The GPI-phospholipase C of Trypanosoma brucei is nonessential but influences parasitemia in mice.

In the mammalian host, the cell surface of Trypanosoma brucei is protected by a variant surface glycoprotein that is anchored in the plasma membrane through covalent attachment of the COOH terminus to a glycosylphosphatidylinositol. The trypanosome also contains a phospholipase C (GPI-PLC) that cleaves this anchor and could thus potentially enable the trypanosome to shed the surface coat of VSG. Indeed, release of the surface VSG can be observed within a few minutes on lysis of trypanosomes in vitro. To investigate whether the ability to cleave the membrane anchor of the VSG is an essential function of the enzyme in vivo, a GPI-PLC null mutant trypanosome has been generated by targeted gene deletion. The mutant trypanosomes are fully viable; they can go through an entire life cycle and maintain a persistent infection in mice. Thus the GPI-PLC is not an essential activity and is not necessary for antigenic variation. However, mice infected with the mutant trypanosomes have a reduced parasitemia and survive longer than those infected with control trypanosomes. This phenotype is partially alleviated when the null mutant is modified to express low levels of GPI-PLC.

A gene from the variant surface glycoprotein expression site encodes one of several transmembrane adenylate cyclases located on the flagellum of Trypanosoma brucei.

The bloodstream form of Trypanosoma brucei contains transcripts of at least four genes showing partial sequence homology to the genes for eucaryotic adenylate and guanylate cyclases (S. Alexandre, P. Paindavoine, P. Tebabi, A. Pays, S. Halleux, M. Steinert, and E. Pays, Mol. Biochem. Parasitol. 43:279-288, 1990). One of these genes, termed ESAG 4, belongs to the polycistronic transcription unit of the variant surface glycoprotein (VSG) gene. Whereas ESAG 4 is transcribed only in the bloodstream form of the parasite, the three other genes, GRESAG 4.1, 4.2, and 4.3, are also expressed in procyclic (insect) forms. These genes differ primarily in a region presumed to encode a large extracellular domain. We show here that ESAG 4-related glycoproteins of about 150 kDa can be found in the trypanosome membrane, that they are detected, by light and electron gold immunocytochemistry, only at the surface of the flagellum, and that the products of at least two of these genes, ESAG 4 and GRESAG 4.1, can complement a Saccharomyces cerevisiae mutant for adenylate cyclase. The recombinant cyclases are associated with the yeast membrane fraction and differ with respect to their activation by calcium: while the GRESAG 4.1 and yeast cyclases are inhibited by calcium, the ESAG 4 cyclase is stimulated. ESAG 4 thus most probably encodes the calcium-activated cyclase that has been found to be expressed only in the bloodstream form of T. brucei (S. Rolin, S. Halleux, J. Van Sande, J. E. Dumont, E. Pays, and M. Steinert. Exp. Parasitol. 71:350-352, 1990). Our data suggest that the trypanosome cyclases are not properly regulated in yeast cells.

BM-520, an original TXA2 modulator, inhibits the action of thromboxane A2 and 8-iso-prostaglandin F2alphain vitro and in vivo on human and rodent platelets, and aortic vascular smooth muscles from rodents.

Thromboxane A(2) (TXA(2)) and 8-iso-PGF(2alpha) are two prostanoid agonists of the thromboxane A(2) receptor (TP), whose activation has been involved in platelet aggregation and atherosclerosis. Agents able to counteract the actions of these agonists are of great interest in the treatment and prevention of cardiovascular events. Here, we investigated in vitro and in vivo the pharmacological profile of BM-520, a new TP antagonist. In our experiments, this compound showed a great binding affinity for human washed platelets TP receptors, and prevented human platelet activation and aggregation induced by U-46619, arachidonic acid and 8-iso-PGF(2alpha). The TP receptor antagonist property of BM-520 was confirmed by its relaxing effect on rat aorta smooth muscle preparations precontracted with U-46619 and 8-iso-PGF(2alpha). Further, its TP antagonism was also demonstrated in vivo in guinea pig after a single intravenous injection (10 mg kg(-1)). We conclude that this novel TP antagonist could be a promising therapeutic tool in pathologies such as atherosclerosis where an increased production of TXA(2) and 8-iso-PGF(2alpha), as well as TP activation are well-established pathogenic events.

Abrupt RNA changes precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro.

We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.

Mild acid stress as a differentiation trigger in Trypanosoma brucei.

In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27 degrees C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37 degrees C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC-) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC+).

Transient adenylate cyclase activation accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms.

Pleomorphic bloodstream forms of Trypanosoma brucei differentiate synchronously into procyclic forms when cultivated at 27 degrees C in the presence of citrate/cis-aconitate. The activity of adenylate cyclase was monitored during this process. Two phases of transient stimulation were observed. The first phase occurred 6-10 h after the triggering of differentiation, a period which immediately follows the release of the bulk of the VSG and immediately precedes both the first cell division and the loss of the bloodstream-specific ESAG 4 transmembrane adenylate cyclase. The second phase occurred between 20 and 40 h, when the cells that emerged from the first division began to proliferate. These observations suggest that cAMP may be involved in differentiation/proliferation of the parasite.

Activity of a novel dual thromboxane A(2)receptor antagonist and thromboxane synthase inhibitor (BM-573) on platelet function and isolated smooth muscles.

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.

Effects of a novel non-carboxylic thromboxane A2 receptor antagonist (BM-531) derived from torasemide on platelet function.

In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function.

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors.

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.

Dexamethasone increases intracellular cyclic AMP concentration in murine T lymphocyte cell lines.

The effects of the synthetic glucocorticoid dexamethasone on the cAMP content of murine T lymphocyte cell lines has been investigated. Incubation of the 3B4.15 T cell hybrids with dexamethasone results in an average 5-fold increase in intracellular cyclic AMP levels after 6 h of treatment. This phenomenon is abolished in the presence of RU486 and of cycloheximide, indicating that it requires binding of the drug to the intracellular glucocorticoids receptor and de novo protein synthesis. Dexamethasone-induced elevation of intracellular cyclic AMP correlates with both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity in T cell hybrids. This modulation of cyclic AMP metabolism is independent of serum-derived factors, suggesting that it is not secondary to transmembrane receptor stimulation by an extracellular ligand. We propose that glucocorticoids interfere with the homeostatic control of intracellular cAMP concentration, leading to a sustained increase in the content of this important second messenger in murine T lymphocyte cell lines. This study suggests that elevation of cAMP levels may represent one way by which glucocorticoids modulate the immune response.

Design, synthesis and biological evaluation of a sulfonylcyanoguanidine as thromboxane A2 receptor antagonist and thromboxane synthase inhibitor.

The synthesis and the structure of N-isopropyl-N'-[2-(3'-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2 (TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 microM) with an IC50 value of 0.45 and 0.15 microM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 microM) completely inhibited TXA2 synthase of human platelets. The pKa value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA2 antagonists related to torasemide and their receptor.

BM 144: an original thromboxane A2 receptor antagonist derived from torasemide.

Torasemide, a new sulfonylurea high ceiling loop diuretic, has been demonstrated to induce a concentration dependent relaxation of canine coronary precontracted with thromboxane A2 (TXA2). With the aim to develop more potent TXA2 receptor antagonists, we investigated a series of torasemide derivatives. This pharmacomodulation led to the discovery of a sulfonyl-cyanoguanidine (BM 144) which presents the same pharmacological profile as sulotroban, a TXA2 receptor antagonist used as reference.

Inhibition of gap-junctional intercellular communication between epithelial cells transformed by the activated H-ras-1 oncogene.

In order to study the effects of an activated H-ras-1 oncogene on gap-junctional intercellular communication, we introduced the EJ/T24 H-ras-1 oncogene into cells of the epithelial Clone 9-3 cell line. Gap-junctional intercellular communication was significantly reduced in H-ras-1-transformed Clone 9-3 derivatives; this result shows that transformation by the activated H-ras-1 oncogene can inhibit gap-junctional intercellular communication. We postulate that the activated H-ras-1 oncogene product could mediate this effect through a change in the phosphorylation of the major gap-junction protein.

Slender and stumpy bloodstream forms of Trypanosoma brucei display a differential response to extracellular acidic and proteolytic stress.

Natural infections of mammals with African trypanosomes, such as Trypanosoma brucei, are generally pleomorphic, the population consisting of different forms, termed slender and stumpy forms, that vary in number as the parasitaemia develops. We show that the differentiation of slender into stumpy forms is characterized by the acquisition by the parasite of the ability to regulate its internal pH, even in the face of a large, inwardly directed gradient of H+, as well as a tolerance towards external proteolytic stress. These adaptations effectively abbrogate cellular stress-activated signalling pathways involving adenylate cyclase and glycosylphosphoinositol-specific phospholipase-C mediated release of the surface coat. Although in metabolic terms stumpy forms of the parasite are considered to be preadapted to life in the arthropod vector, these data clearly demonstrate that these forms also possess additional cellular adaptations designed to deal with the immediate and potentially harmful changes in the extracellular environment that occur upon ingestion of a bloodmeal by the tsetse fly vector.

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines.

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and flavin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.

Structure determination and comparison of BM567, a sulfonylurea, with terbogrel, two compounds with dual action, thromboxane receptor antagonism and thromboxane synthase inhibition.

BM567, a sulfonylurea compound whose crystal structure is here discussed and terbogrel, are both thromboxane receptor antagonists and thromboxane synthase inhibitors. In this paper, their crystallographic and electronic structures are compared and lead to new synthesis prospects among the sulfonylurea series.

BM-573, a dual thromboxane synthase inhibitor and thromboxane receptor antagonist, prevents pig myocardial infarction induced by coronary thrombosis.

The aim of this study was to characterize the effects of BM-573 [N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl] urea], a novel dual thromboxane A2 receptor antagonist and thromboxane synthase inhibitor, on myocardial infarction induced by topical ferric chloride (FeCl3) application to the left anterior descending (LAD) coronary artery in anesthetized pigs. All control animals (n = 6) developed an occlusive thrombus in the LAD coronary artery. The mean infarct size, revealed by triphenyl tetrazolium chloride (TTC), and the area at risk, evidenced by Evans blue, corresponded to 35.3 +/- 2.2 and 36.9 +/- 2.1% of the left ventricular mass, respectively. In the BM-573-treated group (n = 6), a drug infusion (10 mg. kg-1. h-1) started 30 min before FeCl3 application and continued throughout the experimentation. Among the BM-573-treated group, four pigs did not develop coronary artery thrombus and their myocardium appeared healthy. Histopathological examination of FeCl3-injured coronary artery revealed an occlusive and adherent thrombus in control group, while pretreatment with BM-573 prevented thrombus formation. In infarcted zones, lack of desmin staining and muscle structure disorganization were obvious. Depletion of myocardial ATP content was observed in the myocardial necrotic region of the control group, but not in myocardial samples of BM-573-treated pigs that did not develop myocardial infarction. When BM-573 prevented LAD artery occlusion, the area under the curve of plasmatic troponin T was reduced by 77% over 6 h. These data suggest that BM-573 could be useful for the prevention of myocardial infarction.

Expression of a variant surface glycoprotein of Trypanosoma gambiense in procyclic forms of Trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein.

Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.