Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices.
(25/2044)
1. The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. 2. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. 3. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50-500 nM). 8-Phenyltheophylline (10 microM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12 +/- 4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. 4. In the time-interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 microM. 5. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode. (+info)
Caffeine, acting on adenosine A(1) receptors, prevents the extinction of cocaine-seeking behavior in mice.
(26/2044)
Drug-naive DBA/2 mice were trained to self-administer cocaine (40 microgram/kg/infusion) i.v. by nose poking. The number of nose-poke responses was higher in mice receiving response-contingent injections of cocaine (active group) than in yoked controls or in animals receiving response-contingent saline injections. Twenty-four hours after the training session (cocaine or saline self-administration), mice were injected i.p. with saline, cocaine, caffeine, 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX), 8-cyclopentyl theophylline (8-CPT), 5-amino-7-(2-phenylethyl)2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine (SCH 58261), or 9-chloro-2(2-furyl)[1,2, 4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943) and placed again in exactly the same operant boxes as during the training session but without response-contingent i.v. infusions. Saline injection elicited similar responding in animals from the active group and from the yoked control group. A low dose of cocaine (5 mg/kg) or caffeine (3 mg/kg), but not higher doses, produced greater responding in the active group than in the yoked control group during a single extinction trial. The adenosine A(1)-receptor antagonists DPCPX and 8-CPT and the nonselective antagonist CGS 15943 partially reproduced the effect of a low dose of caffeine on the cocaine-associated behavior in a dose-dependent manner and did not alter the nose-poke activity of yoked control mice in the extinction experiment. In contrast, the adenosine A(2A) antagonist SCH 58261, in doses above 1 mg/kg, reduced nose-poke activity equally in active and yoked control animals. This confirms that a drug from a different pharmacological class (adenosine-receptor antagonist) can induce behavior changes similar to the effects of the original self-administered drug (indirect dopamine-receptor agonist). The data also suggest that the effects of caffeine on cocaine-seeking behavior might be related to interaction with adenosine A(1) receptors, but not A(2A) receptors. (+info)
Amplification of the cyclic AMP response to forskolin in pheochromocytoma PC12 cells through adenosine A(2A) purinoceptors.
(27/2044)
In this study, we present evidence on the ability of endogenous adenosine to modulate adenylyl cyclase activity in intact PC12 cells. The adenosine receptor antagonists PD 115199, xanthine amine congener, 8-cyclopentyl-1,3-dipropylxanthine, 8-(p-sulfophenyl)theophylline, and 3,7-dimethyl-1-propargylxanthine inhibited 10 microM forskolin-induced cyclic AMP (cAMP) accumulation, with IC(50) values of 2.76 +/- 1.16 nM, 17.4 +/- 1.08 nM, 443 +/- 1. 03 nM, 2.00 +/- 1.01 microM, and 2.25 +/- 1.05 microM, respectively. Inhibition by 2.5 nM PD 115199 was only partially reversed by increasing forskolin concentrations up to 100 microM. The addition of PD 115199 with or 60 min after forskolin caused a comparable inhibition of forskolin effect over the next hour. Both exogenous adenosine (0.1 microM) and its precursor, AMP (10 and 100 microM), significantly enhanced forskolin-induced cAMP accumulation, whereas inosine was ineffective. Forskolin activity was also potentiated by the hydrolysis-resistant adenosine receptor agonists 5'-N-ethylcarboxamido adenosine and CGS 21680 (8.9- and 12.2-fold increase, respectively). Adenosine deaminase (1 U/ml) and 8-SPT (25 microM), which nearly abolished the response to 1 microM adenosine, also reduced cAMP accumulation caused by AMP (-78 and -54%, respectively). These results demonstrate that in PC12 cells, activation of adenylyl cyclase by forskolin is highly dependent on the occupancy of A(2A) adenosine receptors and that AMP potentially contributes to the amplification of forskolin response. (+info)
Contributions of adenosine receptor activation to the ocular actions of epinephrine.
(28/2044)
PURPOSE: Epinephrine is an effective drug for glaucoma treatment. However, the mechanisms responsible for the ocular hypotensive action of this compound are not completely understood. Adenosine is an autacoid released by all cells. This study evaluated the role of adenosine receptor activation in epinephrine-induced changes in ocular function. METHODS: Rabbits were pretreated topically with the moderately selective adenosine A1 antagonist 8-(p-sulfophenyl)theophylline (8-SPT) or the adenosine A2 antagonist 3,7-dimethyl-l-propargylxanthine (DMPX). Epinephrine (500 microg) was then administered, and intraocular pressures (IOPs), pupil diameters (PDs), or total outflow facility was evaluated. In a separate group of animals, epinephrine or vehicle was administered, and aqueous humor samples obtained to evaluate changes in aqueous humor purine levels by means of high-performance liquid chromatography. RESULTS: In control animals, epinephrine produced a biphasic change in IOP: an initial rise in IOP of approximately 1 mm Hg from 1/2 to 1 hour followed by significant reduction in IOP of 8 to 9 mm Hg from 3 to 5 hours postadministration. These animals also exhibited a significant increase in PD of 2 to 3 mm from 1/2 to 2 hours postadministration. Pretreatment with 8-SPT (1000 microg) enhanced the initial rise in IOP, while significantly inhibiting the ocular hypotensive response. Pretreatment with 8-SPT also significantly enhanced the epinephrine-induced increase in PD. Inhibition of the epinephrine-induced reduction in IOP by 8-SPT was dose-related with an IC50 of 446 microg. Administration of 8-SPT alone did not significantly alter IOP or PD. The A2 antagonist DMPX did not alter the epinephrine-induced change in IOP or PD. In rabbits pretreated with 8-SPT, the epinephrine-induced increase in outflow facility was significantly reduced by 60% when compared with those in rabbits treated with epinephrine alone. In vehicle-treated rabbits, aqueous humor adenosine and inosine levels were 2.7 +/- 0.38 and 29 +/- 4.2 ng/100 microl, respectively. Three hours after epinephrine administration, adenosine and inosine levels had significantly increased to 11 +/- 1.6 and 66 +/- 4.4 ng/100 microl, respectively. CONCLUSIONS: These results support the idea that in rabbits epinephrine administration stimulates adenosine release in the anterior segment. This rise in endogenous levels of adenosine then leads to the activation of ocular adenosine receptors and is in part responsible for the ocular hypotensive action of epinephrine. (+info)
Microembolization in pigs: effects on coronary blood flow and myocardial ischemic tolerance.
(29/2044)
Coronary microembolization has been reported to increase coronary blood flow (CBF) through adenosine release. Because adenosine may increase ischemic tolerance against infarction, we tested the hypothesis that myocardial microembolization, a common finding in patients with ischemic heart disease, induces cardioprotection. Additionally, because the use of microspheres is a common tool to measure tissue perfusion, the effects of small amounts of microspheres on CBF were examined. Using anesthetized pigs, we measured CBF with a transit time flow probe on the left anterior descending coronary artery (LAD). In six pigs the relationship between the amount of injected microspheres (0-40 x 10(6), 15 micrometer in diameter, left atrial injections) and the effect on CBF was examined. Coronary hyperemia occurred, which was linearly related to the amount of microspheres injected: maximal increase in CBF (%) = 2.8 +/- 1.5 (SE) + (5.8 +/- 0.7 x 10(-7) x number of injected microspheres). Because injection of 40 x 10(6) microspheres induced a long-lasting hyperemic response, which could be blocked by 8-p-sulfophenyl theophylline, ischemic tolerance was examined in five other pigs after two injections, each of 40 x 10(6) microspheres, at a 30-min interval. Six control pigs had no injections. Ischemic tolerance was evaluated by measuring infarct size (tetrazolium stain) as the percentage of area at risk (fluorescent particles) after 45 min of LAD occlusion followed by 2 h of reperfusion. Pretreatment by microspheres increased infarct size from 60 +/- 3% of area at risk in control animals to 84 +/- 6% (P < 0.05). The injection of microspheres induced a significant hyperemic flow response without causing necrosis by itself. We conclude that microembolization, evoking coronary hyperemia, does not improve but reduces myocardial ischemic tolerance against infarction in pigs. (+info)
Role of K(+)(ATP) channels and adenosine in regulation of coronary blood flow in the hypertrophied left ventricle.
(30/2044)
In the hypertrophied heart, increased extravascular forces acting to compress the intramural coronary vessels might require augmentation of metabolic vasodilator mechanisms to maintain adequate coronary blood flow. Vascular smooth muscle ATP-sensitive potassium (K(+)(ATP)) channel activity is important in metabolic coronary vasodilation, and adenosine contributes to resistance vessel dilation in the hypoperfused heart. Consequently, this study was performed to determine whether K(+)(ATP) channels and adenosine have increased importance in exercise-induced coronary vasodilation in the hypertrophied left ventricle. Studies were performed in dogs in which banding of the ascending aorta had resulted in a 66% increase in left ventricular mass in comparison with historic normal animals. Treadmill exercise resulted in increases of coronary blood flow that were linearly related to the increase of heart rate or rate-pressure product. During resting conditions, K(+)(ATP) channel blockade with glibenclamide caused a 17 +/- 5% decrease in coronary blood flow, similar to that previously observed in normal hearts. Unlike normal hearts, however, glibenclamide blunted the increase in coronary flow that occurred during exercise, causing a significant decrease in the slope of the relationship between coronary flow and the rate-pressure product. Adenosine receptor blockade with 8-phenyltheophylline did not alter coronary blood flow at rest or during exercise. Furthermore, even after K(+)(ATP) channel blockade with glibenclamide, the addition of 8-phenyltheophylline had no effect on coronary blood flow. This finding was different from normal hearts, in which the addition of adenosine receptor blockade after glibenclamide impaired exercise-induced coronary vasodilation. The data suggest that, in comparison with normal hearts, hypertrophied hearts have increased reliance on opening of K(+)(ATP) channels to augment coronary flow during exercise. Contrary to the initial hypothesis, however, adenosine was not mandatory for exercise-induced coronary vasodilation in the hypertrophied hearts either during control conditions or when K(+)(ATP) channel activity was blocked with glibenclamide. (+info)
Involvement of adenosine receptor, potassium channel and protein kinase C in hypoxic preconditioning of isolated cardiomyocytes of adult rat.
(31/2044)
A possible mechanism for hypoxic preconditioning of adult rat cardiomyocytes was pharmacologically investigated. Isolated cardiomyocytes in all experimental groups were incubated for 120 min under hypoxic conditions followed by 15-min reoxygenation (sustained H/R). Sustained H/R decreased rod-shaped cells. Exposure of the cardiomyocytes to 20-min of hypoxia/30-min reoxygenation (hypoxic preconditioning) attenuated the sustained H/R-induced decrease in rod-shaped cells. The effects of hypoxic preconditioning were abolished by treatment with the protein kinase C (PKC) inhibitor polymyxin B, but abolished by neither the adenosine A1/A2-antagonist sulfophenyl theophylline (SPT) nor the ATP-sensitive potassium channel (K(ATP) channel) blocker glibenclamide. In another series of experiments, cardiomyocytes were incubated without hypoxic preconditioning in the presence of either the PKC activator PMA, adenosine or K(ATP)-channel opener nicorandil and then subjected to sustained H/R. Treatment of the cells with PMA, adenosine or nicorandil mimicked the effects of hypoxic preconditioning. The effects of treatment with adenosine and nicorandil were abolished by polymyxin B treatment. Combined treatment with both SPT and glibenclamide abolished the effects of hypoxic preconditioning, whereas it failed to abolish PMA-induced cytoprotection. These results suggest that the activation of PKC in hypoxic preconditioned cardiomyocytes coupled independently with stimulation of adenosine receptor or opening of K(ATP) channel, either of which is fully enough to exert the cytoprotective effects. (+info)
Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.
(32/2044)
Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) >> N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s). (+info)