The Inorganic and Amino Acid Composition of Some Lamellibranch Muscles

The electrolyte composition of vertebrate striated muscle has been the subject of many investigations, and some of the principles governing the distribution of electrolytes between the muscle fibres and the plasma are fairly well understood. In comparison with the plasma the sarcoplasm of striated muscle contains a low concentration of sodium and chloride and a high concentration of potassium. This unequal distribution is a dynamic equilibrium maintained by the active extrusion of sodium from the cell. The distribution of potassium and chloride is very close to a Donnan equilibrium across the potential gradient produced by the sodium extrusion and by a concentration of indiffusible anions inside the fibres. Less is known about ionic equilibrium in invertebrate muscle, but a recent detailed investigation of the fibres of the muscles of Carcinus maenas suggests that the conditions are similar to those found in the vertebrates (Shaw, 1955). Gross analyses of Loligo muscle (Manery, 1939) would fit into the same picture if allowance is made for a small quantity, about 5%, of extracellular fluid.

Vertebrate smooth muscles (Manery & Bale, 1941 ; Wilkins and Cullen, 1933) contain a much higher proportion of sodium and chloride than striated muscle but this may be due, in part at least, to a higher proportion of extracellular fluid between the fibres. Bulbring & Born, quoted by Holman (1957), estimate that the extracellular fluid in the taenia coli muscle of the guinea-pig amounts to 36% of the total wet weight. A large number of analyses of invertebrate smooth muscle have been published, but as the analyses are rarely accompanied by measurements of the proportion of extracellular fluid it is difficult to draw any firm conclusions about the intracellular concentrations of ions. The analyses of the whole muscle usually show a relatively high concentration of sodium and chloride, sometimes amounting to as much as half the concentrations of the same ions in the blood (Singh, 1938; Krogh, 1939, p. 60; Steinbach, 1940), but it is not clear if this is accounted for by a high proportion of extracellular fluid or if the conditions of ionic equilibrium in invertebrate smooth muscle differ from those found in striated muscle.

To throw further light on this problem, measurements have been made of the concentrations of the chief ions—sodium, chloride and potassium—in the sarcoplasm of a variety of lamellibranch muscles. The lamellibranchs form a convenient source of substantial quantities of muscle uncontaminated by other tissues. Unfortunately, the small size of lamellibranch muscle fibres, usually about 8 p in diameter, makes it impracticable to analyse individual fibres; instead, analyses have been made of the whole muscle and the volume of contaminating blood has been estimated by inulin. The muscles which have been analysed are the anterior byssus retractor and the fast and slow portions of the adductor muscle of Mytilus edulis, and the fast and slow portions of the adductor muscle of Pecten maximus. The fast adductor of Pecten is striated and generally similar in physical properties to crab or frog muscle (Abbott & Lowy, 1956), and therefore provides a convenient link with better known muscles. The freshwater species Anodonta cygnaea has a very low concentration of ions in the blood and in the muscle, and analyses have been made of the two portions of the adductor muscles of this species. In order to widen still further the range of conditions, analyses have been made of Mytilus adductors and byssus retractors when the animals are adapted to 50% sea water, and of Anodonta adductors when the animals are adapted to 18% sea water.

Inorganic ions account for only a fraction of the osmotic pressure of the tissues of the marine species, so measurements have been made of the concentrations of phosphate compounds and free amino acids in order to define more closely the differences between the marine and freshwater species.

The fluxes of sodium through the muscle cells of the ventricles of Mytilus and Anodonta have also been measured and will be described in a later paper, but for convenience the results of the analyses of these two tissues are included here.

The animals were kept in well-aerated tanks between 10° and 16° C. Both Mytilus and Anodonta gain sodium and lose potassium from the tissues when kept under laboratory conditions, so animals were used within 1 week of arrival, unless otherwise stated.

After excision and cleaning the muscles were blotted to remove surface fluid. The water content was taken as the loss of weight after drying overnight at 105° C.

Sodium and potassium were estimated by flame photometry using an ‘EEL’ flame photometer. Measurements were made at maximum dilution, 0-05-0-1 mM./l. The standard solutions used contained both sodium and potassium in similar concentrations to the samples analysed. When small quantities of muscle (5-50 mg.) were dry-ashed with concentrated sulphuric acid significant quantities of sodium were lost at 500° C. Complete solution without loss was obtained by evaporating to dryness on a water-bath with o-i ml. concentrated nitric acid and then dissolving in 5 or 10 ml. of water. Calcium was estimated by precipitating as oxalate and titrating with ceric sulphate (Robertson & Webb, 1939). Magnesium was precipitated as hydroxyquinolate and also titrated with ceric sulphate (Robertson & Webb, 1939). Chloride was estimated by the microdiflFusion method of Conway (1947). Sulphate was estimated gravimetrically as barium sulphate after removing the protein with picric acid. Phosphate was estimated colorimetrically by the method of Fiske & SubbaRow (1925). Total free amino acids were estimated by the method of Van Slyke, MacFadyen & Hamilton (1941). In this method the carbon dioxide evolved on decarboxylation with ninhydrin, indane-trione hydrate, is absorbed by barium hydroxide. Both the carboxyl groups in aspartic acid are decomposed by this method, so the relative amount of aspartic acid was estimated by paper chromatography. Two-dimensional chromatograms were run in phenol/ water and butanol/acetic acid and water, and the spots developed with ninhydrin. After development the spots were extracted with 70% acetone and the relative intensities measured by a ‘Spekker’ absorptiometer. The method is not of high accuracy, but the aspartic acid is only about 5 % of the total amino acids in Mytilus, so the correction is small. The method of Van Slyke et al. does not estimate taurine which was reported to occur in large quantities in Mytilus (Kelly, 1904). Taurine was removed from the muscle by repeated extraction with 70% alcohol and precipitated as barium sulphate, after boiling in bromine water for 4 hr. in alkaline solution. The method is not specific as other alcohol soluble compounds containing sulphur, such as isethionic acid, might also be oxidized to some extent. The chromatograms showed that amino acids containing sulphur were only present in small quantities. Hydroxycarboxylic acids were separated on a column of silica gel and estimated as described by Isherwood (1946), but the quantities found were very small.

The volume of extracellular fluid was estimated by injecting a suitable quantity of a 20% solution of inulin in sea water (or in dilute sea water) into a whole animal, and estimating the concentrations of inulin in the blood and in the muscle the following day. The inulin was estimated by the colorimetric method of Roe, Epstein & Goldstein (1949) using an ‘EEL’ absorptiometer. The blood was collected by hypodermic from the ventricle. The inulin was removed from the muscle by soaking 50-100 mg. portions of muscle, not exceeding 1 mm. in thickness, in a suitable saline for 5 hr., with frequent shaking. Extraction was 95 % complete in an hour.

The adductor muscles of Pecten and Mytilus are large enough to allow several determinations of sodium, potassium and chloride to be made on one animal. Tables 1 and 2 show the results of a number of such analyses. Each figure for sodium and potassium is the mean of the analyses of three separate samples of muscle. Each chloride figure is the mean of two separate determinations. The water contents of the muscles are given in Table 3.

The ventricle of Mytilus, and to some extent the byssus retractor, are rather small for simultaneous determinations of sodium, potassium and chloride, so only the mean values and standard errors of a large number of separate determinations of these quantities are given (Table 4).

The sodium and chloride content of Anodonta muscle is very variable but much of the variation is clearly related to the variation in the blood composition. When expressed as a percentage of the concentration in the blood, the sodium and chloride results are more consistent (Table 5).

The muscles of the marine species show very wide but systematic variations in their sodium, potassium and chloride content. The muscles may be arranged in a series on the basis of their inorganic content: Pecten fast adductor, Pecten slow adductor, Mytilus fast adductor, Mytilus ventricle, Mytilus slow adductor, Mytilus byssus retractor. In this series the sodium and chloride contents of the muscles are steadily increasing and the potassium contents of the muscles are steadily falling. It is also the order of increasing time of stress relaxation (or, more generally, decreasing speed of muscle) where this is known (Abbott & Lowy, 1956, 1957). The fastest muscle, the fast portion of Pecten adductor, is similar to Carcinus striated muscle in that it contains little sodium or chloride, whereas the slowest muscle, the byssus retractor, contains almost half as much sodium and chloride as the blood.

The concentrations of ions found in the Anodonta muscles are necessarily much lower than in the marine species because the blood has a total osmolar concentration of only 4-5 % of sea water, but otherwise the picture is similar. The slower muscle contains the greater quantities of sodium and chloride, amounting in this case to more than half of the blood concentration.

The whole muscle contains considerable quantities of extracellular fluid which must be allowed for when calculating the intracellular concentrations of the ions. The extracellular fluid has been assumed to be identical with the space accessible to inulin (Table 6). The sodium, potassium and chloride contents of the blood of Anodonta are given in Table 5. The sodium and chloride content of the blood of Pecten and of Mytilus is assumed to be the same as that of the sea water in which they had been kept before analysis. The potassium content of the blood of Pecten and Mytilus is rather higher than that of sea water, and was measured at the same time as the potassium content of the muscle (Tables i and 4). The mean sodium content of the blood of Anodonta is rather lower than a previously published figure by the author (Potts, 1954). This is probably because Birmingham tap water has a much lower sodium content than Cambridge tap water.

The intracellular concentrations of sodium, potassium and chloride (Table 7, column 3) are calculated from the analyses of the whole muscles (Table 7, column 1) and from the quantities of ions in the inulin spaces of the muscle (Table 7, column 2). The results are expressed in mM./kg. fibre water. The intracellular concentrations of sodium and chloride are calculated from the small difference between two large and variable quantities, and so are peculiarly subject to error. However, the results are fairly consistent, and show that as the speed of the muscle increases, the intracellular sodium and chloride fall and, to a lesser extent, potassium rises. In the fast adductor of Pecten the intracellular concentrations of sodium and chloride are less than one-tenth of the concentrations of the same ions in the blood, and in this respect the muscle is similar to vertebrate striated muscle. In the slow adductor muscles, especially the slow adductor of Anodonta, the relative intracellular concentrations of chloride are very much higher.

The total osmolar concentration inside the muscle fibres of Anodonta is only about one-twentieth of that of the marine species. Fuller analyses have been made of the ventral adductors of Mytilus and Anodonta to define more clearly the differences between the marine and freshwater species.

The calcium, magnesium and sulphate concentrations in Mytilus (Table 8) are broadly similar to those found in the muscles of other marine animals. Shaw (1955) reported a calcium concentration of 5-4 mM./kg. fibre water in the muscles of Carcinus maenas. The magnesium concentration in the ventral adductor of Mytilus is about twice as great as in Carcinus, 34 mM./kg. fibre water compared with 16 mM./kg. fibre water, but the concentration of magnesium in the blood is correspondingly higher, 56 mM./kg. water compared with 21·2 mM./kg. water. The concentration of magnesium in Anodonta blood, 0·02 mM./kg. water, is exceedingly low, but the muscle still contains 5·2 mM./kg. fibre water. Florkin & Duchâteau (1950) reported a magnesium concentration of 7·1 mM./kg. fibre water; the fibre water was calculated on the assumption that all the chloride was extracellular and the portion of the adductor analysed was not specified.

Phosphagen phosphate, inorganic phosphate and adenosine triphosphate have been measured in both Mytilus and Anodonta (Table 9). The highest concentration of phosphate in whole muscle is found in the fast portion of Mytilus adductor. Some of the difference between the two portions of the adductor is due to the greater proportion of blood in the slow muscle, but the difference is not abolished even when allowance is made for this extracellular fluid. The freshwater animal contains about half the phosphate concentration of the marine animal in spite of its very much lower blood concentration. The phosphate compounds were conserved more effectively than any other measured variable when the Unionidae became adapted to fresh water.

The concentrations of free amino acids in Mytilus and Anodonta were estimated by measuring the amount of carbon dioxide evolved on decarboxylation with ninhydrin. Aspartic acid, but not glutamic acid, decomposes to give 2 mmoles of carbon dioxide for 1 mmole of acid. Taurine, a sulphonic acid, is not estimated by this method. Taurine and aspartic acid were estimated separately by other means, and the total free amino acids calculated by adding taurine to the amount estimated by decarboxylation and subtracting the amount of aspartic acid (Table 10). Kelly (1904) estimated that taurine made up 5% of the dry weight of Mytilus muscle, corresponding to 100 mM./kg. water in the whole muscle. The chromatograms show that Mytilus muscle contains considerable quantities of glycine, alanine, glutamic acid and arginine and some histidine, tyrosine and threonine. Anodonta muscle contains glycine, arginine and glutamic acid. The concentration of amino acids in Mytilus blood is only 2·5 mM./kg. water, and is even lower, 0·5 mM./kg. water, in Anodonta. Duchâteau, Sarlet, Camien & Florkin (1952) estimated the quantities of free amino acids, excluding taurine, in Mytilus and Anodonta by bioassay. They found that Mytilus muscle contained 166-2 mM./kg. total water and Anodonta muscle contained 9·5 mM./kg. total water; the part of the adductor assayed was not defined.

Hydroxycarboxylic acids occur in very low concentrations in lamellibranch muscle. Only 2·4 mM./kg. total water were recovered from Mytilus muscle.

The sea water in which Mytilus had been living contained 573 mM./kg. water of chloride. This corresponds to 19·6‰ chlorinity, a freezing-point depression of 1·89° C. or an osmolar concentration of just over 1. The identified constituents of Mytilus muscle amount to less than 700 mM./kg. fibre water (Table 11). The totals given in Table 11 are themselves too high because in the resting muscle much of the arginine and phosphate, which are included separately in Table 11, are combined. On the other hand, no account is taken in Table 11 of calcium, magnesium or sulphate which may contribute to some extent to the osmotic pressure.

The assayed osmotic constituents of Anodonta muscle amount to a total of 60 mM./kg. fibre water, but the muscle contains at least 8-4 and possibly as much as 10 HIM. arginine phosphate/kg. fibre water in the resting state. The mean freezing-point depression of the blood of a large series of Anodonta amounted to 0·078° C. or 42 mM./kg. water (Potts, 1954). Some of the inorganic phosphate recorded in Table 9 may be in compounds not exerting an equivalent osmotic pressure. Calcium and manesium in Anodonta muscle (Table 8) must be largely combined with organic molecules.

Mytilus occurs in estuaries and can tolerate considerable changes in the composition of the blood. The sodium, potassium and chloride contents of muscles from Mytilus which had been living for at least 2 weeks in 50% sea water are given in Table 12.

The percentage dry weight of the muscles is less than that of muscles from normal sea water (Table 3), and the inulin spaces have also been reduced (Table 6), so the individual muscle fibres must be osmotically swollen. From the figures in Tables 3 and 6 it follows that, when allowance is made for the solids in the blood, 100 g. of solids are associated with 247 g. of water in the fibres of the fast adductor of Mytilus from normal sea water, but are associated with 311 g. of water in animals from 50 % sea water. Similarly, in the byssus retractor 100 g. of solids are associated with 266 g. of water in normal sea water and 319 g. in 50% sea water. In each case the distension is much less than the doubling of water content which would occur if the muscles behaved as simple osmometers. The fall in the potassium concentration of the muscles is a little greater than that caused by the osmotic uptake of water alone, but the fall in the sodium and chloride concentrations is very much greater. In the Mytilus muscles in normal sea water the average intracellular concentrations of sodium and chloride are 88 and 87 mM./kg. water respectively (Table 7). In 50 % sea water these concentrations have fallen to 30 and 22 mM./kg. water.

The amino-acid content of the muscle also falls by an amount greater than that due to water uptake alone. In the normal ventral adductor the intracellular concentration of free amino acids is 295 ±6 mM./kg. water (Table 10). In 50% sea water this falls to 183 ± 6 mM./kg. water. The uptake of water should have reduced the concentration to 234 mM./kg. water ; the remainder of the reduction must be caused by the excretion, polymerization or metabolism of the amino acids. In the byssus retractor the intracellular concentration of free amino acids is 273 ± 6 mM./kg. water when the animals are in sea water, and only 166 ± 6 mM./kg. water when the animals are in 50% sea water (Table 10). The osmotic swelling of the muscle fibres alone would reduce the concentration to 228 mM./kg. water. The concentration of free amino acids in the blood has risen to 5·4 mM./kg. water when the animal is in 50% sea water from 2·5 mM./kg. water in normal sea water. Some of the amino acids in the blood are probably derived from the muscles.

Analyses of Anodonta which had been living for 10 days in 18% sea water are recorded in Tables 3, 6, 7, 10 and 13. The percentage dry weights of the muscles and the inulin spaces have increased in 18% sea water. In an Anodonta from tap water 100 g of dry matter in the ventral adductor are associated with 440 g. of intracellular water. In 18 % sea water this has fallen to 187 g. of intracellular water. In the dorsal adductor the figures are 415 and 204 g. respectively. Exosmosis has removed over half the fibre water. Correspondingly, the intracellular concentrations of potassium have also doubled in 18% sea water (Table 7), but the intracellular concentrations of sodium and chloride have increased eight- or tenfold.

In both Anodonta and Mytilus adaptation to a changed osmolar concentration is brought about partly by water movement into or out of the muscle fibres, and partly by the increase or decrease of the total content of sodium, chloride and free amino acids in the muscles.

In many of the muscles analysed the distribution of potassium and chloride across the fibre membrane appears to differ significantly from a Donnan equilibrium. For example, in the byssus retractor muscle of Mytilus from normal sea water the potassium concentration ratio between the inside and the outside of the fibres is 12·2 ±0’4, while the chloride is 3·9 ±0·3. Similarly, in Anodonta slow adductor the potassium ratio is 38 ± 3·0, while the chloride ratio is only 2·0 ± 0·35 (Table 14). In vertebrate striated muscle, in some invertebrate striated muscle and in many vertebrate and invertebrate nerves it is well established that the distribution of potassium and chloride approximates to a Donnan equilibrium (Boyle & Conway, 1941; Hodgkin, 1951; Shaw, 1955). The last two columns of figures in Table 14 therefore require careful consideration. The fast adductor of Pecten, the only muscle in the table comparable to vertebrate striated muscle or to Carcinus muscle in physical properties, has potassium and chloride concentration ratios close to a Donnan equilibrium. In the remaining muscles it is generally the slower muscles which differ most from the equilibrium conditions. Either the principles governing ionic equilibrium in lamellibranch smooth muscles differ from those in striated muscle, or some important factor has been neglected in calculating the results in Table 7.

In every case the apparent high concentration of chloride inside the fibres is associated with a high concentration of sodium. If the inulin space is not identical with the extracellular fluid, but is for some reason somewhat smaller, then the calculated concentrations of sodium and chloride inside the fibres will be too high. For example, if it is assumed that all the sodium in the byssus retractor muscle of Mytilus is extracellular, then the extracellular space would amount to 43 % of the total water content, the intracellular chloride concentration would be reduced to 46 mM./kg. water and the intracellular potassium would be increased to 191 mM./kg. water which would be close to the conditions of a Donnan equilibrium. This could be the case if the muscle contains a third phase, neither blood nor sarcoplasm, inaccessible to inulin but containing large quantities of sodium and chloride. Such a third phase could be formed either by connective tissue between the fibres or by a specialized region of the fibres such as the sarcolemma. In the vertebrates connective tissue contains large quantities of sodium and chloride (Manery & Hastings, 1939). Olson (1938) described connective tissue between the fibres of Thyone muscle, which he correlated with the high chloride content of the muscle, but an histological examination of Mytilus byssus retractor by the present author, using the same technique as Olsen (that is, fixing with Bouin and staining with Mallory’s triple stain) revealed only negligible amounts of connective tissue.

Carey & Conway (1954) have described experiments on frog sartorii from which they conclude that most of the non-extracellular sodium is in the sarcolemma and only a small quantity is in the sarcoplasm. In order to account for the discrepancy between the observed and the equilibrium concentrations in Mytilus byssus retractor the sarcolemma would have to contain the very large quantity of 100 mM. Cl/kg. muscle. It is difficult to reconcile the potassium and chloride content of lamellibranch smooth muscle with Conway’s model of a simple Donnan equilibrium.

In a recent review Robertson (1957) has discussed the distribution of potassium and chloride ions in the muscles of a number of invertebrates. On the basis of his own analysis of Nephrops, Shaw’s analysis of Carcinus (1955) and Krogh’s analysis of Mytilus and Eriocheir (1939) he concludes that only in Carcinus does the distribution approximate to a Donnan equilibrium. In the remaining animals the chloride concentration inside the fibres exceeds the equilibrium concentration.

I am grateful to the Director of the Marine Biological Association Laboratory, Plymouth, for hospitality and facilities during the course of part of this work.