Role of A1 adenosine receptors in regulation of vascular tone.
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The vascular response to adenosine and its analogs is mediated by four adenosine receptors (ARs), namely, A(1), A(2A), A(2B), and A(3). A(2A)ARs and/or A(2B)ARs are involved in adenosine-mediated vascular relaxation of coronary and aortic beds. However, the role of A(1)ARs in the regulation of vascular tone is less well substantiated. The aim of this study was to determine the role of A(1)ARs in adenosine-mediated regulation of vascular tone. A(1)AR-knockout [A(1)AR((-/-))] mice and available pharmacological tools were used to elucidate the function of A(1)ARs and the impact of these receptors on the regulation of vascular tone. Isolated aortic rings from A(1)AR((-/-)) and wild-type [A(1)AR((+/+))] mice were precontracted with phenylephrine, and concentration-response curves for adenosine and its analogs, 5'-N-ethyl-carboxamidoadenosine (NECA, nonselective), 2-chloro-N(6)-cyclopentyladenosine (CCPA, A(1)AR selective), 2-(2-carboxyethyl)phenethyl amino-5'-N-ethylcarboxamido-adenosine (CGS-21680, A(2A) selective), and 2-chloro-N(6)-3-iodobenzyladenosine-5'-N-methyluronamide (Cl-IBMECA, A(3) selective) were obtained to determine relaxation. Adenosine and NECA (0.1 microM) caused small contractions of 13.9 +/- 3.0 and 16.4 +/- 6.4%, respectively, and CCPA at 0.1 and 1.0 microM caused contractions of 30.8 +/- 4.3 and 28.1 +/- 3.9%, respectively, in A(1)AR((+/+)) rings. NECA- and CCPA-induced contractions were eliminated by 100 nM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, selective A(1)AR antagonist). Adenosine, NECA, and CGS-21680 produced an increase in maximal relaxation in A(1)AR((-/-)) compared with A(1)AR((+/+)) rings, whereas Cl-IBMECA did not produce contraction in either A(1)AR((+/+)) or A(1)AR((-/-)) rings. CCPA-induced contraction at 1.0 microM was eliminated by the PLC inhibitor U-73122. These data suggest that activation of A(1)ARs causes contraction of vascular smooth muscle through PLC pathways and negatively modulates the vascular relaxation mediated by other adenosine receptor subtypes. (+info)
A1 antagonism in asthma: better than coffee?
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Adenosine is a ubiquitous biological mediator with the capacity to produce both pro- and anti-inflammatory effects in tissues. Proinflammatory and bronchoconstrictive actions of adenosine in the asthmatic lung are well recognized, with the latter being mediated, in part, through A(1) receptor activation on airway smooth muscle. In this issue of the JCI, Sun et al. report findings in adenosine deaminase-deficient mice that suggest the occurrence of anti-inflammatory actions of adenosine in the lung, mediated through A(1) adenosine receptors on macrophages. Here we discuss the history of the study of adenosine receptor ligands for asthma and how enhanced understanding of adenosine receptor biology may aid in the rational exploitation of these receptors as therapeutic targets. (+info)
Modulating effect of adenosine deaminase on function of adenosine A1 receptors.
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AIM: To study the modulating effect of adenosine deaminase (ADA) on the adenosine A1 receptor (A1R) in HEK293 cells stably expressing the human A1R. METHODS: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned A1R cDNA was sequenced and stably expressed in HEK293 cells. The modulating effect of adenosine deaminase on A1R was studied by using [3H]DPCPX binding assay and an intracellular calcium assay. RESULTS: HEK293 cells stably expressing human A1R were obtained. Saturation studies showed that the K(D) value and B(max) value of [3H]DPCPX were 1.6+/-0.2 nmol/L and 1.819+/-0.215 nmol/g of protein respectively, in the absence of ecto-ADA respectively, and 1.3+/-0.2 nmol/L and 1.992+/-0.130 nmol/g of protein in the presence of ecto-ADA respectively, suggesting that the K(D) value and B(max) value of [3H]DPCPX were unaffected by ecto-ADA. In the case of [3H]DPCPX competition curves obtained from intact cells or membranes, A1R agonist CCPA/[3H]DPCPX competition curve could be fitted well to a one-site model in the absence of ecto-ADA and a two-site model in the presence of ecto-ADA with a K(H) value of 0.74 (0.11+/-4.8) nmol/L (intact cells) or 1.8 (0.25+/-10) nmol/L (membrane) and a K(L) value of 0.94 (0.62+/-1.41) micromol/L (intact cells) or 0.77 (0.29+/-0.99) micromol/L (membrane). The K(L) value is not significantly different from the IC50 value of 0.84(0.57+/-1.23) micromol/L (intact cells) or 0.84 (0.63+/-1.12) micromol/L (membrane) obtained in the absence of ecto-ADA. Similar results were obtained from the CPA/[3H]DPCPX competition curve in the absence or presence of ecto-ADA on intact cells or membranes. Intracellular calcium assay demonstrated that the EC50 value of CPA were 10 (5+/-29) nmol/L and 94 (38+/-229) nmol/L in the presence or absence of ecto-ADA, respectively. CONCLUSION: A1R stably expressed in the HEK293 cells display a low affinity for agonists in the absence of ADA and high and low affinities for agonists in the presence of ADA. The presence of ADA may promote the signaling through the adenosine A1 receptor in HEK293 cells. (+info)
Effects of adenosine agonist R-phenylisopropyl-adenosine on halothane anesthesia and antinociception in rats.
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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. (+info)
Dynamics of fast synaptic excitation during trains of stimulation in myenteric neurons of guinea-pig ileum.
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Fast excitatory postsynaptic potentials (fEPSPs) occur in bursts in the myenteric plexus during evoked motor reflexes in the guinea-pig ileum in vitro. This study used electrophysiological methods to study fEPSPs during stimulus trains to mimic bursts of synaptic activity in vitro. The amplitude of fEPSPs or fast excitatory postsynaptic currents (EPSCs) declined (rundown) during stimulus trains at frequencies of 0.5, 5, 10 and 20 Hz. At 0.5 Hz, fEPSP or fEPSC amplitude declined by 50% after the first stimulus but remained constant for the remainder of the train. At 5, 10 and 20 Hz, synaptic responses ran down completely with time constants of 0.35, 0.21 and 0.11 s, respectively. Recovery from rundown occurred with a time constant of 7 s. Mecamylamine, a nicotinic cholinergic receptor antagonist, or PPADS, a P2X receptor antagonist, reduced fEPSP amplitude, but they had no effect on rundown. Responses caused by trains of ionophoretically applied ATP or ACh (to mimic fEPSPs) did not rundown. Blockade of presynaptic inhibitory muscarinic, adenosine A1, opioid, alpha2-adrenergic and 5-HT1A receptors or pertussis toxin (PTX) treatment did not alter rundown. Antidromic action potentials followed a 10-Hz stimulus train. Iberiotoxin (100 nM), a blocker of large conductance calcium activated K+ (BK) channels, did not alter rundown. These data suggest that synaptic rundown is not due to: (a) action potential failure; (b) nicotinic or P2X receptor desensitization; (c) presynaptic inhibition mediated by pertussis-toxin sensitive G-proteins, or (d) BK channel activation. Synaptic rundown is likely due to depletion of a readily releasable pool (RRP) of neurotransmitter. (+info)
Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.
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1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery. (+info)
The role of extracellular adenosine in regulating mossy fiber synaptic plasticity.
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Hippocampal mossy fiber synapses show unique molecular features and dynamic range of plasticity. A recent paper proposed that the defining features of mossy fiber synaptic plasticity are caused by a local buildup of extracellular adenosine (Moore et al., 2003). In this study, we reassessed the role of ambient adenosine in regulating mossy fiber synaptic plasticity in mouse and rat hippocampal slices. Synaptic transmission was highly sensitive to activation of presynaptic adenosine A1 receptors (A1Rs), which reduced transmitter release by >75%. However, most of A1Rs were not activated by ambient adenosine. Field potentials increased only by 20-30% when A1Rs were fully blocked with the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM). Moreover, blocking A1Rs hardly altered paired-pulse facilitation, frequency facilitation, or posttetanic potentiation. Frequency facilitation was similar in A1R-/- mice and when measured with NMDA receptor-mediated EPSCs in CA3 pyramidal cells in the presence of DPCPX. Additional experiments suggested that the results obtained by Moore et al. (2003) can partially be explained by their usage of a submerged recording chamber and elevated divalent cation concentrations. In conclusion, a reduction of the basal release probability by ambient adenosine does not underlie presynaptic forms of plasticity at mossy fiber synapses. (+info)
Novel mitogenic effect of adenosine on coronary artery smooth muscle cells: role for the A1 adenosine receptor.
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Adenosine is a vascular endothelial cell mitogen, but anti-mitogenic for aortic smooth muscle cells and fibroblasts when acting via the A2B adenosine receptor. However, we show that adenosine increases porcine coronary artery smooth muscle cell (CASMC) number, cellular DNA content, protein synthesis, and PCNA staining. RT-PCR analysis indicates that porcine CASMC express A1, A2A, A3, and barely detectable levels of A2B receptor mRNAs. The mitogenic effect of adenosine is mimicked by NECA, CCPA, and R-PIA, but not by CGS21680and 2-Cl-IB-MECA, and is inhibited by DPCPX, indicating a prominent role for the A1 receptor. This interpretation is supported by the finding that adenosine- and CCPA-induced DNA synthesis is significantly inhibited by pertussis toxin, but substantially potentiated by PD81723, an allosteric enhancer of the A1 receptor. When a cDNA encoding the porcine A1 receptor was cloned and expressed in COS-1 cells, A1 receptor pharmacology is confirmed. Anti-sense oligonucleotides to the cloned sequence dramatically suppress the mitogenic effect of adenosine and CCPA. Conversely, over-expression of the cloned A1 receptor in CASMC increases adenosine- and CCPA-induced DNA synthesis. Furthermore, stimulation with adenosine or CCPA of intact coronary arteries in an organ culture model of vascular disease increases cellular DNA synthesis, which was abolished by DPCPX. We conclude that adenosine acts as a novel mitogen in porcine CASMC that express the A1 adenosine receptor, possibly contributing to the development of coronary artery disease. (+info)