Adenosine Deaminase Inhibitor Erythro-9 Research Articles

The Effect of Adenosine Signaling on Memory Impairment Induced by Pentylenetetrazole in Zebrafish.

Epilepsy is characterized by the manifestation of spontaneous and recurrent seizures. The high prevalence of comorbidities associated with epilepsy, such as cognitive dysfunction, affects the patients quality oflife. Adenosine signaling modulation might be an effective alternative to control seizures and epilepsy-associated comorbidities. This study aimed to verify the role of adenosine modulation on the seizure development and cognitive impairment induced by pentylenetetrazole (PTZ) in zebrafish. At first, animals were submitted to a training session in the inhibitory avoidance test and, after 10min, they received an intraperitoneal injection of valproate, adenosine A1 receptor agonist cyclopentyladenosine (CPA), adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2A receptor antagonist ZM 241385, adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nony1)-adenine hydrochloride (EHNA) or the nucleoside transporter inhibitor dipyridamole. Thirty min after the intraperitoneal injection, the animals were exposed to 7.5mM PTZ for 10min, where they were evaluated for latency to reach the seizure stages (I, II, and III). Finally, 24h after the training session, the animals were submitted to the inhibitory avoidance test to verify their cognitive performance during the test session. Valproate, CPA, and EHNA showed antiseizure effects and prevented the memory impairment induced by PTZ exposure. DPCPX, ZM 241385, and dipyridamole pretreatments caused no changes in seizure development; however, these drugs prevented memory impairment without altering locomotion. Our results reinforce the antiseizure effects of adenosine signaling and support the idea that the involvement of adenosine in memory processes may be a target for preventive strategies against cognitive impairment associated with epilepsy.

Simultaneous determination of cordycepin and its metabolite 3'-deoxyinosine in rat whole blood by ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry and its application to accurate pharmacokinetic studies.

Cordycepin from Cordyceps possesses excellent pharmacological properties, including anti-inflammation and anti-tumor effects, therefore representing a potential alternative medicine. However, doubts about the pharmacokinetic results of cordycepin had been raised in the previous study due to its rapid deamination. The organic solvent methanol was immediately added to terminate the degradation of cordycepin in anticoagulated blood samples and enable the accurate evaluation of pharmacokinetics in vivo. A sensitive and selective ultra-high-performance liquid chromatography coupled with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry method was developed and validated to simultaneously determine cordycepin and its deamination metabolite 3'-deoxyinosine using 2-chloroadenosine as an internal standard in rat whole blood. The calibration curves of cordycepin and 3'-deoxyinosine showed excellent linearity within the concentration range of 1.05-10 000.00 ng/ml with acceptable accuracy, precision, selectivity, recovery, matrix effect, and stability. This method was successfully applied to the pharmacokinetic study of cordycepin and its metabolite in rat blood. The effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride on the pharmacokinetics of cordycepin was investigated. In summary, the reliable pharmacokinetic parameters of cordycepin and its deamination metabolite 3'-deoxyinosine in rat blood were successfully elucidated. Erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride considerably prolonged the half-life of cordycepin in vivo.

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.

High-intensity swimming exercise reduces neuropathic pain in an animal model of complex regional pain syndrome type I: Evidence for a role of the adenosinergic system

This study investigated the involvement of the adenosinergic system in antiallodynia induced by exercise in an animal model of complex regional pain syndrome type I (CRPS-I). Furthermore, we analyzed the role of the opioid receptors on exercise-induced analgesia. Ischemia/reperfusion (IR) mice, nonexercised and exercised, received intraperitoneal injections of caffeine (10mg/kg, a non selective adenosine receptor antagonist), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1mg/kg, a selective adenosine A1 receptor antagonist), ZM241385 (3mg/kg, a selective adenosine A2A receptor antagonist), adenosine deaminase inhibitor erythro-9-(2-hydroxy-3nonyl) adenine [(EHNA), 5mg/kg, an adenosine deaminase inhibitor] or naloxone (1mg/kg, a nonselective opioid receptor antagonist). The results showed that high-intensity swimming exercise reduced mechanical allodynia in an animal model of CRPS-I in mice. The antiallodynic effect caused by exercise was reversed by pretreatment with caffeine, naloxone, DPCPX but it was not modified by ZM241385 treatment. In addition, treatment with EHNA, which suppresses the breakdown of adenosine to inosine, enhanced the pain-relieving effects of the high-intensity swimming exercise. This is the first report demonstrating that repeated sessions of high-intensity swimming exercise attenuate mechanical allodynia in an animal model of CRPS-I and that the mechanism involves endogenous adenosine and adenosine A1 receptors. This study supports the use of high-intensity exercise as an adjunct therapy for CRPS-I treatment.

Adenosine and inosine exert cytoprotective effects in an in vitro model of liver ischemia-reperfusion injury

Liver ischemia represents a common clinical problem. In the present study, using an in vitro model of hepatic ischemia-reperfusion injury, we evaluated the potential cytoprotective effect of the purine metabolites, such as adenosine and inosine, and studied the mode of their pharmacological actions. The human hepatocellular carcinoma-derived cell line HepG2 was subjected to combined oxygen-glucose deprivation (COGD; 0-14-24 h), followed by re-oxygenation (0-4-24 h). Adenosine or inosine (300–1,000 μM) were applied in pretreatment. Cell viability and cytotoxicity were measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and lactate dehydrogenase methods, respectively. The results showed that both adenosine and inosine exerted cytoprotective effects, and these effects were not related to receptor-mediated actions, since they were not prevented by selective adenosine receptor antagonists. On the other hand, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 10 μM) markedly and almost fully reversed the protective effect of adenosine during COGD, while it did not influence the cytoprotective effect of inosine in the same assay conditions. These results suggest that the cytoprotective effects are related to intracellular actions, and, in the case of adenosine also involve intracellular conversion to inosine. The likely interpretation of these findings is that inosine serves as an alternative source of energy to produce ATP during hypoxic conditions. The protective effects are also partially dependent on adenosine kinase, as the inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, 2HCl (ABT 702, 30 μM) significantly reversed the protective effect of both adenosine and inosine during hypoxia and re-oxygenation. Collectively, the current results support the view that during hypoxia, adenosine and inosine exert cytoprotective effects via receptor-independent, intracellular modes of action, which, in part, depend on the restoration of cellular bioenergetics. The present study supports the view that testing of inosine for protection against various forms of warm and cold liver ischemia is relevant.

Involvement of Adenosine Signaling in Controlling the Release of Ghrelin from the Mouse Stomach

Ghrelin, a potent orexigenic hormone released from the stomach, is important in regulating energy metabolism. Abnormal ghrelin levels are associated with eating disorders and metabolic diseases. However, factors involved in the regulation of ghrelin release remain unclear. Here, we examined the involvement of adenosine signaling in the control of ghrelin release from the perfused mouse stomach. Adenosine stimulated ghrelin release concentration-dependently, and the A(2A) receptor-selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261) abolished the increased release. The A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) augmented ghrelin release concentration-dependently, whereas the A(1) receptor-selective agonist 2-chloro-N(6)-cyclopentyladenosine inhibited ghrelin release. In A(2A) receptor knockout mice, adenosine inhibited ghrelin release, and the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked this inhibition. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride increased ghrelin release in wild-type and A(1) receptor knockout mice but not in A(2A) receptor knockout mice. Colocalization of ghrelin immunoreactivity with A(1) and A(2A) receptor immunoreactivities in the gastric nerve fibers were observed. Colocalization was also detected for ghrelin and A(1) receptor immunoreactivities in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine exerts predominantly a tonic A(2A) receptor-mediated stimulatory action on gastric ghrelin release, whereas an A(1) receptor-mediated inhibitory action is also apparent when the tonic excitatory effect was removed.

Opiates, Sleep, and Pain

Opiates, Sleep, and Pain

Purinergic modulation in the development of the rat uncrossed retinotectal pathway

Adenosine is a neuromodulator implicated in nervous system development and plasticity and its effects are mediated by inhibitory (A1, A3) and excitatory (A2a, A2b) receptors. The role of adenosine in the synaptic activity depends mainly on a balanced activation of A1 and A2a receptors which are activated by various ranges of adenosine concentrations. Herein, we investigated the expression of A1 and A2a receptors and also the accumulation of cAMP in the superior colliculus at different stages of development. Furthermore, we examined the effects of an acute in vivo blockade of adenosine deaminase during the critical period when the elimination of misplaced axons/terminals takes place with a simultaneous fine tuning of terminal arbors into appropriate terminal zones. Lister Hooded rats ranging from postnatal days (PND) 0–70 were used for ontogeny studies. Our results indicate that A1 expression in the visual layers of the superior colliculus is higher until PND 28, while A2a expression increases after PND 28 in a complementary developmental pattern. Accordingly, the incubation of collicular slices with 5′-N-ethylcarboxamido-adenosine, a non-specific adenosine receptor agonist, showed a significant reduction in cAMP accumulation at PND 14 and an increase in adults. For the anatomical studies, the uncrossed retinotectal projections were traced after the intraocular injection of horseradish peroxidase. One group received daily injections of an adenosine deaminase inhibitor (erythro-9(2-hydroxy-3-nonyl adenine), 10 mg/kg i.p.) between PND 10 and 13, while control groups were treated with vehicle injections (NaCl 0.9%, i.p.). We found that a short-term blockade of adenosine deaminase during the second postnatal week induced an expansion of retinotectal terminal fields in the rostrocaudal axis of the tectum. Taken together, the results suggest that a balance of purinergic A1 and A2a receptors through cAMP signaling plays a pivotal role during the development of topographic order in the retinotectal pathway.

Regulation of Somatostatin Release by Adenosine in the Mouse Stomach

Adenosine inhibits gastric acid secretion, either directly by acting on acid-secreting parietal cells or indirectly by stimulating the release of the acid inhibitor, somatostatin. The present study examined the role of adenosine on somatostatin release in an isolated vascularly perfused mouse stomach model. Concentrations of exogenous adenosine >or= 1.0 microM stimulated gastric release of somatostatin-like immunoreactivity (SLI), and this effect was blocked by the A(2A) receptor antagonist ZM 241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol]. The A(2A) receptor agonist CGS 21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride] augmented SLI release in a concentration-dependent manner, suggesting that A(2A) receptor activation is involved in the stimulatory effect of adenosine on SLI release. Conversely, SLI release was inhibited by the A(1) receptor agonists N(6)-cyclopentyladenosine and 2-chloro-N(6)-cyclopentyladenosine and lower concentration of adenosine (0.01 microM). The involvement of specific adenosine receptors in controlling the release of gastric SLI was also examined using A(2A) receptor knockout (A(2A)R-KO) mice. In these mice, adenosine (10 microM) inhibited SLI release, and the effect was abolished by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a link between the selective A(1) activation and inhibition of SLI release. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride augmented SLI release in wild-type controls but not in the presence of ZM 241385 or in A(2A)R-KO mice. We conclude that adenosine has dual actions on regulating mouse gastric SLI release: stimulatory at higher concentrations through the A(2A) receptor and inhibitory at lower concentrations through the A(1) receptor, whereas A(2B) and A(3) receptors have a minimal role.

The adenosine deaminase inhibitor erythro-9-[2-hydroxyl-3-nonyl]-adenine decreases intestinal permeability and protects against experimental sepsis: a prospective, randomised laboratory investigation

IntroductionThe treatment of septic conditions in critically ill patients is still one of medicine's major challenges. Cyclic nucleotides, adenosine and its receptors play a pivotal role in the regulation of inflammatory responses and in limiting inflammatory tissue destruction. The aim of this study was to verify the hypothesis that adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibition by erythro-9-[2-hydroxyl-3-nonyl]-adenine could be beneficial in experimental endotoxicosis/sepsis.MethodWe used two established animal models for endotoxicosis and sepsis. Twenty-four male Wistar rats that had been given intravenous endotoxin (Escherichia coli lipopolysaccharide) were treated with either erythro-9-[2-hydroxyl-3-nonyl]-adenine infusion or 0.9% saline during a study length of 120 minutes. Sepsis in 84 female C57BL/6 mice was induced by caecal ligation and puncture. Animals were treated with repeated erythro-9-[2-hydroxyl-3-nonyl]-adenine injections after 0, 12 and 24 hours or 4, 12 and 24 hours for delayed treatment.ResultsIn endotoxaemic rats, intestinal production of hypoxanthine increased from 9.8 +/- 90.2 μmol/l at baseline to 411.4 +/- 124.6 μmol/l and uric acid formation increased from 1.5 +/- 2.3 mmol/l to 13.1 +/- 2.7 mmol/l after 120 minutes. In endotoxaemic animals treated with erythro-9-[2-hydroxyl-3-nonyl]-adenine, we found no elevation of adenosine metabolites. The lactulose/L-rhamnose ratio (14.3 versus 4.2 in control animals; p = 2.5 × 10-7) reflects a highly permeable small intestine and through the application of erythro-9-[2-hydroxyl-3-nonyl]-adenine, intestinal permeability could be re-established. The lipopolysaccharide animals had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios. Erythro-9-[2-hydroxyl-3-nonyl]-adenine reduced this effect. The mucosa damage score of the septic animals was higher compared with control and therapy animals (p < 0.05). Septic shock induction by caecal ligation and puncture resulted in a 160-hour survival rate of about 25%. In contrast, direct adenosine deaminase-1 inhibition resulted in a survival rate of about 75% (p = 0.0018). A protective effect was still present when erythro-9-[2-hydroxyl-3-nonyl]-adenine treatment was delayed for four hours (55%, p = 0.029).ConclusionsWe present further evidence of the beneficial effects achieved by administering erythro-9-[2-hydroxyl-3-nonyl]-adenine, an adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibitor, in an endotoxicosis and sepsis animal model. This suggests a potential therapeutic option in the treatment of septic conditions.

Adenosine Promotion of Cellular Migration in Bronchial Epithelial Cells Is Mediated by the Activation of Cyclic Adenosine Monophosphate–Dependent Protein Kinase A

Migration of neighboring cells into the injury is important for rapid repair of damaged airway epithelium. We previously reported that activation of the A2A receptors (A2AARs) mediates adenosine-stimulated epithelial wound...

Merits of HPLC-based method over spectrophotometric method for assessing the kinetics and inhibition of mammalian adenosine deaminase

Measurement of adenosine deaminase (ADA) activity using spectrophotometric method presents problem, regarding the quantitative estimation of the substrate degradation and product formation, due to the closely apposed λ max of the substrates, product and the inhibitor. The feasibility of applying reverse-phase HPLC technique, for studying adenosine deaminase-catalyzed reaction product and inhibition study was examined. We have drawn a comparison between the HPLC-based method over the corresponding spectrophotometric method. A gradient elution pattern was used to separate substrate (adenosine and deoxyadenosine), product (inosine and deoxyinosine) and standard adenosine deaminase inhibitor (erythro-9-(3-nonyl- ρ-aminobenzyl)-adenine) in the HPLC method. The product formation was quantitated by monitoring the absorbance at 260 nm with the progress of time. The limit of detection as well as the limit of quantification of the respective enzymatic product were found to be in nano molar (nM) range in the HPLC method. This study was also extended to monitor adenosine deaminase activity in different cancer cells of hematological origin. The HPLC-based method is found to be suitable for the quantitative estimation of adenosine deaminase-catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC-based method has specific advantages over the spectrophotometric method. Moreover, the concentration of different nucleotides in cell lysate and body fluid can be measured using this HPLC method.

Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at −70 mV [18.8 ± 1.1 pA ( n = 98) at 1 mM] in a dose-dependent manner (EC 50 = 177 μM). A similar current was induced by A 1 agonist N 6-cyclopentyladenosine (1 μM), whereas A 1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 μM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K +, and was attenuated by Ba 2+ (100 μM) and 4-aminopyridine (5 mM) but not tetraethylammonium (5 mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 μM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 μM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 μM). We conclude that a Ba 2+- and 4-aminopyridine-sensitive K + channel in SG neurons is opened via the activation of A 1 receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.

Endogenous adenosine modulation of 22Na uptake by rat brain synaptosomes.

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence 22Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1-5 U/ml) caused an excitatory effect on 22Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25 microM). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1-10 microM) and the adenosine deaminase inhibitor EHNA (10-25 microM) inhibited 22Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.

Adenosine regulates the survival of avian retinal neurons and photoreceptors in culture.

Adenosine modulates the survival of chick embryo retinal neurons in culture. When cultures were incubated for 3 days and refed with fresh medium, a large proportion of neurons died in the subsequent 3 days of culture. This cell death was prevented by preincubation of cultures for at least 24 h with adenosine plus the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine uptake blocker nitrobenzylthioinosine (NBI), the adenosine A2A receptor agonist 2-[4-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680), or the permeant cyclic AMP analog 8-bromo cyclic AMP, but not the A1 receptor agonist cyclohexyladenosine (CHA). Adenosine deaminase induced cell death when added to culture medium, and this effect was prevented by EHNA. Cell death was not observed when the medium was replaced by a conditioned medium from sister cultures. The data strongly suggest that adenosine regulates the survival of developing retinal neurons by a long-term activation of A2A receptors and the increase of cyclic AMP levels.

Adenosine A(2a) receptor-mediated inhibition of rod opsin mRNA expression in tiger salamander.

The neuromodulator adenosine mediates dark-adaptive changes in retinal photoreceptors through A(2a) receptors. In cold-blooded vertebrates, opsin mRNA expression is lower at night than during the day. In the present study, we tested whether adenosine could inhibit opsin mRNA expression in cultured rod cells and if endogenous adenosine acts to suppress opsin mRNA in the intact retina at night. Semi-quantitative in situ hybridization showed that treatment with 100 nm of the A(2a)/A(2b) agonist N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) reduced opsin mRNA 41% in cultured rod cells. The effect of DPMA was blocked by 10 microm of the A(2a) antagonist 8-(3-chlorostyryl)caffeine (CSC) but not by 10 microm of the A(2b) antagonist alloxazine. One micromolar adenosine alone had no effect on opsin mRNA. However, in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA), 1 microm adenosine reduced opsin mRNA 61%. EHNA alone reduced opsin mRNA by 26%. Consistent with an A(2a) receptor mechanism, 100 nm forskolin (adenylate cyclase agonist) decreased opsin mRNA 34%. Finally, northern blots showed that intravitreal injection of 10 microm CSC at night increased opsin I mRNA 38%. Thus, endogenous adenosine suppresses rod opsin I mRNA expression at night; in vitro results indicate this reduction occurs through A(2a)-like receptor binding and stimulation of adenylate cyclase activity.

Broad spectrum anticonvulsant activity of BW534U87: possible role of an adenosine-dependent mechanism.

The novel putative anticonvulsant drug 1-[2,6-difluorophenyl)-methyl]-1H-1,2,3-triazolo[4,5-c]) pyridine-4-amine monohydrochloride (BW534U87) effectively reduced seizures induced in rodents by threshold maximal and supramaximal electroshock, electrical kindling, pentylenetetrazole (PTZ) infusion and by vestibular stimulation in the genetically seizure-prone epilepsy-like (EL) mouse. The range of animal seizure models in which BW534U87 was effective is consistent with a broad spectrum anticonvulsant profile. In the EL mouse, the activity of BW534U87 was partially reversed by predosing with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), suggesting that an adenosine-dependent mechanism contributed to the antiseizure activity of the drug. BW534U87 inhibited rat brain homogenate adenosine deaminase activity, thus, raising the possibility that, by blocking the metabolism of endogenous adenosine by this route, BW534U87 limited seizure activity by promoting the inhibitory tone mediated by endogenous adenosine in the brain. The seizure protection conferred by the selective adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) in EL mice and mice infused with PTZ confirms that inhibition of adenosine metabolism by deamination is an effective antiseizure strategy in these models.

Chondrocytes respond to adenosine via A2receptors and activity is potentiated by an adenosine deaminase inhibitor and a phosphodiesterase inhibitor

Objective To test the mechanisms by which adenosine and adenosine analogues stimulate adenylate cyclase and suppress lipopolysaccharide (LPS)-induced production of nitric oxide (NO) by chondrocytes.Methods Primary chondrocytes isolated from equine articular cartilage were plated in monolayer. Intracellular cyclic-AMP (cAMP) accumulation was measured following exposure to medium containing adenosine, the non-hydrolyzable adenosine analogue N6-methyladenosine, the A2Aspecific agonist N6-(dimethoxyphenyl)-ethyl]adenosine (DPMA), the adenosine deaminase inhibitor erythro-9-(2-Hydroxy-3-nonyl)adenine hydrochloride (EHNA), or forskolin, a potent stimulator of adenylate cyclase. Regulation of NO production by LPS-stimulated chondrocytes, as determined by nitrite concentration, was assessed in the presence of adenosine, N6-methyladenosine, DPMA, the broad agonist 5′-N-ethylcarboxamidoadenosine (NECA), or forskolin. Alternatively, LPS-stimulated chondrocytes were exposed to EHNA or the phosphodiesterase inhibitor rolipram in the presence or absence of supplemental adenosine.Results Adenosine, N6-methyladenosine, DPMA, and forskolin each increased intracellular cAMP accumulation in a concentration-dependent manner and suppressed NO production by LPS-stimulated chondrocytes. NECA also decreased NO production by chondrocytes stimulated with LPS. Incubation with EHNA, to protect endogenously produced adenosine, or rolipram, which prevents the degradation of cAMP, similarly suppressed LPS-stimulated NO production. The addition of exogenous adenosine with EHNA or rolipram further suppressed NO production.Conclusions This study documents functional responses to adenosine by articular chondrocytes. These responses are mimicked by the A2Areceptor agonist, DPMA. Effects were enhanced by protecting adenosine using an adenosine deaminase inhibitor or by potentiating the cAMP response with rolipram. These experiments suggest that adenosine may play a physiological role in regulation of chondrocytes and that adenosine pathways could represent a novel target for therapeutic intervention.

Enhanced oral bioavailability of 2'- beta-fluoro-2',3'-dideoxyadenosine (F-ddA) through local inhibition of intestinal adenosine deaminase.

Intestinal enzyme inhibition may be an effective tool to increase the oral bioavailability of compounds that undergo first-pass intestinal metabolism. However, systemic enzyme inhibition may be undesirable and therefore should be minimized. 2-Beta-fluoro-2',3'-dideoxyadenosine (F-ddA) is an adenosine deaminase (ADA) activated prodrug of 2-beta-fluoro-2',3'-dideoxyinosine (F-ddI) with enhanced delivery to the central nervous system (CNS) that has been tested clinically for the treatment of AIDS. Unfortunately, intestinally localized ADA constitutes a formidable enzymatic barrier to the oral absorption of F-ddA. This study explores various factors involved in inhibitor selection and dosage regimen design to achieve local ADA inhibition with minimal systemic inhibition. In situ intestinal perfusions with mesenteric vein cannulation were performed in the rat ileum to determine the lumenal disappearance and venous blood appearance of F-ddA and F-ddI. Coperfusions with the ADA inhibitor erythro9-(2-hydroxy-3-nonyl)adenine [(+)-EHNA] over a range of concentrations were used to monitor inhibitor effects on F-ddA absorption and metabolism. High concentrations of EHNA in coperfusions with F-ddA completely inhibited intestinal ADA, increasing the permeability coefficient of F-ddA by nearly threefold but producing high systemic inhibition of ADA. Mathematical models were utilized to show that in full-length intestinal perfusions an optimal log mean lumenal EHNA perfusate concentration of 0.5 microg/ml could achieve an intestinal bioavailability of 80% with <20% systemic inhibition. Optimizing local enzyme inhibition may require careful selection of a suitable inhibitor, the dose of the inhibitor, and the inhibitor vs. drug absorption profiles.