Acta physiol. scand. 1973. 89. 126-128 From the Department of Physiology, University of Tnrku, Finland

Arginase Activity in Rat Small Intestinal Mucosa BY

hl.-P. HARRI and K. HARTIALA

Received 8 Deccmber 1972

Abstract

HARRI, M.-P. and K. HARTIALA. ArginaJc, activity in rat >mall intcahnal mu( o w . Acta physiol. scand. 1973. 89. 126-129.

When the arginase activity of rat srnall intestinal mucosa was compared with that of the livci. it was found to be 5 to 10 per cent to 1 g of tissue. The inucosa of the glandular part of the stomach showed almost no arginase activity. The intestinal activity was liighest in jejunal iniicosa and as a whole higher in inale than in female rats.

It is now well known that several conjugation reactions may occur in the intestinal mucosa, for instance glucuronide formation, sulphatation and esterification. Non- conjugative metabolic reactions are also found in the mucosa. The formation of urea froni arginine by intestinal arginase is an example of intestinal deconjugation reactions.

Small amounts of arginase activity have been demonstrated in the small intestinal mucosa of dog (Kossel and Dakin 1904, Eldbacher and Boneni 1925), cat and probably also pigeon (Eldbacher and Bonem 1925), rabbit (Roche, Mourgue and Baret 1953) and mouse (Greenstein and Meister 1952). Arginase activity wa5 recently found in rat small intestine by Greengard, Sahib and Knox 1970.

In this study, the duodenal, jejunal and ileal mucosal arginase activities oC i a t are compared with those of the liver and the mucosa of the glandular part of the stomach.

Materials and Methods The experimental animals werc 6 months old Wistar rats. The females weighed 180 g to 240 g and the males 200 g to 360 g. They were fed ad libitum with standard food pellets but fasted before the experiments for one day and night.

The animals were killed by a blow on the neck, and the tissue specimens were removed. The stomach and the sinall intestine were opened and rinsed wilh a cold buffer solution containing 0.067 M KHzPOi-NaHP04, 1.0 mM EDTA and 0.25 M sucrose, pH 7.4. The mucosal specimens were removed from the glandular part of the stornacll and srnall intcstinal serosa with a knife and homogenized, as were the liver specimens, in the buffer solution wing a glass homogenizer with a rotating Teflon pestle.

126

ARGINASE l c r i v i w I N INTESTINAL MUC:OSA 127 T A B L E I . The arginase activities of rat small intestinal niucosa (d~roden~tm, j~~jununi , ileuril}

compared with those of the liver and of the glandular part of the stomach.

Tissue Arginase activity

pmol of urea fornied/min/g of tissue S.E.

a ) Females (7) : Liver Duodenal mucosa Jejunal mucosa Ileal mucosa Glandular ventricular mucosa b) Males ( 12) : Liver Duodenal niucosa Jejunal inucosa Ileal niucosa Glandular ventricular ~nucosa

158.0 9.4

13.2 7.8 0.2

199.3 13.5 18.0 11.6 0.5

8.3 1.3 1.5 I .2 0.1

12.2 1.5 2.1 1 .0 0.1

TABLE 11. Total urea synthesis of rat livcr, intrstinal niucosa (duodcnum, jejunum, ileitni) and glandular ventricular mucosa calculated to the whole organ weights.

Organ Range of arginase activity pniol of urea/min/wholc organ

a ) Females (7) : Liver Duodenal niucosa Jejunal mucosa Ileal niucosa Small intcstinal inucosa (total) Glandular ventricular mucosa b) Sfales (12) : Liver Duodenal mucosa Jejunal mucosa Ileal nii ic~sa Small intestinal iiiucosa (total) Glandular ventricular niucosa

998 - 1260 1 . 7 - 5.3

10.8 - 32.7 5.9-25.1

20.5-60.1 0 .0- 0.3

849 -2400 1.6- 8 .4 7.4-43.5 4.1 -23.3

19.0 - 73.8 0.1 - 0.6

The incul)ation mixtures contained 4.2 mg of arginine dihydrate (20 .0 mill) in 1 nil of the buffer solution. The amounts of the tissue were selected to give a linear correlation of reaction velocity to the corresponding tissue wet weights. They were 2.0 mg of liver, 100 mg of glandular ventricular iniico~a and 10 mg of each rnucosal specimen from the various parts of the small intestine. The incubations were carried out a t 37" C for 10 min.

The formation of iirea was measured with a modified diacetyl tnonoxirne-tliioseinicarbazide method presented by Coulotnbe and Favrean 1963. 0.1 nil samples of the incubation mixtures were added to 0.9 in1 of 5 '%- trichloroacetic acid, and the precipitated material was removed by centrifuging. 0.2 r n l of the clear supernatant was transferred to a tube containing 5 ml of the reagent, freshly prepared from 5 m g of diacetyl monoxirne and 0.25 nig of thiosemi- carbazide in 50 % phosphoric acid. The colour was developed by boiling thc mixture in a water bath for a period of 20 min. The colour intensity of the cooled samples wiis detcrmined with a Hitachi spectrophotometer at 530 rim against a sample takcrt a t the beginning of the incubation just after adding the substratc.

I n order to examine the possible urcasc activity of the samples, incubations with added urea (0.2 mg/ml) and no arginine were also carried out.

128 M.-P. HARRI AND K. HARTIALA

Results T h e results of the determinations of urea formation in the tissues tested are preaent- ed in Table I.

The total urea synthesis in the whole organs tested ranged as presented in Tahlc 11. T h e amount of urea synthesis in the small intestinal mucosa was on an average 3.0 per cent in females and 2.8 per cent in males conipared with the liver.

Under the experimental conditions employed no urease activity could Iw demonstrated.

Discussion T h e arginase activity of various parts of the small intestine was found to be about 5-10 per cent of the liver activity to 1 g of tissue wet weight, but when the total weights are considered the activities remained at about 3 per cent. T h e relative arginase activities of rat liver and small intestine seem to Iw quite i n accordancr ivitli the results of Greengard, Sahib and Knox (1970). The glandiilar part of the stomach s e e m to be practically inactive in urea synthesis.

When both sexes are coriipared it is found that the arginase activity of li\rer i i i

the males is very significantly (2P < 0.001: student t-test) higher than i n the females in this laboratory strain. When the niucosae of various parts of thc small intestine are compared it can be seen (Table I ) that the jejunal mucosa is the most active site of the arginase activity. The activity in the male duodenal niiicosa is quite significantly (2P < 0.05) and in the male ileal mucosa significantly (2P < 0.01 ) higher than in females. This demonstrates some sex difference in the intestinal handling of arginine in the rat.

O u r tentative attempts to further characterize the arginase of rat small intestinal mucosa have shown that it may be associated with mitochondria. Arginasc in I'at liver is one of the soluble enzymes, as shown by Kosenthal ct al . 1956.

'I'his investigation was supported by a grant froin the U S . Public Health Sewice (.lhI-OG018- 09).

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