Cyclic 3′,5′-adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat

Molecular and Biochemical Parasitology, 3 (1981) 19- 31

19

Elsevier/North-Holland Biomedical Press

CYCLIC 3 ' , 5 ' - A D E N O S I N E M O N O P H O S P H A T E L E V E L S D U R I N G T H E D E V E L O P M E N T A L C Y C L E O F TR YPANOSOMA B R UCEI B R UCEI IN T H E R A T

PATRICIA E. MANCINI and CURTIS L. PATTON

Department o f Epidemiology and Public Health, Yale University School o f Medicine, New Haven, CT 06510, U.S.A. (Received 4 June 1980; accepted 24 September 1980)

Intracellular content of cyclic 3',5'-adanosine monophosphate was determined in several strains of Trypanosoma brucei brucei during their growth in rats. In non-relapsing infections with the cloned monomorphic strain ll0M and with the cloned pleomorphic strain YTatl, the amount of cyclic AMP per 109 trypanosomes increased from 30 to over 90 pmol as the parasitemia increased from patancy to o v e r 1 0 9 organisms per ml. This increase was not observed during non-relapsing infections with strain 427. During infections with strain YTatl in both immunocompetent and lethally X-irradiated rats, cyclic AMP content of the parasite increased from 2 0 - 2 0 pmol per 1 0 9 .cells early in logarithmic growth to 6 5 - 70 pmol per 109 cells at peak parasitemia, then decreased as the transition to intermediate and short stumpy forms commenced. At crisis, basal levels were reestablished when long slender forms were the lowest percentage of the total population and intermediate and short stumpy forms predominated, suggesting a correlation between morphologle type and level of cyclic AMP per cell during fluctuations in parasitemia. Increases in intracellular cyclic AMP were measured during in vitro incubation of the parasite in medium containing potential effectors of the trypanosome cyclic AMP system. Sodium fluoride, adenosine and methyl xanthines stimulated increases in cyclic AMP content while isoproterenol, prostaglandin El, serotonin, histamine and several trypanocidal drugs were ineffective. The results are discussed in terms of the possible regulatory role of cyclic AMP in differentiation of trypanosomes. Key words: Trypanosoma brucei brucei; Cyclic 3',5'-adenosine monophosphate; Cellular regulation; Developmental cycle; Long slender trypomastigotes; Short stumpy trypomastigotes.

INTRODUCTION Little is k n o w n

a b o u t the regulatory mechanisms responsible for d e v e l o p m e n t a l

changes in t r y p a n o s o m e s during t h e course o f i n f e c t i o n [1, 2]. In view o f the p o t e n t i a l i m p o r t a n c e o f t r y p a n o s o m e surface changes in the relationship b e t w e e n h o s t and parasite

Abbreviations: PBSG, phosphate-buffered saline and glucose; TG buffer, trypanosome buffer with glucose; TCA, trichloroacetic acid; BSA, bovine serum albumin; IBMX, isobutyl methylxanthine; PGE1, prostaglandin E 1 . 0166-6851/81/0000-0000/$02.50

©Elsevier/North-Holland Biomedical Press

20 [ 3 - 6 ] , it would be of interest to determine how transmission of extracellular signals from the parasite cell surface to intracellular compartments is accomplished. The role of cyclic nucleotides, and especially cyclic 3',5'-adenosine monophosphate (cyclic AMP), as a 'second messenger' [7] is now well established in a variety of prokaryotic and eukaryotic cell systems. Alterations in intracellular content of this nucleotide have been implicated i n c e l l differentiation [8, 9], reproduction [t0], macromolecular synthesis [11], neurotransmission [12] and membrane permeability changes [13, 14]. It is possible that the interactions of trypanosomes with their mammalian and arthropod hosts involve a similar cyclic nucleotide-dependent mechanism. As yet unknown host or parasite factors acting at the trypanosome cell surface could lead to changes in cyclic AMP levels in the parasite which are related to the physiological and developmental changes associated with infection. There have been few published studies concerning the role of cyclic nucleotides in trypanosomatids. Adenylate cyclase activity (EC 4.6.1.1) has been reported in Trypanosoma brucei [15] and in T. cruzi [16]. In addition, cyclic AMP-binding protein [17] and soluble protein kinase activity [18] have been demonstrated in T. gambiense. Elevation of intracellular cyclic AMP has been correlated with decreased cell proliferation in Leishmania tropica promastigotes, and with inhibition of transformation of amastigotes to promastigotes in L. donovani [19]. We have previously shown that this cyclic nucleotide is present in Trypanosoma lewisi and that its concentration increases two-fold during the transition from reproducing to non-reproducing forms [20]. We now report that the intracellular levels of cyclic AMP in YTatl, a cloned pleomorphic variant of T. brucei, and in EATRO 110M, a monomorphic strain of T. brucei, undergo non-random changes during fluctuations in parasitemia in normal and immunosuppressed rats. In addition, certain known effectors of the cyclic AMP system stimulate rapid, transient increases of this cyclic nucleotide in trypanosomes in vitro. MATERIALS AND METHODS Trypanosomes. The majority of the experiments described were performed using a triplecloned pleomorphic variant derived from T. brucei brucei TREU-164 [21 ] now designated YTatl [22]. In certain experiments the cloned monomorphic strains of T. brucei brucei designated EATRO 110M and 427 were also used. EATRO 110M, a highly virulent monomorphic substrain, was derived from the pleomorphic laboratory strain EATRO 110 [23]. Trypanosomes were maintained as stabilates in liquid N2 and infections were initiated by i.p. injection of stabilate cells into adult female Sprague-Dawley rats. Relapsing infections were initiated by injection of 5 - 1 0 × 10 6 YTatl cells/rat. Injection of 5 - 1 0 × 10 7 cells/rat results in a fulminating parasitemia that kills the infected animal at 4 days post-infection. Approximately 1 × 10 7 cells/rat of the ll0M strain or the 427 strain were used to initiate monomorphic non-relapsing infections. For experiments involving immunosuppressed hosts, rats were lethally X-irradiated (930 rad whole body)

21 [21]. All infections were monitored by hemacytometer counts of trypanosomes in infected tail blood. Slender bloodstream trypomastigotes, intermediate forms, and short stumpy forms were assessed during relapsing infection by differential counts on Giemsastained blood smears. In order to assay the cyclic AMP content of trypanosomes during the course of infection, 0 . 5 - 1 . 0 ml of infected blood was withdrawn by cardiac puncture from each rat at specified intervals post-infection. The trypanosomes were isolated by the glycerol lysis technique of Rosen et al. [24], purified by DEAE chromatography [25], and processed for cyclic AMP analysis within 20 rain. of the initial blood collection. Trypanosomes to be used for in vitro experirnents were obtained by cardiac puncture, differential centrifugation and column chromatography [21]. They were washed in phosphate-buffered saline and glucose (PBSG: 47 mM Na2HPO4/3 mM NaH2PO4/100 mM NaC1 containing 0.1% glucose, pH 8.0) before suspension in the appropriate incubation medium.

Cyclic AMP assays. Cyclic AMP was assayed by radioimmunoassay (RIA) [26] as available in kit form from New England Nuclear (Cambridge, Mass.). Trypanosomes were sedimented by centrifugation at 10 000 × g for 15 s and suspended in 1 ml of ice-cold medium or buffer, from which 10-/al aliquots were withdrawn for cell counts. 50/al of 100% (w/v) trichloroacetic acid (TCA) was added immediately to the suspension to precipitate cellular macromolecules. TCA-insoluble material was removed by centrifugation at 15 000 × g for 1 min, and discarded. The supernatant was extracted three times with water-saturated ether to remove TCA, adjusted to pH 5.0 with 1 N NaOH, and immediately assayed for cyclic AMP or frozen at -20°C until assayed. Because the assay is sensitive to femtomolar concentrations of cyclic AMP with little or no cross-reactivity with other nucleotides, it reproducibly detects cyclic AMP in as few as 1 × 107 trypanosomes per ml of cell suspension. Reagents. All chemicals were reagent grade. Bovine serum albumin (BSA) was Cohn Fraction V (Sigma Chemical Co.). Dulbecco's modified Eagle medium containing 4.5 g glucose/1 was purchased from Gibco. Trypanosome buffer with glucose (TG) consisted of 20 mM Na2HPO4/2 mM NaH2PO4" H20/80 mM NaC1/5 mM KCI/1 mM MgSO4" 7H20/ 10 mM glucose, pH 7.4. Cimetidine was a gift of Dr. R. Meyerson of Smith, Kline and French Laboratories (Philadelphia, PA). Fluoxetine was a gift of Dr. R. Shulman of Eli Lilly and Co. (Indianapolis, IN). Pentamidine was a gift of May and Baker Ltd. (Dagenham, England). Isobutyl methylxanthine (IBMX), caffeine, theophylline, isoproterenol, histamine, serotonin and prostaglandin El (PGE1) were purchased from Sigma Chemical Co. Incubation conditions. For in vitro studies, column purified trypanosomes were suspended at 5 × 107 cells/ml in 37°C prewarmed TG Buffer or Dulbecco's modified Eagle medium with 0.1% bovine serum albumin (BSA) and incubated in the presence or absence of the appropriate effector for 15 and 30 min. At the end of the incubation period, an

22 aliquot containing 1 × 108 cells was removed, centrifuged at 12 000 × g for 15 s in the cold and resuspended for assay of cyclic AMP as described above. RESULTS

Cyclic AMP levels in T. brucei during the course o f infection in the rat. We have compared the intracellular levels of cyclic AMP in trypanosomes isolated at various times after infection of immunocompetent and lethally X-irradiated rats. Non-relapsing infections were initiated using the highly virulent monomorphic strains EATRO 110M and 427, each of which produces parasitemias of 1 - 2 × 109/ml by 3 days post-infection. Nonrelapsing infections were also obtained with the pleomorphic variant YTatl by injection of l0 s parasites/rat. A fulminating parasitemia ensued, followed by death of the host by approximately 4 days post-infection. However, if 5 - 1 0 × 106 organisms of this variant were used to initiate infections, parasitemia fluctuations characteristic for a pleomorphic infection were seen, with intermediate forms appearing at 5 - 6 days, short stumpy forms at 6 - 7 days, and death of the host at 8 - 9 days post-infection. In immunocompetent hosts, strain-specific differences are apparent in the levels of trypanosome cyclic AMP during non-relapsing infections (Table I). Trypanosomes of strain 427 maintain relatively low basal levels of this nucleotide throughout the infection. However, in strains 110M and YTatl the content of this cyclic nucleotide increases on a per cell basis from 2 0 - 3 0 pmol per 109 cells to as high as 97 pmol per 109 cells as the infections approach peak parasitemia. It should be noted that trypanosome isolates from individual rats at the same level of parasitemia are subject to differences in absolute amount of cyclic AMP, but the relative amounts are internally consistent for each sample from the same rat. In addition, trypanosomes isolated from moribund animals at maximum parasitemia, that is, 1 - 2 × 109/ml blood, show a precipitous drop in intracellular content of cyclic AMP (data not shown). It is not known at present if this is due to a response of the trypanosomes to metabolic changes in the dying host or to altered membrane permeability of the parasite at high population density which could result in loss of cyclic AMP into the extracellular environment. Control experiments using the YTatl strain indicated that cyclic AMP levels in the parasite are not affected by the isolation procedures used where isolation is completed within 30 min after blood collection and the cell suspensions are maintained at 4°C. Data in Table II demonstrate that the total cyclic AMP detectable in whole blood is the sum of the contributions from blood and trypanosomes. Trypanosomes isolated from infected blood by glycerol lysis and column chromatography show the same content of intracellular cyclic AMP as trypanosomes processed with whole infected blood. In addition, when blood from uninfected rats is processed by glycerol lysis and column chromatography in the same manner as blood from infected rats, the column effluent contains negligible amounts of cyclic AMP. During relapsing infections produced by the YTatl clone in immunocompetent rats, the cyclic AMP per cell rose from 2 0 - 3 0 pmol per 109 cells in early logarithmic growth

23 TABLE I Intracellular content of cyclic AMP in T. brucei during non-relapsing infections,a Strain

Rat

Parasitemiab (× 10 -8 )

Time c (h)

pmol cAMP/109 cells -+ S.D.

427

1

4.2 8.9 5.7 9.4 2.2 4.4 1.1 7.2 9.2 3.0 6.0 3.0 8.2 12.0

65 72 65 72 55 72 72 94 104 74 82 76 98 110

46.4-+ 1.2 27.6 -+ 0.1 38.5 -+ 4.3 26.8 -+ 0.5 30.0 -+ 2.2 84.2 -+ 11.5 152.8 -+ 12.9 46.4 -+ 3.1 87.5 -+ 5.2 51.9 +- 5.6 75.6 -+ 2.2 33.9 -+ 7.7 74.6 -+ 9.0 97.1 -+ 12.1

2 ll0M

1 2

YTatl

1 2

a Each rat received either 5 × 107 T. brucei YTatl, 1 × 107 T. brucei 110M, or 1 × 107 T. brucei 427 i.p. At the designated times post-infection 0.5-1.0 ml of blood was withdrawn by cardiac puncture. Trypanosomes were isolated and column purified as described, and intracellular levels of cyclic AMP (cAMP) determined. b Trypanosomes/ml tail blood c Hours post-infection

TABLE II Effect of isolation procedure on the intracellular content of T. brucei YTatl cyclic AMP. Sample

pmol cAMP/sample

A. Infected whole blood, 1 ml, 6.1 X l0 s trypanosomesa B. Uninfected whole blood, 1 ml a C. (Sample A - Sample B) = pmol cAMP/6.1 x l0 s trypanosomes D. Sample C, normalized to pmol cAMP/109 trypanosomes E. DE-52 column-purified trypanosomes, pmol cAMP/109 trypanosomesb F. DE-52 column effluent of processed, uninfected blood, 1 ml c

63.6 32.0 31.6 51.8 53.6 <0.5

a Whole blood from infected or uninfected rats was precipitated within 30 s of collection by addition of i00/~1 of 100% (w/v) TCA, then processed and assayed for cyclic AMP as described in Materials and Methods. b Trypanosomes were isolated from the blood of the infected rat in Sample A by glycerol lysis and DE-52 column chromatography, then processed for cyclic AMP analysis as described. c Uninfected blood was processed by glycerol lysis and DE-52 column chromatography, and the column effluent analyzed for cyclic AMP content as described.

24

to a high of 65 pmol per l 0 9 cells at peak parasitemia (Fig. 1A). However, as intermediate forms appear in the population and the percentage of long slender forms decreases, there is a decrease in the average cyclic nucleotide content per cell. The lowest cyclic AMP levels occur at approximately the time when long slender forms make up the lowest percentage of the total population and short stumpy forms begin to predominate. As the infection progresses toward the second wave of parasitemia, the cyclic AMP level again begins to rise, preceeding the appearance of the second slender population. Although the time course of infection varies from host to host, the Overall trend in the %LS

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Time pI (h) Fig. 1. Intracellular cyclic AMP c o n t e n t o f T. brucei Y T a t l isolated during the course of pleom o r p h i c infections in i m m u n o c o m p e t e n t and lethally X-irradiated rats, Animals were infected i.p. at zero time with 1 X 10 .7 trypanosomes. At designated times post-infection (pI) 0 . 0 5 - 1 . 0 ml o f infected blood was obtained by cardiac puncture, t r y p a n o s o m e s were purified as described in Materials and Methods, and intracellular cyclic AMP c o n t e n t was determined. Each point represents a sample from a single rat. The histograms represent the percentage o f long slender (LS) bloodstream f o r m s in the population as determined by differential counts on Giemsa-stained blood smears. A. I m m u n o c o m p e t e n t rats. B. Lethally X-irradiated rats.

25 population suggests a correlation between shifts in morphologic type and intracellular concentration of this cyclic nucleotide. In order to investigate the effect of the immune system of the host on intracellular content of trypanosome cyclic AMP, a pleomorphic infection was established in lethally X-irradiated rats using the YTatl clone, and the cyclic AMP content of trypanosomes isolated at various times after infection was determined. Cyclic AMP levels in trypanosomes from irradiated animals follow a pattern similar to that seen in trypanosomes from intact animals (Fig. 1B). The cyclic AMP content per cell rises from 30 pmol to 70 pmol per 109 parasites during the initial phase of the infection then decreases as the morphology of the population shifts from slender to intermediate forms.

In vitro stimulation of the cyclic AMP system in trypanosomes. In order to investigate alterations of cyclic AMP during in vitro growth, we attempted to stimulate increases in intracellular content of this nucleotide by using various effectors known to elevate cyclic AMP levels in mammalian systems. We also examined several drugs that are trypanocidal in vivo and/or in vitro for their possible stimulatory effects. The results of short term incubation of bloodstream slender forms of YTatl in the presence or absence of these agents are shown in Table III. Catecholamines and prostaglandins are natural effectors of the cyclic AMP system in higher eukaryotes. The /3-catecholamine agonist isoproterenol, and PGE1 elevate cyclic AMP levels in cells of the mammalian nervous system [27-29], yet neither of these agents has a short-term effect on cyclic AMP levels in trypanosomes. On the other hand, methyl xanthines such as caffeine, theophyUine and isobutyl methylxanthine, which are potent inhibitors of cyclic-nucleotide phosphodiesterases (EC 3.1.4.17) in many cell systems [7, 29], elevate cyclic AMP levels in this strain of trypanosomes by a factor of 2 after 1 5 - 3 0 min incubation at a concentration of 10 tiM. Serotonin and histamine, whose actions are mediated by cyclic AMP in other cell systems [7], are vasoactive substances released by mast cells at sites of tissue damage and would likely be present at sites of trypanosome invasion of host tissue. These drugs do not have an effect at the concentrations tested. Penfamidine, a trypanocidal drug, has no effect on cyclic AMP levels at sublethal concentrations up to 15 min. A 2-fold stimulation of cyclic AMP levels which occurs after 60 min incubation may actually be a function of drug toxicity. Fluoxetine, an inhibitor of serotonin re-uptake at the synaptic cleft in the mammalian nervous system [30], has in vitro trypanocidal activity (Escobar and Patton, manuscript in preparation). This drug, at concentrations below the LDso (90 /aM), stimulates a slight increase in cyclic AMP levels above control values. However, it is unclear whether this represents a specific stimulation or is an artefact caused by toxicity of the drug. Cimetidine, an H2 receptor antagonist [31], has neither trypanocidal nor cyclic AMP-stimulating activity. Sodium fluoride and adenosine at pharmacologic concentrations cause rapid transient increases in intraceUular levels of cyclic AMP. Since both these agents have stimulatory effects on adenyl cyclase activity in whole cells and in particulate cell fractions in

26 TABLE III Intracellular cyclic AMP in T. brucei YTatl incubated with potential cyclic AMP stimulating agents, a Treatment

Control Isoproterenol Prostaglandin E~ IBMX Theophylline Caffeine Serotonin Histamine Fluoxetine Cimetidine Pentamidine Adenosine Sodium fluoride

Concentration

Relative amount b

(uM)

15 min

30 min

100 100 10 10 i0 10 100 10 10 10 10 10

1.0 1.13 0.95 2.13 1.96 2.47 1.16 0.87 1.67 1.04 1.17 5.63 2.91

1.0 1.29 0.94 2.37 2.18 1.16 1.05 1.73

a Trypanosomes were isolated and column-purified as described in Materials and Methods, then suspended at 5 × 107/ml in pre-warmed 37°C incubation medium. Experiments involving fluoxetine, cimetidine, pentamidine, serotonin and histamine were performed in TG buffer without BSA to eliminate non-specific binding of drugs. Other experiments were performed in Dulbecco's-modified Eagle medium containing 0.1% BSA. Agents were added at zero time and flasks were incubated in a 37°C shaking water bath. Aliquots containing 1 × 10 s cells were withdrawn at 15 and 30 min, centrifuged immediately at 12 000 X g for 15 s, and the cell pellet resuspended in 1 ml of ice-cold incubation medium then processed for cyclic AMP assay as described. b (pmol cAMP/109 treated cells)/(pmol cAMP]109 control cells).

mammalian cells [7, 32, 33], their effect on trypanosome cyclic AMP content may be a consequence o f a similar mode o f action. Preliminary data suggest that high concentrations (5 mM) o f tryptophol, a tryptophan metabolite that is released into the bloodstream o f the host by trypanosomes during infection [34], can elevate intracellular levels o f cyclic AMP in T. brucei 110M by 4- to 5-fold after 30 min exposure in vitro while having no adverse effect on cell viability o r glucose metabolism (unpublished observations). This phenomenon is under further investigation. DISCUSSION The transition from long slender bloodstream trypomastigotes to short stumpy forms during pleomorphic infection with T. brucei involves a series o f morphologic, physiologic and biochemical alterations which, taken together, constitute a major differentiative event. Morphologic changes include a shortening o f flagellum and cell body [ 1], and the

27 formation of extensive smooth endoplasmic reticulum [35], large digestive vacuoles [23] and mitochondrial cristae. Accompanying the morphologic changes are biochemical and physiologic shifts in synthesis of respiratory enzymes and in carbohydrate metabolism toward aerobic pathways of energy utilization [36]. Stumpy forms also exhibit increased protein uptake and digestion relative to slender trypomastigotes [23], as well as decreased proliferative capacity and infectivity for the mammalian host [1], suggesting the occurrence of major changes in protein and nucleic acid metabolism. Nothing is known about the signals which might initiate these developmental changes, or about the mechanisms for transmission of the signals to the appropriate compartment o f the parasite. Using established models of cyclic nucleotide action from other cell systems, we suggest that alterations in the cyclic AMP content of the parasite during the vertebrate phase of the life cycle may be involved in the developmental events associated with infection in the mammalian host. During infections with strain YTatl and strain 110M, but not with strain 427, cyclic AMP levels per cell are relatively low early in infection when the cells are rapidly reproducing and increase as the infection approaches peak parasitemia. This observation is in agreement with our previously reported data for T. lewisi [20], and with data from other eukaryotic cell systems [37] where rapidly dividing cells contain less cyclic AMP than non-reproducing or slowly reproducing cells. At peak parasitemia trypanosomes may be considered a stationary phase population, since an increase in population doubling time is observed. Moreover, this change in population doubling time is seen in immunosuppressed hosts where it is probably not due to removal of cells by trypanocidal antibody. In many prokaryotic and eukaryotic cell systems, developmental changes are initiated as the population reaches stationary phase. For example, Dictyostelium discoideum, a cellular slime mold, normally exists as free-living ameboid cells. Under starvation conditions, the cells aggregate to form a fruiting body, the multicellular structure which expresses differentiated functions necessary for spore formation. The stimulus for this aggregation is the release of cyclic AMP by 'pacemaker' cells in the population and the subsequent stimulation of cyclic AMP synthesis and release by neighboring cells until the entire population becomes aggregation-competent [9]. During infections with T. brucei in the rat, as parasite numbers increase there is a coincident alteration of the surrounding environment [34], and this could affect the parasite cyclic AMP system. Such changes might be either host or parasite directed, and involve either increases or decreases in critical serum components. Some strains of trypanosomes, such as 427, may be incapable of detecting or reacting to such environmental changes while others such as YTatl and 110M respond with an increase in intracellular cyclic AMP. During a relapsing infection with YTatl this increase is concurrent with the initiation of a developmental sequence which culminates in the production of stumpy forms. If cyclic AMP were involved in transmission of a signal for differentiation in these organisms, then it would be expected that their cyclic AMP content should return to basal levels once the signal has been received and processed, and this is observed during crisis in a relapsing infection with YTatl. It should be noted that changes in intracellular content of this cyclic

28 nucleotide could result from altered rates of degradation as well as synthesis;thus, agents which affect phosphodiesterase activity may be as important as those which stimulate a receptor-coupled adenyl cyclase in the parasite membrane. The nature of the inductive stimulus and the regulatory role, if any, played by cyclic AMP in the differentiation process in trypanosomes is unknown. However, based on in vivo experiments, the inductive signal does not appear to be specific anti-trypanosomal antibody since both cyclic AMP changes and morphological transformation occur in trypanosomes isolated from immunoincompetent animals. We are at present examining the cyclic AMP-dependent phosphorylation events in T. brucei to determine if phosphorylation of specific protein substrates within the cell could have regulatory effects on cell differentiation. Attempts to specifically stimulate alterations in cyclic AMP in vitro using effectors known to elevate this cyclic nucleotide in other systems have produced both positive and negative results. Catecholamines, prostaglandins, histamine and serotonin, hormones and vasoactive substances normally present in host plasma or tissues, have no effect on the trypanosome cyclic AMP system. These results agree with those previously reported by Martin et al. [15] who were unable to demonstrate a stimulation of adenyl cyclase activity in membrane preparations from T. brucei 427 using glucagon or adrenaline, known effectors of mammalian adenyl cyclase. Neither were they able to demonstrate stimulation by sodium fluoride or adenosine at millimolar concentrations. We, however, have shown that sodium fluoride or adenosine at micromolar concentrations does stimulate rapid, short-term, 3- and 5-fold increases, respectively, in intracellular levels of cyclic AMP in YTatl (Table II1). Fluoride activation of adenyl cyclase is independent of the presence of a hormone-sensitive receptor in other systems [33]; however, adenosine has been shown to specifically stimulate increases in cyclic AMP in cultured nervous system cells [29, 32]. Strain differences with respect to response to potential effectors may be potentially useful for investigating the cyclic AMP system of trypanosomes. Caffeine-, theophylline- and IBMX-stimulated increases in trypanosome cyclic AMP are probably the result of inhibition of phosphodiesterases [7, 29]. The slight stimulatory effect of fluoxetine on intraceUular cyclic AMP in T. brucei could be the result of specific stimulation of a drug-sensitive receptor or a non-specific increase in cyclic nucleotide caused by a toxic effect of the drug itself. These results demonstrate the necessity of finding non-toxic agents, which can specifically stimulate the adenyl cyclase of the trypanosome membrane. Of several potential effectors studied thus far, no single agent emerges which is both stimulatory and non-toxic. Despite our current lack of information on the subject of an inductive stimulus for cyclic AMP changes, the presence of a cell surface receptor specifically coupled to a membrane-bound adenyl cyclase becomes an attractive possibility. The similarities between the trypanosome cyclic AMP system and that of other eukaryotic cells would suggest that cyclic AMP does have a regulatory function in trypanosomes. These similarities include: (1) fluoride, methyl xanthine and adenosine stimulation of cyclic AMP levels in whole cells; (2) demonstrable membrane-bound adenyl cyclase activity in salivarian and

29 stercorarian trypanosomes [ 15, 16]; (3) developmentally related changes in cellular levels o f cyclic AMP in T. brucei; and (4) reported effects o f cyclic AMP and its analogs on developmental changes in other parasitic protozoa [19]. We have documented a strain-related pattern o f cyclic AMP changes during the developmental cycle o f T. brucei in the rat and suggested a possible mechanism whereby cyclic AMP levels might regulate differentiative events in the parasite. A combination o f in vivo and in vitro approaches should permit further analysis o f the developmental role o f this cyclic nucleotide in the life cycle o f trypanosomes. NOTE ADDED DURING REVISION Kaushal et al. [38] recently demonstrated that an increase in gametocytogenesis is observed in response to the addition o f cyclic AMP to stationary phase cultures of Plasmodium falciparum. However, this effect is not seen when cyclic AMP is added to logarithrnically growing populations. ACKNOWLEDGEMENTS This work was supported b y U.S.P.H.S. Training Grant AI07136 and Research Grant AI15742 from NIH - NIAID. We thank James Strickler for thoughtful discussion, Diane Grabowski for excellent technical assistance and Roberta Belli for help in preparation o f the manuscript. REFERENCES 1

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