Effects of A1 receptor agonist/antagonist on spontaneous seizures in pilocarpine-induced epileptic rats.

Adenosine is an endogenous anticonvulsant that activates pre- and postsynaptic adenosine A1 receptors. A1 receptor agonists increase the latency for the development of seizures and status epilepticus following pilocarpine administration. Although hippocampal adenosine is increased in the chronic phase of the pilocarpine model, it is not known whether the modulation of A1 receptors may influence the frequency of spontaneous recurrent seizures (SRS). Here, we tested the hypothesis that the A1 receptor agonist RPia ([R]-N-phenylisopropyladenosine) and the A1 antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine) administered to chronic pilocarpine epileptic rats would respectively decrease and increase the frequency of SRS and hippocampal excitability. Four months after Pilo-induced SE, chronic epileptic rats were video-monitored for the recording of SRS before (basal) and after a 2-week treatment with RPia (25µg/kg) or DPCPX (50µg/kg). Following sacrifice, brain slices were studied with electrophysiology. We found that rats given RPia had a 93% nonsignificant reduction in the frequency of seizures compared with their own pretreatment baseline. In contrast, the administration of DPCPX resulted in an 87% significant increase in seizure rate. Nontreated epileptic rats had a similar frequency of seizures along the study. Corroborating our behavioral data, in vitro recordings showed that slices from animals previously given DPCPX had a shorter latency to develop epileptiform activity, longer and higher DC shifts, and higher spike amplitude compared with slices from nontreated Pilo controls. In contrast, smaller spike amplitude was recorded in slices from animals given RPia. In summary, the administration of A1 agonists reduced hippocampal excitability but not the frequency of spontaneous recurrent seizures in chronic epileptic rats, whereas A1 receptor antagonists increased both.

Neurabin scaffolding of adenosine receptor and RGS4 regulates anti-seizure effect of endogenous adenosine.

Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A(1) receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G-protein signaling 4 (RGS4), a protein known to turn off G-protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ∼50% rate of death in wild-type (WT) mice did not affect neurabin-null mice or WT mice cotreated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

Differential neuroprotection by A(1) receptor activation and A(2A) receptor inhibition following pilocarpine-induced status epilepticus.

Aiming at a better understanding of the role of A(2A) in temporal lobe epilepsy (TLE), we characterized the effects of the A(2A) antagonist SCH58261 (7-(2-phenylethyl)-5-amino-2(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) on seizures and neuroprotection in the pilocarpine model. The effects of SCH58261 were further analyzed in combination with the A(1) agonist R-Pia (R(-)-N(6)-(2)-phenylisopropyl adenosine). Eight groups were studied: pilocarpine (Pilo), SCH+Pilo, R-Pia+Pilo, R-Pia+SCH+Pilo, Saline, SCH+Saline, R-Pia+Saline, and R-Pia+SCH+Saline. The administration of SCH58261, R-Pia, and R-Pia+SCH58261 prior to pilocarpine increased the latency to SE, and decreased either the incidence of or rate of mortality from SE compared with controls. Administration of R-Pia and R-Pia+SCH58261 prior to pilocarpine reduced the number of Fluoro-Jade B-stained cells in the hippocampus and piriform cortex when compared with control. This study showed that pretreatment with R-Pia and SCH58261 reduces seizure occurrence, although only R-Pia has neuroprotective properties. Further studies are needed to clarify the neuroprotective role of A(2A) in TLE.

Adenosine receptor interactions alter cardiac contractility in rat heart.

1. The effect of the adenosine A(2) receptor (AdoA(2)R) agonist N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA) on adenosine A(1) receptor (AdoA(1)R)-mediated negative inotropic responses was investigated in rat heart. 2. Hearts from male Wistar rats (250-350 g) were perfused with Krebs'-Henseleit solution at constant flow in non-recirculating Langendorff mode. Hearts were paced at 5 Hz (5 ms duration, supramaximal voltage) via ventricular electrodes. After 30 min equilibration, (R)-N(6)-phenylisopropyl adenosine (R-PIA) concentration-response curves were constructed in the absence or presence of DPMA. 3. In paced hearts, R-PIA induced concentration-dependent decreases in triple product (heart rate x peak systolic developed pressure x dP / dt(max)), which were significantly attenuated by 1 nmol / L DPMA with a shift in pEC(50) from 8.0 +/- 0.5 (n = 9) in control hearts to 6.63 +/- 1.03 (n = 5) in treated tissues (P < 0.05). The AdoA(2A)R antagonist 8-(3-chlorostyryl)caffeine (1 micromol / L) and the adenylyl cyclase inhibitor cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL12330A; 100 nmol / L) reversed the effects of DPMA on AdoA(1)R-mediated negative inotropic actions, whereas the AdoA(2B)R antagonist alloxazine (3 micromol / L) had no effect on DPMA activity. 4. The results of the present study show that stimulation of the AdoA(2)R attenuates AdoA(1)R-dependent reductions in inotropic state. The receptor involved appears to be the AdoA(2A)R and its action involves stimulation of adenylyl cyclase activity.

Role of beta-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.

Resistance to the antilipolytic effect of insulin in adipocytes of African-American compared to Caucasian postmenopausal women.

High fatty acid (FA) flux is associated with systemic insulin resistance, and African-American (AA) women tend to be more insulin resistant. We assessed possible depot and race difference in the antilipolytic effect of insulin in adipocytes isolated from abdominal (Abd) and gluteal (Glt) subcutaneous (sc) adipose tissue of overweight, postmenopausal AA and Caucasian (C) women. Percent body fat, fasting insulin, visceral adiposity, and adipocyte size was higher in AA women. Disinhibited lipolysis (presence of adenosine deaminase) per unit adipocyte surface area was similar in Abd and Glt and in AA and C. However, rates of 'basal' [submaximal phenylisopropyl adenosine (PIA)-suppressed] and insulin-suppressed lipolysis were higher in Abd of AA compared with C women even after adjustment for percent fat and visceral fat area. The race difference in rates of PIA- and insulin-suppressed lipolysis in AA were correlated with their hyperinsulinemia, but AA race, independent of fasting insulin, was associated with lower responsiveness (percent suppression) to submaximal insulin concentrations, although sensitivity (ED50) was not affected. Overall, these data are consistent with the hypothesis that decreased responsiveness of Abd adipocytes to antilipolytic effectors may contribute to higher FA availability and thereby to racial differences in insulin resistance.

Reduced expression and desensitization of adenosine A1 receptor/adenylyl cyclase pathway after chronic (-)N6-phenylisopropyladenosine intake during pregnancy.

Little is known about the G protein-coupled receptor desensitization process during pregnancy. Wistar pregnant rats were treated with (-)N(6)-phenylisopropyladenosine (R-PIA), an adenosine A(1) receptor (A(1)R) agonist, in their drinking water during pregnancy, and the effect on A(1)R/adenylyl cyclase system was studied in both maternal and fetal brain. In maternal brain, binding assays revealed a significant decrease in total receptor numbers in plasma membranes (27%, P<0.05), with no significant changes in receptor affinity. The effect of R-PIA on plasma membranes from fetal brains was more marked, with approximately 42% (P<0.05) of the total receptors detected in control fetuses. Real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that chronic R-PIA treatment during the whole gestational period only decreased significantly mRNA level coding A(1)R in maternal brain (P<0.05). alpha Gi(1,2) and alpha Gi(3) subunits were not affected in mothers or fetuses as revealed by immunoblotting. mRNA levels coding these subunits were also unaffected in mothers and fetuses. On the other hand, forskolin- and forskolin-plus guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S)-stimulated adenylyl cyclase activity was decreased in maternal (P<.01) and fetal brain (P<.001). Furthermore, adenylyl cyclase inhibition elicited by N(6)-cyclohexyladenosine (CHA), a selective A(1)R agonist, was significantly decreased in both maternal (P<0.05) and fetal brain (P<.01), suggesting a desensitization of the A(1)R/adenylyl cyclase pathway. Therefore, these results suggest that R-PIA intake during pregnancy causes desensitization of the A(1)R-mediated inhibitory transduction pathway in both maternal and fetal brain, probably due to the decreased density of A(1)R at the cell surface.

Adenosine A1 but not A2a receptor agonist reduces hyperalgesia caused by a surgical incision in rats: a pertussis toxin-sensitive G protein-dependent process.

BACKGROUND: Activation of A1 adenosine receptors (A1Rs) causes antinociception after nerve injury and inflammation. However, the role of A2a adenosine receptors (A2aRs) for pain processing is less clear. In the current study, the authors investigated the role of spinal adenosine A1Rs and A2aRs for the maintenance of mechanical hyperalgesia in an animal model for postoperative pain. METHODS: Rats with intrathecal catheters were anesthetized and underwent plantar incision. Spontaneous pain behavior and withdrawal threshold to punctuate stimulation were measured before and after administration of intrathecal R-phenylisopropyl-adenosine (R-PIA; A1R agonist), 2-w p-2-carbonyl-ethyl-phenylethylaminox-5X-N-ethylcarboxami-doadenosine (CGS21680; A2aR agonist), or vehicle. In separate groups of animals, the effects of pertussis toxin, forskolin, glibenclamide, 4-aminopyridine, tetraethylammonium, apamin, charybdotoxin, or margatoxin on R-PIA-induced antinociception were examined. RESULTS: Intrathecal administration of 5 nmol R-PIA but not 10 nmol CGS21680 decreased nonevoked spontaneous pain behavior. Furthermore, intrathecal administration of R-PIA but not of CGS21680 increased withdrawal thresholds after incision. Pretreatment with pertussis toxin and administration of forskolin, glibenclamide, 4-aminopyridine, and tetraethylammonium inhibited R-PIA-induced antinociception. In addition, intrathecal administration of apamin, charybdotoxin, or margatoxin did not modify mechanical hypoalgesia mediated by R-PIA. CONCLUSIONS: Spinal A1Rs but not A2aRs play an important role in the maintenance of nonevoked and evoked pain behaviors after an incision. Furthermore, A1R-induced spinal antinociception is mediated by interactions with pertussis toxin-sensitive G proteins. In addition, the opening of adenosine triphosphate-sensitive K channels but not of calcium-activated potassium channels and voltage-gated Kv1.3 or Kv1.6 channels contribute to the antinociceptive effect of A1R agonists.

mRNA expression and antilipolytic role of phosphodiesterase 4 in rat adipocytes in vitro.

Adipocyte lipolysis is dependent on an increase in the intracellular concentration of cAMP. Intracellular phosphodiesterases (PDEs) hydrolyze cAMP and limit stimulation of lipolysis. In the present study, the mRNA expression of PDE4 subtypes and the antilipolytic role of PDE4 in rat adipocytes were investigated. Fragments encoding PDE4A (233 bp), PDE4B (786 bp), PDE4C (539 bp), and PDE4D (262 bp) sequences were amplified by RT-PCR. The mRNA expression of PDE4 subtypes (A, B, C, D) determined by real-time quantitative PCR was 7, 18.7, 18.9, and 7.2% relative to PDE3B. Inhibition of PDE4 by rolipram increased basal lipolysis and reversed in part prostaglandin E2 antilipolysis. The combination of PDE3 and PDE4 inhibitors synergistically reversed both prostaglandin E2 and phenylisopropyl adenosine antilipolysis. Stimulation of adipocytes with prostaglandin E2 increased total PDE activity and PDE3 activity measured by hydrolysis of 3[H]cAMP by the particulate fraction of adipocytes. The present study confirmed that mRNAs for all four PDE4 subtypes were expressed in rat adipocytes, with PDE4B and PDE4C predominant. Moreover, PDE4 not only limits the rate of basal lipolysis but also contributes to prostaglandin E2 antilipolysis in rat adipocytes.

Efficacy of an adenosine A1 receptor agonist compared with atropine and pralidoxime in a rat model of organophosphate poisoning.

The objective of this study was to evaluate the effects of an adenosine A1 agonist, phenylisopropyl adenosine (PIA), on metamidophos poisoning compared to specific antidotes. Rats were poisoned with metamidophos (30 mg/kg, oral) and observed for 24 hours. One group received sodium chloride (1 mL/kg) and four experimental groups received atropine (5 mg/kg), pralidoxime (PAM, 20 mg/kg), atropine/PAM (5/20 mg/kg) or PIA (1 mg/kg) intraperitoneally. Atropine reduced salivation and prevented respiratory distress when compared to sodium chloride-treated rats. Treatment with PAM did not cause any suppression of cholinergic signs. Atropine and PAM combination prevented salivation, convulsion and respiratory distress. PIA delayed initial time of the salivation, convulsion and time to death. However, PIA was found ineffective against the metamidophos-induced cholinergic symptoms and mortality. All treatments, except PIA, lead to survival of these animals. Acetylcholinesterase (AChE) activity was not normalized by PIA or PAM. PIA prevented metamidophos-induced diaphragmatic muscle necrosis as much as PAM. In conclusion, a single dose of PIA was unable to protect the rats from metamidophos toxicity. Further studies are needed involving a combination of PAM and/or atropine with repeated doses of PIA to clarify the efficacy of adenosine agonists in OP poisoning.

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 rats.

AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Prolonged treatment with prostaglandin E1 increases the rate of lipolysis in rat adipocytes.

Prolonged treatment of adipocytes with certain inhibitors of lipolysis, including N(6)-phenylisopropyl adenosine (PIA) and prostaglandin E(1) (PGE(1)) leads to down-regulation of G(i). Prolonged treatment with PIA increases the rate of lipolysis, and we have reported that tumor necrosis factor-alpha (TNF alpha) stimulates lipolysis by down-regulating G(i). To determine the relationship between G(i) concentration and lipolysis, we have investigated the effect of two other acute inhibitors of lipolysis; PGE(1), which down-regulates G(i), and nicotinic acid (NA), which does not down-regulate G(i). Rat adipocytes were incubated with PIA (300 nM), PGE(1) (3 microM) or nicotinic acid (1 mM) for 24 h. The rate of lipolysis (glycerol release) was increased approximately 2 to 3-fold in PIA-treated cells, and in PGE(1)-treated cells. Conversely, the rate of lipolysis was not altered by the prolonged nicotinic acid treatment. These findings support the hypothesis that the rate of lipolysis in adipocytes is determined, at least partly, by the cellular concentration of G(i) proteins.

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.

Regulation of renal A(1) adenosine receptors in vivo.

We have compared renal A(1) adenosine receptor (AR) regulations in rats after chronic agonist and antagonist treatments. In one group, R-phenylisopropyladenosine (R-PIA), a selective A(1) AR agonist, was infused subcutaneously for 7 days. Another group was fed theophylline, a non-selective AR antagonist, for 2 weeks. Renal cortex membrane A(1) AR binding with 1,3-[(3)H]-dipropyl-8-cyclopentylxanthine demonstrated approximately 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(i)alpha-proteins from the R-PIA treated rats decreased by approximately 30%. Renal G(i)alpha levels did not change in theophylline-treated rats. Consistent with the A(1) AR desensitization, R-PIA-treated rats had significantly higher basal renin release and showed attenuated A(1) AR-mediated inhibition of renin release. These data suggest that prolonged A(1) AR stimulation results in downregulation of renal A(1) ARs and G(i)alpha, accompanied by desensitization of A(1) AR-mediated inhibitory effects on renin release. Unlike cardiac and brain A(1) ARs, renal A(1) receptors are not subject to up-regulation following chronic antagonist treatment.

Inhibition of proteasome activity blocks the ability of TNF alpha to down-regulate G(i) proteins and stimulate lipolysis.

Prolonged treatment of rat adipocytes with TNF alpha increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (G(i)). Separately, down-regulation of G(i) by prolonged treatment with an A(1)-adenosine receptor agonist, N(6)-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF alpha and PIA-mediated G(i) down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 microM) or calpeptin (50 microM), before 30-h treatment with either TNF alpha (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of alpha-subunits of G(i)1 and G(i)2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF alpha and PIA stimulated lipolysis approximately 2-fold and caused G(i) down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNF alpha and PIA-stimulated lipolysis (the 50% inhibitory concentration was approximately 2 microM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF alpha-induced G(i) down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF alpha and PIA is secondary to G(i) down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.

Adenosine A(1) receptors mediate plasma exudation from the oral mucosa.

The purpose of this study was to pharmacologically characterize the adenosine receptor subtype(s) that mediates adenosine-induced increases in macromolecular efflux from the intact hamster cheek pouch. Using intravital microscopy, we found that 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX), a selective adenosine receptor-1 antagonist, but not 3,7-dimethyl-1-propargylxanthine (DMPX), a selective adenosine receptor-2 antagonist, significantly attenuated adenosine-induced leaky site formation and increased clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the intact hamster cheek pouch (P < 0.05). Both compounds had no significant effects on bradykinin-induced responses. Nanomolar concentrations of R(-)-N(6)-(2-phenylisopropyl)-adenosine [R(-)-PIA], a selective adenosine A(1) agonist, evoked significant, concentration-dependent increases in macromolecular efflux. This response was significantly attenuated by PACPX but not by DMPX. In contrast, CGS-21680, a selective adenosine A(2) agonist, increased macromolecular efflux but only at micromolar concentrations. This response was significantly attenuated by DMPX but not by PACPX. Suffusion of nitroglycerin had no significant effects on R(-)-PIA- and CGS-21680-induced responses. In addition, suffusion of N(G)-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, had no significant effects on adenosine-induced responses. Indomethacin had no significant effects on adenosine-, R(-)-PIA-, and CGS-21680-induced increases in macromolecular efflux. Collectively, these data indicate that adenosine increases macromolecular efflux from the intact hamster cheek pouch by stimulating high-affinity adenosine A(1) receptors in a specific, nitric oxide- and prostaglandin-independent fashion.

Protective effect of adenosine receptor agonists in a new model of epilepsy--seizures evoked by mitochondrial toxin, 3-nitropropionic acid, in mice.

The role of adenosine receptor agonists in the convulsant activity of mitochondrial toxin, 3-nitropropionic acid (3-NPA), was studied in mice. The occurrence of seizures evoked by peripheral application of 3-NPA was inhibited with the use of A1 adenosine receptor agonist, R-N6-phenylisopropyladenosine and A1/A2 agonist, 2-chloroadenosine. Moreover, both drugs prevented 3-NPA-induced mortality. Similarly, A1/A2 agonist, 5'-N-ethylcarboxamidoadenosine, protected against seizures evoked by the intracerebral administration of 3-NPA, and this effect was reversed by the co-application of adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline. Obtained results suggest that A1 adenosine receptor activation may modulate the chain of events leading to the development of 3-NPA-induced seizures.

Increase of waking and reduction of NREM and REM sleep after nitric oxide synthase inhibition: prevention with GABA(A) or adenosine A(1) receptor agonists.

The effect of N(G)-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of enzyme nitric oxide synthase (NOS), on spontaneous sleep during the light period, was studied in adult rats implanted for chronic sleep recordings. L-NAME was injected by subcutaneous (s.c.) route or was infused directly into the dorsal raphe nucleus (DRN). Subcutaneous (46.0--185.0 micromol/kg) administration of L-NAME increased waking (W), slow wave sleep (SWS) and rapid-eye-movement sleep (REMS) latency, whereas SWS, REMS and the number of REM periods were reduced. Direct application of L-NAME into the DRN (0.37--1.1 micromol) induced an increment of W and a reduction of SWS and REMS. Values corresponding to SWS and REMS latency, and the number of REM periods remained within control levels. Subcutaneous administration of the GABA(A) receptor agonist muscimol (1.7--3.5 micromol/kg) or the adenosine A(1) receptor agonist L-PIA [L(-)N(6)-(2-phenylisopropyl)adenosine] (0.1--0.3 micromol/kg) induced slight but inconsistent changes of W, light sleep (LS), SWS and REMS that did not attain significance. Pretreatment with muscimol (1.7--3.5 micromol/kg, s.c.) or L-PIA (0.1--0.3 micromol/kg, s.c.) antagonized the increase of W and reduction of SWS and REMS induced by s.c. (92.0 micromol/kg) or intra-DRN (0.74 micromol) administration of L-NAME. However, neither muscimol nor L-PIA prevented the increase of REMS latency induced by L-NAME 92.0 micromol/kg, s.c. Our findings tend to indicate that the change of behavioral state observed after systemic or intra-DRN administration of L-NAME is partly related to the reduction of GABA and adenosine at critical sites in the CNS.

Adenosine inhibits norepinephrine release in the postischemic rat heart: the mechanism of neuronal stunning.

OBJECTIVE: Numerous studies support the concept of impaired postischemic sympathetic neurotransmission in the heart. We hypothesized that postischemic neuronal dysfunction (neuronal stunning) is caused by a transient suppression of exocytotic norepinephrine (NE) release from sympathetic nerve terminals. Furthermore, we assessed the role of presynaptic adenosine-receptors and alpha2-adrenoceptors in neuronal stunning. METHODS AND RESULTS: Exocytotic NE release was induced by two electrical field stimulations (S(1) and S(2)) in isolated perfused rat hearts. S(1) was performed under baseline conditions and S(2) either during or following intervention. Results are expressed as mean S(2)/S(1) ratios+/-S.E.M. Stepwise increase of global ischemic periods (10, 20, and 30 min) induced a progressive suppression of NE release in the postischemic hearts, which was reversible during reperfusion. Both the degree and duration of NE suppression was dependent on the extent of the preceding ischemic period. Following 10-min ischemia complete recovery of NE release was achieved after 5-min reperfusion (1.07+/-0.12), whereas 5-min reperfusion did not restore NE release after 30 min (0.36+/-0.07) of ischemia. The adenosine-receptor antagonists 8-phenyltheophylline (8-PT; non-selective) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; adenosine A1-receptor subtype selective) significantly increased NE release after 30-min ischemia and 5-min reperfusion (0.78+/-0.06 and 0.64+/-0.07), while in the same experimental protocol blockade of alpha2-adrenoceptors by yohimbine failed to restore the postischemic release (0.24+/-0.06). In non-ischemic hearts the adenosine analogue R(-)N(6)-(2-phenylisopropyl)adenosine (R-PIA) resulted in a marked suppression of NE release (0.61+/-0.07). The inhibitory effect of R-PIA and 2-chloro-N(6)-cyclopentyladenosine (CCPA; adenosine A1-receptor subtype selective agonist) persisted 5 min after cessation of R-PIA (0.62+/-0.05) and CCPA (0.58+/-0.04). Activation of alpha2-adrenoceptors by 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14,304) also caused a reduction of NE release (0.50+/-0.02), but the release increased to control levels 5 min after cessation of UK 14,304 (0.90+/-0.06). CONCLUSIONS: The results establish the phenomenon of neuronal stunning in terms of a postischemic suppression of exocytotic NE release and provide evidence that neuronal stunning is mediated by endogenous adenosine through activation of presynaptic adenosine A1-receptors.