Analgesic properties of aqueous leaf extract of Haematostaphis barteri: involvement of ATP-sensitive potassium channels, adrenergic, opioidergic, muscarinic, adenosinergic and serotoninergic pathways.

BACKGROUND: Pain is the most common cause of patients seeking medical advice as a result of its association with different pathologies. This study evaluated the antinociceptive property of Haematostaphis barteri as well as the possible mechanism(s) associated with its antinociceptive property. METHODS: Mice were administered H. barteri (30-300 mg kg-1; p.o.), followed by intraplantar injection of 10 µL of 5% formalin into the hind paws. The pain score was determined for 1 h in the formalin test. The possible nociceptive pathways involved in the antinociceptive action of H. barteri were determined by pre-treating mice with theophylline (5 mg kg-1, a non-selective adenosine receptor antagonist), naloxone (2 mg kg-1, a non-selective opioid receptor antagonist), glibenclamide (8 mg kg-1; an ATP-sensitive K+ channel inhibitor), and atropine (3 mg kg-1; non-selective muscarinic antagonist). RESULTS: H. barteri (30-300 mg kg-1) significantly and dose dependently precluded both first and second phases of nociception. Pre-treatment with naloxone had no effect on the analgesic activities of H. barteri in the first phase. Again, pre-treatment with atropine and glibenclamide did not significantly reverse the neurogenic antinociception of the extract in phase 1. However, theophylline reversed the analgesic effect of the extract in the first phase. In phase 2, theophylline had no effect on the analgesic activities of the extract. Naloxone, atropine, and glibenclamide significantly blocked the antinociception of H. barteri in the inflammatory phase of the formalin test. CONCLUSIONS: H. barteri possesses antinociceptive property mediated via the opioidergic, adrenergic, muscarinic, ATP-sensitive K+ channels, and adenosinergic nociceptive pathways.

The potential of alkaline phosphatase as a treatment for sepsis-associated acute kidney injury.

Sepsis-associated acute kidney injury (AKI) is associated with a high attributable mortality and an increased risk of developing chronic kidney failure in survivors. As a successful therapy is, as yet, unavailable, a pharmacological treatment option is clearly warranted. Recently, two small phase II clinical trials demonstrated beneficial renal effects of bovine-derived alkaline phosphatase administration in critically ill patients with sepsis-associated AKI. The rationale behind the renal protective effects remains to be fully elucidated, but is likely to be related to dephosphorylation and thereby detoxification of detrimental molecules involved in the pathogenesis of sepsis-associated AKI. A potent candidate target molecule might be endotoxin (lipopolysaccharide) from the cell wall of Gram-negative bacteria, which is associated with the development of sepsis and becomes nontoxic after being dephosphorylated by alkaline phosphatase. Another target of alkaline phosphatase could be adenosine triphosphate, a proinflammatory mediator released during cellular stress, which can be converted by alkaline phosphatase into the tissue-protective and anti-inflammatory molecule adenosine. Human recombinant alkaline phosphatase, a recently developed replacement for bovine-derived alkaline phosphatase, has shown promising results in the preclinical phase. As its safety and tolerability were recently confirmed in a phase I clinical trial, the renal protective effect of human recombinant alkaline phosphatase in sepsis-associated AKI shall be investigated in a multicenter phase II clinical trial starting at the end of this year.

[Adenosine and homeostatic control of sleep. Actions in target structures of the sleep-wake circuits]. / Adenosina y control homeostático del sueño. Acciones en estructuras diana de los circuitos de vigilia y sueño.

Sleep homeostasis occurs during prolonged wakefulness. Drowsiness and sleep pressure are its behavioral manifestations and, when sleep is allowed, there is a sleep rebound of sufficient duration and intensity to compensate for the previous deprivation. Adenosine is one of the molecules involved in sleep homeostasic regulation. Caffeine and theophylline, stimulants widely consumed by the humans, are antagonists. It is an endogenous factor, resulting from ATP metabolism in neurons and glia. Adenosine accumulates in the extracellular space, where it can exert regulatory actions on the sleep-wakefulness cycle circuits. Adenosine acts through the purinergic receptors A1 and A2. This paper reviews: 1) the metabolic pathways of cerebral adenosine, and the mechanisms of its release by neurons and glia to the extracellular space; 2) the actions of adenosine and its antagonists in regions of the central nervous system related to wakefulness, non-REM sleep, and REM sleep, and 3) the synaptic mechanisms involved in these actions.

Adenosine sensory transduction pathways contribute to activation of the sensory irritation response to inspired irritant vapors.

The molecular mechanisms through which sensory irritants stimulate nasal trigeminal nerves are poorly understood. The current study was aimed at evaluating the potential contribution of purinergic sensory transduction pathways in this process. Aerosols of 4-36 mM adenosine 5'-triphosphate (ATP) and adenosine both acted as sensory irritants. Large dose capsaicin pretreatment to induce degeneration of transient receptor potential vanilloid type-1 (TRPV1)-expressing C fibers greatly reduced, but did not abolish, the sensory irritation response to ATP aerosol and was without effect on the response to adenosine aerosol, indicating that ATP acts largely on capsaicin-sensitive (primarily C fibers) and adenosine acts on capsaicin-insensitive (primarily Adelta fibers) nerves. The response to adenosine was diminished by pretreatment with the broad-based adenosine receptor antagonist theophylline (20 mg/kg) and A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 mg/kg), providing evidence that adenosine stimulates capsaicin-insensitive nerves via the A1 receptor. The sensory irritation responses to 275 ppm styrene and 110 ppm acetic acid vapors were significantly reduced by theophylline pretreatment suggesting a role for adenosine signaling pathways in activation of the sensory irritant response by these vapors. If sensory nerves are activated by mediators that are released from injured airway mucosal cells, then nasal sensory nerve activation may be a reflection of irritant-induced alterations in airway cell integrity.

Purine P1 receptor-dependent immunostimulatory effects of antiviral acyclic analogues of adenine and 2,6-diaminopurine.

Acyclic nucleoside phosphonates are widely recognised antivirals. The oral prodrugs of prototype compounds, e.g., 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), and 9-(R)-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] were approved by FDA for treatment of hepatitis B (Hepsera), and acquired immunodeficiency syndrome (AIDS) (Viread), respectively. A number of acyclic nucleoside phosphonates possess immunostimulatory activity. The present experiments demonstrate that activation of cytokine and chemokine secretion is mediated by adenosine receptors. Included in the study were 9-(R)-[2-(phosphonomethoxy)propyl]adenine [tenofovir], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, and N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine. All of them activate secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5), and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) in murine macrophages. With exception of MIP-1alpha, the effects were inhibited by antagonists of adenosine A(1), A(2B), and A(3) receptors (not by adenosine A(2A) receptor antagonist). The adenosine A(1) receptor antagonist inhibited TNF-alpha, IL-10, and RANTES, adenosine A(2B) receptor antagonist inhibited TNF-alpha and RANTES, and adenosine A(3) receptor antagonist inhibited IL-10 and RANTES. The suppression is due to decreased transcription of cytokine mRNA. It may be suggested that acyclic nucleoside phosphonates are nonspecific ligands for purine P(1) receptors.

P2Y purinergic potentiation of glucose-induced insulin secretion and pancreatic beta-cell metabolism.

Purine nucleotides and their analogs increase insulin secretion through activation of pancreatic beta-cell P2Y receptors. The present study aimed at determining the role of glucose metabolism in the response to P2Y agonists and whether ATP-activated K+ channels (KATP channels) are involved in this response. The experiments were performed in the rat isolated pancreas, perfused with a Krebs-bicarbonate buffer supplemented with 2 g/l bovine serum albumin under dynamic glucose conditions from 5 mmol/l baseline to 11 mmol/l. ADPbetaS (0.5 micromol/l) was selected as a stable and selective P2Y agonist. This compound, ineffective on the 5 mmol/l glucose background, induced a significant threefold increase in insulin release triggered by the glucose challenge. The effect of ADPbetaS was markedly reduced (P <0.001) in the presence of an inhibitor of glucose metabolism. In addition to glucose, the ADP analog also amplified the beta-cell insulin response to 15 mmol/l methyl pyruvate (P <0.05), but it was ineffective on the insulin response to 2.5 mmol/l methyl succinate. A nonmetabolic stimulus was applied using tolbutamide (185 micromol/l). Insulin secretion induced by the KATP channel blocker was strongly reinforced by ADPbetaS (P <0.001), which prompted us to check a possible interplay of KATP channels in the effect of ADPbetaS. In the presence of diazoxide 250 micromol/l and 21 mmol/l KCl, ADPbetaS still amplified the second phase of glucose-induced insulin secretion (P <0.001). We conclude that P2Y receptor activation is able to promote insulin secretion through a mechanism, involving beta-cell metabolism and a rise in intracellular calcium; this effect does not result from a direct inhibitory effect on KATP channels.

Reduced startle habituation and prepulse inhibition in mice lacking the adenosine A2A receptor.

Adenosine and dopamine receptors interact in the CNS to modulate behaviour, including sensorimotor gating. Prepulse inhibition (PPI) has been suggested to be an operational measure of sensorimotor gating. PPI and startle habituation are disrupted in patients with schizophrenia. In experimental animals, both parameters are modulated by dopaminergic and adenosine receptor agonists and antagonists. In the present study, we measured PPI and startle habituation in mice that lack functional adenosine A(2A) receptors. Startle amplitudes, startle habituation and PPI were significantly reduced in mice homozygous null for the adenosine A(2A) receptor (A(2A)(-/-)). In addition, differential effects of amphetamine and MK-801 on startle amplitude, startle habituation and PPI were observed between A(2A)(-/-) and wildtype controls. These data support the involvement of adenosine A(2A) receptors in regulation of PPI and startle habituation.

Effect of taurine deficiency on adenosine receptor-mediated relaxation of the rat aorta.

We recently demonstrated that chronic taurine supplementation or deficiency causes alterations in reactivity of the rat aorta to several vasoactive agents. In the present investigation, we examined the effects beta-alanine-induced endogenous taurine deficiency on the mechanical responsiveness of the isolated rat aorta to adenosine receptor stimulation with 2-chloroadenosine (CAD), 5'-N-ethylcarboxyamidoadenosine (NECA), and N(6)-cyclopentyladenosine (CPA). The adenosine analogs produced concentration-dependent (1 x 10(-9)-3 x 10(-3) M) relaxations of aortas from both control and beta-alanine-treated rats with the rank order of potencies NECA>CAD>CPA, which was consistent with A(2) receptor identification. CAD and NECA induced both endothelium-dependent and -independent relaxations of the aortas. The endothelium-dependent responses to both agents and the independent responses to CAD were significantly attenuated by beta-alanine treatment. The relaxation responses of the aortas from control and taurine-deficient rats to CAD and NECA were markedly antagonized by ZM241385 (10(-5) M), suggesting the involvement of A(2A) adenosine receptors. Further, N-nitro-L-arginine methyl ester (L-NAME; 10(-5) M) significantly attenuated the endothelium-mediated relaxation produced by CAD and NECA in both groups. However, the inhibitory effect of L-NAME was less on the beta-alanine-treated tissues, providing evidence that the effect of taurine deficiency was linked to a reduction in nitric oxide generation. As in the aorta, CAD produced both endothelium-dependent and -independent relaxation responses in the rat superior mesenteric artery, and both responses were inhibited by chronic beta-alanine treatment, suggesting that not only similar responses can be generated by a given adenosine agonist in different vascular beds, but also beta-alanine treatment modulates these responses. On the other hand, while CPA elicited only endothelium-independent aortic relaxation, this response was not altered by taurine deficiency. The results indicate that endogenous taurine deficiency causes differential inhibitory effects on adenosine receptor-mediated vasorelaxation, depending upon the agonists used. Given the recognized role of adenosine in the vasculature, these alterations suggest taurine-mediated modulation of blood flow regulation.

Study of an adenosine A1 receptor agonist on trigeminally evoked dural blood vessel dilation in the anaesthetized rat.

The purpose of this study was to use intravital microscopy to determine the effect of a selective adenosine A1 receptor agonist, GR79236 (1, 3 and 10 microg/kg i.v.), on neurogenic dural blood vessel dilation in anaesthetized rats. Vasodilation was evoked either by electrical stimulation of perivascular trigeminal nerves or by intravenous CGRP. GR79236 (1-10 microg/kg i.v.) caused a dose-dependent inhibition of neurogenic vasodilation, but had no significant effect on dural vasodilation caused by CGRP. GR79236 (1-3 microg/kg i.v.) had no effect on basal dural vessel diameter, but caused transient dose-dependant bradycardia and hypotension. Bradycardia was more prolonged following 10 microg/kg i.v. GR79236. Pre-treatment with the adenosine A1 receptor antagonist DPCPX (1 mg/kg i.v.) prevented the inhibitory effect of GR79236 (10 microg/kg i.v.) on neurogenic vasodilation as well as GR79236-induced bradycardia and hypotension. These data suggest that the inhibition of neurogenic vasodilation by GR79236 is mediated via the activation of prejunctional adenosine A1 receptors. Provided the systemic cardiovascular effects could be limited, such a mechanism may offer a novel approach to migraine therapy.

The involvement of nitric oxide on the adenosine A(2) receptor-induced cardiovascular inhibitory responses in the posterior hypothalamus of rats.

This study was performed to investigate the putative relationship between nitric oxide (NO) and adenosine A(2) receptors on central cardiovascular regulation in the posterior hypothalamus of rats. Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague-Dawley rats. Injection of adenosine A(2) receptor agonist 5'-(N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 5 nmol) produced a dose-dependent decrease of blood pressure and heart rate. Pretreatment with adenosine A(2) receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 nmol) blocked the depressor and bradycardiac effects of CPCA (5 nmol). Pretreatment with soluble guanylate cyclase inhibitor LY-83,583 (5 nmol) attenuated the depressor and bradycardiac effects of CPCA (5 nmol). In addition, pretreatment with NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (40 nmol) attenuated the depressor and bradycardiac responses of CPCA (5 nmol). These results suggest that adenosine A(2) receptor in the posterior hypothalamus plays an inhibitory role in central cardiovascular regulation and that NO participates in the inhibitory response induced by adenosine A(2) receptor stimulation in the posterior hypothalamus.

Neuroprotective effects of hyperthermic preconditioning on infarcted volume after middle cerebral artery occlusion in rats: role of adenosine receptors.

OBJECTIVE: There are still only a limited number of studies regarding the neuroprotective effects of hyperthermic preconditioning on regional brain ischemia or regarding the role of adenosine A1 receptors in such pretreatment. We examined the effects of hyperthermic pretreatment on infarcted volume after middle cerebral artery occlusion (MCAO), as well as the contribution of A1 receptors, to the responses in rats. DESIGN: Prospective, randomized animal study. SETTINGS: An animal research laboratory in a medical university. SUBJECTS: Male Wistar rats (200-250 g). INTERVENTION: All animals were anesthetized with isoflurane during each pretreatment, as well as for MCAO. The animals were assigned as follows: (i) sham-control group (n = 8), which was maintained at normothermia (37 +/- 0.2 degrees C pericranial temperature) for 15 mins, then kept in an awake state for 0.5, 3, 6, 18, 24, or 48 hrs before 2-hr MCAO; (ii) hyperthermia group (n = 8), which was subjected to 42 +/- 0.5 degrees C for 15 mins, and then received the same treatment as the sham group; (iii) DPCPX (a selective central adenosine receptor antagonist)-treated control group, which was given the agent before normothermia pretreatment, then kept for a recovery time of 0.5 or 24 hrs (n = 8 in each group) before MCAO; (iv) DPCPX plus hyperthermia-treated group, which was administered the agent at the same dose as the control before hyperthermic exposure, then selected for each recovery time (n = 8 in each group) before MCAO; (v) DPCPX-ischemic group, to which the agent was administered before MCAO (n = 8); and (vi) vehicle-ischemic group, in which peanut oil as a vehicle, instead of DPCPX, was injected before MCAO (n = 8). Values are expressed as mean +/- se. Statistical analysis was done by analysis of variance, followed by Scheffe's F test, Mann-Whitney U test, or the chi-square test as appropriate (p <.05). MAIN RESULTS: The infarcted volume in hyperthermic animals kept for 18 or 24 hrs before the occlusion procedure was significantly smaller than in the sham controls, but not in rats kept for 0.5, 3.0, 6.0, and 48 hrs. DPCPX partially reversed the reduction in infarcted volume that was induced by hyperthermic preconditioning after focal ischemia, whereas the agent itself did not affect the volume after ischemia. CONCLUSION: These data indicate that hyperthermic pretreatment reduces the effects on MCAO-induced cerebral infarction, possibly via a partial mediation of the central adenosine receptors in the brain. The results also suggest a need for further studies to define the relationship between heat shock proteins and central adenosine receptors in preconditioning.

Adenosine receptor-mediated control of in vitro release of pain-related neuropeptides from the rat spinal cord.

Although it is well established that adenosine exerts antinociceptive effects at the spinal level in various species including human, the mechanisms responsible for such effects are still a matter of debate. We presently investigated whether adenosine-induced antinociception might possibly be related to an inhibitory influence of this neuromodulator on the spinal release of neuropeptides implicated in the transfer and/or control of nociceptive signals. For this purpose, the K(+)-evoked overflow of substance P-, calcitonin gene-related peptide (CGRP)- and cholecystokinin-like materials was measured from slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid supplemented with increasing concentrations of various adenosine receptor ligands. The data showed that stimulation of adenosine A(1) and (possibly) A(3) receptors, but not A(2A) receptors, exerted an inhibitory influence on the spinal release of CGRP-like material. In contrast, none of the adenosine A(1), A(2A) and A(3) receptor agonists tested within relevant ranges of concentrations significantly affected the release of substance P- and cholecystokinin-like materials. These results support the idea that adenosine-induced antinociception at the spinal level might possibly be caused, at least partly, by the stimulation of inhibitory adenosine A(1) receptors located presynaptically on primary afferent fibres containing CGRP but not substance P.

Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart.

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (SPT; 100 microM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 microM) or reactive blue (RB; 10 microM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (50 microM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5'-nucleotidase inhibitor, alpha,beta-methylene adenosine diphosphate (AMP-CP; 100 microM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.

Adenosine attenuates reperfusion-induced apoptotic cell death by modulating expression of Bcl-2 and Bax proteins.

This study tests the hypothesis that infarct reduction with adenosine (Ado) is associated with inhibition of apoptotic cell death by modulating expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and reducing neutrophil accumulation. In three groups of dogs, the left anterior descending coronary artery was occluded for 60 min and reperfused for 6 h. Either saline (Control, n=8), Ado (140 microg/kg/min, n=8) or CGS21680, an adenosine A2A receptor analogue, (0.2 microg/kg/min, n=7) were infused during the first 2 h of reperfusion. Myocardial apoptosis was detected by histological TUNEL staining and DNA laddering. Expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. Neutrophil localization was detected by immunohistochemistry with monoclonal anti-neutrophil CD18 antibody. There was no group difference in collateral blood flow (colored microspheres) during ischemia. Intra-left atrial administration of Ado and CGS21680 significantly decreased infarct size from 26+/-2% in Control to 13+/-1%* and 16+/-3%*, respectively. TUNEL positive cells in the peri-necrotic zone of the ischemic myocardium were also significantly reduced from 16+/-2% in Control group to 9+/-1%* and 10+/-2%*, respectively, consistent with the absence of DNA laddering in these two groups. Densitometrically, Ado and CGS21680 at reperfusion significantly increased the expression (% of normal myocardium) of downregulated Bcl-2 from 45+/-6% in Control group to 78+/-12%* and 69+/-10%*, respectively, and attenuated expression of upregulated Bax from 198+/-16% in Control group to 148+/-10%* and 158+/-12%*, respectively. Furthermore, the number of positive CD18 cells (mm(2) myocardium), which was significantly correlated with TUNEL positive cells in peri-necrotic zone, was significantly reduced from 403+/-42 in Control group to 142+/-18* in Ado group and 153+/-20%* in CGS21680 group, respectively. In conclusion, the present study suggests that inhibition of apoptosis by Ado at reperfusion involves alterations in anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and neutrophil accumulation, primarily mediated by an adenosine A2A receptor. * P<0.05 v Control group.

Stimulation of adenosine A1 receptors attenuates dopamine D1 receptor-mediated increase of NGFI-A, c-fos and jun-B mRNA levels in the dopamine-denervated striatum and dopamine D1 receptor-mediated turning behaviour.

Adenosine A1 receptors antagonistically and specifically modulate the binding and functional characteristics of dopamine D1 receptors. In the striatum this interaction seems to take place in the GABAergic strionigro-strioentopeduncular neurons, where both receptors are colocalized. D1 receptors in the strionigro-strioentopeduncular neurons are involved in the increased striatal expression of immediate-early genes induced by the systemic administration of psychostimulants and D1 receptor agonists. Previous results suggest that a basal expression of the immediate-early gene c-fos tonically facilitates the functioning of strionigro-strioentopeduncular neurons and facilitates D1 receptor-mediated motor activation. The role of A1 receptors in the modulation of the expression of striatal D1 receptor-regulated immediate-early genes and the D1 receptor-mediated motor activation was investigated in rats with a unilateral lesion of the ascending dopaminergic pathways. The systemic administration of the A1 agonist N6-cyclopentyladenosine (CPA, 0.1 mg/kg) significantly decreased the number of contralateral turns induced by the D1 agonist SKF 38393 (3 mg/kg). Higher doses of CPA (0.5 mg/kg) were necessary to inhibit the turning behaviour induced by the D2 agonist quinpirole (0.1 mg/kg). By using in situ hybridization it was found that CPA (0.1 mg/kg) significantly inhibited the SKF 38393-induced increase in the expression of NGFI-A and c-fos mRNA levels in the dopamine-denervated striatum. The increase in jun-B mRNA expression could only be inhibited with the high dose of CPA (0.5 mg/kg). A stronger effect of the A1 agonist was found in the ventral striatum (nucleus accumbens) compared with the dorsal striatum (dorsolateral caudate-putamen). The results indicate the existence of antagonistic A1-D1 receptor-receptor interactions in the dopamine-denervated striatum controlling D1 receptor transduction at supersensitive D1 receptors.

Paeoniflorin reverses guanethidine-induced hypotension via activation of central adenosine A1 receptors in Wistar rats.

1. Intravenous injection of paeoniflorin, a glycoside purified from the root of Paeonia lactiflora, reversed guanethidine-induced hypotension in Wistar rats. 2. Pretreatment with the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine inhibited this effect of paeoniflorin in a dose-dependent manner. 3. The action of paeoniflorin was not modified by 8-(p-sulfophenyl)theophylline, the polar antagonist of the adenosine A1 receptor, which is not able to enter the central nervous system. 4. We conclude that paeoniflorin can reverse guanethidine-induced hypotension via activation of adenosine A1 receptors in the brain of Wistar rats.

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery.

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.

Adenosine A1 receptor-dependent and -independent effects of the allosteric enhancer PD 81,723.

The 2-amino-3-benzoylthiophene PD 81,723 has been shown to exhibit allosteric enhancement of adenosine A1 receptor binding and function. The aim of this study was to clarify the mechanism of this effect using membranes purified from rat brain and Chinese hamster ovary (CHO)-A1 cells that stably express the rat adenosine A1 receptor as well as intact CHO-A1 and nontransfected CHO cells. In membranes containing 100 microM magnesium, (2-amino-4, 5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81, 723) significantly increased the affinity of the adenosine A1 receptor agonist, cyclopentyladenosine, for the low-affinity receptor without affecting high-affinity binding or Bmax. In intact cells, PD 81,723 inhibited basal adenylyl cyclase (AC) activity as well as forskolin-, cholera toxin-, and pertussis toxin-stimulated AC activity in CHO-A1 and CHO cells. Basal AC activity was inhibited 49% in CHO and 82% in CHO-A1 cells by 30 microM PD 81,723. In CHO-A1 cells, half-maximal inhibition of forskolin-stimulated AC occurred at 5 microM PD 81,723 compared to 10 microM in CHO cells. Cholera toxin-stimulated AC was reduced 90% in both CHO and CHO-A1 cells by 30 microM PD 81,723. At the same concentration of PD 81,723, pertussis toxin-stimulated AC activity was reduced 86% (CHO-A1) and 77% (CHO). [3H]forskolin was displaced from purified rat liver AC by PD 81,723 with an IC50 of 96 microM. These results demonstrate that two mechanisms appear to contribute to the observed effects of PD 81, 723. One mechanism is allosteric enhancement of adenosine A1 receptor function. Results from transfected and nontransfected cells suggest that PD 81,723 also inhibits AC directly by binding to the catalytic unit at or near the forskolin-binding site.

Adenosine A1 receptor-mediated excitation of nociceptive afferents innervating the normal and arthritic rat knee joint.

We tested the hypothesis that adenosine excites nociceptive primary afferents innervating the knee joint. Neuronal recordings were made from fine nerve filaments innervating the knee joint in rats anaesthetized with pentobarbitone. Drugs were injected close-arterially (i.a.) or into the articular space (i.art.). We studied normal and chronically inflamed arthritic joints, the latter 14-21 days after a single intra-articular injection of Freund's Complete Adjuvant, performed under halothane anaesthesia. Adenosine injected i.a. caused delayed (approximately 10 s) excitation of the majority of polymodal C-fibre afferents, and had similar effects when injected directly into the joint. Adenosine triphosphate (ATP) had biphasic effects on discharge, a fast (<1 s) excitation was followed by a delayed increase similar to that seen with adenosine. The adenosine A1 receptor agonists N6-cyclopentyladenosine (CPA) and N-[(1S,trans)-2-hydroxypentyl] adenosine (GR79236) also excited the C-fibre afferents. The A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) antagonized the responses evoked by adenosine, CPA, and the delayed increase seen after ATP, indicating that excitation of the nociceptive afferents was mediated via adenosine A1 receptors. Adenosine and ATP evoked delayed excitatory effects of similar magnitude, regardless of whether or not the knee joint was chronically inflamed. The increased basal discharge observed in arthritic joints was unaffected by DPCPX, which implies that the increase in spontaneous activity associated with arthritis is unlikely to involve tonically released adenosine. The results support the hypothesis that adenosine excites primary afferent nociceptive nerve terminals in the rat knee joint, an effect mediated by adenosine A1 receptors. ATP, adenosine, and A1 receptors may play a role in generating the peripheral nociceptive (pain) signal.