Ammonia formation in the medicinal leech, Hirudo medicinalis—in vivo and in vitro investigations

0300-9629/89$3.00 + 0.00 0 1989 Pergamon Press plc

Camp. Lfiochem. Physiol. Vol. 94A, No. 2, pp. 187-194, 1989 Printed in Great Britain

AMMONIA HIRUDO

FORMATION IN THE MEDICINAL LEECH, MEDICINALIS-IN VIVO AND IN VITRO INVESTIGATIONS PETER TSCHOERNER

and

ERNST ZEBE*

Zoologisches Institut, Lehrstuhl fur Zoophysiologie, Universitlt Mtinster, Hindenburgplatz D-4400 Miinster, FRG. Telephone: (0251) 83-3851

55,

(Received 6 February 1989) The excretion of N compounds was investigated in leeches fed various test solutions. 2. Ingestion was followed by a striking increase of NH, release exhibiting a characteristic time-course. 3. The NH, excreted resulted from the degradation of N compounds present in the test solutions. 4. Formation of NH, from proteins was inhibited by kanamycin, but was unaffected in the case of amino acids. 5. Symbiotic microorganisms do not significantly contribute to NH, formation. 6. Glutamate dehydrogenase and AMP deaminase are the enzymes most likely to be responsible for NH, formation in Hirudo.

Abstract-l.

INTRODUCTION

the alimentary tract which receives the blood stored in the foregut. Protein digestion in Hirudo seems to be confined to this strikingly tiny section of the alimentary system in which bacteria are only infrequently observed (Zebe et al., 1986). The ingestion of blood by the leech also causes the oxygen consumption and the excretion of NH, (and/or NH:) to rise dramatically. The rates of both processes were found to increase to eight times their previous level within a few days. Evidently, the NH, does not originate from excretory products of the host taken up with blood such as urea or NH,. Although oxygen consumption and NH3 release gradually decreased from their peak values observed a few days after feeding, comparatively high rates of both processes were maintained during a period of several weeks. It seems remarkable that hypoxia caused an immediate drop of NH, excretion to less than 25% of its previous level (Zebe et al., 1986) The investigations also revealed that, after feeding, the proteolytic activity in the intestinum slowly started to rise. After several weeks it was at a maximum from which it decreased gradually to the level typical of starving animals. However, in some experiments, the proteolytic activity failed to rise after feeding: when the antibiotic kanamycin was added to the blood or serum prior to the ingestion by the leech. In this case it remained as low as in starving animals. In contrast, oxygen consumption and NH, release seemed to be unaffected by the antibiotic, since the rates of both processes rose in a manner similar to that observed in the absence of kanamycin. However, in this case a long-term effect was found: oxygen uptake and NH, excretion decreased substantially after several days, although their rates remained clearly above the level measured in starving leeches (Zebe et al., 1986). In conclusion, it must be stated that many details of the processes involved in the digestion of blood by

The medicinal leech, Hirudo medicinalis, is specialized for feeding on vertebrate blood. As opportunities to do this occur rather rarely, the leech has squired the ability to take up very large volumes of food-up to eight times its own body weight-once it has found

a host. The worm starts concentrating the ingested blood immediately and, while still sucking, can be observed to excrete large drops of fluid. Within 24 hr, most of the water and the ions taken up with the blood are eliminated, so that the osmotic balance is restored. The foregut in which the ingested blood is stored has an enormous capacity due to 10 pairs of diverticula. The foregut thus occupies a major proportion of the total body volume. The blood stored in it does not change in appearance for weeks or even months. The degradation of the blood proteins proceeds very slowly and the food stored may last for several months. Until recently, the physiology of digestion in Hirudo was largely unknown despite of the fact that leeches have been used in medicine for several centuries. Proteolysis was presumed to result from the activity of symbiotic bacteria. This was postulated by Busing et al. (1951, 1953) because they had found that, in the foregut of the medical leech, only one species of bacterium is present, Pseudomonas hirudinis. These investigators also observed that, in culture, Pseudomonas hirudinis inhibits the growth of other species of bacteria and induces haemolysis as well as proteolysis (reviewed by Sawyer, 1986). In previous investigations we succeeded in demonstrating for the first time that (1) the foregut of Hirudo contains potent inhibitors of proteolytic enzymes and (2) at least two endopeptidases and one exopeptidase are present in the intestinum, the part of *Author to whom all correspondence

should be addressed. 187

PEER TSCHOERNIB

188

and

ERNST ZEBE

the medicinal leech still have to be elucidated so that the chain of events which eventually result in the release of NH, will be completely understood. This includes a hypothetical role which the bacteria present in the contents of the foregut may play in the metabolism of their host. In view of this situation, we have conducted several in vivo and in vitro experiments to find answers to the following questions:

detach themselves after unsuccessfully trying to continue sucking.

(1) Does the dramatic increase in the formation of NH, starting immediately after feeding result from the degradation of endogenous compounds or from the breakdown of certain components contained in the ingested blood? (2) Which of the reactions known to be involved in the formation of NH1 in other animals are used in Hirudo? Does the leech have a special way of nitrogen excretion possibly correlated to its specializing in feeding on vertebrate blood? To what extent do the bacteria contribute to the production of NH,? (3) Are there compounds which are deaminated particularly rapidly and, therefore, might function as favourite substrates resulting in high rates of NH, formation? (4) Does a spatial correlation exist in the body of the leech between protein digestion and the formation of NH,?

Measurement of excretion products

MATERIALS

AND METHODS

Animals Leeches (0.61.2g

weight) were purchased from a commercial supplier. They were kept in chlorine-free tap water in the dark at room temperature (18-25°C) and the water was changed once a week. Only animals that had been starving for several months were used in the experiments. (Under natural conditions starvation periods of such length are not unusual.) Feeding procedure

Special vials (volume 20ml), fitted with a synthetic membrane (“Paratilm”) instead of a glass bottom, were used for feeding the leeches. The test solutions were prepared from a mixture confining arginine (1 .O~01~1) and glucose (5.5 mmol/l) by adding the N compound to be tested and adjusting the osmotic value to approx. 3OOmosmol/l by adding NaCl and the pH to 7.4. Albumin and globulin were dialysed (2 x 4 hr against water) prior to their adding to the basic mixture. In addition to these test solutions. the following native media were used: serum (freshly prepared from pig blood obtained from a slaughterhouse), deproteinated serum (heated 10min in boiling water and centrifuged) and fetal calf serum (Fa. SERVA, Heidelberg), A defined volume of a test solution was pipetted into a vial and a leech was held close to the bottom membrane. As soon as the animal had attached to the membrane and started sucking (usually after 1 or 2min), the vial with the leech was transferred to a somewhat larger vessel to keep the animal in a moist atmosphere and to prevent it from crawling away after it had finished the meal. This set-up was also necessary to check whether the test solution was properly and quantitatively ingested by individual animals and also to detect occasional troubles such as the leaking of the test solution through the membrane after repeated puncturing by the leech. All animals which had not properly ingested the solution offered were eliminated. Ingestion of 2.0, 2.5 or 3.0ml of the test solutions was usually accomplished in less than 10 min. The leeches would

Incubation

After the animals had ingested the test solutions they were transferred to plastic vessels containing a defined volume of

water to be incubated at constant tem~rat~e (2O”C),either jnd~vjd~lly or in groups, as indicated Mow. The water was changed after 1, 2 or more days and frozen to be stored at - 18°C until it was analysed. NH, and urea in the test solutions and in the water were determined according to Da Fonseca-Wollheim (1973), Kerscher and Ziegenhorn (1985) or Lauber (1976). A calibration curve was obtained from standard solutions of (NH&SO, or urea. Preparation of extracts and assays of enzyme activities

After dissecting the animals tissue samples were transferred to preweighed vessels containing ice-cold buffer (phosphate or Tris-HCl, SOmmol/l, pH 7.5). They were weighed and buffer was added to a tissue/buffer ratio of 1:9. An Ultra-Turrax homogenizer was used for homogenization (3 x 20 set at maximum speed). The contents of the foregut were sonicated to break up the cells of the microorganisms. The resulting homogenates were centrifuged (20 mitt, 30,~g) and the su~matants were used for assaying the activities of the following enzymes, as indicated: (1) Glutamate dehydrogenase (GLDH, EC 1.4.1.2): Schmidt and Schmidt (1985). (2) Glutaminase (EC 351.2) and asnaraainase t’EC 3.5.1.1): van Waarde‘and Kesbeke (1982).* (3) AMP deaminase (ADA, EC 354.6): van Waarde (1981). (4) Serine dehydratase (EC 4.2.1.14): Bresnick et al. (1971). (5) Aspartate aminotransferase (GOT, EC 2.6.1.1): Rej and Horder (1983). (6) Alanine aminotransferase (GPT, EC 2.6.1.2): Horder and Rej (1983). (7) Arginase (EC 3.5.3.1) and urease (EC 3.5.1.5): Bergmeyer et al. (1983). (8) L-Amino oxidase (EC 1.4.3.2) and o-amino oxidase (EC 1.4.3.3): Bergmeyer et al. (1983). Stat~stiea~ treatment

The significance of differences was evaluated by Student’s z-test (P > 95%).

RESULTS

To test their ability for utilizing specific components of vertebrate blood and/or degradation products thereof, leeches were fed different types of “artificial diet”. The rates of NH, excretion, the time-course and the proportion of N excreted of the total N ingested were used as criteria to determine the extent of the utilization of the respective compound. The mean quantities of NH,, released within 24 hr, during an experimental period of several weeks following feeding are shown in Fig. 1. The curves represent the data obtained with the different test solutions. They exhibit striking differences in height and shape. NH, release by ieeches fed native serum rose steeply and, after approximately 1 week, was at a peak at eight times the level prior to feeding. Thereafter, it declined slowly and in 3 or 4 weeks

Ammonia

formation in

Hirudo

601

189

,

(b)

aspartate

x

al40 \ m E 5 20 I

glucose

0

days after

feeding

0

2

4

6

8

10

days after

12

14

16

18

20

feeding

Fig. I(a) and (b). Release of NH, by leeches fed test solutions containing various N compounds (100 mmol/I), In one experiment kanamycin (1 mg/ml) was added to a solution of alanine (lrmol/g/24 hr; mean values of five individuals f SD).

it had returned to the level prior to feeding. In contrast, the increase in the formation of NH, by leeches fed deproteinated serum was more modest. It is also evident from Fig. la that ingesting a test solution without a N compound added (i.e. glucose dissolved in saline) did not affect the release of NH3. Therefore, the leeches of this group can be used as controls. Utilization of specific amino acids by Him& was investigated employing the same method. Although certain differences in the rates and in the proportion of deamination seem to exist in Fig. la and b, the data of NH, fo~ation obtained after feeding various amino acids should be interpreted with caution. Usually, large variations between the individuals of the same experimental group were found. Furthermore, for technical reasons, it was necessary to conduct three series of experiments successively. As a consequence, leeches used in different series may have differed with respect to their physiological state and, in particular, to the length of the starvation period prior to the feeding. As Fig. la and b demonstrates, alanine, glycine, serine, glutamine and arginine were utilized rapidly, although the quantity of NH3 arising almost never amounted to 100% of the N taken up. The data shown should not be interpreted to indicate differences in the utilization of these amino acids. However, the rate of NH3 formation by leeches fed

~utamate and aspartate was significantly less (P > 98%) than that observed with the amino acids mentioned above and, in addition, the ratio of N excreted to N ingested was also comparatively low. In all experiments, most of the NH3 was excreted in the 2 weeks following the ingestion of the test solutions. As reported above, kanamycin had been shown to exert a long-term inhibitory effect on the excretion of NH, in leeches fed serum or blood (Zebe et al., 1986). In this respect, a new experiment was conducted using a mixture of alanine and kanamycin instead of a protein solution. Contrary to the previous investigation, NH, release in the presence and in the absence of kanamycin did not differ significantly (Figs lb and 4). Ingestion of glycylglycine also resulted in a clearly increased rate of NH, excretion (Fig. 1b), whereas in the case of glutathion (not shown) NH, formation was minimal. Altogether, these results seem important in the following respects: (1) The quantity of NH3 formed from deproteinated serum was substantially lower than that arising from native serum. This indicates that the components which, in vertebrate blood, constitute the so-called rest-N fraction do not contribute significantly to the formation of NH,.

190

PETERTSCHOERNERand ERNST ZEBE

(2) Glycine and glycylglycine were utilized at similar rates although the dipeptide contains twice as much N as the amino acid. (3) Kanamycin did not affect the deamination of alanine, contrary to the deamination of serum proteins observed previously (Zebe et al., 1986). It also seemed important to study the formation and excretion of urea in addition to that of NH,, because an unknown proportion of the NH, measured in the water could have resulted from the hydrolysis of urea by microbial activity either within the body of the leech or in the ambient water. The excretion of urea was checked by feeding leeches native serum, deproteinated serum or a solution of arginine and sub~quently monito~ng the urea present in the water in addition to NH,. Figure 2 shows the results. Urea excretion after the ingestion of native serum exhibited a characteristic time-course: large quantities appeared in the water shortly after feeding but, within 2 days, the release dropped to a negligible level. It is noteworthy that the amounts of urea excreted by leeches fed deproteinated serum as well as the time-course were similar to those given native serum. In the animals fed arginine, the rate of urea excretion was lower than in the former experimental groups, but the release of urea continued during the entire period of monitoring. In all feeding experiments described to this point, NH, release exhibited maximal rates between 7 and 10 days after the uptake of the test solutions

TI60 u q4L-l f =20 Ti E S.0

I

serum/serum

0

+r60 x 1 ‘11140 f ;20 E t0

10

5%

0

IO

20

30

albumin/5%

20

30

40

50

60

albumin

40

SO

60

'='60 x <40 f = 20 zi E LO - 60 x 240 f ; 20 E zo

1

1% globulin/5%

0

10

20

30

globulin

40

50

60

14_~~~~~~I ~, 0

10

20 30 40 50 days after feeding

60

Fig. 3. Release of NH, by leeches fed test solutions twice with an interval of 4 weeks between the meals ~mol/g~24 hr; mean values of five individuals & SD).

16%

serum

16 t

0

2

4

6 8 10 12 14 16 18 20 days afterfeeding

Fig. 2. Release of urea by leeches fed serum, deproteinated serum or arginine (100 nmol/l) bmol/individual/24 hr; mean values of five individuals f SD).

(although they differed in absolute terms and appeared to specifically depend on the compound ingested). This observation raises the question whether the decline of NH, formation 2 weeks after the feeding might be due to a lack of substrate. To test this possibility, another series of feeding experiments was conducted. Leeches were fed a particular test solution (2.Oml~individual) and after 28 days they were offered a second meal of 2.0 ml/individual, either of the same or of another solution. The daily NH3 release and. in some cases, the excretion of urea also were monitored as usual. The results are shown in Fig. 3. As it has been already reported above, the ingestion of serum was followed by a steep increase of NH, excretion which continued for about 10 days. Thereafter, the rate declined rapidly. A repeated ingestion of serum resulted in a similar time-course of NH, excretion. However, the total quantities excreted seemed to be lower than after the first meal. Essentially the same results were obtained in a parallel experiment in which the leeches were fed successively two meals of pure albumin solution (5%, which is the normal plasma content) instead of serum. On the other hand, substantially less NH3 was excreted by animals fed globulin (1% solution) and the amounts of NH3 were still lower after the second feeding (although a 5% solution was used in this case). After the ingestion of glycine (50 mmoljl), the time-course of deamination was similar to that observed in the serum experiment

Ammonia

0

= 60 x

10

20

30

40

formation

50

60

50

60

serum+kanamycin/serum

?40 f ; 20 E ‘LO 0

lo

20

days

30

after

40

feeding

Fig. 4. Release of NH, by leeches fed test solutions with or without kanamycin (1 mg/ml) added and repeated feeding after 4 weeks &mol/g/24 hr; mean values of five individuals + SD). again NH, release seemed reduced in absolute quantities after the second meal consisting of 100 mmol/l glycine. Some groups of leeches were fed different solutions at the first and the second meal. In one experiment, glycine with kanamycin added was offered for the first feeding and albumin for the second one. This resulted in the excretion of large amounts of NH, following the first meal, whereas NH, release from the protein was very low (Fig. 4). In a parallel experiment, serum plus kanamycin was used for the first feeding and native serum for the second. In this case, NH, excretion was high on the days after the first meal (very likely resulting from non-protein N taken up with the serum), but thereafter NH, release declined dramatically. After ingesting normal serum at the second meal, there was also a short phase of high-rate release of NH,, followed by a steep decrease. It is noteworthy that urea excretion exhibited a similar pattern, except that on the second or third day after each meal it actually declined to zero (Fig. 2). The experiments described in the following section are an attempt to elucidate the mode(s) of NH, release in Hirudo. In the formation of NH, resulting from the utilization of amino acids generally three different ways seem possible: and

(1) Oxidative deamination of glutamate which frequently arises by transamination, i.e. by transforming an amino acid into an 0x0 acid by transferring the amino group to oxoglutarate in a first step resulting in the synthesis of glutamate (“transdeamination”). (2) Deamination of adenosine monophosphate (AMP) in the so-called purine nucleotide cycle. (3) Deamination of certain amino acids by specific enzymes such as histidase or serine dehydratase. Table I. Maximal

activities

of deaminating

in Hirudo

191

In vertebrates, NH3 may also arise from the deamidation of glutamine and occasionally of asparagine. To find out which of these alternative pathways may be involved in the formation of NH, by the leech the maximal activities of the enzymes catalysing the respective reactions were assayed in different parts of alimentary tract as well as in other tissues. The results of the tests using conventional spectrophotometric methods are compiled in Table 1. Evidently, only two NH, generating enzymes are present in substantial activities: glutamate dehydrogenase (GlDH, NAD specific) and AMP deaminase (ADA). In addition to these enzymes, very low activities of serine dehydratase were measured, whereas the test for asparaginase, glutaminase, L-amino oxidase (substrate leucine and L-alanine), D-amino oxidase (substrate D-&ink), arginase and urease were negative. Table 1 shows that fairly high activities of GlDH were assayed in the body-wall and also in the epithelial lining of the foregut and its diverticula. However, the values found in the intestinum greatly exceeded those in the former tissues. In contrast, the contents of the foregut were virtually free of GlDH. The distribution of ADA was clearly different from that of GlDH. The highest values were measured in the body-wall, whereas the activities assayed in the intestinum were strikingly low and once more no activity was detected in the contents of the foregut. Since, indirectly, transaminases could also be involved in the generation of NH, (transdeamination), aspartate amino transferase (GOT) and alanine amino transferase (GPT) activities were assayed in the tissues of Hirudo. The data in Table 1 show that in all extracts the former enzyme was found to be more active than the latter. The distribution of both transaminases in the organs tested was rather uniform. In conclusion, it has to be emphasized that very large individual variations in the activities of all enzymes were observed. DISCUSSJON

Because of its highly specialized feeding habit, the medicinal leech is totally dependent on the degradation of blood proteins for both anabolic and catabolic metabolism. However, to be utilized as substrates for the production of energy, the amino acids arising from protein digestion have first to be deaminated. As a consequence, the rate of NH, formation in Hirudo should correspond to the actual rate of catabolism. A contribution of the endogenous reserves such as glycogen seems negligible except in particular situations including anaerobiosis and muscular activity (but replenishment of these stores

and transaminating

enzymes in some tissues of Hirudo (U/g W.W. f SD,

numbers of extracts assayed in parentheses) GlDH Body-wall Foregut epithelium Foregut contents Intestinum

9.60 6.69 0.12 78.57

f k + +

2.90 (16) 2.54 (17) 0.17 (8) 43.37 (7)

ADA 6.36 f 1.84 (14) 2.67 k 0.85 (13) 0 (4) 1.12 kO.79 (3)

GOT 4.17+ 1.43 3.35 f 0.87 0.16+0.12 6.52 f 2.77

GPT (8) (8) (3) (3)

1.04 1.00 0.13 1.60

* i f f

0.33 0.36 0.04 0.44

(7) (8) (3) (3)

192

PETERTSCHOERNER and

during subsequent recovery would again require amino acids as substrates and thus contribute to the formation of NH,). The steep rise of NH3 release following the ingestion of proteins or amino acids, which failed to occur in the case of a test solution containing only glucose in addition to saline, clearly demonstrates the significance of the components of the normal food for the dramatic increase of the metabolic rate in Hirudo following the ingestion of blood. It must be emphasized that feeding experiments are especially suited for studying the digestive physiology of the leech, because the animals voluntarily ingest large volumes of the test solutions and, in every respect, exhibit a normal behaviour. Nevertheless, very large individual variations were observed in all parameters measured while investigating the metabolism of Hirudo. A steep rise of NH, excretion, following the ingestion of the test solutions containing various amino acids, was clearly evident after 24 hr. If the absorption of amino acids in Hirudo indeed proceeds solely in the intestinum, the ingested solutions must have been forwarded rather rapidly from the foregut to this part of the alimentary tract. This was actually demonstrated in special experiments by adding labelled inulin to a test solution prior to its ingestion. Within 24 hr, some radioactivity appeared in the intestinum (Roters, personal communication). Five to 7 days after feeding, NH3 excretion was maximal and subsequently it decreased rather quickly, until, after 2 or 3 weeks. it had returned to the level typical of starvation. This time-course of NH, formation differs from that observed after the ingestion of serum, since. in this case, very high (although not maximal) rates were maintained for extended periods, indicating continuing digestion of substantial amounts of proteins (Zebe et al., 1986). Deamination of most of the amino acids tested was similar in rate and in time-course except for proline and particularly for glutamate and aspartate, from which distinctly smaller amounts of NH, resulted. Perhaps this was due to a slow absorption of these acidic amino acids, because in these cases the reduction of the body volume after feeding was strikingly delayed and also decreased in extent. Uniformly, the quantity of NH, released was clearly less than the total N taken up in the test solution. It is very likely that a certain proportion of the ingested amino acids was utilized in anabolic processes. The concentrations of amino acids arising in the normal digestion of blood proteins are substantially lower than those in the test solutions. Therefore, the results reported cannot be taken to represent the processes which occur under natural conditions; but they demonstrate that Hirudo possesses an enormous capacity for deaminating various amino acids. Strikingly different amounts of NH, were observed after feeding leeches the peptides, glycylglycine and glutathion. In the case of the former, the NH, released into the water roughly amounted to that observed after feeding glycine, whereas NH, formation from glutathion was minimal. It seems possible that glycylglycine was deaminated without being hydrolysed, whereas glutathion certainly was not split, because otherwise substantial quantities of NH,

ERNSTZEBE

would have been formed by the deamination of the arising glycine. After the ingestion of serum, the formation of NH, was shown to occur in two distinct phases. The first one is characterized by a peak attained within 24 hr after feeding and a rapidly decreasing rate on the second day. This early NH3 excretion probably results from the elimination of NH, (and urea) taken up with the serum (which contained 2.2 mmol NH3 and 6.6 mmol urea/l). The second phase starts after 3 or 4 days and shows peak values of NH, excretion after 5-7 days. It is very likely that this indicates the digestion of serum proteins. This is evident if NH, excretion following the ingestion of deproteinated serum and of pure dialysed albumin solution are compared. In the first case, NH, release was virtually limited to a short period of approximately 2 days after feeding whereas, in the second case, it started after some delay, probably due to the absence of preformed NH, and the rather slow initiation of albumin digestion. Previous investigations have shown that, in starving leeches, the activity of proteinases is very low; but, according to in vitro measurements, it would be still sufficient to digest 2 nmol of haemoglobin/hr/mg intestinum (epithelium plus contents) which amounts to 50&1000 nmol/day and individual (Zebe et al. 1986). As low as the proteolytic activity may appear, it would suffice for the rate of protein degradation and subsequent deamination of the amino acids to account for the formation of the quantities of NH, actually measured during the first week following feeding. After reaching a peak, 7-10 days after a meal, the excretion of NH, started decreasing, although the maximum proteolytic activity assayed in intestinum extracts continued to rise gradually, showing peak values approximately 4 weeks after feeding. On the other hand, it appears that the rate of protein degradation remains approximately constant for periods of many weeks. Generally, the processing of food in the alimentary system of Hirudo was shown to proceed very slowly indeed, as it amounted to less than 0.5% of the weight of the animals. prior to ingesting several ml of serum (Roters, personal communication). When the leeches were offered a second meal 4 weeks after the first one, however, characteristic changes in the rate of NH, excretion were observed, which were very likely to correspond to changes in the rate of protein digestion. There was a sharp increase to a maximum followed by a relatively slow return to the previous “normal” level. Presumably, this increase in protein degradation was due to a rising demand for energy resulting from the muscular work of sucking and from the osmotic work of restoring the osmotic balance after ingesting a large volume of hyperosmotic fluid. From the enzymatic activities assayed in different tissues of Hirudo, it can be concluded that either GlDH or ADA (or both) should be responsible for the release of NH,. A direct (02 dependent) oxidation of amino acids, especially by symbiotic microorganisms, seems highly unlikely. In the intestinum, GlDH activity was maximal, whereas the level of ADA was strikingly low. In the body-wall and in the foregut epithelium. the activities of both enzymes

Ammonia formation in Hirudo

193

were rather similar and, with regard to GlDH, considerably lower than in the intestinum. The transaminases, GOT and GPT, were also found in substantial activities but, contrary to the desaminases, they were rather evenly distributed in the tissues studied. At this point, it has to be emphasized that assaying maximal enzymatic activities reveals only relative and hypothetical capacities which do not allow any conclusions as to the significance of a particular enzyme in the metabolism of an intact tissue or even organism. Nevertheless, it seems justified to expect a central role of GlDH in the deamination of amino acids by the leech; but, at present, it appears premature to infer from the finding of strikingly high activities in the epithelial lining of the intestinum that ammonia arises mainly in this location. [Preliminary investigations have shown the GlDH of Hirudo is not exceptional, but it has catalytic and physical properties which are similar to those of the GlDHs of other animals including the bivalve Mya arenaria (Moyes et al., 1985) and the channel catfish. Zct~furus punctatus (Casey et al., 1983).] The significance of ADA in Hirudo is difficult to assess, since it is not clear whether the enzyme is involved in the purine nucleotide cycle or whether it has another function still unknown. While there is convincing evidence for the operation of this cycle in vertebrate muscle, its functioning or its significance in other vertebrate tissues seems still in question (for review, see van Waarde, 1988). This also holds for invertebrates generally. Although high ADA activities have been found in some annelids, including earthworms and the lugworm, and, in addition, high rates of NH, formation from AMP by the homogenized gut of Lumbricus have been demonstrated, the physiological role of ADA in these cases is anything but clear (Bishop and Barnes, 1971, MacDonnell and Tillinghast, 1973). Generally, the contents of the foregut exhibited only minimal enzymatic activities (GOT and GPT were probably vertebrate enzymes present in the serum ingested). Therefore, a significant contribution of the symbiotic microorganisms to the formation of NH, in Hirudo is unlikely. In conclusion, the results reported seem to provide answers to the questions formulated in the introduction as follows:

high ADA activity). Since glutaminase and amino oxidases are virtually absent, no special way of N excretion seems to exist in Hirudo. The cont~bution of microorganisms to the degradation of amino acids and to their deamination seems negligible. (3) Several of the amino acids tested in feeding experiments were deaminated at high rates; but NH3 formation from glutamate and aspartate proceeded particularly slowly, which could also be due to a slow absorption of these amino acids from the alimentary tract. NH3 release after the ingestion of blood (or various test solutions) exhibited a typical time-course: very high rates during the 2 weeks following a meal and gradually decreasing rates thereafter. This probably reflects the changing in the rate of catabolic processes, (4) Although some NH, is very likely to arise in the intestinum as soon as the amino acids produced in protein digestion are absorbed, it is not possible to assess, even approximately, its proportion of the total NH, formation, i.e. to evaluate the role of specific tissues in the deamination (and utilizations of amino acids.

(1) The increase of NHs formation after the ingestion of blood by Hirudo results mainly from the degradation of proteins and the subsequent deamination of the amino acids arising. Only a small proportion of the NH, released originates directly from the blood of the host, i.e. is already ingested as NH,. There is no rise in the breakdown of endogenous reserves evoked by the feeding behaviour. Hydrolysis of urea taken up with the blood is insi~ificant. (2) Transdeamination of glutamate appears to be a particularly important way of NH3 formation. The high activity of ADA in some tissues of the leech may be interpreted as to indicate that this enzyme too could be involved in large-style deamination although, at present, this is only hypothetical (which also holds for all tissues of invertebrates exhibiting

Casey C. A., Perlman D. F., Vorhaben J. E. and Campbell J. W. (1983) Hepatic ammoniagenesis in the channel catfish Ictalurus punctatus. Molec. Physiol. 3,

Acknowledgements-The

authors are indebted to F. J. Roters for many stimulating discussions, for permission to quote some of his unpublished data and for his help in preparing the graphs. The support of the investigation by the Deutsche Forschungsgemeinschaft (Grant Ze 40,’15-3) is gratefully acknowledged. REFERENCES Bergmeyer H. U., Grass1 M. and Waiter H. E. (1983) In Methods of Enzymatic Analysis (Edited by Bergmeyer H. U.), 3rd Edn. Vol. II, pp. 148-151, 155-156 and 320-32 1. Verlag Chemie, Weinheim. Bishop S. H. and Barnes L. B. (1971) Ammonia-forming mechanisms: deamination of 5’-adenvlic acid (AMP) bv some poiychaete annelids. Comp. Bidchem. Phisiof. 408, 407-422.

Bresnick E., Maytield E. D., Liebelt A. G. and Liebelt R. A. (1971) Enzyme patterns in a group of transplantable mouse hepatomas of different growth rates. Cancer Res. 31, 743-751. Busing K. H. (I 95 1) Pseudomonas hirudinis, ein bakterieller Darmsymbiont des Blutegels (Hirudo oficinalis). Zbl. Bakt. 157, 478-484.

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