Role Of Adenosine Receptor Subtypes Research Articles

Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

BackgroundThe development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses.ResultsHere we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses.ConclusionThe three receptor sets considered (mAChR, AR and TrkB receptors) intervene in modulating the conditions of the competition between nerve endings, possibly helping to determine the winner or the lossers but, thereafter, the final elimination would occur with some autonomy and independently of postsynaptic maturation.

Role of adenosine receptor subtypes in methamphetamine reward and reinforcement

The neurobiology of methamphetamine (MA) remains largely unknown despite its high abuse liability. The present series of studies explored the role of adenosine receptors on MA reward and reinforcement and identified alterations in the expression of adenosine receptors in dopamine terminal areas following MA administration in rats. We tested whether stimulating adenosine A1 or A2A receptor subtypes would influence MA-induced place preference or MA self-administration on fixed and progressive ratio schedules in male Sprague–Dawley rats. Stimulation of either adenosine A1 or A2A receptors significantly reduced the development of MA-induced place preference. Stimulating adenosine A1, but not A2A, receptors reduced MA self-administration responding. We next tested whether repeated experimenter-delivered MA administration would alter the expression of adenosine receptors in the striatal areas using immunoblotting. We observed no change in the expression of adenosine receptors. Lastly, rats were trained to self-administer MA or saline for 14 days and we detected changes in adenosine A1 and A2A receptor expression using immunoblotting. MA self-administration significantly increased adenosine A1 in the nucleus accumbens shell, caudate-putamen and prefrontal cortex. MA self-administration significantly decreased adenosine A2A receptor expression in the nucleus accumbens shell, but increased A2A receptor expression in the amygdala. These findings demonstrate that MA self-administration produces selective alterations in adenosine receptor expression in the nucleus accumbens shell and that stimulation of adenosine receptors reduces several behavioral indices of MA addiction. Together, these studies shed light onto the neurobiological alterations incurred through chronic MA use that may aid in the development of treatments for MA addiction.

Cardioprotection of controlled and cardiac-specific over-expression of A(2A)-adenosine receptor in the pressure overload.

Adenosine binds to three G protein-coupled receptors (R) located on the cardiomyocyte (A1-R, A2A-R and A3-R) and provides cardiac protection during both ischemic and load-induced stress. While the role of adenosine receptor-subtypes has been well defined in the setting of ischemia-reperfusion, far less is known regarding their roles in protecting the heart during other forms of cardiac stress. Because of its ability to increase cardiac contractility and heart rate, we hypothesized that enhanced signaling through A2A-R would protect the heart during the stress of transverse aortic constriction (TAC). Using a cardiac-specific and inducible promoter, we selectively over-expressed A2A-R in FVB mice. Echocardiograms were obtained at baseline, 2, 4, 8, 12, 14 weeks and hearts were harvested at 14 weeks, when WT mice developed a significant decrease in cardiac function, an increase in end systolic and diastolic dimensions, a higher heart weight to body weight ratio (HW/BW), and marked fibrosis when compared with sham-operated WT. More importantly, these changes were significantly attenuated by over expression of the A2A-R. Furthermore, WT mice also demonstrated marked increases in the hypertrophic genes β-myosin heavy chain (β-MHC), and atrial natriuretic factor (ANF) – changes that are mediated by activation of the transcription factor GATA-4. Levels of the mRNAs encoding β-MHC, ANP, and GATA-4 were significantly lower in myocardium from A2A-R TG mice after TAC when compared with WT and sham-operated controls. In addition, three inflammatory factors genes encoding cysteine dioxygenase, complement component 3, and serine peptidase inhibitor, member 3N, were enhanced in WT TAC mice, but their expression was suppressed in A2A-R TG mice. A2A-R over-expression is protective against pressure-induced heart failure secondary to TAC. These cardioprotective effects are associated with attenuation of GATA-4 expression and inflammatory factors. The A2A-R may provide a novel new target for pharmacologic therapy in patients with cardiovascular disease.

Roles of adenosine receptor subtypes on the antinociceptive effect of sildenafil in rat spinal cord

We recently found that the antinociceptive effects produced by intrathecal administration of sildenafil, a phosphodiesterase 5 inhibitor, were reversed by a nonspecific adenosine receptor antagonist, suggesting that adenosine receptors are involved in sildenafil-induced antinociception. Four adenosine receptor subtypes have been identified: A 1, A 2A, A 2B, and A 3. We examined the involvement of spinal adenosine receptor subtypes in the antinociceptive effects of intrathecal sildenafil. Intrathecal catheters were implanted in male SD rats, and nociception was assessed using the formalin test, which consisted of a subcutaneous injection of 50 μl of 5% formalin solution into the hind paw. We examined the effects of an adenosine A 1 receptor antagonist (CPT), an adenosine A 2A receptor antagonist (CSC), an adenosine A 2B receptor antagonist (alloxazine), and an adenosine A 3 receptor antagonist (MRS 1220) on sildenafil-induced antinociception. Intrathecal sildenafil suppressed formalin-induced flinching during phases 1 and 2 of the test in a dose-dependent manner. Intrathecal CPT, CSC, alloxazine, and MRS 1220 all suppressed the antinociceptive effects of sildenafil during both phases of the formalin test. These results suggest that sildenafil is an effective treatment for acute pain and the facilitated pain state at the spinal level. Additionally, spinal adenosine A 1, A 2A, A 2B, and A 3 receptors may play a role in sildenafil-induced antinociception.

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS‐1 cells: The possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.

In vivo adenosine A2B receptor desensitization in guinea-pig airway smooth muscle: Implications for asthma

This study was aimed at characterizing the role of adenosine receptor subtypes in the contractility modulation of guinea-pig airway smooth muscle in normal and pathological settings. In vitro and in vivo experiments were performed by testing selective agonists and antagonists on isolated tracheal smooth muscle preparations and pulmonary inflation pressure, respectively, under normal conditions or following ovalbumin-induced allergic sensitization. In normal and sensitized animals, the adenosine A(2A)/A(2B) receptor agonist, NECA, evoked relaxing responses of isolated tracheal preparations precontracted with histamine, and such an effect was reversed by the adenosine A(2B) antagonist, MRS 1706, in the presence or in the absence of epithelium. The expression of mRNA coding for adenosine A(2B) receptors was demonstrated in tracheal specimens. In vitro desensitization with 100 microM NECA markedly reduced the relaxing effect of the agonist. In vivo NECA or adenosine administration to normal animals inhibited histamine-mediated bronchoconstriction, while these inhibitory effects no longer occurred in sensitized guinea-pigs. Adenosine plasma levels were significantly higher in sensitized than normal animals. In conclusion, our data demonstrate that: (i) adenosine A(2B) receptors are responsible for the relaxing effects of adenosine on guinea-pig airways; (ii) these receptors can undergo rapid adaptive changes that may affect airway smooth muscle responsiveness to adenosine; (iii) ovalbumin-induced sensitization promotes a reversible inactivation of adenosine A(2B) receptors which can be ascribed to homologous desensitization. These findings can be relevant to better understand adenosine functions in airways as well as mechanisms of action of asthma therapies targeting the adenosine system.

Role of Adenosine Receptor Subtypes in Rat Jejunum in Unfed State Versus Glucose-Induced Hyperemia

Adenosine is a key mediator in intestinal absorptive hyperemia. This study examines the role of adenosine receptor subtypes in the intestinal microvasculature at rest (unfed) and during glucose exposure. Intravital video microscopy was used to record vascular responses in the rat jejunum in unfed resting states versus active glucose absorption. Two series of experiments were performed: topical adenosine alone and with adenosine receptor antagonists, and topical glucose alone and with adenosine receptor antagonists. We found that distal premucosal arterioles were more reactive to adenosine than were larger inflow arterioles. The selective A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (200 nm), and the A2b receptor antagonist, alloxazine (60 microm), decreased the sensitivity and reactivity of the inflow and premucosal arterioles to adenosine, whereas the selective A2a receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) (200 nm) had no effect on inflow arteriole diameter and only slightly reduced the premucosal arteriolar sensitivity to adenosine. As previously observed, isotonic glucose caused vasodilation (24 +/- 3.4% of the control) in the distal premucosal arterioles. Conversely, premucosal arterioles did not dilate during exposure of the intestine to isotonic mannitol solution that is not actively absorbed. Adenosine A2a RA CSC and A2b RA alloxazine attenuated glucose-induced vasodilation, whereas adenosine A1 RA DPCPX completely abolished glucose-induced dilation. These findings suggest that resting tone in premucosal vessels appears to be responsive to adenosine mediation rather than inflow arteriolar tone; the adenosine A1, A2a, and A2b receptors all contribute to adenosine-mediated vasodilation in the intestine, with the greatest attenuation seen with A1 receptor antagonism; and other vasoactive mediators might also contribute to glucose-induced jejunal vasodilation, and interaction might exist between adenosine receptors and other mediators.

Specific subclass of adenosine receptors modulate release of EETs and superoxide in brain tissues

Three adenosine receptor types (A1, A2 and A3) are distributed in neurons, glia, endothelium and vascular smooth muscle. Two A2 receptor subclasses A2a and A2b are associated with vascular function. The adenosine receptor type mediating the formation of EETs or superoxide in the brain is unknown. We examined the role of adenosine receptor subtypes in EETs release and generation of superoxide in cultured astrocytes and in brains of A2a receptor knock mouse. Stimulation of astrocytes with the A2a agonist CGS-21680 (1 μM) induced a greater production of superoxide than that induced by the non-selective A2a and A2b agonist NECA (1 μM). The A2a receptor antagonist ZM 241385 (1 μM) reduced the CGS 21680-induced but not the NECA-induced superoxide generation. A2a receptor knockout reduced basal level of superoxide, whereas this A2a knock down had no effect on the increase in superoxide stimulated by CGS-21680 or NECA. Results of LC-MS analysis revealed that A2a receptor knock out markedly reduced basal release of EETs compared to that in the wild type. Stimulation of astrocytes with the A2a agonist CGS-21680 (1 μM) or with adenosine (1 μM) increased the release of EETs that was attenuated by the A2a receptor blockade but not by A2a knock out. These results indicate that A2a adenosine receptor is not the only subtype that is important for basal or adenosine evoked release of EETs or superoxide in the brain.

Roles of Adenosine Receptor Subtypes in the Antinociceptive Effect of Intrathecal Adenosine in a Rat Formalin Test

The contributions of adenosine receptor subtypes to antinociception produced by adenosine were determined at the spinal level. There are 4 types of adenosine receptors, namely A₁, A_2A, A_2B and A₃. The authors investigated the properties of the subtypes of spinal adenosine receptors in terms of nociceptive modulation. The nociceptive state was induced by subcutaneously injecting formalin solution (5%, 50 µl) into the hind paws of male Sprague-Dawley rats. After observing the effect of intrathecal adenosine during the formalin test, the effects of intrathecal adenosine A₁(CPT), A_2A (CSC), A_2B (alloxazine) and A₃(MRS 1220) receptor antagonists on the action of adenosine were examined. Intrathecal adenosine inhibited phase 2 flinching response without affecting phase 1 response. CPT, CSC, alloxazine and MRS 1220 antagonized the antinociceptive action of adenosine during phase 2 of the formalin test. These results suggest that spinal adenosine A₁, A_2A, A_2B and A₃receptors may play an important role in the antinociception of adenosine in the formalin-induced facilitated state.

Adenosine and cerebral ischemia: therapeutic future or death of a brave concept?

Numerous studies have consistently shown that agonist stimulation of adenosine A 1 receptors results in a significant reduction of morbidity and mortality associated with global and focal brain ischemia in animals. Based on these observations, several authors have suggested utilization of adenosine A 1 receptors as targets for the development of clinically viable drugs against ischemic brain disorders. Recent advent of adenosine A 1 receptor agonists characterized by lowered cardiovascular effects added additional strength to this argument. On the other hand, although cardioprotective, adenosine A 3 receptor agonists proved severely cerebrodestructive when administered prior to global ischemia in gerbils. Moreover, stimulation of adenosine A 3 receptors appears to reduce the efficacy of some of the neuroprotective actions mediated by adenosine A 1 receptors. The review discusses the possible role of adenosine receptor subtypes (A 1, A 2, and A 3) in the context of their involvement in the pathology of cerebral ischemia, and analyzes putative strategies for the development of clinically useful strategies based on adenosine and its receptors. It also stresses the need for further experimental studies before definitive conclusions on the usefulness of the adenosine concept in the treatment of brain ischemia can be made.

Differing Roles of Adenosine Receptor Subtypes in Retinal Ischemia-Reperfusion Injury in the Rat

Adenosine has been shown to be a major component of the retina's endogenous reaction to ischemia. In earlier studies, the significant changes in adenosine concentration that occur during ischemia and the ensuing reperfusion period were documented. While previous studies have shown that adenosine is a mediator of the changes in blood flow that occur in response to ischemia, hypoxia, and hypoglycemia in the retina, little is known about other functional effects that result from these changes in adenosine concentration. Accordingly, the influence of adenosine receptor blockade on the functional and histological outcome following ischemia in rats was examined. Specific antagonists of the adenosine A1 and A2a receptors were injected systemically, prior to ischemia of either 5, 30, or 60 min. The recovery of the electroretinogram a and b waves was followed for up to 7 days after ischemia, and retinal structure was examined by light microscopy. The adenosine A1 receptor antagonist DPCPX attenuated recovery after retinal ischemia of either 5 or 30 min, while the A2a receptor antagonist CSC dramatically protected retinal function and structure even with ischemia lasting up to 60 min. It was concluded that blockade of the A2a receptor, possibly combined with stimulation of the A1 receptor, may represent a potential new strategy for the prevention of ischemic damage in the retina.

Adenosine and cerebral ischemia: therapeutic future or death of a brave concept?

Numerous studies have consistently shown that agonist stimulation of adenosine A 1 receptors results in a significant reduction of morbidity and mortality associated with global and focal brain ischemia in animals. Based on these observations, several authors have suggested utilization of adenosine A 1 receptors as targets for the development of clinically viable drugs against ischemic brain disorders. Recent advent of adenosine A 1 receptor agonists characterized by lowered cardiovascular effects added additional strength to this argument. On the other hand, although cardioprotective, adenosine A 3 receptor agonists proved severely cerebrodestructive when administered prior to global ischemia in gerbils. Moreover, stimulation of adenosine A 3 receptors appears to reduce the efficacy of some of the neuroprotective actions mediated by adenosine A 1 receptors. The review discusses the possible role of adenosine receptor subtypes (A 1, A 2, and A 3) in the context of their involvement in the pathology of cerebral ischemia, and analyzes putative strategies for the development of clinically useful strategies based on adenosine and its receptors. It also stresses the need for further experimental studies before definitive conclusions on the usefulness of the adenosine concept in the treatment of brain ischemia can be made.

Role of adenosine receptor subtypes in neural stunning of sympathetic coronary innervation.

Adenosine plays an important role in postischemic dysfunction of cardiac sympathetic nerves because exogenously infused adenosine produces and adenosine deaminase prevents "neural stunning." We examined whether adenosine acts via a specific receptor mechanism to produce neural stunning. Anesthetized dogs were treated with propranolol to attenuate increases in coronary flow due to adrenergic stimulation of myocardial metabolism. A 15-min occlusion of the left anterior descending coronary artery (LAD) attenuated subsequent LAD coronary vasoconstriction to bilateral sympathetic stimulation during reperfusion by 75% (P < 0.05). Coronary infusion of the adenosine-receptor antagonist 8-p-sulfophenyltheophylline (nonspecific), 8-cyclopentyl-1,3-dipropylxanthine (A1 specific), or 3,7-dimethyl-1-propagylxanthine (A2 specific) during LAD occlusion prevented the attenuation of sympathetic coronary constriction. In separate experiments, either the specific adenosine agonist N6-cyclopentyl-adenosine (A1 specific) or CGS-21680 (A2 specific) or a combination of both agonists was infused into the LAD for 15 min. Neither agonist alone attenuated subsequent sympathetic coronary constriction. In contrast, 15 min after the combined administration of both agonists, sympathetic vasoconstriction was reduced. We conclude that adenosine is capable of attenuating neurogenic coronary constriction through a receptor-mediated mechanism. Activation of more than one receptor subtype is necessary to produce neural stunning.

Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.

Preconditioning with brief ischemia before a sustained period of ischemia reduces infarct size in the perfused heart. A cultured chick ventricular myocyte model was developed to investigate the role of adenosine receptor subtypes in cardiac preconditioning. Brief hypoxic exposure, termed preconditioning hypoxia, prior to prolonged hypoxia, protected myocytes against injury induced by the prolonged hypoxia. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with C1-IB-MECA can replace preconditioning hypoxia and simulate preconditioning, with a maximal effect at 100 nM. While activation of the A2a receptor by 1 microM CGS21680 could not mimic preconditioning, its stimulation during preconditioning hypoxia, however, attenuated the protection against hypoxia-induced injury. Blockade of A2a receptors with the selective antagonist CSC (1 microM) during preconditioning hypoxia enhanced the protective effect of preconditioning. Nifedipine, which blocked the A2a receptor-mediated calcium entry, abolished the A2a agonist-induced attenuation of preconditioning. Isoproterenol, forskolin, and BayK 8644, which stimulated calcium entry, also attenuated preconditioning. Nifedipine blocked the increase in calcium uptake by these agents as well as their attenuating effect on preconditioning. The present study provides the first evidence that the adenosine A3 receptor is present on ventricular myocytes and can mediate simulation of preconditioning. The data demonstrate, for the first time, that activation of the A2a receptor antagonizes the preconditioning effect of adenosine, with increased calcium entry during the preconditioning stimuli as a novel mechanism.

Adenosine receptor specificity in preconditioning of isolated rabbit cardiomyocytes: evidence of A3 receptor involvement.

The aim was to further characterise an experimental model of preconditioning of isolated rabbit cardiomyocytes and to determine the role of adenosine receptor subtypes in initiation of the protective response. Isolated myocytes were subjected to 5 min preincubation in the presence or absence of glucose and various agonists and antagonists of adenosine receptors. Ischaemic pelleting was preceded by a 30 min postincubation period. Rate and extent of injury during ischaemia was determined by sequential sampling of the pelleted cells and assessment of trypan blue permeability following 85 mOsm swelling. Myocytes were preconditioned with a 30-50% reduction of injury by a 5 min glucose-free preincubation. Substitution of 5 mM pyruvate for glucose during preincubation did not prevent the protective response. Protection was maintained over a 60-180 min postincubation period. Protection was blocked by 100 microM of the non-specific adenosine A1/A2 antagonist SPT, both when added only during preincubation or only into the ischaemic pellet. Calphostin C, a specific protein kinase C inhibitor at 200 nM, added to the ischaemic pellet blocked protection. Preincubation with R-PIA, the adenosine A1 agonist, did not precondition at an A1 selective dose of 1 microM, but did at 100 microM. The selective A2 agonist CGS 12680 (1 microM) did not precondition. The selective A1/A3 adenosine agonist, APNEA, preconditioned at 1 microM and 200 nM dose levels. Preconditioning induced either by 200 nM APNEA or by glucose-free preincubation was not blocked by 200 nM or 10 microM of the A1 antagonist DPCPX, which has extremely low affinity for A3 receptors, but was blocked by 1 microM of the A1/A3 adenosine antagonist BW 1433U83. Preconditioning can be induced in isolated myocytes by a 5 min preincubation/30 min postincubation protocol, and a similar protection induced by adenosine agonists with A3, but not A1 selectivity. Preconditioning is blocked by non-selective or selective A1/A3 adenosine antagonists and a specific protein kinase C inhibitor, but not by A1 antagonists with little affinity for A3 receptors. The results suggest that preconditioning in isolated rabbit myocytes requires participation of adenosine receptors with agonist/antagonist binding characteristics of the A3 subtype, and is likely to be mediated by activation of protein kinase C.