Comp. Biochem. Physiol. Vol. 75B, No. 3, pp. 471473, 1983 0305-0491/8353.00+ .00 Printed in Great Britain. © 1983 Pergamon Press Ltd HEPATIC ARGINASE ACTIVITY IN INTRA- AND EXTRAUTERINE LARVAE OF THE OVOVIVIPAROUS SALAMANDER, SALAMANDRA SALAMANDRA (L.) (AMPHIBIA, URODELA) JOCHEN SCHINDELMEISER*, INGRID SCHINDELMEISERt a n d HARTMUT GREVENt *Anatomical and tZoological Institutes, University of Miinster, D-4400 Miinster, FRG (Tel.: 0251 831) (Received 29 November 1982) Abstract--l. The hepatic arginase activity of Salamandra salamandra was determined at three different stages of intra- and extrauterine larval development and at fully metamorphosed juveniles. 2. The highest enzymatic activity was found in intrauterine larvae in November, the lowest in intrauterine larvae in June of the following year. 3. These data can be correlated with the ureotelism of intrauterine larvae previously described and are discussed with respect to the metabolism of larval and juvenile fire salamanders. INTRODUCTION Environmental conditions strongly influence the type of nitrogen excretion in amphibian larvae. When living in a sufficient amount of water, ammonotelism is clearly predominant (Cohen, 1966; Balinsky, 1970; and others). However, restriction of water availability may result in a graduated shift to complete ureotelism (e.g. larvae of land-nesting leptodactylids: Balinsky et al., 1961; Candelas and Gomez, 1963; Martin and Cooper, 1972; Shoemaker and McClanahan, 1973; Scaphiopus-larvae developing in ephemeral ponds: Jones, 1980). As to the larvae of the terrestrial, ovoviviparous fire salamander, Salamandra sal- amandra, which develops in the uterus with only a restricted amount of fluid at their disposal, strong indications for ureotelism could recently be obtained (Schindelmeiser and Greven, 1981). Usually the transition from ammonotelism to ureo- telism occurs during metamorphosis and is accom- panied by the extreme development of the hepatic ornithine-urea cycle (e.g. Cohen, 1966; Frieden, 1967) with a strong enhancement of activity of the re- spective enzymes (Brown, 1964). The activity of arginase seems to reflect the level of the complete cycle, although it should be considered that the enzyme is presumably not rate limiting; possibly it is still serving other functions (Balinsky, 1970; Shoe- maker and McClanahan, 1982). To our knowledge the hepatic activity of the ornithine-urea cycle enzymes has only been deter- mined in adult amphibia under conditions of water shortage and osmotic stress (Balinsky et al., 1967b; Balinsky, 1981; Jones, 1982). Larval amphibia, in particular those developing in a very restricted vol- ume of fluid within the female genital tract, have never been examined from this point of view. MATERIAL AND METHODS Fully developed larvae (for definition see Joly, 1968) of S. salamandra were obtained after natural or artificial birth in November respectively May-June. Some larvae were used 471 immediately after birth for biochemical determinations, some were placed into tap water at 17°C and fed with Tubifex. They were kept for different periods under these conditions up to the end of metamorphosis. For the determination of hepatic arginase (EC 3.5.3.1), the larvae were sacrificed, their livers removed and stored at 18°C (not longer than 2-3 hr). Before homogenization the fresh weight of all liver preparations belonging to one stage was determined and then the tissue homogenized in 9 vol of a 12mmol/l sodium maleate buffer, pH 7.5, containing 10mmol/l MnC12 and 0.01~ Triton X-100, with a Potter- Elvehjem homogenisator (2 min at 0°C) (Riiegg and Russell, 1980). The homogenates were centrifugated for 15min at 4000 rpm and the supernatant immediately used for en- zymatic determination. Arginase was determined applying a modification of the method of Middelhoven (1964). Incu- bation took place for 30 min at 37°C with 1.0 ml 0.2 mol/l sodium glycinate, pH 9.5, 0.5 ml 0.2 mol/1 arginine hydro- chloride (pH 9.5 with NaOH), 0.4 ml of water and 0.1 ml supernatant respectively a solution of pure arginase (from bovine liver, Sigma, specific activity 50 units/mg, used for calibrations). The reaction was stopped by incubating the reaction mixture for 5 min at 95°C to destroy enzymatic activities. As control, the boiling step was carried through with the complete mixture directly after addition of the supernatant. After cooling and centrifugation, the undiluted super- natant was tested with a slightly modified test kit of Boehringer for detection of urea (Schindelmeiser and Greven, 1981). RESULTS Hepatic arginase activity was determined at four different stages during intra- and extrauterine life: Stage 1, end of November, intrauterine larvae immediately after artificial birth. Stage 2, beginning of June in the following year, intrauterine larvae immediately after artificial birth (females were kept on moist filter paper only; in presence of water natural birth usually takes place in early spring). Stage 3, middle of June, extrauterine larvae 14 days after artificial birth.