Isosmotic Regulation in Various Tissues of the Diamondback Terrapin Malaclemys Centrata Centrata (Latreille)^*

Diamondback terrapins are brackish-water turtles that can be caught wild in regions of different or varying salinity (Dunson, 1970). When acclimatized either to fresh water or to sea water they show modifications in the osmotic pressure and composition of their blood (Bentley, Bretz & Schmidt-Nielsen, 1967; Gilles-Baillien, 1970). During summer months terrapins collected in sea water have a higher blood osmotic pressure (460 m-osmoles/1) than animals acclimatized to fresh water for several months (310 m-osmoles/l). This higher osmotic pressure is due to greater Na and Cl concentrations but also and mainly to a higher content of urea (Gilles-Baillien, 1970). As it is usually taken for granted that cells are isosmotic to the blood, our purpose was to investigate which ions or molecules are implicated in the intracellular isosmotic regulation of the tissues. This paper reports the modifications in the inorganic ions and in some nitrogen compounds in various tissues of diamondback terrapins acclimatized either to fresh water or to sea water.

The tissue sampling was carried out during summer months (from May till the end of September) in diamondback terrapins collected and kept in sea water on the one hand and in others acclimatized for several months to fresh water on the other hand. Several tissues were selected. The muscle tested was the one joining the humerus to the clavicle. Mucosae of bladder, jejunum and colon were excised and by dissection cleared of muscular and connective tissues.

Na, K and Cl were determined, after extraction with HNO₃ 0·08 N and adequate dilution, either with a Beckman flame photometer for Na and K or with a Buchler-Cotlove chloridometer for Cl. Amino acids and taurine were estimated with a Beckman amino acid autoanalyser. The urea concentration was measured according to a colorimetric technique involving a digestion by urease (SIGMA Technical Bulletin No. 14).

The inorganic ion content of the muscle and of the bladder jejunum and colon mucosae has been estimated in diamondback terrapins collected and kept in sea water and in others acclimatized to fresh water for several months. Table 1 shows the results obtained. In the muscle there is no significant difference in the inorganic ion content. In the bladder mucosa the only significant variation is a higher content in K for the sea-water animals; but in absolute value this variation is far from being important. In the colon mucosa higher concentrations in Na and Cl are found for the terrapins in sea water, but the K concentrations are similar for animals in both conditions. The reverse is the case for the jejunum mucosa: Na and Cl concentrations are comparable for animals in both conditions while the K content is much higher in the sea-water terrapins.

A comparison of the free amino acids has also been carried out in the same tissues between a diamondback in sea water and another in fresh water (Table 2). Considering first the total of the free amino acids estimated, one can say that in the four tissues investigated the free amino acid pool is higher for the animals kept in sea water and the difference is especially important in the muscle and in the jejunum mucosa. In the muscle all amino acids are implicated in the variation except aspartate. In the jejunum mucosa all of them without exception are involved. But while the muscle of sea-water animals shows a higher content not only in NH₈ but also (and mainly) in taurine and urea, in the case of the jejunum NH₃ is lower in sea-water animals, the taurine content is similar in both conditions and the urea concentration is much higher in sea-water animals.

In the colon mucosa and in the bladder mucosa the variation in the amino acid content is less impressive. Nonetheless, in the case of the bladder the total content is more than doubled, the glutamate is tripled; the taurine concentration is also approximately doubled. In the colon mucosa, the glutamate content is also higher in sea-water animals, the taurine content in contrast is lower. In both bladder and colon urea is the compound showing the widest variation.

Our aim in this paper was to search for the compounds involved in the isosmotic regulation of the diamondback terrapin. Indeed when animals in sea water and in fresh water are compared some modifications of the inorganic ion content as well as of the amino acid content in response to the change in the osmolarity of the blood are seen. However, in the muscle no significant change in the inorganic ion content could be detected. This is at variance with results obtained in two euryhaline species of teleosts, Paralichthys lethostigma and Crenimugil labrosus, where the inorganic ion content varies according to the salinity in their environment (Lasserre & Gilles, 1971). In these same species all the non-essential amino acids especially are subject to changes while in the diamondback terrapin all the amino acids except aspartate are involved. This illustrates once more that the cells of invertebrates are not the only ones where amino acids vary during acclimatization to different salinities (see Schoffeniels & Gilles, 1970). Indeed the variations expressed as percentages are here also of great extent but they refer to values which are much lower than those found in the cells of invertebrates. Therefore when considering the final balance of osmotic pressure that must be achieved by the muscle when the diamondback terrapin goes from fresh water to sea water neither inorganic ions nor amino acids are the important effectors in the osmotic regulation.

But taurine and urea appear to be more important osmo-effectors. For urea one can assume that it slowly diffuses from the blood. As to taurine it seems to be implicated in the osmotic adjustment of the muscle in all animals investigated.

In the bladder mucosa as far as inorganic ions are concerned only a very small increase in K is recorded in sea-water animals. Amino acids, taurine and NH₃ are also weak osmo-effectors. Only urea appears to play an important part in the intracellular osmotic pressure.

In the colon, Na, Cl and urea are associated with a larger osmotic pressure but other unknown compounds must be involved to a greater extent.

In the jejunum amino acids together with urea are greatly implicated in the osmotic adjustment. Moreover, there is an important increase in K. In a terrestrial tortoise (Testudo hermanni) during hibernation there is also an increase in the osmotic pressure of the blood due to Na, Cl and mainly urea (Gilles-Baillien & Schoffeniels, 1965-6). In this species an important increase in K of the jejunum mucosa in response to hibernation has also been recorded (Gilles-Baillien, 1969). In contrast, in other tissues inorganic ions appear to be more involved in osmotic adjustment than they are in the diamondback terrapin.

Among vertebrates it seems therefore that the mechanisms of adjustment of osmotic pressure by amino acids are still present at least in certain teleosts (see for instance Lasserre & Gilles, 1971) in some amphibians (see, for instance, Schoffeniels & Tercafs, 1965) in a chelonian reptile Malaclemys contrata (this paper). But their important role in invertebrates is partially supplanted in vertebrates by urea in species that have developed the system of ureogenesis (see Cohen & Brown, 1960; Florkin, 1966) or by devices specialized in achieving salt/water balance, thus ensuring variations of smaller amplitude in osmolarities of the blood and tissues.

We wish to thank Professor D. C. Tosteson (Pharmacology and Physiology Department of Duke University, Durham, N.C.) and Professor E. Schoffeniels (Department of Biochemistry, Liège University, Liège, Belgium) for providing us with facilities and for their interest in this work. Our sincerest thanks are also due to Dr Costlow, Director of the Duke University Marine Laboratory for his hospitality.