The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages.

Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages.

Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-alpha) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-alpha production. For this purpose, omp25 and omp31 null mutants of B. suis (Deltaomp25 B. suis and Deltaomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-alpha. We showed that, in contrast to WT B. suis or Deltaomp31 B. suis, Deltaomp25 B. suis induced TNF-alpha production when phagocytosed by human macrophages. The complementation of Deltaomp25 B. suis with WT omp25 (Deltaomp25-omp25 B. suis mutant) significantly reversed this effect: Deltaomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-alpha than did macrophages infected with the Deltaomp25 B. suis mutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-alpha production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-alpha production upon infection of human macrophages.

Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes.

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Yersinia outer protein P of Yersinia enterocolitica simultaneously blocks the nuclear factor-kappa B pathway and exploits lipopolysaccharide signaling to trigger apoptosis in macrophages.

Exposure of macrophages to bacteria or LPS mediates activation of signaling pathways that induce expression of self defense-related genes. Pathogenic Yersinia species impair activation of transcription factor NF-kappaB and trigger apoptosis in macrophages. In this study, we dissected the mechanism of apoptosis induction by Yersinia. Selectively, Yersinia enterocolitica strains producing the effector protein Yersinia outer protein P (YopP) hampered NF-kappaB activation and subsequently conferred apoptosis to J774A.1 macrophages. Thereby, YopP bound and inhibited the macrophage NF-kappaB-activating kinase IKKbeta. YopP- and Yersinia-, but not Salmonella-induced apoptosis was specifically prevented by transient overexpression of NF-kappaB p65, giving evidence that YopP mediates cell death by disrupting the NF-kappaB signaling pathway. Transfection of J774A.1 macrophages with YopP induced a moderate, but significant degree of apoptosis (40-50% of transfected cells). This effect was strongly enhanced by additional initiation of LPS signaling (80-90%), indicating a synergism between LPS-induced signal transduction and inhibition of NF-kappaB by YopP. This reflects a strategy of a bacterial pathogen that takes advantage of LPS, serving as cofactor, to impair the macrophage.

The cytotoxin YopT of Yersinia enterocolitica induces modification and cellular redistribution of the small GTP-binding protein RhoA.

Pathogenic Yersinia enterocolitica produces two virulence plasmid-encoded cytotoxins, YopE and YopT, that are translocated into target cells where they disrupt the actin cytoskeleton. Here we show that infection of cells with wild type Y. enterocolitica and a yopE mutant, but not with a yopT mutant, induces an increase in the electrophoretic mobility of the small GTPase RhoA. As tested by isoelectric focusing, YopT-dependent modification resulted in an acidic shift of RhoA. Furthermore, RhoA modification induced by YopT was accompanied by redistribution of membrane-bound RhoA toward the cytosol. Finally, a yopE mutant of Y. enterocolitica expressing the cytotoxic activity of YopT specifically disrupted RhoA-controlled actin stress fibers. These findings provide evidence for inactivation of RhoA by the translocated Y. enterocolitica cytotoxin YopT and suggest a novel inhibitory modification of RhoA by a bacterial virulence factor.

Cell-specific localization of G protein alpha-subunits in the islets of Langerhans.

G protein alpha-subunits are involved in the transduction of receptor-mediated regulation of insulin and glucagon secretions. To get further insight into the status of G proteins in alpha- and beta-cells of the Langerhans islets, we have used immunohistochemistry to study the distribution of alpha-subunits in pancreas sections from the rat. Our results show that only insulin-immunoreactive beta-cells display immunoreactivity for selective antibodies directed against the different members of the Galphas and Galpha12-families (alphas, alphaolf, and alpha12, alpha13 respectively). Immunoreactivities for antibodies directed against members of the Galphaq- and Galphai-families showed a more diverse localization: alpha11 and alphao2 were only detected in glucagon-immunoreactive alpha-cells, whereas alphai1 was detected in all beta-cells but only in a few alpha-cells. Even though beta-cells showed immunoreactivities for alphao-non-isoform-selective antibodies, we could not identify the isoform(s) present using selective alphao1 and alphao2 antibodies. Other members of the Galphai- and Galphaq-families (alphai3, alphat2, alphaz and alphaq) were detected in both alpha- and beta-cells. In conclusion, our findings demonstrate a clear difference in the localization of G protein alpha-subunits between alpha- and beta-cells, suggesting the involvement of specific receptor transduction pathways for the neuronal/hormonal regulation of alpha- and beta-cell functions.

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

Expression and bactericidal activity of nitric oxide synthase in Brucella suis-infected murine macrophages.

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-gamma)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-gamma enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-alpha) mRNAs was induced in both c-Brucella- and ops-Brucella-infected cells and was strongly potentiated by IFN-gamma. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-gamma-treated cells infected with c-Brucella or ops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-gamma-treated cells infected with ops-Brucella. These discrepancies between iNOS mRNA and protein levels were not due to differences in TNF-alpha production. The iNOS inhibitor N omega-nitro-L-arginine methyl ester increased B. suis multiplication specifically in IFN-gamma-treated cells infected with ops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcgamma receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-gamma and antibrucella antibodies are present, i.e., following expression of acquired immunity.

The Raf-1/mitogen-activated protein kinase kinase-1/extracellular signal-regulated-2 signaling pathway as prerequisite for interleukin-2 gene transcription in lectin-stimulated human primary T lymphocytes.

It has been shown that stimulation of lymphoid cells causes the activation of the extracellular signal-regulated-2 (ERK-2) which activates nuclear factor of activated T cells (NF-AT), a transcription factor involved in the regulation of interleukin-2 (1L2) gene transcription. ERK-2 is activated via a kinase cascade initiated by activation of the G protein p21Ras followed by phosphorylation and activation of Raf-1 and mitogen-activated protein kinase kinase-1 (MEK-1). Activation of this pathway has been described primarily in human T cell lines; however, using primary T lymphocytes from transgenic mice, a recent study has shown that a blockade of this cascade did not perturb lymphocyte stimulation and proliferation. In the present paper, we studied in human primary T cells the possible involvement of the Raf-1/MEK-1/ERK-2 pathway upon stimulation by jacalin, a mitogenic lectin which specifically stimulates CD4+ lymphocytes. We show here that the mitogen-activated protein (MAP) kinase pathway was stimulated in human purified lymphocytes upon activation with jacalin. Moreover, activation of this pathway appeared to be essential, since its blockade by a specific inhibitor of the MEK-1 kinase abolished IL2 gene transcription; in contrast, in T cells stimulated with phytohemagglutinin M(PHA), another potent T cell mitogenic lectin, blockade of MEK-1 reduced but did not totally inhibit either ERK-2 phosphorylation or IL2 mRNA expression. This shows, as already suggested, that another pathway in addition to the Raf-1/MEK-1/ERK-2 kinase cascade could be triggered in T cell activation. Jacalin stimulation therefore appeared to be a good model for the specific activation of the MAP kinase pathway in human primary T lymphocytes, which would allow the characterisation of drugs specifically targeted to this particular pathway.

Interaction of Yersinia enterocolitica with macrophages leads to macrophage cell death through apoptosis.

Suppression of the host defense is one of the hallmarks of Yersinia enterocolitica infection. This enteric pathogen resists phagocytosis and interferes with macrophage functions from an extracellular localization (oxidative-burst generation and tumor necrosis factor alpha production). In this study, we investigated the fate of the Y. enterocolitica-infected macrophage. We found that murine J774A.1 macrophages and macrophages derived from human monocytes were killed by infection with Y. enterocolitica. Analysis of cellular morphology and DNA fragmentation revealed that macrophage cell death occurs through the induction of apoptosis. A total of 92% +/- 5% (mean +/- standard deviation) of murine J774A.1 macrophages and 74% +/- 6% of human monocyte-derived macrophages underwent apoptosis upon Yersinia infection after 4 and 20 h, respectively. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone blocked completion of the Yersinia-induced apoptotic program but not the surface exposure of phosphatidylserine as an early-stage apoptotic event. Analysis of different Yersinia mutants showed that macrophage apoptosis depends on a functional Y. enterocolitica type III protein secretion system. Apoptotic cell death of macrophages was not related to the YopE-mediated cytotoxic effect of Yersinia, since disruption of actin microfilaments by a Y. enterocolitica strain expressing a restricted repertoire of yop genes, including YopE, did not result in macrophage apoptosis. Furthermore, Yersinia-induced cytotoxic alterations in epithelial HeLa cells, which are conferred by YopE, did not lead to apoptosis. Our data demonstrate for the first time that Y. enterocolitica promotes the apoptosis of macrophages, an effect which is clearly distinct from the morphological alterations mediated by Yersinia on epithelial HeLa cells.

Evidence for a CD4-associated calcium influx independent of the phosphoinositide transduction pathway in human T cells.

We recently showed, using human Jurkat T cell variants lacking the T cell receptor (TCR)/CD3 complex, that the lectin jacalin is able to trigger intracellular calcium increase provided that CD4 is expressed on the cell surface. Involvement of the CD4 molecule in jacalin-induced biological effects was furthermore demonstrated in differentiated U937 myelomonocytic cells expressing or not expressing CD4, and is confirmed here in human CD4-transfected mouse thymoma cells. In the present paper, we analyze the CD4-associated calcium response triggered by jacalin independently of the TCR/CD3 complex. We show that the observed calcium rise results from a direct long-lasting calcium influx from the outside without release of calcium from intracellular stores. We demonstrate that it is independent of the phosphoinositide phospholipase C transduction pathway. Moreover, we show that this peculiar calcium response can be blocked by protein tyrosine kinase inhibitors (herbimycin and genistein) giving evidence of the involvement of a protein tyrosine kinase, the best candidate of which is the CD4-associated p56lck. Altogether, our results suggest that, independently of the TCR/CD3 complex, CD4 may be involved in the triggering of a calcium signal dependent on a protein tyrosine kinase and independent of the phosphoinositide transduction pathway.

Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH2-terminal kinase. Correlation with its inhibitory effect on tumor necrosis factor-alpha production.

The enteropathogenic bacterium Yersinia enterocolitica counteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocolitica prevents macrophage tumor necrosis factor-alpha (TNFalpha) production. Murine J774A.1 macrophages responded to Y. enterocolitica infection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocolitica strain harboring the Y. enterocolitica virulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocolitica strain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocolitica mutants revealed that (i) MAPK inactivation parallels the inhibition of TNFalpha release, (ii) the suppressor effect on TNFalpha production, which originates from the lack of TNFalpha mRNA, is distinct from the ability of Y. enterocolitica to resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocolitica virulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocolitica possesses one or more virulence proteins that suppress TNFalpha production by inhibiting ERK1/2, p38, and JNK kinase activities.

Involvement of G alpha q/11 in the contractile signal transduction pathway of muscarinic M3 receptors in caecal smooth muscle.

The nature of the pertussis toxin-insensitive G-protein involved in muscarinic-mediated phosphoinositides breakdown and contraction of isolated smooth muscle cells from the circular layer of the rabbit caecum was investigated. Immunoblotting of membrane proteins using affinity purified antibodies directed against different G-protein alpha-subunits revealed the expression of G alpha q/11, G alpha 11 and G alpha 12 in these cells. The carbachol-mediated [3H]inositol phosphates accumulation in saponin-permeabilized cells was abolished by anti-G alpha q/11-antibodies whereas anti-G alpha i1,2-antibodies were ineffective. Moreover, the carbachol-induced contraction of permeabilized cells, as determined by videomicrocopic measurements, was reversed by anti-G alpha q/11-antibodies but not affected by anti-G alpha i1,2-antibodies. From these data, we conclude that carbachol stimulates phosphoinositides hydrolysis and cell contraction through activation of specific muscarinic M3 receptors coupled to the pertussis toxin-insensitive G alpha q/11-protein. This is the first demonstration of G alpha q/11 implication in the contractile signal transduction pathway of muscarinic M3 receptors in smooth muscle cells.

Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes.

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37 degrees C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30 degrees C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37 degrees C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.

Cells retrovirally transfected with fibroblast growth factor-2 isoforms exhibit altered adenylate cyclase activity and G-protein functionality.

Basic fibroblast growth factor (FGF-2) is synthesized as different molecular mass isoforms all lacking the signal-peptide sequence. The high molecular-mass isoforms (21-24 kDa) possess a signal sequence directing their nuclear translocation. The role of each isoform is still poorly understood, however, modifications in intracellular signalling pathways could explain some effects of these peptides. In order to evaluate the role of FGF-2 isoforms on the adenylate cyclase (AC) signalling pathway, we retrovirally infected a rat pancreatic cell line (AR4-2J) with point-mutated FGF-2 cDNAs, allowing the expression of the 18 (A5 cells) or 22.5 kDa isoform (A3 cells) at a low level. In membrane preparations of A3 cells, unscheduled expression of the 22.5 kDa FGF-2 isoform induced a 2-fold decrease in both basal and forskolin-stimulated AC activity. Studies carried out on intact cells also showed decreased accumulation of cAMP in A3 cells in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Both FGF-2 peptides also induced functional modifications of G-proteins without affecting their levels. The 22.5 kDa peptide led to enhanced ADP-ribosylation of both alpha(s)-subunits in vitro, whereas the expression of the low molecular-mass 18 kDa peptide resulted in a 2-fold increase in alpha12 and alpha0 ADP-ribosylations. Furthermore, control CAT cells (AR4-2J cells transfected with the retrovirus containing the chloramphenicol acetyltransferase gene) and A5 cells were growth-inhibited by 8-Br-cAMP, in contrast to A3 cells. These data provide evidence that the expression of FGF-2 peptides could play a role in cell functions by modifying the AC signalling pathway. FGF-2 peptides are able to modulate both AC activity and the regulatory G-proteins. Finally FGF-2 expression may interfere with cAMP-regulated cell proliferation.

Prolonged elevation of intracellular cyclic AMP levels in U937 cells increases the number of receptors for and the responses to formylmethionyl-leucylphenylalanine, independently of the differentiation process.

The effects of elevated levels of cyclic AMP induced by cholera toxin (CTx) were investigated on the differentiated promyelomonocytic cell line U937. After CTx treatment, the initial inhibition of the oxidative burst induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) was followed by a progressive increase over 20 h, resulting in 4-6-fold potentiation of the initial burst. Various cyclic-AMP-elevating agents produced similar potentiation of the FMLP- or C5a-induced oxidative burst, but the phorbol 12-myristate 13-acetate-induced oxidative burst was not affected by CTx pretreatment of cells. Furthermore, the increase in arachidonate release and intracellular Ca2+ triggered by FMLP were amplified after CTx treatment. ADP-ribosylation of Gi alpha subunits catalysed by pertussis toxin was slightly increased after CTx treatment, despite similar immunoreactivity of the alpha subunit of Gi2. FMLP binding sites present in CTx-treated membranes were 3-6 times more abundant than in control membranes. Expression of mRNAs encoding the FMLP receptor and one of its related receptors were enhanced after CTx treatment of both undifferentiated and undifferentiated U937 cells. In parallel, after undifferentiated cells were treated with CTx, they were able to increase intracellular Ca2+, but not the oxidative burst, in response to FMLP. These data demonstrate that CTx, by increasing cyclic AMP, enhances the expression of chemotactic receptors independently of U937 cell differentiation.

IL-1 stimulates a diverging signaling pathway in EL4 6.1 thymoma cells. IL-2 release, but not IL-2 receptor expression, is sensitive to pertussis toxin.

We reassessed the involvement of Bordetella pertussis toxin (PTX)-sensitive proteins in the IL-1 signaling pathway on the responses induced by IL-1 on the murine thymoma cell line EL4 6.1. We demonstrate that the ADP-ribosyltransferase activity of PTX, and not its cell-anchoring B oligomer part, is responsible for the inhibition of IL-1-induced IL-2 release, since 1) the concentration of PTX (< or = 1 ng/ml) required to block the secretion is 100 to 1000 times lower than the concentration needed with the B oligomer; and 2) the mutated PT-9K/129G, devoid of ADP-ribosyltransferase activity, was inactive at 100 ng/ml. We found that partial ADP-ribosylation of the Gi2/Gi3 proteins before stimulation with IL-1 was sufficient to obtain full inhibition of IL-2 release. PTX did not however inhibit the appearance on the cell surface of the high affinity IL-2 receptors or the IL-2 release induced by PMA. In addition, we show that PTX prevented the expression of the IL-2 mRNA induced by IL-1, without affecting the binding of IL-2 specific nuclear factors to the T cell distal element of the IL-2 promoter. Furthermore, PTX also inhibited IL-1-induced proliferation of non-transformed thymocytes. In conclusion, our results demonstrate that IL-1-induced IL-2 release is sensitive to PTX-catalyzed ADP-ribosylation and that IL-1 activates a diverging pathway on EL4 6.1 cells.

A GTP-binding protein modulates a Ca2+ pump present in reticulocyte endocytic vesicles.

Rat reticulocytes were used to prepare endocytic vesicles to study calcium fluxes across the endosomal membrane. We used 45Ca2+, and found that endocytic vesicles from reticulocytes present a Ca(2+)-ATPase that pumps Ca2+ into the lumen of vesicles. This activity was sensitive to vanadate and the calmodulin antagonists: trifluoperazine and calmidazolium. Western blot analysis using a monoclonal antibody evidenced that Ca(2+)-ATPase present in reticulocyte endocytic vesicles is probably the same as the erythrocyte Ca2+ pump. Ca2+ pump activity was shown to be partially inhibited by GTP gamma S. Moreover, mastoparan and benzalkonium chloride, both activators of heterotrimeric G proteins, were found to decrease 45Ca2+ uptake by endocytic vesicles. These results suggest the involvement of a trimeric G protein in the modulation of Ca(2+)-ATPase.

Up-regulation in late pregnancy of both Go1 alpha and Go2 alpha isoforms in human myometrium.

The nature of the 39 kDa pertussis toxin substrate previously detected in human pregnant myometrium was investigated. Comparison of membranes from non-pregnant and from 39-40 week pregnant myometrium revealed a higher level at 39 kDa of pertussis toxin ADP-ribosylation and Go alpha immunoreactivity in late pregnancy. Furthermore, quantification of both Go alpha isoforms with specific anti-alpha o1- and alpha o2-antibodies revealed an increase in their expression in late pregnancy. At the same time, only limited changes in the levels of Gi1,2, Gi3 and Gq were observed. In rat myometrial membranes, neither pertussis toxin substrate nor Go alpha immunoreactivity could be detected at the 39 kDa level. These results demonstrate that, in the human myometrium in late pregnancy, there is an increase in the expression of both isoforms of the Go alpha subunits, suggesting a role for these proteins throughout gestation and/or near term for parturition.

Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o).

The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, we have examined the effect of mastoparan, an activator of heterotrimeric G proteins, on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibited catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibited calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that was similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocked both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reversed in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibited calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. We conclude that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells.