Concentration-response relationships for adenosine agonists during preconditioning of rabbit cardiomyocytes

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine), APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 microM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 microM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 microM CCPA. The APNEA curve appeared moriophasic, the major slope occurring between 1-100 nM; DPCPX (1 microM) shifted the concentration-response curve approximately 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of approximately 5 nM; DPCPX shifted the BNECA concentration-protection relationship approximately 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.     

Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat

1. The receptors mediating the vasodilator responses to adenosine in the isolated mesenteric arterial bed of the rat were identified by use of selective agonists and antagonists and the involvement of the endothelium was examined. 2. Adenosine-mediated dilatation of the mesentery was potentiated by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but in contrast, removal of the endothelium substantially reduced the responses to adenosine. 3. The order of potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-p-(-2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > 2-chloro-N6-cyclopentyl-adenosine (CCPA) > or = adenosine, suggesting the presence of A2A receptors. 4. Adenosine-mediated dilatation was inhibited by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (3 microM) and by the A2A receptor antagonist 8-(3-chlorostyryl)caffeine (500 nM), but was unaffected by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM). 5. Reducing the pH of the perfusate to 6.8 potentiated the actions of both CGS 21680 and adenosine, but the vasodilator effects of carbachol were the same at both pH values. The adenosine response at the lower pH as at pH 7.4, was unaffected by DPCPX. The actions of the nitrovasodilator, sodium nitroprusside, were also potentiated at pH 6.8 relative to those at the higher pH value but smaller responses were obtained at the lower pH value with forskolin, a stimulator of adenylyl cyclase, than at pH 7.4. 6. It is concluded that the adenosine receptor mediating dilatation of the rat mesenteric arterial bed is of the A2A subtype, that the response, under the conditions used, is apparently partly dependent on the endothelium (but not due to the release of nitric oxide), and that the response to activation of this receptor is potentiated by a reduction in pH which is similar to that seen in ischaemic conditions.     

Antagonists of the platelet P2T receptor: a novel approach to antithrombotic therapy

The platelet P2T receptor plays a major role in platelet aggregation, and its antagonists are predicted to have significant therapeutic potential as antithrombotic agents. We have explored analogues of adenosine triphosphate (ATP), which is a weak, nonselective but competitive P2T receptor antagonist. Modification of the polyphosphate side chain to prevent breakdown to the agonist adenosine diphosphate (ADP) and substitution of the adenine moiety to enhance affinity and selectivity for the P2T receptor led to the identification of 10e (AR-C67085MX), having an IC50 of 2.5 nM against ADP-induced aggregation of human platelets. Compound 10e was the first very potent antagonist of the P2T receptor, with a selectivity for that subtype of the P2 receptor family of >1000-fold. Further modification of the structure produced compound 10l (AR-C69931MX) having an IC50 of 0.4 nM. In vivo, at maximally effective antithrombotic doses, there is little prolongation of bleeding time (1.4-fold), which is in marked contrast to the 5-6-fold found with GPIIb/IIIa antagonists.     

Functional characterization of adenosine receptors in the nucleus tractus solitarius mediating hypotensive responses in the rat

1. The aim of this study was to characterize adenosine receptors located in the nucleus tractus solitarius (NTS) that mediate decreases in blood pressure in the anaesthetized rat. To determine the adenosine receptor subtype involved, a range of selective agonists and antagonists were studied and their relative potencies evaluated. 2. The rank order of agonist potency in inducing decreases in diastolic blood pressure was N6-cyclopentyladenosine (CPA) > N6-cyclohexyladenosine (CHA) > N-ethyl-carboxamidoadenosine (NECA) > or = 2-phenylaminoadenosine (CV1808) > 2-p-(carboxyethyl)phenethylamino-5' N-ethylcarboxamidoadenosine (CGS 21680) > N6-(2-(4-aminophenyl)ethyl)-adenosine (APNEA). 3. The hypotensive action of CPA following microinjection into the NTS was antagonized by i.v. infusions (50 micrograms kg-1 min-1) of adenosine receptor antagonists, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), 8-phenyltheophylline (8-PT), 8-(p-sulphophenyl)theophylline (8-SPT), and 1,3-dipropyl-8-N-(2-diethylamino)ethyl)-N methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo) benzenesulphonamidexanthine (PD 115199). The antagonist potency order was DPCPX > PD115199 > or = 8-PT. Intravenous infusion of 8-SPT had no effect on blood pressure responses to microinjection of CPA into the NTS. 4. The results suggest that adenosine A1 receptors in the NTS mediate hypotensive responses in the anaesthetized rat preparation.     

Differential expression of adenosine A1 and A2 receptors in preadipocytes and adipocytes

Multiple physiological functions have been described to be affected by adenosine in numerous cell types. A comparative study of the expression of adenosine receptors has been performed in preadipocytes and adipocytes from rat epididymal fat pad. The results show that, in agreement with its well known antilipolytic effect, adenosine induces a negative modulation of adenylate cyclase via the A1 receptor present in adipocytes. By contrast, the A2 receptor subtype, which is positively coupled to adenylate cyclase, is herein demonstrated to be only expressed in adipose precursor cells. This expression allows, in chemically defined medium, the adenosine analogue NECA, by means of its ability to elevate cAMP concentration, to potentiate differentiation. These findings emphasize the role that adenosine might play as a bimodal regulatory extracellular signal in adipose tissue development.     

Activation of various subtypes of G-protein alpha subunits by partial agonists of the adenosine A1 receptor

The activation of different G protein subtypes by the rat adenosine A1 receptor initiated by stimulation with the full agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and by six structurally distinct partial agonists of this receptor was investigated. Endogenous G protein alpha subunits in rat cortical membranes were inactivated by N-ethylmaleimide (NEM). Activation of rat recombinant myristoylated alpha(o), alpha(i1), alpha(i2) and alpha(i3) by partial agonists in comparison to the full agonist was assessed by guanosine-5'-(gamma-[35S]thio)triphosphate ([35S]GTPgammaS) binding after reconstitution of G protein alpha subunits with the adenosine A1 receptor in N-ethylmaleimide-treated membranes. 2-Chloro-N6-cyclopentyladenosine and 3' -deoxy-N6-cyclopentyladenosine (3'-d-CPA), the partial agonist with the highest intrinsic activity, were significantly more potent in activation of alpha(i) subtypes than alpha(o). In contrast, 5'-methylthioadenosine (MeSA), 2'-deoxy-2-chloroadenosine (cladribine), 2'-deoxy-N6-cyclopentyladenosine (2'-d-CPA), 2-phenylaminoadenosine (CV 1808) and C8-aminopropyl-N6-cyclopentyladenosine (C8-aminopropyl-CPA) did not exhibit higher potency for Go or any Gi subtype. All partial agonists, although carrying structurally different modifications, showed higher relative intrinsic activities in activation of Gi than of Go, indicating that Gi-coupled pathways may be activated selectively via the A1 receptor by partial agonists, but not Go-mediated responses.     

Adenosine receptor mediates motility in human melanoma cells

Cell motility is an essential component of tumor progression and metastasis. A number of factors, both autocrine and paracrine, have been found to influence cell motility. In the present study, adenosine and adenine nucleotides directly stimulated chemotaxis of A2058 melanoma cells in the absence of exogenous factors. Three adenosine receptor agonists stimulated motility in the melanoma cells and two adenosine receptor antagonists strongly inhibited the chemotactic response to both adenosine and AMP. The chemotactic stimulation by adenosine and AMP was pertussis toxin sensitive. Otherwise unresponsive Chinese hamster ovary cells which were transfected with the adenosine A1 receptor cDNA acquired the direct, pertussis toxin sensitive, chemotactic response to adenosine, and this response was inhibited by adenosine receptor antagonists. These findings demonstrate that adenosine and adenine nucleotides are capable of stimulating chemotaxis of tumor cells mediated through an adenosine receptor, probably of the A1 subtype. The possibility of antimetastatic therapies based on inhibition of adenosine receptor activity is raised.     

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.     

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.     

Peritoneal malformation with intestinal dystopia--a case report

Four subtypes of adenosine receptors have recently been cloned from thyroid, brain and testis. In this review we have summarised properties of these purinergic receptors. The cloned A1 and A2 subtypes are probably similar or identical to receptors that exist on cardiac and vascular tissues, respectively. A comparison of the amino acid sequences of A1, A2a, and A2b receptors reveals several stretches of conserved amino acids that are unique to adenosine receptors, primarily in the membrane spanning regions. Species differences in A1 receptors indicate that minor changes in receptor structure can produce marked changes in ligand binding properties and may facilitate the identification of amino acids involved in ligand recognition. A confusing A1 receptor subclassification system of putative A1a, A1b, and A3 subtypes has emerged based on subtle rank order potency differences for various ligands among tissues. cDNAs corresponding to these A1 subtypes have not yet been isolated. Atrial A1 receptors activate K+ channels and inhibit adenylyl cyclase. These two pathways appear to be independently up and down regulated, suggesting the existence either of atrial A1 receptor subtypes or of differential regulation of the coupling of a single receptor to distinct GTP binding proteins. An adenosine receptor distinct from A1 and A2 receptors has been cloned from testis and designated TGPCR, or A3, although it differs from the pharmacologically defined A3 receptor. We suggest that the current A1/A3 receptor subtype nomenclature be abandoned and superseded by a nomenclature based solely on receptor cDNAs. In addition to the cloned adenosine receptors, a novel A4 subtype has been proposed based on pharmacological and electrophysiological criteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA

Among the recently reported 2-(ar)alkynyl derivatives of 5'-N-ethylcarboxamidoadenosine (NECA), the (R,S)-2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA [(R,S)-PHPNECA or SCH 59761] was found to be a very potent agonist at A1 and A2A receptor subtypes, with a Ki of 2.5 nM and 0.9 nM, respectively. Furthermore, this compound showed an inhibitory activity on platelet aggregation 16-fold higher than NECA, being the most potent anti-aggregatory nucleoside reported so far. Since this compound bears a chiral carbon in the side chain, the diastereoisomer separation was undertaken both by chiral HPLC and by a stereospecific synthetic method. Binding assays have shown that the (S)-diastereomer is about fivefold more potent and selective than the (R)-diastereomer as agonist of the A2A receptor subtype [(S)-PHPNECA, KiA2A = 0.5 nM; (R)-PHPNECA, KiA2A = 2.6 nM]. Functional studies indicated that (S)-PHPNECA possesses marked vasodilating activity and produces a relevant decrease in heart rate. Moreover, the (S)-diastereomer proved to be about ten times more potent than the (R)-diastereomer in inducing cardiovascular effects, in in vivo hemodynamic studies. However, the greatest difference between these two enantiomers resulted in the platelet aggregation test: in fact, the (R)-diastereomer displayed an inhibitory activity similar to that of NECA, whereas the (S)-diastereomer was 37-fold more active than NECA as an inhibitor of rabbit platelet aggregation, induced by ADP. These data suggest that (S)-PHPNECA could be a useful tool to investigate the mode of binding of agonists to the platelet adenosine receptor subtype.     

X-linked recessive spondyloepiphyseal dysplasia tarda. Clinical and radiographic evolution in a 6-generation kindred and review of the literature

Numerous studies have consistently shown that agonist stimulation of adenosine A1 receptors results in a significant reduction of morbidity and mortality associated with global and focal brain ischemia in animals. Based on these observations, several authors have suggested utilization of adenosine A1 receptors as targets for the development of clinically viable drugs against ischemic brain disorders. Recent advent of adenosine A1 receptor agonists characterized by lowered cardiovascular effects added additional strength to this argument. On the other hand, although cardioprotective, adenosine A3 receptor agonists proved severely cerebrodestructive when administered prior to global ischemia in gerbils. Moreover, stimulation of adenosine A3 receptors appears to reduce the efficacy of some of the neuroprotective actions mediated by adenosine A receptors. The review discusses the possible role of adenosine receptor subtypes (A1, A2, and A3) in the context of their involvement in the pathology of cerebral ischemia, and analyzes putative strategies for the development of clinically useful strategies based on adenosine and its receptors. It also stresses the need for further experimental studies before definitive conclusions on the usefulness of the adenosine concept in the treatment of brain ischemia can be made.     

Genetic risk of neuropsychological impairment in schizophrenia: a study of monozygotic twins discordant and concordant for the disorder

We investigated the effect of the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30-40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 +/- 3.3%), slowly developing inhibition of [Ca2+]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid-transfer proteins encoded by the barley low-temperature-inducible gene family, blt4

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A1 and A2A receptors whereas some differences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A2A and A2B subtypes (Ki = 0.052 and 0.84 microM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A3 receptor (4d, Ki = 0.62 microM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A2B adenosine receptors.     

Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The influence of adenosine and selective A1 and A2 agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC50 = 90 +/- 14 microM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 microM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC50 = 10 +/- 1 microM), suggesting an interaction with extracellular adenosine receptors of A1 and/or A2 subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A1 and A2 adenosine receptors, 5'-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A1 agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC50 = 0.10 +/- 0.04 and 0.60 +/- 0.12 microM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A2 receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 microM) (EC50 = 1.4 +/- 0.5 microM) as well as by the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 microM) (EC50 = 0.7 +/- 0.3 microM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC50 > 22 +/- 2.5 microM). The effect of R-PIA was also shifted to the right by DPCPX (EC50 = 8.2 +/- 1.6 microM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 microM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A1 and A2 adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.     

Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists

A series of 8-substituted derivatives of 3,7-dimethyl-1-propargylxanthine (DMPX) was synthesized and investigated as A2A adenosine receptor antagonists. Different synthetic strategies for the preparation of DMPX derivatives and analogues were explored. A recently developed synthetic procedure starting from 3-propargyl-5,6-diaminouracil proved to be the method of choice for the preparation of this type of xanthine derivatives. The novel compounds were investigated in radioligand binding studies at the high-affinity adenosine receptor subtypes A1 and A2A and compared with standard A2A adenosine receptor antagonists. Structure-activity relationships were analyzed in detail. 8-Styryl-substituted DMPX derivatives were identified that exhibit high affinity and selectivity for A2A adenosine receptors, including 8-(m-chlorostyryl)-DMPX (CS-DMPX, Ki A2A = 13 nM, 100-fold selective), 8-(m-bromostyryl)-DMPX (BS-DMPX, Ki A2A = 8 nM, 146-fold selective), and 8-(3,4-dimethoxystyryl)-DMPX (Ki A2A = 15 nM, 167-fold selective). These and other novel compounds are superior to the standard A2A adenosine receptor antagonists KF17837 (4) and CSC (5) with respect to A2A affinity and/or selectivity.     

Optimized visualization of vessels in contrast enhanced intracranial MR angiography

Agonist-induced desensitization has been described for the A1, A2A, and A3 adenosine receptor subtypes of the G protein-coupled receptor superfamily. Desensitization of the fourth adenosine receptor subtype, the A2B adenosine receptor (A(2B)R), has not been studied extensively. We sought to determine whether the A(2B)R is subject to agonist-induced desensitization. COS 7 cells, which exhibit endogenous expression of the A(2B)R, and transfected CHO cells, which stably express a modified rat A(2B)R bearing a 5' FLAG epitope tag, were studied. Cyclic AMP (cAMP) responsiveness to an acute challenge was measured after pretreating (desensitizing) cells with the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). Incubation with NECA resulted in hyporesponsiveness to acute agonist challenge in both COS 7 and transfected CHO cells. Desensitized cells exhibited restoration of cAMP responses after recovery for 24 hr in growth medium. Choleratoxin-induced cAMP responses were preserved in desensitized cells, and high concentrations of NECA were unable to overcome the desensitization. Membrane levels of the epitope-tagged A(2B)R were assessed by western blot in transiently transfected COS 7 cells. The expression of epitope-tagged A(2B)Rs was not different between control and desensitized cells. In northern blot analysis, levels of endogenous A(2B)R mRNA were similar in control and desensitized COS 7 cells. We conclude that the A(2B)R is subject to agonist-induced desensitization with preserved expression of A(2B)R mRNA and protein. Uncoupling of the A2B adenosine receptor from the G protein complex may contribute to the mechanism of desensitization.     

Evidence for the presence of both P1 and P2 purinoceptors in the rat myometrium

1. Adenosine, adenosine triphosphate (ATP) and some stable analogues of adenosine inhibited field stimulation-induced contractions of the uterus from rats treated with oestradiol cypionate (20 micrograms/kg, s.c.) 1 day previously. Adenosine was twice as potent as ATP; both were potentiated by dipyridamole (10 mumol/L). 2. The order of agonist potency of adenosine and its analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) > N6-cyclohexyladenosine > or = R-phenylisopropyladenosine = S-phenylisopropyladenosine = 2-chloroadenosine > or = adenosine > or = ATP > > 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine. This order suggests the presence of P1 purinoceptors of the A2B subtype. 3. Responses to agonists were antagonized to differing extents by the P1 purinoceptor antagonist 8-phenyltheophylline (10 mumol/L). 4. In uterine preparations from rats pretreated for 2 days with oestrogen (20 micrograms/kg, s.c.) and for 1 day with progesterone (3 mg/animal, s.c.), the inhibitory potencies of adenosine and NECA were reduced, indicating hormonal regulation of uterine responsiveness to P1 purinoceptor agonists. 5. Stable analogues of ATP caused contractions of unstimulated myometrial preparations from oestrogen-treated animals, indicating activation of a P2 purinoceptor, possibly of the P2X subtype, because of the relative order of potency was alpha, beta-methylene ATP > beta, gamma-methylene ATP = ATP = 2-methylthio ATP.     

Differential activation of adenosine receptors decreases N-type but potentiates P-type Ca2+ current in hippocampal CA3 neurons

Adenosine is released in the brain in significant quantities in response to increased cellular activity. Adenosine has been shown either to decrease synaptic transmission or to produce an excitatory response in hippocampal synapses, resulting in increased glutamate release. Previous reports have shown that adenosine or its analogs reduced Ca2+ current in dorsal root ganglion and hippocampal neurons. Here we show that the selective activation of adenosine receptor subtypes has different effects on Ca2+ channels from acutely isolated pyramidal neurons from the CA3 region of guinea pig hippocampus. Activation of A1 receptors inhibited primarily N-type Ca2+ current. In contrast, activation of A2b receptors resulted in significant potentiation of P-type but not N-type Ca2+ current. This potentiation could be inhibited by blocking the cAMP-dependent protein kinase. Because of the ubiquity of adenosine, the differential effects on Ca2+ channels of adenosine receptor subtype activation may have significant implications for neuronal excitability.