Erythro-9-(2-hydroxy-3-nonyl)adenine Research Articles

Dimethyl Sulfoxide Induces Hemolysis and Pulmonary Hypertension.

Vascular and lung injury are well established complications associated with hemolytic disorders, and hemolysis associated pulmonary hypertension (PH) has emerged as the most serious complication of sickle cell disease. The causal relationship between intravascular hemolysis and the development of PH is still under investigation. Previously we have shown that repetitive administration of hemolyzed autologous blood causes PH in rats. Dimethyl sulfoxide (DMSO), a widely used solvent and anti-inflammatory agent, induces hemolysis in vivo. We hypothesized that repetitive administration of DMSO would induce PH in rats. We also examined hemolysis-induced release of adenosine deaminase (ADA) and arginase from red blood cells, which may amplify hemolysis-mediated vascular injury. Acute administration of DMSO (1.5ml/30 min into the right atrium) induced intravascular hemolysis and pulmonary vasoconstriction. DMSO-induced increase in right ventricular peak systolic pressure (RVPSP) was associated with increased release of ADA. Notably, the acute increase in RVPSP was attenuated by administration of an adenosine A2A receptor agonist or by pretreatment of animals with ADA inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Repetitive administration of DMSO for 10 days produced anemia, hemoglobinuria, hemoglobinemia, splenomegaly, and development of PH. Histopathological analysis revealed pulmonary vascular remodeling. The presented data describe a new model of hemolysis induced PH, suggesting that hemolysis is mechanistically related to pulmonary hypertension, and pointing to a potential pathogenic role that adenosine deaminase and accelerated adenosine metabolism may play in hemolysis associated pulmonary hypertension.

Role of the nucleoside-metabolizing enzymes on pain responses in zebrafish larvae

Purinergic signaling is a pathway related to pain underlying mechanisms. Adenosine is a neuromodulator responsible for the regulation of multiple physiological and pathological conditions. Extensive advances have been made to understand the role of adenosine in pain regulation. Here we investigated the effects of purinergic compounds able to modulate adenosine production or catabolism on pain responses induced by Acetic Acid (AA) in zebrafish larvae. We investigated the preventive role of the ecto-5′-nucleotidase inhibitor adenosine 5′-(α,β-methylene)diphosphate (AMPCP) and adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)­adenine (EHNA) on the AA-pain induced model. The pain responses were evaluated through exploratory and aversive behaviors in zebrafish larvae. The exploratory behavior showed a reduction in the distance covered by animals exposed to 0.0025% and 0.050% AA. The movement and acceleration were reduced when compared to control. The treatment with AMPCP or EHNA followed by AA exposure did not prevent behavioral changes induced by AA for any parameter tested. There were no changes in aversive behavior after the AA-induced pain model. After AA-induced pain, the AMP hydrolysis increased on zebrafish larvae. However, the AMPCP or EHNA exposure did not prevent changes in AMP hydrolysis induced by the AA-induced pain model in zebrafish larvae. Although AMPCP or EHNA did not show differences in the AA-induced pain model, our results revealed changes in AMP hydrolysis, suggesting the involvement of the purinergic system in zebrafish larvae pain responses.

Biochemical characterization of adenosine deaminase (CD26; EC 3.5.4.4) activity in human lymphocyte-rich peripheral blood mononuclear cells.

The conversion of adenosine to inosine is catalyzed by adenosine deaminase (ADA) (EC 3.5.4.4), which has two isoforms in humans (ADA1 and ADA2) and belongs to the zinc-dependent hydrolase family. ADA modulates lymphocyte function and differentiation, and regulates inflammatory and immune responses. This study investigated ADA activity in lymphocyte-rich peripheral blood mononuclear cells (PBMCs) in the absence of disease. The viability of lymphocyte-rich PBMCs isolated from humans and kept in 0.9% saline solution at 4-8°C was analyzed over 20 h. The incubation time and biochemical properties of the enzyme, such as its Michaelis-Menten constant (Km) and maximum velocity (Vmax), were characterized through the liberation of ammonia from the adenosine substrate. Additionally, the presence of ADA protein on the lymphocyte surface was determined by flow cytometry using an anti-CD26 monoclonal human antibody, and the PBMCs showed long-term viability after 20 h. The ADA enzymatic activity was linear from 15 to 120 min of incubation, from 2.5 to 12.5 µg of protein, and pH 6.0 to 7.4. The Km and Vmax values were 0.103±0.051 mM and 0.025±0.001 nmol NH3·mg-1·s-1, respectively. Zinc and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) inhibited enzymatic activity, and substrate preference was given to adenosine over 2′-deoxyadenosine and guanosine. The present study provides the biochemical characterization of ADA in human lymphocyte-rich PBMCs, and indicates the appropriate conditions for enzyme activity quantification.

Characterization of total adenosine deaminase activity (ADA) and its isoenzymes in saliva and serum in health and inflammatory conditions in four different species: an analytical and clinical validation pilot study

BackgroundMeasurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report’s objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated.ResultstADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibition in canine and porcine samples (serum and saliva) and bovine saliva, whereas for equine saliva 0.94 mM was needed. ADA2 activity was not detected in bovine serum and was very low or absent in equine serum and bovine saliva. An automated procedure to measure ADA2 consisting of adding EHNA to a commercial reagent for tADA measurement provided repetitive (coefficients of variation < 8.8% in serum and < 10% in saliva) and accurate (linearity of serial sample dilutions with R2 > 0.90) results, being equivalent to a manual incubation of the sample with EHNA at a similar concentration. Salivary tADA, as well as ADA1 and ADA2, were higher in dogs with leishmaniosis, horses with acute abdominal disease and pigs with lameness than in healthy animals. tADA and isoenzymes in saliva showed a positive significant correlation with serum ferritin in dogs (r = 0.602, P < 0.01; r = 0.555, P < 0.05; and r = 0.632, P < 0.01; respectively for tADA, ADA1 and ADA2) and serum C-reactive protein in pigs (r = 0.700, P < 0.01, for both tADA and ADA1; r = 0.770, P < 0.001, for ADA2), whereas salivary ADA2 significantly correlated with serum amyloid A in horses (r = 0.649, P < 0.01). In cows, salivary tADA and ADA1 significantly increased after calving, correlating with total white blood cell count (r = 0.487, P < 0.05, for both tADA and ADA1).ConclusionsThe activity of total ADA and its different isoenzymes, can be measured in serum and saliva of dogs, horses, pigs and cows by a simple and fast procedure described in this report. When measured in saliva, these analytes correlated with other biomarkers of inflammation and it could potentially be used as a biomarkers of inflammation and immune activation in the species of this study.

Development of dried blood spot quality control materials for adenosine deaminase severe combined immunodeficiency and an LC-MS/MS method for their characterization

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is an autosomal recessive disorder in which a lack of ADA enzyme prevents the maturation of T- and B-cells; early intervention is crucial for restoring immune function in affected neonates. ADA is responsible for purine metabolism and—in its absence—adenosine, deoxyadenosine, and S-adenosylhomocysteine build up and can be detected in the blood. Preparing dried blood spot (DBS) quality control (QC) materials for these analytes is challenging because enrichments are quickly metabolized by the endogenous ADA in normal donor blood. Adding an inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), has been previously reported to minimize enzyme activity, although this adds additional cost and complexity. We describe an alternative method using unnatural L-enantiomer nucleosides (L-adenosine and L-2′-deoxyadenosine) which eliminates the need for enzyme inhibition. We also present a novel method for characterization of the materials using liquid chromatography mass spectrometry to quantify the analytes of interest.

Role of Phosphodiesterase2A in Proliferation and Migration of Human Osteosarcoma Cells.

The prognosis of patients with osteosarcoma is poor; therefore, new treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of the 11 families (PDE1-PDE11) of the phosphodiesterase superfamily that regulates the intracellular concentrations and effects of cAMP and cGMP. This in vitro study was performed to investigate the role of PDE2 in human oral osteosarcoma HOSM-1 cells. PDE2 expression was measured by a cAMP-PDE assay and real-time-PCR. The effects of the PDE2-specific inhibitors, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), 8-bromo-cAMP, and 8-bromo-cGMP on cell proliferation and migration were assessed. PDE2 activity and PDE2A mRNA expression were detected in HOSM-1 cells. Cell proliferation was inhibited by EHNA and 8-bromo-cAMP but not by 8-bromo-cGMP. Cell migration was stimulated by EHNA and 8-bromo-cGMP, but it was inhibited by 8-bromo-cAMP. Cell proliferation is regulated by PDE2-cAMP signaling and cell migration is regulated by PDE2-cGMP signaling in HOSM-1 cells.

A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition.

Adenosine deaminase (ADA), able to catalyze the irreversible deamination of adenosine into inosine, can be found in almost all tissues and plays an important role in several diseases. In this work, we developed a label-free fluorescence method for the detection of adenosine deaminase activity and inhibition. In the presence of ADA, ATP has been shown to be hydrolyzed. The ATP aptamer was shown to form a G-quadruplex/thioflavin T (ThT) complex with ThT and exhibited an obvious fluorescence signal. However, the ATP aptamer could bind with ATP and exhibited a low fluorescence signal because of the absence of ADA. This assay showed high sensitivity to ADA with a detection limit of 1 U/L based on an SNR of 3 and got a good linear relationship within the range of 1–100 U/L with R2 = 0.9909. The LOD is lower than ADA cutoff value (4 U/L) in the clinical requirement and more sensitive than most of the reported methods. This technique exhibited high selectivity for ADA against hoGG I, UDG, RNase H and λexo. Moreover, this strategy was successfully applied for assaying the inhibition of ADA using erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and, as such, demonstrated great potential for the future use in the diagnosis of ADA-relevant diseases, particularly in advanced drug development.

Clearance of rapid adenosine release is regulated by nucleoside transporters and metabolism.

Adenosine is a neuromodulator that regulates neurotransmission in the brain and central nervous system. Recently, spontaneous adenosine release that is cleared in 3–4 sec was discovered in mouse spinal cord slices and anesthetized rat brains. Here, we examined the clearance of spontaneous adenosine in the rat caudate‐putamen and exogenously applied adenosine in caudate brain slices. The V max for clearance of exogenously applied adenosine in brain slices was 1.4 ± 0.1 μmol/L/sec. In vivo, the equilibrative nucleoside transport 1 (ENT1) inhibitor, S‐(4‐nitrobenzyl)‐6‐thioinosine (NBTI) (1 mg/kg, i.p.) significantly increased the duration of adenosine, while the ENT1/2 inhibitor, dipyridamole (10 mg/kg, i.p.), did not affect duration. 5‐(3‐Bromophenyl)‐7‐[6‐(4‐morpholinyl)‐3‐pyrido[2,3‐d]byrimidin‐4‐amine dihydrochloride (ABT‐702), an adenosine kinase inhibitor (5 mg/kg, i.p.), increased the duration of spontaneous adenosine release. The adenosine deaminase inhibitor, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA) (10 mg/kg, i.p.), also increased the duration in vivo. Similarly, NBTI (10 μmol/L), ABT‐702 (100 nmol/L), or EHNA (20 μmol/L) also decreased the clearance rate of exogenously applied adenosine in brain slices. The increases in duration for blocking ENT1, adenosine kinase, or adenosine deaminase individually were similar, about 0.4 sec in vivo; thus, the removal of adenosine on a rapid time scale occurs through three mechanisms that have comparable effects. A cocktail of ABT‐702, NBTI, and EHNA significantly increased the duration by 0.7 sec, so the mechanisms are not additive and there may be additional mechanisms clearing adenosine on a rapid time scale. The presence of multiple mechanisms for adenosine clearance on a time scale of seconds demonstrates that adenosine is tightly regulated in the extracellular space.

Role of adenosine signaling on pentylenetetrazole-induced seizures in zebrafish.

Adenosine is a well-known endogenous modulator of neuronal excitability with anticonvulsant properties. Thus, the modulation exerted by adenosine might be an effective tool to control seizures. In this study, we investigated the effects of drugs that are able to modulate adenosinergic signaling on pentylenetetrazole (PTZ)-induced seizures in adult zebrafish. The adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) decreased the latency to the onset of the tonic-clonic seizure stage. The adenosine A1 receptor agonist cyclopentyladenosine (CPA) increased the latency to reach the tonic-clonic seizure stage. Both the adenosine A2A receptor agonist and antagonist, CGS 21680 and ZM 241385, respectively, did not promote changes in seizure parameters. Pretreatment with the ecto-5'nucleotidase inhibitor adenosine 5'-(α,β-methylene) diphosphate (AMPCP) decreased the latency to the onset of the tonic-clonic seizure stage. However, when pretreated with the adenosine deaminase (ADA) inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), or with the nucleoside transporter (NT) inhibitors, dipyridamole and S-(4-Nitrobenzyl)-6-thioinosine (NBTI), animals showed longer latency to reach the tonic-clonic seizure status. Finally, our molecular analysis of the c-fos gene expression corroborates these behavioral results. Our findings indicate that the activation of adenosine A1 receptors is an important mechanism to control the development of seizures in zebrafish. Furthermore, the actions of ecto-5'-nucleotidase, ADA, and NTs are directly involved in the control of extracellular adenosine levels and have an important role in the development of seizure episodes in zebrafish.

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N6-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.

Functional Evidence for the Involvement of Microtubules and Dynein Motor Complexes in TRIM5α-Mediated Restriction of Retroviruses

The tripartite motif (TRIM) family of proteins includes the TRIM5α antiretroviral restriction factor. TRIM5α from many Old World and some New World monkeys can restrict the human immunodeficiency virus type 1 (HIV-1), while human TRIM5α restricts N-tropic murine leukemia virus (N-MLV). TRIM5α forms highly dynamic cytoplasmic bodies (CBs) that associate with and translocate on microtubules. However, the functional involvement of microtubules or other cytoskeleton-associated factors in the viral restriction process had not been shown. Here, we demonstrate the dependency of TRIM5α-mediated restriction on microtubule-mediated transport. Pharmacological disruption of the microtubule network using nocodazole or disabling it using paclitaxel (originally named taxol) decreased the restriction of N-MLV and HIV-1 by human or simian alleles of TRIM5α, respectively. In addition, pharmacological inhibition of dynein motor complexes using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and small interfering RNA-mediated depletion of the dynein heavy chain (DHC) similarly decreased TRIM5α-mediated restriction. The loss in restriction resulting from either the disassembly of microtubules or the disruption of dynein motor activity was seen for both endogenous and overexpressed TRIM5α and was not due to differences in protein stability or cell viability. Both nocodazole treatment and DHC depletion interfered with the dynamics of TRIM5α CBs, increasing their size and altering their intracellular localization. In addition, nocodazole, paclitaxel, and DHC depletion were all found to increase the stability of HIV-1 cores in infected cells, providing an alternative explanation for the decreased restriction. In conclusion, association with microtubules and the translocation activity of dynein motor complexes are required to achieve efficient restriction by TRIM5α. The primate innate cellular defenses against infection by retroviruses include a protein named TRIM5α, belonging to the family of restriction factors. TRIM5α is present in the cytoplasm, where it can intercept incoming retroviruses shortly after their entry. How TRIM5α manages to be present at the appropriate subcytoplasmic location to interact with its target is unknown. We hypothesized that TRIM5α, either as a soluble protein or a high-molecular-weight complex (the cytoplasmic body), is transported within the cytoplasm by a molecular motor called the dynein complex, which is known to interact with and move along microtubules. Our results show that destructuring microtubules or crippling their function decreased the capacity of human or simian TRIM5α to restrict their retroviral targets. Inhibiting dynein motor activity, or reducing the expression of a key component of this complex, similarly affected TRIM5α-mediated restriction. Thus, we have identified specific cytoskeleton structures involved in innate antiretroviral defenses.

Hot shot induction and reperfusion with a specific blocker of the es-ENT1 nucleoside transporter before and after hypothermic cardioplegia abolishes myocardial stunning in acutely ischemic hearts despite metabolic derangement: Hot shot drug delivery before hypothermic cardioplegia

Simultaneous inhibition of the cardiac equilibrative-p-nitrobenzylthioinosine (NBMPR)-sensitive (es) type of the equilibrative nucleoside transport 1 (ENT1) nucleoside transporter, with NBMPR, and adenosine deaminase, with erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), prevents release of myocardial purines and attenuates myocardial stunning and fibrillation in canine models of warm ischemia and reperfusion. It is not known whether prolonged administration of hypothermic cardioplegia influences purine release and EHNA/NBMPR-mediated cardioprotection in acutely ischemic hearts. Anesthetized dogs (n=46), which underwent normothermic aortic crossclamping for 20 minutes on-pump, were divided to determine (1) purine release with induction of intermittent antegrade or continuous retrograde hypothermic cardioplegia and reperfusion, (2) the effects of postischemic treatment with 100 μM EHNA and 25 μM NBMPR on purine release and global functional recovery, and (3) whether a hot shot and reperfusion with EHNA/NBMPR inhibits purine release and attenuates ventricular dysfunction of ischemic hearts. Myocardial biopsies and coronary sinus effluents were obtained and analyzed using high-performance liquid chromatography. Warm ischemia depleted myocardial adenosine triphosphate and elevated purines (ie, inosine>adenosine) as markers of ischemia. Induction of intermittent antegrade or continuous retrograde hypothermic (4°C) cardioplegia releases purines until the heart becomes cold (<20°C). During reperfusion, the levels of hypoxanthine and xanthine (free radical substrates) were >90% of purines in coronary sinus effluent. Reperfusion with EHNA/NBMPR abolished ventricular dysfunction in acutely ischemic hearts with and without a hot shot and hypothermic cardioplegic arrest. Induction of hypothermic cardioplegia releases purines from ischemic hearts until they become cold, whereas reperfusion induces massive purine release and myocardial stunning. Inhibition of cardiac es-ENT1 nucleoside transporter abolishes postischemic reperfusion injury in warm and cold cardiac surgery.

Characterization of phosphodiesterase 2A in human malignant melanoma PMP cells

The prognosis for malignant melanoma is poor; therefore, new diagnostic methods and treatment strategies are urgently needed. Phosphodiesterase 2 (PDE2) is one of 21 phosphodiesterases, which are divided into 11 families (PDE1-PDE11). PDE2 hydrolyzes cyclic AMP (cAMP) and cyclic GMP (cGMP), and its binding to cGMP enhances the hydrolysis of cAMP. We previously reported the expression of PDE1, PDE3 and PDE5 in human malignant melanoma cells. However, the expression of PDE2 in these cells has not been investigated. Herein, we examined the expression of PDE2A and its role in human oral malignant melanoma PMP cells. Sequencing of RT-PCR products revealed that PDE2A2 was the only variant expressed in PMP cells. Four point mutations were detected; one missense mutation at nucleotide position 734 (from C to T) resulted in the substitution of threonine with isoleucine at amino acid position 214. The other three were silent mutations. An in vitro migration assay and a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay revealed that suppressing PDE2 activity with its specific inhibitor, erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA), had no impact on cell motility or apoptosis. Furthermore, the cytotoxicity of EHNA, assessed using a trypan blue exclusion assay, was negligible. On the other hand, assessment of cell proliferation by BrdU incorporation and cell cycle analysis by flow cytometry revealed that EHNA treatment inhibited DNA synthesis and increased the percentage of G2/M-arrested cells. Furthermore, cyclin A mRNA expression was downregulated, while cyclin E mRNA expression was upregulated in EHNA-treated cells. Our results demonstrated that the PDE2A2 variant carrying point mutations is expressed in PMP cells and may affect cell cycle progression by modulating cyclin A expression. Thus, PDE2A2 is a possible new molecular target for the treatment of malignant melanoma.

Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects.

In asthmatic patients, inhalation of hyperosmolar saline or D-mannitol (D-M) elicits bronchoconstriction, but in healthy subjects exercise causes bronchodilation. Hyperventilation causes drying of airway surface liquid (ASL) and increases its osmolarity. Hyperosmolar challenge of airway epithelium releases epithelium-derived relaxing factor (EpDRF), which relaxes the airway smooth muscle. This pathway could be involved in exercise-induced bronchodilation. Little is known of ASL hyperosmolarity effects on epithelial function. We investigated the effects of osmolar challenge maneuvers on dispersed and adherent guinea-pig tracheal epithelial cells to examine the hypothesis that EpDRF-mediated relaxation is associated with epithelial cell shrinkage. Enzymatically-dispersed cells shrank when challenged with ≥10 mOsM added D-M, urea or NaCl with a concentration-dependence that mimics relaxation of the of isolated perfused tracheas (IPT). Cells shrank when incubated in isosmolar N-methyl-D-glucamine (NMDG) chloride, Na gluconate (Glu), NMDG-Glu, K-Glu and K2SO4, and swelled in isosmolar KBr and KCl. However, isosmolar challenge is not a strong stimulus of relaxation in IPTs. In previous studies amiloride and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells. Confocal microscopy in tracheal segments showed that adherent epithelium is refractory to low hyperosmolar concentrations that induce dispersed cell shrinkage and relaxation of IPT. Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells. Our studies dissociate relaxation of IPT from cell shrinkage after hyperosmolar challenge of airway epithelium.

A new strategy based on pharmacophore-based virtual screening in adenosine deaminase inhibitors detection and in-vitro study

Background and the purpose of the studyAdenosine deaminase (ADA) inhibition not only may be applied for the treatment of ischemic injury, hypertension, lymphomas and leukaemia, but also they have been considered as anti- inflammatory drugs. On the other hand according to literatures, ADA inhibitors without a nucleoside framework would improve pharmacokinetics and decrease toxicity. Hence we have carried out a rational pharmacophore design for non-nucleoside inhibitors filtration.MethodsA merged pharmacophore model based on the most potent non-nucleoside inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and natural products were generated and applied for compounds filtration. The effects of filtrated compounds based on pharmacophore and docking studies investigated on ADA by UV and Fluorescence spectroscopy techniques.ResultsExtracted compounds were find efficiently inhibit ADA, and the most potent (2) shows an inhibition constant equal to 20 μM. Besides, Fluorescence spectroscopy studies revealed that enzyme 3D structure bear further change in lower concentrations of compound 2.Conclusion3 non-nucleoside inhibitors for ADA are presented. According to obtained results from UV and fluorescence spectroscopy, such interesting pharmacophore template with multiple approaches will help us to extract or design compound with desired properties.

Distinct Patterns of Constitutive Phosphodiesterase Activity in Mouse Sinoatrial Node and Atrial Myocardium

Phosphodiesterases (PDEs) are critical regulators of cyclic nucleotides in the heart. In ventricular myocytes, the L-type Ca2+ current (ICa,L) is a major target of regulation by PDEs, particularly members of the PDE2, PDE3 and PDE4 families. Conversely, much less is known about the roles of PDE2, PDE3 and PDE4 in the regulation of action potential (AP) properties and ICa,L in the sinoatrial node (SAN) and the atrial myocardium, especially in mice. Thus, the purpose of our study was to measure the effects of global PDE inhibition with Isobutyl-1-methylxanthine (IBMX) and selective inhibitors of PDE2, PDE3 and PDE4 on AP properties in isolated mouse SAN and right atrial myocytes. We also measured the effects of these inhibitors on ICa,L in SAN and atrial myocytes in comparison to ventricular myocytes. Our data demonstrate that IBMX markedly increases spontaneous AP frequency in SAN myocytes and AP duration in atrial myocytes. Spontaneous AP firing in SAN myocytes was also increased by the PDE2 inhibitor erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA), the PDE3 inhibitor milrinone (Mil) and the PDE4 inhibitor rolipram (Rol). In contrast, atrial AP duration was increased by EHNA and Rol, but not by Mil. IBMX also potently, and similarly, increased ICa,L in SAN, atrial and ventricular myocytes; however, important differences emerged in terms of which inhibitors could modulate ICa,L in each myocyte type. Consistent with our AP measurements, EHNA, Mil and Rol each increased ICa,L in SAN myocytes. Also, EHNA and Rol, but not Mil, increased atrial ICa,L. In complete contrast, no selective PDE inhibitors increased ICa,L in ventricular myocytes when given alone. Thus, our data show that the effects of selective PDE2, PDE3 and PDE4 inhibitors are distinct in the different regions of the myocardium indicating important differences in how each PDE family constitutively regulates ion channel function in the SAN, atrial and ventricular myocardium.

On-pump inhibition of es-ENT1 nucleoside transporter and adenosine deaminase during aortic crossclamping entraps intracellular adenosine and protects against reperfusion injury: role of adenosine A1 receptor.

The inhibition of adenosine deaminase with erythro-9 (2-hydroxy-3-nonyl)-adenine (EHNA) and the es-ENT1 transporter with p-nitro-benzylthioinosine (NBMPR), entraps myocardial intracellular adenosine during on-pump warm aortic crossclamping, leading to a complete recovery of cardiac function and adenosine triphosphate (ATP) during reperfusion. The differential role of entrapped intracellular and circulating adenosine in EHNA/NBMPR-mediated protection is unknown. Selective (8-cyclopentyl-1,3-dipropyl-xanthine) or nonselective [8-(p-sulfophenyl)theophyline] A1 receptor antagonists were used to block adenosine A1-receptor contribution in EHNA/NBMPR-mediated cardiac recovery. Anesthetized dogs (n = 45), instrumented to measure heart performance using sonomicrometry, were subjected to 30 minutes of warm aortic crossclamping and 60 minutes of reperfusion. Three boluses of the vehicle (series A) or 100 μM EHNA and 25 μM NBMPR (series B) were infused into the pump at baseline, before ischemia and before reperfusion. 8-Cyclopentyl-1,3-dipropyl-xanthine (10 μM) or 8-(p-sulfophenyl)theophyline (100 μM) was intra-aortically infused immediately after aortic crossclamping distal to the clamp in series A and series B. The ATP pool and nicotinamide adenine dinucleotide was determined using high-performance liquid chromatography. Ischemia depleted ATP in all groups by 50%. The adenosine/inosine ratios were more than 10-fold greater in series B than in series A (P < .001). ATP and function recovered in the EHNA/NBMPR-treated group (P < .05 vs control group). 8-Cyclopentyl-1,3-dipropyl-xanthine and 8-(p-sulfophenyl)theophyline partially reduced cardiac function in series A and B to the same degree but did not abolish the EHNA/NBMPR-mediated protection in series B. In addition to the cardioprotection mediated by activation of the adenosine receptors by extracellular adenosine, EHNA/NBMPR entrapment of intracellular adenosine provided a significant component of myocardial protection despite adenosine A1 receptor blockade.

Dynein regulates cell migration depending on substrate rigidity

The mechanical environment in combination with biochemical signaling is an important regulatory factor for cellular physiology including tissue development, cell motility and differentiation. Exerting a tractional force triggered by myosin-dependent cell contractility is known to be an indispensible element of cell migration in a mechanically stiff environment such as a 2D planar surface. However, a number of reports have argued that the requirement of myosin activity for cell migration is limited by cell type and the environment. In this study, we present evidence that dynein, a minus end-directed microtubule motor, plays a central role in cell migration in the absence of tractional force. Interfering with the dynein activity through a dynein-specific inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dramatically inhibited 2D migration of the fibroblast when cell contractility was blocked by Rho kinase or a myosin inhibitor, although EHNA itself did not affect cell migration. Cell migration in 3D soft collagen matrices, where the cell exerts a relatively low tractional force compared to that on a 2D stiff surface, is also profoundly inhibited by dynein intermediate chain (DIC) silencing regardless of the presence of myosin activity. In addition, DIC-silenced cells on a soft acrylamide surface show decreased migration without blockade of myosin activity. Taken together, our results suggest that dynein may be a primary regulatory factor for cell migration when a cell is in a mechanically low-tension environment, such as in a 3D matrix.

Adenosine-mediated cardiovascular toxicity in amitriptyline-poisoned rats

We investigated the contribution of endogenous adenosine to amitriptyline-induced cardiovascular toxicity in rats. A control group of rats was pretreated with intraperitoneal (i.p.) 5% dextrose and received intravenous 0.94 mg/kg/min of amitriptyline for 60 minutes. The second and third groups of rats pretreated with i.p. 10 mg/kg of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase inhibitor, and i.p. 1 mg/kg of S-(4-nitrobenzyl)-6-thioinosine (NBTI), a facilitated adenosine transport inhibitor, received 5% dextrose and amitriptyline infusion, respectively. Outcome parameters were mean arterial pressure (MAP), heart rate (HR), QT and QRS durations, and plasma adenosine concentrations. Plasma adenosine concentrations were increased in all groups. In the control group, amitriptyline decreased MAP and HR and prolonged QT and QRS durations after 10 minutes of infusion. In EHNA/NBTI-pretreated rats, amitriptyline prolonged QRS duration at 10 and 20 minutes. In EHNA/NBTI pretreated rats, amitriptyline-induced MAP, HR reductions, and QRS prolongations were more significant than that of dextrose-infusion–induced changes. Our results indicate that amitriptyline augmented the cardiovascular effects of endogen adenosine by increasing plasma levels of adenosine in rats.

Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

A novel strategy for the preparation of in-column adenosine deaminase (ADA) microreactor and rapid screening of enzyme inhibitors in natural extracts was demonstrated. In this approach, ADA was encapsulated in anionic polyelectrolyte alginate that was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte polyethylenimine (PEI). On-line enzyme inhibition study was performed by capillary electrophoresis (CE). The substrate and product were baselined separated within 75 s. The enzyme activity was determined by the quantification of peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The inhibition percentage was used to evaluate relative activity of ADA microreactor. A known ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was employed as a model compound for the validation of the inhibitor screening method, and the screening of ADA inhibitor in 19 traditional Chinese herbal medicines was performed.