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The antagonistic interactions between adenosine A1 and dopamine D1 receptors were studied in a mouse Ltk− cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In membrane preparations, both the adenosine A1receptor agonist N 6-cyclopentyladenosine and the GTP analogue guanyl-5′-yl imidodiphospate induced a decrease in the proportion of dopamine D1 receptors in a high affinity state. In the cotransfected cells, the adenosine A1 agonist induced a concentration-dependent inhibition of dopamine-induced cAMP accumulation. Blockade of adenosine A1 receptor signal transduction with the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine or with pertussis toxin pretreatment increased both basal and dopamine-stimulated cAMP levels, indicating the existence of tonic adenosine A1 receptor activation. Pretreatment with pertussis toxin also counteracted the effects of low concentrations of the A1 agonist on D1 receptor-agonist binding. The results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the generation of second messengers.

We have investigated how glucose deprivation in vitro influences the basal and electrically evoked release of dopamine and acetylcholine from rat striatal slices and the role of endogenous activation of NMDA receptors and adenosine A1 receptors in determining the magnitude of this response. Rat striatal slices, preincubated with [ 3 H ]dopamine and [ 14 C ]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with glucose-free Krebs' solution. Such glucose deprivation caused a 2 to 3-fold increase of the electrically evoked, calcium-dependent release of endogenous adenosine (but not hypoxanthine and inosine) and [ 3 H ]dopamine and a 30% increase in release of [ 14 C ]acetylcholine. Glucose deprivation also caused a delayed increase in the release of [ 3 H ]dopamine, but not of [ 14 C ]acetylcholine. The dopamine release was not calcium dependent. The addition of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM), a selective adenosine A1 receptor antagonist, slightly enhanced the glucose deprivation-induced stimulatory effect on the evoked release of these two transmitters, whereas the NMDA receptor antagonist dizocilpine((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine; 3 μM) markedly attenuated the stimulatory effect of glucose deprivation. The change in basal dopamine release was not influenced by DPCPX, but was slightly attenuated by dizocilpine. In summary, the results suggest that lack of substrate induces release of both glutamate, which by actions on presynaptic NMDA receptors causes the release of dopamine, and of adenosine, which via adenosine A1 receptors reduces the electrically evoked release of both dopamine and acetylcholine.

1. Rat striatal slices, preincubated with [3H]-dopamine and [14C]-choline, were continuously superfused and electrically stimulated. Electrically evoked release of [3H]-dopamine and [14C]-acetylcholine (ACh) was not significantly changed by elimination of Mg2+ from superfusion buffer, but the basal release of [3H]-dopamine was doubled. 2. Kynurenic acid (100-800 microM) caused, in the absence but not presence of Mg2+, a concentration-dependent decrease in the evoked release of these two transmitters. The addition of glycine reversed the inhibition of the evoked release of both transmitters caused by kynurenic acid (400 microM) in a concentration-dependent manner. In addition, glycine increased the evoked release of [3H]-dopamine via a site inhibitable by strychnine (1 microM). 3. Another two antagonists at N-methyl-D-aspartate (NMDA) receptors, 2-amino-5-phosphonovaleric acid and dizocilpine, also decreased significantly the evoked release of the two transmitters in a concentration-dependent manner in the absence, but not presence of Mg2+. By contrast, an antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) significantly decreased the evoked release of the two transmitters in the presence, but not in the absence of Mg2+. 4. Electrical field stimulation evoked release of endogenous adenosine, and this release tended to be higher in the absence of Mg2+. However, the addition of a selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (200 nM) did not influence the evoked release of the two transmitters, showing that the released adenosine is of little importance in controlling ACh and dopamine release from striatal slices. Non-NMDA receptors may play a similar role when Mg2+ ions are present. 5. The results indicate that NMDA receptors activated in the absence of Mg2+ participate in the electrically-evoked release of [3H]-dopamine and [14C]-ACh from the striatum.

We have examined the role of adenosine in mediating effects of mild hypoxia on electrically evoked transmitter release. Rat striatal slices, preincubated with [3H]dopamine and [14C]choline, were superfused continuously and stimulated electrically. Before and during the second stimulation, some slices were superfused with Krebs' solution with lowered oxygen. This mild hypoxia caused a significant increase of the electrically evoked outflow of endogenous adenosine, hypoxanthine and inosine into the superfusion buffer, whereas electrically evoked release of [3H]dopamine and [14C]acetylcholine was significantly decreased. The addition of 8-cyclopentyl-1,3-dipropylxanthine, a selective adenosine A1 receptor antagonist, blocked the hypoxia-induced inhibitory effect on the evoked release of these two transmitters in a concentration-dependent manner. In summary, the results show that reduction of the oxygen supply to striatal slices results in an increased release of endogenous adenosine, which, by acting on adenosine A1 receptors, decreases the electrically evoked release of dopamine and acetylcholine.

1. We have characterized the binding of the new potent and selective antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o[1,5- c]pyrimidine, [3H]-SCH 58261, to human cloned A2A adenosine receptors. 2. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]-SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (KD = 2.3 nM) and limited capacity (apparent Bmax = 526 fmol mg-1 protein). 3. Competition experiments revealed that binding of 0.5 nM [3H]-SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) = 2-phenylaminoadenosine (CV 1808) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > R-N6-phenylisopropyladenosine (R-PIA) > or = N6-cyclohexyladenosine (CHA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine receptor antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > SCH 58261 > xanthine amine congener (XAC) > (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3- dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > theophylline. 5. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. 6. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]-CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.

1. 1. Adenosine is a normal constituent of all body fluids and its levels are raised, for example, by hypoxia and ischemia. In addition, both adenosine and ATP can be released by endothelial cells and neutrophils in response to physiologic stimulation. 2. 2. Human neutrophil leukocytes possess multiple adenosine receptors and P 2 purinoceptors. 3. 3. ATP can increase intracellular Ca 2+ levels in neutrophils, cause degranulation and enzyme release, potentiate the oxidative burst and enhance their adhesion to the endothelium. ATP is broken down to adenosine by ecto -enzymes. Via A 1 receptors, adenosine can increase neutrophil chemotaxis and, via A 2A receptors, it can decrease the oxidative burst, degranulation and adhesion to endothelium. 4. 4. Adenosine and adenine nucleotides are important endogenous modulators of neutrophil functions, and drugs may exert important actions via purinoceptors on neutrophil leukocytes.

Three clones of PC12 cells that differ with respect to their nerve growth factor (NGF) receptors were examined: wild-type PC12 cells that have both trkA and p75LNTR receptors; the MR-1 clone that possesses a normal trkA receptor and a truncated form of p75LNTR without the extracellular NGF-binding part; and a new PC12 variant, called v-clone, that is partly characterized here. The v-clone had no demonstrable binding to trkA, but displayed binding to p75LNTR as assessed by chemical crosslinking. NGF did not induce any change in the tyrosine phosphorylation of phosphatidy-3'-kinase in the v-clone. NGF induced neurite extension in wild-type cells, induced it more rapidly in mR-1, but not at all in v-clone cells. The v-clone lacked the b-form of protein kinase C, but transfection with this enzyme did not restore responsiveness to NGF. Neurite extension in response to staurosporine and basic fibroblast growth factor was equal in wild-type and v-clone cells. All three clones responded to forskolin, with the mR-1 clone the most responsive. NGF stimulated AP 1 binding activity in all clones. The response was transient in the MR-1 clone but prolonged in the wild-type and v-clone cells. In the wild-type and MR-1 clone cells, AP 1 binding activity was reduced by a tyrphostin analog, whereas in the v-clone cells it was inhibited by staurosporine. NGF increased inositol (1,4,5)-trisphosphate (InsP3) formation in all clones. In the wild-type and v-clone cells the InsP3 responses were followed by [Ca2+]i increases. It is concluded that although trkA is required for differentiation in response to NGF in PC12 cells, the concomitant stimulation, by NGF, of p75LNTR may affect phospholipase C and AP 1. This may be important for the reported ability of p75LNTR to modify the phenotypic changes induced in PC12 cells by NGF.

This minireview briefly summarizes the evidence that adenosine, acting on four G-protein coupled receptors, can play physiological roles, but is also critically involved in pathological processes. The factors that decide which of these is the more important in a specific cell or organ are briefly summarized. The fact that drugs that target adenosine receptors in disease will also hit the physiological processes will make drug development more tricky.

Rat striatal and hippocampal slices, preincubated with [3H] dopamine (DA) ¿or [3H] noradrenaline (NA)] and [14C] choline, were superfused continuously and stimulated electrically. 2-chloroadenosine (2-CADO 0.001-100 microM), a non-selective adenosine receptor agonist, produced a concentration-dependent inhibition of the electrically evoked DA and acetylcholine (ACh) release from the striatal slices and of the electrically evoked NA and ACh release from the hippocampal slices. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 3, 30 and 200 nM), a selective adenosine A1 receptor antagonist, caused a concentration-dependent, parallel, rightward shift of the 2-CADO concentration-response curve, consistent with competitive antagonism. The pA2 values ranged between 8.4 and 8.8. In the case of ACh release from the hippocampus, but in no other case, was there an increase in release of radioactivity at low concentrations of 2-CADO in the presence of DPCPX. The stimulation in the hippocampus could be blocked by a selective adenosine A2A receptor antagonist KF 17837. By itself KF 17837 (0.1-100 microM) had no effect on electrically evoked NA release from hippocampal slices, but decreased electrically evoked ACh release. This inhibition was counteracted by DPCPX (1 microM). These results show that, under the conditions used, DA release in the striatum, and NA release in the hippocampus, as well as ACh release from the striatum are regulated by adenosine A1 but not by adenosine A2A receptors. By contrast, ACh release from the hippocampus is tonically regulated both by adenosine A1 receptors, which inhibit release, and by adenosine A2A receptors which stimulate release.

The effects of long-term oral administration of low doses of caffeine (0.3 g/l) and its metabolites theophylline, theobromine and paraxanthine (each at 0.5 g/l in drinking water) on bicuculline- and pentylenetetrazol (PTZ)-induced seizures and c-fos expression were studied in mice. In addition, adenosine and benzodiazepine receptor density was examined. The plasma levels of the methylxanthines were much higher during the active period at night than during the day. The maximal level of caffeine was 14 microM. Brain theophylline levels (8-13 nmol/g) tended to be higher and more constant than brain caffeine levels in caffeine-consuming mice. Clonic seizures induced by bicuculline (4 mg/kg i.p.) were significantly reduced in severity by 14 day caffeine treatment and mortality was also reduced. Long-term treatment with caffeine metabolites was less effective. The seizures induced by PTZ (60 mg/kg i.p.) were also significantly reduced by long-term caffeine treatment. After bicuculline or PTZ treatment, c-fos mRNA expression was weaker in the cerebral cortex in animals receiving caffeine, irrespective of whether the animals had seizures or not. No significant changes in the binding of adenosine receptor ligands or benzodiazepines were seen after long-term caffeine treatment. These results show that long-term treatment with caffeine in a dose that is commonly seen in humans decreases the seizures induced by bicuculline, and to a lesser extent, those induced by PTZ. This may be related to a decreased neuronal excitability. The effect is due to the combined effects of theophylline, to which caffeine is metabolized in brain, and caffeine itself, but could not be ascribed to changes in A1 and A2A adenosine or benzodiazepine receptors.

The coupling of the four defined types of adenosine receptors to G proteins and the consequent activation of effector pathways is briefly summarized. It is pointed out that the G proteins are able to influence many types of cellular effector systems, and, in particular, that the a and the β,γ-subunits may activate different signalling pathways that may either act synergistically or antagonistically in the cell. Because adenosine physiologically plays the role of a modulator, particular emphasis is placed on the interactions with parallel signalling pathways.

Adenosine A 2A receptors, which have been cloned from several mammalian species, are activated by physiological concentrations of adenosine to stimulate the formation of cAMP and other mediators. The A 2A receptors are found on neutrophil leukocytes, platelets, blood vessels and, very abundantly, on some cells in the CNS. In the caudate nucleus they coexist with dopamine D 2 receptors, and stimulation of A 2A receptors causes a decrease in D 2 receptor-mediated neurotransmission Thus, drugs that act on A 2A receptors can be used to modify dopaminergic neurotransmission known to be important in neurological and psychiatric disorders. In this review, Ennio Ongini and Bertil Fredholm describe how recently developed potent and selective A 2A receptor antagonists can be used to delineate the physiological and pathological processes regulated by A 2A receptors. Results from in vitro and in vivo pharmacology studies strengthen the notion that A 2A receptors are an interesting target for drug development.

Since adenosine has been shown to mediate some actions of ethanol we have examined the effect of ethanol (20 and 80 mM) or its metabolite acetate (5 and 20 mM) on the formation and release of adenosine by rat hippocampal slices. The ATP pool of the slices was radioactively labelled by preincubation with [3H]-adenine. The efflux of radioactivity under basal conditions and following ATP breakdown induced by combined hypoxia/hypoglycaemia was examined. Ethanol or acetate did not increase the total efflux of [3H]-purines, but changed the composition to a larger proportion of [3H]-adenosine. The release of endogenous adenosine was also increased. This type of effect exactly mirrors that previously reported for purine nucleoside transport inhibitors. The present results thus show that ethanol (20 mM) can increase adenosine release from a brain slice by a mechanism that probably involves transport inhibition.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Since the P 2X purinoceptor has been shown to be homologous with RP-2, a gene activated in thymocytes undergoing apoptosis after glucocorticoid treatment, we have investigated the possible role of P 2X purinoceptors in thymocyte apoptosis. We report that P 2X purinoceptor mRNA is expressed both in untreated rat thymocytes and thymocytes treated with the synthetic glucocorticoid. 6 α -methylprednisolone, in a dose that can induce apoptosis. Furthermore, the P 2X receptor agonist, extracellular ATP, was unable to trigger apoptosis in rat thymocytes and had no effect on methylprednisolone-induced DNA fragmentation. Finally, the P 2X purinoceptor antagonists, suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, had no inhibitory effects on DNA fragmentation caused by methylprednisolone. Thus, our data demonstrate that P 2X purinoceptors are not directly involved in glucocorticoid-mediated thymocyte apoptosis.

Adenosine receptors have been the target of intense research with respect to potential use of selective ligands in a variety of therapeutic areas. Caffeine and theophylline are adenosine receptor antagonists, and over the past three decades a wide range of selective agonists and antagonists for adenosine receptor subtypes have been developed. A complication to the therapeutic use of adenosine receptor ligands is the observation that the effects of acute administration of a particular ligand can be diametrically opposite to the chronic effects of the same ligand. This 'effect inversion' is discussed here by Ken Jacobson and colleagues, and has been observed for effects on cognitive processes, seizures and ischaemic damage.

The binding of the adenosine A2A receptor selective agonist 2-[4-(2-p-carboxyethyl)phenylamino] -5'-N-ethylcarboxamidoadenosine (CGS 21680) to the rat hippocampal and cerebral cortical membranes was studied and compared with that to striatal membranes. [3H] CGS 21680, in the concentration range tested (0.2-200 nM), bound to a single site with a Kd of 58 nM and a Bmax of 353 fmol/mg protein in the hippocampus, and with a Kd of 58 nM and a Bmax of 264 fmol/mg protein in the cortex; in the striatum, the single high-affinity [3H] CGS 21680 binding site had a Kd of 17 nM and a Bmax of 419 fmol/mg protein. Both guanylylimidodiphosphate (100 microM) and Na+ (100 mM) reduced the affinity of [3H] CGS 21680 binding in the striatum by half and virtually abolished [3H] CGS 21680 binding in the hippocampus and cortex. The displacement curves of [3H] CGS 21680 binding with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), N6-cyclohexyladenosine (CHA), 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CADO) were biphasic in the hippocampus and cortex as well as in the striatum. The predominant [3H]CGS 21680 binding site in the striatum (80%) had a pharmacological profile compatible with A2A receptors and was also present in the hippocampus and cortex, representing 10-25% of [3H]CGS 21680 binding. The predominant [3H]CGS 21680 binding site in the hippocampus and cortex had a pharmacological profile distinct from A2A receptors: the relative potency order of adenosine antagonists DPCPX, 1,3-dipropyl- 8-¿4-[(2-aminoethyl)amino]carbonylmethyl- oxyphenyl¿ xanthine (XAC), 8-(3-chlorostyryl)caffeine (CSC), and (E)-1,3-dipropyl-8-(3,4-dimethoxystyryl)- methylxanthine (KF 17,837) as displacers of [3H] CGS 21680 (5 nM) binding in the hippocampus and cerebral cortex was DPCPXXACCSC approximately KF 17,837, and the relative potency order of adenosine agonists CHA, NECA, CADO, 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC), and 2-phenylaminoadenosine (CV 1808) was CHA approximately NECAor = CADOAPEC approximately CV1808CGS 21680. In the presence of DPCPX (20 nM), [3H] CGS 21680 (0.2-200 nM) bound to a site (A2A-like) with a Kd of 20 nM and a Bmax of 56fmol/mg protein in the hippocampus and with a Kd of 22 nM and a Bmax of 63fmol/mg protein in the cortex. In the presence of CSC (200 nM), [3H]CGS 21680(0.2-200 nM) bound to a second high-affinity site with a Kd of 97 nM and a Bmax of 255 fmol/mg protein in the hippocampus and with a Kd of 112 nM and a Bmax of 221 fmol/mg protein in the cortex. Two pharmacologically distinct [3H]CGS 21680 binding sites were found in synaptosomal membranes of the hippocampus and cortex and in the striatum, one corresponding to A2A receptors and the other to the second high-affinity [3H]CGS 21680 binding site. In contrast, the pharmacology of [3H]CHA binding was similar in synaptosomal membranes of the three brain areas. The present results establish the existence of at least two high-affinity [3H]CGS 21680 binding sites in the CNS and demonstrate that the [3H]CGS 21680 binding site predominant in the hippocampus and cerebral cortex has different binding characteristics from the classic A2A adenosine receptor, which predominates in the striatum.

Changes in brain a 1 and A 2A receptors and in the corresponding mRNA were studied using quantitative receptor autoradiography and in situ hybridisation. [ 3 H]-DPCPX was used as an antagonist ligand at A 1 receptors and [ 1 H]-CGS 21680 as an agonist ligand at A 2A receptors. Treatment of rats with the relatively a 1 receptor selective adenosine analogue R-PIA (0.3 mg/kg) for 7 days in the presence of the peripherally acting antagonist 8-p-sulfophenyltheophylline (8-PST;10 mg/kg) caused a decrease in the binding of the a 1 receptor ligand, but not in that of the A 2A receptor ligand. The effect on a 1 receptors was also seen in the presence of 100 μM GTP that decreases agonist binding to insignificant levels. There was no change in either a 1 or A 2A receptor mRNA. No significant changes were detected following administration of either R-PIA or 8-PST alone. These results thus demonstrate an effect on brain a 1 receptors after systemic administration of R-PIA in the presence of a peripherally acting adenosine antagonist, demonstrating that, under these conditions, the agonist reaches receptors in significant amounts.

Caffeine (100 mg/kg, i.p.) induces a rapid increase in the expression of mRNA for the immediate early genes (IEGs) c-fos and NGFI-A in rat striatum. We have examined how this response is affected by pretreatment with either the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (1 and 3 mg/kg, i.p.), the competitive NMDA receptor antagonist D-CPP (6 mg/kg, i.p.), or the non-selective excitatory amino acid receptor antagonist kynurenic acid (300 mg/kg, i.p). The two NMDA receptor antagonists significantly reduced the caffeine-induced expression of both c-fos mRNA and NGFI-A mRNA in the medial part of the caudate putamen. The effect was less pronounced in the lateral part of the caudate putamen. MK-801 caused an enhancement of c-fos and NGFI-A mRNA expression in nucleus accumbens. Pretreatment with kynurenic acid caused no marked alterations in the caffeine-induced expression of c-fos mRNA and NGFI-A mRNA in any brain region. These findings suggest that glutamatergic transmission via NMDA receptors contributes to the induction of c-fos mRNA and NGFI-A mRNA by caffeine in striatum. In addition we show that MK-801 can either increase or decrease the caffeine effect on IEGs depending on the region studied.

The time course of expression of mRNA for NGFI-A and NGFI-B after a single intraperitoneal injection of saline or caffeine was examined using in situ hybridization. Administration of a high dose of caffeine (100 mg/kg) decreased locomotor behavior and increased NGFI-A and NGFI- B mRNA in the entire striatum. A lower dose of caffeine (50 mg/kg) caused a weak enhancement of both messages, which was confined to the lateral part of caudate-putamen. This dose increased horizontal, but not vertical, movement. In rats treated with the lowest dose of caffeine (25 mg/kg), the expression of both investigated genes tended to be lower than for saline-treated rats and both horizontal and vertical locomotor activity increased markedly. The reduction in the number of labeled neurons seemed to occur predominantly in enkephalin- containing neurons, which coexpress adenosine A2A receptors and dopamine D2 receptors. The decrease of mRNA for NGFI-A, NGFI-B, and jun B caused by caffeine (25 mg/kg) could be mimicked by the D2 agonist quinpirole (1 and 3 mg/kg). Moreover, caffeine could significantly decrease the expression seen following treatment with the D2 antagonist raclopride (2 mg/kg). In addition, in the parietal cortex, 25 mg/kg of caffeine caused a significant elevation of both examined immediate early genes. Thus, biphasic changes in locomotion induced by caffeine are paralleled by biphasic changes in mRNA for NGFI-A, NGFI-B, and jun B. The results also provide additional support for a functionally important interaction between adenosine and dopamine D2 receptors.

Adenosine is present in the brain under physiological conditions and acts on G protein-coupled receptors. Adenosine regulates neurotransmitter release and neuronal firing rate, dopaminergic neurotransmission, glial cell function, and cerebral blood flow. Adenosine is implicated in sleep/wakefulness and protection against ischemic brain damage, epilepsy, and pain.

2-[p-(2-carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadeno sine (CGS 21680) is considered a selective ligand for adenosine A2A receptors, which are known to be enriched in striatum and olfactory tubercle. We have investigated the characteristics of [3H]CGS 21680 binding in several brain regions using quantitative autoradiography. In agreement with previous data the radioligand was found to label the caudate-putamen, accumbens nucleus, olfactory tubercle and globus pallidus, but also many other structures, e.g. cerebral and cerebellar cortex, hippocampus, thalamus and some brainstem nuclei, were labelled. Cortical and striatal binding of [3H]CGS 21680 was unaltered by high concentrations of the adenosine transport inhibitor dipyridamole or the phosphodiesterase inhibitor rolipram but was displaced by 1,3-diethyl-8-phenylxanthine, the A2 selective adenosine antagonist CP 66,713, and the A2A selective agonist SHA 118. These three agents were approximately equipotent in striatum, cortex and hippocampus. The A2 selective agonist CV 1808 was a 4-5 times more potent displacer in cortex and hippocampus than in the striatum. [3H]CGS 21680 binding was strongly magnesium-dependent in all the studied brain regions, in contrast to the binding of adenosine A1 agonists. The binding of [3H]CGS 21680 to cerebral cortex and hippocampus, but not the binding to striatum, was displaced by the adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine in nanomolar concentrations. The present study provides evidence that in cerebral cortex and hippocampus, most of the [3H]CGS 21680 binds to a receptor site that is distinct from the striatal A2A receptor and the classical adenosine A1 receptor and may represent a hitherto unrecognized binding site.

Of the known biochemical actions of caffeine, only inhibition of adenosine receptors occurs at concentrations achieved during normal human consumption of the drug. Under normal physiological conditions, adenosine is present in sufficient concentrations to activate A1 and A2a receptors. Via actions on A, receptors, adenosine decreases neuronal firing and the release of neurotransmitters. The exact mechanisms are not known, but several possibilities are discussed. Via actions on A2a receptors, adenosine - and hence caffeine - can influence dopaminergic neurotransmission. Caffeine can induce rapid changes in gene expression and, somewhat later, marked adaptive changes. These include antiepileptic and neuroprotective changes. Thus, caffeine has a number of central effects directly or indirectly related to adenosine receptors. Some of these are potentially useful, and drug development based on the actions of caffeine should be interesting.

Research on adenosine has a long history, and since the beginning more than 80 years ago there has been a link to energy metabolism. This is due to the fact that levels of adenosine increase under conditions of relative energy deficiency and that adenosine can have actions that limit this: increase respiration, increase blood flow, and reduce cellular work. There are also effects on intermediary metabolism and on overall energy homeostasis. This minireview provides a brief and personal historical background to these developments.

Role of Adenosine A1 Receptors in Regulation of Arteriolar Responses to Adenosine and Angiotensin II

The rat D2 receptor and the dog A2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D2 receptors/mg protein and approx. 5 pmol/mg protein of the A2a receptor as judged by binding experiments with [3H]raclopride [or[3H]-N-propyl-apomorphine (NPA)] and [3H]-CGS 21680, respectively. The high affinity KD values were 0.43 and 19 nM for D2 and A2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A2b receptors known to be present on virtually every cell. The two A2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D2 receptors in the co-transfected COS-7 cells in contrast to the increased KH, KL and RH values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A2a and D2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of Gs protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A2a receptors and D2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.

Adenosine is released during brain ischemia and provides neuroprotection by actions on nerve and glial cells. Activation of the adenosine A1 receptor enhances the K+ and Cl- conductance in neurons, leading to membrane hyperpolarization and postsynaptic reduction of neuronal Ca2+ influx through voltage- and NMDA receptor-dependent channels. In addition adenosine A1 receptor activation decreases excitatory amino acid release, possibly via inhibition of N- and P-type Ca2+ channels. The A1 and A2 receptors, coupled to Gi/G(o) and Gs proteins respectively, often co-exist and interact with the phospholipase C-dependent activation of the protein kinase C and the adenylyl cyclase. Activation of the A1 receptor may mimic metabotropic receptor stimulation in activating intracellular Ca2+ mobilization and PKC. A2 receptor mediated cAMP formation is depressed by high intracellular Ca2+ but enhanced by PKC activation. By modulating these metabolic signaling events, adenosine may influence acute cell functions, gene transcription and sustained changes of nerve and glial cells relevant for the development of ischemic damage. The neuroprotective adenosine effect seems to be amplified by treatment with propentofylline, which enhances adenosine release, influences the balance between A1 and A2 receptor mediated actions, depresses the free radical formation in activated microglia and influences astrocyte reactions.

1. Adenosine is an endogenous neuroprotective agent; stimulation of A1 receptors decreases excitatory amino acid neurotransmission and stimulation of A2 receptors inhibits platelet and neutrophil activation and promotes vasodilation. 2. Post-ischemic administration of propentofylline (HWA 285) reduces neuronal damage in gerbils and improves glucose metabolism in all regions of brain in acute stroke patients. 3. Propentofylline inhibits the transport of adenosine into cultured cells and increases extracellular adenosine concentrations in ischemic brain. Thus, enhanced stimulation of adenosine receptors may account for some of the neuroprotective effects of this compound. 4. Propentofylline inhibits free radical production by cultivated microglia cells, stimulates nerve growth factor production and inhibits cAMP-phosphodiesterase activity. These effects may also be important for neuroprotection.

We used quantitative in situ hybridization to study changes in the expression of c-fos following hypoxic-ischemia (H-I) in the neonatal rat brain. 7-day-old rat pups were subjected to a unilateral ligation of the common carotid artery followed by a 2 h 15 min hypoxic period (7.7% O2 in N2). This resulted in the expected ipsilateral infarction of cortex, lateral hippocampus, lateral-superior aspects of the striatum and the white matter of the corpus callosum. Brain damage was not seen in the contralateral hemisphere subjected only to hypoxia. c-fos mRNA levels increased in the contralateral hemisphere immediately after the hypoxia and had returned towards normal levels 2 h thereafter. In the ipsilateral hemisphere, the expression of c-fos was delayed but very marked at 2 h. Animals subjected only to hypoxia showed little or no increase in c-fos mRNA. Thus the earliest recorded increase in c-fos after hypoxic ischemia, which occurred on the non-ischemic, contralateral side, may represent a generalized response to a more localized insult.

Male rats were treated i.p. with either 5 mg/kg amphetamine, 3 and 30 mg/kg cocaine or 100 mg/kg caffeine and killed after 30 min. Brains were sectioned and processed for radioactive in situ hybridization histochemistry for the labelling of either c-fos, enkephalin, substance P, neurokinin B, choline acetyltransferase, somatostatin or adenosine A2A receptor messenger RNA. The distribution of c-fos messenger RNA was investigated both at the regional level using film autoradiography, and at the cellular level using emulsion autoradiography. All drug treatments except 3 mg/kg cocaine induced an increased level of c-fos messenger RNA in cells that had a neuron-like morphology. The cells that contained the c-fos messenger RNA were identified by making pairs of 5-μm sections in which one section was processed for c-fos messenger RNA and the other was processed for one of the other messenger RNA species. After amphetamine treatment, only some 10% of the cells in the striatum were labelled, and to a variable extent. Instead there was prominent labelling of a band in the cortex that runs parallel to the cortical surface. There was also a moderate degree of labelling in the nucleus accumbens. c-fos -positive cells were substance P-positive and negative for enkephalin or A2A receptor messenger RNA. Cocaine (30 mg/kg) induced a modest labelling in the caudate-putamen, as well as in the accumbens. With cocaine treatment (30 mg/kg), about 30% of striatal neuron-like cells were c-fos labelled. Most c-fos -positive cells were substance P-positive, but none of the c-fos -positive cells were enkephalin-positive or A2A-receptor-positive. Cocaine (3 mg/kg) had no significant effect on c-fos. Caffeine gave rise to a strong hybridization signal in the caudate-putamen, particularly the dorsolateral part. No other region examined differed significantly from control. With caffeine treatment, about 73% of neuron-like cells were c-fos labelled in the lateral striatum, but labelling was much less pronounced in the medial part or in the accumbens. c-fos -labelled cells were found in enkephalin-positive and enkephalin-negative, substance P-positive and substance P-negative, neurokinin B-positive and neurokinin B-negative groups. No choline acetyltransferase-positive or somatostatin-positive cells were found that were also c-fos -positive with any of the treatments. We conclude that each of the different CNS stimulant drugs induces a highly specific pattern of c-fos messenger RNA. Furthermore, caffeine induces a pattern of c-fos induction that differs markedly from that of amphetamine and cocaine.

We analysed the expression of protein kinase C (PKC) isoforms in smooth muscle cells (SMC) in various states of differentiation, using Western blots and thermocycle amplification of mRNA. SMC isolated from intact tissues express three isoforms of PKC, namely alpha, delta, and zeta. Following cell culture, SMC additionally express mRNA for PKC-epsilon, but significant amounts of the corresponding protein were not detected. Transformed SMC, such as the cell line DDT1 MF-2, express both mRNA and protein for PKC-epsilon, lack the delta isoenzyme, whilst maintaining the expression of the alpha and zeta isoforms. Thus PKC-delta and epsilon isoenzyme expression appears to vary with the state of differentiation of these cells, with PKC-epsilon expression increasing as the cells become proliferative.

Adenosine mediates tubuloglomerular feedback responses via activation of A(1)-receptors on the renal afferent arteriole. Increased preglomerular reactivity, due to reduced nitric oxide (NO) production or increased levels of ANG II and reactive oxygen species (ROS), has been linked to hypertension. Using A(1)-receptor knockout (A(1)(-/-)) and wild-type (A(1)(+/+)) mice we investigated the hypothesis that A(1)-receptors modulate arteriolar and blood pressure responses during NO synthase (NOS) inhibition or ANG II treatment. Blood pressure and renal afferent arteriolar responses were measured in nontreated mice and in mice with prolonged N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or ANG II treatment. The hypertensive responses to L-NAME and ANG II were clearly attenuated in A(1)(-/-) mice. Arteriolar contractions to L-NAME (10(-4) mol/l; 15 min) and cumulative ANG II application (10(-12) to 10(-6) mol/l) were lower in A(1)(-/-) mice. Simultaneous treatment with tempol (10(-4) mol/l; 15 min) attenuated arteriolar responses in A(1)(+/+) but not in A(1)(-/-) mice, suggesting differences in ROS formation. Chronic treatment with L-NAME or ANG II did not alter arteriolar responses in A(1)(-/-) mice, but enhanced maximal contractions in A(1)(+/+) mice. In addition, chronic treatments were associated with higher plasma levels of dimethylarginines (asymmetrical and symmetrical) and oxidative stress marker malondialdehyde in A(1)(+/+) mice, and gene expression analysis showed reduced upregulation of NOS-isoforms and greater upregulation of NADPH oxidases. In conclusion, adenosine A(1)-receptors enhance preglomerular responses during NO inhibition and ANG II treatment. Interruption of A(1)-receptor signaling blunts l-NAME and ANG II-induced hypertension and oxidative stress and is linked to reduced responsiveness of afferent arterioles.

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A(2A) receptors (A(2A)Rs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A(2A)Rs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A(2A)Rs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A(2A)Rs were focally removed from the NAc core or other A(2A)R-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

Adenosine levels are increased under conditions of energy deprivation, both because intracellular energy stores are reduced and because ATP is released. The adenosine thus formed can serve to influence energy homeostasis in a number of different ways, besides alterations in blood supply and cellular work (including contraction, maintenance of membrane potential, and biosynthesis), which will be covered in other chapters. Here, effects on energy homeostasis will be briefly reviewed. Adenosine acting at the A(1) receptor is a powerful and nonredundant inhibitor of lipolysis. It increases glucose uptake in fat and muscle, but its effects on insulin secretion may be even more important than the actions at insulin target tissues. Glucagon is also influenced. In addition to these peripheral actions, adenosine acts in the brain to regulate sleep-wakefulness, food intake, and body temperature. These effects are both direct at the relevant neurons and indirect by influences on regulatory transmitters and hormones.

OBJECTIVE To determine the mechanisms by which blockade of adenosine A2B receptors (A2BRs) reduces insulin resistance. RESEARCH DESIGN AND METHODS We investigated the effects of deleting or blocking the A2BR on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A2BR transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ∼42 kb encompassing the A2BR gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation. RESULTS Treatment of mice with the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A2BR deletion or blockade and minimally affected by deletion of A1Rs or A2ARs. During hyperinsulinemic-euglycemic clamp of diabetic KKAY mice, A2BR antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A2BR mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status. CONCLUSIONS Diabetes affects the production of adenosine and the expression of A2BRs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A2BR signaling in diabetes increases insulin resistance in part by elevating proinflammatory mediators. Selective A2BR blockers may be useful to treat insulin resistance.

Adenosine levels are increased under conditions of energy deprivation, both because intracellular energy stores are reduced and because ATP is released. The adenosine thus formed can serve to influence energy homeostasis in a number of different ways, besides alterations in blood supply and cellular work (including contraction, maintenance of membrane potential, and biosynthesis), which will be covered in other chapters. Here, effects on energy homeostasis will be briefly reviewed. Adenosine acting at the A(1) receptor is a powerful and nonredundant inhibitor of lipolysis. It increases glucose uptake in fat and muscle, but its effects on insulin secretion may be even more important than the actions at insulin target tissues. Glucagon is also influenced. In addition to these peripheral actions, adenosine acts in the brain to regulate sleep-wakefulness, food intake, and body temperature. These effects are both direct at the relevant neurons and indirect by influences on regulatory transmitters and hormones.

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In this study, the basal and evoked release of [3H]- and endogenous adenosine, inosine and hypoxanthine from rat hippocampal slices, labelled with [3H]adenine, was investigated. Evoked release was brought about by either electrical stimulation or energy depletion. The aim was to determine whether adenosine is formed intracellularly, and released as adenosine or extracellularly, from sequential extracellular hydrolysis of released ATP. All measurements were made in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA) to inhibit the enzyme adenosine deaminase. It was found that electrical field stimulation (5 min) increased the release of endogenous adenosine from hippocampal slices 10-fold and increased the proportion of [3H]-label associated with adenosine from approx 7% of the total released to 13% after the first stimulation and 20% after the second stimulation. Removal of oxygen and glucose from the superfusion medium (energy depletion) increased the release rate of endogenous adenosine 16-fold and increased the proportion of [3H]-label associated with [3H]adenosine from approx 10% of the total released to 50%. In order to prevent extracellular formation of adenosine, experiments were carried out in the presence of 50 microM alpha, beta-methylene ADP (AOPCP), an inhibitor of ecto-5'-nucleotidase. AOPCP was found to be without effect on either the basal or evoked release of adenosine. In contrast, L-homocysteine thiolactone (0.1-1.0 mM) which was used to "trap" intracellular adenosine reduced both the basal and evoked release of adenosine by 70-85%. This effect of L-homocysteine thiolactone also occurred in the presence of adenosine uptake inhibitors. It is concluded from these results that adenosine is formed predominantly intracellularly in hippocampal slices and is released as adenosine as a result of either tissue depolarisation or energy depletion. Furthermore, the finding that during energy depletion there is a proportionally greater release of adenosine than other ATP breakdown products, such as inosine and hypoxanthine, indicates that energy depletion is both a potent and selective stimulus for adenosine formation and release.

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca2+ and a sustained inhibition of forskolin-, dopamine- and 5'-N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca2+]i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca2+ (10(-7) to 10(-3) M), both in ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca2+]i and subsequent entry of Ca2+ which directly interacts with a component of the adenylyl cyclase system.

The effects of long-term caffeine treatment onN-methyl- d -aspartate (NMDA)-induced seizures in mice were studied. Caffeine was added (0.3 g/1) to drinking water for 14 days and the mice ingested 60–70 mg/kg/day. During the treatment, the plasma concentrations of methylxanthines (caffeine, theophylline and/or paraxanthine, theobromine) were measured. NMDA (150 mg/kg i.p.) was administered to control mice and to mice during and after the caffeine administration. A1 adenosine receptor density in the gyrus dentatus of hippocampus, measured by quantitative receptor autoradiography with [3H]cyclohexyl adenosine as the ligand, was not significantly altered after long-term caffeine treatment. NMDA-induced clonic seizures, wet dog shakes and mortality were significantly reduced at the end of long-term caffeine treatment but returned towards control at 1 and 2 days after withdrawal. At the end of caffeine treatment, tonic seizures were also absent. These results show that long-term treatment with caffeine in a dose that gives plasma levels of 6–10 μM decreases the effects of NMDA on e.g. seizure susceptibility, and that this effect cannot be ascribed to changes of A1 adenosine receptor density.

In the present overview we focus on two aspects: (a) the role of adenosine as an endogenous cerebroprotective agent and (b) the possible significance of adenosine-dopamine interactions. Adenosine is a normal constituent of the brain extracellular environment. Its levels greatly increase upon neuronal excitation or as a consequence of decreased supply of metabolic substrates. Via its receptors it tends to limit its own formation in several ways. By increasing blood flow and glucose uptake it increases substrate supply. By decreasing the release of excitatory neurotransmitters and by causing hyperpolarization it decreases energy demand. This suggests a role as an endogenous cerebroprotective agent, something which is supported by several lines of pharmacological evidence. The adenosine A2a receptor is enriched in dopamine rich-areas of the brain and it has been found to be colocalized with dopamine D2 receptors on medium-sized spiny neurons in the striatum. Recent evidence shows that A2 agonists decrease agonist binding to dopamine D2 receptors. Furthermore, adenosine receptor antagonists have been shown to produce behavioural effects that are due to enhanced dopaminergic neurotransmission. It is suggested that the A2- D2-receptor interactions may explain some of the stimulatory effects of low doses of caffeine. © 1993 Wiley-Liss, Inc.

There is a need to reduce the amount of fossil energy used for transport, both because of the easily available fossil fuel is becoming sparser and because of climate concerns. In this article, the concept of “peak oil” is briefly presented. Second, a practical approach to reduction of fossil fuel use for transport elaborated by two British commissions is presented. A key feature is the introduction of electric cars. This raises the third issue covered in this article: namely, how battery technology is going to meet the increasing needs posed by the transport sector.

Ghrelin, a potent orexigenic hormone released from the stomach, is important in regulating energy metabolism. Abnormal ghrelin levels are associated with eating disorders and metabolic diseases. However, factors involved in the regulation of ghrelin release remain unclear. Here, we examined the involvement of adenosine signaling in the control of ghrelin release from the perfused mouse stomach. Adenosine stimulated ghrelin release concentration-dependently, and the A(2A) receptor-selective antagonists 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) and 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH 58261) abolished the increased release. The A(2A) receptor-selective agonist 2-p-(2-carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680) augmented ghrelin release concentration-dependently, whereas the A(1) receptor-selective agonist 2-chloro-N(6)-cyclopentyladenosine inhibited ghrelin release. In A(2A) receptor knockout mice, adenosine inhibited ghrelin release, and the A(1) receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked this inhibition. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride increased ghrelin release in wild-type and A(1) receptor knockout mice but not in A(2A) receptor knockout mice. Colocalization of ghrelin immunoreactivity with A(1) and A(2A) receptor immunoreactivities in the gastric nerve fibers were observed. Colocalization was also detected for ghrelin and A(1) receptor immunoreactivities in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine exerts predominantly a tonic A(2A) receptor-mediated stimulatory action on gastric ghrelin release, whereas an A(1) receptor-mediated inhibitory action is also apparent when the tonic excitatory effect was removed.

A meeting was held May 19, 2010 at the Karolinski Institute on Nomenclature in Pharmacology. This meeting occurred in conjunction with the Symposium The Changing World of G Protein Coupled Receptors: From Monomers to Dimers and Receptor Mosaics (Higher-order Oligomers) held the previous day at the Royal Swedish Academy of Science. Two broad topics of nomenclature were discussed; ligand nomenclature and the definition of ‘receptor–receptor’ interactions. This paper summarizes discussions on these topics along with a consensus definition of the term ‘receptor–receptor’ interaction.

As behooves something so deeply entrenched in culture, the historical origins of the use of methylxanthines are unknown and dressed in myth. This is true for coffee as well as tea, and for both it is interesting to note that their common use is really very recent. For coffee we know that its use became more widespread in the fifteenth and sixteenth centuries, and in Europe this occurred in the eighteenth and nineteenth centuries. The use of tea became more common during the Ming Dynasty in China and during the eighteenth century in Britain. Coffee was mostly an upper-class drink in Arabia, and remained a relative luxury in Europe until quite recently. The use of other methylxanthine-containing beverages, such as maté, is even less well known. It is interesting to note that before these drinks were commonly used on a daily basis they were used for medicinal purposes, indicating that their pharmacological actions had long been noted.

A high protein intake is associated with increased glomerular filtration rate (GFR), which has been suggested to be mediated by reduced signaling of the tubuloglomerular feedback (TGF) mechanism. Nitric oxide (NO) has been shown to contribute to high protein-induced glomerular hyperfiltration, but the specific NO synthase (NOS) isoform responsible is not clear. In this study, a model for high-protein-induced hyperfiltration in conscious mice was developed. Using this model, we investigated the role of TGF using adenosine A1-receptor knockout mice lacking the TGF mechanism. Furthermore, the role of the different NOS isoforms was studied using neuronal-, inducible-, and endothelial-NOS knockout mice, and furthermore, wild-type mice acutely administered with the unspecific NOS inhibitor Nω-nitro-l-arginine methyl ester (100 mg/kg). GFR was measured consecutively in mice given a low-protein diet (8% casein) for 10 days, followed by a high-protein diet (50% casein) for 10 days. All mice developed high protein-induced hyperfiltration to a similar degree. These results demonstrate that high protein-induced glomerular hyperfiltration is independent of the TGF mechanism and NOS isoforms.

As behooves something so deeply entrenched in culture, the historical origins of the use of methylxanthines are unknown and dressed in myth. This is true for coffee as well as tea, and for both it is interesting to note that their common use is really very recent. For coffee we know that its use became more widespread in the fifteenth and sixteenth centuries, and in Europe this occurred in the eighteenth and nineteenth centuries. The use of tea became more common during the Ming Dynasty in China and during the eighteenth century in Britain. Coffee was mostly an upper-class drink in Arabia, and remained a relative luxury in Europe until quite recently. The use of other methylxanthine-containing beverages, such as mate, is even less well known. It is interesting to note that before these drinks were commonly used on a daily basis they were used for medicinal purposes, indicating that their pharmacological actions had long been noted.

Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-κB ligand in the presence or absence of the A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A1R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A1R antagonist DPCPX inhibits osteoclast formation (IC50=1 nM), with altered morphology and reduced ability to resorb bone. A1R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A1R and further suggest that A1R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.—Kara, F. M., Chitu, V., Sloane, J., Axelrod, M., Fredholm, B. B., Stanley, R., Cronstein, B. N. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function.