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G L Stiles

Publications and source records attributed to G L Stiles.

At least 55 records · Page 3Linked to original sources
PubMed1994

A binding site model and structure-activity relationships for the rat A3 adenosine receptor.

A novel adenosine receptor, the A3 receptor, has recently been cloned. We have systematically investigated the hitherto largely unexplored structure-activity relationships (SARs) for binding at A3 receptors, using 125I-N6-2-(4-aminophenyl)ethyladenosine as a radioligand and membranes from Chinese hamster ovary cells stably transfected with the rat A3-cDNA. As is the case for A1 and A2a receptors, substitutions at the N6 and 5' positions of adenosine, the prototypic agonist ligand, may yield fairly potent compounds. However, the highest affinity and A3 selectivity is found for N6,5'-disubstituted compounds, in contrast to A1 and A2a receptors. Thus, N6-benzyladenosine-5'-N-ethylcarboxamide is highly potent (Ki, 6.8 nM) and moderately selective (13- and 14-fold versus A1 and A2a). The N6 region of the A3 receptor also appears to tolerate hydrophilic substitutions, in sharp contrast to the other subtypes. Potencies of N6,5'-disubstituted compounds in inhibition of adenylate cyclase via A3 receptors parallel their high affinity in the binding assay. None of the typical xanthine or nonxanthine (A1/A2) antagonists tested show any appreciable affinity for rat A3 receptors. 1,3-Dialkylxanthines did not antagonize the A3 agonist-induced inhibition of adenylate cyclase. A His residue in helix 6 that is absent in A3 receptors but present in A1/A2 receptors may be causal in this respect. In a molecular model for the rat A3 receptor, this mutation, together with an increased bulkiness of residues surrounding the ligand, make antagonist binding unfavorable when compared with a previously developed A1 receptor model. Second, this A3 receptor model predicted similarities with A1 and A2 receptors in the binding requirements for the ribose moiety and that xanthine-7-ribosides would bind to rat A3 receptors. This hypothesis was supported experimentally by the moderate affinity (Ki 6 microM) of 7-riboside of 1,3-dibutylxanthine, which appears to be a partial agonist at rat A3 receptors. The model presented here, which is consistent with the detailed SAR found in this study, may serve to suggest future chemical modification, site-directed mutagenesis, and SAR studies to further define essential characteristics of the ligand-receptor interaction and to develop even more potent and selective A3 receptor ligands.

Adenylyl Cyclase Inhibitors↗
PubMed1994

125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine, a high affinity radioligand for the rat A3 adenosine receptor.

The rat A3 adenosine receptor (AR) is a recently characterized AR subtype cloned from testis and brain cDNA libraries. N6-2-(4-Amino-3-[125I]iodophenyl)ethyladenosine, a high affinity A1AR agonist, has served as the only radioligand available for study of the A3AR. The relatively low affinity of N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine for the A3AR and its greater A1AR selectivity necessitate the development of more appropriate radioligands for A3AR analysis. This report characterizes 125I-4-aminobenzyl-5'-N-methylcarboxamidoadenosine (125I-AB-MECA), a high affinity radioligand for the A3AR, in two cell lines that express this AR subtype. Membranes from Chinese hamster ovary (CHO) cells expressing the rat A3AR and from the rat mast cell line RBL-2H3 bound 125I-AB-MECA with Kd values of 1.48 +/- 0.33 nM and 3.61 +/- 0.30 nM, respectively. As determined by 125I-AB-MECA binding, levels of A3AR expression in the A3AR-CHO cell line and RBL-2H3 cells were 3.06 +/- 0.21 pmol/mg and 1.02 +/- 0.13 pmol/mg, respectively. Binding of 125I-AB-MECA was characterized in competition assays. In the A3AR-CHO cell line a potency order of cyclohexyl-5'-N-ethylcarboxamidoadenosine (cyclohexyl-NECA) = benzyl-NECA > (-)-N6-[(R)-phenylisopropyl]adenosine = NECA was observed, and in RBL-2H3 cells (-)-N6-[(R)-phenylisopropyl]adenosine and NECA were equipotent. Xanthine amine congener (XAC) and 8-cyclopentyl-1,3-dipropylxanthine did not significantly inhibit 125I-AB-MECA binding. The parent compound, AB-MECA, dose-dependently inhibited forskolin-stimulated adenylyl cyclase activity in A3AR-CHO cell membranes. 125I-AB-MECA bound to the rat A1AR and canine A2aAR expressed in COS-7 cells with Kd values of 3.42 +/- 0.43 nM and 25.1 +/- 12.6 nM, respectively. This binding was significantly reduced in the presence of 1 microM XAC. In RBL-2H3 cells, XAC had no effect on 125I-AB-MECA affinity and reduced the level of radioligand binding by approximately 5%.

Adenosine↗
PubMed1993

A role for central A3-adenosine receptors. Mediation of behavioral depressant effects.

The behavioral effects of a selective A3 adenosine receptor agonist 3-IB-MECA (N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high-affinity A3-agonist radioligand [125I]AB-MECA (N6-(3-iodo-4-aminobenzyl)-5'-N-methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the Kd value for binding to A3 receptors was 1.39 +/- 0.04 nM with a Bmax of 14.8 +/- 2.1 fmol/mg protein. 3-IB-MECA at 0.1 mg/kg i.p. was a locomotor depressant with > 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3-IB-MECA were unaffected. 3-IB-MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.

Adenosine↗
PubMed1993

Functional consequences of A1 adenosine-receptor phosphorylation by the beta-adrenergic receptor kinase.

Treatment of smooth-muscle cells with R-phenylisopropyladenosine (R-PIA) leads to a loss of A1 adenosine receptor (A1AR)-mediated inhibition of adenylate cyclase, a decrease in receptor number and an increase in receptor phosphorylation. In this study, the role of the beta-adrenergic receptor kinase (beta ARK) in the phosphorylation and inactivation of the A1AR was examined. A1ARs were purified from bovine brain and reconstituted into phospholipid vesicles, with or without a 10-fold excess of Gi/Go (a 50:50 mixture). The reconstituted receptor preparations were phosphorylated with beta ARK in the absence (control) or presence (treated) of R-PIA. R-PIA stimulated A1AR phosphorylation by 2-3-fold over control. Phosphorylation of the A1AR was blocked by XAC, and A1AR antagonist, underscoring its agonist dependence. The stoichiometry of phosphorylation obtained was approx. 1.3 mol of phosphate per mol of A1AR. Phosphorylation of the A1AR by beta ARK was enhanced by an additional 42% when G beta gamma (30 nM) was included in the phosphorylation mixture. In order to test the role of phosphorylation on receptor function, the purified A1AR was reconstituted with a mixture of Gi/Go, phosphorylated with beta ARK and used to determine high-affinity [125I]APNEA (A1AR agonist) binding. Agonist binding was reduced by about 50% in the treated preparations compared to control. In contrast, antagonist ([3H]XAC) binding was increased by about 50%. These data are consistent with an uncoupling of the A1AR from G proteins following receptor phosphorylation. In control preparations, R-PIA stimulated GTPase activity from 0.08 to 0.164 pmol Pi released/pmol Gi/Go per min. Phosphorylation of receptor by beta ARK reduced R-PIA-stimulated GTPase activity by 35%. In addition, phosphorylation of the A1AR by beta ARK decreased R-PIA-stimulated GTP gamma S binding by 62%. These data provide evidence that A1AR phosphorylation by beta ARK results in a diminished receptor-G-protein interaction.

Animals↗
PubMed1993

The A3 adenosine receptor is the unique adenosine receptor which facilitates release of allergic mediators in mast cells.

Mast cells release the mediators of the immediate hypersensitivity reaction. Adenosine is known to modulate this process, but the receptor responsible for this is not the classical A1 or A2 adenosine receptors. This study was undertaken to determine whether the unique adenosine receptor (AR) previously postulated in a cultured mast cell line (RBL-2H3 cells) is the recently cloned A3AR. The receptors were quantitated by the agonist 125I-labeled APNEA (aminophenylethyladenosine), an A3AR agonist, which yielded Bmax and Kd values of 826 fmol/mg protein and 34 nM, respectively. A variety of adenosine analogs competed for 125I-APNEA binding sites with the following potency series: (R)-phenylisopropyladenosine = 5'-N-ethylcarboxamide adenosine > (S)-phenylisopropyladenosine. 125I-APNEA binding was relatively insensitive to the xanthine amine congener (XAC, 1 microM), a selective antagonist for the A1AR. Functionally, activation of these A3AR stimulated the production of inositol 1,4,5-triphosphate, leading to an increase in the level of intracellular Ca2+. Furthermore, while activation of these receptors alone produced little secretory response in RBL-2H3 cells, it enhanced antigen-induced secretion by 2-2.5-fold. Northern blotting studies using poly(A+) RNA from RBL-2H3 cells detected two transcripts of 2.0 and 3.5 kilobases, which hybridized to an A3AR cDNA but not to the A1 or A2AR cDNA probes. These data indicate that the unique AR that potentiates the secretory response to antigen in RBL-2H3 cells is exclusively the A3AR.

Amino Acid Sequence↗
PubMed1993

Solubilization and characterization of the A2-adenosine receptor.

Binding of [3H]CGS 21680, an agonist radioligand selective for A2-adenosine receptors (A2AR), to membranes and solubilized preparations from bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffective in modulating agonist binding whereas cations, Na+ and Mg++, had distinct effects. The addition of NaCl (200 mM) as well as the Mg(++)-free preparation of membranes led to a significant decrease in binding affinity and the number of binding sites. Moreover, the presence of Na+ was required for the demonstration of a guanine nucleotide effect, i.e. a decrease in maximal binding. Following solubilization, agonist-A2AR interactions were sensitive to guanine nucleotides even in the absence of Na+; guanine nucleotides and Na+ had additive effects in reducing the number of binding sites. Moreover, the effect of GTP was reversible, i.e. binding returned to control levels upon removal of the nucleotide. This suggests the A2AR and its G protein (presumably Gs) are solubilized as a functional unit and may not dissociate even in the presence of GTP following solubilization. We, therefore, believe that a "tight" association exists between receptor and G protein (Gs), and that guanine nucleotides and sodium act at different sites on the R-G complex. Drawing an analogy with similar observations on the avian beta-adrenergic receptor (Hertel et al, J. Biol. Chem. 265:17988-94, 1990; Parker & Ross, J. Biol. Chem. 266:9987-96, 1991) we postulate that the regulatory features of the A2AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.

Adenosine↗
PubMed1992

Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor.

We have previously reported the selective amplification of several rat striatal cDNA sequences that encode guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. One of these sequences (R226) exhibited high sequence identity (58%) with the two previously cloned adenosine receptors. A full-length cDNA clone for R226 has been isolated from a rat brain cDNA library. The cDNA clone encodes a protein of 320 amino acids that can be organized into seven transmembrane stretches. R226 has been expressed in COS-7 and CHO cells and membranes from the transfected cells were screened with adenosine receptor radioligands. R226 could bind the nonselective adenosine agonist tritiated N-ethyladenosine 5'-uronic acid ([3H]NECA) and A1-selective agonist radioiodinated N6-2-(4-amino-3-iodophenyl)-ethyladenosine ([125I]APNEA) but not A1-selective antagonists tritiated 1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) and 8-(4-[([[(2-aminoethyl)amino]carbonyl]methyl)oxy]-phenyl)-1, 3-dipropylxanthine ([3H]XAC) or the A2-selective agonist ligands tritiated 2-[4-(2-carboxyethyl)phenyl]ethyl-amino 5'-N-ethylcarboxamidoadenosine ([3H]CGS21680) and radioiodinated 2-[4-([2-[(4-aminophenyl)methylcarbonylamino] ethylaminocarbonyl]ethyl)phenyl]ethylamino 5'-N-ethylcarboxamidoadenosine. Extensive characterization with [125I]APNEA showed that R226 binds [125I]APNEA with high affinity (Kd = 15.5 +/- 2.4 nM) and the specific [125I]APNEA binding could be inhibited by adenosine ligands with a potency order of (R)-N6-phenyl-2-propyladenosine (R-PIA) = NECA greater than S-PIA greater than adenosine greater than ATP = ADP but not by antagonists XAC, isobutylmethylxanthine, and DPCPX. In R226 stably transfected CHO cells, adenosine agonists R-PIA, NECA, and CGS21680 inhibited by 40-50% the forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive G protein with an EC50 of 18 +/- 5.6 nM, 23 +/- 3.5 nM, and 144 +/- 34 nM, respectively. Based on these observations we conclude that R226 encodes an adenosine receptor with non-A1 and non-A2 specificity, and we thus name it the A3 adenosine receptor. mRNA analyses revealed that the highest expression of R226 was in the testis and low-level mRNAs were also found in the lung, kidneys, heart, and some parts of the central nervous system such as cortex, striatum, and olfactory bulb. The high-expression level of the A3 receptor in the testis suggests a possible role for adenosine in reproduction.

Adenosine↗
PubMed1992

Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Adenosine receptors.

With the recent purification and cloning of the A1AR and the cloning of the A2AR in association with the development of selective radioligands, we are now poised to begin to understand at the most fundamental level the structure, function, and regulation of adenosine receptors. Although adenosine's physiological effects have been appreciated for more than 60 years, we are only now ready to address questions at the biochemical and molecular biological levels. We are likely to begin to see evidence for a whole group of adenosine receptors undetected by previous technology. One era of adenosine receptor research has just ended, and we now enter the new with anxious anticipation.

Amino Acid Sequence↗
PubMed1992

Adenosine receptors.

Our knowledge of A1 and A2ARs has grown dramatically since they were first defined by Burnstock. With the recent purification of the A1AR and the cDNA cloning of the A2AR, an even more rapid expansion of information regarding their structure, function, and regulation should now ensue. We may then be able to return to the intact cell and organ system and better understand the physiological role of these ubiquitous receptors.

Adenylyl Cyclases↗
PubMed1992

Characterization of human striatal A2-adenosine receptors using radioligand binding and photoaffinity labeling.

The adenosine agonist [3H]CGS21680 (2-[4-[[2-carboxyethyl]phenyl]ethylamino]-5'- N-ethylcarboxamidoadenosine) bound to A2 receptors in human striatal membranes with a Kd of 17.8 +/- 1.1 nM and a Bmax of 313 +/- 10 fmol/mg protein. The addition of 100 microM GTP diminished both the affinity of agonist radioligand for A2 adenosine binding sites and the total binding, resulting in Kd and Bmax values of 28.6 +/- 1.0 nM and 185 +/- 22 fmol/mg of protein. Adenosine ligands competed for [3H]CGS21680 with the expected potency order. The adenosine antagonist [3H]XAC (8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl] oxy]phenyl]-1,3-dipropylxanthine), although A1-selective in the rat, binds to human striatal A2 receptors with high affinity. 25 nM CPX (8-cyclopentyl-1,3-dipropylxanthine), an A1-selective antagonist, was added to the incubation medium and effectively eliminated 91% of [3H]XAC (1 nM) binding to human A1 receptors, yet preserved 90% of binding to A2 receptors. [3H]XAC exhibited saturable, specific binding (50% of total) to A2 sites with a Kd of 2.98 +/- 0.54 nM and a Bmax of 0.71 +/- 0.23 pmol/mg protein (25 degrees C, non-specific binding defined with 100 microM NECA). The potency order for antagonists against 1 nM [3H]XAC was CGS15943A greater than XAC approximately PD115,119 greater than PAPA-XAC greater than CPX greater than HTQZ approximately XCC approximately CP-66,713 greater than theophylline approximately caffeine, indicative of an A2-type binding site. A2a-receptors were found to be present in the human cortex, albeit at a much lower density than in the striatum. Photoaffinity labeling using 125I-PAPA-APEC revealed a molecular weight of 45K, but proteolytic cleavage was observed, resulting in fragments of MW 43K and 37K. In the absence of proteolytic inhibitors the 37K fragment, which still bound 125I-PAPA-APEC, was predominant.

Adenosine↗
PubMed1992

Immunological identification of A2 adenosine receptors by two antipeptide antibody preparations.

Two antipeptide antibody preparations were raised against deduced amino acid sequences within the presumed second extra-cellular loop (antibody TP/1) and the carboxyl-terminal domain (antibody TP/2) of the canine-derived A2 adenosine receptor (A2AR) cDNA species termed RDC8. Immunoblotting of canine liver plasma membranes with both TP/1 and TP/2 identified a single band of 52 kDa, which co-migrated with 125I-2-[4-[2-[2-[(4- azidophenyl)methylcarbonylamino]ethylaminocarbonyl]ethyl] phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine-labeled receptor. However, in membranes prepared from canine striatum, photoaffinity labeling and immunoblotting with TP/2, but not TP/1, revealed a single band of 34 kDa; the identity of the band observed on the immunoblot as an A2AR was confirmed by the ability of TP/2 to specifically immunoprecipitate photoaffinity-labeled receptor from crude canine striatal membranes. The size difference between liver and striatal A2ARs was not due to tissue-specific proteolysis, because membranes from striatum were prepared with a protease inhibitor cocktail previously shown to be effective in inhibiting endogenous A2AR proteolysis during membrane preparation. Also, the protease-sensitive carboxyl-terminal region of the receptor had remained intact, because the peptide used to raise TP/2 antibodies resides in this domain of the molecule. The difference in size was also not due to a greater carbohydrate content of the liver receptor, because treatment of liver and striatal membranes with endoglycosidase F produced small mobility shifts for both receptors. Removal of N-linked carbohydrate chains also did not alter the inability of TP/1 to recognize the striatal A2AR. Hence, we conclude that the A2AR present in liver, which displays the predicted immunoreactivity of RDC8, is immunologically distinct from the A2AR expressed in striatum and that the latter may represent an additional A2AR subtype.

Amino Acid Sequence↗
PubMed1992

Chemical modification and irreversible inhibition of striatal A2a adenosine receptors.

The ligand recognition site of A2a-adenosine receptors in rabbit striatal membranes was probed using non-site-directed labeling reagents and specific affinity labels. Exposure of membranes to diethylpyrocarbonate at a concentration of 2.5 mM, followed by washing, was found to inhibit the binding of [3H]CGS 21680 and [3H]xanthine amine congener to A2a receptors, by 86 and 30%, respectively. Protection from diethylpyrocarbonate inactivation by an adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine, and an antagonist, theophylline, suggested the presence of two histidyl residues on the receptor, one associated with agonist binding and the other with antagonist binding. Binding of [3H]CGS 21680 or [3H]xanthine amine congener was partially restored after incubation with 250 mM hydroxylamine, further supporting histidine as the modification site. Preincubation with disulfide-reactive reagents, dithiothreitol or sodium dithionite, at greater than 5 mM inhibited radioligand binding, indicating the presence of essential disulfide bridges in A2a receptors, whereas the concentration of mercaptoethanol required to inhibit binding was greater than 50 mM. A number of isothiocyanate-bearing affinity labels derived from the A2a-selective agonist 2-[(2-aminoethylamino) carbonylethylphenylethylamino]-5'-N- ethylcarboxamidoadenosine (APEC) were synthesized and found to inhibit A2a receptor binding in rabbit and bovine striatal membranes. Binding to rabbit A1 receptors was not inhibited. Preincubation with the affinity label 4-isothiocyanatophenylaminothiocarbonyl-APEC (100 nM) diminished the Bmax for [3H]CGS 21680 binding by 71%, and the Kd was unaffected, suggesting a direct modification of the ligand binding site. Reversal of 4-isothiocyanatophenylaminothiocarbonyl-APEC inhibition of [3H]CGS 21680 binding with hydroxylamine suggested that the site of modification by the isothiocyanate is a cysteine residue. A bromoacetyl derivative of APEC was ineffective as an affinity label at submicromolar concentrations.

Adenosine↗
PubMed1991

Adrenergic receptor responsiveness and congestive heart failure.

Our knowledge and understanding of the structure, mechanism of action and regulation of receptor-adenylate cyclase systems have increased dramatically in the last few years. A family of receptors (including the beta-adrenergic receptors) and guanine nucleotide regulatory proteins (G proteins) have been purified and cloned. Structure-function studies are beginning to provide insight into how the various components of the transmembrane signaling apparatus interact to promote alterations in the activity of the effector systems. Much effort has been devoted to understanding how various pathophysiologic conditions, such as ischemia or congestive heart failure, and the therapeutic methods used to treat such conditions perturb or regulate receptor systems. It has become abundantly clear that such regulation does occur but is not restricted to simple alterations in receptor number, and may well involve covalent modification (phosphorylation) of receptors or alteration in the ability of receptors to interact with G proteins. In addition, regulation of the quantity or functionality of the various G proteins and the catalytic unit of adenylate cyclase itself appear to occur. For example, recent evidence suggests that congestive heart failure in humans is associated with a decreased number of beta-adrenergic receptors as well as an increased quantity of the inhibitory G protein (Gi). These alterations may provide important insight into how to develop new therapeutic methods. Mechanisms generally responsible for transmembrane signaling, how the components are regulated by pathophysiologic conditions, and drugs used to treat disease states are discussed in detail.

Adenylyl Cyclases↗
PubMed1991

Methylxanthine treatment of smooth muscle cells differentially modulates adenylate cyclase responsiveness.

The DDT1 MF-2 smooth muscle cell line was used to study regulation of the A1 and A2 adenosine receptor (AR)-adenylate cyclase system by two different methylxanthines. 3-isobutyl 1-methylxanthine (IBMX) is both an AR antagonist and a phosphodiesterase inhibitor, while xanthine amine congener is an AR antagonist without phosphodiesterase activity. Incubation of cells for 18 hr with 100 microM IBMX produced a significant (P less than .05) decrease in the basal, isoproterenol- and sodium fluoride-stimulated adenylate cyclase activity. This generalized decrease in adenylate cyclase activity was associated with a significant decrease in the quantity of alpha s (Gs) as determined by Western blotting. In contrast, no alteration in alpha i (Gi) was observed in these same membranes. A significant increase in both the quantity of A1AR and the receptors' affinity for agonist occurred; however, no alteration in the ability of an A1AR selective agonist to inhibit adenylate cyclase activity was observed. Treatment for 18 hr with 50 nM xanthine amine congener, conversely, resulted in an increase in basal and isoproterenol stimulated adenylate cyclase activity with no change in membrane alpha s (Gs). With IBMX, there was an increase in agonist affinity for the A1AR without an associated change in the effect of adenosine agonists on adenylate cyclase activity. These data indicate that methylxanthine analogs which lack the ability to inhibit phosphodiesterases regulate receptor-mediated transmembrane signaling systems quite differently from those possessing such characteristics. The more prototypic methylxanthines regulate both receptors and G proteins in these smooth muscle cells.

1-Methyl-3-isobutylxanthine↗
PubMed1991

The A2 adenosine receptor: guanine nucleotide modulation of agonist binding is enhanced by proteolysis.

Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125I-2-[4-(2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminnocarbonyl]ethyl)phenyl]ethylamino-5'-N- ethylcarboxamidoadenosine (125I-PAPA-APEC) and its photoaffinity analog 125I-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is labeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125I-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr approximately 47,000 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory GS-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit striatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5'-(beta,gamma-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein. Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the GS protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.

Adenosine↗
PubMed1991

Distinct pathways of desensitization of A1- and A2-adenosine receptors in DDT1 MF-2 cells.

Desensitization of adenosine receptors (ARs) was studied in DDT1 MF-2 cells, which possess both A1- and A2AR, differentially coupled to adenylate cyclase. (-)-N6-(R)-Phenylisopropyladenosine (R-PIA), an A1AR-selective agonist at the appropriate concentrations, desensitized A1AR-mediated inhibition of adenylate cyclase activity in a time- (t1/2, 8 hr) and dose-dependent and reversible fashion. This was associated with significant decreases in total A1AR number and in the number of receptors possessing a high affinity for agonist in membrane preparations. The decrease in total A1AR in the membranes from the desensitized cells (approximately 40%) was associated with a 37% increase in A1AR measured in light vesicle preparations, compared with control cells. To test a possible role of phosphorylation in A1AR desensitization, cells were incubated with [32P]orthophosphate, followed by exposure to R-PIA for 18 hr. Subsequent purification of the A1AR indicated a 3-4-fold increase in phosphorylation of A1AR in cells treated with R-PIA, compared with control cells. Desensitization of the A1AR did not alter the levels of alpha s and alpha 12 proteins or affect the ability of stimulatory effectors, such as isoproterenol, sodium fluoride, and forskolin, to activate adenylate cyclase. These results suggest that uncoupling, down-regulation, and phosphorylation of the A1AR contribute, at least in part, to desensitization of this inhibitory receptor. Desensitization of the A2AR was characterized using an A2-selective agonist, 2-[4-(2-(4-aminophenyl]methylcarbonyl)ethyl)phenyl]ethylamino- 5'-N-ethylcarboxamidoadenosine (PAPA-APEC). Pretreatment of cells with PAPA-APEC (100 nM) resulted in a rapid loss of agonist stimulation of adenylate cyclase activity (t1/2 of this effect, 45 min). This effect was dose dependent (EC50, approximately 10 nM) and rapidly reversible. Interestingly, desensitization of the A2AR resulted in no change in receptor number, affinity, or mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Taken together, these data suggest distinct mechanisms of desensitization of A1- and A2ARs in a single cell type.

Adenosine↗