Regulation of Cyclic AMP by the μ-Opioid Receptor in Human Neuroblastoma SH-SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and pharmacological studies of new hybrid derivatives of fentanyl active at the mu-opioid receptor and I2-imidazoline binding sites

Two series of fentanyl-derived hybrid molecules bearing potent I2-imidazoline binding site (IBS) ligands (i.e., guanidine and BU224 moieties) linked with an aliphatic (m=2, 3, 4, 6, 7, 8, 9 and 12 methylene units) or aromatic spacer were prepared. Their affinities for the mu-opioid receptors and for the I2-IBS were determined through competition binding studies on human postmortem brain membranes. Whereas the BU224 hybrid molecules bound to the mu-opioid receptor and the I2-IBS in the micromolar to low micromolar range, the alkaneguanidine series exhibited remarkable affinities in the nanomolar range for both receptors. [35S]GTPgammaS functional assays were performed on human postmortem brain membranes with selected ligands from each series (4f and 8g) showing the highest dual affinity for the mu-opioid receptor and I2-IBS affinities. Both compounds displayed agonist properties: at the mu-opioid receptor for the alkaneguanidine derivative 4f (spacer: six methylene units) and at a G-protein coupled receptor (GPCR) which remains to be determined for 8g. The lack of analgesic properties of 4f in vivo (i.e., hot plate and writhing tests in mice), discordant with the good in vitro binding data (Ki mu=1.04+/-0.28 nM, Ki I2=409+/-238 nM), may possibly be due to the low intrinsic efficacy of the compound. Alternatively, a low access to the central nervous system for this kind of hybrid molecules cannot be ruled out. Two new compounds reported here (9f and 13), which were not dual acting, are worth mentioning for their outstanding binding affinities; 9f bound to the mu-opioid receptor with a picomolar affinity (Ki=0.0098+/-0.0033 nM), whereas 13 presented an I2-IBS affinity (Ki=18+/-11 nM) similar to the reference compound BU224.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Potassium- and Carbachol-Evoked Release of [3H]Noradrenaline from Human Neuroblastoma Cells, SH-SY5Y

The human neuroblastoma clone SH-SY5Y expresses potassium-, carbachol-, and calcium ionophore A23187-evoked, calcium-dependent release of [3H]noradrenaline. Release in response to carbachol and potassium was greater than additive. Atropine (Ki = 0.33 nM), hexahydrosiladifenidol (Ki = 18 nM), and pirenzepine (Ki = 1,183 nM) completely inhibited the carbachol-evoked noradrenaline release, an order of potency suggesting that an M3 receptor was linked to release. In contrast, noradrenaline release was only partially inhibited by the M2-selective antagonists methoctramine (10(-4) M) and AFDX-116 (10(-4) M), by approximately 14 and 46%, respectively. The nicotinic antagonist d-tubocurarine (10(-4) M) resulted in a partial inhibition of release, a finding suggesting that a nicotinic receptor may also be involved. SH-SY5Y provides a suitable cell line in which to study the biochemical mechanisms underlying the cholinergic receptor regulation of noradrenaline release.     

Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor.

A number of studies have demonstrated that a major portion of the ligand binding site of the Torpedo nicotinic acetylcholine receptor is near cysteines 192 and 193 of the alpha subunit. The role of conserved tyrosine and aspartate residues within this region in ligand binding and receptor activation was investigated using a combination of site-directed mutagenesis and expression in Xenopus oocytes. Wild-type receptors are half-maximally activated (K1/2) by 20 microM acetylcholine with a Hill coefficient, n, of 1.9. Substitution of alpha Y190 and alpha Y198 with phenylalanines (alpha Y190F, alpha Y198F) or alpha D200 with asparagine (alpha D200N) shifts the K1/2 to 408, 117, and 75 microM, respectively, with no effect on the Hill coefficient. To further study the effects of these mutations on activation, the responses of the receptors to the partial agonists phenyltrimethylammonium (PTMA) and tetramethylammonium (TMA) were examined. Wild-type receptors are half-maximally activated by 73 microM PTMA and 2 mM TMA. In contrast, alpha Y190F, alpha Y198F, and alpha D200N receptors are not activated by PTMA and TMA by concentrations of up to 500 microM or 5 mM, respectively. However, PTMA and TMA do act as competitive antagonists of the mutant receptors, an indication that the binding of these compounds is not abolished by these mutations. Comparison of the the Ki values for TMA and PTMA inhibition with the K 1/2 values for TMA and PTMA activation of wild-type receptors indicates that the affinities of these compounds are similar in wild-type and mutant receptors. Therefore, alpha Y190F, alpha Y198F, and alpha D200N mutations do not significantly alter the affinity of the ligand binding site; rather, these mutations appear to interfere with the coupling of ligand binding to channel opening.     

D1 Dopamine Receptors of NS20Y Neuroblastoma Cells Are Functionally Similar to Rat Striatal D1 Receptors

Dopamine or agonists with D1 receptor potency stimulated cyclic AMP (cAMP) accumulation in whole cell preparations of NS20Y neuroblastoma cells. The accumulation of cAMP after D1 stimulation was rapid and linear for 3 min. Both dopamine and the novel D1 receptor agonist dihydrexidine stimulated cAMP accumulation two- to three-fold over baseline. The pseudo-Km for dopamine was approximately 2 microM, whereas for dihydrexidine it was approximately 30 nM. The effects of both drugs were blocked by either the D1-selective antagonist SCH23390 (Ki, 0.3 nM) or the nonselective antagonist (+)-butaclamol (Ki, 5 nM). Both (-)-butaclamol and the D2-selective antagonist (-)-sulpiride were ineffective (Ki greater than 3 microM). Forskolin (10 microM), prostaglandin E1 (1 microM), and adenosine (10 microM) also stimulated cAMP accumulation, but none were antagonized by SCH23390 (1 microM). Finally, muscarinic receptor stimulation (100 microM carbachol) inhibited both D1- and forskolin-stimulated increases in cAMP accumulation by 80%. The present results indicate that NS20Y neuroblastoma cells have D1 receptors that are coupled to adenylate cyclase, and that these receptors have a pharmacological profile similar to that of the D1 receptor(s) found in rat striatum.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Differences in the efficiency of their interaction with membrane receptors of the dialkylaminoalkyl and dialkylaminoacyl derivatives of phenothiazine

New dialkylaminoacyl phenothiazine derivatives (DAC) were compared with their dialkylaminoalkyl analogues (neuroleptics chlorpromazine, trifluoperazine and fluphenazine) as well as with anti-arrhythmia drugs ethmozine and ethacizine for their receptor-blocking potencies. It was established that DAC are significantly less potent with dopamine alpha 1-adrenergic and H1-histamine receptors of calf and rabbit brain, which can explain the absence of neuroleptic effect of DAC drugs. DAC affinities to muscarinic and alpha-adrenergic receptors of both types are very similar to those of ethmozine and ethacizine. New DAC substance G-512 (chlorpromazine analogue) demonstrated high affinity to M1-muscarinic receptors of rabbit brain cortex (Ki = 4.2 nM) and to M2-muscarinic receptors of the rabbit heart (Ki = 48 nM).     

Assessment of delta-opioid receptor activation by a series of peptides in cultured cells.

This study was designed to investigate the relative ability of a series of cyclic opioid peptides to initiate the first activation steps following their binding of delta-opioid receptors. The extent of stimulation of low Km guanosine-triphosphatase (GTPase activity) and inhibition of hormonally-stimulated cAMP accumulation in the NG108-15 (neuroblastoma-glioma) hybrid cell line were determined and compared for six closely related peptides. In addition, their binding affinity was assessed by competition with 3H-[D-Pen2D-Pen5]-enkephalin (3H-DPDPE) in membranes from these cells. All peptides tested elicited comparable maximal effects for both functional responses. Different potencies in stimulating the low Km GTPase was observed at sub-maximal agonist concentrations, although the shallow dose-response behavior did not allow accurate determination of ED50s. Estimation of ED50s for inhibition of cAMP accumulation could be made by curve fitting and were similar for four of these peptides, while DCDPE and 3R-methylDCDPE, the highest affinity analogs, were considerably more potent. In general, the observed differences in hormonal activity somewhat parallel the rank order of binding affinities, but no strict relationship was found between receptor binding and activation.     

Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Growth, Receptor Phosphorylation, and Dimerization in Neuroblastoma SH-SY5Y Cells

Abstract: SH-SY5Y is a thrice cloned cell line originally derived from the human neuroblastoma cell line SK-N-SH. It grows well in serum-containing medium and undergoes neuritogenesis in response to several trophic factors. Because it has been reported that this clonal line does not have receptors for platelet-derived growth factor (PDGF), it has been unclear what the major mitogenic factor in serum is for these cells. In competitive binding studies using radiolabeled PDGF-BB, we found that SH-SY5Y cells specifically bind PDGF with a K _D = 0.14 ± 0.06 n M and B _max = 7.3 ± 2.3 p M . Functionality of these receptors was demonstrated by an increased [³H]-thymidine incorporation in response to PDGF (stimulation index = 2.5). At concentrations of PDGF-BB between 5 and 100 ng/ml, maximum stimulation occurred with 20 ng/ml. Maximum DNA synthesis occurred after 12–24-h exposure to PDGF. Gangliosides GM3 and GT1b greatly inhibited [³H]thymidine incorporation, which was also inhibited to a lesser extent by GM1. Phosphorylation on tyrosine of a 170-kDa protein in response to PDGF stimulation of intact cells was demonstrated by western blot analysis probing with anti-phosphotyrosine antibody. Immunoprecipitation with anti-PDGF β-receptor antibody and visualization on a western blot with an anti-phosphotyrosine antibody also revealed a 170-kDa protein. Maximum phosphorylation of the 170-kDa protein occurred after 5-min exposure to 20 ng/ml PDGF. This phosphorylation was inhibited by gangliosides GM1, GM2, GD1a, and GT1b but not by GM3. Receptor dimerization was also inhibited by GM1. These results show that SH-SY5Y cells have specific receptors for PDGF-BB that are functional, and can be modulated by gangliosides.     

Synthesis of beta-substituted naphth-1-yl ethylamido derivatives as new melatoninergic agonists

Naphthalene melatoninergic ligands with alkyl groups (Me, Et, Pr, Bz) in the beta position of the ethylamido chain were synthesised. The affinity of the compounds for chicken brain melatonin receptors was evaluated using 2-[125I]-iodomelatonin as the radioligand. An increase in the affinity was observed with the beta-methyl derivatives and the greatest increase was seen with the (-) enantiomers. The introduction of a 2- or 7-MeO group on the naphthalene ring and the lengthening (Et, Pr) of the alkylamido chain gave potent compounds such as (-)1h (Ki = 24 pM). The functional activity of these compounds was evaluated by the aggregation of melanophores in Xenopus laevis tadpoles. The potency to produce lightening of the skin of Xenopus laevis was related to the affinities values of the molecules at melatonin chicken brain receptors. The most potent ligands were found to be full agonists and compound 1h was 25 fold more potent than melatonin in this bioassay.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.     

Identification of high affinity endothelin-1 receptor subtypes in human tissues.

Two subtypes of the high affinity endothelin-1 (ET-1) receptor were identified in human tissue, and were distinguished by their differential affinities for sarafotoxin S6c (S6c). Uterus contains mostly the ETA subtype, with low affinity for S6c (Ki greater than 7300 nM), while the predominant subtype in hippocampus is ETB, with high affinity for S6c (Ki = 0.25 nM). These subtypes also have different affinities for [Ala1,3,11,15]-ET-1, which was found to be ETB selective. The two subtypes distinguished by these ligands in human tissue correspond to the subtypes previously identified in rat. Differential stimulation of phosphatidyl inositol turnover in rat tissue slices by ET-1 and S6c indicates that both ETA and ETB subtypes represent functional receptors.     

Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine.

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.     

Covalent crosslinking analysis of angiotensin receptors on differentiated NG108-15 cells

Neuroblastoma x glioma hybrid cells (NG108-15) were used as a model system to characterize neuronal-glial type angiotensin (ANG) receptors by covalent crosslinking analysis. After differentiation with 1.5% DMSO and 0.5% fetal bovine serum for four to five days, saturation analysis revealed a single high affinity site with a Kd = 1.35 +/- 0.42 nM and a Bmax = 468 +/- 106 fmol/mg protein. Using the homobifunctional crosslinking reagent bis(sulfosuccinimidyl) suberate (BS3), a site with an estimated Mr of 78 kDa was specifically labeled with 125I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10(-6) M) inhibited specific labeling. The Ki for ANG III binding was similar by both pharmacologic (Ki = 3.33 +/- 0.98 nM) and gel densitometric (Ki = 2.65 +/- 0.32 nM) analyses. We conclude that the 78 kDa protein represents a high affinity ANG binding site with similar affinities for both ANG II and ANG III.     

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.     

Carbachol enhances forskolin-stimulated cyclic AMP accumulation via activation of calmodulin system in human neuroblastoma SH-SY5Y cells.

We have investigated the modulatory action of carbachol on intracellular cAMP levels in human neuroblastoma SH-SY5Y cells. Carbachol enhanced forskolin-stimulated cAMP levels in a dose-dependent manner (EC50 = 3 microM). The enhancing effect of carbachol was completely inhibited by pirenzepine and atropine. Pertussis toxin treatment of the cells partially affected the ability of carbachol. Furthermore, carbachol also enhanced the effect of vasoactive intestinal peptide (EC50 = 3 microM)-, adenosine- and prostaglandin E1-stimulated cAMP levels. The enhancing response of carbachol was sensitive to trifluoperazine but insensitive to calphostin C. These results suggest that the mechanism for carbachol-induced cAMP levels may act, at least in part, through the activation of calmodulin system in SH-SY5Y cells. Hence we describe for the first time a synergistic interaction between calmodulin- and cAMP-dependent signal transduction pathway mediated by carbachol in neuron-derived cell line.     

2,8-Disubstituted adenosine derivatives as partial agonists for the adenosine A2A receptor

Novel 2,8-disubstituted adenosine derivatives were synthesized in good overall yields starting from 2-iodoadenosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. Some compounds displayed good adenosine A(2A) receptor affinities, with most of the 2-(1-hexynyl)- and 2-[(E)-1-hexenyl]-substituted derivatives having K(i) values in the nanomolar range. Although the introduction of an 8-alkylamino substituents decreased the affinity for the adenosine A(2A) receptor somewhat, the selectivity for this receptor compared to A(3) was improved significantly. The 8-methylamino (12) and 8-propylamino (14) derivatives of 2-(1-hexynyl)adenosine (3), showed reasonable A(2A) receptor affinities with K(i) values of 115 and 82nM, respectively, and were 49- and 26-fold selective for the adenosine A(2A) receptor compared to the A(3) receptor. The compounds were also evaluated for their ability to stimulate the cAMP production in CHO cells expressing the human adenosine A(2A) receptor. 2-(1-Hexynyl)adenosine (3) and 2-[(E)-1-hexenyl]adenosine (4) both showed submaximal levels of produced cAMP, compared to the reference full agonist CGS 21680, and thus behaved as partial agonists. Most 8-alkylamino-substituted derivatives of 3, displayed similar cAMP production as 3, and behaved as partial agonists as well. Introduction of alkylamino groups at the 8-position of 4, showed a slight reduction of the efficacy compared to 4, and these compounds were partial agonists also.