A large number of species of Amphibia, both Anura and Urodela, are capable of tolerating a moderately saline environment. Rana cancrivora, Bufo uiridis, and Xenopus laevis are among the most euryhaline frogs so far studied. Rana cancriuora can tolerate undiluted seawater. In a saline environment, Amphibia show raised plasma sodium and chloride and raised intracellular potassium and chloride. In the adults, plasma and tissue urea are elevated, especially in the more euryhaline species. Free amino acids contribute negligibly to plasma osmolarity, but are very important in intracellular fluids. By these various means, the osmotic pressure of body fluids is always maintained at a higher level than that of the surroundings. Larval Amphibia, however, do not make urea; Rana cancriuora tadpoles can live in saltwater, but maintain the osmolarity of their body fluids below that of their surroundings.Adaptive responses to hyperosmolar environment include decreased skin sodium transport, greatly reduced urine flow, and release of posterior pituitary hormones. After the initial response, the release of these hormones declines, and urine flow increases. Accumulation of urea occurs slowly, but this substance plays an increasingly important role as adaptation proceeds. Accumulation is due initially to urea retention, and possibly to greater synthesis due to a high concentration of precursors of the urea cycle. In later stages of adaptation, increased urea synthesis is due to elevated levels of urea cycle enzymes, especially those that appear to have been rate-limiting. Liver glutamate dehydrogenase is also elevated. In animals subjected to pure osmotic stress, in solutions not containing sodium, responses are similar to, but not identical with, those caused by a medium containing sodium chloride.

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1. An improved radioassay for glutathione synthetase and gamma-glutamylcysteine synthetase was developed. 2. Xenopus laevis liver gamma-glutamylcysteine synthetase was purified 324-fold by saline-bicarbonate extraction, protamine sulphate precipitation, CM-cellulose and DEAE-cellulose column chromatography, and gel filtration. 3. Rat liver gamma-glutamylcysteine synthetase was purified 11400-fold by a procedure similar to that employed for the Xenopus laevis enzyme. 4. Rat liver gamma-glutamylcysteine synthetase activity was inhibited by GSH and activated by glycine. These effects, which were not found in the enzyme from Xenopus laevis, may have a regulatory significance. 5. Isotope-exchange experiments revealed fundamental differences in the partial reactions catalysed by the rat and Xenopus laevis synthetases. The enzyme from Xenopus laevis appears to follow a Bi Bi Uni Uni Ping Pong mechanism, with glutamyl-enzyme as intermediate before the addition of cysteine and the release of gamma-glutamylcysteine. The results for the rat liver enzyme are consistent with a Tri Tri sequential mechanism.

A technique for the organ culture of postnatal and adult rat liver has been developed. Liver slices, 0.3 mm thick, were maintained in Conway units at the interphase between medium and a 95% O2:5% CO2 atmosphere. Postnatal liver in culture for up to 72 h had healthy hepatocytes throughout the explants; if adult liver was used the upper 0.2 mm was healthy after 24 h. These slices incorporated tritiated orotate and leucine into trichloroacetic acid-precipitable material. Incorporation of orotate was shown to be spread over the entire slice of neonatal liver. Culturing did not alter the potassium ion content of postnatal liver. Tyrosine aminotransferase activity in liver slices from postnatal, adult, and adrenalectomized adult rats was stimulated by glucocorticoids and dibutyryl cyclic AMP. Cycloheximide and actinomycin D prevented this response. Further, cortisol exerted a permissive effect on the stimulation of tyrosine aminotransferase activity by dibutyryl cyclic AMP in slices from adrenalectomized rats. Induction of urea cycle enzymes by cortisol was demonstrated in cultures of liver from adrenalectomized adult animals.