Adenosine deaminase and A1 adenosine receptors internalize together following agonist-induced receptor desensitization

A1 adenosine receptors (A1Rs) and adenosine deaminase (ADA; EC 3.5.4. 4) interact on the cell surface of DDT1MF-2 smooth muscle cells. The interaction facilitates ligand binding and signaling via A1R, but it is not known whether it has a role in homologous desensitization of A1Rs. Here we show that chronic exposure of DDT1MF-2 cells to the A1R agonist, N6-(R)-(phenylisopropyl)adenosine (R-PIA), caused a rapid aggregation or clustering of A1 receptor molecules on the cell membrane, which was enhanced by pretreatment with ADA. Colocalization between A1R and ADA occurred in the R-PIA-induced clusters. Interestingly, colocalization between A1R and ADA also occurred in intracellular vesicles after internalization of both protein molecules in response to R-PIA. Agonist-induced aggregation of A1Rs was mediated by phosphorylation of A1Rs, which was enhanced and accelerated in the presence of ADA. Ligand-induced second-messenger desensitization of A1Rs was also accelerated in the presence of exogenous ADA, and it correlated well with receptor phosphorylation. However, although phosphorylation of A1R returned to its basal state within minutes, desensitization continued for hours. The loss of cell-surface binding sites (sequestration) induced by the agonist was time-dependent (t1/2= 10 +/- 1 h) and was accelerated by ADA. All of these results strongly suggest that ADA plays a key role in the regulation of A1Rs by accelerating ligand-induced desensitization and internalization and provide evidence that the two cell surface proteins internalize via the same endocytic pathway.     

Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus

The adenosine A3 receptor is expressed in brain, but the consequences of activation of this receptor on electrophysiological activity are unknown. We have characterized the actions of a selective adenosine A3 receptor agonist, 2-chloro-N6-(3-lodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), and a selective A3 receptor antagonist, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191), in brain slices from rat hippocampus. In the CA1 region, activation of A3 receptors had no direct effects on synaptically evoked excitatory responses, long-term potentiation, or synaptic facilitation. However, activation of A3 receptors with Cl-IB-MECA antagonized the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission. The effects of Cl-IB-MECA were blocked by pretreatment with MRS 1191, which by itself had no effect on A1 receptor-mediated responses. The presynaptic inhibitory effects of baclofen and carbachol, mediated via GABA(B) and muscarinic receptors, respectively, were unaffected by Cl-IB-MECA. The maximal response to adenosine was unchanged, suggesting that the primary effect of Cl-IB-MECA was to reduce the affinity of adenosine for the receptor rather than to uncouple it. Similar effects could be demonstrated after brief superfusion with high concentrations of adenosine itself. Under normal conditions, endogenous adenosine in brain is unlikely to affect the sensitivity of A1 receptors via this mechanism. However, when brain concentrations of adenosine are elevated (e.g., during hypoxia, ischemia, or seizures), activation of A3 receptors and subsequent heterologous desensitization of A1 receptors could occur, which might limit the cerebroprotective effects of adenosine under these conditions.     

Binding affinity of adenosine receptor agonists and antagonists at human cloned A3 adenosine receptors

The A3 adenosine receptor is one of the four adenosine receptors which have thus far been identified. Cloning of the A3 receptor from animal species such as rat, sheep and human has shown that there are interspecies differences in its peripheral distribution, and binding affinity for various adenosine receptor ligands. The adenosine derivative, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA), is a potent A3 receptor agonist which is used as a reference drug. In this report we have characterized the binding of selected adenosine receptor agonists and antagonists to HEK 293 cells transfected with the human A3 adenosine receptor using [125I]AB-MECA as radioligand. HE-NECA and NECA were the most potent compounds showing Ki values in the low nanomolar range, while the recently discovered non-xanthine A2A receptor antagonists ZM 241385, SCH 58261 and SCH 63390 showed affinity values in the micromolar range. These data further indicate the need to examine the affinity of new adenosine receptor ligands directly in human A3 receptors.     

The unpredicted high affinities of a large number of naturally occurring tachykinins for chimeric NK1/NK3 receptors suggest a role for an inhibitory domain in determining receptor specificity

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.     

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.     

N6,C8-distributed adenosine derivatives as partial agonists for adenosine A1 receptors

The synthesis and biological evaluation of N6, C8-disubstituted derivatives of adenosine as potential partial agonists for adenosine receptors is described. Via three routes, two series of compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a-e and C8-(cyclopentylamino)adenosine analogs 3e and 9a-d, respectively. The X-ray structure determination of one of these compounds, N6-ethyl-8(cyclopentylamino)adenosine (9b), was carried out (orthorhombic, space group P2(1)2(1)2(1) (No. 19) with a = 11.039(3), b = 8.708(2), and c = 24.815(12) angstrom, Z=4,R1=0.0974,R2(W) = 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety of this compound is in an anti conformation. The compounds were tested in vitro in radioligand binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1 receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and 3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in conscious, normotensive rats.     

Molecular structural basis of ligand selectivity for 5-HT2 versus 5-HT1C cortical receptors

A molecular structural criterion of ligand selectivity for the 5-HT2 versus 5-HT1C receptor was hypothesized on the basis of radioligand binding data. Despite the large number of compounds which have been tested at both receptors, analysis of published data led to the identification of only five agents which are greater than 10-fold selective for the 5-HT2 versus the 5-HT1C receptor. Comparison of the two-dimensional structures revealed that, although these five compounds represent three distinct structural classes, they share a common structural feature located in the region hypothesized to be involved in receptor binding: a carbonyl or carboxyl oxygen interposed spatially between an aromatic ring and nitrogen atom. This structural feature was used to predict the relative selectivity of compounds that had not previously been analyzed at both the 5-HT2 and 5-HT1C receptors. All six drugs tested which contain the identified reactive carbonyl or carboxyl group were found to be selective for the 5-HT2 versus the 5-HT1C receptor with selectivity ratios ranging from 26 to 380. By contrast, three agents which are structurally similar but do not contain the reactive carbonyl or carboxyl group displayed equally high affinity for both receptor binding sites. Since the physiological roles of the 5-HT2 and 5-HT1C receptor are markedly different, it would be of potential clinical and scientific value to utilize this molecular structural feature to further identify chemical compounds which would selectively interact with only one of the two receptors.     

Homologous desensitization of the D1A dopamine receptor: efficacy in causing desensitization dissociates from both receptor occupancy and functional potency

The role of drug efficacy in agonist-induced desensitization was studied in C-6 glioma cells transfected with the monkey dopamine D1A (mD1A) receptor. Dopamine pretreatment for 2 hr produced greater than 80% loss of responsiveness in the stimulation of cAMP accumulation that was blocked by the D1 antagonist SCH23390. A series of full and partial D1 agonists from structurally dissimilar classes were then examined. Three full agonists (dihydrexidine, SKF82958, A77636) desensitized the receptor to the same extent as dopamine, whereas two other full agonists (dinapsoline and A68930) and all the partial agonists tested (SKF38393, pergolide and d-lysergic acid diethylamide tartrate) produced only partial desensitization (i.e., 50% that of dopamine). Whereas partial agonists (i.e., SKF38393, pergolide and d-lysergic acid diethylamide tartrate) caused no alteration in ligand-accessible mD1A receptors, four of the full agonists (dopamine, dihydrexidine, dinapsoline, A68930) caused a 30 to 40% reduction in receptor number. One full agonist, A77636, caused nearly an 80% decrease in receptor number, despite the fact that the degree of functional desensitization was similar to the other full agonists. The desensitization of the D1 receptor was homologous, not affecting beta-2 adrenergic receptors endogenous to C-6 cells. Neither incubation with cAMP analogs, nor inhibition of protein kinase A, affected dopamine-induced desensitization, suggesting a cAMP-independent mechanism in this cell line. Together, these data suggest that functional desensitization of the mD1A receptor expressed in C-6 glioma cells is a cAMP-independent mechanism, cannot be predicted reliably from agonist efficacy for stimulating adenylate cyclase and can occur in the absence of changes in receptor number.     

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

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

Cardiac myocytes rendered ischemia resistant by expressing the human adenosine A1 or A3 receptor

Adenosine is an important mediator of the endogenous defense against ischemia-induced injury in the heart. Adenosine can achieve cardioprotection by mediating the effect of ischemic preconditioning and by protecting against myocyte injury when it is present during the infarct-producing ischemia. A novel adenosine A3 receptor can mediate this protective function. One approach to achieve cardioprotection is to enhance myocardial sensitivity to the endogenous adenosine by increasing the number of adenosine receptors instead of administering an adenosine receptor agonist. The objective of the present study was to investigate whether genetic manipulation of the cardiac myocyte, achieved by gene transfer and overexpression of the human A3 receptor cDNA, renders the myocytes resistant to the deleterious effect of ischemia. Prolonged hypoxia with glucose deprivation, causing myocyte injury and adenosine release, was used to simulate ischemia in cultured chick embryo ventricular myocytes. During simulated ischemia, cultured myocytes with enhanced expression of the human A3 receptor and showed significantly higher ATP content, fewer cells killed, and less creatine kinase released into the medium than either control or mock-transfected myocytes. Also, increased expression of the A3 receptor caused an enhanced cardioprotective effect by the preconditioning ischemia. Overexpressing the adenosine A1 receptor also led to increased protection against ischemia-induced myocyte injury as well as an enhanced preconditioning effect. Thus, increasing the receptor level improves the myocyte sensitivity to the endogenous adenosine, which in turn causes all of the cardioprotective effects found for exogenously administered adenosine agonists. The study provides the first proof for the new concept that an increased expression of the human A3 receptor in the cardiac myocyte can be an important cardioprotective therapeutic approach.     

Loss of A1 adenosine receptors in human temporal lobe epilepsy

Using quantitative receptor autoradiographic methods we have examined A1 adenosine receptors, adenosine uptake sites, benzodiazepine receptors, NMDA, AMPA, and kainic acid receptors in temporal lobes removed from patients suffering from complex partial seizures and in normal control post-mortem temporal cortex. Binding to A1 adenosine receptors and NMDA receptors was reduced in epileptic temporal cortex, while the other neurochemical parameters were unchanged. The reason for this A1 receptor loss is unclear as it occurred in both idiopathic and symptomatic cases and thus may be a consequence rather than an initial cause of seizures. However, because adenosine is a powerful anticonvulsant substance, loss of anticonvulsant A1 receptors may contribute to the human epileptic condition. It is also possible that the observed differences in A1 binding are due to autopsy vs. biopsy changes in the levels of A1 adenosine receptors.     

Modulation of [3H]quinpirole binding to dopaminergic receptors by adenosine A2A receptors

Alteration of ligand binding to dopamine D2 receptors through activation of adenosine A2A receptors in rat striatal membranes has been studied by means of kinetic analysis. The binding of dopaminergic agonist [3H]quinpirole to rat striatal membranes was characterized by the constants Kd = 1.50+/-0.09 nM and Bmax = 115+/-2 fmol/mg of protein. The kinetic analyses revealed that the binding had at least two consecutive and kinetically distinguishable steps, the fast equilibrium of complex formation between receptor and agonist (KA = 5.9+/-1.7 nM), followed by a slow isomerization equilibrium (Ki = 0.06). Activation of adenosine A2A receptors by CGS 21680 caused enhancement of the rate [3H]quinpirole binding, altering mainly the formation of the receptor-ligand complexes (KA) as well as the isomerization rate of this complexes (ki), while the deisomerization rate (k[-i]) and the apparent dissociation rate remained unchanged.     

Constitutive activity of a chimeric D2/D1 dopamine receptor

Chimeric D1/D2 receptors were constructed to identify structural determinants of drug affinity and efficacy. We previously reported that chimeras that had D1 receptor transmembrane domain VII together with amino-terminal sequence from the D2 receptor were nonfunctional. D2/D1 chimeras were constructed that contained D2 receptor sequence at the amino- and carboxyl-terminal ends and D1 receptor sequence in the intervening region. Chimeric receptors with D2 sequence from transmembrane domain 7 to the carboxyl terminus together with D2 receptor sequence from the amino terminus through transmembrane helix 4 (D2[1-4,7]) and 5 (D2[1-5,7]) bound [3H]spiperone with high affinity, consistent with the hypothesis that D2 receptor transmembrane domain I or II is incompatible with D1 receptor transmembrane domain VII. D2[1-4,7] and D2[1-5,7] had affinities similar to D1 and D2 receptors for most nonselective dopamine antagonists and had affinities for most of the selective antagonists that were intermediate between those of the parent receptors. D2[1-4,7] and D2[1-5,7] mediated dopamine receptor agonist-induced stimulation and inhibition, respectively, of cAMP accumulation. The more efficient coupling of D2[1-5,7] to inhibition of cAMP accumulation, compared with the coupling of D2[5-7] and D2[3-7], supports the view that multiple D2 receptor cytoplasmic domains acting in concert are necessary for receptor activation of Gi. In contrast, D2[1-4,7], which contains only one cytoplasmic loop (the third) from the D1 receptor, is capable of activating Gs. D2[1-4,7] exhibited several characteristics of a constitutively active receptor, including enhanced basal (unliganded) stimulation of cAMP accumulation, high affinity for agonists even in the presence of GTP, and blunted agonist-stimulated cAMP accumulation. A number of dopamine receptor antagonists were inverse agonists at D2[1-4,7], inhibiting basal cAMP accumulation. Some of these drugs were also inverse agonists at the D1 receptor. Interestingly, several antagonists also potentiated forskolin-stimulated cAMP accumulation via D2[1-5,7] and via the D2 receptor, which could reflect inverse agonist inhibition of native constitutive activity of this receptor.     

Modulation of [3H]acetylcholine release from cultured amacrine-like neurons by adenosine A1 receptors

We have investigated the effect of endogenous adenosine on the release of [3H]acetylcholine ([3H]ACh) in cultured chick amacrine-like neurons. The release of [3H]ACh evoked by 50 mM KCl was mostly Ca2+ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A1 receptor antagonist. The effect of adenosine on [3H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca2+ channels. Ligand binding studies using [3H]DPCPX confirmed the presence of adenosine A1 receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A1 receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca2+-dependent release of [3H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A1 receptors. The effect of adenosine on the [3H]ACh release may be due to a direct inhibition of N-type Ca2+ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

The structure and function of A1 and A2B adenosine receptors

Of the four G protein coupled adenosine receptor (AR) subtypes, the A1 is best suited for studies of reconstitution with G proteins. Recombinant A1 receptors extended with hexahistidine and FLAG have been purified to near homogeneity. In reconstitution assays using pure recombinant G protein subunits, the composition of the gamma subunit influences coupling to purified A1ARs. The least well characterized AR is the A2B. New data indicate that A(2B)ARs can trigger the degranulation of canine and human mast cell lines. Recombinant human A(2B)ARs are blocked by the anti-asthma drugs theophylline and enprofylline at concentrations that are used therapeutically to treat asthma. Although A(2B)ARs have long been known to stimulate adenylyl cyclase, they also can activate phospholipase C and mobilize Ca2+ by signaling through Gq/11. There is great potential for new therapies based on compounds that selectively target individual AR subtypes.     

A physiological role of the adenosine A3 receptor: sustained cardioprotection

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart. A newly recognized adenosine receptor, the A3 subtype, is expressed on the cardiac ventricular cell, and its activation protects the ventricular heart cell against injury during a subsequent exposure to ischemia. A cultured chicken ventricular myocyte model was used to investigate the cardioprotective role of a novel adenosine A3 receptor. The protection mediated by prior activation of A3 receptors exhibits a significantly longer duration than that produced by activation of the adenosine A1 receptor. Prior exposure of the myocytes to brief ischemia also protected them against injury sustained during a subsequent exposure to prolonged ischemia. The adenosine A3 receptor-selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) caused a biphasic inhibition of the protective effect of the brief ischemia. The concomitant presence of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) converted the MRS1191-induced dose inhibition curve to a monophasic one. The combined presence of both antagonists abolished the protective effect induced by the brief ischemia. Thus, activation of both A1 and A3 receptors is required to mediate the cardioprotective effect of the brief ischemia. Cardiac atrial cells lack native A3 receptors and exhibit a shorter duration of cardioprotection than do ventricular cells. Transfection of atrial cells with cDNA encoding the human adenosine A3 receptor causes a sustained A3 agonist-mediated cardioprotection. The study indicates that cardiac adenosine A3 receptor mediates a sustained cardioprotective function and represents a new cardiac therapeutic target.