Presence Of Adenosine Receptors Research Articles

Adenosine receptor distribution in Rhesus monkey ocular tissue

Adenosine receptor (ADOR) antagonists, such as 7-methylxanthine (7-MX), have been shown to slow myopia progression in humans and animal models. Adenosine receptors are found throughout the body, and regulate the release of neurotransmitters such as dopamine and glutamate. However, the role of adenosine in eye growth is unclear. Evidence suggests that 7-MX increases scleral collagen fibril diameter, hence preventing axial elongation. This study used immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to examine the distribution of the four ADORs in the normal monkey eye to help elucidate potential mechanisms of action. Eyes were enucleated from six Rhesus monkeys. Anterior segments and eyecups were separated into components and flash-frozen for RNA extraction or fixed in 4% paraformaldehyde and processed for immunohistochemistry against ADORA1, ADORA2a, ADORA2b, and ADORA3. RNA was reverse-transcribed, and qPCR was performed using custom primers. Relative gene expression was calculated using the ΔΔCt method normalizing to liver expression, and statistical analysis was performed using Relative Expression Software Tool. ADORA1 immunostaining was highest in the iris sphincter muscle, trabecular meshwork, ciliary epithelium, and retinal nerve fiber layer. ADORA2a immunostaining was highest in the corneal epithelium, trabecular meshwork, ciliary epithelium, retinal nerve fiber layer, and scleral fibroblasts. ADORA2b immunostaining was highest in corneal basal epithelium, limbal stem cells, iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells and scattered scleral fibroblasts. ADORA3 immunostaining was highest in the iris sphincter, ciliary muscle, ciliary epithelium, choroid, isolated retinal ganglion cells, and scleral fibroblasts. Compared to liver mRNA, ADORA1 mRNA was significantly higher in the brain, retina and choroid, and significantly lower in the iris/ciliary body. ADORA2a expression was higher in brain and retina, ADORA2b expression was higher in retina, and ADORA3 was higher in the choroid. In conclusion, immunohistochemistry and RT-qPCR indicated differential patterns of expression of the four adenosine receptors in the ocular tissues of the normal non-human primate. The presence of ADORs in scleral fibroblasts and the choroid may support mechanisms by which ADOR antagonists prevent myopia. The potential effects of ADOR inhibition on both anterior and posterior ocular structures warrant investigation.

Expression of adenosine receptor subclasses in malignant and adjacent normal human prostate tissues.

Adenosine, a purine nucleoside plays important roles in the pathogenesis of cancer initiation and promotion via interaction with four adenosine receptors. In the present study we examined the differential expression pattern of adenosine receptors in the malignant and adjacent normal human prostate tissues. Prostate cancer tissue samples and adjacent normal tissues were obtained from 20 patients undergoing radical prostatectomy and histopathological diagnosis was confirmed for each sample. Total RNA was extracted and reverse transcribed into cDNA and the mRNA expression levels of adenosine receptors were investigated by Taq-man real-time RT-PCR experiment. Quantitative protein analysis was done by Western blotting experiment. Moreover, the mRNA and protein expression levels of adenosine receptors were measured after androgen treatment. Taq-man real-time RT-PCR measurements show different expression levels of adenosine receptor transcripts. A2B adenosine receptor was predominantly expressed in tumor tissues (2.4-fold) followed by significantly expression of A3 (1.6-fold) and A2A adenosine receptors (1.5-fold) compared to adjacent normal tissues. The presence of adenosine receptors at protein levels in prostate cancer tissues compared with normal tissues was shown the following rank order: A2B > A3 > A2A > A1 . Androgen receptor regulates adenosine receptors mRNA and protein expression in AR-positive LNCaP cells, which was not seen in AR-negative PC-3 cells. These results indicated for the first time, the differential mRNA expression profile and protein levels of adenosine receptors in the human prostate cancer. Interestingly, the A2B adenosine receptor followed by A3 is highly expressed in prostate tumor samples in comparison with the adjacent normal tissues. The findings support the possible key role of A2B adenosine receptor in promoting cancer cell growth and suggest that A2B may be a novel target for prostate cancer treatment.

Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression.

Abnormal alveolar development associated with elevated adenine nucleosides.

Adenosine signaling has been characterized in various physiologic systems, but little is known about the role of adenosine signaling in lung development. Alveogenesis and microvascular maturation are the final stages in lung development in mammals. Alveogenesis in the mouse begins on Postnatal Day 5, when the process of secondary septation plays a pivotal role in the expansion of the alveolar sacs and microvascular maturation. Adenosine deaminase null mice (ADA-/-) exhibit abnormalities in alveogenesis in association with elevated lung adenosine levels. Large-scale gene expression analysis of ADA-/- lungs using oligonucleotide-based microarrays revealed novel relationships between gene expression patterns and elevated lung adenosine during the stages of alveolar maturation. Genes regulating apoptosis, proliferation, and vascular development were shown to be altered, and decreased cell proliferation in association with increased alveolar type II cell apoptosis was shown to contribute to abnormal secondary septation in these mice. ADA enzyme therapy allowed for normal patterns of apoptosis, proliferation, and alveolar development in association with prevention of adenosine elevations. These findings were correlated with the presence of adenosine receptors in the developing lung, suggesting the involvement of receptor signaling. These studies provide evidence that elevated lung adenosine can lead to abnormal alveogenesis by disrupting patterns of cell proliferation and apoptosis.

Modulatory effect of adenosine receptors on the ascending and descending neural reflex responses of rat ileum

BackgroundAdenosine is known to act as a neuromodulator by suppressing synaptic transmission in the central and peripheral nervous system. Both the release of adenosine within the small intestine and the presence of adenosine receptors on enteric neurons have been demonstrated. The aim of the present study was to characterize a possible involvement of adenosine receptors in the modulation of the myenteric reflex. The experiments were carried out on ileum segments 10 cm in length incubated in an single chambered organ bath, and the reflex response was initiated by electrical stimulation (ES).ResultsES caused an ascending contraction and a descending relaxation followed by a contraction. All motility responses to ES were completely blocked by tetrodotoxin, indicating that they are mediated by neural mechanisms. Atropine blocked the contractile effects, whereas the descending relaxation was significantly increased. The A1 receptor agonist N6-cyclopentyladenosine increased the ascending contraction, whereas the ascending contraction was reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Activation of the A1 receptor further reduced the descending relaxation and the latency of the peristaltic reflex. The A2B receptor antagonist alloxazine increased ascending contraction, whereas descending relaxation remained unchanged. For A2A and A3 receptors, we found contradictory effects of the agonists and antagonists, thus there is no clear physiological role for these receptors at this time.ConclusionsThis study suggests that the myenteric ascending and descending reflex response of the rat small intestine is modulated by release of endogenous adenosine via A1 receptors.

Adenosine responses in experimental vein bypass grafts

Purpose: Veins develop unique endothelial and smooth muscle cell physiologic phenotypes after implantation as vein grafts in the arterial circulation. Receptor-mediated relaxation is reduced or absent in these grafts. This study examines the responses of vein grafts to adenosine, a known potent endogenous vasodilator, and compares these responses with the native veins and arteries. Methods: The presence of adenosine receptors (A 1 and A 2) by radioligand binding and the in vitro responses to the adenosine analogue N-ethyl-carboxyamido-adenosine were assessed in precontracted common carotid jugular vein bypass grafts placed in New Zealand white rabbits for 28 days. Results were compared with those obtained in precontracted jugular veins and carotid arteries. Both endothelialized and de-endothelialized vessels were studied. The contribution of nitric oxide (NO) and prostanoid production to relaxation was also determined by preincubation with the specific inhibitors <!--**Check here**-->l-monomethylarginine and indomethacin, respectively. Finally, the in vitro relaxation in response to the respective A 1 and A 2 adenosine receptor agonists R-phenyl-isopropyl-adenosine and CGS-21680 was also examined. Results: The results show that the adenosine-induced responses of the vein grafts differ from those of the jugular vein and carotid artery. First, in contrast to the carotid artery, vein graft adenosine-mediated relaxation is NO and prostanoid dependent, similar to the response of the jugular vein. Second, A 1 receptor activation in the vein graft produces an endothelium-dependent contractile response. Third, the A 2 receptor-mediated responses in the vein grafts appear to be independent of the endothelium. Fourth, radioligand studies show the presence of both receptor subtypes (A 1 and A 2) on the vein grafts with a ratio (A 1/A 2 = 1.4) closer to that of the jugular vein (A 1/A 2 = 1.8) than to that seen in the carotid artery (A 1/A 2 = 0.5). Conclusions: Vein graft adenosine responses appear to be unique in that they neither maintain a venous phenotype nor acquire an arterial phenotype. In particular, endothelial A 1 receptor-mediated responses change from relaxation to contraction, and receptor activated NO-mediated relaxation is preserved within the vein grafts probably via A 2 receptor signalling. (J Vasc Surg 1998;28:929-38.)

Expression and function of adenosine receptors in the chinchilla cochlea

Previous studies indicate the presence of adenosine receptors in the cochlear tissues obtained from different animals. This study was initiated to determine the subtypes of adenosine receptor (AR) present in the chinchilla cochlea and to assess their function. Radioligand binding studies demonstrate the presence of both the A1 AR and A3AR in membranes prepared from the cochlea, using the radioligands [3H]DPCPX and [125I]APNEA. Estimates of the number (Bmax) of A1AR and A1AR plus A3AR by saturation curves were 118 ± 13 and 417 ± 120 fmol/mg, respectively, with the respective equilibrium dissociation constants (Kd) averaging 2.7 ± 0.2 and 26.3 ± 13.8 nM. No significant number of A2aAR were detected using [3H]CGS21680. The nonhydrolyzable adenosine analog R-phenylisopropyladenosine (R-PIA, 1 μM) elicited a small but significant degree of inhibition of forskolin-stimulated adenylyl cyclase activity (10.4 ± 2.5%) in cochlear membrane preparations, which was insensitive to blockade by theophylline (100 μM). Furthermore, R-PIA elicited an increase in inositol 1,4,5-trisphosphate production in dissociated cell preparations obtained from the cochlea. No significant effect of R-PIA was observed on auditory measures such as auditory brainstem evoked response, cochlear action potential and endocochlear potential following round window application. However, round window application of R-PIA elicited significant increases in the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase and significantly reduced the levels of malondialdehyde, a marker of lipid peroxidation. These results suggest a potential cytoprotective role of adenosine in the cochlea against oxidative damage.

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The 'R'-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 microM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 microM respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.

Adenosine Receptors: Autoradiographic Evidence for Their Location on Axon Terminals of Excitatory Neurons

Adenosine receptors were made visible on light microscopy by autoradiography with tritiated cyclohexyladenosine. In the cerebellum, adenosine receptors were absent in Weaver mice, which lack granule cells, and were displaced in Reeler mice, which have displacements of granule cells. Thus, adenosine receptors appear to be located on the axon terminals of excitatory granule cells in the cerebellum. Removal of one eye of a rat depleted adenosine receptors in the contralateral superior colliculus, suggesting that the receptors occur on axon terminals of excitatory projections from retinal ganglion cells. The presence of adenosine receptors on excitatory axon terminals may explain synaptic inhibition by adenosine and the behavioral effects of xanthines.

Effects of Adenosine and Its Derivatives on the Canine Intestinal Vasculature

The effects of adenosine and two of its structural analogs on small intestinal blood flow, flow distribution, oxygen extraction, and uptake were studied in anesthetized dogs. Adenosine induced dose-dependent increases in intestinal blood flow and oxygen consumption but decreased oxygen extraction. These effects were significantly attenuated by theophylline, an adenosine antagonist. Propranolol did not influence the responses of the intestinal circulation to adenosine suggesting that beta-adrenergic receptors are not involved in the action of adenosine. 2-Chloroadenosine, one of the most potent adenosine analogs, produced changes very similar to adenosine but was about six times more potent on a molar basis. Effects of 2-chloroadenosine were attenuated by a 10-fold smaller dose of theophylline. Adenosine-9 beta-D-arabinofuranoside, another analog, was ineffective in the mesenteric circulation. Characteristic activity of both analogs and the antagonism of the effects of adenosine and 2-chloroadenosine by theophylline suggest the presence of adenosine-sensitive binding sites in precapillary resistance vessels of the intestinal circulation. The presence of adenosine receptors in intestinal vascular smooth muscle is further supported by our in vitro results in which all three agents induce patterns of response similar to those noted under in vivo conditions. However, concentrations necessary to obtain relaxation of isolated muscle strips were much greater than those sufficient to produce mesenteric vasodilation in vivo. It was also noted that strips obtained from branches of the superior mesenteric artery were more reactive to adenosine and its analogs than were strips prepared from the main trunk of the vessel.

Specific binding of 2-[3H]chloroadenosine to rat brain cortical membranes.

2-[3H]Chloroadenosine was used as a ligand to investigate the presence of adenosine receptors on rat brain cortical membranes. The binding studies reveal the presence of a single binding site which has an apparent dissociation constant (KD) of 23.5 nM and a maximal binding capacity (Bmax) of 476 fmol/mg protein. This binding can be inhibited by adenosine, adenine, nicotinamide adenine dinucleotide phosphate, inosine, theophylline, and isobutylmethylxanthine. These findings strongly support the suggestion that there is an adenosine receptor on brain cell membranes.