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Background Studies investigating the correlation between spinal adenosine A1 receptors and vincristine-induced peripheral neuropathy (VIPN) are limited. This study explored the role of intrathecal N6-(2-phenylisopropyl)-adenosine R-(-)isomer (R-PIA) in the rat model of VIPN. Methods Vincristine (100 μg/kg) was intraperitoneally administered for 10 days (two 5-day cycles with a 2-day pause) and VIPN was induced in rats. Pain was assessed by evaluating mechanical hyperalgesia, mechanical dynamic allodynia, thermal hyperalgesia, cold allodynia, and mechanical static allodynia. Biochemically, tumor necrosis factor-alpha (TNF-α) level and myeloperoxidase (MPO) activity were measured in the tissue from beneath the sciatic nerve. Results Vincristine administration resulted in the development of cold allodynia, mechanical hyperalgesia, thermal hyperalgesia, mechanical dynamic allodynia, and mechanical static allodynia. Intrathecally administered R-PIA (1.0 and 3.0 μg/10 μl) reversed vincristine-induced neuropathic pain (cold and mechanical static allodynia). The attenuating effect peaked 15 min after intrathecal administration of R-PIA after which it decreased until 180 min. However, pretreatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 μg/10 μl) 15 min before intrathecal R-PIA administration significantly attenuated the antiallodynic effect of R-PIA. This antiallodynic effect of intrathecal R-PIA may be mediated through adenosine A1 receptors in the spinal cord. Intrathecally administered R-PIA also attenuated vincristine-induced increases in TNF-α level and MPO activity. However, pretreatment with intrathecal DPCPX significantly reversed this attenuation. Conclusions These results suggest that intrathecally administered R-PIA attenuates cold and mechanical static allodynia in a rat model of VIPN, partially due to its anti-inflammatory actions.

Introduction: It is well known that exposure to different stressors caused a sequence of biochemical, physiological and behavioural changes including analgesia which is realised by activating multiple endogenous pain inhibitory systems. Although the existing data reveal the important role of the adenosine modulatory system in the processing of nociceptive information, scarce data are available for its role in stress-induced changes in nociception. Materials and Methods: Male ICR mice – controls and exposed to acute (120 minutes) restraint stress (RS) were used; Acetic acid-induced nociception (writhing test); Adenosine A1 receptor agonist N6-R phenylisopropil-adenosine (R-PIA, 0.5 mg/kg, intraperitoneally, IP), nonselective adenosine receptor antagonist theophylline (acute 75mg/kg, IP and chronic 75 mg/kg/day/14 days, IP). Results: RS and R-PIA decreased the visceral nociception in mice. Acute and chronic treatment with theophylline did not influence the pain reactions neither in nonstressed nor in stressed animals. R-PIA administered before restraint stress fully reversed stress–induced antinociception. Single dose of theophylline antagonized the effects of R-PIA both in nonstressed and stressed animals. Chronic theophylline did not antagonize the antinociceptive effect of R-PIA, but abolished R-PIA induced reversion of SIA. Conclusion: Endogenous adenosine is not able to change the RS-induced visceral antinociception, as theophylline did not show any effects on the stress. However, adenosine A1 receptors are involved both in the antinociception in the healthy mice and RS-induced antinociception, perhaps by different mechanism.

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out ( Adora1 −/− , Adora3 −/− ) mice. Confirming prior data, stimulation of the A 3 adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H 1 receptors. In contrast, A 1 AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A 1 AR agonists, including N 6 -cyclopentyladenosine (CPA), N 6 -cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A 1 AR and A 3 AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy- N 6 - endo -norbornyladenosine], or using Adora3 −/− mice allowed selective stimulation of A 1 AR. AMP-stimulated hypothermia can occur independently of A 1 AR, A 3 AR, and mast cells. A 1 AR and A 3 AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A 1 AR nor A 3 AR was required for fasting-induced torpor. A 1 AR and A 3 AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms. [ABSTRACT FROM AUTHOR]

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 µM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

G protein-coupled A2B adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A2BAR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A2BAR. In cAMP accumulation assays, both 5′-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5′-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A2BAR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A2BAR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative ‘operational model' characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N6-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A2BAR antagonist in MIN-6 mouse pancreatic β cells expressing low A2BAR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target. [ABSTRACT FROM AUTHOR]

Involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations encountered in newborns following an in utero caffeine exposure has been investigated on pontomedullary–spinal cord, caudal pons–medullary–spinal cord and medullary–spinal cord preparations isolated from newborn rats. According to the drinking fluid of dams (tap water or 0.02% caffeine), two groups of preparations were distinguished, no-caffeine and caffeine. In the no-caffeine group, adenosine A1 receptor activation induces a decrease in respiratory frequency (Rf) in caudal pons–medullary–spinal cord and medullary–spinal cord preparations whereas, in presence of the rostral pons, an increase is observed. A parallel Fos detection indicates that this discrepancy may be due to the excitatory action of the medial parabrachial nucleus at the rostral pontine level that surpasses inhibitory influence of the adenosine A1 receptor activation at the medullary level particularly in the ventrolateral reticular nucleus of the medulla. In caffeine group, an increase in the baseline Rf in presence of the pons and no change in medullary–spinal cord preparations have been observed. Depending on Fos detection, we assume that the medial parabrachial nucleus is the main region involved in the exaggeration of Rf. Moreover, adenosine A1 receptor activation was modified by in utero caffeine exposure with an overcharge of the Rf increase in pontomedullary–spinal cord preparations and an exaggeration of the Rf decrease in medullary–spinal cord preparations. Based on Fos detection, we link the overcharge in Rf of pontomedullary spinal cord preparations to an increase in the medial parabrachial nucleus neuronal activity. Similarly, exaggeration of Rf decrease observed without the pons is linked with a decrease in activity of the ventrolateral reticular neurons.This study brings evidence for the involvement of adenosinergic A1 systems in the occurrence of respiratory perturbations in newborns following in utero caffeine exposure and the importance of rostral pons in the adenosinergic A1 modulation of the respiratory control. [Copyright &y& Elsevier]

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 A1 adenosine receptor (A1AR) 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 A1AR agonists R-PIA or CCPA significantly reduced cisplatin-induced threshold changes at all but the highest test frequency. In addition, A1AR agonists protected against cisplatin-induced hair cell damage and significantly reduced cisplatin-induced lipid peroxidation. Co-administration of the A1AR antagonist, DPCPX, completely reversed the protective effects of R-PIA. In contrast, pretreatment with CGS-21680, an A2A adenosine receptor (A2AAR) agonist, significantly increased cisplatin-induced threshold changes. Our findings are consistent with the notion that the A1AR contributes significantly to cytoprotection in the cochlea, and thereby protects against hearing loss. [Copyright &y& Elsevier]

The effects of adenosine A and A receptors on catalepsy were studied in mice. The adenosine agonists 5-N′-ethylcarboxamide-adenosine (NECA), N-phenylisopropyladenosine (PIA) and N-cyclohexyladenosine (CHA) induced dose dependent catalepsy. The A adenosine antagonist 8-phenyltheophylline (8-PT) potentiated catalepsy induced by NECA, R-PIA and CHA. However, theophylline did not potentiate but inhibited the responses induced by NECA, R-PIA and CHA. Neither 8-PT nor theophylline alone has any effect on catalepsy in mice. It is concluded that catalepsy induced by the adenosine agonists may be due to A receptor stimulation and that the A antagonism may potentiate the response. [ABSTRACT FROM AUTHOR]

Previous studies in pigs have shown that the A1-adenosine receptor agonist, R-PIA, is a potent antifibrillatory agent during myocardial ischaemia and that this effect can be overriden by atrial pacing. However, reports of A1 -adenosine receptor down-regulation following chronic exposure to A1 -receptor agonists suggest that this may limit their use as potential antiarrhythmic therapy. The acute and chronic effects of R-PIA were examined on ventricular arrhythmias and hemodynamics in Langendorff-perfused rat isolated hearts subjected to acute regional myocardial ischemia in an attempt to confirm the heart rate dependency of the antifibrillatory mechanism of R-PIA and to assess the effects of chronic treatment on this protection. Acute challenge with R-PIA (10-10 to 5 × 10-8 M; n = 10 for all groups) produced a concentration-dependent bradycardia prior to coronary occlusion. Coronary artery occlusion in control hearts (n= 20) resulted in an immediate increase in perfusion pressure, from61 ± 6 to 87 ± 7 mmHg within 5 minutes, followed by a gradual continued rise reaching a maximum of 123 ± 9 mmHg by the end of the 30-minute experimental period. R-PIA significantly attenuated the sustained increase in perfusion pressure in a non–concentration-dependent manner. A concentration of 10-10 M R-PIA had no effect on the incidence of ventricular fibrillation (VF), while all higher concentrations reduced the incidence of VF to a similar degree (from 60% in controls to10%, 20%, 10%, and 0% with 10-9, 5× 10-9, 10-88, and 5 × 10-8 M R-PIA, respectively). R-PIA also reduced the total ventricular premature beat (VPB) count, but in a concentration-dependent manner. Chronic treatment of the rats with R-PIA (50 µg/kg ip, bd; n = 10) for 7 days caused a significant attenuation of the bradycardic response to acute perfusion in vitro with R-PIA (10-8 M) and abolished the attenuation of the sustained rise in perfusion pressure during myocardial ischemia. The antifibrillatory effect of R-PIA, however, was unaffected by chronic pretreatment (VF incidence 0% vs. 70% in control hearts from rats that had been given chronic saline ip injections n = 10; P < 0.01). These results suggest that the bradycardia induced by acute R-PIA may not be the mechanism underlying the antifibrillatory effect of R-PIA, while the reduction in the less severe arrhythmias is heart rate dependent. Furthermore, while chronic treatment with R-PIA significantly attenuates the heart rate response to acute R-PIA challenge, the antifibrillatory properties remain intact [ABSTRACT FROM AUTHOR]

Objective: Adenosine has previously been shown to protect against ischaemia induced ventricular arrhythmias. The aim of this study was to determine whether stimulation of A1 adenosine receptors with a selective agonist also protects against arrhythmias, and to attempt to elucidate the underlying mechanisms. Methods: Large White/Welsh Landrace cross breed domestic pigs (25-40 kg) subjected to left anterior descending coronary artery occlusion were used. R-PIA [R(-)N6-(2-phenylisopropyl) adenosine; 5 μg·kg−1] was given prior to coronary artery occlusion and the effects on haemodynamic variables and early ischaemic arrhythmias in the absence and presence of right atrial pacing were studied. The three experimental groups were: solvent controls, n=10; R-PIA without pacing, n=10; R-PIA with atrial pacing, n=10. Ex vivo assessment of platelet aggregation was also performed to determine any inhibitory effects of R-PIA on platelets. Results: Administration of R-PIA without atrial pacing reduced the total number of ventricular ectopic beats induced by coronary occlusion, from 326(SEM 71) in controls to 121(30) (p<0.05) and the incidence of ventricular fibrillation from 70% to 20% (p<0.05). At the dose used, R-PIA reduced heart rate from 102(7) to 74(3) beats·min−1, with a consequent reduction in mean arterial blood pressure from 95(4) to 81(3) mm Hg. Atrial pacing following drug administration, at a rate of 109(5) beats·min−1, restored blood pressure and abolished the antiarrhythmic effects of R-PIA. Drug intervention had no effect on either ex-vivo platelet aggregation or coronary sinus oxygen content, suggesting a lack of activity at A2 adenosine receptors. Conclusions: An A1 adenosine receptor agonist exerts a marked protection against ischaemia induced ventricular arrhythmias and fibrillation in pigs. The reversal of this effect by restoring heart rate suggests that the drug induced bradycardia may be important in the antiarrhythmic action of R-PIA, possibly via an anti-ischaemic effect.Cardiovascular Research 1993;27:84-89 [ABSTRACT FROM PUBLISHER]

This symposium provided a forum for presentations by the relevant groups on ligand design and ligand binding on the adenosine A 1 , receptor. Agreement appears to exist that the "N 6 -C 8 " model of ligand binding to the receptor is the preferred mode. A consensus has not yet been reached on the actual placement of the ligand in the receptor and the exact amino acids which interact in its binding. Two viable models exist at present. Both can be tested with selective site-directed mutagenic studies on the A 1 receptor as well as with additional designed ligands.