J. Prolozool., 39(5), 1992, pp. 613-618 0 I992 by the Society of Protozoologists
Arginine Catabolism by Leishmania donovani Promastigotes J . J O S E P H BLUM Division of Physiology, Department of Cell Biology, Duke UniversityMedical Center, Durham, North Carolina 2771 0
ABSTRACT. Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of a-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than a-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine. It is suggested that the effects of glucose and 2-deoxyglucose on arginine catabolism depend largely upon the nature of their metabolites,whereas the effects of the various amino acids examined depend largely on the extent to which they interfere with or enhance arginine transport into the cells. Key words. a-aminoisobutyrate, 2-deoxyglycose, osmotic stress, urea.
A
previous study showed that when Leishmania donovani promastigotes are grown in a defined medium, they consume, in varying amounts, 13 of the 19 amino acids studied, and produced five others to various extents [24]. Arginine was one of the amino acids apparently consumed, its concentration decreasing very slightly (from 0.84 to 0.79 mM) during a fiveday experiment. Arginine was also consumed by L. mexicana [ 10, 131 and by L. braziliensis when growing in medium supplemented with arginine [27]. It was subsequently shown that L. donovani and L. braziliensis contained arginase but no other enzymes of the ornithine-arginine pathway [7]. More recently, Bera [ 11 demonstrated the presence of the five enzymes of the y-guanidobutyramide pathway in L. donovani and provided evidence suggesting that it is the primary pathway for arginine catabolism in intact promastigotes. Although Bera [I] showed that arginine could stimulate the rate of oxygen consumption by promastigotes resuspended in a buffered salt solution to the same extent as glucose, no further information is available about any factors that might regulate its utilization by L. donovani. In this study the effects of glucose and two of its analogues, 2-deoxyglucose and 6-deoxyglucose, on the rate of urea formation from promastigotes resuspended in a buffer solution containing arginine were examined. Next the effects of several amino acids including leucine, proline, lysine, glycine, threonine, and glutamate as well as alanine and its non-metabolizable analogue, a-aminoisobutyrate, on the rate of urea formation were examined. The results obtained from these studies are discussed in terms of the effects of metabolites of glucose and 2-deoxyglucose on urea formation and the effects of the amino acids themselves on arginine transport into cells. Since earlier studies had shown interesting effects of osmotic conditions on cell shape [ l l , 121 and metabolism [3, 4, 6, 261 the effects of osmotic conditions on arginine catabolism were also examined. MATERIALS AND METHODS
Leishmania donovani promastigotes, strain Sudan 1S, were obtained from Dr. A. Mukkada (University of Cincinnati, Cincinnati, OH) and grown in modified Pan's medium as described [3]. Three milliliters of a stock culture were inoculated into 50 ml of medium in a 500-ml capacity Erlenmeyer flask and grown for 48 h at 26" C under air. An additional 50 ml of medium was then added and the flasks transferred to a shaker bath (80 strokedmin) maintained at 26" C for about 17 h (late log phase) 4 1 h, 65 h, or 89 h (1-day, 2-day, or 3-day stationary phase, respectively). The cultures were then centrifuged for 4 min at
1,400 g at room temperature, resuspended in Hanks' Balanced Salt Solution excluding glucose or phenol red (HBSS-), washed twice, and resuspended in a suitable volume of HBSS-. Samples ofthe twice-washed cells were taken for determination ofprotein content [I91 and then aliquots of 1 ml were added to 50-ml Erlenmeyer flasks containing L-arginine and other reagents as desired, such that the total volume was 3 ml. Unless otherwise specified, all reagents were made up in HBSS-, which has an osmolality of 305 mosmlkg [l 11. The flasks were incubated under air in a shaker bath maintained at 26 k 0.5" C. Samples of 0.7 ml were taken at 0.3, 25.3, 50.3, and 75.3 rnin after the addition of the cells to the flasks, and placed in a boiling water bath for 1.5 rnin and then in ice. The urea content of the samples was measured by the diacetyl monoxime method (Sigma procedure #535, Sigma Company, St. Louis, MO). The L-isomers of arginine, alanine, proline, leucine, lysine, glycine, threonine, and glutamate were obtained from Sigma as were y-aminobutyric acid, y-guanidinobutyric acid, mannitol, phorbol myristic acid, phorbol monoacetate, A23 187, 3',5'-cyclic GMP, dibutyrylcyclic AMP, mannose, 2-deoxyglucose, 6-deoxyglucose, and a-aminoisobutyric acid. RESULTS Arginine is oxidized to y-guanidobutyramide (GGBM), which is then hydrolyzed to y-guanidobutync acid (GGBA), which in turn is hydrolyzed to y-aminobutyric acid and urea [ 11. Preliminary experiments showed that washed promastigotes produced no urea during a 75-min incubation in the presence of added GGBA (data not shown), consistent with Bera's [ 11 observation that GGBA did not cause an increase in oxygen consumption. As can be seen in Fig. 1, however, L-arginine is catabolized to urea at a rate (about 70 nmole/mg proteinlh) that is approximately constant for at least 75 min. In a separate experiment it was found that the rate did not decrease significantly if the concentration of arginine was reduced from 2 mM to 1 mM, although a decrease in the rate of urea formation occurred if the initial concentration ofarginine was 0.5 mM (data not shown). Effects of glucose and alanine on urea formation from arginine. It has been shown that leucine oxidation by L. donovani promastigotes is inhibited by glucose and by alanine, but not by glutamate [4]. It was of interest, therefore, to begin this study of arginine catabolism by ascertaining whether the rate of urea formation was affected by glucose or by alanine. Figure 1 shows that glucose significantly increased and alanine significantly decreased the rate of urea formation by late log phase promasti-
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Minutes Fig. 1. Rate of urea formation from arginine by L. donovani proCombined effects of alanine and glucose on urea formation Fig. 2. mastigotes from cultures in the late log phase of growth. Cells were washed and resuspended in HBSS- and incubated in the presence of from arginine. Late log phase promastigotes were treated as described in Fig. 1. Cells were incubated with arginine (M arginine ), plus 4 mM arginine (-), arginine plus 4 mM glucose (G---O), arginine plus 4 mM alanine (A---A), arginine plus 2 mM leucine alanine (A- --A), arginine plus glucose (G- - -O), or arginine plus glu(7-- -V), 4 mM alanine alone (A)or 2 mM leucine alone (V).Samples cose and alanine (P- -V). Values are means f SD (n = 4). were collected at the times shown on the abscissa and assayed for urea. Data shown are the means +. SD (n = 4). Effect of culture age on arginine catabolism. An earlier study showed that L. mexicana promastigotes resuspended in fresh gotes. Leucine, however, did not alter the rate of urea formation. growth medium secrete four times as much urea as do amasPractically no urea was formed if the cells were incubated with tigotes [lo]. To ascertain whether arginine catabolism to urea alanine or leucine alone (Fig. l), and, similarly, no urea was by L. donovani promastigotes changed with growth phase, cells formed by cells incubated with glucose alone (data not shown). were collected from 3-day stationary phase cultures. Although Since glucose enhanced and alanine inhibited the rate of urea these cells were shorter and more ellipsoidal, they did not lose formation from arginine, the combined effect of these modifiers their flagella and did not look like amastigotes. The rate of urea on arginine catabolism was examined. Figure 2 shows that the formation was much lower than that of late log phase cells (Fig. activating effect of glucose and the inhibiting effect of alanine 4).As in log phase cells, glucose caused an increase and alanine canceled each other so that the rate of formation of urea from a decrease in the rate of arginine catabolism, and leucine had arginine alone did not differ significantly from the rate when no effect. The rate of formation of urea from alanine alone or from leucine alone was very low. It was also apparent that, both glucose and alanine were present. Experiments were also performed to examine the effects of especially in the presence of glucose, the rate of urea formation acetate (which is metabolized via the intramitochondrial Krebs appeared to increase during the course of the incubation (Fig. cycle), proline, and glutamate on the catabolism of arginine. 4). It therefore seemed worthwhile to examine the time course Neither acetate (3 mM) nor proline (8 mM) had any significant of reduction in the rate of urea formation from arginine as a effect on the rate of urea formation from 4 mM arginine (data function of culture age. In this set of experiments we also exnot shown), but glutamate caused a significant increase in urea amined whether the effects of glucose or alanine varied with formation (Fig. 3). When both alanine and glutamate were add- culture age. In addition, the washed cells of each age were ined, the inhibitory effect of alanine was stronger than the acti- cubated with mannose and 2-deoxyglucose (2-DG), to detervating effect of glutamate, causing a small net decrease in the mine whether mannose could substitute for glucose and whether rate of urea production. An experiment was also performed in 2-DG, which is phosphorylated but not further metabolized which urea formation from 4 m M arginine was measured in the [22], had the same activating effect on arginine catabolism as presence of 4 mM lysine, 4 m M glycine, or 4 m M threonine. did glucose. Figure 5 shows results obtained with cells from late log phase Neither lysine nor glycine had any effect on the rate of urea formation, but threonine caused a small inhibition (- 15%, data and 1-,2-, and 3-day stationary phase cultures. Cells from late not shown). The rate of urea formation from each of these three log phase cultures behaved as expected, i.e. glucose activated urea formation, while alanine inhibited it (Fig. 5A). It was also amino acids alone was barely detectable.
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found that mannose, which is transported by the glucose camer [23,28], activated urea formation to the same extent as glucose. In cells from I-day stationary phase cultures, exactly the same pattern was observed except that the rate of urea formation in the presence of arginine alone was reduced from approximately 90 to 55 nmoles/h.mg protein (Fig. 5B). Cells from 2-day stationary phase cultures produced urea at about 20 nmoles/h.mg protein, and there was no further decline in cells from 3-day stationary phase cultures (Fig. 5C, D). At each culture age, glucose (and, equally, mannose) activated urea formation, while alanine inhibited. Of particular interest was the effect of 2-DG, which, at each culture age, inhibited urea formation to approximately the same extent as did alanine (Fig. 5). It should also be noted that whereas in cells from log phase cultures (Fig. 14) urea production from arginine was approximately linear with time of incubation in the presence of added glucose, in cells from 2- and 3-day stationary phase cultures, the rate of urea formation increased during incubation in the presence of glucose. Effects of 6-deoxyglucose and of a-aminoisobutyric acid on arginine catabolism. The finding that 2-DG inhibited urea formation whereas glucose activated it suggested that the effect of accumulation of glucose or 2-DG inside the cell might depend on the extent to which the compound was further metabolized. An experiment was therefore performed to ascertain whether 6-deoxyglucose (6-DG), an analogue of glucose which, in Trypanosoma brucei, is actively transported but not phosphorylated or further metabolized [9], or a-aminoisobutyric acid (AIB), which is actively transported by L. tropica promastigotes [ 181, would affect the rate of urea formation from arginine.
Fig. 4 . Arginine catabolism by promastigotes from stationary phase cultures. Cells from 3-day stationary phase cultures were washed and resuspended in HBSS- and incubated in the presence of 2 mM arginine (M arginine ), plus 4 mM glucose (C---O), arginine plus 4 mM alanine (A- - -A), arginine plus 2 mM leucine (V- - -V),alanine alone (A),or leucine alone (7).Values are means f SD (n = 4). Figure 6 shows that whereas 2-DG caused an appreciable inhibition of urea formation, 6-DG had no effect. It is also apparent that AIB inhibited urea formation, though not as strongly as alanine. Effect of possible second messengers. Dibutyryl CAMP(0.75 mM) had no effect on the rate of urea formation from arginine at either 305 or 153 mosm/kg (Fig. 4). Experiments were also performed at 153 mosm/kg in which phorbol myristic acid (2.16 pM), A23187 (2.77 wM), and 3',5'-cyclic G M P (1 mM) were added to separate flasks. None of these compounds had any significant effect on the rate of urea formation from late log phase promastigotes incubated with 4 mM arginine (data not shown). Effects of osmotic pressure on urea formation from arginine. It has become apparent that osmotic pressure affects intermediary metabolism in many cells (see, e.g. Haussinger and Lang [14]), including Leishmania. The rate of formation of I4CO, from a number of I4C-labeled substrates including glutamate, alanine, and leucine was inhibited by hyperosmotic stress, but hypo-osmotic stress had n o appreciable effect [4, 61. Hypo-osmotic stress did, however, have some effects on the pathways ofglucose catabolism [26]. An initial study showed that whereas hyperosmotic conditions had a small inhibitory effect on arginine catabolism, hypo-osmotic conditions (1 53 mosm/kg) caused a strong inhibition. It was then realized that, since hypo-osmotic stress causes a very rapid release of alanine from the large internal pool present in these cells [12], it was possible that the inhibition was a secondary effect due to the alanine. To test this hypothesis, a set of experiments was performed in which the cells were collected, washed twice in HBSS-, and then split into two portions. One portion was mixed with an equal volume of HBSS-, the other with an equal volume ofwater. The two flasks were the incubated for 4 min, a time sufficient for release of most of the alanine pool [ 121, and then centrifuged. The super-
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Minutes Minutes Fig. 5. Effects of alanine, glucose, 2-deoxyglucose, mannose, and acute hypo-osmotic stress on arginine catabolism as a function ofculture age. Cells from A) late log phase, B) 1-day stationary, C ) 2-day stationary, and D) 3-day stationary cultures were collected and washed in HBSS-. The washed cells were then incubated in HBSS- containing 4 mM arginine alone (M 4) mM, arginine supplemented with 8 mM 8 mM alanine (A- - -A), or 8 mM 2-deoxyglucose glucose (0--a), (U- - a ). At the times indicated on the abscissa, samples were taken and assayed for urea. As in all preceding figures, the urea concentration measured after 20 sec of incubation has been subtracted from all subsequent values.
natants were removed and the cell pellets resuspended in HBSS-. The rates of urea production from added arginine were then measured under hypo-, iso-, and hyperosmotic conditions. Control cells, that had been pre-incubated under iso-osmotic conditions, were inhibited to a small but significant extent by hyperosmotic stress, and to a larger extent by hypo-osmotic stress (Fig. 7). Cells that had been depleted of their alanine by hypoisomotic pretreatment were now no longer significantly inhibited by hypo-osmotic stress. They were, however, still inhibited to a small extent by hyperosmotic stress (Fig. 7). It thus appears that whereas hyperosmotic stress causes appreciable inhibition of the rate of urea formation from arginine, hypo-osmotic stress acts largely via the inhibitory effects of the alanine released. DISCUSSION Although Bera's [ 11data support the view that urea formation from arginine by L. donovani promastigotes is entirely via the GGBM pathway, the present data, in which only urea formation was measured, cannot exclude the possibility that arginase is also active in the intact cell under some of the conditions examined. Regardless of the pathway(s) used, there is no obvious
0
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MINUTES Fig. 6 . Effect of 6-deoxyglucose and AIB on arginine catabolism. Cells from late log phase cultures were collected and washed in HBSS-. The washed cells were then incubated in HBSS- containing 4 mM arginine alone (M 4) mM , arginine supplemented with 8 mM alanine (A---A), 8 mM AIB (A---A), 8 mM 2-deoxyglucose ( I 3 - U),or 8 mM 6-deoxyglucose (0- - -0).For further details, see legend to Fig. 5 . explanation why the rate of urea formation should be increased by glucose and mannose but inhibited by 2-DG. Glucose and mannose are strong inhibitors offatty acid oxidation by L. major promastigotes, but 2-DG has no effect [2]. This is consistent with regulation by a metabolite of glucose or mannose (e.g. fructose-2,6-bisphosphate[25]) that is not formed from 2-DG. The inhibition of leucine oxidation by glucose is also likely to result from a metabolite of glucose beyond the level of glucose6-phosphate [4].The identity of the metabolite(s) of glucose responsible for the inhibition of fatty acid and leucine oxidation and for the activation of arginine catabolism remains to be investigated. The failure of added dibutyryl CAMP, cGMP, phorbol myristic acid, or A23 187 to affect the rate of urea formation suggests that CAMP, cGMP, diacylglycerol, and Ca++ are not involved in the regulation of arginine (or leucine [4]) catabolism. Arginine catabolism is inhibited by 2-DG but not affected by 6-DG. Since 6-DG is actively transported by T. brucei but is unlikely to be phosphorylated, it is perhaps not surprising that it does not affect the rate of urea formation in L. donovani (assuming that the glucose transport system is similar to that of T. brucei), especially since there i s kinetic symmetry for its transport, so that the intracellular concentration is not likely to
BLUM-ARGININE CATABOLISM BY LEISHMANIA D O N 0 VANI
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Fig. 7. Effect of hypo- and hyperosmotic conditions on arginine catabolism. Late log phase promastigotes were washed twice in HBSS-. The washed cell pellet was resuspended in 21 ml HBSS- and 10 ml were added to 10 ml of HBSS- and 10 ml were added to 10 ml of H,O. The separate flasks were incubated in a 26" C shaker bath for 4 min and then centrifuged, the supernatants removed by suction, and the pellets resuspended in identical volumes of HBSS-. Aliquots of 1 ml of the control and hypo-osmotically pretreated cells were then added to 50-ml flasks containing 0.5 ml of 24 mM arginine dissolved in HBSSand 1.5 ml of either water, HBSS-, or 610 mM mannitol dissolved in HBSS-, so that the final osmolalities were as indicated on the graph. Samples were taken for assay of urea content at the times shown on the abscissa.
greatly exceed that ofthe extracellular fluid [9]. However, 2-DG is phosphorylated but not further metabolized [21]. About twothirds of the intracellular pool consists of 2-deoxy-D-glucose phosphate (2-DGP) and the remainder is free 2-DG; the intracellular concentration (sum of 2-DG 2DGP) is about 20-fold higher than the extracellular concentration [22]. The gradual accumulation of this large intracellular pool of 2-DG plus 2-DGP could account for the slow (- 20 min) change in cell shape that occurs when washed promastigotes are incubated with 2-DG (or glucose), since the shape change is prevented by addition of mannitol and mimicked (albeit very rapidly) by addition of water [l 1, 121. It therefore seemed likely that the inhibitory effect of 2-DG on urea formation from arginine might be due to a slow increase in cell volume. This possibility was reinforced when it was found that addition of water (i.e. hypo-osmotic stress) caused an inhibition of urea formation. That inhibition, however, appears to result largely from the release of the internal pool of alanine; in cells that had been depleted of alanine by a
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brief exposure to hypo-osmotic conditions, there was little inhibition of urea formation during a subsequent 1-h exposure to hypo-osmotic conditions (Fig. 7). Thus it seems likely that 2-DG and/or 2-DGP inhibit urea formation by a direct effect on the arginine catabolism pathway(s) rather than via triggering a volume sensor which in turn affects this pathway. It should also be noted that hyperosmotic stress, which would tend to decrease cell volume, inhibits urea formation. The amount of inhibition, however, is relatively small (Fig. 7) and could be due to an increase in the concentration of one or more cytoplasmic components that affect the arginine catabolic pathway(s) in a relatively non-specific way rather than via a specific signal released by a sensor responding to the decrease in cell volume. Leucine, alanine, proline, and glutamate are oxidized at high rates by promastigotes resuspended in a buffer solution [4, 15, 161. It is not known to what extent leucine accumulates intracellularly in L. donovani, but the products of its catabolismacetyl CoA and, presumably acetoacetate [4]-are unlikely to affect the pathway(s) of arginine catabolism, consistent with the finding that acetate has no effect on urea formation. Although threonine had a small inhibiting effect on arginine metabolism, neither glycine nor lysine had any effect. The failure of lysine to inhibit the rate of arginine catabolism suggests that, if arginine is transported into L. donovani, it is not via a transporter comparable to the Ly+ system of many mammalian cells [S]. Aminoisobutyrate, like alanine, is actively transported into L. tropica promastigotes, but, unlike alanine, is not further metabolized [ 181. Proline, although it accumulates intracellularly by active transport [17, 291 had no effect on urea formation despite the fact that glutamate, which is formed from proline, increased the rate of urea formation. One possible hypothesis to explain the inhibitory effects of alanine and AIB, the activating effect of glutamate, and the partial cancellation of the inhibitory effect of alanine by the activating effect of glutamate (Fig. 5B) is that, unlike leucine or proline or lysine or glycine, alanine and glutamate interact with a transport system(s) that carries arginine. According to this hypothesis, alanine and AIB would interfere with arginine uptake, while glutamate would enhance it. The rate of uptake of AIB via the proton gradientdriven alanine transporter is inhibited only about 20% by 1 mM arginine [ 181, so it is not likely that alanine and arginine compete for this transporter. It is known that there are two systems responsible for the uptake of alanine, proline, and phenylalanine in promastigotes [ S ] , but it is not known whether glutamate is taken up by either of these transporters or by other amino acid transporting systems such as are present in most mammalian cells [8]. Further studies on the amino acid transport systems of promastigotes will be required to more fully understand the way in which various amino acids affect arginine uptake and/ or catabolism. Although fatty acid oxidation capacity increases with culture age [2] the capacity to oxidize a number of other substrates such as glucose, alanine, and acetate decreases [ 151, as does that of arginine (Fig. 5). The decrease is already evident within 1 day after entering the late log phase ofgrowth, and is largely complete in 2-day stationary phase cultures. Whether this decrease is related to a decrease in the rate of putrescine synthesis as the cells stop growing is unknown. This decrease is not, however, accompanied by any qualitative change in the effects of glucose, mannose, 2-deoxyglucose or alanine on the rate of urea formation. The primary effect of increasing culture age is therefore likely to be a reduction in activity of one or more of the enzymes of the arginine catabolic pathway and/or of the arginine uptake system(s). In log and 1 -day stationary phase cells, urea formation from arginine was linear with time, and the addition of glucose caused, if anything, a slight deceleration in the rate of urea
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formation (Fig. 5A, B). I n 3-day stationary phase cells, however, whereas the rate o f urea formation in the absence of glucose was constant throughout the 75-min assay period, the presence o f glucose caused an accelerating rate (Fig. 5D).Although there are no a m i n o acids present in HBSS-, proteolysis could provide a source of a m i n o acids for de n o v o synthesis (fueled by A T P from glucose catabolism) of an arginine transporter. Although this explanation is consistent with the increased level of proteinase activity in stationary a s compared t o log phase promastigotes of L. mexicana mexicana [20], other explanations are also possible. Further experiments will be required t o determine whether a reduction in activity o f an enzyme(s) o f the catabolic pathway and/or a transporter is responsible for t h e reduced rate o f urea formation from arginine i n 3-day as compared t o log phase promastigotes, a n d t o understand the factors responsible for the accelerating rate of urea formation in 3-day stationary phase cells i n the presence of glucose. ACKNOWLEDGMENTS This work was supported by NIH G r a n t R 0 1 A126534. I am grateful t o A. Hayes for excellent technical assistance. LITERATURE CITED 1. Bera, T. 1987. The y-guanidinobutyramide pathway of L-argi-
nine catabolism in Leishmania donovani promastigotes. Mol. Biochem. Parasitol., 23: 183-192. 2. Blum, J. J. 1990. Effects of culture age and hexoses on fatty acid oxidation by Leishmania major. J. Protozool., 37:505-5 10. 3. Blum, J. J. 1991a. Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes. J. Protozool.. 38:229233. 4. Blum, J. J. 1991b. Oxidation of leucine by Leishmania donovani. J. Protozool., 38:527-531. 5. Bonay, P. & Cohen, B. E. 1983. Neutral amino acid transport in Leishmania promastigotes. Biochim. Biophys., 731 :222-228. 6. Burrows, C. M. & Blum, J. J. 1991. Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes. J. Protozool., 38:47-5 2. 7. Camargo, E. P., Coelho, J. A,, Moraes, G. & Figueiredo, E. N. 1978. Trypanosoma spp., Leishmania spp. and Leptomonas spp.; enzymes of ornithine-arginine metabolism. Exp. Parasitol., 46: 14 1-1 44. 8. Christensen, H. N. 1984. Organic ion transport during seven decades; the amino acids. Biochim. Biophys. Acta, 779:255-269. 9. Conroy, K., Eisenthal, R., Game, S. & Holman, G. 1987. Sugar transport in Trypanosoma brucei. Biochem. SOC.Trans., 15:1073. 10. Coombs, G. H. & Sanderson, B. E. 1985. Amine production by Leishmania mexicana. Ann. Trop. Med. Parasitol., 79:4094 15. 11. Darling, T. N. & Blum, J. J. 1990. Changes in the shape of Leishmania major promastigotes in response to hexoses, proline, and hypo-osmotic stress. J. Protozool., 371267-272. 12. Darling, T. N., Burrows, C. M. & Blum, J. J. 1990. Rapid shape change and release of ninhydrin-positive substances by Leishmania ma-
jor promastigotes in response to hypo-osmotic stress. J. Protozool., 37: 49 3 4 9 9 . 13. Hart, D. T. & Coombs, G. H. 1982. Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp. Parasitol., 54:397409. 14. Haussinger, D. & Lang, F. 1991. Cell volume in the regulation of hepatic function: a mechanism for metabolic control. Biochim. Biophys. Acta, 107:331-350. 15. Keegan, F. & Blum, J. J. 1990. Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes. Mol. Biochem. Parasitol., 39:235-246. 16. Krassner, S. M. & Flory, B. 1972. Proline metabolism in Leishmania donovani promastigotes. J. Protozool., 19:682-685. 17. Law, S. S. & Mukkada, A. J. 1979. Transport of L-proline and its regulation in Leishmania tropica promastigotes. J. Protozool., 26: 295-30 I . 18. Lepley, P. R. & Mukkada, A. J. 1983. Characteristics of an uptake system for a-aminoisobutyric acid in Leishmania tropica promastigotes. J. Protozool., 30:4146. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. I. & Randall, R. J. 1950. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193:265-275. 20. Mallinson, D. J. & Coombs, G. H. 1986. Molecular characterisation of the metacyclic forms of Leishmania. ZRCS Med. Sci., 14: 557-558. 21. Mukkada, A. J., Schaefer, F. W., 111, Simon, M. W. & Neu, C. 1974. Delayed in vitro utilization of glucose by Leishmania tropica promastigotes. J. Protozool., 21:393-397. 22. Schaefer, F. W., 111, Martin, E. & Mukkada, A. J. 1974. The glucose transport system in Leishmania tropica promastigotes. J. Protozool., 21 :592-5 96. 23. Schaefer, F. W., I11 & Mukkada, A. J. 1976. Specificity of the glucose transport system in Leishmania tropica promastigotes. J. Protozool., 23:446449. 24. Steiger, R. F. & Meshnick, S. R. 1977. Amino-acid and glucose utilization of Leishmania donovani and L. braziliensis. Trans. Roy. SOC. Trop. Med. Hygiene, 71:441443. 25. Van Schaftingen, E., Opperdoes, F. R. & Hers, H.-G. 1985. Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6bisphosphate. Eur. J. Biochern., 153:403406. 26. Walsh, M. & Blum, J. J. 1991. Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes. Mol. Biochem. Parasitol., 50:205-2 14. 27. Yoshida, N. & Camargo, E. P. 1978. Ureotelism and ammonotelism in trypanosomatids. J. Bacteriol., 136: 1 184-1 186. 28. Zilberstein, D. & Dwyer, D. M. 1984. Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Parasitol., 12:327336. 29. Zilberstein, D. & Dwyer, D. M. 1985. Proton motive forcedriven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc. Natl. Acad. Sci. USA, 82:17 161720. Received 1-17-92, 5-18-92; accepted 4-29-92
Arginine Catabolism by Leishmania donovani Promastigotes J . J O S E P H BLUM Division of Physiology, Department of Cell Biology, Duke UniversityMedical Center, Durham, North Carolina 2771 0
ABSTRACT. Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of a-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than a-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine. It is suggested that the effects of glucose and 2-deoxyglucose on arginine catabolism depend largely upon the nature of their metabolites,whereas the effects of the various amino acids examined depend largely on the extent to which they interfere with or enhance arginine transport into the cells. Key words. a-aminoisobutyrate, 2-deoxyglycose, osmotic stress, urea.
A
previous study showed that when Leishmania donovani promastigotes are grown in a defined medium, they consume, in varying amounts, 13 of the 19 amino acids studied, and produced five others to various extents [24]. Arginine was one of the amino acids apparently consumed, its concentration decreasing very slightly (from 0.84 to 0.79 mM) during a fiveday experiment. Arginine was also consumed by L. mexicana [ 10, 131 and by L. braziliensis when growing in medium supplemented with arginine [27]. It was subsequently shown that L. donovani and L. braziliensis contained arginase but no other enzymes of the ornithine-arginine pathway [7]. More recently, Bera [ 11 demonstrated the presence of the five enzymes of the y-guanidobutyramide pathway in L. donovani and provided evidence suggesting that it is the primary pathway for arginine catabolism in intact promastigotes. Although Bera [I] showed that arginine could stimulate the rate of oxygen consumption by promastigotes resuspended in a buffered salt solution to the same extent as glucose, no further information is available about any factors that might regulate its utilization by L. donovani. In this study the effects of glucose and two of its analogues, 2-deoxyglucose and 6-deoxyglucose, on the rate of urea formation from promastigotes resuspended in a buffer solution containing arginine were examined. Next the effects of several amino acids including leucine, proline, lysine, glycine, threonine, and glutamate as well as alanine and its non-metabolizable analogue, a-aminoisobutyrate, on the rate of urea formation were examined. The results obtained from these studies are discussed in terms of the effects of metabolites of glucose and 2-deoxyglucose on urea formation and the effects of the amino acids themselves on arginine transport into cells. Since earlier studies had shown interesting effects of osmotic conditions on cell shape [ l l , 121 and metabolism [3, 4, 6, 261 the effects of osmotic conditions on arginine catabolism were also examined. MATERIALS AND METHODS
Leishmania donovani promastigotes, strain Sudan 1S, were obtained from Dr. A. Mukkada (University of Cincinnati, Cincinnati, OH) and grown in modified Pan's medium as described [3]. Three milliliters of a stock culture were inoculated into 50 ml of medium in a 500-ml capacity Erlenmeyer flask and grown for 48 h at 26" C under air. An additional 50 ml of medium was then added and the flasks transferred to a shaker bath (80 strokedmin) maintained at 26" C for about 17 h (late log phase) 4 1 h, 65 h, or 89 h (1-day, 2-day, or 3-day stationary phase, respectively). The cultures were then centrifuged for 4 min at
1,400 g at room temperature, resuspended in Hanks' Balanced Salt Solution excluding glucose or phenol red (HBSS-), washed twice, and resuspended in a suitable volume of HBSS-. Samples ofthe twice-washed cells were taken for determination ofprotein content [I91 and then aliquots of 1 ml were added to 50-ml Erlenmeyer flasks containing L-arginine and other reagents as desired, such that the total volume was 3 ml. Unless otherwise specified, all reagents were made up in HBSS-, which has an osmolality of 305 mosmlkg [l 11. The flasks were incubated under air in a shaker bath maintained at 26 k 0.5" C. Samples of 0.7 ml were taken at 0.3, 25.3, 50.3, and 75.3 rnin after the addition of the cells to the flasks, and placed in a boiling water bath for 1.5 rnin and then in ice. The urea content of the samples was measured by the diacetyl monoxime method (Sigma procedure #535, Sigma Company, St. Louis, MO). The L-isomers of arginine, alanine, proline, leucine, lysine, glycine, threonine, and glutamate were obtained from Sigma as were y-aminobutyric acid, y-guanidinobutyric acid, mannitol, phorbol myristic acid, phorbol monoacetate, A23 187, 3',5'-cyclic GMP, dibutyrylcyclic AMP, mannose, 2-deoxyglucose, 6-deoxyglucose, and a-aminoisobutyric acid. RESULTS Arginine is oxidized to y-guanidobutyramide (GGBM), which is then hydrolyzed to y-guanidobutync acid (GGBA), which in turn is hydrolyzed to y-aminobutyric acid and urea [ 11. Preliminary experiments showed that washed promastigotes produced no urea during a 75-min incubation in the presence of added GGBA (data not shown), consistent with Bera's [ 11 observation that GGBA did not cause an increase in oxygen consumption. As can be seen in Fig. 1, however, L-arginine is catabolized to urea at a rate (about 70 nmole/mg proteinlh) that is approximately constant for at least 75 min. In a separate experiment it was found that the rate did not decrease significantly if the concentration of arginine was reduced from 2 mM to 1 mM, although a decrease in the rate of urea formation occurred if the initial concentration ofarginine was 0.5 mM (data not shown). Effects of glucose and alanine on urea formation from arginine. It has been shown that leucine oxidation by L. donovani promastigotes is inhibited by glucose and by alanine, but not by glutamate [4]. It was of interest, therefore, to begin this study of arginine catabolism by ascertaining whether the rate of urea formation was affected by glucose or by alanine. Figure 1 shows that glucose significantly increased and alanine significantly decreased the rate of urea formation by late log phase promasti-
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Minutes Fig. 1. Rate of urea formation from arginine by L. donovani proCombined effects of alanine and glucose on urea formation Fig. 2. mastigotes from cultures in the late log phase of growth. Cells were washed and resuspended in HBSS- and incubated in the presence of from arginine. Late log phase promastigotes were treated as described in Fig. 1. Cells were incubated with arginine (M arginine ), plus 4 mM arginine (-), arginine plus 4 mM glucose (G---O), arginine plus 4 mM alanine (A---A), arginine plus 2 mM leucine alanine (A- --A), arginine plus glucose (G- - -O), or arginine plus glu(7-- -V), 4 mM alanine alone (A)or 2 mM leucine alone (V).Samples cose and alanine (P- -V). Values are means f SD (n = 4). were collected at the times shown on the abscissa and assayed for urea. Data shown are the means +. SD (n = 4). Effect of culture age on arginine catabolism. An earlier study showed that L. mexicana promastigotes resuspended in fresh gotes. Leucine, however, did not alter the rate of urea formation. growth medium secrete four times as much urea as do amasPractically no urea was formed if the cells were incubated with tigotes [lo]. To ascertain whether arginine catabolism to urea alanine or leucine alone (Fig. l), and, similarly, no urea was by L. donovani promastigotes changed with growth phase, cells formed by cells incubated with glucose alone (data not shown). were collected from 3-day stationary phase cultures. Although Since glucose enhanced and alanine inhibited the rate of urea these cells were shorter and more ellipsoidal, they did not lose formation from arginine, the combined effect of these modifiers their flagella and did not look like amastigotes. The rate of urea on arginine catabolism was examined. Figure 2 shows that the formation was much lower than that of late log phase cells (Fig. activating effect of glucose and the inhibiting effect of alanine 4).As in log phase cells, glucose caused an increase and alanine canceled each other so that the rate of formation of urea from a decrease in the rate of arginine catabolism, and leucine had arginine alone did not differ significantly from the rate when no effect. The rate of formation of urea from alanine alone or from leucine alone was very low. It was also apparent that, both glucose and alanine were present. Experiments were also performed to examine the effects of especially in the presence of glucose, the rate of urea formation acetate (which is metabolized via the intramitochondrial Krebs appeared to increase during the course of the incubation (Fig. cycle), proline, and glutamate on the catabolism of arginine. 4). It therefore seemed worthwhile to examine the time course Neither acetate (3 mM) nor proline (8 mM) had any significant of reduction in the rate of urea formation from arginine as a effect on the rate of urea formation from 4 mM arginine (data function of culture age. In this set of experiments we also exnot shown), but glutamate caused a significant increase in urea amined whether the effects of glucose or alanine varied with formation (Fig. 3). When both alanine and glutamate were add- culture age. In addition, the washed cells of each age were ined, the inhibitory effect of alanine was stronger than the acti- cubated with mannose and 2-deoxyglucose (2-DG), to detervating effect of glutamate, causing a small net decrease in the mine whether mannose could substitute for glucose and whether rate of urea production. An experiment was also performed in 2-DG, which is phosphorylated but not further metabolized which urea formation from 4 m M arginine was measured in the [22], had the same activating effect on arginine catabolism as presence of 4 mM lysine, 4 m M glycine, or 4 m M threonine. did glucose. Figure 5 shows results obtained with cells from late log phase Neither lysine nor glycine had any effect on the rate of urea formation, but threonine caused a small inhibition (- 15%, data and 1-,2-, and 3-day stationary phase cultures. Cells from late not shown). The rate of urea formation from each of these three log phase cultures behaved as expected, i.e. glucose activated urea formation, while alanine inhibited it (Fig. 5A). It was also amino acids alone was barely detectable.
615
BLUM-ARGININE CATABOLISM BY LEISHMANIA DONOVAN1
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found that mannose, which is transported by the glucose camer [23,28], activated urea formation to the same extent as glucose. In cells from I-day stationary phase cultures, exactly the same pattern was observed except that the rate of urea formation in the presence of arginine alone was reduced from approximately 90 to 55 nmoles/h.mg protein (Fig. 5B). Cells from 2-day stationary phase cultures produced urea at about 20 nmoles/h.mg protein, and there was no further decline in cells from 3-day stationary phase cultures (Fig. 5C, D). At each culture age, glucose (and, equally, mannose) activated urea formation, while alanine inhibited. Of particular interest was the effect of 2-DG, which, at each culture age, inhibited urea formation to approximately the same extent as did alanine (Fig. 5). It should also be noted that whereas in cells from log phase cultures (Fig. 14) urea production from arginine was approximately linear with time of incubation in the presence of added glucose, in cells from 2- and 3-day stationary phase cultures, the rate of urea formation increased during incubation in the presence of glucose. Effects of 6-deoxyglucose and of a-aminoisobutyric acid on arginine catabolism. The finding that 2-DG inhibited urea formation whereas glucose activated it suggested that the effect of accumulation of glucose or 2-DG inside the cell might depend on the extent to which the compound was further metabolized. An experiment was therefore performed to ascertain whether 6-deoxyglucose (6-DG), an analogue of glucose which, in Trypanosoma brucei, is actively transported but not phosphorylated or further metabolized [9], or a-aminoisobutyric acid (AIB), which is actively transported by L. tropica promastigotes [ 181, would affect the rate of urea formation from arginine.
Fig. 4 . Arginine catabolism by promastigotes from stationary phase cultures. Cells from 3-day stationary phase cultures were washed and resuspended in HBSS- and incubated in the presence of 2 mM arginine (M arginine ), plus 4 mM glucose (C---O), arginine plus 4 mM alanine (A- - -A), arginine plus 2 mM leucine (V- - -V),alanine alone (A),or leucine alone (7).Values are means f SD (n = 4). Figure 6 shows that whereas 2-DG caused an appreciable inhibition of urea formation, 6-DG had no effect. It is also apparent that AIB inhibited urea formation, though not as strongly as alanine. Effect of possible second messengers. Dibutyryl CAMP(0.75 mM) had no effect on the rate of urea formation from arginine at either 305 or 153 mosm/kg (Fig. 4). Experiments were also performed at 153 mosm/kg in which phorbol myristic acid (2.16 pM), A23187 (2.77 wM), and 3',5'-cyclic G M P (1 mM) were added to separate flasks. None of these compounds had any significant effect on the rate of urea formation from late log phase promastigotes incubated with 4 mM arginine (data not shown). Effects of osmotic pressure on urea formation from arginine. It has become apparent that osmotic pressure affects intermediary metabolism in many cells (see, e.g. Haussinger and Lang [14]), including Leishmania. The rate of formation of I4CO, from a number of I4C-labeled substrates including glutamate, alanine, and leucine was inhibited by hyperosmotic stress, but hypo-osmotic stress had n o appreciable effect [4, 61. Hypo-osmotic stress did, however, have some effects on the pathways ofglucose catabolism [26]. An initial study showed that whereas hyperosmotic conditions had a small inhibitory effect on arginine catabolism, hypo-osmotic conditions (1 53 mosm/kg) caused a strong inhibition. It was then realized that, since hypo-osmotic stress causes a very rapid release of alanine from the large internal pool present in these cells [12], it was possible that the inhibition was a secondary effect due to the alanine. To test this hypothesis, a set of experiments was performed in which the cells were collected, washed twice in HBSS-, and then split into two portions. One portion was mixed with an equal volume of HBSS-, the other with an equal volume ofwater. The two flasks were the incubated for 4 min, a time sufficient for release of most of the alanine pool [ 121, and then centrifuged. The super-
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J. PROTOZOOL., VOL. 39, NO. 5, SEPTEMBER-OCTOBER 1992
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Minutes Minutes Fig. 5. Effects of alanine, glucose, 2-deoxyglucose, mannose, and acute hypo-osmotic stress on arginine catabolism as a function ofculture age. Cells from A) late log phase, B) 1-day stationary, C ) 2-day stationary, and D) 3-day stationary cultures were collected and washed in HBSS-. The washed cells were then incubated in HBSS- containing 4 mM arginine alone (M 4) mM, arginine supplemented with 8 mM 8 mM alanine (A- - -A), or 8 mM 2-deoxyglucose glucose (0--a), (U- - a ). At the times indicated on the abscissa, samples were taken and assayed for urea. As in all preceding figures, the urea concentration measured after 20 sec of incubation has been subtracted from all subsequent values.
natants were removed and the cell pellets resuspended in HBSS-. The rates of urea production from added arginine were then measured under hypo-, iso-, and hyperosmotic conditions. Control cells, that had been pre-incubated under iso-osmotic conditions, were inhibited to a small but significant extent by hyperosmotic stress, and to a larger extent by hypo-osmotic stress (Fig. 7). Cells that had been depleted of their alanine by hypoisomotic pretreatment were now no longer significantly inhibited by hypo-osmotic stress. They were, however, still inhibited to a small extent by hyperosmotic stress (Fig. 7). It thus appears that whereas hyperosmotic stress causes appreciable inhibition of the rate of urea formation from arginine, hypo-osmotic stress acts largely via the inhibitory effects of the alanine released. DISCUSSION Although Bera's [ 11data support the view that urea formation from arginine by L. donovani promastigotes is entirely via the GGBM pathway, the present data, in which only urea formation was measured, cannot exclude the possibility that arginase is also active in the intact cell under some of the conditions examined. Regardless of the pathway(s) used, there is no obvious
0
20
40
60
80
MINUTES Fig. 6 . Effect of 6-deoxyglucose and AIB on arginine catabolism. Cells from late log phase cultures were collected and washed in HBSS-. The washed cells were then incubated in HBSS- containing 4 mM arginine alone (M 4) mM , arginine supplemented with 8 mM alanine (A---A), 8 mM AIB (A---A), 8 mM 2-deoxyglucose ( I 3 - U),or 8 mM 6-deoxyglucose (0- - -0).For further details, see legend to Fig. 5 . explanation why the rate of urea formation should be increased by glucose and mannose but inhibited by 2-DG. Glucose and mannose are strong inhibitors offatty acid oxidation by L. major promastigotes, but 2-DG has no effect [2]. This is consistent with regulation by a metabolite of glucose or mannose (e.g. fructose-2,6-bisphosphate[25]) that is not formed from 2-DG. The inhibition of leucine oxidation by glucose is also likely to result from a metabolite of glucose beyond the level of glucose6-phosphate [4].The identity of the metabolite(s) of glucose responsible for the inhibition of fatty acid and leucine oxidation and for the activation of arginine catabolism remains to be investigated. The failure of added dibutyryl CAMP, cGMP, phorbol myristic acid, or A23 187 to affect the rate of urea formation suggests that CAMP, cGMP, diacylglycerol, and Ca++ are not involved in the regulation of arginine (or leucine [4]) catabolism. Arginine catabolism is inhibited by 2-DG but not affected by 6-DG. Since 6-DG is actively transported by T. brucei but is unlikely to be phosphorylated, it is perhaps not surprising that it does not affect the rate of urea formation in L. donovani (assuming that the glucose transport system is similar to that of T. brucei), especially since there i s kinetic symmetry for its transport, so that the intracellular concentration is not likely to
BLUM-ARGININE CATABOLISM BY LEISHMANIA D O N 0 VANI
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Fig. 7. Effect of hypo- and hyperosmotic conditions on arginine catabolism. Late log phase promastigotes were washed twice in HBSS-. The washed cell pellet was resuspended in 21 ml HBSS- and 10 ml were added to 10 ml of HBSS- and 10 ml were added to 10 ml of H,O. The separate flasks were incubated in a 26" C shaker bath for 4 min and then centrifuged, the supernatants removed by suction, and the pellets resuspended in identical volumes of HBSS-. Aliquots of 1 ml of the control and hypo-osmotically pretreated cells were then added to 50-ml flasks containing 0.5 ml of 24 mM arginine dissolved in HBSSand 1.5 ml of either water, HBSS-, or 610 mM mannitol dissolved in HBSS-, so that the final osmolalities were as indicated on the graph. Samples were taken for assay of urea content at the times shown on the abscissa.
greatly exceed that ofthe extracellular fluid [9]. However, 2-DG is phosphorylated but not further metabolized [21]. About twothirds of the intracellular pool consists of 2-deoxy-D-glucose phosphate (2-DGP) and the remainder is free 2-DG; the intracellular concentration (sum of 2-DG 2DGP) is about 20-fold higher than the extracellular concentration [22]. The gradual accumulation of this large intracellular pool of 2-DG plus 2-DGP could account for the slow (- 20 min) change in cell shape that occurs when washed promastigotes are incubated with 2-DG (or glucose), since the shape change is prevented by addition of mannitol and mimicked (albeit very rapidly) by addition of water [l 1, 121. It therefore seemed likely that the inhibitory effect of 2-DG on urea formation from arginine might be due to a slow increase in cell volume. This possibility was reinforced when it was found that addition of water (i.e. hypo-osmotic stress) caused an inhibition of urea formation. That inhibition, however, appears to result largely from the release of the internal pool of alanine; in cells that had been depleted of alanine by a
+
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brief exposure to hypo-osmotic conditions, there was little inhibition of urea formation during a subsequent 1-h exposure to hypo-osmotic conditions (Fig. 7). Thus it seems likely that 2-DG and/or 2-DGP inhibit urea formation by a direct effect on the arginine catabolism pathway(s) rather than via triggering a volume sensor which in turn affects this pathway. It should also be noted that hyperosmotic stress, which would tend to decrease cell volume, inhibits urea formation. The amount of inhibition, however, is relatively small (Fig. 7) and could be due to an increase in the concentration of one or more cytoplasmic components that affect the arginine catabolic pathway(s) in a relatively non-specific way rather than via a specific signal released by a sensor responding to the decrease in cell volume. Leucine, alanine, proline, and glutamate are oxidized at high rates by promastigotes resuspended in a buffer solution [4, 15, 161. It is not known to what extent leucine accumulates intracellularly in L. donovani, but the products of its catabolismacetyl CoA and, presumably acetoacetate [4]-are unlikely to affect the pathway(s) of arginine catabolism, consistent with the finding that acetate has no effect on urea formation. Although threonine had a small inhibiting effect on arginine metabolism, neither glycine nor lysine had any effect. The failure of lysine to inhibit the rate of arginine catabolism suggests that, if arginine is transported into L. donovani, it is not via a transporter comparable to the Ly+ system of many mammalian cells [S]. Aminoisobutyrate, like alanine, is actively transported into L. tropica promastigotes, but, unlike alanine, is not further metabolized [ 181. Proline, although it accumulates intracellularly by active transport [17, 291 had no effect on urea formation despite the fact that glutamate, which is formed from proline, increased the rate of urea formation. One possible hypothesis to explain the inhibitory effects of alanine and AIB, the activating effect of glutamate, and the partial cancellation of the inhibitory effect of alanine by the activating effect of glutamate (Fig. 5B) is that, unlike leucine or proline or lysine or glycine, alanine and glutamate interact with a transport system(s) that carries arginine. According to this hypothesis, alanine and AIB would interfere with arginine uptake, while glutamate would enhance it. The rate of uptake of AIB via the proton gradientdriven alanine transporter is inhibited only about 20% by 1 mM arginine [ 181, so it is not likely that alanine and arginine compete for this transporter. It is known that there are two systems responsible for the uptake of alanine, proline, and phenylalanine in promastigotes [ S ] , but it is not known whether glutamate is taken up by either of these transporters or by other amino acid transporting systems such as are present in most mammalian cells [8]. Further studies on the amino acid transport systems of promastigotes will be required to more fully understand the way in which various amino acids affect arginine uptake and/ or catabolism. Although fatty acid oxidation capacity increases with culture age [2] the capacity to oxidize a number of other substrates such as glucose, alanine, and acetate decreases [ 151, as does that of arginine (Fig. 5). The decrease is already evident within 1 day after entering the late log phase ofgrowth, and is largely complete in 2-day stationary phase cultures. Whether this decrease is related to a decrease in the rate of putrescine synthesis as the cells stop growing is unknown. This decrease is not, however, accompanied by any qualitative change in the effects of glucose, mannose, 2-deoxyglucose or alanine on the rate of urea formation. The primary effect of increasing culture age is therefore likely to be a reduction in activity of one or more of the enzymes of the arginine catabolic pathway and/or of the arginine uptake system(s). In log and 1 -day stationary phase cells, urea formation from arginine was linear with time, and the addition of glucose caused, if anything, a slight deceleration in the rate of urea
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formation (Fig. 5A, B). I n 3-day stationary phase cells, however, whereas the rate o f urea formation in the absence of glucose was constant throughout the 75-min assay period, the presence o f glucose caused an accelerating rate (Fig. 5D).Although there are no a m i n o acids present in HBSS-, proteolysis could provide a source of a m i n o acids for de n o v o synthesis (fueled by A T P from glucose catabolism) of an arginine transporter. Although this explanation is consistent with the increased level of proteinase activity in stationary a s compared t o log phase promastigotes of L. mexicana mexicana [20], other explanations are also possible. Further experiments will be required t o determine whether a reduction in activity o f an enzyme(s) o f the catabolic pathway and/or a transporter is responsible for t h e reduced rate o f urea formation from arginine i n 3-day as compared t o log phase promastigotes, a n d t o understand the factors responsible for the accelerating rate of urea formation in 3-day stationary phase cells i n the presence of glucose. ACKNOWLEDGMENTS This work was supported by NIH G r a n t R 0 1 A126534. I am grateful t o A. Hayes for excellent technical assistance. LITERATURE CITED 1. Bera, T. 1987. The y-guanidinobutyramide pathway of L-argi-
nine catabolism in Leishmania donovani promastigotes. Mol. Biochem. Parasitol., 23: 183-192. 2. Blum, J. J. 1990. Effects of culture age and hexoses on fatty acid oxidation by Leishmania major. J. Protozool., 37:505-5 10. 3. Blum, J. J. 1991a. Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes. J. Protozool.. 38:229233. 4. Blum, J. J. 1991b. Oxidation of leucine by Leishmania donovani. J. Protozool., 38:527-531. 5. Bonay, P. & Cohen, B. E. 1983. Neutral amino acid transport in Leishmania promastigotes. Biochim. Biophys., 731 :222-228. 6. Burrows, C. M. & Blum, J. J. 1991. Effects of osmotic pressure on the oxidative metabolism of Leishmania major promastigotes. J. Protozool., 38:47-5 2. 7. Camargo, E. P., Coelho, J. A,, Moraes, G. & Figueiredo, E. N. 1978. Trypanosoma spp., Leishmania spp. and Leptomonas spp.; enzymes of ornithine-arginine metabolism. Exp. Parasitol., 46: 14 1-1 44. 8. Christensen, H. N. 1984. Organic ion transport during seven decades; the amino acids. Biochim. Biophys. Acta, 779:255-269. 9. Conroy, K., Eisenthal, R., Game, S. & Holman, G. 1987. Sugar transport in Trypanosoma brucei. Biochem. SOC.Trans., 15:1073. 10. Coombs, G. H. & Sanderson, B. E. 1985. Amine production by Leishmania mexicana. Ann. Trop. Med. Parasitol., 79:4094 15. 11. Darling, T. N. & Blum, J. J. 1990. Changes in the shape of Leishmania major promastigotes in response to hexoses, proline, and hypo-osmotic stress. J. Protozool., 371267-272. 12. Darling, T. N., Burrows, C. M. & Blum, J. J. 1990. Rapid shape change and release of ninhydrin-positive substances by Leishmania ma-
jor promastigotes in response to hypo-osmotic stress. J. Protozool., 37: 49 3 4 9 9 . 13. Hart, D. T. & Coombs, G. H. 1982. Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp. Parasitol., 54:397409. 14. Haussinger, D. & Lang, F. 1991. Cell volume in the regulation of hepatic function: a mechanism for metabolic control. Biochim. Biophys. Acta, 107:331-350. 15. Keegan, F. & Blum, J. J. 1990. Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes. Mol. Biochem. Parasitol., 39:235-246. 16. Krassner, S. M. & Flory, B. 1972. Proline metabolism in Leishmania donovani promastigotes. J. Protozool., 19:682-685. 17. Law, S. S. & Mukkada, A. J. 1979. Transport of L-proline and its regulation in Leishmania tropica promastigotes. J. Protozool., 26: 295-30 I . 18. Lepley, P. R. & Mukkada, A. J. 1983. Characteristics of an uptake system for a-aminoisobutyric acid in Leishmania tropica promastigotes. J. Protozool., 30:4146. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. I. & Randall, R. J. 1950. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193:265-275. 20. Mallinson, D. J. & Coombs, G. H. 1986. Molecular characterisation of the metacyclic forms of Leishmania. ZRCS Med. Sci., 14: 557-558. 21. Mukkada, A. J., Schaefer, F. W., 111, Simon, M. W. & Neu, C. 1974. Delayed in vitro utilization of glucose by Leishmania tropica promastigotes. J. Protozool., 21:393-397. 22. Schaefer, F. W., 111, Martin, E. & Mukkada, A. J. 1974. The glucose transport system in Leishmania tropica promastigotes. J. Protozool., 21 :592-5 96. 23. Schaefer, F. W., I11 & Mukkada, A. J. 1976. Specificity of the glucose transport system in Leishmania tropica promastigotes. J. Protozool., 23:446449. 24. Steiger, R. F. & Meshnick, S. R. 1977. Amino-acid and glucose utilization of Leishmania donovani and L. braziliensis. Trans. Roy. SOC. Trop. Med. Hygiene, 71:441443. 25. Van Schaftingen, E., Opperdoes, F. R. & Hers, H.-G. 1985. Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6bisphosphate. Eur. J. Biochern., 153:403406. 26. Walsh, M. & Blum, J. J. 1991. Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes. Mol. Biochem. Parasitol., 50:205-2 14. 27. Yoshida, N. & Camargo, E. P. 1978. Ureotelism and ammonotelism in trypanosomatids. J. Bacteriol., 136: 1 184-1 186. 28. Zilberstein, D. & Dwyer, D. M. 1984. Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Parasitol., 12:327336. 29. Zilberstein, D. & Dwyer, D. M. 1985. Proton motive forcedriven active transport of D-glucose and L-proline in the protozoan parasite Leishmania donovani. Proc. Natl. Acad. Sci. USA, 82:17 161720. Received 1-17-92, 5-18-92; accepted 4-29-92
