Presence Of EHNA Research Articles

Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5′-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity.

PDE2 activity differs in right and left rat ventricular myocardium and differentially regulates β2 adrenoceptor-mediated effects.

The important regulator of cardiac function, cAMP, is hydrolyzed by different cyclic nucleotide phosphodiesterases (PDEs), whose expression and activity are not uniform throughout the heart. Of these enzymes, PDE2 shapes β1 adrenoceptor-dependent cardiac cAMP signaling, both in the right and left ventricular myocardium, but its role in regulating β2 adrenoceptor-mediated responses is less well known. Our aim was to investigate possible differences in PDE2 transcription and activity between right (RV) and left (LV) rat ventricular myocardium, as well as its role in regulating β2 adrenoceptor effects. The free walls of the RV and the LV were obtained from Sprague-Dawley rat hearts. Relative mRNA for PDE2 (quantified by qPCR) and PDE2 activity (evaluated by a colorimetric procedure and using the PDE2 inhibitor EHNA) were determined in RV and LV. Also, β2 adrenoceptor-mediated effects (β2-adrenoceptor agonist salbutamol + β1 adrenoceptor antagonist CGP-20712A) on contractility and cAMP concentrations, in the absence or presence of EHNA, were studied in the RV and LV. PDE2 transcript levels were less abundant in RV than in LV and the contribution of PDE2 to the total PDE activity was around 25% lower in the microsomal fraction of the RV compared with the LV. β2 adrenoceptor activation increased inotropy and cAMP levels in the LV when measured in the presence of EHNA, but no such effects were observed in the RV, either in the presence or absence of EHNA. These results indicate interventricular differences in PDE2 transcript and activity levels, which may distinctly regulate β2 adrenoceptor-mediated contractility and cAMP concentrations in the RV and in the LV of the rat heart.

Repetitive systemic morphine alters activity-dependent plasticity of Schaffer-collateral-CA1 pyramidal cell synapses: involvement of adenosine A1 receptors and adenosine deaminase.

The effectiveness of O-pulse stimulation (TPS) for the reversal of O-pattern primed bursts (PB)-induced long-term potentiation (LTP) were examined at the Schaffer-collateral-CA1 pyramidal cell synapses of hippocampal slices derived from rats chronically treated with morphine (M-T). The results showed that slices derived from both control and M-T rats had normal field excitatory postsynaptic potential (fEPSP)-LTP, whereas PS-LTP in slices from M-T rats was significantly greater than that from control slices. When morphine was applied in vitro to slices derived from rats chronically treated with morphine, the augmentation of PS-LTP was not seen. TPS given 30 min after LTP induction failed to reverse the fEPSP- or PS-LTP in both groups of slices. However, TPS delivered in the presence of long-term in vitro morphine caused the PS-LTP reversal. This effect was blocked by the adenosine A1 receptor (A1R) antagonist CPX (200 nM) and furthermore was enhanced by the adenosine deaminase (ADA) inhibitor EHNA (10 μM). Interestingly, TPS given 30 min after LTP induction in the presence of EHNA (10 μM) can reverse LTP in morphine-exposed control slices in vitro. These results suggest adaptive changes in the hippocampus area CA1 in particular in adenosine system following repetitive systemic morphine. Chronic in vivo morphine increases A1R and reduces ADA activity in the hippocampus. Consequently, adenosine can accumulate because of a stimulus train-induced activity pattern in CA1 area and takes the opportunity to work as an inhibitory neuromodulator and also to enable CA1 to cope with chronic morphine. In addition, adaptive mechanisms are differentially working in the dendrite layer rather than the somatic layer of hippocampal CA1.

FK506 binding protein (FKBP12) may contribute to 4‐chloro‐m‐cresol induced adenosine in human B cells

IntroductionRyanodine receptor (RyR1) agonist 4‐chloro‐m‐cresol (4CmC) stimulates normoxic adenosine (ADO) production in B cells, especially from malignant hyperthermia susceptible individuals. In an attempt to identify a pathway for this action of 4CmC, FK506, an immunosuppressant drug that disrupts the interaction of FK506‐binding protein (FKBP12) with RyR1, and Xestospongin C (XeC), a specific inhibitor of IP3R were tested. FKBP12 forms a complex with the RyR and IP3R to regulate their physiological release of intracellular Ca2+.MethodsB cells were suspended in HBSS + ADO deaminase inhibitor (EHNA) followed by addition of FK506 or XeC and incubated at 37°C for 10 min. In a 2nd experiment; 10min after addition of FK506 or XeC, 4CmC (2mM) was applied and incubation continued for 30 min. After cellular reactions termination, supernatant [ADO] was assayed using HPLC.Results(means ± SEM for 106 cells): ADO basal level was 0.422 ± 0.097μM (n=7). FK506 (2, 20, 200μM) increased ADO levels by 59 ± 4, 207 ± 21, and 240 ± 24% respectively. FK506 (2, 20, 200μM) increased the sensitivity of B cells to 4CmC by 4±5, 13±4, and 49±10%. XeC did not alter basal or 4CmC‐induced ADO in B cells.ConclusionFK506 likely displaces FKBP12 from the RyR1 receptor in B cells resulting in leaky Ca2+ channels and to cytosolic Ca2+ accumulation. This stimulates Ca2+ pumping, generating ADP and AMP leading to formation of ADO in the presence of EHNA.

Inhibition of lung phosphodiesterase improves responsiveness to inhaled nitric oxide in isolated-perfused lungs from rats challenged with endotoxin.

To investigate the ability of phosphodiesterase (PDE) selective inhibitors to improve responsiveness to inhaled nitric oxide (NO) in isolated-perfused lungs of rats pretreated with endotoxin/lipopolysaccharide (LPS). Prospective, controlled animal study in the animal research facility of a university hospital. Sixteen hours after adult Sprague-Dawley rats were injected intraperitoneally with 0.4 mg/ kg E. coli 0111:B4 LPS administration, lungs were isolated and perfused, and the thromboxane mimetic U46619 was employed to increase the mean pulmonary artery pressure by 5-7 mmHg. The lungs were then ventilated with or without 0.4 ppm NO, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA; PDE type 2 inhibitor), milrinone (PDE type 3 inhibitor), or zaprinast (inhibitor of PDE types 5 and 9) were added to the perfusate. In the presence of EHNA (12.5, 25, 50 microM) the vasodilator response to inhaled NO was not greater than in its absence (0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg vs. 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg, respectively). In the presence of milrinone (125, 250, 500 nM), the vasodilator response to inhaled NO was also not improved. In contrast, zaprinast (3.7, 7.4, 14.8 microM) augmented the pulmonary vasodilatory effect of inhaled NO in lungs from LPS-pretreated rats from 0.25 +/- 0.25, 0.5 +/- 0.25, 0.75 +/- 0.25 mmHg to 0.75 +/- 0.25, 1.5 +/- 0.5, 1.75 +/- 0.75 mmHg, respectively (p < 0.05). Our results demonstrate that inhibition of pulmonary PDE enzyme activity with zaprinast increases vasodilator responsiveness to inhaled NO in lungs obtained from rats 16 h after LPS challenge.

Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth

Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum-induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([3H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [3H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A2 adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation.

2′-deoxy-2′-methylene derivatives of adenosine, guanosine, tubercidin, cytidine and uridine as inhibitors of L1210 cell growth in culture

The 2′-deoxy-2′-methylene derivatives of adenosine (MdAdo), guanosine (MdGuo), tubercidin (MdTu), cytidine (MdCyd) and undine (MdUrd) were synthesized as mechanism-based inhibitors directed at ribonucleotide reductase. It was shown that MdCyd 5′-diphosphate irreversibly inactivated ribonucleotide reductase from Escherichia coli (Baker et al., J Med Chem 34: 1879–1884, 1991). In studies reported here, MdAdo/EHNA, MdGuo and MdCyd inhibited L1210 cell growth with IC 50 values of 3.4,10.6 and 1.4 μM, respectively. Since MdAdo is a substrate for adenosine deaminase, the presence of EHNA was required to give maximal growth inhibition. 8-Aminoguanosine was not required to maximize the cytotoxic effects of MdGuo. The 2′-deoxy-2′-methylene derivatives of tubercidin and uridine did not inhibit L1210 cell growth at concentrations as high as 50 μM (MdTu) or 100 μM (MdUrd). L1210 cell lines resistant to hydroxyurea (directed at the non-heme iron subunit of ribonucleotide reductase) or deoxyadenosine (directed at the effector binding subunit of ribonucleotide reductase) were not resistant to MdCyd. An L1210 cell line that. was highly resistant to dGuo due to the loss of a relatively specific deoxyribonucleoside kinase (Cory et al., J Biol Chem 268: 405–409, 1993) had a 6.6 fold increase in the IC 50 value toward MdCyd, but showed only a 2-fold increase in resistance to MdGuo. Another L1210 cell line that was markedly deficient in adenosine kinase activity was highly resistant to MdAdo. Analysis by now cytometry showed that MdCyd slowed the transit of the cells through the G 2/M phase of the cell cycle resulting in the buildup of the G 2/M population. MdAdo, MdGuo and MdCyd inhibited the incorporation of [ 14C]cytidine into DNA without an effect on RNA synthesis or total cellular uptake of [ 14C]cytidine. The conversion of [ 14C]cytidine to deoxycytidine nucleotides was partially inhibited by MdGuo, but not by MdAdo or MdCyd. These data show that the 2′-deoxy-2′-methylene derivatives of adenosine, guanosine and cytidine are activated via specific nucleoside kinases and that the modes of action of these compounds are not identical.

Decreased 5′nucleotidase activity in suppressor (OKT 8) T lymphocytes from homosexuals with AIDS-related complex: Nonassociation with enhanced deoxynucleoside toxicity

The purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′nucleotidase (5NT) have been shown to be important for normal lymphocyte maturation. Abnormalities of these enzymes have been associated with hereditary as well as acquired immunodeficiency states. Enzyme activity was measured in helper (OKT 4) and suppressor (OKT 8) lymphocyte subsets from 10 homosexuals with AIDS-related complex (ARC) and in 10 healthy controls. There were no significant differences in either mean ADA activity or mean PNP activity between ARC OKT 4 cells and control OKT 4 cells and between ARC OKT 8 cells and control OKT 8 cells. By contrast, mean 5NT activity was slightly decreased in OKT 4 cells from ARC patients compared with that of controls and more significantly diminished in ARC OKT 8 cells compared with that of controls. Both deoxyadenosine and deoxyguanosine, when incubated separately with OKT 4 and OKT 8 cells in the presence of EHNA, and ADA inhibitor, did not significantly inhibit lymphocyte blastogenesis to a greater extent in ARC patients than in controls. Hence, the decreases in 5NT activity most likely reflect lymphocyte immaturity and are not associated with biochemical abnormalities leading to increased deoxynucleoside toxicity.

ADENOSINE DEAMINASE DEFICIENCY AND CHONDRO-OSSEOUS DYSPLASIA: 230

Purine metabolism was investigated in an in vitro model of adenosine deaminase (ADA) deficiency in rabbit epiphyseal growth plate (EGP) and articular cartilage (AC) to elucidate the biochemical basis for the poorly understood chondro-osseous dysplasia that occurs with ADA deficiency.Deoxyadenosine (dado) in the presence of EHNA, an ADA inhibitor, is toxic in a dose dependent fashion to EGP in organ culture as determined by LDH release. Dado or EHNA alone as well as adenosine, homocysteine thiolactone and deoxyguanosine alone or in combination are not toxic. The model is not toxic to mature AC.The combination of 100 μM dado and 5 μM EHNA causes decreased radiolabeled thymidine, glycine and SO4 incorporation (92%, 64%, and 36%, respectively) by EGP cartilage in organ culture for 5 days but does not affect isotope incorporation in AC.Incubations of released chondrocytes in the model causes deoxyATP accumulation in EGP and AC cells at 48 hours. ATP concentrations are undetectable at 48 hours in EGP cells but 1.4 ± 0.4 nmol/106 AC cells.These data demonstrate a selective toxic effect of the combination of dado and an ADA inhibitor to EGP cartilage and suggest that the chondro-osseous dysplasia of ADA deficiency is the consequence of the deficiency state. ADA deficiency may cause altered chondrocyte function and viability by ATP depletion.

Aedes albopictus cells resistant to adenosine because of a defect in nucleoside transport.

By growing Aedes albopictus mosquito cells in media containing increasing concentrations of adenosine and subsequently plating low numbers of cells in the presence of EHNA (an inhibitor of adenosine deaminase), three clones were obtained which were resistant to adenosine. The adenosine-resistant clones contained level of adenosine and thymidine kinase similar to those in the parental cells, but were unable to incorporate labeled nucleotides (adenosine, uridine, thymidine, or guanosine) into TCA-precipitable material. The inability to incorporate nucleosides was also reflected in an enhanced resistance to several nucleoside analogs such as 5-fluorodeoxyuridine and tubercidin but not to the unribosylated base, 5-fluorouracil. Direct measurements over short time intervals indicated that the primary defect in these cells was at the level of nucleoside transport.