Biomedical subjects

J Linden

Publications and source records attributed to J Linden.

At least 19 recordsLinked to original sources

PubMed2006

Mouse spinal cord compression injury is reduced by either activation of the adenosine A2A receptor on bone marrow-derived cells or deletion of the A2A receptor on non-bone marrow-derived cells.

Activation of the adenosine A(2A) receptor (A(2A)R) at the time of reperfusion has been shown to reduce ischemia-reperfusion injury in peripheral tissues and spinal cord. In this study we show that treating mice with the A(2A)R agonist, 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester for four days beginning before or just after the onset of reperfusion after compression-induced spinal cord injury rapidly (within 1 day) and persistently (>42 days) reduces locomotor dysfunction and spinal cord demyelination. Protection is abolished in knockout/wild type bone marrow chimera mice selectively lacking the A(2A)R only on bone marrow-derived cells but retaining receptors on other tissues including blood vessels. Paradoxically, reduced spinal cord injury is also noted in A(2A)R -/- mice, and in wild type/knockout bone marrow chimera mice selectively lacking the A(2A)R on non-bone marrow-derived cells, or in mice treated with the A(2A) antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol. The greatest protection is seen in knockout/wild type bone marrow chimera mice treated with 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-piperidine-1-carboxylic acid methyl ester, i.e. by activating the A(2A)R in mice expressing the receptor only in bone marrow-derived cells. The data suggest that inflammatory bone marrow-derived cells are the primary targets of A(2A) agonist-mediated protection. We conclude that A(2A) agonists or other interventions that inhibit inflammation during and after spinal cord ischemia may be effective in reducing spinal cord injury in patients, but excessive or prolonged stimulation of the A(2A)R may be counterproductive. It may be possible to devise strategies to produce optimal spinal cord protection by exploiting temporal differences in A(2A)R-mediated responses.

Adenosine A2 Receptor Agonists↗

PubMed2006

Characterization of [125I]ZM 241385 binding to adenosine A2A receptors in the pineal of sheep brain.

Adenosine is a ubiquitous neuromodulator and homeostatic regulator that exerts its physiologic actions through activation of A(1), A(2A), A(2B) and A(3) adenosine receptor subtypes. In the central nervous system, adenosine's action in neurons is manifested in its modulation of tonic inhibitory control. Adenosine released in the brain during hypoxia has critical depressant effects on breathing in fetal and newborn mammals, an action suggested to be mediated by A(2A) receptors in the posteromedial thalamus. In an effort to more accurately define the spatial distribution of adenosine A(2A) receptors in fetal sheep diencephalon, we have used a receptor autoradiographic technique utilizing an iodinated radioligand [(125)I]ZM 241385, which has greater sensitivity and resolution than the tritiated compound. The distribution of ligand binding sites in the fetal sheep diencephalon indicated that the highest levels of binding were in select thalamic nuclei, including those implicated in hypoxic depression of fetal breathing, and the pineal. Given the high density of labeled A(2A) receptors in the pineal, these sites were characterized more fully in homogenate radioligand binding assays. These data indicate that [(125)I]ZM 241385 binding sites display a pharmacological signature consistent with that of adenosine A(2A) receptors and are expressed at similar levels in fetal, lamb and adult ovine brain. The adenosine A(2A) receptor pharmacologic signature of the [(125)I]ZM 241385 binding site in pineal cell membranes generalized to the site characterized in membranes derived from other portions of the lamb thalamus, including the sector involved in hypoxic inhibition of fetal breathing. These results have important implications for the functional roles of adenosine A(2A) receptors in the thalamus and pineal of sheep brain.

Adenosine↗

PubMed2006

Effects of cis-9,trans-11 CLA in rats at intake levels reported for breast-fed infants.

CLA intake in exclusively breast-fed infants is close to levels found to have physiological effects in animals. However, in the majority of studies mixtures of CLA isomers have been used and the independent effects of the major CLA isomer in human milk, cis-9,trans-11 CLA, at the intake level in exclusively breast-fed infants have hardly been studied. We therefore studied the effects of cis-9,trans-11 CLA on plasma lipids and glucose, immune function, and bone metabolism in growing rats. Thirty male Sprague-Dawley rats (n = 10/group) were fed either 20 mg/kg/d cis-9,trans-11 CLA and 20 mg/kg/d sunflower oil (CLA20), 40 mg/kg/d cis-9,trans-11 CLA (CLA40), or 40 mg/kg/d sunflower oil (placebo) for 8 wk. No significant differences between groups were found in plasma lipids, glucose, insulin, C-reactive protein, or lipid peroxidation. Liver fat content was lowest in the CLA20 group. In vitro interleukin 2 (IL-2) production increased, and tumor necrosis factor alpha, IL-1beta, prostaglandin E2, and leukotriene B4 production decreased in the CLA20 group. No differences between groups were detected in IL-4, IL-6, or interferon gamma production, plasma osteocalcin, insulin-like growth factor, or urinary deoxypyridinoline crosslinks. Plasma tartrate-resistant acid phosphatase 5b activity was significantly increased in the CLA40 group. The results indicate anti-inflammatory effects and enhanced T-cell function for the CLA20 group. No adverse effects were seen in the CLA20 group, whereas indications of increased bone resorption rate were observed in the CLA40 group.

Animals↗

PubMed2006

Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia.

The aim of the current study was to clone the equine adenosine A(2A) receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A(2A)-R agonists revealed that the eA(2A)-R functionally coupled to Galpha(s) as indicated by an increase in intracellular [(3)H]cAMP upon receptor activation. Finally, NF-kappaB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-kappaB activity. These results indicate that the heterologously expressed eA(2A)-R has a pharmacological profile similar to that of other mammalian A(2A) receptors and thus can be utilized for further characterization of the eA(2A)-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.

Adenosine↗

PubMed2006

Effect of novel A2A adenosine receptor agonist ATL 313 on Clostridium difficile toxin A-induced murine ileal enteritis.

Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 microg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-alpha) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF-alpha production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.

Adenosine A2 Receptor Agonists↗

PubMed2001

Pharmacological stress myocardial perfusion imaging with the potent and selective A(2A) adenosine receptor agonists ATL193 and ATL146e administered by either intravenous infusion or bolus injection.

BACKGROUND: Adenosine (Ado) and dipyridamole are alternatives to exercise stress for myocardial perfusion imaging. Though generally safe, side effects frequently occur that cause patient discomfort and sometimes lead to premature termination of the study or require aminophylline administration. Recently, a new class of A(2A) Ado receptor agonists was synthesized. ATL193 and ATL146e are 2-propynylcyclohexyl-5'-N-ethylcarboxamido derivatives of Ado. The study goals were to evaluate the potency and selectivity of these new compounds on recombinant canine Ado receptors and to evaluate their hemodynamic properties in dogs to assess their usefulness as vasodilators for myocardial perfusion imaging. METHODS AND RESULTS: In assays of recombinant canine Ado receptors, ATL-193 and ATL-146e were highly selective for the A(2A) over the A(1) and A(3) receptors and were more potent than MRE-0470 and CGS-21680. In 16 anesthetized dogs, the agonists were administered by infusion (ATL-193; n=7 normal) or bolus injection (ATL-146e; n=9 critical left anterior descending coronary artery stenosis), and hemodynamic responses were compared with those of Ado. Both agonists produced dose-dependent coronary flow (CF) elevation without provoking the hypotension observed with Ado. After an ATL-146e bolus, the CF increase was sustained for several minutes, providing ample time for injection and myocardial uptake of (99m)Tc-sestamibi, and CF returned to baseline within 20 minutes. The CF increase was completely blocked by the selective A(2A) antagonist ZM241385 (3 microgram. kg(-1). min(-1)). CONCLUSIONS: ATL-193 and ATL-146e are highly potent and selective Ado A(2A) receptor agonists with excellent potential for use as vasodilators for myocardial perfusion imaging. An important advantage of ATL-146e is the ability to administer it by bolus injection.

Adenosine↗

PubMed2001

[3H]MRS 1754, a selective antagonist radioligand for A(2B) adenosine receptors.

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B) adenosine receptors. This is the least well-defined adenosine receptor subtype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K(D) value of 1.13 +/- 0.12 nM and a B(max) value of 10.9 +/- 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1) and A(3) adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A(2A) receptors was of low affinity (K(D) > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure-activity relationship for agonists and antagonists at A(2B) receptors. The K(i) values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a K(i) of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A(2B) receptors.

Acetamides↗

PubMed2001

Ultrastructural localization of adenosine A2A receptors suggests multiple cellular sites for modulation of GABAergic neurons in rat striatum.

Activation of adenosine A2A receptors (A2AR) has been shown to antagonize the function of D2 dopaminergic regulation of striatal gamma-aminobutyric acid (GABA)-ergic output and, thus, locomotor activity. Adenosine A2A receptor immunoreactivity (A2A-LI) has been localized to rat striatum by light microscopy by using a previously characterized human A2AR monoclonal antibody. In this study, we evaluated the localization of A2A-LI and its colocalization with GABA immunoreactivity (GABA-LI) in dorsolateral rat striatum by immunoelectron microscopy to further characterize the potential mechanism of purinergic control of striatal output. Ultrastructural analysis demonstrated A2A-LI associated with the plasma membrane and cytoplasmic membranous structures of striatal neurons. A2A-LI was prevalent in dendrites and dendritic spines ( approximately 70% of total A2A-profiles counted) and less prevalent in axons and axon terminals (23%), soma (3%), and glia (3%). Cellular elements exhibiting both A2A-LI and GABA-LI comprised 23% of the total profiles counted; colabeling was most common in dendrites. A2A-LI was observed primarily at asymmetric synapses (n = 70) (both pre- and postsynaptically but predominantly in the postsynaptic element) and less frequently at symmetric synapses (n = 17). Of the 714 A2A-immunoreactive profiles examined, 37% were apposed to GABA-labeled profiles. The most common appositions were A2A-labeled dendrites apposed to GABA-immunoreactive dendrites (n = 132), axon terminals (n = 28), and somata (n = 22) and A2A-labeled axons apposed to GABA-labeled dendrites (n = 58), axon terminals (n = 14), and somata (n = 9). Our findings suggest that adenosine may play an important role in modulating excitatory input to striatal neurons and that A2AR may modulate GABAergic signaling at several cellular sites within the rat striatum.

Animals↗

PubMed2001

Design, synthesis, and evaluation of novel A2A adenosine receptor agonists.

We have been interested in the design, synthesis, and evaluation of novel adenosine A2A agonists. Through the use of comparative molecular field analysis (CoMFA) we have generated a training model that includes 78 structurally diverse A2A agonists and correlated their affinity for isolated rat brain receptors with differences in their structural and electrostatic properties. We validated this model by predicting the activity of a test set that included 24 additional A2A agonists. Our CoMFA model, which incorporates the physiochemical property of dipole and selects against A1 receptor activity, generated a correlated final model (r2 = 0.891) that provides for enhanced A2A selectivity and predictability. Synthesis, pharmacological evaluation, and modeling of four novel ligands further validate the utility and predictive power (r2 = 0.626) of the CoMFA model.

Alkynes↗

PubMed2001

Gene dose effect reveals no Gs-coupled A2A adenosine receptor reserve in murine T-lymphocytes: studies of cells from A2A-receptor-gene-deficient mice.

Agonist binding to extracellular A2A adenosine receptors (A2ARs) inhibits the activation of virtually all tested functions of T-cells and can induce apoptosis in thymocytes. The evaluation of levels of expression of these immunosuppressive receptors is expected to clarify whether the absence of spare A2ARs (no 'receptor reserve') might be one of the mechanisms of attenuation of the effects of extracellular adenosine on T-cells. A2A transcript is found in T-cells and functional receptors can be demonstrated, but the density of receptor on T-cells is too low to be detected by radioligand binding. Studies of direct radioligand binding to murine brain with the selective A2AR agonist [3H]CGS21680 (2-(4-[(2-carboxyethyl)-phenyl]ethylamino)-5'-N-ethylcarboxamidoadenosine) established that striata levels of A2AR are virtually absent from A2A knock-out mice. Mice that are heterozygous (A2AR+/-) for the A2AR express significantly decreased levels of A2AR. To test for the presence of spare receptors in T-cells we took advantage of this gene dose effect and examined whether the decrease in the number of receptors in thymocytes from A2AR+/- mice was proportionately reflected in a decrease in the functional cAMP response of T-cells to adenosine. cAMP accumulation and apoptosis induced by adenosine and by A2AR agonist are of a lower magnitude in T-cells from A2AR+/- heterozygous mice than in T-cells from A2AR+/+ littermate control mice. These results indicate that there is no A2AR reserve in murine T-cells. Strongly decreased adenosine-triggered cAMP increases were detected in thymocytes from A2AR-/- mice, suggesting that A2B adenosine receptors cannot fully compensate for the loss of A2ARs in murine T-cells. We conclude that the number of A2ARs is the limiting factor in determining the maximal cAMP response of T-lymphocytes to extracellular adenosine, thereby minimizing the immunosuppressive effects of extracellular adenosine.

Animals↗

PubMed2001

Systemic adenosine A2A agonist ameliorates ischemic reperfusion injury in the rabbit spinal cord.

BACKGROUND: The adenosine A2A agonist ATL-146e (4-[3-[6-Amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylic acid methyl ester) has been shown to prevent reperfusion injury in multiple organ systems through inhibition of activated leukocyte-endothelial interaction. We hypothesized that systemic ATL-146e could reduce spinal cord reperfusion injury after aortic clamping. METHODS: Twenty-six rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group received intravenous ATL-146e for 3 hours during reperfusion. A second cohort received only vehicle and served as controls. Animals were assessed at 24 and 48 hours using the Tarlov (0 to 5) scoring system for hind limb function. To evaluate neuronal attrition, immunostaining of lumbar spinal cord sections was performed using anti-SMI 33 antibody against neurofilament. RESULTS: Systemic ATL-146e was tolerated without hemodynamic lability. Animals that received ATL-146e had significantly improved neurologic outcomes 24 and 48 hours after spinal cord ischemia (p < 0.001). There was preservation of neuronal architecture in the ventral horn of spinal cord sections from animals receiving ATL-146e compared with control animals. CONCLUSIONS: Intravenous ATL-146e given during reperfusion is tolerated without hemodynamic lability, and results in substantially improved spinal cord function after ischemia by preservation of ventral horn neurons.

Animals↗

PubMed2001

Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists.

Novel 2-propynylcyclohexyl-5'-N:-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-(3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl] -prop-2-ynyl)-cyclohexylmethyl ester (ATL193) were >50 x more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-N:-ethylcarboxamidoadenosine (CGS21680) for human A(2A) AR binding. Human A(2A) AR affinity for substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A(2A) AR agonist stimulation of human neutrophil [cyclic AMP](i), and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were approximately equipotent agonists of human A(3) ARs. We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP](i) and the effect of PKA inhibition on A(2A) AR agonist activity. ATL193-stimulated neutrophil [cyclic AMP](i) was blocked by antagonists with the potency order: ZM241385 (A(2A)-selective)>MRS1220 (A(3)-selective)>>N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A(2B)-selective) approximately 8-(N-methylisopropyl)amino-N(6)-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A(1)-selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89. The data indicate that activation of A(2A)ARs inhibits neutrophil oxidative activity by activating [cyclic AMP](i)/PKA.

Adenosine↗

PubMed2001

Home-based factor infusion therapy and hospitalization for bleeding complications among males with haemophilia.

Information from the medical records of 2650 US males with haemophilia living in six states was used to examine the influence of infusing factor concentrate at home (home therapy) and other variables on rates of hospitalization for a haemorrhagic bleeding complication (HBC) over a 4-year period. Bleeding complications included actual and suspected haemorrhagic events but excluded elective admissions for procedures necessitated by haemorrhage (e.g. joint synovectomy). Other risk determinants considered in the analyses included age, race, employment status, health insurance type, care received in federally funded haemophilia treatment centres (HTCs), factor deficiency type and severity, amount of factor prescribed, prophylactic treatment, and presence of inhibitors at baseline. Survival analysis methods were used to evaluate relationships between baseline risk factors and subsequent hospitalization rates. During 8708 person years (PYs) of follow-up, 808 subjects (30.5%) had a total of 1847 bleeding-related hospitalizations; an overall rate of 21.2 admissions per 100 PYs. Using proportional hazards regression to adjust for all of the studied factors, we found that home therapy use (among residents of four of the states) and care in HTCs were independently associated with a decreased risk for a first HBC. Patients who had government-sponsored health insurance or who had no insurance, those of minority race or ethnicity, those with higher levels of factor use, and those with inhibitors were at increased HBC risk. We conclude that the use of home therapy and receipt of care in HTCs are each associated with a substantially lower risk for HBC among males with haemophilia.

Adolescent↗

PubMed2001

Enhanced protection from renal ischemia-reperfusion [correction of ischemia:reperfusion] injury with A(2A)-adenosine receptor activation and PDE 4 inhibition.

BACKGROUND: We previously demonstrated in rats and mice that agonists of A(2A)-adenosine receptors (A(2A)-ARs) reduce renal injury following ischemia-reperfusion. We now extend these studies and examine the effects of ATL-146e (formerly DWH-146e), an A(2A)-AR agonist, and rolipram, a type IV phosphodiesterase (PDE 4) inhibitor, on murine renal injury following ischemia-reperfusion. METHODS: C57BL/6 mice were treated with rolipram, ATL-146e, or both compounds combined and were subjected to renal ischemia for 32 minutes and reperfusion for 24 to 48 hours. In vitro studies were performed on suspended and adhering human neutrophils. RESULTS: Continuous delivery of rolipram or ATL-146e during reperfusion reduced renal injury in a dose-dependent manner. Maximal protection was observed when ATL-146e was infused for six hours during reperfusion. Elevated plasma creatinine and myeloperoxidase activity produced by ischemia-reperfusion were reduced by rolipram (0.1 ng/kg/min) and ATL-146e (10 ng/kg/min) by up to approximately 60% and 70%, respectively. Co-infusion of both compounds produced a maximum reduction of plasma creatinine of approximately 90% and myeloperoxidase activity. In vitro studies on suspended and adhering human neutrophils demonstrated that selective stimulation of A(2A)-ARs by ATL-146e increased cAMP accumulation, reduced oxidative activity of activated neutrophils, and decreased activated neutrophil adherence. These responses were potentiated by rolipram. CONCLUSIONS: We conclude that the combined infusion of ATL-146e and rolipram leads to enhanced renal tissue protection from ischemia-reperfusion by mechanisms that may include reduced neutrophil adherence/recruitment and release of reactive oxygen species.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Adenosine analogue reduces spinal cord reperfusion injury in a time-dependent fashion.

BACKGROUND: We hypothesized that inflammation during spinal cord reperfusion worsens ischemic injury. ATL-146e, an adenosine A(2A) agonist with known anti-inflammatory properties, was used to test this hypothesis at varied intervals to determine the time course of reperfusion injury. METHODS: Forty rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group (n = 14 animals) received 0.06 microg/kg/min systemic ATL-146e over 3 hours, beginning after 30 minutes of ischemic time. A second group (n = 6 animals) received ATL-146e over 1.5 hours. A third group (n = 3 animals) received ATL-146e over 1 hour, and a fourth group (n = 17 animals) received saline solution. All animals were assessed at 48 hours for hind limb motor function (Tarlov scale, 0-5). RESULTS: Animals that received ATL-146e for 3 hours (Tarlov score, 4.3 +/- 0.22; P <.001) or 1.5 hours (Tarlov score, 2.7 +/- 0.6; P <.05) had improved neurologic outcomes compared with rabbits that received saline solution (Tarlov score, 0.6 +/- 0.29). Animals that received ATL-146e for 1 hour (Tarlov score, 0.7 +/- 0.8) were not significantly different from those animals that received saline solution. CONCLUSIONS: Systemic ATL-146e, given during reperfusion, results in time-dependent improvement in spinal cord function after ischemia. This implies that the mechanism of spinal reperfusion injury includes leukocyte-mediated inflammation at a critical post-ischemic time interval.

Adenosine↗

PubMed2001

An adenosine A2A agonist, ATL-146e, reduces paralysis and apoptosis during rabbit spinal cord reperfusion.

BACKGROUND: We hypothesized that systemic ATL-146e, an adenosine A(2A) agonist, would decrease spinal cord reperfusion inflammatory stress and inhibit apoptosis and that these effects would correlate with improved neurologic functional outcome. METHODS: Thirty rabbits underwent cross-clamping of the infrarenal aorta for 45 minutes. One group of animals (n = 14) received 0.06 microg/kg per minute of ATL-146e infused intravenously for 3 hours, beginning 15 minutes before reperfusion. A second group of animals (n = 16) underwent spinal cord ischemia with saline vehicle alone and served as ischemic controls. Animals (n = 9, 11) from each group survived for 48 hours and assessed for neurologic impairment with the Tarlov (0-5) scoring system. Four animals from each group were humanely killed at the end of the 3-hour treatment period, and the remainder killed after 48 hours' survival. In all animals, lumbar spinal cord tissue specimens were frozen for subsequent Western blot analysis of heat shock protein 70 (HSP 70), and for the p85 fragment of poly (ADP-ribose) polymerase (PARP). Neuronal viability indices were determined at 48 hours with hematoxylin and eosin staining. RESULTS: There was improvement in neurologic function in rabbits receiving ATL-146e (P <.001) compared with ischemic controls. At the end of the 3-hour treatment period there was a 46% (P <.05) decrease in HSP 70 expression in the ATL-146e group compared with the control group, but no difference in PARP expression. At 48 hours, there was no difference between control and ATL-146e groups in HSP 70 expression, but there was a 65% (P <.05) reduction in PARP in the spinal cords of animals that had received ATL-146e. There was a significant improvement in neuronal viability indices in animals receiving ATL-146e compared with ischemic controls (P <.05). CONCLUSIONS: Systemic ATL-146e infusion during reperfusion after spinal cord ischemia results in preservation of hindlimb motor function. There is evidence of decreased spinal cord inflammatory stress immediately after treatment with ATL-146e as indicated by reduced HSP 70 induction. Treatment with ATL-146e is associated with a reduction in neuronal apoptosis as suggested by a substantial decrease in the fragmentation of PARP at 48 hours. These results suggest that inflammation during reperfusion and subsequent apoptosis contribute to paralysis after restoration of blood flow to the ischemic spinal cord.

Animals↗

PubMed2001

A3 adenosine receptor activation triggers phosphorylation of protein kinase B and protects rat basophilic leukemia 2H3 mast cells from apoptosis.

Adenosine accumulates to high levels in inflamed or ischemic tissues and activates A3 adenosine receptors (ARs) on mast cells to trigger degranulation. Here we show that stimulation of rat basophilic leukemia (RBL)-2H3 mast-like cells with the A3 AR agonists N6-(3-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA; 10 nM) or inosine (10 microM) stimulates phosphorylation of protein kinase B (Akt). IB-MECA (1 microM) also causes a >50% reduction in apoptosis caused by exposure of RBL-2H3 cells to UV light. Akt phosphorylation is not stimulated by 100 nM N6-cyclopentyladenosine (A1-selective) or CGS21680 (A2A-selective) and is absent in cells pretreated with wortmannin or pertussis toxin. The KI values of the AR antagonists BW-1433 and 8-sulfophenyltheophylline (8-SPT) were determined in radioligand binding assays for all four subtypes of rat ARs: BW-1433 (A1, 5.8 +/- 1.0 nM; A2A, 240 +/- 37; A2B, 30 +/- 10; A3, 12,300 +/- 3, 700); 8-SPT (A1, 3.2 +/- 1.2 microM; A2A, 57 +/- 4; A2), 2.2 +/- 0.8; A3, >100). BW-1433 and the A3-selective antagonist MRS1523 (5 microM), but not 8-SPT (100 microM), block IB-MECA-induced protection from apoptosis, confirming the A3 AR as the mediator of the antiapoptotic response. The data suggest that adenosine and inosine activate Gi-coupled A3 ARs to protect mast cells from apoptosis by a pathway involving the betagamma subunits of Gi, phosphatidylinositol 3-kinase beta, and Akt. We speculate that activation of A3 ARs on mast cells or other cells that express A3 ARs (e.g., eosinophils) may facilitate their survival and accumulation in inflamed tissues.

Adenosine↗

PubMed2001

Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection.

Adenosine accumulation during ischemia and inflammation protects tissues from injury. In ischemic tissues adenosine accumulates due to inhibition of adenosine kinase, and in inflamed tissues adenosine is formed from adenine nucleotides that are released from many cells including platelets, mast cells, nerves, and endothelium. Nucleotides are rapidly converted to adenosine by a family of ecto-nucleotidases including CD39 and CD73. Activation of A(1) and possibly A(3) adenosine receptors (ARs) protects heart and other tissues by preconditioning through a pathway including protein kinase C and mitochondrial K(ATP) channels. Activation of A(2A) receptors limits reperfusion injury by inhibiting inflammatory processes in neutrophils, platelets, macrophages and T cells. Adenosine produces proinflammatory responses mediated by receptors that vary among species; A(3) and A(2B) receptors mediate degranulation of rodent and human or canine mast cells, respectively. Novel adenosine receptor subtype-selective ligands have recently been developed. These include MRS1754 (A(2B) blocker), MRS1220 (A(3) blocker), MRE 3008F20 (human A(3) blocker), MRS1523 (rat A(3) blocker), and ATL146e (A(2A) agonist). These new pharmacologic tools will help investigators to sort out how adenosine protects tissues from injury and to identify new therapeutic agents that hold promise for the treatment of inflammatory and ischemic diseases.

Adenosine↗