Hydrolysis of Extracellular ATP by Ectonucleoside Triphosphate Diphosphohydrolase (ENTPD) Establishes the Set Point for Fibrotic Activity of Cardiac Fibroblasts

The establishment of set points for cellular activities is essential in regulating homeostasis. Here, we demonstrate key determinants of the fibrogenic set point of cardiac fibroblasts (CFs) by focusing on the pro-fibrotic activity of ATP, which is released by CFs. We tested the hypothesis that the hydrolysis of extracellular ATP by ectonucleoside triphosphate diphosphohydrolases (ENTPDs) regulates pro-fibrotic nucleotide signaling. We detected two ENTPD isoforms, ENTPD-1 and -2, in adult rat ventricular CFs. Partial knockdown of ENTPD-1 and -2 with siRNA increased basal extracellular ATP concentration and enhanced the pro-fibrotic effect of ATP stimulation. Sodium polyoxotungstate-1, an ENTPD inhibitor, not only enhanced the pro-fibrotic effects of exogenously added ATP but also increased basal expression of α-smooth muscle actin, plasminogen activator inhibitor-1 and transforming growth factor (TGF)-β, collagen synthesis, and gel contraction. Furthermore, we found that adenosine, a product of ATP hydrolysis by ENTPD, acts via A2_B receptors to counterbalance the pro-fibrotic response to ATP. Removal of extracellular adenosine or inhibition of A2_B receptors enhanced pro-fibrotic ATP signaling. Together, these results demonstrate the contribution of basally released ATP in establishing the set point for fibrotic activity in adult rat CFs and identify a key role for the modulation of this activity by hydrolysis of released ATP by ENTPDs. These findings also imply that cellular homeostasis and fibrotic response involve the integration of signaling that is pro-fibrotic by ATP and anti-fibrotic by adenosine and that is regulated by ENTPDs.     

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis.     

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.     

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A_2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A_2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A_2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

Intracellular adenosine formation and release by freshly-isolated vascular endothelial cells from rat skeletal muscle: effects of hypoxia and/or acidosis

Previous studies suggested indirectly that vascular endothelial cells (VECs) might be able to release intracellularly-formed adenosine. We isolated VECs from the rat soleus muscle using collagenase digestion and magnetic-activated cell sorting (MACS). The VEC preparation had >90% purity based on cell morphology, fluorescence immunostaining, and RT-PCR of endothelial markers. The kinetic properties of endothelial cytosolic 5′-nucleotidase suggested it was the AMP-preferring N-I isoform: its catalytic activity was 4 times higher than ecto-5′nucleotidase. Adenosine kinase had 50 times greater catalytic activity than adenosine deaminase, suggesting that adenosine removal in VECs is mainly through incorporation into adenine nucleotides. The maximal activities of cytosolic 5′-nucleotidase and adenosine kinase were similar. Adenosine and ATP accumulated in the medium surrounding VECs in primary culture. Hypoxia doubled the adenosine, but ATP was unchanged; AOPCP did not alter medium adenosine, suggesting that hypoxic VECs had released intracellularly-formed adenosine. Acidosis increased medium ATP, but extracellular conversion of ATP to AMP was inhibited, and adenosine remained unchanged. Acidosis in the buffer-perfused rat gracilis muscle elevated AMP and adenosine in the venous effluent, but AOPCP abolished the increase in adenosine, suggesting that adenosine is formed extracellularly by non-endothelial tissues during acidosis in vivo. Hypoxia plus acidosis increased medium ATP by a similar amount to acidosis alone and adenosine 6-fold; AOPCP returned the medium adenosine to the level seen with hypoxia alone. These data suggest that VECs release intracellularly formed adenosine in hypoxia, ATP during acidosis, and both under simulated ischaemic conditions, with further extracellular conversion of ATP to adenosine.     

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.     

Adenine nucleotide metabolism by chondrocytes in vitro: Role of ATP in chondrocyte maturation and matrix mineralization

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism. © 1995 Wiley-Liss Inc.     

The signaling effects of ATP on melanoma-like skin cancer

Melanoma is a type of skin cancer originated by the malignant transformation of melanocytes. Increasing incidence and mortality require efforts focused on studies and research about this cancer. Its microenvironment is rich in extracellular ATP, but there are no studies evaluating the ectonucleotidases and ATP effects on tumor-derived melanoma cells with known amounts of ATP. This way, the objective of this work was to evaluate the purinergic signaling in the pathophysiology of in vivo melanoma and the in vitro effects of ATP signaling. We found increased and effective extracellular ATP hydrolysis in platelets and a significant decrease of extracellular ATP levels and adenosine hydrolysis. In addition, we cultured PBMCs of melanoma patients and used ATP salt with specific concentrations to evaluate its signaling effects. The enzymatic activity analysis revealed that even with higher ATP doses cells metabolize adenine nucleotides less efficiently, and present low ATP, ADP and AMP hydrolytic activity in CM compared to CT cells. In summary, we showed for the first time important data about the purinergic signaling in the pathophysiology of melanoma and ATP signaling exercising immunosuppressive effects. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for melanoma management and treatment and could offer novel therapeutic prospects.     

The relationship between constitutive ATP release and its extracellular metabolism in isolated rat kidney glomeruli.

ATP and adenosine are important extracellular regulators of glomerular functions. In this study, ATP release from glomeruli suspension and its extracellular metabolism were investigated. Basal extraglomerular ATP concentration (1nM) increased several fold during inhibition of ecto-ATPase activity, reflecting the basal ATP release rate. Mechanical perturbation increased the amounts of ATP released from glomeruli. ATP added to glomeruli was almost completely degraded within 20 minutes. In that time, AMP was the main product of extracellular ATP metabolism. Significant accumulation of AMP was observed after 5 min (194 +/-16 microM) and 20 min (271 +/-11 microM), whereas at the same time concentration of adenosine was only 10 muM. A competitive inhibitor of ecto-5-nucleotidase alpha-beta-methylene-ADP (AOPCP), decreased extraglomerular ATP and adenosine concentration by 80% and 50%, respectively. Similarly, AMP (100 microM) also markedly reduced extraglomerular ATP accumulation, whereas IMP, its deamination product, was not effective. P1, P5-diadenosine pentaphosphate (Ap5A) - an inhibitor of ecto-adenylate kinase prevented significantly the disappearance of ATP from extraglomerular media caused by AMP. These findings demonstrate that the decrease in extracellular ATP concentration observed after addition of AOPCP or AMP is caused by the presence of ecto-adenylate kinase activity in the glomeruli. The enzyme catalyses reversible reaction 2ADP<->ATP+AMP, and a rise in the AMP concentration can lead to fall in ATP level. The present study provides evidence the extraglomerular accumulation of ATP reflects both release of ATP from glomeruli cells and its metabolism by ecto-enzymes. Our data suggest that AMP, produced from ATP in the Bowman's capsular space, might plays a dual role as a substrate for ecto-adenylate kinase and ecto-nucleotidase reactions being responsible for the regulation of intracapsular ATP and adenosine concentration. We conclude that AMP degrading and converting ecto-enzymes effectively determine the balance between ATP and adenosine concentration and thus the activation of P2 and/or adenosine receptors.     

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.     

Modification by arachidonic acid of extracellular adenosine metabolism and neuromodulatory action in the rat hippocampus

Adenosine and arachidonate (AA) fulfil opposite modulatory roles, arachidonate facilitating and adenosine inhibiting cellular responses. To understand if there is an inter-play between these two neuromodulatory systems, we investigated the effect of AA on extracellular adenosine metabolism in hippocampal nerve terminals. AA (30 microm) facilitated by 67% adenosine evoked release and by 45% ATP evoked release. These effects were not significantly modified upon blockade of lipooxygenase or cyclooxygenase and were attenuated (52-61%) by the protein kinase C inhibitor, chelerythrine (6 microm). The ecto-5'-nucleotidase inhibitor, alpha,beta-methylene ADP (100 microm), caused a larger inhibition (54%) of adenosine release in the presence of AA (30 microm) compared with control (37% inhibition) indicating that the AA-induced extracellular adenosine accumulation is mostly originated from an increased release and extracellular catabolism of ATP. This AA-induced extracellular adenosine accumulation is further potentiated by an AA-induced decrease (48%) of adenosine transporters capacity. AA (30 microm) increased by 36-42% the tonic inhibition by endogenous extracellular adenosine of adenosine A(1) receptors in the modulation of acetylcholine release and of CA1 hippocampal synaptic transmission in hippocampal slices. These results indicate that AA increases tonic adenosine modulation as a possible feedback loop to limit AA facilitation of neuronal excitability.     

Plant population size and isolation affect herbivory of <Emphasis Type="Italic">Silene latifolia</Emphasis> by the specialist herbivore <Emphasis Type="Italic">Hadena bicruris</Emphasis> and parasitism of the herbivore by parasitoids

Habitat fragmentation can affect levels of herbivory in plant populations if plants and herbivores are differentially affected by fragmentation. Moreover, if herbivores are top–down controlled by predators or parasitoids, herbivory may also be affected by differential effects of fragmentation on herbivores and their natural enemies. We used natural Silene latifolia populations to examine the effects of plant population size and isolation on the level of herbivory by the seed predating noctuid Hadena bicruris and the rate of parasitism of the herbivore by its parasitoids. In addition, we examined oviposition rate, herbivory and parasitism in differently sized experimental populations. In natural populations, the level of herbivory increased and the rate of parasitism decreased with decreasing plant population size and increasing degree of isolation. The number of parasitoid species also declined with decreasing plant population size. In the experimental populations, the level of herbivory was also higher in smaller populations, in accordance with higher oviposition rates, but was not accompanied by lower rates of parasitism. Similarly, oviposition rate and herbivory, but not parasitism rate, increased near the edges of populations. These results suggests that in this system with the well dispersing herbivore H. bicruris, habitat fragmentation increases herbivory of the plant through a behavioural response of the moth that leads to higher oviposition rates in fragmented populations with a reduced population size, increased isolation and higher edge-to-interior ratio. Although the rate of parasitism and the number of parasitoid species declined with decreasing population size in the natural populations, we argue that in this system it is unlikely that this decline made a major contribution to increased herbivory.     

Reactive oxygen species contribute to the promotion of the ATP-mediated proliferation of mouse skeletal myoblasts

Reactive oxygen species (ROS) and extracellular adenosine 5'-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10μM ATP increased proliferation of mouse myoblasts by ～20%. ATP also induced intracellular Ca(2+) oscillations, which were independent of extracellular Ca(2+). Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H(2)O(2)) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H(2)O(2) mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation.     

Ecto-Nucleotidase Activities of Promastigotes from Leishmania (Viannia) braziliensis Relates to Parasite Infectivity and Disease Clinical Outcome

Background

Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease.

Methodology/Principal Findings

Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages.

Conclusions/Significance

Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.     

Activity-dependent feedback modulation of spike patterning of supraoptic nucleus neurons by endogenous adenosine

Neuropeptide secretion from the dendrites of hypothalamic magnocellular supraoptic nucleus (SON) neurons contributes to the regulation of neuronal activity patterning, which ultimately determines their peptide output from axon terminals in the posterior pituitary gland. SON dendrites also secrete a number of other neuromodulators, including ATP. ATP degrades to adenosine in the extracellular space to complement transported adenosine acting on pre- and postsynaptic SON A1 receptors to reduce neuronal excitability, measured in vitro. To assess adenosine control of electrical activity in vivo, we made extracellular single-unit recordings of the electrical activity of SON neurons in anesthetized male rats. Microdialysis application (retrodialysis) of the A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT) increased phasic vasopressin cell intraburst firing rates progressively over the first 5 s by 4.5 +/- 1.6 Hz (P < 0.05), and increased burst duration by 293 +/- 64% (P < 0.05). Hazard function plots were generated from interval interspike histograms and revealed that these effects were associated with increased postspike excitability. In contrast, CPT had no effect on the firing rates and hazard function plot profiles of continuously active vasopressin and oxytocin cells. However, CPT significantly increased clustering of spikes, as quantified by the index of dispersion, in oxytocin cells and continuously active vasopressin cells (by 267 +/- 113% and 462 +/- 67%, respectively, P < 0.05). Indeed, in 4 of 5 continuously active vasopressin cells, CPT induced a pseudophasic activity pattern. Together, these results indicate that endogenous adenosine is involved in the local control of SON cell activity in vivo.     

LmxPK4, a mitogen-activated protein kinase kinase homologue of Leishmania mexicana with a potential role in parasite differentiation

Members of the mitogen-activated protein (MAP) kinase cascade are important for the establishment of a Leishmania mexicana infection and are involved in flagellar length control, although the underlying molecular mechanisms remain to be elucidated. This study reports the cloning and characterization of LmxPK4, a MAP kinase kinase homologue of L. mexicana displaying putative plant-like regulatory phosphorylation sites. The recombinant protein has autophosphorylating activity and phosphorylates myelin basic protein. An LmxPK4 gene deletion mutant showed a proliferation defect after infection of macrophages and no or delayed lesion development in mice. Irrespective of the onset of lesion development parasites showed an early and homogeneous lesion development in re-infection experiments. This is indicative for a compensation of the null mutant phenotype. Additionally, this phenotype could be reverted by reintroduction of the wild-type gene into the deletion background. Mutants expressing loss-of-function or N-terminally truncated versions of LmxPK4 retained the null mutant phenotype. LmxPK4 is stage-specifically expressed in promastigotes and during differentiation to amastigotes, but is not detectable in amastigotes isolated from the mammalian host. Moreover, its in vitro kinase activity increases with temperature rise up to 40 degrees C. Our results suggest that LmxPK4 is involved in the differentiation process and affects virulence of Leishmania mexicana.     

Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation

Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 microM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50-3500 microM) showed a biphasic dose response. Up to 800 microM, ATP was cytotoxic in a dose-dependent manner (EC(50) 90 microM). In a range between 800 and 2500 microM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A(3) receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A(1-3) antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 microM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 microM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 microM) a reversible G(1) phase arrest of the cell cycle was induced, whereas higher concentrations (1000 microM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.