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K A Jacobson

Publications and source records attributed to K A Jacobson.

At least 181 records · Page 10Linked to original sources
PubMed1992

High affinity acylating antagonists for muscarinic receptors.

The muscarinic antagonists pirenzepine and telenzepine were derivatized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors.

Acylation↗
PubMed1992

In vivo imaging of insulin receptors in monkeys using 18F-labeled insulin and positron emission tomography.

We previously described a prosthetic group methodology for incorporating 18F into peptides and showed that 18F-labeled insulin (18F-insulin) binds to insulin receptors on human cells (IM-9 lymphoblastoid cells) with affinity equal to that of native insulin (1). We now report studies using 18F-insulin with positron emission tomography to study binding to insulin receptors in vivo. Positron emission tomography scans were performed in six rhesus monkeys injected with 0.3-1.4 mCi of 18F-insulin (approximately 0.1 nmol, SA 4-11 Ci/mumol). Integrity of the tracer in blood, determined by immunoprecipitation, was 94% of control for the first 5 min and decreased to 31% by 30 min. Specific, saturable uptake of 18F was observed in the liver and kidney. Coinjection of unlabeled insulin (200 U, approximately 1 nmol) with the 18F-insulin reduced liver and increased kidney uptake of the labeled insulin. Liver radioactivity was decreased by administration of unlabeled insulin at 3 min, but not 5 min, after administration of the tracer, while some kidney radioactivity could be displaced 5 min after injection. Clearance of 18F was predominantly in bile and urine. 18F-insulin is a suitable analogue for studying insulin receptor-ligand interactions in vivo, especially in the liver and kidney.

Animals↗
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

A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase.

Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.

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

Molecular modeling of adenosine receptors. I. The ligand binding site on the A1 receptor.

The amino acid sequence of the canine adenosine A1 receptor and the atomic coordinates of a structurally related protein, bacteriorhodopsin, were combined to generate a three-dimensional model for the adenosine A1 receptor. This model consists of seven amphipathic alpha-helices, forming a pore that has a rather distinct partition between hydrophobic and hydrophilic regions. Subsequently, a highly potent and selective ligand, N6-cyclopentyladenosine, was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand, obtained from indirect modeling studies by the 'active analog approach'. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Finally, the deduced binding site was used to model other potent ligands that could all be accommodated consistent with earlier biochemical and pharmacological findings.

Adenosine↗
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

Direct observation of brownian motion of lipids in a membrane.

Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results. Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.

Antibodies, Monoclonal↗
PubMed1991

Apparent heterogeneity of cardiac A1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes.

While G protein-coupled receptors are often studied by analyzing antagonist radioligand: "cold" agonist inhibition curves using an independent site model, it is now clear that KL and KH values determined in these analyses are not reliable estimates of the affinities of the agonists for "free" and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: Gi/Go protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes. As A1 adenosine receptors couple to Gi, and perhaps to Go, we have performed A1 adenosine receptor radioligand "competition" studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand [3H]xanthine amine congener appeared to be confounded by ligand binding to A2 adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A1-specific radioligand [3H]1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the KL values determined by computer analysis of "competition" curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1991

Spatial Fourier analysis of video photobleaching measurements. Principles and optimization.

The major use of the fluorescence recovery after photobleaching (FRAP) technique is to measure the translational motion of the molecular components in various condensed media. In a conventional laser spot photobleaching experiment, a photomultiplier is used to measure the total brightness levels of the bleached region in the sample, so no spatial information can be directly obtained. In video-FRAP, a series of images after photobleaching is acquired, allowing the spatial character of the recovery to be determined; this permits direct detection of both anisotropic diffusion and flow. To utilize all of the available image data to determine the transport coefficients, a two-dimensional spatial Fourier transform analysis of the images after photobleaching was employed. The change in the transform between two time points reflects the action of diffusion during the interim. An important advantage of this method, which involves taking the ratio of image transforms at different time points, is that it does not require a specific initial condition to be created by laser photobleaching. The ability of the analysis to extract transport coefficients from computer-simulated diffusional recovery is assessed in the presence of increasing amounts of noise. Experimental data analysis from the diffusion of proteins in viscous solutions and from the diffusion of protein receptors on cell surfaces demonstrate the feasibility of the Fourier analysis to obtain transport coefficients from the video FRAP measurement.

Biophysical Phenomena↗
PubMed1991

Muscarinic receptor binding and activation of second messengers by substituted N-methyl-N-[4-(1-azacycloalkyl)-2-butynyl]acetamides.

A series of substituted azacycloalkyl analogues of the muscarinic agonist UH 5 (N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]acetamide, 1a) were synthesized and evaluated pharmacologically. These compounds were developed as intermediates for further derivatization leading to functionalized congeners of 1a. The compounds were synthesized by using a Mannich-type condensation of N-acetyl-N-methylpropargylamine to various substituted saturated azaheterocycles. The compounds were screened at a single concentration in competitive binding assays in rat cerebral cortical membranes against either [3H]N-methylscopolamine (at 100 microM) or [3H]oxotremorine-M (at 1 microM) labels. Candidates were then selected for further evaluation of their effect on phosphoinositide (PI) turnover in membranes from A9L cells transfected with cDNA of either m1-muscarinic cholinergic receptors (m1AChRs) or m3AChRs. The analogues were also tested for the inhibition of adenylate cyclase in NG108-15 cells expressing m4AChRs. The azetidine analogue of 1a had a Ki value of 12 nM for the inhibition of [3H]oxotremorine-M binding in rat brain and had an agonist potency at m1-,m3-, and m4AChRs comparable to 1a. The substituted 5- and 6-member ring analogues generally had lower binding affinities and were less potent than 1a in stimulating PI turnover. Several compounds were moderately effective in inhibiting cyclic AMP production in NG108-15 cells.

Acetamides↗
PubMed1991

Functionalized congener approach to muscarinic antagonists: analogues of pirenzepine.

The M1-selective muscarinic receptor antagonist pirenzepine 6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) was derivatized to explore points of attachment of functionalized side chains for the synthesis of receptor probes and ligands for affinity chromatography. The analogues prepared were evaluated in competitive binding assays versus [3H]-N-methylscopolamine at four muscarinic receptor subtypes (m1AChR-m4AChR) in membranes from rat heart tissue and transfected A9L cells. 9-(Hydroxymethyl)pirenzepine, 8-(methylthio)pirenzepine, and a series of 8-aminosulfonyl derivatives were synthesized. Several 5-substituted analogues of pirenzepine also were prepared. An alternate series of analogues substituted on the 4-position of the piperazine ring was prepared by reaction of 4-desmethylpirenzepine with various electrophiles. An N-chloroethyl analogue of pirenzepine was shown to form a reactive aziridine species in aqueous buffer yet failed to affinity label muscarinic receptors. Within a series of aminoalkyl analogues, the affinity increased as the length of the alkyl chain increased. Shorter chain analogues were generally much less potent than pirenzepine, and longer analogues (7-10 carbons) were roughly as potent as pirenzepine at m1 receptors, but were nonselective. Depending on the methylene chain length, acylation or alkyl substitution of the terminal amine also influenced the affinity at muscarinic receptors.

Animals↗
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

Behavioral effects of A1- and A2-selective adenosine agonists and antagonists: evidence for synergism and antagonism.

The locomotor effects in mice of selective A1 and A2 adenosine agonists, antagonists and combinations of agonists were investigated using a computerized activity monitor. The A2-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino[-5'-N- ethylcarboxamidoadenosine (APEC), an amine derivative of 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide, was a more potent locomotor depressant than its amide conjugates. The rank order of potency after i.p. injection for adenosine agonists was 5'-N-ethylcarboxamidoadenosine (NECA) (ED50, 5.8 nmol/kg) greater than APEC (ED50, 25 nmol/kg) greater than N6-cyclohexyladenosine (CHA) (ED50, 270 nmol/kg). An A1-selective, centrally acting, adenosine antagonist, 8-cyclopentyltheophylline (10 mg/kg), completely reversed the locomotor depressant effects of CHA (A1-selective) and NECA (nonselective) at doses of agonists as high as twice the ED50, and shifted the dose-response curves to the right, suggesting a primary involvement of A1 receptors. 8-cyclopentyltheophylline did not affect the depressant effects of APEC at the ED50, consistent with the A2-selectivity of APEC. The locomotor effects of APEC and CHA were completely reversed by theophylline, but not by the peripherally active 8-p-sulfophenyltheophylline, indicating central action of the adenosine agonists. The depressant effects of APEC, but not of NECA or CHA, were reversed significantly by an A2-selective adenosine receptor antagonist, 4-amino-8-chloro-1-phenyl-[1,2,4]triazol[4,3-a]quinoxaline. Low or subthreshold doses of CHA potentiated the depressant effects of APEC. A subthreshold dose of CHA did not alter the depressant effect of NECA, whereas a subthreshold dose of APEC increased the depressant effects of low doses of NECA. Thus, it appears that A1- and A2-selective adenosine agonists have separate central depressant effects, which can be potentiative.(ABSTRACT TRUNCATED AT 250 WORDS)

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↗
PubMed1990

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.

Adenosine↗