R-phenylisopropyladenosine Research Articles

Cantharidin and sodium fluoride attenuate the negative inotropic effect of the A1-adenosine receptor agonist N6-(R)-phenylisopropyl adenosine in isolated human atria.

Cantharidin and sodium fluoride inhibit the activity of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) and increase the force of contraction in human atrial preparations. R-phenylisopropyl adenosine (R-PIA) acts as an agonist at A1-adenosine receptors. R-PIA exerts a negative inotropic effect on human atria. The effect of R-PIA-and its various manifestations-are currently explained as a function of the inhibition of sarcolemmal adenylyl cyclase activity and/or opening of sarcolemmal potassium channels. We hypothesise that cantharidin and sodium fluoride may attenuate the negative inotropic effect of R-PIA. During open heart surgery, trabeculae carneae from the right atrium were obtained for human atrial preparations (HAPs). These trabeculae were mounted in organ baths and electrically stimulated at 1Hz. Furthermore, we studied isolated electrically stimulated left atrial (LA) preparations from female wild-type mice (CD1). The force of contraction was recorded under isometric conditions. R-PIA (1µM) exerted a rapid negative inotropic effect in the HAPs and mice LA preparations. These negative inotropic effects of R-PIA were attenuated by pre-incubation for 30min with 100-µM cantharidin in HAPs, but not in mice LA preparations. Adenosine signals via A1 receptors in a species-specific pathway in mammalian atria. We postulate that R-PIA, at least in part, exerts a negative inotropic effect via activation of serine/threonine phosphatases in the human atrium.

A nonolfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features.

Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea.

Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Adenosine Triphosphate–Sensitive Potassium Channel Blockers Attenuate the Antiallodynic Effect of R-PIA in Neuropathic Rats

Nerve injury can generate neuropathic pain. The accompanying mechanical allodynia may be reduced by the intrathecal administration of adenosine. The neuroprotective effects of adenosine are mediated by the adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channel. We assessed the relationship between the adenosine A1 receptor agonist, N⁶-(R)-phenylisopropyl adenosine (R-PIA), and K(ATP) channels to determine whether the antiallodynic effects of R-PIA are also mediated through K(ATP) channels in a rat nerve ligation injury model of neuropathic pain. Mechanical allodynia was induced by tight ligation of the left lumbar fifth and sixth spinal nerves. Mechanical allodynia in the left hindpaw was evaluated using von Frey filaments to measure withdrawal thresholds. R-PIA (0.5, 1, or 2 μg) was administered intrathecally to induce antiallodynia. We assessed whether pretreatment with the K(ATP) channel blockers glibenclamide or 5-hydroxydecanoate reversed the antiallodynic effect of R-PIA. Also, we evaluated whether diazoxide, a K(ATP) channel opener, had an antiallodynic effect and promoted the antiallodynic effect of R-PIA. Lastly, we investigated whether the voltage-activated K channel blocker 4-aminopyridine attenuated the effect of R-PIA. Intrathecal R-PIA produced maximal antiallodynia at 2 μg (P < 0.05). Intrathecal pretreatment with glibenclamide and intraperitoneal pretreatment 5-hydroxydecanoate significantly reduced the antiallodynic effect of R-PIA. Diazoxide produced an antiallodynic effect and also enhanced the antiallodynic action of R-PIA. 4-Aminopyridine had no effect on the antiallodynic action of R-PIA. The antiallodynic effects of adenosine A1 receptor stimulation may be related to K(ATP) channel activity in a rat model of nerve ligation injury.

Evidence for a Role of Cyclic AMP and Endothelium in Rat Aortic Relaxation Induced by R-PIA

It is known that vasodilator effects of adenosine are mediated by A 2 receptors in various animal species. Never- theless, in several blood vessels there is evidence for vasodilator responses to some adenosine analogues, which are resis- tant to the antagonists of the different adenosine receptor subtypes, suggesting that they are not mediated by any of the known adenosine receptors. There are contradictory reports about the effects produced by adenosine and its analogues on aortic vasodilation. The results obtained in the present study dealing with rat aortic rings and the relaxant effects induced by the selective adenosine A 1 receptor agonist N 6 -R-phenylisopropyladenosine (R-PIA) provide evidence that cyclic AMP and the presence of endothelium are possibly involved in rat aortic relaxation induced by this adenosine analogue.

Effects of Conformational Restriction of 2-Amino-3-benzoylthiophenes on A1 Adenosine Receptor Modulation

2-Amino-3-benzoylthiophenes (2A3BTs) have been widely reported to act as allosteric enhancers (AEs) at the A(1) adenosine receptor (A(1)AR). Herein we describe the synthesis of a series of 1-aminoindeno[1,2-c]thiophen-8-ones and a series of (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones as conformationally rigid analogues of the 2A3BTs. These compounds were screened using a functional assay of A(1)AR-mediated phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in intact Chinese hamster ovary (CHO) cells to identify both potential agonistic effects as well as the ability to allosterically modulate the activity of the orthosteric agonist, N(6)-(R-phenylisopropyl)adenosine (R-PIA). All of the 1-aminoindeno[1,2-c]thiophen-8-ones (14a-c and 17a-f) proved either to be inactive or behaved as antagonists in the functional assay. However, the (2-aminoindeno[2,1-b]thiophen-3-yl)(phenyl)methanones with para-chloro substitution (compounds 25b, 25d, and 25f) did significantly augment the R-PIA response, indicating a positive allosteric effect.

Role of β-arrestin1/ERK MAP kinase pathway in regulating adenosine A1 receptor desensitization and recovery

Exposure of cells to adenosine receptor (AR) agonists leads to receptor uncoupling from G proteins and downregulation of the A(1)AR. The receptor levels on the cell surface generally recover on withdrawal of the agonist, because of either translocation of the sequestered A(1)AR back to plasma membrane or de novo synthesis of A(1)AR. To examine the mechanism(s) underlying A(1)AR downregulation and recovery, we treated ductus deferens tumor (DDT(1) MF-2) cells with the agonist R-phenylisopropyladenosine (R-PIA) and showed a decrease in membrane A(1)AR levels by 24 h, which was associated with an unexpected 11-fold increase in A(1)AR mRNA. Acute exposure of these cells to R-PIA resulted in a rapid translocation of beta-arrestin1 to the plasma membrane. Knockdown of beta-arrestin1 by short interfering RNA (siRNA) blocked R-PIA-mediated downregulation of the A(1)AR, suppressed R-PIA-dependent ERK1/2 and activator protein-1 (AP-1) activity, and reduced the induction of A(1)AR mRNA. Withdrawal of the agonist after a 24-h exposure resulted in rapid recovery of plasma membrane A(1)AR. This was dependent on the de novo protein synthesis and on the activity of ERK1/2 but independent of beta-arrestin1 and nuclear factor-kappaB. Together, these data suggest that exposure to A(1)AR agonist stimulates ERK1/2 activity via beta-arrestin1, which subserves receptor uncoupling and downregulation, in addition to the induction of A(1)AR expression. We propose that such a pathway ensures both the termination of the agonist signal and recovery by priming the cell for rapid de novo synthesis of A(1)AR once the drug is terminated.

Effect of chronic gestational treatment with the adenosine A1 receptor agonist R‐phenylisopropyladenosine on metabotropic glutamate receptors/phospholipase C pathway in maternal and fetal brain

Pregnant Wistar rats were orally treated with the adenosine receptor agonist R-phenylisopropyladenosine (R-PIA) throughout the gestational period, and the status of the metabotropic glutamate (mGlu) receptor/phospholipase C transduction pathway from maternal and fetal brain was analyzed. In mothers' brains, radioligand binding assays revealed a significant decrease in the Bmax value, without any significant alteration in Kd value. Similar results were observed in the steady-state level of mGlu(1) and mGlu(5) receptors assayed by Western blot, suggesting that both receptor subtypes were modulated by chronic R-PIA treatment. mRNA coding mGlu(1) or mGlu(5) receptors was not altered, suggesting a posttranscriptional modulation as a possible mechanism of the loss of mGlu(1) and mGlu(5) receptors at the membrane surface. Moreover, phospholipase C stimulated by (R,S)-3,5-dihydroxyphenylglycine (DHPG), a selective agonist of group I mGlu receptors, was also significantly decreased after R-PIA treatment, suggesting a heterologous desensitization of group I mGlu/PLC pathway in maternal brain. Western blot and RT-PCR assays showed that neither alphaG(q/11) nor PLCbeta(1) was affected by R-PIA treatment. In fetal brain, no significant differences in mGlu receptors/PLC transduction pathway were observed after R-PIA treatment. These results suggest that chronic R-PIA intake during pregnancy modulates group I mGlu receptor signalling pathway in maternal brain, promoting a down-regulation of mGlu(1) and mGlu(5) receptors and a heterologous desensitization of the mGlu/PLC system.

The Mechanism of R-PIA Induced Mechanical Antiallodynia in a Peripheral Neuropathic Rat

The injury by a nerve ligation produces a mechanical allodynia. The antiallodynic effect resulted from intrathecal administration of the adenosine analogues has been well known. ATP-sensitive potassium channel blockers have been known to reverse the effect of some antinociceptive drugs in animal and human studies. Therefore, the present study is to assess the relationship between antiallodynic effect of N6-(R)-phenylisopropyl adenosine (R-PIA) and mitochondrial ATP-sensitive potassium (mKATP) channel in a neuropathic pain model. Methods: Allodynia was induced in male Sprague Dawley rats by the tight ligation of the left lumbar 5th and 6th spinal nerves. We tested the mechanical allodynia by pricking von Frey filaments to the left hind paw and assessed withdrawal thresholds of paw with up-down method. For the estimation of the antiallodynic effect of R-PIA, R-PIA (0.5, 1 and 2μg) or saline were administered intrathecally.To investigate the reversal effect on antiallodynic effect of R-PIA, variable amounts of 5-hydroxydecanoate (5-HD, 20, 30 and 40 mg), mKATP channel blocker were administered intraperitoneally at 5 min prior to the intrathecal injection of 2μg of R-PIA, and the degree of allodynia was assessed. Results: The paw withdrawal threshold was gradually increased with increased dose of R-PIA and reached the maximum level with 2μg R-PIA (P ＜ 0.05). The increase of paw withdrawal threshold with 2μg R-PIA was significantly reversed dose-dependently by intraperitoneal pretreatment of 20, 30 and 40 mg/kg 5-HD (P ＜ 0.05). Conclusions: In our results, intraperitoneal injection of 5-HD before intrathecal injection of R-PIA had reversed the antiallodynic effect of R-PIA. This results suggest that the mechanism of mechanical antiallodynia induced by intrathecal injection of R-PIA may relate with the mKATP channel in a rat model of nerve ligation injury.

Radioligand binding and functional responses of ligands for human recombinant adenosine A3 receptors

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.

Effects of A 1 adenosine receptor stimulation on the expression of genes involved in calcium homeostasis

We investigated whether A 1 adenosine receptor stimulation affects expression of genes involved in calcium homeostasis, including sarcolemmal L-type Ca 2+ channel, Na +/Ca 2+ exchanger, sarcoplasmic reticulum (SR) Ca 2+-ATPase, phospholamban, or ryanodine receptor. Three models of A 1 stimulation were used: i) an acute model, i.e. isolated perfused rat hearts treated for 120 min with 15 nM R-phenylisopropyladenosine (R-PIA), an A 1 receptor agonist; ii) a subacute model, i.e. rats treated with 1.5 mg/kg R-PIA e.v. and sacrificed after 24 h; iii) a transgenic model, i.e. mice overexpressing A 1 adenosine receptors. In all models gene expression was determined by RT-PCR, and oxalate-supported Ca 2+ uptake, representing SR Ca 2+ uptake, was measured in the crude homogenate. Significant increase in the expression of the phospholamban gene was observed in each model of A 1 stimulation, while the expression of the other four genes was not significantly modified. In the acute model, SR Ca 2+ uptake was unaffected, however in the subacute and transgenic models uptake rate was significantly reduced. In parallel experiments, hearts obtained from the subacute model demonstrated a significant reduction in irreversible tissue injury from 30 min of ischemia and 120 min of reperfusion. Increased resistance to ischemia has already been reported also in our transgenic model. In conclusion, A 1 adenosine receptor stimulation up-regulates phospholamban gene expression, which leads within 24 h to a reduced rate of SR Ca 2+ uptake. Changes in Ca 2+ homeostasis might contribute to the delayed cardioprotective effect of adenosine.

Interaction of exercise and adenosine receptor agonist and antagonist on rat heart antioxidant defense system

This study investigated the interactive effects of acute exercise and adenosine receptor agonist and antagonist on antioxidant enzyme activities, glutathione and lipid peroxidation in the heart of the rat. Male Fisher-344 rats were divided into six groups and treated as follows: (1) saline control; (2) acute exercise (100% VO2max); (3) R-Phenyl isopropyl adenosine (R-PIA) (3.46 micromol/kg, i.p.); (4) theophylline (1.70 micromol/kg, i.p.) plus acute exercise; (5) theophylline plus R-PIA; and (6) theophylline. Animals were sacrificed 1 h after treatments; hearts were isolated and analyzed. The results show that acute exercise as well as adenosine receptor agonist R-PIA significantly enhanced cardiac superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) activity by 36-135% and 16-51%, respectively. Adenosine receptor agonist R-PIA significantly decreased cardiac GSSG concentration and enhanced GSH/GSSG ratio by 22 and 30%, respectively. Whereas theophylline treatment blocked the activation of antioxidant enzyme activities enhanced by acute exercise and R-PIA. Theophylline treatment significantly increased lipid peroxidation by 43% in the heart of exercised rats. The study concluded that the adenosine receptors are involved in the upregulation of cardiac antioxidant defense system and attenuation of lipid peroxidation due to acute exercise in rats.

Effects of adenosine agonist R-phenylisopropyl-adenosine on halothane anesthesia and antinociception in rats1

To investigate the antinociceptive effect of adenosine agonist R-phenylisopropyl-adenosine (R-PIA) given to conscious rats by intracerebroventricular (ICV) and intrathecal (IT), and identify the effect of R-PIA on minimum alveolar concentration (MAC) of halothane with pretreatment of A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or K+ channel blocker 4-aminopyridine (4-AP). Sprague-Dawley rats were implanted with 24-gauge stainless steel guide cannula using stereotaxic apparatus and ICV method, and an IT catheter (PE-10, 8.5 cm) was inserted into the lumbar subarachnoid space, while the rats were under pentobarbital anesthesia. After one week of recovery from surgery, rats were randomly assigned to one of the following protocols: MAC of halothane, or tail-flick latency. All measurements were performed after R-PIA (0.8-2.0 microg) microinjection into ICV and IT with or without pretreatment of DPCPX or 4-AP. Microinjection of adenosine agonist R-PIA in doses of 0.8-2.0 microg into ICV and IT produced a significant dose- and time-dependent antinociceptive action as reflected by increasing latency times and ICV administration of adenosine agonist R-PIA (0.8 microg) reducing halothane anesthetic requirements (by 29%). The antinociception and reducing halothane requirements effected by adenosine agonist R-PIA was abolished by DPCPX and 4-AP. ICV and IT administration of adenosine agonist R-PIA produced an antinociceptive effect in a dose-dependent manner and decreased halothane MAC with painful stimulation through activation of A1 receptor subtype, and the underlying mechanism involves K+ channel activation.

Phospholipase C is involved in the adenosine-activated signal transduction pathway conferring protection against iodoacetic acid-induced injury in primary rat neuronal cultures

We have demonstrated before that exposure of neuronal cultures to poisoning by iodoacetic acid, followed by “reperfusion” (iodoacetate-“reperfusion” insult; IAA-R insult), results in severe cytotoxicity. This insult was found to be associated with ATP depletion and generation of reactive oxygen species. The cultured neurons could be protected against the insult by activation of the adenosine A 1 receptors and by presence of antioxidants. Previous studies in our laboratory demonstrated that the adenosine-activated signal transduction pathway (Ado-STP) conferring protection against the IAA-R insult, involves activation of protein kinase C-epsilon (PKCɛ) and opening of ATP sensitive potassium (K ATP) channels. In this respect, the adenosine-activated protective mechanism against the IAA-R insult is similar to the Ado-STP in the neurons and in cardiomyocytes against ischemia-reperfusion injury. Phospholipase C (PLC) is an additional component demonstrated recently to participate in the myocardial Ado-STP protecting against ischemia-reperfusion. Here we provide proof for the involvement of PLC also in the neuronal Ado-STP protecting against the IAA-R insult. Primary rat neuronal cultures were exposed to the IAA-R insult. The neurons could be protected against this insult by activation of the adenosine A 1 receptors by N 6-(R)-phenylisopropyladenosine (R-PIA), a specific A 1 adenosine receptor agonist. Exposure of the cultures to the PLC inhibitor U73122, abrogated the protection. The exposure of the cultures to R-PIA was found to enhance PLC activity, an effect that could be abrogated by presence of U73122. The R-PIA-induced increase in PLC activity was short-lived, in the range of minutes. These results demonstrate that activation of PLC is a vital step in the neuronal protective Ado-STP, but that it does not contribute directly to the relatively long time window of the protection signal shown previously to characterize the neuronal mechanism. The results also support the suggestion that the Ado-STP protecting against the IAA-R insult and that protecting against ischemia-reperfusion may represent the same mechanism.

Protection against cisplatin ototoxicity by adenosine agonists

Cisplatin is a commonly used antineoplastic agent that causes ototoxicity through the formation of reactive oxygen species (ROS). Previous studies have shown that cisplatin causes an upregulation of A 1 adenosine receptor (A 1AR) in the cochlea, and that application of the adenosine agonist, R-phenylisopropyladenosine ( R-PIA), to the round window (RW) results in significant increases in cochlear glutathione peroxidase and superoxide dismutase. These data suggest that adenosine receptors (ARs) are an important part of the cytoprotective system of the cochlea in response to oxidative stress. The purpose of the current study was to investigate the effect of various adenosine agonists on cisplatin ototoxicity using RW application. Auditory brainstem response (ABR) thresholds were recorded in anesthetized chinchillas at 1, 2, 4, 8 and 16 kHz. The auditory bullae were surgically opened, and 1 mM R-PIA, 10 μM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)/ R-PIA (1 mM) cocktail, 100 μM 2-chloro- N-cyclopentyladenosine (CCPA), 2-[4-(2- p-carboxy-ethyl)phenylamino]-5′- N-ethylcarboxamidoadenosine (CGS) or vehicle were applied to the RW. After 90 min, the remaining solution was removed and cisplatin was applied to the RW. The bullae were closed and the animals recovered for 72 h, after which, follow-up ABRs were performed. Cochleae were harvested for scanning electron microscopy (SEM) and for lipid peroxides. Pre-administration of the A 1AR agonists R-PIA or CCPA significantly reduced cisplatin-induced threshold changes at all but the highest test frequency. In addition, A 1AR agonists protected against cisplatin-induced hair cell damage and significantly reduced cisplatin-induced lipid peroxidation. Co-administration of the A 1AR antagonist, DPCPX, completely reversed the protective effects of R-PIA. In contrast, pretreatment with CGS-21680, an A 2A adenosine receptor (A 2AAR) agonist, significantly increased cisplatin-induced threshold changes. Our findings are consistent with the notion that the A 1AR contributes significantly to cytoprotection in the cochlea, and thereby protects against hearing loss.

The protective effect of Allium sativum L. clove aqueous and methanolic extracts against hypoxia-induced lethality in mice.

The antihypoxic activity of Allium sativum clove (garlic) aqueous and methanolic extracts was studied in mice. The extracts of garlic showed that the antihypoxic effect was dose-dependent. The minimum effective doses of aqueous and methanolic extracts were 0.2 g/kg and 5.12 g/kg, respectively. Phenytoin, 50 mg/kg, and R-phenylisopropyladenosine (R-PIA), 1.6 mg/kg (R-PIA) as positive controls increased survival time up to 52.5 +/- 2.9 min and 120.5 +/- 6 min, respectively, compared to normal saline (34.73 +/- 0.71 min). The high doses of aqueous (16.9 g/kg) and methanolic (12.8 g/kg) extracts increased survival time up to 73.17 +/- 4.9 and 68.41 +/- 3.7, respectively. These results indicated that the extracts of A. sativum cloves have a protective effect against hypoxia-induced lethality in mice.

Decreased inflammatory pain due to reduced carrageenan-induced inflammation in mice lacking adenosine A 3 receptors

Mice with a targeted disruption of adenosine A 3 receptor (A 3AR) gene were assessed for their nociceptive threshold and for their localized inflammatory response following carrageenan injected into the hindpaw. Under basal conditions no difference was seen between A 3AR knock-out (A 3AR −/−) and wild-type (A 3AR +/+) mice in nociceptive response to mechanical or heat stimuli. The antinociceptive response to the intrathecal adenosine analogue R-phenylisopropyl adenosine (R-PIA) was also unchanged in the A 3AR −/− mice. In contrast, heat hyperalgesia, plasma extravasation and edema following carrageenan-induced inflammation in the hind paw were significantly reduced in A 3AR −/− mice compared to the A 3AR +/+ controls. Thus, mice lacking A 3AR had deficits in generating the localized inflammatory response to carrageenan, supporting a pro-inflammatory role of A 3AR in peripheral tissues. However, no evidence for a role of A 3AR in nociception and the antinociceptive effect of R-PIA was found.

Regulation of Renal A1 Adenosine Receptors in vivo

We have compared renal A₁ adenosine receptor (AR) regulations in rats after chronic agonist and antagonist treatments. In one group, R-phenylisopropyladenosine (R-PIA), a selective A₁ AR agonist, was infused subcutaneously for 7 days. Another group was fed theophylline, a non-selective AR antagonist, for 2 weeks. Renal cortex membrane A₁ AR binding with 1,3-[³H]-dipropyl-8-cyclopentylxanthine demonstrated ∼40% reduction in the B_max for the R-PIA group without any changes in the K_d values. Neither the B_max nor the K_d changed following chronic theophylline treatment. Renal cortex G_iα-proteins from the R-PIA treated rats decreased by ∼30%. Renal G_iα levels did not change in theophylline-treated rats. Consistent with the A₁ AR desensitization, R-PIA-treated rats had significantly higher basal renin release and showed attenuated A₁ AR-mediated inhibition of renin release. These data suggest that prolonged A₁ AR stimulation results in downregulation of renal A₁ ARs and G_iα, accompanied by desensitization of A₁ AR-mediated inhibitory effects on renin release. Unlike cardiac and brain A₁ ARs, renal A₁ receptors are not subject to up-regulation following chronic antagonist treatment.

Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

Tissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5'-N'-methyluronamide (IB-MECA) inhibited LPS-induced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A 1 receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at −60 mV that was inhibited by pre-application of barium or Rp-adenosine 3′, 5′-cyclic monophosphothioate triethylamine. In contrast, the A 2a receptor agonist 2-[ p-(2-carboxyethyl)phenylethylamino]-5′- N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at −60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.