1. The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity Patricia Wright, Paul Anderson, Lei Weng, Natasha Frick, Wei Peng Wong, Yuen Kwong Ip. Journal of Experimental Zoology 301A:559-568 (2004)

2. INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

3. Rana cancrivora accumulate urea in body fluids when acclimated to saline water. High internal urea balances the osmotic stress of the external environment. (Gordon et al., 1961) (Smith, 1936)

4. It is a strategy similar to marine elasmobranchs. However, unlike elasmobranchs, Rana cancrivora do not efficiently reabsorb urea from the renal tubules.

5. Gordon and Tucker(1968) reported a very high urea excretion rates. It would be futile to synthesize urea at relatively high rates only to lose it to the environment.

6. Question I Does the rate of total urea excretion change with increased salinity?

7. Urea influx across the pelvic region of the ventral skin is dramatically enhanced in salt acclimated amphibians. An enhanced rate of urea influx across the skin in Rana cancrivora would facilitate whole body urea retention. (Garcia-Romeu et al.,1981; Rapoport et al., 1988; Lacoste et al., 1991; Dyktoet al., 1993)

8. Question II Does skin urea permeability change with increased salinity?

9. The increase in tissue urea levels may be due to an increase in hepatic urea synthesis. Saline environment had no significant effect Key urea cycle enzyme, carbamoyl phosphate synthetase I (CPSase I). (Balinsky et al. 1972; Colley et al. 1972)

10. Progressive water depletion in muscle and liver was observed on exposure to the hyperosmotic environment. Any changes in enzyme activity detected may be simply due to an overall concentration of cellular constituents. (Colley et al., 1972)

11. Question III Is there an induction of hepatic CPSase I specific activity?

12. Skeletal muscle tissue urea levels increase with increased external salinity. One possibility is that urea is not the only osmolyte that increases in muscle tissue under these conditions. (Gordon and Tucker, 1968)

13. 在體外蛋白質吸收過程中，小腸酵素將其分解成 為胺基酸，再藉由肝細胞轉化為體內蛋白質，而 過多的氨基酸則會隨尿液排出體外。 但是這個過程會附帶產生有毒物質「氨」。而肝 臟可將氨轉變為尿素，並與尿液排出體外。 胺基酸 蛋白質分解 氨尿素

14. There was no clear increase in the levels of six different amino acids in skeletal muscle tissue, when Rana cancrivora were acclimated to 26ppt. A complete analysis of all the individual amino acids has not been performed on R. cancrivora tissues, to our knowledge. (Gordon and Tucker, 1968)

15. Question IV Do amino acids co-accumulate with urea in intracellular compartments?

16. Urea excretion rates Urea flux rates across the ventral pelvic skin The concentrations of urea in plasma The levels of individual amino acids in liver and muscle Liver CPSase I activity

17. INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

18. Before stared the exp The frogs used in the present study were juveniles. Frogs were raise in 20-L plastic chambers(1 ppt, 27 ℃ ). Separate groups of frogs were acclimated to either 10 or 20 ppt for one month. The frogs were fed red worms and the photoperiod was 12 h dark: 12 h light.

19. The concentrations of urea in plasma 1ppt 10ppt 20ppt Frogs were sacrificed, and blood samples were collected 20ppt, 4days 25ppt, 4days

20. Urea excretion rates Chamber water was replaced of autoclaved water (1ppt control or 20ppt) After 4 & 8 hours, water samples were removed and frozen until later analysis

21. Urea flux rates across the ventral pelvic skin Influx rate isosmolarUrea 1, 2, 5, 100, 200, 300 mmol/L Mucosal side Serosal side 30 min

22. Liver CPSase I activity and the levels of individual amino acids in liver and muscle 10ppt, 2days 1ppt 13ppt, 1days 17ppt, 2days20ppt, 2days Frogs were sacrificed, and liver and muscles samples were collected

23. INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

24. Question I Does the rate of total urea excretion change with increased salinity?

25. There was no significant difference between urea excretion rates.

27. Plasma Na+ and Cl concentrations were significantly elevated in frogs at 20 and 25ppt

28. The dramatic elevation of plasma and tissue urea levels may be the result of an increase in the rate of urea synthesis.

29. Question II Does skin urea permeability change with increased salinity?

30. There were no significant differences between urea influx at 1 and 20ppt acclimation, nor urea efflux.

31. A linear relationship between J urea and the urea concentration. Urea influx at 20ppt acclimation

32. Question III Is there an induction of hepatic CPSase I specific activity?

33. There was a significant increase in CPSase I activity when frogs were exposed to saline waters,

34. The key urea cycle enzyme, CPSase I,is induced in a hyperosmotic environment, suggesting a stimulation of the rate of urea synthesis.

35. Question IV Do amino acids co-accumulate with urea in intracellular compartments?

36. In liver

37. In muscle

38. INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

39. Urea is a far more important osmolyte than amino acids in the tissues.

40. The crab-eating frog Rana cancrivora is very tolerant of saline water, because of their rapid accumulation of organic osmolytes, urea.

41. The difference between the urea excretion rates presented by Gordon and Tucker (1968) and the present study probably reflect the addition of toluene( 甲苯 ) to the water which may have altered the properties of the skin.