Administration Of CPA Research Articles

Effect of simultaneous application of adenosine A1 receptor agonist and A2A receptor antagonist on memory, inflammatory factors, and PSD-95 in lipopolysaccharide-induced memory impairment

The potential role of adenosine, a natural neuroprotective agent, and its receptors in the pathogenesis of Alzheimer's disease has been proposed. The present study aims to examine the effect of administering both an A1 receptor agonist and an A2A adenosine receptor antagonist simultaneously on memory, inflammatory factors, and PSD-95 in an LPS-induced Alzheimer's disease model in rats. Fifty-six male Wistar rats were randomly divided into seven groups: Saline, LPS, Saline + Vehicle, LPS + Vehicle, LPS + SCH58261 (A2A receptor antagonist), LPS + CPA (A1 receptor agonist), LPS + SCH58261+CPA. LPS (3 mg/kg/ip) was used to cause memory impairment. Treatment was performed by intraventricular injection of CPA at a dose of 700 μg and SCH-58261 at 40 μg for ten days. Passive avoidance and Y-maze tests were performed to examine animals’ memories. IL-10, TNF-α, and PSD-95 levels were measured in the brain using ELISA and western blot, respectively. Compared to the groups receiving each medication separately, the simultaneous administration of CPA and SCH58261 improved memory (P<0.05). Additionally, compared to the single medication groups, there was a significant increase in IL-10, PSD-95, and a significant decrease in TNF-α in the brain tissue (P<0.05). These findings suggest that the activation of A1 receptors along with A2A receptor inhibition could be a potential therapeutic strategy for Alzheimer's disease. These findings suggest that A1 receptor activation combined with A2A receptor inhibition may be a promising therapeutic approach for Alzheimer's disease.

Oral delivery of chitosan-coated PLGA nanoemulsion loaded with artesunate alleviates ulcerative colitis in mice

Artesunate (ARS) has been shown to have a protective effect on ulcerative colitis (UC) in mice. However, its lack of targeting and short half-life severely hamper its efficacy. In this study, polylactic acid-glycolic acid copolymer (PLGA) and chitosan (CS) double emulsification solvent volatilisation method was used to prepare a stable nanoemulsion loaded with ARS (CPA). The in vitro drug release profile was detected using dialysis and the potential protective effect was evaluated in an experimental ulcerative colitis (UC) model induced by oral administration of dextran sulphate sodium (DSS). The results suggested that the mean droplet diameter of CPA nanoemulsion is 409.9 ± 9.21 nm, the polydispersity index is 0.17 ± 0.01 and the zeta potential is 40.07 ± 1.65 mV. The cumulative release curve showed the ARS was mainly released at pH 7.4, which is similar to the colonic environment. Oral administration of CPA effectively relieved DSS-induced clinical symptoms by lowering the body weight loss, disease activity index (DAI) score and impressively maintained tight junction protein expression in colon tissue when compared to the blank nanoemulsion control. Meanwhile, CPA remarkably suppressed TLR4/NF-κB pathway activation and mRNA levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhanced levels of IL-10 and CD206. In addition, the effect of CPA was slightly better than that of injecting ARS. Therefore, this study demonstrates a convenient drug delivery system for oral administration of ARS that potentially helps to target colonic tissue and alleviate UC.

Central Adenosine A1 Receptors Arbitrate the Nicotine Counteraction of Cardiovascular and Autonomic Dysfunctions in Septic Rats

The cholinergic anti‐inflammatory pathway plays a crucial role in improving end‐organ damage provoked by inflammatory disorders. Given the interplay between cholinergic and adenosinergic neuronal circuits in the central nervous system, we tested the hypothesis that central adenosine A1 receptors (A1Rs) modulate the offsetting action of nicotine on cardiovascular and inflammatory insults induced by sepsis. Pharmacologic agonistic and antagonistic studies were conducted in conscious rats, pre‐instrumented with femoral vessel and intracisternal (i.c.) catheters. Sepsis was induced by cecal ligation and puncture (CLP) 24 hr before cardiovascular measurements. Nicotine (25 or 100 µg/kg i.v.) dose‐dependently reversed septic manifestations of hypotension and concomitant reductions in time and spectral measures of heart rate variability (HRV) and cardiac sympathovagal balance (low‐frequency/high‐frequency ratio). Like nicotine, the i.c. administration of N(6)‐cyclopentyladenosine (CPA, AR1 agonist) to CLP rats caused significant increases in indices of HRV and sympathovagal balance and greater surges in these parameters were noted upon simultaneous administration of CPA plus nicotine. We also show that the favorable influences of nicotine on blood pressure and HRV in septic rats were significantly inhibited after central blockade of A1Rs by i.c. 8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Immunohistochemically, the alleviation by nicotine of the upregulated myocardial expression of NFκB in septic rats disappeared upon prior exposure to DPCPX. The data suggest that central adenosine A1Rs act tonically to facilitate the ameliorative effect of nicotine against the inflammatory response of sepsis and interrelated cardiomyopathic and neuropathic hitches.

α-D-1,6-glucan from Castanea mollissima Blume alleviates dextran sulfate sodium-induced colitis in vivo

A homogenous α-D-1,6-glucan (CPA) was extracted from Castanea mollissima Blume. The effect of CPA on ameliorating dextran sulfate sodium induced colitis was investigated. CPA repressed TNF-α and IL-1β level in LPS stimulated RAW264.7 cells. After the intragastric administration of CPA (200 or 400 mg/kg/day), the colon length and body weights of mice with colitis increased and the disease activity index reduced. CPA alleviated colon tissue damage by elevating ZO-1 and occludin protein levels and regulating TNF-α and IL-1β by inhibiting the protein expression of NLPR3 and NF-κB p65. The abundance of Bacteroidetes and Firmicutes was altered and short-chain fatty acid (SCFA) levels, especially propionic, butyric, and isovaleric acids increased significantly. These results indicated that CPA could alleviate colitis by protecting mucosal barriers, reducing inflammation, and regulating intestinal microbiota and SCFA levels. Thus, CPA can be developed as a functional food for the prevention and treatment of colitis.

The role of adenosine A1 receptors in the nucleus accumbens during morphine withdrawal.

Opioids are effective analgaesic agents, but serious adverse effects such as tolerance and withdrawal contribute to opioid dependence and limit their use. Opioid withdrawal is a common occurrence in human opiate addicts that is not life-threatening. Studies have shown that the mesocorticolimbic system, especially the nucleus accumbens, is an important region in drug addiction and adenosinereceptors play a modulatory role in the mechanism of action of drug dependence and withdrawal. The aim of this study was to investigate the effects of the selective A1 receptor agonist CPA (N6 -cyclopentyladenosine) on withdrawal symptoms, and the concentration of dopamine and noradrenaline in the nucleus accumbens and locomotor activity behaviour during naloxone-precipitated withdrawal in morphine-dependent rats. The local administration of CPA (1.5, 3.0, and 6.0mmol/L bilateral 250nL) into the nucleus accumbens decreased the Gellert-Holtzman withdrawal scale, and increased concentrations of dopamine and noradrenaline in the same region during naloxone-induced withdrawal. Our findings suggest that administration of the A1 receptor agonist significantly decreased withdrawal behaviours and increased dopamine and noradrenaline concentrations in opioid withdrawal in a dose-dependent manner. These results demonstrate that adenosine receptors should be examined as a potential mechanism that could be exploited for the treatment of morphine withdrawal.

Cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice

Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.

Use of 11C-MPDX and PET to Study Adenosine A1 Receptor Occupancy by Nonradioactive Agonists and Antagonists

Adenosine A1 receptors (A1Rs) in human and rodent brains can be visualized with the radioligand 8-dicyclopropylmethyl-1-(11)C-methyl-3-propylxanthine ((11)C-MPDX) and PET. Here we investigated whether A1R occupancy by nonradioactive agonists and antagonists can be assessed with this technique. Small-animal PET scans with arterial blood sampling were obtained for 4 groups of isoflurane-anesthetized Wistar rats: controls (n = 7); pretreated with a centrally active A1R agonist, N(6)-cyclopentyladenosine (CPA; 0.25 mg/kg intraperitoneally; dissociation constant, 0.48 nM; n = 7); pretreated with a moderate dose of caffeine (antagonist for A1Rs and adenosine A2A receptors; 4 mg/kg intraperitoneally; dissociation constant, 11 μM; n = 6); and pretreated with a high dose of caffeine (40 mg/kg intraperitoneally; n = 6). The administration of CPA resulted in a strong reduction (>50%) in the heart rate, and caffeine administration resulted in a small increase (10%-15%). A caffeine dose of 4 mg/kg (n = 6) resulted in 65.9% A1R occupancy, and a dose of 40 mg/kg (n = 6) resulted in 98.5% occupancy (calculated from a modified Lassen plot). However, the administration of CPA resulted in an increase in (11)C-MPDX binding in the brain. Small-animal PET with (11)C-MPDX can be used to assess antagonist but not agonist binding at A1Rs. Changes in tracer uptake after the administration of CPA resembled previously reported changes induced by treatment of rats with ethanol and an adenosine kinase inhibitor (ABT702). Thus, the administration of an exogenous agonist or increasing the level of an endogenous agonist have similar effects. Agonists and antagonists may bind to different sites on the A1R protein having allosteric interactions.

AB0066 Cyclic phosphatidic acid (cpa) stimulates hyaluronic acid production in human osteoarthritic articular chondrocytes, and intra-articular administration of cpa suppresses pain, swelling, and cartilage destruction in rabbit experimental osteoarthritis.

BackgroundCyclic phosphatidic acid (cPA) is one of lipid mediators, has been shown various biological effects1. On human skin fibroblasts, cPA stimulates high molecular hyaluronic acid (HA) production through up-regulating HA...

H1 but not H2 histamine antagonist receptors mediate anxiety-related behaviors and emotional memory deficit in mice subjected to elevated plus-maze testing

This study investigated the role of H1 and H2 receptors in anxiety and the retrieval of emotional memory using a Trial 1/Trial 2 (T1/T2) protocol in an elevated plus-maze (EPM). Tests were performed on 2 consecutive days, designated T1 and T2. Before T1, the mice received intraperitoneal injections of saline (SAL), 20 mg/kg zolantidine (ZOL, an H2 receptor antagonist), or 8.0 or 16 mg/kg chlorpheniramine (CPA, an H1 receptor antagonist). After 40 min, they were subjected to the EPM test. In T2 (24 h later), each group was subdivided into two additional groups, and the animals from each group were re-injected with SAL or one of the drugs. In T1, the Student t-test showed no difference between the SAL and ZOL or 8 mg/kg CPA groups with respect to the percentages of open arm entries (%OAE) and open arm time (%OAT). However, administration of CPA at the highest dose of 16 mg/kg decreased %OAE and %OAT, but not locomotor activity, indicating anxiogenic-like behavior. Emotional memory, as revealed by a reduction in open arm exploration between the two trials, was observed in all experimental groups, indicating that ZOL and 8 mg/kg CPA did not affect emotional memory, whereas CPA at the highest dose affected acquisition and consolidation, but not retrieval of memory. Taken together, these results suggest that H1 receptor, but not H2, is implicated in anxiety-like behavior and in emotional memory acquisition and consolidation deficits in mice subjected to EPM testing.

Cyclic phosphatidic acid (cPA) stimulates the production of hyaluronic acid (HA) in human osteoarthritic articular chondrocytes, and intra-articular ADMINISTRATION OF CPA suppresses pain, swelling, and cartilage destruction in rabbit experimental osteoarthritis

Cyclic phosphatidic acid (cPA) stimulates the production of hyaluronic acid (HA) in human osteoarthritic articular chondrocytes, and intra-articular ADMINISTRATION OF CPA suppresses pain, swelling, and cartilage destruction in rabbit experimental osteoarthritis

Protection of Neuroblastoma Neuro2A Cells from Hypoxia-Induced Apoptosis by Cyclic Phosphatidic Acid (cPA)

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl2) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl2 induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl2-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl2. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA1, LPA2, and LPA6, which are G-protein coupled receptors that can be activated by cPA. To date, LPA1 and LPA2 have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA1 and LPA2 on cPA-induced neuroprotective functions, Ki16425, a selective LPA1 and LPA3 antagonist, was adopted to know the LPA1 function and siRNA was used to knockdown the expression of LPA2. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl2-induced hypoxia damage is mediated via LPA2.

Effects of cyclic phosphatidic acid on delayed neuronal death following transient ischemia in rat hippocampal CA1

Cyclic phosphatidic acid (cPA) is a lipid mediator that elicits a neurotrophin-like action in embryonic hippocampal neurons in vitro. In this study, we investigated the effects of cPA and 2- O-carba-oleoyl-cPA (2ccPA), a metabolically stabilized cPA derivative, on ischemia-induced delayed neuronal death in the rat hippocampal CA1 region. Transient occlusion for 8 min of bilateral carotid arteries besides permanent ligation of bilateral vertebral arteries was performed and morphological changes of the neurons were examined histologically 5 days after occlusion. cPA or 2ccPA was continuously administered for 5 days by means of an osmotic pump that was implanted subcutaneously before occlusion. Five days after occlusion, delayed neuronal death occurred in approximately 85% of the CA1 hippocampal neurons in the 0.2–2% bovine serum albumin vehicle control group. However, administration of cPA significantly increased the number of undamaged neurons in a dose-dependent manner. At the most effective concentration (18 μg/kg/5d), the number of undamaged neurons was increased to 4 times of that in the vehicle control group. 2ccPA also showed a neuroprotective effect, but it was less potent than that of natural cPA. These results indicate that systemic administration of both cPA and 2ccPA can protect neurons from ischemia-induced delayed neuronal death in the hippocampus.

Differential effects of adenosine A1 receptor on pain-related behavior in normal and nerve-injured rats

This study investigated the effects of N6-cyclopentyladenosine (CPA), a potent and selective adenosine A1 receptor (A1R) agonist in normal and nerve-injured rats and mechanisms of its action by behavioral tests and electrophysiological technique. The results showed: (1) In normal rats, intraperitoneal administration of CPA (1 mg/kg) increased paw withdrawal latencies, in a way blocked by a selective A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 3 mg/kg, i.p.), but had no influence on the threshold of mechanical stimulation. (2) In rats with neuropathic pain induced by spinal nerve ligation (SNL), CPA reduced thermal hyperalgesia and mechanical allodynia, which could last 6 h and 10 h, respectively ( n = 6/group, P < 0.05). Both of the effects could be blocked by pretreatment of DPCPX intraperitoneally. (3) The baseline of C-fiber but not A-fiber evoked field potentials was depressed by spinal application of CPA (0.01 mM), and this effect was prevented by application of DPCPX (0.02 mM) 30 min before CPA. (4) Spinal application of CPA depressed long-term potentiation (LTP) of A- and C-fiber evoked field potentials, and both the depression could be blocked by pretreatment of DPCPX 30 min before CPA. These results suggested that the activation of A1R has different influences on normal and neuropathic rats probably due to the absence and presence of central sensitization in spinal dorsal horn.

Direct blockade of inflammatory hypernociception by peripheral A1 adenosine receptors: Involvement of the NO/cGMP/PKG/KATP signaling pathway

Through activation of the A1 adenosine receptors (A1Rs) at both the central and peripheral level, adenosine produces antinociception in a wide range of tests. However, the mechanisms involved in the peripheral effect are still not fully understood. Therefore, the mechanisms by which peripheral activation of A1Rs reduces inflammatory hypernociception (a decrease in the nociceptive threshold) were addressed in the present study. Immunofluorescence of rat dorsal root ganglion revealed significant expression of A1Rs in primary sensory neurons associated with nociceptive pathways. Functionally, peripheral activation of A1Rs reduced inflammatory hypernociception because intraplantar (i.pl.) administration of an A1R antagonist (DPCPX) enhanced carrageenan-induced hypernociception. On the other hand, local (paw) administration of CPA (a selective A1R agonist) reversed mechanical hypernociception induced by carrageenan or by the directly acting hypernociceptive mediator prostaglandin E2 (PGE2). Down-regulation of A1Rs expression in primary nociceptive neurons by intrathecal treatment with antisense oligodeoxinucleotides significantly reduced peripheral antinociceptive action of CPA. Direct blockade of PGE2 inflammatory hypernociception by the activation of A1Rs depends on the nitric oxide/cGMP/Protein Kinase G/KATP signaling pathway because the peripheral antinociceptive effect of CPA was prevented by pretreatment with inhibitors of neuronal nitric oxide synthase (N-propyl-l-arginine), guanylyl cyclase (ODQ), and Protein Kinase G (KT5823) as well as with a KATP blocker (glibenclamide). However, this effect of CPA was not reduced by naloxone, excluding the participation of endogenous opioids. These results suggest that the peripheral activation of A1R plays a role in the regulation of inflammatory hypernociception by a mechanism that involves the NO/cGMP/PKG/KATP intracellular signaling pathway.

Intra-VTA adenosine A 1 receptor activation blocks morphine stimulation of motor behavior and cortical and limbic Fos immunoreactivity

Drugs of abuse produce psychomotor stimulation as one of their characteristic behavioral effects. Single administration of opiates stimulates motor activity via effects on gamma-aminobutyric acid (GABA) and dopamine transmission in the ventral tegmental area (VTA). Adenosine A 1 receptor agonists inhibit VTA GABAergic and dopaminergic transmission and are predicted to alter the behavioral effects of opiates. This study examined the effects of intra-VTA administration of selective adenosine A 1 receptor agonist N 6-cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1, 3-dimethylxanthine (CPT) on morphine-induced motor stimulation in C57BL/6 mice. It also examined the effects of CPA on morphine's activation of VTA neurons projecting to limbic and cortical regions including the nucleus accumbens (NAc), anterior cingulate cortex (ACg) and prelimbic cortex (PrL) via quantitation of immediate-early gene c-Fos protein in these regions. Mice received subcutaneous morphine and intra-VTA administration of CPA and then automated motor activity was measured. Morphine treatment induced both motor activity and Fos immunoreactivity in the NAc, ACg and PrL suggesting that behavioral stimulation is produced by neural activation in these regions. Intra-VTA CPA administration produced a dose-dependent inhibition of morphine-induced motor stimulation and blocked c-Fos induction in all three regions. Intra-VTA CPT treatment had no effects on motor activity or on morphine-induced motor stimulation. VTA adenosine A 1 agonist inhibition of morphine's effects on motor activity and of neural activation of VTA projections suggests that these neurons and their regulation are critical to morphine's stimulant effects. Adenosine A 1 receptor agonists and purinergic modulators may represent useful treatment approaches for blocking the behavioral effects of opiates.

Cyclic phosphatidic acid stimulates respiration without producing vasopressor or tachycardiac effects in rats

The effects of a novel lipid mediator, cyclic phosphatidic acid (cPA), on respiratory and cardiovascular functions were examined in anesthetized rats. Intravenous (i.v.) administration of 3- O-carba-oleoyl-cPA at doses of 130 and 390 μg/kg produced dose-dependent increases in tidal volume and respiratory frequency, resulting in an increase in total ventilation. Heart rate was slightly decreased at a dose of 390 μg/kg, while systemic arterial pressure was not affected. Bilateral section of vagi and carotid sinus nerves designed to eliminate major regulatory inputs from the peripheral afferents to the respiratory center reduced these responses, but did not abolish them. These results indicate that cPA stimulates respiration, via central and peripheral mechanisms acting on the central respiratory rhythm generator in the brain stem. Administration of cPA may be of therapeutic value as a respiratory stimulant without producing vasopressor or tachycardiac effects, for treatment of respiratory disorders.

The Effect of Repeated Administration of Cyclophosphamide on CYP2B1 and 2B2 in Rat.

Cyclophosphamide is used in high doses as a part of the conditioning regimen prior to stem cell transplantation. It is usually given for two or four consecutive days, primarily to facilitate engraftment of donor cells. Cyclophosphamide is a prodrug that is activated in liver by a 4-hydroxylation reaction catalyzed by cytochrome P450 (CYP) enzymes. Several studies have shown that cyclophosphamide induces its own metabolism, which affects its pharmacokinetic parameters after repeated dose (Chang TK, Yu L et al. Cancer Res 1997; 57:1946–54 and Schuler U, Ehninger G et al. Cancer Chemother Pharmacol 1987; 20:248–52). In the present study, we aimed to investigate the effect of repeated doses of cyclophosphamide on the CYPs in rat. mRNA, protein, and enzyme activity levels were investigated. Animals received (200 mg/kg, i.v.) at time 0, 20, 48 h. Additionally another group of animals was treated at 0, 6, 20, 26, 48 and 54 h. At each time point three animals were killed 30 min after the administration (Xie H, Afsharian P et al. Xenobiotica 2005; 35:239–51). mRNAs of CYP2B1 and 2B2 were significantly induced up to 458- and 8.3-fold at 6h, 983- and 102-fold at 26 h, and 342- and 33-fold at 54 h. CYP2B protein levels were increased and their peaks was observed at 20 and 48 h. Microsomal activity of CYP2B was determined at three different concentration of cyclophosphamide (1, 0.5, 0.1 mM) by measuring the formation rate of 4-hydroxy-cyclophosphamide (4-OH-CPA). The microsomal activity increased as reflected in an increase in cyclophosphamide 4-hydroxylation at all concentrations used using microsomes from rats treated at 6, 20 and 48 h. A significant increase of 4-hydroxylation of cyclophosphamide (0.1 mM) by 2.9-, 4.4- and 4.2- fold, respectively compared to the control rats. However, a decrease in the hydroxylation rate was observed using microsomes from rats treated at 26 and 54 h and in all concentrations used that might due to hepatotoxicity effect of cyclophosphamide when administered in short time period of repeated dose. A significant (p<0.05) decrease in CPA concentration and a significant (p<0.05) increase in its metabolite (4-OH-CPA) level in plasma were observed with repeated administration of CPA. In conclusion, an induction effect on CYP2B and a substantial up regulation of its mRNA expression were observed after repeated administration of CPA. This information can have a high clinical importance when a dosing schedule for cyclophosphamide considering the polymorphism of CYPs in cancer patient.

Enhancement of wind-up by the combined administration of adenosine A 1 receptor ligands on spinalized rats with carrageenan-induced inflammation

The adenosine A 1 receptor agonist N6-cyclopentyladenosine (CPA) is very effective in reducing wind-up in intact but not in spinalized adult rats with carrageenan-induced inflammation, suggesting an adenosine-mediated supraspinal modulation. Since wind-up is a spinal cord mediated phenomenon but highly influenced by descending modulatory systems, especially in situations of sensitization, we assessed the possible involvement of adenosine in the modulation of wind-up. We studied the effect of the adenosine A 1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) in the presence and in the absence of the adenosine A 1 receptor agonist CPA. The experiments were carried out in spinalized male Wistar rats under α-chloralose anaesthesia. Withdrawal reflexes, studied as single motor units, were activated by noxious mechanical and high-intensity repetitive electrical stimulation (wind-up). While CPA and CPT were not able to induce any change on wind-up when injected alone, the combination of the two drugs, in any order, lead to an important enhancement of wind-up. This enhancement not always paralleled an increase of responses to noxious mechanical stimulation, indicating that the effect is mainly located in the spinal cord. In addition, the enhancement of wind-up was not further increased by the administration of the opioid receptor antagonist naloxone. We conclude that the depression of the wind-up phenomenon observed in spinalized animals is, at least in part, dependent of adenosine systems and can be relieved by the combined administration of CPA and CPT.

N6-Cyclopentyladenosine inhibits proliferation of murine haematopoietic progenitor cells in vivo

Effects of N 6-cyclopentyladenosine (CPA), the selective adenosine A 1 receptor agonist, on bone marrow haematopoietic progenitor cells for granulocytes and macrophages (CFC-GM) were investigated by utilizing the model of haematopoietic damage induced by 5-fluorouracil. Experiments were performed in vivo on B10CBAF 1 mice. A single i.p. injection of CPA at the optimum dose of 200 nmol/kg administered 22 h before a single injection of 5-fluorouracil (100 mg/kg, i.p.) protected CFC-GM against the cytotoxic damage as determined 4 days later. Isomolar doses of the selective agonists for adenosine A 2A receptors, i.e. 2- p-(2-carboxyethyl)-phenethylamino-5′- N-ethylcarboxamidoadenosine, and for adenosine A 3 receptors, i.e. N 6-(3-iodobenzyl)adenosine-5′- N-methyluronamide, did not induce such effects. Because 5-fluorouracil is a cell cycle-specific drug damaging mainly cells in the S-phase, protective effects of CPA can be explained by its inhibitory action on the cell cycling. This interpretation was confirmed by experiments demonstrating that repeated administration of CPA in the hyperproliferation phase of the recovering haematopoiesis after 5-fluorouracil treatment inhibited transiently restoration of CFC-GM counts.

Effects of stable adenosine receptor agonists on bone marrow haematopoietic cells as inferred from the cytotoxic action of 5-fluorouracil

The aim of the study was to investigate the effects of stable adenosine receptor agonists on bone marrow hematopoiesis by utilizing the model of hematopoietic damage induced by 5-fluorouracil (5-FU), a cycle-specific cytotoxic agent. Effects of a non-selective agonist NECA activating all the known adenosine receptors (A1, A2A, A2B, A3) and of the selective agonists for A1 (CPA), A2A (CGS 21680), and A3 (IB-MECA) adenosine receptors were investigated. Experiments were performed with B10CBAF1 mice under in vivo conditions. Adenosine receptor agonists were given in single injections before 5-FU administration and the effects were determined 4 days later. The numbers of femoral marrow nucleated cells and hematopoietic progenitor cells (CFC-GM and BFU-E) were taken as indices of the effects. The non-selective agonist NECA given at a dose of 200 nmol/kg induced biphasic time-dependent effects, i.e. protection and sensitization, when given 10 h and 22 h before 5-FU administration, respectively. The use of isomolar doses of selective receptor agonists indicated that the protective effects of NECA were induced by activation of A2A and A2B receptors, while the sensitizing action of NECA was mediated via A3 receptors. In addition, it was observed that A1 receptors induced protection when activated by administration of CPA 22 h before 5-FU. These findings are discussed with respect to the action of adenosine receptor agonists on the cell cycle state and on the cell cycle-independent cellular protective mechanisms.