Action potentials can be recorded from the 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. I

6 356 HELEN LE B. SKAER Normal lh 3h 5h 6h 7h Normal 5h 6h 7h 50 mv L 200 msec Fig. 3. Electrical activity recorded from the longitudinal muscle cells of Merderella enigtnatica at various times after io~ 4 M ouabain had been introduced into the bathing medium. A, class I cells. B, class II cells. C, control. Animals were dissected and left in normal saline for a week before the electrical activity was recorded. Normal lomin lh 3h 6h 10 h ttt Normal 10 min lh 3h XXX **= *= 6h 10 h 24 h 33 h 45 h 50 mvl 200 msec Fig. 4. Electrical activity recorded from the longitudinal muscle cells of Merderella enigtnatica at 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 were dissected and left in dilute saline + sucrose for a week before the electrical activity was recorded.

7 Water balance of a serpulid polychaete M. enigmatica. IV 357 Normal saline 50 HIM Na + 50 mv 200 msec Na + 4- free MUIUUUU: 940 mm Na + Fig. 5. Electrical activity recorded from the longitudinal muscle cells to show the effects of altered 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 types of electrical activity becomes blurred as the resting potentials decline. Nevertheless, it seems likely that the activity of class I cells (Fig. 3 A) declines faster than that of class II cells (Fig. 3 B). The control animals were dissected and left in normal saline; the record in Fig. 3 C was taken one week after dissection. Animals that have been dissected and left in dilute saline + sucrose containing 10-4 M ouabain continue to move for about 24 h, as was found for normal saline with added ouabain. The pattern of action potentials recorded from the muscle cells is also very similar (see Fig. 4) except that in dilute saline with sucrose and ouabain the action potentials show an initial increase in size (Fig. 4 A and B; to 3 h). The control animals were dissected and left in dilute saline + sucrose for a week before the record in Fig. 4 C was taken. Cyanide Animals dissected and left in normal saline, with 2Xio" 3 M sodium cyanide added, continue to move for four days after exposure to the cyanide solution. Action potentials r

8 358 HELEN LE B. SKAER 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/ml TTX R.P (129) ±0-63 ( J 5) (33) 1931 ±o-s7 (16) ±068 (16) Overshoot ±0-42 (138) 927 ±1-13 ds) (35) 9-3S ±i-33 (17) ±1-09 (20) Undershoot ±0-38 (116) I-OO (14) 2429 ±065 (31) (14) (20) Rate of rise (V/s) ±065 (46) IO-IO + o-6o (34) 8-85 ±0-79 (17) Rate of fall (V/s) (47) ±1-18 (34) (17) The figures are the means ± the standard error. The figures in brackets show the numbers in each sample. tfc= 1U-1t: Normal 3 min 6 min 1 h 50 mv L 200 msec Saline 2mVL 5 min Fig. 6. A. Electrical activity recorded from the longitudinal muscle cells of Mercierella enigmatica to show the effect of 2 x io~ 5 g/ml of tetrodotoxin (TTX) in normal saline. B. Extracellular records of electrical activity of the ventral nerve cord of Periplaneta before and after 2 x io~ 6 g/ml TTX had been introduced into the bathing medium.

9 Water balance of a serpulid polychaete M. enigmatica. IV [Ca] o (mm) Fig. y. Graph to show the relationship between the height of the action-potential overshoot of class I cells and the concentration of calcium in the bathing medium. The insets show electrical activity recorded from animals bathed in solutions of calcium concentration marked by the point on the graph nearest each inset. Where the records show three traces, the lowest displays the differentiated signal derived from the action potential. obtained from cells up to 11 h after exposure to cyanide appear to be undiminished in size. 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 concentration are shown in Fig. 5. The resting potential and the characteristics of the action potential appear to be remarkably little affected by the concentration of sodium in the extracellular medium (see Table 3). Moreover, tetrodotoxin, a poison which specifically 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 concentration of calcium in the medium is varied. The overshoot in 3 mm calcium is significantly smaller than in normal saline (31 mm calcium) and is significantly larger in 80 mm calcium saline. The results are shown in Fig. 7 and Table 4. The fourth point on the graph is for undissected aquarium animals in which the unbound calcium concentration in the blood is 17-5 mm (Skaer, 1974ft). The slope of the line drawn is mv for a ten-fold change of external calcium concentration. The resting potentials of cells in 80 mm and 3 mm calcium are not significantly different from those of cells in normal saline. The maximum rate of rise and fall of the action potential is substantially reduced when the external calcium concentration is lowered. 100

10 360 HELEN LE B. SKAER Saline Normal 80 mm-ca 2+ 3 mm-ca mm-mn mm-co mm-co 2+ Table 4. The influence of external calcium concentration on the action potentials of the longitudinal muscle cells of Mercierella enigmatica R.P ±0-24 (129) (34) ±0-30 (101) (69) 1794 ±0-56 (34) (12) Overshoot II - I (138) ±0-94 (34) 0-85 ±0-52 ("4) (70) ±O-93 (34) 5'42 ±1-77 (12) Undershoot ±0-38 (116) (32) 23-SS (106) ±0-49 (64) ±0-83 (33) I-8I (12) Rate of rise (V/s) (46) 226 ±0-15 (109) (II) Rate of fall (V/s) 2481 ±1-24 (47) 8-85 ±0-54 (109) 5-22 ±1-23 (II) Length (ms) 974 ±0-30 (76) IO-I2 ±O-23 (8l) The figures are the means + the standard error. The figures in brackets are the numbers in each sample. Action potentials persist in dissected animals that are bathed with salines containing 15 mm manganese but the height of the overshoot is significantly lower than in animals dissected under normal saline. The addition of 30 mm cobalt to the normal saline bathing the muscle cells has no significant effect on the size of the overshoot but the addition of 90 mm cobalt reduces it from to 5-42 mv. In fact the effect of 90 mm cobalt is very similar to that of reducing the external calcium concentration; all parameters of the action potential are reduced. 90 mm cobalt, however, also causes a decrease in the level of the resting potential (Table 4), a characteristic that is shared to a lesser 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 in Fig. 8 and in Table 5. In salines containing twice the normal concentration of chloride the size of the overshoot is significantly decreased. On the other hand a significant increase in the height of the overshoot is obtained when the concentration of chloride outside the cell is decreased, but the graph in Fig. 8 clearly does not describe a single straight-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 slope abruptly decreases to 0-75 mv for a similar concentration change and finally there is a slight increase to 2-7 mv. There are no changes in the other parameters of the action potential that correlate completely with the alterations in the size of the overshoot

11 Water balance of a serpulidpolychaete M. enigmatica. IV j O [Cl] 0 (mm) 1000 Fig. 8. Graph to show the relationship between the height of the action-potential overshoot of class I cells and the concentration of chloride in the bathing medium. The insets show electrical activity recorded from animals bathed in solutions of chloride concentration marked by the point on the graph nearest each inset. Where the records show three traces, the lowest displays the differentiated signal derived from the action potential. except for the changes in the length of the action potential (measured from the threshold to the lowest point of the undershoot). Increasing the external chloride concentration appears to decrease the length of the action potential, the reverse being found when the 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 calcium in the external medium is reduced to a tenth of the normal level. If the chloride concentration is also reduced by a similar amount the overshoot of the action potential is 7-40 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 the overshoot (Table 5), i.e. there is a significant increase in height compared with the size in normal saline. There is also a significant increase in the length of the action potential.

12 362 HELEN LE B. SKAER Saline Normal 1030 mm-cl~ 50 mm-cl- Cl- free 5omM-Cl~; 3 mm-ca 2+ Hypersaline control Table 5. The influence of external chloride concentration on the action potentials of the longitudinal muscle cells of Mercierella enigmatica Resting potential (129) (18) i8-35 ±0-32 (79) ±o-43 (22) (13) 2O-OO ±0-56 (II) Overshoot II-I (138) S-72 ±i-3s (18) (80) I-IO (22) (IS) OO (») Undershoot ±0-38 (116) ±0-79 (17) (83) (22) (14) (9) Rate of rise (V/s) (46) (18) 7-34 ±o-35 (80) (15) 996 ±069 (II) Rate of fall (V/s) (47) 1288 ±1-30 (18) ±0-72 (81) O-86 (15) (11) Length (ms) 9-74 ±0-30 (76) 7-76 ±0-50 (17) ±0-25 (7i) 1572 ±0-51 (23) (13) II-OO + O-I2 (12) The figures are the means ± the standard error. The figures in brackets are the number in each sample. 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 electrical activity of the longitudinal muscles of Mercierella enigmatica does not diminish when the 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 in dilute saline (with or without sucrose) show if anything an increase in overshoot rather than the decrease that might be expected. The effect on isolated muscle preparations of rapidly changing the ionic content of the bathing medium has been studied by Wells & Ledingham (1940). They found that a rapid decrease in concentration caused inhibition of spontaneous contraction of isolated muscles of Arenicola marina and Nereis diversicolor. In contrast, the ability of Mercierella 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 are swollen (Skaer, 1974a) and therefore more easily damaged. The persistence of electrical and mechanical activity over such a wide range of osmotic and ionic concentration of the bathing medium is clearly advantageous to an osmoconformer such as M. enigmatica, living in a wide range of salinities and often in fluctuating conditions. However, the ability of the longitudinal musclefibresto support action potentials when the ionic concentration of the fluids bathing them varies so widely presents the electrophysiologist with an intriguing problem. There are several theoretically possible ways of overcoming such a problem.

13 Water balance of a serpulid polychaete M. enigmatica. IV Compensating water movements If the cells simply swell when the bathing medium is diluted and shrink in hyperosmotic salines, the internal concentrations of ions in the cell will change so that the normal 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 muscle cells changed quite substantially when the animals were exposed to diluted (40 %) sea water. Although the new concentrations of some ions (Ca 2+ and Mg 2+ ) could be explained by assuming that the cells were swollen by 25 %, the concentrations of some others (notably Na + and K + ) could not. Even where the new internal concentrations could be explained by water influx, the dilution was insufficient to preserve the normal ion gradient across the cell membrane. The maintenance of normal or near normal gradients could not therefore be the result simply of dilution of the cell contents. In Mercierella enigmatica the extent to which the maintenance of normal ionic gradients across the cell membrane results from passive osmotic effects is probably quite small. Measurements described previously (Skaer, 1974 a) indicate that the cells are 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 maintained by active regulation must therefore be considered. 2. Active regulation The normal ionic gradients across the excitable membrane could be maintained by the activity of ion pumps. If this were the case the persistence of electrical activity after changing the composition of the bathing medium might be upset by poisons that inhibit the pumps. Ouabain, a cardiac glycoside, specifically inhibits the sodiumpotassium exchange pump (Schatzman, 1953). When dissected animals are exposed to dilute saline with sucrose in the presence of ouabain, the action potentials continue and are enlarged in size, as they are in isotonic dilute saline alone. The electrical activity does eventually diminish after many hours, as it does in control preparations exposed to io" 4 M ouabain in normal saline. It is clear that the adaptation of the muscle cells to a dilute ionic environment does not depend on the activity of a sodium-potassium exchange pump. The normal ionic gradients across the muscle cell membranes might be maintained after changes in the composition of the bathing medium by ion pumps other than the ouabain-sensitive sodium-potassium pump. However, the time course of cyanide poisoning is very long, indicating that the muscle cells either contain large stores of ATP or can resynthesize ATP without recourse to respiration. In view of this, further experiments using general metabolic poisons have not been pursued. 3. Low intracellular concentrations If the intracellular concentration of the ion that carries the inward current was extremely low, the gradient across the cell membrane might be sufficient to give a full action potential overshoot over the range of external concentration of that ion experienced by the animal. 24 E X B 60

14 364 HELEN LE B. SKAER 4. Variation in 'channel' density If the number of' channels' allowing the ion carrying the inward current to cross the membrane was normally limiting and if the number were to increase as the concentration of the ion in the external medium decreased, the action potential overshoot might remain stable. If any of these three possibilities (2-4) were true, one would not expect the overshoot to be any more sensitive to the alteration of the external concentration of a single ion than to the alteration of the concentrations of several or all the ions. In fact the sizes of the overshoot and undershoot would not be expected to change at all and the Nernst slopes 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, such that the net inward and outward currents do not change as long as the relative concentrations of the ions in the extracellular medium remains constant, no matter what their absolute concentrations may be. The ionic basis underlying such effects can be studied by varying the concentration of one ion alone in the bathing medium. These experiments would also differentiate between the possibilities summarized in sections 2-4 and antagonistic ion effects. In this study discussion will be restricted to the overshoot of 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 the action potential either by altering the sodium concentration of the bathing medium over a very wide range or by introducing tetrodotoxin into the external solution suggests that sodium ions play no part in the passage of the inward pulse of current across the cell membrane. On the other hand, altering the external calcium concentration does produce an effect on the size of these parameters. If the height of the overshoot of the action potential is plotted against the log of the external calcium concentration (Fig. 7), a straight-line graph is obtained whose slope gives afigureof m^ change in height of the overshoot for a ten-fold change in external calcium concentration. If the membrane were permeable only to calcium ions, when the inward current of the action potential isflowing, the Nernst equation predicts that the slope for a ten-fold concentration change would be 29 mv. Thus, the passage of calcium ions into the cell does not account entirely for the inward current. The influence of competitors for the calcium channels in the active membrane is not large unless they are present in rather high concentration. However, Geduldig & Junge (1968) working with Aplysia neurones found that a concentration of cobalt approximately three times greater than the calcium concentration in the medium was necessary to block the calcium component of the overshoot. This is also found in Mercierella enigmatica, though even a 90 mm concentration of cobalt in the medium does 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 the action potential but that calcium ions probably do, though there is a component of the

15 Water balance of a serpulid polychaete M. enigmatica. IV 365 inward current that must be carried by another ion. The occurrence of calciummediated 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 by more than one ion have also been recorded in other preparations (smooth muscle: Biilbring & Kuriyama, 1963; frog heart: Niedergerke & Orkand, 1966a, b; snail neurones: Kerkut & Gardner, 1967; Geduldig & Junge, 1968; Krishtal & Magura, 1970; Jerelova, Krasts & Veprintsev, 1971; Sattelle, 1974) although in all of these preparations the inward current is carried partly by sodium ions. Chloride Alterations in the chloride concentration of the bathing medium have a profound effect on the size of the overshoot if the concentration of chloride is increased but only a small effect if the concentration is reduced. The size of the overshoot is significantly reduced when the chloride concentration in the bathing medium is doubled. The osmotic pressure of the high-chloride saline is large (1800 mosm) and it is conceivable that the reduction is due to an osmotic effect on the cell. If the muscle cells shrink in hyperosmotic media, the intracellular concentration of ions would be increased and thus the gradient of calcium across the cell membrane would be reduced. This would tend to reduce the size of the overshoot. However, if the osmotic pressure of normal saline is increased by adding sucrose to it (2020 mosm) and animals are perfused with this hypertonic saline, the size of the overshoot does not decrease, but rather shows a tendency to increase slightly in size. It seems then that the reduction of the overshoot in 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 the normal 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 increase in overshoot produced by reducing chloride alone (0-69 mv). It is possible that the influence of external chloride concentration on the size of the overshoot is enhanced if the calcium concentration is also reduced. Such an interdependence of ion permeabilities 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 and chloride in the external medium is that the height of the action potential is not markedly altered compared with the height in normal saline. The insensitivity of the overshoot can 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 in opposite senses. This means that the size of the overshoot is not very much altered if the ratio of calcium ions and chloride ions in the medium bathing the muscle cells remains unchanged. Although the concentration of the body fluids of Mercierella enigmatica varies over an enormously wide range, the ratios of the concentrations of ions in the blood do not change very much as is shown in Table

16 366 HELEN LE B. SKAER Table 6 Equilibration medium Glass-distilled water Aquarium sea water 150% sea water Dilute saline in blood or saline bathing the tissues I The Normal 1030 mm-cl~ 50 mm-cl- Cl- free 5omM-Cl~; 3 mm-ca a+ 3 mm-ca' + Distilled water adapted (blood Cl- = 67-8 HIM) Dilute saline + sucrose Hypersaline adapted (blood Cl- = 843 HIM) Hypersaline control Table 7 Action-potential Action-potential length overshoot Saline (ms) N II-OO + O-I figures are the means ± the standard error. N is the number in> each sample II-I6±O ±i ± ! I-IO 7-40 ± When animals were dissected and perfused with a dilute saline (made up on the basis of 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 ratio of 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 different salines is shown in Table 7. These results can be interpreted in terms of the distribution of chloride across the membrane. If it is assumed that the external concentration of chloride is normally greater than the internal concentration, then an increase in the length of the action potential would be correlated with a decrease in the size of the gradient (salines 2, 3, 4, 6, 7; by reducing external chloride) and vice versa (salines 1 and 8). Moreover, the magnitude of the change in duration is correlated with the degree to which the chloride gradient is altered (cf. 2 and 3 or 1 and 8). The increase in the duration of the action potential in the hypersaline control (9) (2020 mosm) can also be explained in terms of the chloride gradient. If the cells shrink, the internal chloride concentration might increase and the gradient might therefore be reduced. The chloride concentration in 3 mm-ca 2+ (5) is not different from that in normal saline and this is reflected in the unaltered length of the action potential. Similarly the durations of the action potentials recorded from muscles bathed in salines 2 and 4 are not significantly different and these two salines both contain 50 mm-cl~.

17 Water balance ofa serpulidpolychaete M. enigmatica. IV 367 Table 8 Tissue Frog muscle Crab muscle (Carcinus maenas) Squid nerve Toad nerve Human erythrocyte Alga (Nitella translucens) Intracellular chloride concentration (mm) IS' Extracellular chloride concentration (mm) 775 (plasma) 524 (blood) 520 (sea water) 520 (sea water) in (interfibrillar connective tissue) in (plasma) 1-3 (pond water) Source Conway, 1957 Shaw, Steinbach, 1941 Koechlin, 1954 Shanes & Berman, 1955 a, b Davson, 1964 MacRobbie, 1962 It does seem then that there is quite a good correlation between the size of the chloride gradient and the duration of the action potential and, when the concentrations of other ions are unaltered, between the duration of the action potential and the size of the overshoot (Table 7). The chloride effect Calcium and chloride ions appear to interact in the generation of the overshoot. The involvement of chloride could either be indirect, for example, by influencing the calcium conductance, 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 calcium influx and a chloride efflux. However, if an efflux of chloride leading to an overshoot is postulated, it is necessary to assume that the concentration of chloride in the muscle cells is greater than that in the extracellular fluids. This is not the case generally in animal cells; the concentrations of chloride for some animal and plant cells are shown in Table 8. Only in the large algal plant cells (e.g. Nitella translucens) would an efflux of chloride be favoured (Gaffey & Mullins, 1958) where, since the cells lie naked in pond 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 frog muscle fibres is carried by an influx of chloride ions. If such an influx carried the repolarizing current in the muscle of Mercierella enigmatica, an alteration in the chloride gradient would have an effect both on the rate of fall of the action potential and on the size of the undershoot. Neither of these parameters changes in a way that is consistent with 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 it would occur before the undershoot is expressed. Such an influx of chloride could antagonize the effect of a calcium influx and the peak of the overshoot would be reached when the calcium and chloride currents were balanced so that the result of an increase in either the chloride conductance or the gradient of chloride across the membrane would be an increase in the chloride current and so a reduction in the size of the overshoot (see Fig. 9). Thus an increase in the external chloride concentration would tend to reduce the height of the action potential and conversely a reduction in the chloride

18 368 HELEN LE B. SKAER Normal saline Normal Ca" 1/10 Cl- 1/10 Ca 2+ Normal Cl" 1/10 Ca 2 + 1/10 Cl- Overshoot 1116mV mv 0-85 mv 7-40 mv H- 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 actionpotential overshoot in different external media. concentration would increase it. In fact this increase of the overshoot is only seen when the external calcium is also reduced and not when the external chloride is reduced in otherwise normal saline (Table 5). This effect could be explained by supposing that the chloride conductance is small compared with the calcium conductance so that the enhancing effects of reduced external chloride are normally swamped by the calcium current. These ideas are summarized in Fig. 9. It is not immediately clear how this hypothesis fits the data concerning the duration of the action potentials. Since the chloride current is not the principal inward or outward current, changes in its expression will probably have little effect on the maximum rates of rise and fall of the action potential. The present results certainly do not show any clear relationships between the chloride gradient and the signals from the differentiating circuit. But even if the rates of rise and fall are unaltered by the size of the chloride gradient, the duration of the action potential will be related to the height of the overshoot, 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 potential in M. enigmatica. The first hypothesis could contribute to but not account for its stability. Hypotheses 2-4 all predict horizontal Nernst slopes for the ions involved in carrying the inward and outward currents of the action potential. This prediction is not borne out by experiment. Evidence has been presented to show that the height of the overshoot of the action potential results from the combined effects of calcium and chloride currents. These currents interact in such a way that the overshoot does not change in height as long as the ratio of calcium and chloride in the medium bathing the muscle cells remains unaltered (Table 6, Fig. 9). Such interacting ion effects might account for the stability of the whole of the resting and action potential of the longitudinal muscle cells of M. enigmatica. When the concentration of the external environ-

19 Water balance of a serpulidpolychaete M. enigmatica. IV 369 ment is changed the ratios of the concentrations of the ions in the blood are remarkably little altered despite large changes in their absolute concentrations (see Skaer, 19746). Thus the persistence of electrical activity in Merrierella, and possibly in other osmoconforming species, could depend on the maintenance of the ratios of concentrations of ions in the body fluids rather than on the preservation of fixed concentrations of each ion species. SUMMARY 1. The electrical activity of the two types of longitudinal muscles of an osmoconforming polychaete worm, Merrier ella enigmatica, have been studied in media of widely varying osmotic and ionic composition. Activity persists practically unaltered in both types of muscle cell. 2. The possible effects of osmotically induced changes in cell volume on the ionic gradients across the cell membranes are considered. It is concluded that the normal gradients are unlikely to be maintained as a result of such changes. 3. The involvement of ion pumps in the maintenance of the normal gradients across the muscle cell membranes has been studied using specific and metabolic poisons. It is evident that the persistence of electrical activity in media of altered ionic content does not depend on the sodium-potassium exchange pump. 4. The ionic basis of the overshoot of action potentials recorded from cells of the small resting potential type has been studied. It is concluded that calcium ions but not sodium ions are responsible for the inward current although there is a component of the inward current carried by some other as yet unidentified ion. 5. Alterations in the external concentrations of chloride ions are found to alter both the height of the overshoot and the length of the action potential. 6. Profound alterations in the overshoot height are produced only when the normal ratio of calcium to chloride concentration in the external medium is altered. Possible mechanisms 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, depends on the constancy of the ratios between the concentrations of the ions in the fluids bathing the cells and not on the absolute concentrations of the ions. This work was supported by a Science Research Council grant and a studentship from Girton College, Cambridge. I am very grateful to my supervisor, Dr J. E. Treherne 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 grateful to Mr J. Rodford for help in drawing the figures. REFERENCES ABBOTT, B. D. & PARNAS, I. (1965). Electrical and mechanical responses in deep abdominal extensor muscles of crayfish and lobster. J. gen. Physiol. 48, BOLBRING, E. & KURIYAMA, H. (1963). Effects of changes in the external sodium and calcium concentrations on spontaneous electrical activity in smooth muscle of guinea-pig taenia coli. J. Physiol. 166, CONWAY, E. J. (1957). Metabolic Aspects of Transport across Cell Membranes, pp Ed. Murphy. Univ. of Wise. Press, Madison. DAVSON, H. (1964). Textbook of General Physiology. 3rd Ed. Churchill.

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