Acta physiol. scand. 1979. 105. 508-512 From the Department of Physiology and Biophysics, University of Lund, Sweden

Formation of cadaverine in the pregnant rat BY

ANNE-CHARLOTTE ANDERSON,STIGHENNINGSSON and ELSAROSENGREN Received 3 October 1978

Abstract ANDERSON,A.-CH., S. HENNINGSSON and E. ROSENGREN. Formation of cadaverine in the pregnant rat. Acta physiol. scand. 1979. 105. 508-512. The formation as well as the content of cadaverine were determined in different tissues of pregnant and non-pregnant rats. The placenta and ovary were most potent in the ability to form cadaverine. To our knowledge this is the first report of an in vitro formation of cadaverine linked to a normal physiological process, i.e. pregnancy. The highest concentration of cadaverine was found in the placenta and ovary of the pregnant rat. Treatment with aminoguanidine generally elevated the content of cadaverine, indicating a role of diamine oxidase as a regulator of diamine content. Key words: Cadaverine formation, content of cadaverine, rat pregnancy

Evidence of in vitro formation of cadaverine in mammalian tissue was originally reported in the mouse kidney after the injection of the anabolic steroid Durabolin (Henningsson, Persson and Rosengren 1976). An in vivo format'ion of cadaverine is supported by the observation that rats regularly excrete cadaverine in the urine. The amine has also been detected in human urine (Bremer and Kohne 1971). In the pregnant rat the urinary excretion of cadaverine together with other diamines as well as the polyamine spermidine is greatly increased (Andersson, Henningsson and Rosengren 1978). On the 15th day of pregnancy the cadaverine excretion began to rise and hence increased continuously to day 19-20 of pregnancy when peak values up to 10 times the non-pregnant level were observed. Thereafter the cadaverine excretion fell towards the non-pregnant level which after the birth of the young was resumed. The purpose of the present study was to investigate in which tissue(s) of the pregnant rat an increased formation of cadaverine takes place. In addition the content of cadaverine was determined in various tissues of the pregnant and non-pregnant rat. As stated above, very little is known about the synthesis of cadaverine in mammalian tissues. Regarding the close congener of cadaverine, i.e. putrescine, it has been shown that its synthesis is controlled by the level of the amine itself in different cell culture systems and in intact animals (for ref. see Janne, Poso and Raina 1978). The diamine oxidase inhibitor aminoguanidine might a 508

CADAVERINE IN RAT PREGNANCY

509

priori increase the level of tissue cadaverine and such an effect might influence its synthesis. The diamine oxidase activity is known to be high in the placenta and plasma of pregnant rats (Roberts and Robson 1953, Kobayashi 1964).

Methods Adult female rats, weight 240-340 g, of the Sprague-Dawley strain were used. They were fed a standard pellet diet and water ad libitum. Their estrus cycles were followed by vaginal cytology. Smears were taken in the morning and stained with methylene blue. Only rats with normal estrus cycles were used. Rats selected to become pregnant were allowed to mate in the pro-estrus or early estrus phase. They were then transferred to a male for 24 h. The next morning when sperm was found in the vaginal smear was considered as the first day of pregnancy. On the 19th day of pregnancy the rats were killed by cervical dislocation and exsanguinated. Concomitantly control rats were sacrificed. One group of rats was studied under undisturbed conditions and another group after the injection of the diamine oxidase inhibitor aminoguanidine (10 mg aminoguanidine hemisulphate/kg) for 10 consecutive days before the actual experiment. Tissues of pregnant rats (ovary, uterus, kidney, liver, fetus and placenta) and of non-pregnant rats (ovary, uterus, kidney and liver) were rapidly removed, cooled on ice, minced with scissors and gently homogenized in a Dounce type homogenizer in 4 volumes of cold 0.1 M sodium phosphate buffer (pH 7.2) M EDTA and 2 % (w/v) glucose. The homogenate was centrifuged containing 5 x M dithiothreitol, a t 20 000 x g for 20 min a t 4°C. The supernatant was used as enzyme source. Supernatant corresponding to 160 mg of tissue (uterus, kidney, liver and fetus), 60 mg of tissue (placenta in its whole, maternal and fetal forms) and 16-57 mg (ovary) was incubated in the presence of pyridoxal-5-phosphate (final conc. M), DL-(lJ4C)-lysine monohydrochloride (final conc. M, sp. act. 10 pCi/mmole) and the same buffer as used for homogenization, the total finally made up to 1.0 ml. The incubation was carried out at 37°C for 60 min with agitation. The reaction was stopped by the tipping of 0.7 ml of 2 M citric acid from a side arm of the incubation vessel. Expelled 14C0, was trapped on a 1 0 x 2 5 mm piece of No. 005 Munktell’s filter paper prepared with 1OOpl hydroxide of Hyamine (1 M solution in methanol). For maximal absorption of the 14C0, the shaking was continued for another 45 min. After completed incubation the filter paper was dropped into a scintillation vessel containing 8 ml of scintillation solution (Bray 1960) and the radioactivity was measured in a Packard Tri-Carb liquid scintillation spectrometer. For stoichiometric determinations of cadaverine formation supernatants of the placenta and ovary were M to prevent oxidative incubated in the presence of aminoguanidine in a final concentration of 2 x deamination of the formed cadaverine by diamine oxidase. The reaction was stopped by the tipping of 0.7 ml 9.7 % sulfosalicylic acid from the side arm of the incubation vessel. The amount of 14C02expelled was compared with the amount of the cadaverine formed in the same incubate. For the preparation of the sample for analysis of its cadaverine content 0.4 mg EDTA was added to each ml of the incubate. The incubates were then centrifuged at 800 x g for I5 min whereafter the samples were adjusted to a p H value of 2.0-2.5 and filtered through a filter with a pore size of 0.22 p m (Millipore Corp.). The separation and quantitative estimation of cadaverine in the incubates were carried out on a 6 x 78 mm column of Durrum D C 6A using an automatic amino acid analyzer (LKB-BIOCAL, 3201). The procedure as described by Henningsson (1978) was used with minor modifications. For determination of the content of cadaverine in the tissues of pregnant and non-pregnant rats about 200 mg of tissue was homogenized in a Duran 50 type homogenizer in 3.5 ml sulfosalicylic acid (40 mg sulfosalicylic acid and 0.4 mg EDTA/ml). The extracts were then boiled o n a water-bath for 30 min, whereafter they where prepared and assayed in the same way as described above for determination of cadaverine content in incubates.

Results Cadaverine formation in various tissues from pregnant and non-pregnant rats

The formation of cadaverine in vitro in various tissues from pregnant rats was examined at times when, from a n earlier investigation (Anderson et al. 1978), the urinary excretion of

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ANNE-CHARLOTTE ANDERSON, STIG HENNINGSSON AND ELSA ROSENGREN

TABLE I. Formation of cadaverine (nmol/g x h) in different tissues of pregnant and non-pregnant rats. The tissues of the pregnant rats were excised on the 19th day of pregnancy. The figures are mean values* S.E. of mean of 8 observations (tissues from 4 untreated rats and 4 rats treated with aminoguanidine). Tissue

Non-pregnant

Pregnant

Ovary Uterus Kidney Liver Fetus Whole placenta Fetal placenta Maternal placenta

25 f7.47 22 Ifr3.40 2.1 1.03 2.7k2.11

110 j 1 6 . 3 * * * 9 . 0 k 1.49** 3.9+ 1.38 1.8f 1.01 5.9f 1.94 250 k20.9 350 k29.0 52 f 10.0

*

**=p

Formation of cadaverine in the pregnant rat.

Acta physiol. scand. 1979. 105. 508-512 From the Department of Physiology and Biophysics, University of Lund, Sweden Formation of cadaverine in the p...
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