Species differences in structure-activity relationships of adenosine agonists and xanthine antagonists at brain A1 adenosine receptors

A series of 28 adenosine analogs and 17 xanthines has been assessed as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine binding to A1 adenosine receptors in membranes from rat, calf, and guinea pig brain. Potencies of N6-alkyl- and N6-cycloalkyladenosines are similar in the different species. However, the presence of an aryl or heteroaryl moiety in the N6 substituent results in marked species differences with certain such analogs being about 30-fold more potent at receptors in calf than in guinea pig brain. Potencies at receptors in rat brain are intermediate. Conversely, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine are about 10-fold less potent at receptors in calf brain than in guinea pig brain. Potencies of xanthines, such as theophylline, caffeine and 1,3-dipropylxanthine are similar in the different species. However, the presence of an 8-phenyl or 8-cycloalkyl substituent results in marked species differences. For example, a xanthine amine conjugate of 1,3-dipropyl-8-phenylxanthine is 9-fold more potent at receptors in calf than in rat brain and 110-fold more potent in calf than in guinea pig brain. Such differences indicate that brain A1 adenosine receptors are not identical in recognition sites for either agonists or antagonists in different mammalian species.     

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.     

Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain

A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.     

Prolactin and cyclosporine modulate adenosine transporters and adenosine A1 receptors in the rat brain

The existence of adenosine A1 receptors and adenosine transporters in the central nervous system has been well demonstrated, although their possible modulation by hormones and/or exogenous drugs is poorly understood. To further analyze these modulatory mechanisms, the effects of prolactin and cyclosporine (CyA) on adenosine A1 receptors and transporters were analyzed in the central nervous system. For this purpose the number and affinity of adenosine A1 receptors were measured using the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and the transporters with the high affinity ligand nitrobenzylthioinosine (NBTI). This procedure was carried out in hyperprolactinemic and control male rats treated with CyA or its vehicle for 8 days. As expected, pituitary grafting increased plasma prolactin levels (p<0.01). CyA treatment reduced but did not normalize (p<0.05) this parameter in hyperprolactinemic rats and did not modify circulating prolactin in control animals. Both hyperprolactinemia and CyA treatment reduced the number of adenosine transporters by 70% and by 40% the number of A1 receptors. The Kd for transporters was also reduced in all experimental groups. Hyperprolactinemia increased the affinity of A1 receptors (p<0.01) and CyA treatment did not further modify this parameter. These data demonstrated that prolactin and CyA influence adenosine transporters and A1 receptors at the central nervous system and suggest the existence of an interaction between prolactin and CyA may be operating to modulate these processes.     

Synthesis of di- and tri-substituted adenosine derivatives and their affinities at human adenosine receptor subtypes

The synthesis of 2-(hex-1-ynyl)adenosine derivatives substituted at the N6- and/or 5'-position was carried out on the basis that 2-(hex-1-ynyl)adenosine-5'-N-ethyluronamide (HENECA, 2) showed good affinity and different degree of selectivity for rat adenosine receptors. All new compounds were tested in radioligand binding and adenylyl cyclase assays with recently cloned human A1, A2A, A2B, and A3 adenosine receptors.     

Characterization of the locomotor depression produced by an A2-selective adenosine agonist

Adenosine analogs, such as N6-cyclohexyladenosine (CHA) that are selective for A1-adenosine receptors, and analogs, such as 5'-N-ethylcarboxamidoadenosine (NECA) that are active at both A1 and A2 receptors, cause a profound depression of locomotor activity in mice via a central mechanism. The depression is effectively reversed by non-selective adenosine antagonists such as theophylline. We report that 2-([2-aminoethylamino) carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC), an amine derivative of the A2-selective agonist, CGS21680, is a potent locomotor depressant in mice. The in vivo pharmacology is consistent with A2-selectivity at a central site of action. Two parameters indicative of locomotor activity, horizontal activity and total distance travelled, were measured using a computerized activity monitor. From dose-response curves it was found that APEC (ED50 16 micrograms/kg) is more potent than CHA (ED50 60 micrograms/kg) and less potent than NECA (ED50 2 micrograms/kg). The locomotor depression by APEC was reversible by theophylline, but not by the A1-selective antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1, 3-dipropyl-2-thioxanthine, nor by the peripheral antagonists 8-p-sulfophenyltheophylline (8-PST) and 1,3-dipropyl-8-p-sulfophenylxanthine. The locomotor activity depression elicited by NECA and CHA was reversed by A1-selective antagonists. These results suggest that the effects of APEC are due to stimulation of A2 adenosine receptors in the brain.     

Multiple neurotransmitter receptors in the brain: amines, adenosine, and cholecystokinin

Radioligand binding studies of neurotransmitter receptors have provided discrimination at the molecular level, permitting the differentiation of multiple receptor subtypes for several biogenic amines. Using this paradigm we have labeled two distinct receptors each for cholecystokinin (CCK) and for adenosine. Adenosine receptors were labeled in brain with [3H]N6-cyclohexyladenosine (3H-CHA) and [3H]1,3-diethyl-8-phenylxanthine (3H-DP). The adenosine receptor labeled by 3H-CHA appears to be an A1 site, associated with reduction of adenylate cyclase activity, while 3H-DP sites resemble A2 receptors linked to adenylate cyclase enhancement. Cholecystokinin-33 labeled by the Bolton-Hunter procedure with 125I(125I-BH-CCK) labels different receptors in brain and pancreas. The pancreatic receptor does not react with CCK derivatives of fewer than eight amino acids, while the brain receptor does recognize pentagastrin, the carboxyl-terminal five amino acids of CCK. The "brain type" CCK receptor may normally interact with CCK-4, the carboxyl-terminal tetrapeptide of CCK, recently identified as a unique neuropeptide highly concentrated in the brain. CCK-8, the other major molecular form of CCK, may be the endogenous ligand for the "pancreatic type" receptor.     

Correlation of adenosine receptor affinities and cardiovascular activity

Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [3H]NECA) were compared to their EC25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity (r2 = 0.71, p less than 0.0001) but not with A2 binding affinity (r2 = 0.007, n.s.); conversely, coronary flow (an A2 response) correlated with A2 binding affinity (r2 = 0.83, p less than 0.0001) but not with A1 binding affinity (r2 = 0.05, n.s.). These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses.     

Effect of temperature on atrial and ventricular adenosine A1 receptors of the guinea pig

Cardiac adenosine receptors are coupled to adenylate cyclase inhibition. In the guinea pig heart, the relative agonist potencies observed for adenylate cyclase inhibition were R-N6-phenylisopropyladenosine (R-PIA) = N6-cyclohexyladenosine greater than 5'-N-ethylcarboxamidoadenosine much greater than S-PIA. In both atrial and ventricular membranes, the antagonists 8-phenyltheophylline (8-PT) and isobutylmethylxanthine (IBMX) also showed similar affinities for atrial and ventricular adenosine receptors. The same pattern of relative agonist potencies was observed in experiments performed at either 25 or 37 degrees C. However, the maximal inhibition produced by R-PIA in atrial membranes decreased from 30.8 +/- 3.2% (n = 7) at 25 degrees C to 18.8 +/- 1.6% (n = 4) at 37 degrees C. No such difference in maximal inhibition was observed with ventricular membranes at these two temperatures (34.5 +/- 1.6%, n = 6 at 25 degrees C and 35.3 +/- 0.9%, n = 11 at 37 degrees C). While there was no change in agonist potencies, the affinities of the antagonists 8-PT and IBMX at cardiac adenosine A1 receptors were affected by temperature. At 25 degrees C, the pKD values for 8-PT and IBMX in ventricular membranes were 4.65 +/- 0.21 (n = 3) and 4.55 +/- 0.20 (n = 3), respectively. Their affinities were 7- to 19-fold higher at 37 degrees C, the pKD values being 5.93 +/- 0.12 (n = 7) (p less than 0.02) and 5.38 +/- 0.18 (n = 3) (p less than 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Internalization and desensitization of adenosine receptors

Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A(1), A(2A), A(2B) and A(3) receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A(1)Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A(2A)R and A(2B)R undergo much faster downregulation, usually shorter than 1 h. The A(3)R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A(3)R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.     

Pharmacological profile of adenosine A2 receptor in PC12 cells

The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-(-)-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6-Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8-phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1-methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors.     

Selective effects of adenosine receptor agonists upon coronary resistance and heart rate in isolated working rabbit hearts

Dose dependent changes in heart rate (HR) and coronary resistance (CR) were determined for adenosine, 5'-N-ethylcarboxamide adenosine (NECA) and l-N6-phenylisopropyladenosine (l-PIA) over a dose range of 1 X 10(-9) to 1 x 10(-5) M. Changes in CR were determined under controlled metabolic demand conditions (constant mean aortic pressure, constant mean left atrial pressure, and constant HR). Decreases in HR were determined by allowing the paced hearts to beat spontaneously between doses for 15 seconds. Adenosine significantly decreased CR and HR at greater than or equal to 1 X 10(-5) M. NECA significantly decreased both CR and HR at greater than or equal to 3 X 10(-8) M. l-PIA significantly decreased HR at greater than or equal to 3 X 10(-8) M; however a dose at greater than or equal to 3 X 10(-6) M was required to significantly decrease CR. These results provide evidence that the coronary vasodilator action of adenosine may primarily be mediated by A2 receptors. Furthermore, the data are in support of previous observations that the bradycardic action of adenosine is principally mediated via A1 receptors.     

The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation

Statins activate phosphatidylinositol-3-kinase, which activates ecto-5'-nucleotidase and phosphorylates 3-phosphoinositide-dependent kinase-1 (PDK-1). Phosphorylated (P-)PDK-1 phosphorylates Akt, which phosphorylates endothelial nitric oxide synthase (eNOS). We asked if the blockade of adenosine receptors (A(1), A(2A), A(2B), or A(3) receptors) could attenuate the induction of Akt and eNOS by atorvastatin (ATV) and whether ERK1/2 is involved in the ATV regulation of Akt and eNOS. In protocol 1, mice received intraperitoneal ATV, theophylline (TH), ATV + TH, or vehicle. In protocol 2, mice received intraperitoneal injections of ATV, U0126 (an ERK1/2 inhibitor), ATV + U0126, or vehicle; 8 h later, hearts were assessed by immunoblot analysis. In protocol 3, mice received intraperitoneal ATV alone or with 8-sulfophenyltheophylline (SPT); 1, 3, and 6 h after injection, hearts were assessed by immunoblot analysis. In protocol 4, mice received intraperitoneal ATV alone or with SPT, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), alloxazine, or MRS-1523; 3 h after injection, hearts were assessed by immunoblot analysis. ATV increased P-ERK, P-PDK-1, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels. TH blocked ATV-induced increases in P-ERK, Ser(473) P-Akt, Thr(308) P-Akt, and P-eNOS levels without affecting the induction of P-PDK-1 by ATV. U0126 blocked the ATV induction of Ser(473) P-Akt and Thr(308) P-Akt while attenuating the induction of P-eNOS. A detectable increase in P-ERK, Ser(473) P-Akt and P-eNOS was seen 3 and 6 h after injection but not at 1 h. DPCPX, CSC, and alloxazine partially blocked the ATV induction of P-ERK, Ser(473) P-Akt, and P-eNOS. In conclusion, blockade of adenosine A(1), A(2A), and A(2B) receptors but not A(3) receptors inhibited the induction of Akt and eNOS by statins. Adenosine was required for ERK1/2 activation by statins, which resulted in Akt and eNOS phosphorylation.     

Effects of the allosteric modulator SCH-202676 on adenosine and P2Y receptors

The G protein-coupled receptor allosteric modulator SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine), which affects a wide range of structurally unrelated G protein-coupled receptors, has highly divergent effects on purine receptors. SCH-202676 inhibited radioligand binding to human adenosine A(1), A(2A), and A(3) receptors (IC(50) = 0.5-0.8 microM) and affected dissociation kinetics, but at the human P2Y(1) nucleotide receptor it had no effect. SCH-202676 (10 microM) selectively accelerated agonist dissociation at adenosine A(3) receptors and either slowed (adenosine A(1) receptors) or accelerated (adenosine A(2A) receptors) antagonist dissociation. Thus, SCH-202676 differentially modulated A(1), A(2A), and A(3) receptors as well as agonist- and antagonist-occupied receptors.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.     

A2B adenosine receptor agonists: synthesis and biological evaluation of 2-phenylhydroxypropynyl adenosine and NECA derivatives

In the search for agonists for the elusive A2B adenosine receptor subtypes, 2-phenylhydroxypropynyl-5'-N-methylcarboxamido adenosine (PHPMECA, 14), 2-phenylhydroxypropynyl-5'-N-propylcarboxamido adenosine (PHPPECA, 15), and N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine (19) were synthesized on the basis that introduction of alkynyl chains in 2-position of adenosine derivatives resulted in reasonably good A2B potency compared to NECA [see N6-ethyl-2-phenylhydroxypropynyl adenosine (5) EC50 = 1,700 nM and 2-phenylhydroxypropynyl-5'-N-ethylcarboxamido adenosine (PHPNECA, 8) EC50 = 1,100 nM, respectively]. Radioligand binding studies and adenylyl cyclase assays, performed with recently cloned human A1, A2A, A2B, and A3 adenosine receptors, showed that these modifications produced a decrease in potency at A2B receptor, as well as a general reduction in affinity at the other receptor subtypes. On the other hand, the contemporary presence of an ethyl substituent in N6-position and of a 4'-ethylcarboxamido group in the same compounds led to (R,S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine and (S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine, which did not show the expected increase in potency at A2B subtype. Hence, (S)-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine [(S)-PHPNECA] with EC50 A2B = 220 nM remains the most potent agonist at A2B receptor reported so far.     

Solubilization of stable adenosine A1 receptors from rat brain.

Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.     

Stimulation of glycogenolysis and vasoconstriction by adenosine and adenosine analogues in the perfused rat liver.

Infusion of adenosine into perfused rat livers resulted in transient increases in glucose output, portal-vein pressure, the effluent perfusate [lactate]/[pyruvate] ratio, and O2 consumption. 8-Phenyltheophylline (10 microM) inhibited adenosine responses, whereas dipyridamole (50 microM) potentiated the vasoconstrictive effect of adenosine. The order of potency for adenosine analogues was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than L-phenylisopropyladenosine greater than cyclohexyladenosine greater than D-phenylisopropyladenosine greater than 2-chloroadenosine greater than adenosine, consistent with adenosine actions modulated through P1-purine receptors of the A2-subtype. Hepatic responses exhibited homologous desensitization in response to repeated infusion of adenosine. Adenosine effects on the liver were attenuated at lower perfusate Ca2+ concentrations. Indomethacin decreased hepatic responses to both adenosine and NECA. Whereas adenosine stimulated glycogen phosphorylase activity in isolated hepatocytes, NECA caused no effect in hepatocytes. The response to adenosine in hepatocytes was inhibited by dipyridamole (50 microM), but not 8-phenyltheophylline (10 microM). The present study indicates that, although adenosine has direct effects on parenchymal cells, indirect effects of adenosine, mediated through the A2-purinergic receptors on another hepatic cell type, appear to play a role in the perfused liver.     

Receptor-G protein gamma specificity: gamma11 shows unique potency for A(1) adenosine and 5-HT(1A) receptors

G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.     

A [3H]amine congener of 1,3-dipropyl-8-phenylxanthine. A new radioligand for A2 adenosine receptors of human platelets

A xanthine amine congener (XAC), an amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine, is an antagonist ligand for A2 adenosine receptors of human platelets. XAC inhibited 5'-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of adenylate cyclase activity with a KB of 24 nM. [3H]XAC exhibits saturable, specific binding with a Kd of 12 nM and a Bmax of 1.1 pmol/mg protein at 37 degrees C. [3H]XAC binding in platelets is the first example of labeling of A2 adenosine receptors in which the potencies of adenosine agonists and antagonists in inhibiting binding are commensurate with their potencies at these receptors in functional studies. Furthermore, [3H]XAC is the first antagonist radioligand with high affinity at A2 adenosine receptors.