Testicular adenosine acts as a pro-inflammatory molecule: role of testicular peritubular cells.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesized that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Adenosine receptors enhance the ATP-induced odontoblastic differentiation of human dental pulp cells.

Purinergic signaling regulates various biological processes through the activation of adenosine receptors (ARs) and P2 receptors. ATP induces the odontoblastic differentiation of human dental pulp cells (HDPCs) via P2 receptors. However, there is no information available about the roles of ARs in HDPC odontoblastic differentiation induced by ATP. Here, we found that HDPCs treated with ATP showed higher activity of ADORA1 (A1R), ADORA2B (A2BR), and ADORA3 (A3R). Inhibition of A1R and A2BR attenuated ATP-induced odontoblastic differentiation of HDPCs, whereas activation of the two receptors enhanced the odontoblastic differentiation induced by ATP. However, activation of ARs by adenosine did not induce the odontoblastic differentiation of HDPCs independently without induction of ATP. Our study indicates a positive role for ARs in ATP-induced odontoblastic differentiation of HDPCs, and demonstrates that ATP-induced odontoblastic differentiation of HDPCs may be due to the combined administration of ARs and P2 receptors. This study provides new insights into the molecular mechanisms of pulpal injury repair induced by ATP.

Adenosine receptor ligands and PET imaging of the CNS.

Advances in radiotracer chemistry have resulted in the development of novel molecular imaging probes for adenosine receptors (ARs). With the availability of these molecules, the function of ARs in human pathophysiology as well as the safety and efficacy of approaches to the different AR targets can now be determined. Molecular imaging is a rapidly growing field of research that allows the identification of molecular targets and functional processes in vivo. It is therefore gaining increasing interest as a tool in drug development because it permits the process of evaluating promising therapeutic targets to be stratified. Further, molecular imaging has the potential to evolve into a useful diagnostic tool, particularly for neurological and psychiatric disorders. This chapter focuses on currently available AR ligands that are suitable for molecular neuroimaging and describes first applications in healthy subjects and patients using positron emission tomography (PET).

Adenosine receptors and the central nervous system.

The adenosine receptors (ARs) in the nervous system act as a kind of "go-between" to regulate the release of neurotransmitters (this includes all known neurotransmitters) and the action of neuromodulators (e.g., neuropeptides, neurotrophic factors). Receptor-receptor interactions and AR-transporter interplay occur as part of the adenosine's attempt to control synaptic transmission. A(2A)ARs are more abundant in the striatum and A(1)ARs in the hippocampus, but both receptors interfere with the efficiency and plasticity-regulated synaptic transmission in most brain areas. The omnipresence of adenosine and A(2A) and A(1) ARs in all nervous system cells (neurons and glia), together with the intensive release of adenosine following insults, makes adenosine a kind of "maestro" of the tripartite synapse in the homeostatic coordination of the brain function. Under physiological conditions, both A(2A) and A(1) ARs play an important role in sleep and arousal, cognition, memory and learning, whereas under pathological conditions (e.g., Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, epilepsy, drug addiction, pain, schizophrenia, depression), ARs operate a time/circumstance window where in some circumstances A(1)AR agonists may predominate as early neuroprotectors, and in other circumstances A(2A)AR antagonists may alter the outcomes of some of the pathological deficiencies. In some circumstances, and depending on the therapeutic window, the use of A(2A)AR agonists may be initially beneficial; however, at later time points, the use of A(2A)AR antagonists proved beneficial in several pathologies. Since selective ligands for A(1) and A(2A) ARs are now entering clinical trials, the time has come to determine the role of these receptors in neurological and psychiatric diseases and identify therapies that will alter the outcomes of these diseases, therefore providing a hopeful future for the patients who suffer from these diseases.

PET Imaging of Adenosine Receptors in Diseases.

Adenosine receptors (ARs) are a class of purinergic G-protein-coupled receptors (GPCRs). Extracellular adenosine is a pivotal regulation molecule that adjusts physiological function through the interaction with four ARs: A1R, A2AR, A2BR, and A3R. Alterations of ARs function and expression have been studied in neurological diseases (epilepsy, Alzheimer's disease, and Parkinson's disease), cardiovascular diseases, cancer, and inflammation and autoimmune diseases. A series of Positron Emission Tomography (PET) probes for imaging ARs have been developed. The PET imaging probes have provided valuable information for diagnosis and therapy of diseases related to alterations of ARs expression. This review presents a concise overview of various ARs-targeted radioligands for PET imaging in diseases. The most recent advances in PET imaging studies by using ARs-targeted probes are briefly summarized.

Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains.

Adenosine and ATP modulate cellular and tissue functions via specific P1 and P2 receptors, respectively. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the direct interaction between P1 and P2 receptors. We recently demonstrated that the G(i/o)-coupled adenosine A1 receptor (A1R) and G(q/11)-coupled P2Y1 receptor (P2Y1R) form a heteromeric complex with a unique pharmacology in cotransfected HEK293T cells using the coimmunoprecipitation of differentially epitope-tagged forms of the receptor [Yoshioka et al. (2001) Proc. Natl. Acad. Sci. USA 98, 7617-7622], although it remained to be determined whether this hetero-oligomerization occurs in vivo. In the present study, we first demonstrated a high degree of colocalization of A1R and P2Y1R by double immunofluorescence experiments with confocal laser microscopy in rat cortex, hippocampus and cerebellum in addition to primary cultures of cortical neurons. Then, a direct association of A1R with P2Y1R was shown in coimmunoprecipitation studies using membrane extracts from these regions of rat brain. Together, these results suggest the widespread colocalization of A1R and P2Y1R in rat brain, and both receptors can exist in the same neuron, and therefore associate as hetero-oligomeric complexes in the rat brain.

Cellular localization of adenosine A1 receptors in rat forebrain: immunohistochemical analysis using adenosine A1 receptor-specific monoclonal antibody.

Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT(1)( )MF-2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2-CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence-labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R-immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs.

Ultrastructural localization of adenosine A2A receptors suggests multiple cellular sites for modulation of GABAergic neurons in rat striatum.

Activation of adenosine A2A receptors (A2AR) has been shown to antagonize the function of D2 dopaminergic regulation of striatal gamma-aminobutyric acid (GABA)-ergic output and, thus, locomotor activity. Adenosine A2A receptor immunoreactivity (A2A-LI) has been localized to rat striatum by light microscopy by using a previously characterized human A2AR monoclonal antibody. In this study, we evaluated the localization of A2A-LI and its colocalization with GABA immunoreactivity (GABA-LI) in dorsolateral rat striatum by immunoelectron microscopy to further characterize the potential mechanism of purinergic control of striatal output. Ultrastructural analysis demonstrated A2A-LI associated with the plasma membrane and cytoplasmic membranous structures of striatal neurons. A2A-LI was prevalent in dendrites and dendritic spines ( approximately 70% of total A2A-profiles counted) and less prevalent in axons and axon terminals (23%), soma (3%), and glia (3%). Cellular elements exhibiting both A2A-LI and GABA-LI comprised 23% of the total profiles counted; colabeling was most common in dendrites. A2A-LI was observed primarily at asymmetric synapses (n = 70) (both pre- and postsynaptically but predominantly in the postsynaptic element) and less frequently at symmetric synapses (n = 17). Of the 714 A2A-immunoreactive profiles examined, 37% were apposed to GABA-labeled profiles. The most common appositions were A2A-labeled dendrites apposed to GABA-immunoreactive dendrites (n = 132), axon terminals (n = 28), and somata (n = 22) and A2A-labeled axons apposed to GABA-labeled dendrites (n = 58), axon terminals (n = 14), and somata (n = 9). Our findings suggest that adenosine may play an important role in modulating excitatory input to striatal neurons and that A2AR may modulate GABAergic signaling at several cellular sites within the rat striatum.

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system.

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

Medicinal chemistry of adenosine A3 receptor ligands.

A(3) Adenosine receptors (ARs) exhibit large species differences. Potent, selective agonists for rat (e.g. Cl-IB-MECA, 5) and human A(3) ARs (e.g. PENECA, 17, and analogs) have been developed during the past years. Potent, selective antagonists for human A(3) ARs include the imidazopurinones PSB-10 (28) and PSB-11 (29), the pyrazolotriazolopyrimidines MRE-3005F20 (38) and analogs, and the dihydropyridines (e.g. MRS-1334, 50). For rat A(3) ARs only moderately potent antagonists have been identified, such as the pyridine derivative MRS-1523 (51) and the flavonoid MRS-1067 (52), both of which exhibit only a low degree of selectivity versus the other AR subtypes. Selective antagonist radioligands for the human A(3) receptor, [(3)H]MRE-3008F20 and [(3)H]PSB-11, have been prepared, while A(3)-selective agonist radioligands are still lacking. Recent developments also include allosteric modulators, irreversibly binding antagonists, fluorescence-labelled agonists, partial agonists and inverse agonists for A(3)ARs. Site-directed mutagenesis and molecular modeling studies have been performed in order to obtain information about the ligand binding site and the process of receptor activation. A(3)Adenosine receptors have recently attracted considerable interest as novel drug targets. A(3) Agonists may have potential as cardioprotective and cerebroprotective agents, for the treatment of asthma, as antiinflammatory and immunosuppressive agents, and in cancer therapy as cytostatics and chemoprotective compounds. A(3) AR antagonists might be therapeutically useful for the acute treatment of stroke, for glaucoma, and also as antiasthmatic and antiallergic drugs, since A(3)receptors cannot only mediate antiinflammatory, but also proinflammatory responses. The future development of further pharmacological tools, including potent, selective antagonists for rat A(3) receptors and selective agonist radioligands for rat and human receptors will facilitate the evaluation of the (patho)physiological roles of A(3) receptors and the pharmacological potential of their ligands.

High level of adenosine A1 receptor-like immunoreactivity in the CA2/CA3a region of the adult rat hippocampus.

We describe the immunocytochemical distribution of adenosine A1 receptors in the rat hippocampus. Adenosine A1 receptor-like immunoreactivity was seen on the cell soma and dendrites of pyramidal cells and the cell soma and proximal part of dendrites of granule cells, but not on glial cells. Developmentally, adenosine A1 receptor-like immunoreactivity was diffuse on postnatal day 7 and increased in intensity in individual cells by day 21. In the CA2/CA3a region, the adult pattern of A1 receptor distribution was established by day 28. In the adult rat hippocampus, rostrocaudal inspection revealed that immunoreactivity in CA2/CA3a was greatest. Confocal microscopy revealed differences in the staining patterns for the adenosine A receptor and synaptophysin, a marker of presynaptic terminals. This result suggests that the adenosine A1 receptor might have postsynaptic physiological functions. Double-labeling of adenosine A1 receptors and anterogradely-labeled fibers from the supramammillary nucleus showed that the fibers from the supramammillary nucleus terminate directly on the cell soma of the A1 receptor-immunopositive neurons in CA2/CA3a and the dentate gyrus. These results indicate that the adenosine A 1 receptor in CA2/CA3a and the dentate gyrus are in a position to regulate hippocampal theta activity and that resultant strong synaptic depression in CA2/CA3a could play a role in regulating the intrinsic signal flow between CA3 and CA1.

Evaluation of carbon-11-labeled KF17837: a potential CNS adenosine A2a receptor ligand.

UNLABELLED: The 11C-labeled KF17837 ([7-methyl-11C](E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxa nthine) was evaluated as a PET ligand for mapping adenosine A2a receptors in the central nervous system (CNS). METHODS: The regional brain distribution of [11C]KF17837 and the effect of adenosine antagonists on the distribution were measured in mice by the tissue sampling method. In rats, the regional brain uptake of [11C]KF17837 and the effect of carrier KF17837 was visualized by autoradiography. Imaging of the monkey brain with [11C]KF17837 was performed by PET. RESULTS: In mice, a high uptake of [11C]KF17837 was found in the striatum in which A2a receptors were highly enriched. The uptake was decreased by co-injection of carrier KF17837 or a xanthine-type A2a antagonist CSC but not by nonxanthine-type A2a antagonists ZM 241385 or SCH 58261, or an A1 antagonist KF15372. In the rat brain, [11C]KF17837 was accumulated higher in the striatum than in other brain regions, and the uptake was blocked by co-injection of carrier KF17837. In a monkey PET study, a high striatal uptake of radioactivity was observed. CONCLUSION: Carbon-11-KF17837 binds to adenosine A2a receptors in the striatum. However, the presence of an unknown but specific binding site for xanthine-type compounds also was suggested in the other brain regions. The results also suggested that the in vivo receptor-binding sites of xanthine-type ligands are slightly different from those of nonxanthine-type A2a antagonists.

11C-labeled KF18446: a potential central nervous system adenosine A2a receptor ligand.

UNLABELLED: To develop PET ligands for mapping central nervous system (CNS) adenosine A2a receptors that are localized in the striatum and are coupled with dopamine receptors, 3 11C-labeled xanthine-type adenosine A2a antagonists, [11C]KF18446 ([7-methyl-11C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthin e), [11C]KF19631 ([7-methyl-11C]-(E)-1,3-diallyl-7-methyl-8-(3,4,5-trimethoxystyryl)xanth ine), and [11C]CSC ([7-methyl-11C]-8-chlorostyrylcaffeine), were compared with [11C]KF17837 ([7-methyl-11C]-(E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylx anthine). METHODS: The regional brain uptake of the tracers, the effect of the coinjected adenosine antagonists on the uptake, and the metabolism were studied in mice. In rats, the regional brain uptake of the tracers was visualized by ex vivo autoradiography (ARG). The A2a receptor binding of antagonist 1 was also measured by in vitro ARG. Imaging of the monkey brain was performed with PET with antagonist 1. RESULTS: In mice, the highest striatal uptake was found for antagonist 1 followed by antagonists 2 and 4. The uptake was inhibited by each of 3 KF compounds and by CSC, but not by an A1 antagonist KF15372. Another selective nonxanthine-type A2a antagonist SCH 58261 significantly decreased the striatal uptake of only antagonist 1, the labeled metabolites of which were less than 20% in the plasma 30 min postinjection, but were negligible in the brain tissue. In ex vivo ARG, antagonist 1 showed the highest striatal uptake and the highest uptake ratio of the striatum to the other brain regions. A high and selective binding of antagonist 1 to the striatum was also confirmed by in vitro ARG. PET with antagonist 1 visualized adenosine A2a receptors in the monkey striatum. CONCLUSION: These results indicate that antagonist 1 ([11C]KF18446) is the most suitable PET ligand for mapping adenosine A2a receptors in the CNS.

Quantitative autoradiography of adenosine receptors in brains of chronic naltrexone-treated mice.

1. Manipulation of micro opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding micro opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of micro receptor binding in the brain. 2. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg(-1) kg(-1) day(-1)), administered subcutaneously via osmotic minipump. 3. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [(3)H] D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H] DAMGO), [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H] DPCPX) or [(3)H] 2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H] CGS21680) to micro, A(1) and A(2A) receptors, respectively. 4. A significant increase in micro and A(1) receptor binding was detected in chronic naltrexone-treated brains. The changes in micro receptors were significant in several regions, but changes in A(1) were relatively smaller but showed significant upregulation collectively. No significant change in A(2A) receptor binding was detected in chronic naltrexone-treated brains. 5. The results show that blockade of opioid receptors causes upregulation of A(1) receptors, but not A(2A) receptors, by as yet undefined mechanisms.

Immunolocalisation of adenosine A(1) receptors in the rat kidney.

The location of adenosine A(1) receptors in the rat kidney was investigated using immunolabelling with antibodies raised to a 15-amino-acid sequence near the C-terminus of the receptor (antibody I) and to a 14-amino-acid sequence in the second extracellular loop (antibody II). In the cortex, antibody I bound to adenosine A(1) receptors in mesangial cells and afferent arterioles, whilst antibody II bound to receptors in proximal convoluted tubules. In the medulla, both antibodies bound to receptors in collecting ducts and the papillary surface epithelium. These observations provide support for the diverse functional roles previously proposed for the adenosine A(1) receptor in the kidney. The labelling of distinct but different structures in the cortex by antibodies raised to different amino acid sequences on the A(1) receptor protein suggests that differing forms of the receptor are present in this region of the kidney.

Biochemical and pharmacological evidence for the presence of A1 but not A2a adenosine receptors in the brain of the low vertebrate teleost Carassius auratus (goldfish).

In whole brain membranes of goldfish, 3H-chlorocyclopentyladenosine bound to adenosine A1 receptors. The A1 receptors were ubiquitously distributed in the brain with a maximum in the hypothalamus and a minimum in the spinal cord. In superfused goldfish cerebellar slices, cyclohexyladenosine inhibited the cyclic AMP accumulation stimulated by forskolin and the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline, reversed this effect. In the same brain preparation, 30 mM K+ stimulated the release of glutamate, glutamine, glycine and GABA in a Ca(2+)-dependent manner, whereas the aspartate and taurine release was Ca(2+)-independent. Cyclohexyladenosine, in a dose-dependent manner, inhibited the 30 mM K(+)-evoked release of glutamate whereas that of aspartate was unaffected. The CHA inhibition of glutamate-evoked release was reversed by 8-cyclopentyltheophylline. The adenosine A2a receptors were not detectable in whole brain membranes of goldfish either using the specific agonist 3H-CGS 21680 or 3H-5'-N-ethylcarboxamidoadenosine. The presence of A2b seems to be suggested by the NECA stimulation of cyclic AMP accumulation, which was reversed by 8-cyclopentyltheophylline. The results, taken together, indicate that adenosine has a neuromodulatory function in the nervous system of lower vertebrates which is comparable to that described in mammalian brain.

Detection of adenosine receptor antagonists in rat brain using a modified radioreceptor assay.

The present study describes a modified radioreceptor binding assay using brain homogenate or serum from drug treated animals as the 'competing drug' in a conventional in vitro radioligand binding assay. Method validation involved measurement of the brain and serum concentration of three adenosine receptor antagonists following systemic administration, using a [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) binding assay. The intrinsic [3H]DPCPX binding capacity of test samples was abolished by protein denaturation (80 degrees C, 15 min) and, endogenous ligand was depleted enzymatically, prior to determination of drug concentration. Brain and serum concentrations of the adenosine A1 receptor antagonist, DPCPX increased in a dose related manner when measured 20 min after intraperitoneal injection. Estimated brain concentrations were 13.8, 87.7 and 288 nM following injection of 0.01, 0.1 and 1.0 mg/kg DPCPX, and serum concentrations were 26.5, 195 and 1370 nM respectively. A time dependent decrease in both brain and serum concentration was noted 20-180 min following injection of 1.0 mg/kg DPCPX. The peripheral adenosine receptor antagonists, 1,3-dipropyl-8-p-sulphophenylxanthine (DPSPX; 5.6 mg/kg) and 8-(p-sulphophenyl)theophylline (8-PST; 20 mg/kg), were not detected in brain tissue 20 min after intraperitoneal injection, despite serum concentrations of 56 and 52 microM respectively. This assay provides a useful and versatile method for determining the central penetration of neuroactive drugs.

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia.

Polyclonal antisera were generated against two identical regions of rat and human A1 adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A1 receptors. Immunohistochemistry of CHO cells transfected with the rat or human A1 adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A1 adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A1 receptor expression and support the concept of cellular specificity of A1 adenosine receptor action.

Quantitative autoradiography of adenosine receptors and NBTI-sensitive adenosine transporters in the brains and spinal cords of mice deficient in the mu-opioid receptor gene.

There is a large body of evidence indicating important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed and the animals show complete loss of analgesic responses to morphine as well as differences in pain sensitivity. To investigate if there are any compensatory alterations in adenosine systems in mutant animals, we have carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors and nitrobenzylthioinosine (NBTI) sensitive adenosine transporters in the brains and spinal cords of wild type, heterozygous and homozygous mu-opioid receptor knockout mice. Adjacent coronal sections were cut from the brains and spinal cords of +/+, +/- and -/- mice for the determination of binding of [3H]DPCPX, [3H]CGS21680 or [3H]NBTI to A(1) and A(2A) adenosine receptors and NBTI-sensitive adenosine transporters, respectively. A small but significant reduction in [3H]DPCPX and [3H]NBTI binding was detected in mutant mice brains but not in spinal cords. No significant change in A(2A) binding was detected in mu-opioid receptor knockout brains. The results suggest there may be functional interactions between mu-receptors and A(1) adenosine receptors as well as NBTI-sensitive adenosine transporters in the brain but not in the spinal cord.

Effects of repeated administration of selective adenosine A1 and A2A receptor agonists on pentylenetetrazole-induced convulsions in the rat.

The protective effects of the selective adenosine A1 receptor agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA), the selective adenosine A2A receptor agonist, 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA), and the non-selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA) were studied against lethal seizures induced by intraperitoneal (i.p.) injection of pentylenetetrazole (80 mg/kg). In acute studies there was a dose-dependent reduction of lethal seizures, as shown by the low dose's protecting 50% of animals (PD50): 0.11, 0.05 and 0.05 mg/kg i.p. for CCPA, 2HE-NECA and NECA, respectively. In the repeated administration studies the animals received either vehicle or drug i.p. twice daily for 12 days. The drug doses were twice the PD50 value: 0.3 mg/kg for CCPA or 0.1 mg/kg for both 2HE-NECA and NECA. 2HE-NECA and NECA maintained their protective activity against pentylenetetrazole-induced seizures (63% or 60% vs. 60% or 58% in acute studies, respectively). Conversely, repeated treatment with CCPA resulted in a marked decrease of its effects (67% vs. 30% in acute studies; P < 0.05). The data indicate that in addition to adenosine A1 the A2A receptors also appear to be involved in the protection from seizures. The anticonvulsant effects induced by repeated stimulation of adenosine A1 receptors are subject to tolerance, whereas effects depending on adenosine A2A receptor activation are maintained.