5'-Nucleotidase as a marker of both general and local inflammation in rheumatoid arthritis patients.
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OBJECTIVES: To evaluate measurements of serum and synovial fluid 5'-nucleotidase (5'N) activity as a marker of general and local inflammation in arthritis, and to resolve a contradiction in the literature as to whether or not the activity of 5'N in the synovial fluids of rheumatoid arthritis (RA) patients is raised in comparison with that in the synovial fluids of other arthritis patients. METHODS: Assays for 5'N were carried out in the presence of inhibitors of other phosphatases, AMP deaminase and of 5'N itself. RESULTS: The 5'N activity in the synovial fluid of RA patients was both significantly higher (mean 1.7-fold) and had a greater variance than that in the synovial fluids of other arthritis patients, and the contradiction in the literature was resolved. There was a strong correlation between the 5'N activity in the sera of RA patients and their erythrocyte sedimentation rate. There was no significant correlation between the 5'N in the serum and synovial fluid for the RA patients, in marked contrast to the strong correlation between the two 5'N activities shown by the osteoarthritis patients. The 5'N activity was greater in the synovial fluid than in the serum for virtually all the patients, showing that it was being made locally. CONCLUSIONS: The 5'N activity in the serum (which came mostly from the liver) could be used as a marker of general inflammation, whereas the 5'N in the synovial fluid was mostly produced locally, and could be used as a marker of joint inflammation, particularly for the RA patients. (+info)
Adenosine-mediated killing of cultured epithelial cancer cells.
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Because micromolar concentrations of adenosine (Ado) have been documented recently in the interstitial fluid of carcinomas growing in animals, we examined the effects of low concentrations of Ado on the growth of cultured human carcinoma cells. Ado alone had little effect upon cell growth. In the presence of one of a number of Ado deaminase (ADA) inhibitors, Ado led to significant growth inhibition of all cell lines tested. Similar effects were found when ATP, ADP, or AMP was substituted for Ado. Surprisingly, the ADA inhibitor coformycin (CF) had a much greater potentiating effect than did 2'-deoxycoformycin (DCF), although DCF is a more potent ADA inhibitor. The growth inhibition of the Ado/CF combination was not abrogated by pyrimidines or caffeine, a nonspecific Ado receptor blocker. Toxicity was prevented by the addition of the Ado transport inhibitor dipyridamole or the Ado kinase inhibitor 5'-amino 5'-deoxyadenosine. S-Adenosylhomocysteine hydrolase is not involved because neither homocysteine thiolactone nor an S-adenosylhomocysteine hydrolase inhibitor (adenosine dialdehyde) potentiated toxicity of the Ado/CF combination. Unexpectedly, substitution of 2'-deoxyadenosine (the toxic moiety in congenital ADA deficiency) for Ado, did not lead to equivalent toxicity. The Ado/CF combination inhibited DNA synthesis and brought about morphological changes consistent with apoptosis. Together, these findings indicate that the Ado-mediated killing proceeds via an intracellular route that requires the action of Ado kinase. The enhanced cofactor activity of CF may be attributable to its being a more potent inhibitor of AMP deaminase than is DCF. (+info)
Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice.
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We have used an enzyme-based, twin-barrelled sensor to measure adenosine release during hypoxia in the CA1 region of rat hippocampal slices in conjunction with simultaneous extracellular field recordings of excitatory synaptic transmission. When loaded with a combination of adenosine deaminase, nucleoside phosphorylase and xanthine oxidase, the sensor responded linearly to exogenous adenosine over the concentration range 10 nM to 20 microM. Without enzymes, the sensor when placed on the surface of hippocampal slices recorded a very small net signal during hypoxia of 40 +/- 43 pA (mean +/- s.e.m.; n = 7). Only when one barrel was loaded with the complete sequence of enzymes and the other with the last two in the cascade did the sensor record a large net difference signal during hypoxia (1226 +/- 423 pA; n = 7). This signal increased progressively during the hypoxic episode, scaled with the hypoxic depression of the simultaneously recorded field excitatory postsynaptic potential and was greatly reduced (67 +/- 6.5 %; n = 9) by coformycin (0.5-2 microM), a selective inhibitor of adenosine deaminase, the first enzyme in the enzymic cascade within the sensor. For 5 min hypoxic episodes, the sensor recorded a peak concentration of adenosine of 5.6 +/- 1.2 microM (n = 16) with an IC(50) for the depression of transmission of approximately 3 microM. In slices pre-incubated for 3-6 h in nominally Ca(2+)-free artificial cerebrospinal fluid, 5 min of hypoxia resulted in an approximately 9-fold greater release of adenosine (48.9 +/- 17.7 microM; n = 6). High extracellular Ca(2+) (4 mM) both reduced the adenosine signal recorded by the sensor during hypoxia (3.5 +/- 0.6 microM; n = 4) and delayed the hypoxic depression of excitatory synaptic transmission. (+info)
Enhancement of cellular adenosine triphosphate levels in PC12 cells by extracellular adenosine.
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To elucidate the biological significance of extracellular adenine compounds, the effects of adenosine (Ado) on cellular levels of adenine compounds, especially adenosine triphosphate (ATP), in PC12 cells were studied. Ado and inosine but not adenosine 5'-monophosphate, adenosine 5'-diphosphate, ATP, guanosine, cytosine, thymidine, and uridine, significantly enhanced cellular ATP levels in PC12 cells in time- and dose-dependent manners. Various P1 receptor agonists of Ado did not enhance the ATP level. In addition, theophylline, an antagonist of P1 receptors, did not inhibit the Ado-evoked ATP enhancement. These results suggest that the Ado receptor is not involved in the augmentation of the cellular ATP level induced by Ado in PC12 cells. The ATP-enhancing effect of Ado was potentiated by dipyridamole, an inhibitor of Ado uptake, or coformycin, an inhibitor of Ado deaminase. The effect of Ado on the ATP level was also observed when PC12 cells were incubated in glucose-free medium. Together these results suggest that enhancement of cellular ATP levels in PC12 cells by extracellular Ado might be acceleration of ATP synthesis through the Ado salvage system using hypoxanthine-guanine phosphoribosyltransferase rather than Ado kinase since 5'-iodotubercidin, an inhibitor of Ado kinase, had no effect on the enhancement elicited by Ado. (+info)
Extracellular ATP and adenosine induce cell apoptosis of human hepatoma Li-7A cells via the A3 adenosine receptor.
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1. Extracellular ATP is a potent signaling molecule that modulates a myriad of cellular functions through the activation of P2 purinergic receptors and is cytotoxic to a variety of cells at higher concentrations. The mechanism of ATP-elicited cytotoxicity is not fully understood. In this study, we investigated the effect of extracellular ATP on the human hepatoma Li-7A cells. 2. We observed a time- and dose-dependent growth inhibition of Li-7A cells by ATP, which is accompanied by an increase in the active form of caspase-3 as well as increased cleavage of its substrate, poly (ADP-ribose) polymerase. The cytotoxic effect of extracellular ATP was not mediated by the P2X7 receptor, since (1).the effect was not abolished by the P2X7 receptor antagonists oxidized ATP and KN-62, and (2).extracellular ADP, AMP, and adenosine were also cytotoxic. 3. We found that ATP and ADP were degraded to adenosine by Li-7A cells and that treatment of Li-7A cells by adenosine resulted in growth inhibition and caspase-3 activation, indicating that adenosine is the apoptotic agent. Using adenosine receptor agonists and antagonists, as well as inhibitors of adenosine transport and deamination, we showed that the cytotoxic effect of adenosine is specifically mediated by the A3 receptor even though transcripts of A1, A2A, A2B, and a splice variant of the P2X7 receptors were detected in Li-7A cells by RT-PCR. 4. Cytotoxicity caused by exogenous ATP and adenosine was completely abolished by the caspase-3 inhibitor Z-DEVD-FMK, demonstrating the central role of caspase-3 in apoptosis of Li-7A cells. (+info)
Relatively small increases in the steady-state levels of nucleobase deamination products in DNA from human TK6 cells exposed to toxic levels of nitric oxide.
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Nitric oxide (NO) is a physiologically important molecule that has been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While the complicated chemistry of NO-mediated genotoxicity has been extensively study in vitro, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem by exposing human TK6 lymphoblastoid cells to controlled steady-state concentrations of 1.75 or 0.65 microM NO along with 186 microM O2 in a recently developed reactor that avoids the anomalous gas-phase chemistry of NO and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nucleobase deamination products was defined using a recently developed liquid chromatography/mass spectrometry (LC/MS) method, and the results were correlated with cytotoxicity and apoptosis. A series of control experiments revealed the necessity of using dC and dA deaminase inhibitors to avoid adventitious formation of 2'-deoxyuridine (dU) and 2'-deoxyinosine (dI), respectively, during DNA isolation and processing. Exposure of TK6 cells to 1.75 microM NO and 186 microM O2 for 12 h (1260 microM x min dose) resulted in 32% loss of cell viability measured immediately after exposure and 87% cytotoxicity after a 24 h recovery period. The same exposure resulted in 3.5-, 3.8-, and 4.1-fold increases in dX, dI, and dU, respectively, to reach the following levels: dX, 7 (+/- 1) per 10(6) nt; dI, 25 (+/- 2.1) per 10(6) nt; and dU, 40 (+/- 3.8) per 10(6) nt. dO was not detected above the limit of detection of 6 lesions per 10(7) nt in 50 microg of DNA. A 12 h exposure to 0.65 microM NO and 190 microM O2 (468 microM x min dose) caused 1.7-, 1.8-, and 2.0-fold increases in dX, dI, and dU, respectively, accompanied by a approximately 15% (+/- 3.6) reduction in cell viability immediately after exposure. Again, dO was not detected. These results reveal modest increases in the steady-state levels of DNA deamination products in cells exposed to relatively cytotoxic levels of NO. This could result from limited nitrosative chemistry in nuclear DNA in cells exposed to NO or high levels of formation balanced by rapid repair of nucleobase deamination lesions in DNA. (+info)
Crystallization and preliminary X-ray crystallographic analysis of adenosine 5'-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5'-phosphate.
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Adenosine 5'-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5'-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6(2)22, with unit-cell parameters a = b = 131.325, c = 208.254 A, alpha = beta = 90, gamma = 120 degrees. Diffraction data were collected to 3.34 A resolution from a crystal in complex with coformycin 5'-phosphate and to 4.05 A resolution from a crystal of a mercury derivative. (+info)
AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside.
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AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution. (+info)