PATTERNS OF WATER AND SOLUTE REGULATION IN THE MUSCLE FIBRES OF OSMO- CONFORMING MARINE DECAPOD CRUSTACEANS

Transcription

1 J. exp. Biol. (1978), T* Ity7 With 8 figures Printed in Great Britain PATTERNS OF WATER AND SOLUTE REGULATION IN THE MUSCLE FIBRES OF OSMO CONFORMING MARINE DECAPOD CRUSTACEANS BY ROBERT W. FREEL Department of Biology, University of California, Los Angeles, California (Received 26 May 1977) SUMMARY 1. The patterns of myoplasmic water and solute regulation were examined in several species of marine decapod crustaceans acclimated to various salinities. Three osmoconformers (Callianassa cahformemsis, Cancer antennarius, Emerita analoga) and one weak osmoregulator {Pachygrapsus crassipes) were studied. 2. Although haemolymph water and solute activities varied with adaptational salinity in these species, regulation of cell water content was apparent in all but 25 % s.w. acclimated Pachygrapsus. Cell water regulation was correlated with the regulation of ninhydrinpositive solutes. An estimate of the extent of the regulation was achieved by comparing the observed cell hydration changes to that predicted for an ideal osmometer and to that of a perfect volume regulator. It was concluded that the extent of cell volume regulation was variable and incomplete, particularly in dilute salinities. 3. Net losses of myoplasmic sodium, chloride, and ninhydrinpositive nitrogen were observed in animals acclimated to dilute sea water, while net gains of these solutes were noted in crabs acclimated to concentrated sea water. In contrast, the amounts of cell potassium and calcium plus magnesium were relatively constant fractions of the cell dry weight at any salinity. 4. A simple mechanism, br.sed upon Donnan considerations, is sufficient to account for the constancy of myoplasmic potassium and the variability of cell chloride with changing extracellular osmolarity. INTRODUCTION A variety of marine decapod crustaceans survive substantial changes in the osmotic pressure of their environment (Kinne, 1971). Such tolerance is not restricted to those with moderately developed capacities of extracellular osmoregulation, but is also observed in strictly osmoconforming species. In both osmoconforming and weakly osmoregulating crustaceans the haemolymph osmotic pressure and ionic composition will vary with external salinity. Studies of the consequences of such variable haemolymph osmotic composition have, for the most part, centered about the phenomenon of isosmotic intracellular volume regulation (Schoffeniels & Gilles, 1970). Since the cell volume regulation process is not necessarily complete (Lange, 1970), alterations in

2 108 ROBERT W. FREEL the concentrations of intracellular solutes not involved in the regulation process may also be observed. With few exceptions (Shaw, 1955, 1958; Hays, Lang & Gainer, 1968), little attention has been directed to the description and ultimate understanding of the effects of alterations in the ratios (haemolymph/cellular) of inorganic solutes in osmoconforming or weakly osmoregulating marine crustaceans. The potential physiological consequences of such water and solute adjustments are manifold, ranging from the disruption of normal membrane electrophysiological properties (Pichon & Treherne, 1976) to alterations in the activities of intracellular biochemical pathways (Hochachka & Somero, 1973). The present study was initiated to provide a comparative description of the patterns and magnitudes of intracellular water and solute regulation in decapod crustaceans inhabiting marine environments of varying degrees of osmotic stability. The ultimate aim of such studies is to provide a unified theoretical account for the observed patterns of intracellular water and solute adjustments in the tissues of these and other osmoconforming marine organisms. The results of this study also serve as a foundation for another report dealing with some passive membrane electrophysiological properties (Freel, 1977a) and for future studies on the active membrane properties of muscle tissue from these crustaceans. MATERIALS AND METHODS Experimental animals Ghost shrimp, Callianassa californiensis Dana, were collected from the mud flats at Morro Bay, California, and purchased from bait dealers in San Diego, California. The subtidal rock crab, Cancer antennarius Stimpson, the semiterrestrial shore crab, Pachygrapsus crassipes Randall, and the sandy intertidal mole crab, Emerita analoga Rathbun, were collected at various localities in Los Angeles County, California. Crabs were maintained in a recirculating sea water system containing natural sea water (100% s.w. 2: 1000 mosmol) at 18 C. Mature, intermoult specimens of either gender were used in the following analyses. Experimental animals were challenged with stepwise changes in salinity (ca. 100 mosmol per day) and subsequently maintained at the final acclimation salinity for a minimum of 5 days. The final lower acclimation salinity, selected on the basis of preliminary tolerance studies, varied for each species. These final dilutions were chosen such that the survivorship and activity of the experimental group was comparable to control animals in natural sea water. An upper limit of approximately 1500 mosmol was arbitrarily chosen as the other experimental extreme. Reduced salinities were made by tap water dilution of natural sea water, and 150% s.w. was prepared by the addition of a synthetic marine salt mixture (Instant Ocean). Analytical methods The following analyses were carried out on the carpopodite flexor and extensor muscles dissected from the meropodites of the walking legs of the two brachyuran crabs (Cancer and Pachygrapsus). In the two anomurans (Callianassa and Emerita) the abdominal flexor and extensor musculature was used. Excised tissue samples,

3 Water and solute regulation in muscle fibres 109 weighing mg, were vigorously rinsed in an isotonic sucrose solution for 5 s to remove adherent haemolymph, then blotted on filter paper prior to wet weight determination. After overnight drying at 105 C, the inorganic solutes were extracted from the dried tissues in o1 NHNO 3 in sealed tubes for 24 h at 60 C. This procedure proved to be the most efficient and economical of the several commonly employed extraction techniques. Sodium and potassium were determined with a Beckman DU flame spectrophotometer. Standard solutions were prepared to contain the appropriate levels of interfering ions. Total calcium plus magnesium (Ca + Mg) was determined by microtitration with EDTA in buffered alkaline solutions using Eriochrome Black T as an indicator (Walser, i960). The osmolarities of the haemolymph and sea water samples were measured with a Mechrolab vapour pressure osmometer. Free amino acids were determined as ninhydrinpositive nitrogen (Nin + N) on ethanolic extracts of fresh muscle (25100 mg) following the method of Clark (1973). Glycine was used as a standard. Since proline, taurine, and other low molecular weight nitrogenous solutes are not detected by ninhydrin, the Nin + N levels reported here are minimal estimates of the free amino acid pools of these crustacean tissues. The extracellular space (ECS) of the muscle tissue was determined by injecting the crabs with 2550 /A of artificial haemolymph containing 14 Cinulin (ICN Radiochemicals). After equilibration of the probe (about 4 h), haemolymph samples were taken and dispensed into scintillation vials. of the same size and type as used for the chemical analyses was excised and vigorously rinsed for 5 sec in labelfree saline before blotting and weighing. Both muscle and haemolymph samples were prepared for liquid scintillation counting by digesting in 05 ml of a tissue solubilizer (ICN Tissue Solubilizer) for several days at room temperature. Ten ml of a scintillation cocktail (Freel, Medler & Clark, 1973) were added to the digested samples. The samples were later counted in a Beckman liquid scintillation counter. All count rates were quench corrected. RESULTS solutes and osmotic pressure Both anomurans, Callianassa and Emerita, are essentially complete osmotic and ionic conformers over the imposed salinity ranges (Tables 2, 3). Cancer is also an osmoconformer (Table 4), but it strongly regulates haemolymph Ca + Mg levels, as does the other brachyuran, Pachygrapsus (Table 5). In addition, Pachygrapsus is a weak osmoregulator in both concentrated and dilute sea water. The osmoregulatory capacity of this species is the result of the moderate regulation of blood sodium and chloride. It is evident, therefore, that haemolymph water activity varies considerably with adaptational salinity in each of the species considered here. Muscle extracellular space The 14 Cinulin spaces for the different species and osmotic conditions are presented in Table 1. The size of the extracellular space of Callianassa abdominal musculature is independent of haemolymph osmolarity. The effect of salinity on the ECS of muscle from Cancer, Pachygrapsus, and Emerita was not determined. Therefore, in

4 no ROBERT W. FREEL Table i. Extracellular space (ECS) estimates of the muscle tissue from various decapods at different acclimation salinities. X±i s.e. (number of animals measured in triplicate) Species CaUianatta Callianatsa CalUantuta Pachygrapsut Emerita Cancer Sea water (%) 35 IOO 150 IOO IOO IOO ECS (gm H,O/gm tissue H,O) oiai (5) 0125±0009 (7) (3) (9) x4(3) (8) the calculations of intracellular water and solute levels which follow the 100% s.w. ECS estimates were used for all salinity conditions in these three species. The interspecific variation in the ECS measurements of muscle from 100% s.w. acclimated crabs is a clear demonstration of the morphological differences of the muscle masses examined. Callianassa and Emerita abdominal musculature consists of large, compact associations of many, relatively small fibres. The extensor muscles of the walking legs of Packygrapsus are composed of loosely associated bundles of fewer, but larger muscle fibres. The bulk of Cancer carpopodite muscles are, in contrast, essentially single fibres which are readily separated in the tissue rinsing process. As shown in Table 1, even these single fibres have a measurable extracellular space. Total intracellular water Muscle cell water contents were determined by subtracting the contribution of extracellular water from the muscle tissue water values in Tables 25. It is apparent that the changes in haemolymph water activity, incurred during salinity adaptation, produce changes in the muscle fibre water contents of these species (Tables 25). The magnitude of the observed change in cell water content is best expressed as the relative change in cell hydration (i? H,o) *& follows: _ (gmff 2 O/gm DW), where the control (100% s.w.) and experimental values (subscripts 1 and 2, respectively) are given in grams of cell water per gram of dry cell weight (DW). In Fig. 1 the calculated values of i? Hl o for the various species have been plotted as a function of the observed relative change in haemolymph osmotic pressure (CJCj). The data are presented on loglog coordinates to emphasize the linear behaviour expected of a perfect osmometer (solid line in Fig. 1) in which i? H o = CJC t. An i? H,o = I " implies strict regulation of both cell water and solids. A more realistic situation is described by the hatched curve in Fig. 1. This curve describes the behaviour of a perfect volume regulator in which the cell volume is maintained by the accumulation or release of a solution isosmotic to C a. In this model the weight of solids lost or gained in the regulation process is considered to be replaced by an equivalent amount of water. The /? H,o value for such a perfect volume regulator is dependent upon both the initial cell water content (here taken to be 233 gm H 2 0/gm DW) and the average molecular weight of the solutes involved in the regulation process. In Fig.

5 Table 2. Solute concentrations in Callianassa californiensis acclimated to various salinities. Concentrations are expressed as mmolfl sea water or haemolymph and mmoljkg tissue or cell water (Where applicable ± I S.E. about the mean (duplicates from > 6 animals) U presented) ^ 35% sw. 100% s.w. 150% s.w. [C] (mosmol) ± ± ±228 H,O 826 ± ±o ± [Na] ± ± ± ± ± ± [K] ± ±o ±O ±2'O I5I ± ± [Ca + Mg] ± ±i ±i3 397 ±39 37O ± [Cl] ± ± ±6i 863 ± ± ± [Nin + N] 1580 ± ± ±

6 Table 3. Solute concentrations in Emerita analoga acclimated to various salinities. Concentrations are expressed as mmol\l sea water or haemolymph and mmoljkg tissue or cell water 50% s.w. 100% s.w. 150% s.w. (Where applicable ± i S.B. about the mean (duplicates from > 6 animals) is presented) [C] H,O (mosmol) (%) [Na] [K] [Ca + Mg] [Cl] [Nin + N] ±n ± ± ± ±03 7O ± ± O ± ±30 III ± ±o ±17 I2OI ± ± ± ± ± oo ±to 681 ± ± ± ± ± ± ± ± O ±33'9 4833

7 Table 4. Solute concentrations in Cancer antennarius acclimated to various salinities. Concentrations are expressed as mrnoljl sea water or haemolymph and as mmoljkg tissue or cell water (Where applicable ± i s.e. about the mean (duplicates from > 6 animals) is presented) a c. c 1 a «<> s" 6o% s.w. I OO% S.W. 150% s.w. [C] (mosmol) 6ooo 6131 ± ± ±35 H,O (%) 765 ± ± ± [Na] I ±23 IOI ± ± ±87 55'4 ± [K] 6o 77 ± ±34 "55 IOI 125 ± ± ± ±3» 1750 [Ca + Mg] ± ± oo 361 ±i3 329 ± ± ± [Cl] ± ±i <H ± ± ± ±26 61o [Nin + N] 2747 ± IOI ± ±

8 Table 5. Solute concentrations in Pachygrapsus crassipes acclimated to various salinities. Concentrations are expressed as mmoljl sea water or haemolymph and as mmollkg tissue or cell water (Where applicable ± 1 s.e. about the mean (duplicates from > 6 animals) is presented) [Nin + T 2217 ± ± ± % 8.W. 100% s.w. ISO% 8.W. [C] (mosmol) ± ± ±183 H,O (%) 784 ± *3 ± ± [Na] ± ±48 2IO a ± ±a ±99 IOIO ±5i 508 [K] 25 6i ± ± ± ± u4 ± ± [Ca + Mg] ± ±i »58 ±3* 346 ±i ± IIO 425 ±27 4i 4 [cq ± ± O ± ± O 6887 ±181 m3 ±53 562

9 Water and solute regulation in muscle fibres > OR u o O / / Complete volume regulation X + \ 1 0 o c A + Callianassa Cancer Pachygrapsus Emerita c s 70 2 cd \^ i eye, i i i i i\i Fig. i. The relative change in cell hydration as a function of the relative change in extracellular osmolarity. The corresponding cell water contents are presented on the right ordinate. The lines describing perfect volume regulation (hatched curve) and an ideal osmometer (diagonal line) were generated assuming an initial water content of 70%. The closed and numbered circles in this and the following figures represent: 1, Carcitau maenat (Shaw, 195s, 1958); a, Potamon nhoticut (Shaw, 1959); 3, IJbima emargmata (Gilles, 1970); 4, CalUnectes tapidus (Gerard & Gilles, 1972); 5, Nereis succmea (Freel et a!. 1973); 6, Bttfo vtridis (Gordon, 1965). the limits on the hatched curve were generated by considering solutes having molecular weights ranging between 50 and 100 grams per mole. The utility of this approach lies in the fact that reasonable limits may be applied to the osmotic behaviour of a given cell type. These then permit a more realistic assessment of the magnitude of cell volume regulation within and between species subjected to various osmotic conditions. Two major conclusions may be drawn from the results presented in Fig. 1. First, all of the crustaceans examined in the present study (with the exception of 25 % s.w. acclimated Pachygrapsus) regulate the water content of their muscle to varying degrees, particularly when exposed to hyperosmotic conditions. Secondly, the volume regulation process is not necessarily complete, but even when it is, significant changes in cell water content would be observed. Intracellular solute concentrations The muscle tissue solute concentrations and apparent intracellular solute concentrations are presented in Tables 25 for the different species and osmotic conditions. The apparent intracellular solute concentrations were computed from the following relation: [ (ECS) (2) 1(ECS) ' where [C^] = apparent intracellular solute concentration, mmol/kg cell H 2 O; [C t ] = tissue solute concentration, mmol/kg tissue H 2 O; [C /( ] = haemolymph solute concen

10 n " 0 #5 0 ) c A* 4 3 1« eye, Fig. a 0 ROBERT» w n FREEL 3 D A eye, Fig. 3 Fig. a. The relative change in cell sodium as a function of the relative change in extracellular osmolarity. See Fig. i for symbol identification. Fig. 3. The relative change in cell chloride as a function of the relative change in extracellular ownolarity. See Fig. 1 for symbol identification. z DC A » C 2 /C, Fig. 4. The relative change in cell ninhydrinpositive nitrogen as a function of the relative change in extracellular osmolarity. See Fig. 1 for symbol identification. tration, DIM; and ECS = the extracellular space of the tissue, kg/kg tissue H 2 O. For each species examined the apparent intracellular concentrations of organic and inorganic solutes vary directly with haemolymph osmolarity (Tables 25). The extent to which these changes can be attributed simply to variations in cell hydration may be estimated by comparing the cell solute concentrations on a dry weight basis. As was done for cell water, the relative change for a given cell solute (^solute) n^y be expressed as the ratio of the initial to final cell solute concentrations on a dry weight basis: (mmol/kg 80lute ~ (mmol/kg DW) 2 " (3) Figs. 25 depict the observed variation in as a function of the relative change in

11 Water and solute regulation in muscle fibres ftd J *+ (a) +.5. o.3 A 1* «4 i i i i i I i A *. 6 i i i o 10 D i i CJC, Fig. 5. The relative change in cell potassium (a) and cell calcium plus magnesium (6) as a function of the relative change in extracellular osmolarity. See Fig. 1 for symbol identification. extracellular osmolarity (CJCj) for Na, Cl, K, Ca + Mg, and Nin + N for the different species and osmotic situations. It should be noted that no account has been made for changes in cell solids in the calculation of these ^goiute values. For comparative purposes these figures contain the results of other studies on incomplete osmoregulators and osmoconformers. Consideration of the /? aolnte values presented in these figures shows that there are two major patterns of cell solute response to altered haemolymph osmotic pressure. First, there is a net loss of Na, Cl, and Nin + N from muscle fibres of crabs acclimated to dilute sea water and a net gain of these solutes after acclimation to 150% s.w. (Figs. 2, 3, 4). The sole exception is seen in the Nin + N levels of 25% s.w. adapted Pachygrapsus (Fig. 4). In this particular case R mn +s io, indicating no net loss of amino nitrogen upon dilution. This lack of Nin + N regulation is coincident with the lack of muscle fibre water regulation observed in 25 % s.w. acclimated Pachygrapsus. Intracellular K and Ca + Mg comprise the second major pattern of muscle solute adjustments. These solutes are, to a large extent, simply diluted or concentrated by changes in cell hydration, since in all species and osmotic conditions (Fig. 50, b) the concentrations of these solutes per gram dry cell weight is essentially constant (R K and R^+iu ^ io). DISCUSSION Regulation of intracellular volume The analysis of the relative cell hydration changes as a function of the relative change in extracellular osmolarity presented in Fig. 1 provides a convenient method for assessing the extent of cell volume regulation in euryhaline osmoconforming rganisms. Besides the recent work of Fyhn (1976) on Balanus improvisus and the

12 n8 ROBERT W. FREEL earlier studies of Lange & Mostad (1967) on euryhaline bivalves, no attempt has been made to quantify the capacity of intracellular volume regulation. The analysis of Lange & Mostad (1967) is more precise than that presented here in that the bounds of the regulation process are experimentally defined. Their method is, however, limited to the few situations where tissue densities have been determined (Lange & Mostad, 1967; Lange, 1970; Staaland, 1970). The major advantage of the present approach lies in its applicability to a rather large body of literature regarding cell or tissue water contents of different organisms exposed to various osmotic conditions. The major disadvantage to this approach is in the estimation of the cell dry weight changes accompanying the volume regulation process. The maximum capacity presented in Fig. i was based upon the assumption that the average molecular weight of the solutes employed during regulation ranged between 50 and 100 grams per mole. This is a reasonable approximation since previous studies have documented the quantitative significance of the common, low molecular weight nitrogenous solutes in the regulatory process (Schoffeniels & Gilles, 1970). It is generally assumed in any analysis of cell volume regulation that all intracellular water is osmotically active. However, in crustacean muscle fibres it has been repeatedly demonstrated that only about 70% of the total intracellular water is osmotically active (Hays et ah 1968; Hinke, 1970; Freel, 19776). If the osmotically active fraction represents a constant percentage of the total cell water, independent of extracellular osmolarity or cell hydration, then complications arising from the heterogeneous nature of cell water are resolved. This appears to be the case for isolated nerve from Eriocheir smensis (Gilles, 1973) and in Callianassa muscle (Freel, 19776). In the latter crustacean, single fibre preparations from both 35% s.w. and 150% s.w. acclimated specimens have osmotically active volumes of about 65%. From the foregoing discussion it is concluded that the relative change in cell hydration (#H,O) i 8 a useful and reasonable approximation of the magnitude of cell volume regulation. Based on such an analysis the results presented in Fig. 1 indicate that the cell volume regulation process is not necessarily complete. This feature has also been observed in the isolated muscle fibres of Callinectes sapidus (Lang & Gainer, 1969) and in the muscle of several bivalves (Lange, 1970). It may also be concluded from Fig. 1 that the magnitude of the regulatory response to hyposmotic stress is not necessarily similar to that observed for an equivalent hyperosmotic stress. This implies that a unified mechanism for cell volume control in hypo and hyperosmotic situations may not exist for these organisms. Distinct mechanisms are further indicated by the differences in the time courses for volume control of isolated tissues challenged with hypo or hyperosmotic salines (Lang & Gainer, 1969; Gilles, 1973). In the general scheme proposed by Schoffeniels (Schoffeniels & Gilles, 1970) the accumulation and release of intracellular free amino acids is considered to be governed by the activity of key enzymes involved in amino metabolism. It is further proposed that the activities of these enzymes are in turn modulated by the levels of intracellular ions. In the case of Pachygrapsus this type of unified mechanism is not indicated since this crab cell volume regulates in concentrated salines, but not in dilute sea water, even though the changes in the intracellular inorganic ions are similar to those of the cell volume regulating species. It is also interesting to pointout that the ability to regulate cell volume is noi

13 Water and solute regulation in muscle fibres 119 necessarily restricted to those crustaceans inhabiting osmotically unstable environments, but is also found among the inhabitants of more strictly marine environments such as Cancer and Emerita. Like the tolerances of the latter species to gradually imposed salinity stresses, the cell volume regulating abilities of these typically opencoast crustaceans is interesting. It is difficult to imagine natural situations where environmental osmolarities would ever reach the extremes imposed here experimentally. Ltbirna emarginata is another subtidal and reportedly stenohaline marine decapod which has been shown to cell volume regulate in sea water concentrations as low as 500 mosmol (Gilles, 1970). The above observations are consistent with the notion that laboratory salinity tolerances are not necessarily related to the salinity ranges encountered or tolerated by marine organisms in their natural environment (Kinne, 1971). Thus, it would appear that distinctions between ecological and physiological potential must be made when discussing the biological significance of cell volume regulation. The observations presented here, together with the paucity of evidence documenting nonregulation, suggests that the physiological potential for cell volume regulation is more fundamental to marine organisms than is generally appreciated. Patterns of intracellular electrolyte regulation Besides the regulation of cellular water contents by net amino acid exchanges, other features of intracellular solute regulation are apparent. The general pattern which has emerged from the present study is that there is a net exchange of cellular Na and Cl during adaptation to new osmotic environments. In sharp contrast, cellular K and, to a large extent, cellular Ca + Mg are relatively constant per gram dry cell weight. These results are in accord with observations made on a variety of other organisms for which the relevant data are available (Figs. 25). Such consistent patterns of intracellular electrolyte regulation possibly reflect the passive reestablishment of equilibria which had been disrupted by changes in the activities of plasma water and solutes during salinity adaptation. If the transmembrane ionic gradients of K and Cl are maintained by a Donnan equilibrium, then a model may be proposed here to account for several of the observed patterns of inorganic solute regulation. What is first required is a physical explanation for the experimental observation that potassium, the major diffusible cation within the myoplasma, is a relatively constant fraction of the cell dry weight (R K ~ io). The fact that intracellular K concentrations (mmol/kg cell water) lie within a relatively narrow range has been recognized for many years (Schlieper, 1958; Steinbach, 1962; Burton, 1968, 1973) and various proposals have been presented to account for this phenomenon. For example, Steinbach (1962) considered the ubiquity of the 150 IBM value for [K<] in terms of the evolution of cell systems, arguing that this particular cation is the most benevolent to protoplasmic machinery. Such hypotheses provide little insight to the physical nature of K accumulation in cells. Burton (1973) has provided a most interesting account of the significance of ion concentrations in animal body fluids and tissues. The starting point for his discussion is the classic analysis of Boyle & Con way (1941). However, the Burton model also considers the possibility that potassium is not in passive equilibrium across the membrane, but rather that it is actively accumulated

14 120 ROBERT W. FREEL in exchange for sodium. As a result of this Na/K exchange the ionic gradients are considered to be dependent upon the critical energy barrier to Na excretion from the cell as well as on the permeability characteristics of the cell membrane and the concentration and valence of the nondiffusible solutes within the myoplasm. The following analysis of the patterns of cellular ion adjustments observed in the decapod crustaceans studied here is also based upon the model of Boyle & Conway (1941). In contrast to Burton's representation, the distribution of potassium is considered to be entirely passive. Therefore, the major factors governing the ionic gradients across resting cell membranes are the concentration and charge of nondiffusible solutes within the myoplasm. Aside from the fact that this simplified scheme is sufficient to account for the experimental observations presented here and elsewhere (Freel, 1977 a) for the behaviour of K and Cl, the quantitative significance of a Na/K exchange pump in tissues such as crustacean muscle, where calcium carries the inward active current (Reuter, 1975), may be questionable. Furthermore, based upon the experimental evidence presented here, it appears that the absolute amount of potassium per gram dry cell weight is more constant than is its concentration per se. Indeed, the variations in cell K concentration can be quite large (86191 mmol/kg cell water in Callianassa, Table 2), but the bulk of such changes are brought about chiefly by water movements. The present model considers the myoplasm of a marine osmoconforming organism to be in double Donnan equilibrium with the extracellular milieu. A specific and simplified situation for an ideal muscle fibre in equilibrium with a solution resembling sea water is depicted in Fig. 6. The definitions of the symbols used in this figure and in the equations which follow are given below. [A~ n ] = concentration of intracellular, nondiffusible anion with a valence of n (mmol/kg cell H a O). For the present analysis n = 1. [K,].= total intracellular K concentration (mmol/kg cell H a O). [K,] A = intracellular K concentration acting as counterion to [A~ n ] (mmol/kg cell H 2 0). = intracellular K concentration acting as counterion to [Clj (mmol/kg cell H 2 0). = intracellular Cl concentration (mmol/kg cell H 8 0). [N] = concentration of intracellular nondiffusible, neutral solutes (mmol/kg cell H 2 0). V = volume of intracellular water (kg). [K o ], [Na 0 ], and [Cl 0 ] = extracellular ion concentrations (mm). [Q] and [C o ] = molar sum of intra and extracellular solutes, respectively. For such an ideal system the following equilibrium relations are applicable (Boyle & Conway, 1941): Osmotic equilibrium [CJ = [Ai + [K,] + [Cy + [N] = [K o ] + [Na J + [Cl 0 ] = [C o ]. (4) Macroelectroneutrality (56)

15 Water and solute regulation in muscle fibres 121 Intracellular Extracellular Nondiffusible ^~ Diffusible *~ Nondiffusible [A"] (1167) t K i] A (1167) (10) [ci] (10) K ICI7] (333) P^c (333) [ci] (490) Na [NaJ] (490) [N] (700) Fig. 6. A simplified model of an ideal cell in osmotic, electrical, and Donnan equilibrium with a sea water composed of only sodium, potassium, and chloride. The notation is explained in the text. The numbers in parentheses refer to the concentrations (mm or mmol/kg cell water) of the various components of this hypothetical system. The water content of this cell is 3 gm H t O/gm dry weight (75%). Note that when extracellular osmolarity is altered N becomes diffusible and represents the solute source for volume regulation. Donnan equilibrium [KJ = [KJ [C1J. (6) For the following it will also be convenient to describe the extracellular product of [KJ [Cl 0 ] in terms of [C o ]. For a perfect osmoconformer [C o ] = [K o ] + [Na 0 ] + [Cl 0 ] and [KJ = [ClJ[NaJ. Thus [Cl 0 ] = [CJ. Also, [K o ] = io» [CJ for normal sea water ([KJ = 10 mm; [CJ = 1 M). Thus the [KJ [C1J product may be given as: [KJ[C1J = 5XIO3[CJ'. ( 7 ) How are the concentrations of potassium and chloride expected to change with adaptational salinity in an ideal osmoconformer? Given the equilibrium conditions (equations (4), (5 a), (56) and (6)) the variations in [Cy and [KJ may be calculated strictly in terms of [A~] and [CJ. By definition we have the following relationships: where Therefore, [KJci = [OJ and [KJ A = [A]. [Kj[cy = ([A]+[cy)[cy. From this relation, together with equations (6) and (7), we have the following: [Cl i ]» + [A][Cy S xio»[cj«= o. Solving for [C1J we obtain the internal chloride concentration as a function of the extracellular osmotic concentration: [Cy = (8)

16 122 ROBERT W. FREEL Constant hydration Constant volume CJC, Fig. 7. Theoretical relative changes in cell potassium and chloride as a function of the relative change in extracellular osmotic concentration for cells which maintain constant hydration or constant volume. Also, it follows from equation (6^ that intracellular potassium may be similarly defined: rr MM (9) Since the parameters of equations (8) and (9) are given in concentration terms (mmol/kg cell H 2 O) a knowledge of the volumetric changes of the cell as [C o ] is varied is essential. It will be convenient to consider only the ideal limits of perfect volume regulation and perfect osmoconformity. In the latter case, where the relative cell water content is proportional to the relative change in extracellular water activity, the solutions to equations (8) and (9) are trivial, since [KJ, [Cl<], and [A~] will be proportional to [CJ. The end result is that for ideal osmometers the values of R K and R 01 will be equal to one over the salinity range. For a perfect volume regulator the cell volume may be considered to be maintained by the isosmotic accumulation or release of nitrogenous solutes ([N]). At first, for simplicity, it is assumed that the molecular weight of the solute N is so small that the changes in cell dry weight are negligible. Values for R K and 7?^ may be calculated using the initial quantitative conditions presented in Fig. 6 together with equations (8) and (9). In Fig. 7 these values are presented as a function of the relative change in external osmotic concentration as was done previously (Figs. 25). From Fig. 7 it is apparent that the model predicts net changes in both cell potassium and chloride per gram dry weight. However, it is significant that the change in cell potassium is

17 Water and solute regulation in muscle fibres 123 considerably less than that of cell chloride. Any factor which increases the cell water content during adaptation to dilute sea water will tend to bring the R K and R O1 values closer to unity. It has already been noted that in a more realistic situation the cell dry weight will change with adaptational salinity, since the molecular weight of N cannot be ignored. The dashed lines in Fig. 7 describe the behaviour of R K and R^ for a perfect volume regulator in which the dry weight is changing as a result of the accumulation or release of a solute having a molecular weight of 100 grams/mole. In addition, it is assumed that water replaces or is displaced by such changes in cell dry weight. Clearly, this more reasonable model for perfect volume regulation predicts that cell potassium per gram dry cell weight will remain relatively constant, while chloride will vary with external osmotic concentration. This is precisely what was observed here experimentally (Figs. 3, 5 a). In this analysis of ion gradient adjustments in osmoconforming marine organisms the assumption has been made that the nondiffusible anion fraction (A~) and the free amino acid pool (N) are not related. In striated muscle it can be expected that cellular protein, together with the various phosphate fractions, comprise the bulk of the nondiffusible anion fraction, since the majority of the amino acids (glycine, alanine, and proline) should be neutral under normal cellular ph conditions. The situation for the nervous tissues of marine osmoconformers is, however, likely to be quite different. Thus, in CalUnectes sapidus nerve, 94 % of the total free amino acid composition is supplied by aspartate and glutamate (Gerard & Gilles, 1972). These negatively charged solutes represent the bulk of the anion fraction required to balance the intracellular potassium concentration. If these amino acids were also utilized in the volume regulation process, then the potassium concentration per gram dry cell weight would be expected to be more variable than that observed for muscle. Unfortunately, it is difficult to state at the present time whether or not this is the case, since no detailed studies have been directed towards this end. The chemical analyses of CalUnectes nerves at 50% and 100% s.w. supplied by Gerard & Gilles (1972) are difficult to interpret in this respect, since the magnitude of the relative water exchange for these tissues was close to that expected for an osmometer and also because the total solute change in these fibres was negligible, indicating little capacity for cell volume control. Certainly, cellular solute adjustments in the nervous tissues of osmoconforming marine organisms deserves further experimental attention and would serve as a reasonable test of the model presented here. As a final comment on the utility of the present model it is worth emphasizing that in their original development Boyle & Conway (1941) were able to demonstrate that in the frog sartorius muscle the molar sum of all the nondiffusible solutes in the muscle fibre (7/V) was approximately equal to the molar sum of the nondiffusible anion fraction (e/v). In terms of the present discussion of the adaptation of marine osmoconformers to dilute situations the significance of this observation (17/V = e/v) may be generalized as follows. Combining the definitions utilized here with those of Boyle & Conway (1941) we have: and 7/V=[C 0 ](K,/V)(Cl,/V) (10) (11)

18 124 ROBERT W. FREEL 3500 r 3000 ^ 2500 a f 2000 o E 1500 Q Z 1000 Constant volume Constant hydration [C o Fig. 8. Theoretical relation between intracellular amino acid levels (N) and extracellular osmotic concentration for cells which maintain constant hydration or constant volume. Multiplying both (io) and (n) by the cell water content per gram dry cell weight (V/DW), and subtracting (n) from (io) one obtains the following linear relation between (ije/dw) and [C<J: 7e/DW = [CJ (V/DW)2 (K./DW). In equation (12) rj e/dw is equivalent to N/DW of the present model and represents the solute source for volume regulation. The relation given in equation (12) may be depicted graphically as in Fig. 8 where N/DW is presented as a function of [C o ] for an ideal cell volume regulator. The solid line in the figure describes the variation of N/DW with [C o ] when cell hydration (V/DW) is constant. The dashed curve presents the more realistic situation where cell volume is regulated, resulting in changes in cell dry weight and hydration. Dry weight and cell water variations were calculated as before. In the case of a euryhaline osmoconformer (having initially 75% muscle fibre water, [K^] = 150 mmol/kg cell H a O, and [Cl<] = 333 mmol/kg cell H 2 O when acclimated to 100% s.w.) cell volume can be maintained until N/DW is exhausted. The external concentration at which this occurs is 220 mm. At this particular point [KJ = 110 mmol/kg cell H a O, [C1J = 24 mmol/kg cell H a O, and cell water = 81% of the fibre fresh weight. These values are roughly similar to those of 35 % s.w. acclimated Callianassa (Table 2) as well as to those of a variety of freshwater organisms having plasma osmolarities between 200 and 300 mosmol (Prosser, 1973). Thus, this simple reiteration of the fundamental characteristics of the Boyle & Conway model of ion accumulation serves to emphasize the potential theoretical basis for the similarity of cell water and electrolyte levels between fresh water and marine organisms. The model presented here has omitted the behaviour of cell sodium and the calcium plus magnesium fraction. These solutes have been neglected primarily

19 Water and solute regulation in muscle fibres 125 because of the uncertainty of their physical status within the myoplasm and the mechanism of their equilibrium disposition across the membrane. Equations similar to (8) and (9) cannot be developed for [Na^] without introducing additional variables. It should also be pointed out that predicting [Na<] changes from relations such as the GoldmanHodgkinKatz equation (Hodgkin & Katz, 1949) is useless, since [Na^] can take on a wide range of values without altering the [K^] or [CLJ values when the permeability of the fibre to Na is low. A most appropriate test of the Donnan model discussed here requires that the products of the concentrations (or more accurately, activities) of the diffusible ions in the myoplasm equals their product on the outside (equation (6)). Previous attempts to relate the concentration gradients of K and Cl across crustacean muscle fibre membranes, in terms of the Donnan principle, have met with varying success (see Freel, 1977 a). The major difficulty arises from the use of apparent intracellular concentrations without regard for the heterogeneous nature of cell water and solutes in these tissues (Hays et al. 1968; Hinke & Gayton, 1971; Freel, 19776). A quantitative appraisal of the potassium and chloride concentration gradients observed in the present study is likewise inadequate. Therefore, definitive statements concerning the role of the Donnan principle in the transmembrane distributions of potassium and chloride can only be achieved by considering the thermodynamic activity gradients of these ions. In another report (Freel, 1977 a) the transmembrane activity gradients for potassium and chloride are presented for several of the marine decapods studied here. The results of this study support the hypothesis that these ions are distributed in accordance with the Donnan principle and that during the adaptation to new osmotic environments adjustments in the internal product of [K^] [CLJ are met chiefly by net changes in the intracellular chloride level. This paper represents part of a Ph.D. dissertation submitted to the Department of Biology, University of California at Los Angeles. I would like to thank Drs M. S. Gordon, G. N. Somero, S. Hagiwara, M. E. Clark, S. S. Hillman and Messrs C. Loretz and R. Putnam for their interest and helpful discussions throughout the development of this report. Part of this work was supported by the University of California Patent Foundation and by the UCLA Laboratory of Fisheries and Marine Biology. REFERENCES BOYLE, P. J. & CONWAY, E. J. (1941). Potassium accumulation in muscle and associated changes. y. Pkyriol., Lond. 101, 163. BURTON, R. F. (1968). Cell potassium and the significance of osmolarity in vertebrates. Comp. Biochem. Pkytiol. xj, nbzmi. BURTON, R. F. (1973). The significance of ionic concentrations in the internal media of animals. Biol. Rev. 48, CLARK, M. E. (1973). Amino acids in osmoregulation. In Experiments in Physiology and Biochemistry, vol. 6 (ed. G. A. Kerkut), pp London: Academic Press. FREEL, R. W. (10770). Transmembrane activity gradients for K and Cl in the muscle fibres of osmoconforming marine crustaceans. J. exp. Biol. 73, FREEL, R. W. (19776). Cellular hydration and solvent volumes of crustacean muscle fibres. In preparation. FREEL, R. W. MEDLER, S. G. & CLARK, M. E. (1073). Solute adjustments in the coelomic fluid and muscle fibres of a euryhaline polychaete, Neanthes sucdnea, adapted to various salinities. Biol. Bull. 144, EXB 27

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