Ecto-5′-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5′-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5′-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5′-AMP. EPEC infection triggered a release of ecto-5′-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5′-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5′-nucleotidase was susceptible to inhibition by zinc acetate and by α,β-methylene-adenosine diphosphate (α,β-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5′-AMP. In addition, α,β-methylene-ADP and zinc blocked the ability of 5′-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5′-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5′-nucleotidase, such as α,β-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.     

Feedback effects of host-derived adenosine on enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) is a common cause of diarrhea in children in developing countries. After adhering to intestinal cells, EPEC secretes effector proteins into host cells, causing cell damage and eventually death. We previously showed that EPEC infection triggers the release of ATP from host cells and that ATP is broken down to ADP, AMP, and adenosine. Adenosine produced from the breakdown of extracellular ATP triggers fluid secretion in intestinal monolayers and may be an important mediator of EPEC-induced diarrhea. Here we examined whether adenosine has any effects on EPEC bacteria. Adenosine stimulated EPEC growth in several types of media in vitro . Adenosine also altered the pattern of EPEC adherence to cultured cells from a localized adherence pattern to a more diffuse pattern. Adenosine changed the expression of virulence factors in EPEC, inhibiting the expression of the bundle-forming pilus (BFP) and enhancing expression of the EPEC secreted proteins (Esps). In vivo , experimental manipulations of adenosine levels had strong effects on the outcome of EPEC infection in rabbit intestinal loops. In addition to its previously reported effects on host tissues, adenosine has strong effects on EPEC bacteria, stimulating EPEC growth, altering its adherence pattern, and changing the expression of several important virulence genes. Adenosine, like noradrenaline, is a small, host-derived molecule that is utilized as a signal by EPEC.     

Two pathways for ATP release from host cells in enteropathogenic Escherichia coli infection

We previously reported that enteropathogenic Escherichia coli (EPEC) infection triggered a large release of ATP from the host cell that was correlated with and dependent on EPEC-induced killing of the host cell. We noted, however, that under some circumstances, EPEC-induced ATP release exceeded that which could be accounted for on the basis of host cell killing. For example, EPEC-induced ATP release was potentiated by noncytotoxic agents that elevate host cell cAMP, such as forskolin and cholera toxin, and by exposure to hypotonic medium. These findings and the performance of the EPEC espF mutant led us to hypothesize that the CFTR plays a role in EPEC-induced ATP release that is independent of cell death. We report the results of experiments using specific, cell-permeable CFTR activators and inhibitors, as well as transfection of the CFTR into non-CFTR-expressing cell lines, which incriminate the CFTR as a second pathway for ATP release from host cells. Increased ATP release via CFTR is not accompanied by an increase in EPEC adherence to transfected cells. The CFTR-dependent ATP release pathway becomes activated endogenously later in EPEC infection, and this activation is mediated, at least in part, by generation of extracellular adenosine from the breakdown of released ATP.     

Role of EspF in host cell death induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.     

Sensitization of cultured Chinese hamster cells to 42 degrees C hyperthermia by pentalenolactone, an inhibitor of glycolytic ATP synthesis

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42 degrees C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 microM pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (less than 20 per cent) was observed. Hypoxic cultures were obtained by incubating dense cell suspensions (2 X 10(6)/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42 degrees C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.     

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.     

Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height,Cell-Substrate Interactions,and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell''s height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.     

Cell surface complexes (''cortices'') isolated from Paramecium tetraurelia cells as a model system for analysing exocytosis in vitro in conjunction with microinjection studies.

Cortex preparations isolated from Paramecium tetraurelia cells consist of surface with secretory organelles (trichocysts) still attached. In the absence of nucleotides, in media with a pCa of 5-5.5 and a pH of greater than or equal to 6.5, maximal exocytosis occurred when the Mg2+ concentration was lowered from 10 to 0.5 mM. ATP, as well as its non-hydrolysable analogues adenosine 5'-[gamma-thio]triphosphate (ATP[S]) and adenosine 5'[beta gamma-imido]triphosphate (App[NH]p), inhibited exocytosis at a concentration equivalent to that occurring in vivo (as determined by h.p.l.c.), but preincubation with ATP augmented the exocytotic response. GTP and its analogues only slightly stimulated exocytosis in vitro, but sensitivity to Ca2+ was increased significantly, in particular with GTP. These effects of nucleotides were rapidly reversible. Intracellular GTP concentrations (0.35 mM) would suffice for full activation with the pCai values assumed to occur in these cells during activation. On microinjection, ATP inhibited the secretagogue response in intact cells. Whereas microinjected GTP stimulated exocytosis (membrane fusion) without a secretagogue added, Gpp[NH]p remained without any effect; GTP[S] permanently abolished any triggered secretory response. Concomitantly, h.p.l.c. analysis of triggered and untriggered cells showed that GTP hydrolysis occurs immediately after synchronous (1 s) exocytosis in vivo. The precise site(s) of action of GTP during signal transduction in Paramecium cells remain to be determined.     

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.     

Oral infection with enteropathogenic Escherichia coli triggers immune response and intestinal histological alterations in mice selected for their minimal acute inflammatory responses

Enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea, is an important public health problem in Brazil and other developing countries. In vitro assays of bacterial adhesion to cultured cells are important tools for studying bacterial pathogenicity but do not reproduce all the events that occur in natural infections. In this study, the effects of oral infection with EPEC on mice selected for their minimal acute inflammatory response (AIRmin) were evaluated. Mice were orally infected with EPEC and variations in body weight, bacterial shedding and antibody production observed. The infected animals developed seric and secretory anti‐EPEC antibodies; however, neither mortality nor diarrhea was observed. Light microscopy of their intestines demonstrated histological modifications that were not present in controls. However, electron microscopy did not show bacteria attached to the intestinal epithelia to form attaching and effacing lesions, characteristic of EPEC in humans. The bacteria were detected in Peyer's patches and intestinal contents up to 5 hr post‐infection. When human anti‐EPEC secretory immunoglobulin A or avian immunoglobulin Y antibodies were administered to infected animals, they developed minor histological alterations compared with non‐treated animals. In summary, it was found that EPEC triggers immune responses and intestinal histological alterations but does not produce evidence of diarrheal disease in mice infected by the oral route. This study of EPEC experimental infection provides a better understanding of the effects of antibodies on bacterial infections and may provide a suitable model for the design and testing of immunobiological products for active or passive immunization.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Differential regulation of Na+/H+ exchange isoform activities by enteropathogenic E. coli in human intestinal epithelial cells

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal foodborne pathogen associated with diarrhea, especially in infants and young children. Although EPEC produces characteristic attaching and effacing lesions and loss of microvilli, the pathophysiology of EPEC-associated diarrhea, particularly during early infection, remains elusive. The present studies were designed to examine the direct effects of EPEC infection on intestinal absorption via Na(+)/H(+) exchanger (NHE) isoforms. Caco-2 cells were infected with EPEC strain E2348/69 or nonpathogenic E. coli HB101 for a period of 60 to 120 min. Total NHE activity was significantly increased at 60 min, reaching approximately threefold increase after 90 min of EPEC infection. Similar findings were seen in HT-29 cells and T84 cells indicating that the response was not cell-line specific. Most surprising was the differential regulation of NHE2 and NHE3 by EPEC. Marked activation of NHE2 (300%) occurred, whereas significant inhibition ( approximately 50%) of NHE3 activity was induced. The activity of basolateral isoform NHE1 was also significantly increased in response to EPEC infection. Mutations that disrupted the type III secretion system (TTSS) ablated the effect of EPEC on the activity of both NHE2 and NHE3. These results suggest that EPEC, through a TTSS-dependent mechanism, exerts differential effects on NHE isoform activity in intestinal epithelial cells. Additionally, NHEs do not appear to play any role in EPEC-mediated inflammation, because the NHE inhibitors amiloride and 5-(N-ethyl-N-isopropyl)amiloride did not prevent EPEC-mediated IkappaBalpha degradation.     

Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y<Subscript>1</Subscript> receptor-cAMP signal pathway

Extracellular adenosine-5′-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y₁ receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y₁ receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.     

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.     

Redistribution of cyclic GMP in response to sodium butyrate in colon cells

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.     

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.     

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.     

A hemolysin of Vibrio mimicus (VMH) stimulates cells to produce ATP and cyclic AMP which appear to be secretory mediators

The hemolysin of Vibrio mimicus(VMH) is a pore-forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP-dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.