Biomedical subjects

J Linden

Publications and source records attributed to J Linden.

At least 73 records · Page 4Linked to original sources

PubMed1996

Adenosine-induced vasoconstriction in vivo. Role of the mast cell and A3 adenosine receptor.

Adenosine, a vasodilator metabolite, is often produced in tissues where the demand for oxygen exceeds the supply. We have recently demonstrated in isolated cannulated arterioles that adenosine and its metabolite, inosine, can also cause vasoconstriction by stimulation of mast cells. Secondary release of histamine and thromboxane is responsible for the inosine-induced constriction in vivo. In the present study, we explored the vasomotor effects of adenosine in vivo and investigated the role of the A3 adenosine receptor in mediating vasoconstriction. In vivo, local application of adenosine (10-6 to 10-4 mol/L) to arterioles consistently caused dose-dependent vasodilation. A fraction of arterioles, however, exhibited a biphasic response, with constriction following dilation. This, too, was dose dependent; 37% of arterioles constricted by 12.7 +/- 4.3% of the initial diameter in response to 10-4 mol/L adenosine. In the presence of 8-(p-sulfophenyl)theophylline (8-SPT), an antagonist of A1 and A2 adenosine receptors, dilation in response to the same dose of adenosine was reduced, and constriction was enhanced; 85% of the tested arterioles constricted by -44.3 +/- 6.0% of the initial diameter. The A3 adenosine receptor has been shown to facilitate mediator release from mast cells, and its role was also examined. N6-(3-Iodo-4-aminobenzyl)adenosine (I-ABA), an agonist of A1 and A3 adenosine receptors, produced dose-dependent vasoconstriction. 1,3-Dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433), an antagonist of A1, A2, and A3 receptors, significantly reduced the vasoconstrictor response to adenosine, which was unmasked during treatment with 8-SPT. In addition, both adenosine and I-ABA stimulated mast cell uptake of ruthenium red, indicating degranulation. The I-ABA-induced constriction was abolished by combined histamine and thromboxane receptor antagonists. We conclude that adenosine can cause vasoconstriction in vivo, which is often masked by A2 receptor-mediated vasodilation. Mast cells are stimulated in the course of the response, and the A3 adenosine receptor is involved in mediating constriction.

Adenosine↗

PubMed1996

PD 81,723, an allosteric enhancer of the A1 adenosine receptor, lowers the threshold for ischemic preconditioning in dogs.

PD 81,723 (PD) acts allosterically to increase agonist binding to A1 adenosine receptors and to enhance functional A1 receptor-mediated responses in the heart and other tissues. To determine if PD lowers the threshold for ischemic preconditioning (PC), pentobarbital-anesthetized dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. Ischemic PC was produced by either 2.5 or 5 minutes of LAD occlusion 10 minutes before the 60-minute occlusion. PD (100 micrograms/kg total dose, 5 to 50 mumol/L in coronary arterial blood) or vehicle was infused intracoronarily for 17.5 minutes before the 60-minute occlusion period in non-PC dogs or in dogs preconditioned with 2.5 minutes of ischemia. Myocardial infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Compared with the control group (26.3 +/- 3.6%, mean +/- SEM), infarct size was not significantly affected by 2.5 minutes of PC alone (23.4 +/- 4.2%) or by PD alone (26.5 +/- 1.7%) but was decreased by PD + PC (14.6 +/- 1.7%, P < .05) or by a longer period (5 minutes) of PC alone (12.5 +/- 3.3%). The intravenous administration of the selective antagonist of A1 adenosine receptors, 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg), or the ATP-sensitive K+ channel blocker, glibenclamide (0.3 mg/kg), for 15 minutes before PD + PC blocked the protection (23.6 +/- 2.3% or 25.9 +/- 3.3%, respectively). None of the compounds studied affected systemic hemodynamics, collateral blood flow, or AAR. To determine which subtypes of canine adenosine receptors were affected by 10 mumol/L PD, radioligand binding studies were conducted using membranes derived from COS-7 cells expressing recombinant canine receptors and agonist radioligands. PD enhanced the binding of [125I]N6-4-amino-3-iodobenzyladenosine (125I-ABA) to A1 receptors by increasing the t1/2 for dissociation by 2.18-fold, but PD had no effect on the dissociation kinetics of 125I-ABA from A3 receptors or [125I]-[2-(4-amino-3-iodo-phenyl)ethylamino] adenosine from A2A receptors. Glibenclamide at concentrations up to 10 mumol/L had no effect on the binding of radioligands to recombinant canine A1, A2A, or A3 receptors. These data suggest that PD reduces the amount of time required for ischemia to produce preconditioning by enhancing adenosine binding to its A1 receptor. Glibenclamide prevents the protection afforded by A1 receptor activation by a mechanism not involving adenosine receptor blockade.

Allosteric Regulation↗

PubMed1996

125I-APE binding to adenosine receptors in coronary artery: photoaffinity labeling with 125I-azidoAPE.

Coronary arteries are known to contain adenosine receptors that elicit vasodilation. Past attempts to characterize these receptors by radioligand binding have been unsuccessful. In the present study, a newly synthesized iodinated adenosine analogue, [125I]2-[2-(4-amino-3-iodophenhyl)ethylamino]adenosine (125I-APE), was found to bind to adenosine receptors in porcine coronary artery smooth muscle membranes. Specific 125I-APE binding is temperature sensitive with maximal binding detected at 4 degrees C. 125I-APE binds to a high affinity low density site with a KD of 0.59 +/- 0.11 nM and a Bmax 7 +/- 0.8 fmoles/mg protein. A high abundance lower affinity site is suggested by the fact that APE competes for 125I-APE binding with a concentration that inhibits 50% (IC50) of 0.96 microM. Competition with various other adenosine receptor agonists results in a potency order of (IC50, microM): 2-phenylaminoadenosine (CV 1808, 0.34) > APE (0.96) > CGS 22988 (5.2) > 2-chloroadenosine (30) > CGS 21680 and NECA (> 100). Agonist binding is not affected by GppNHp (10(-7)-10(-3) M). Among antagonists the potency order is (microM): CGS 15943 (1.1) > 8-(3-chlorostyryl)-caffeine (CSC, 5.3) > 8-sulfophenyltheophylline (SPT, 86) > theophylline (> 100). These binding characteristics are similar to the properties of a putative A4 binding site characteristic of A2a receptors assayed at a low temperature. Photoaffinity labeling of porcine coronary artery membrane proteins with the azide derivative of 125I-APE revealed a 45,000-Da binding site. Photolabeling is prevented by coincubation of membranes at 4 degrees C with various adenosine receptor antagonists (1 microM CSC, 1 microM CGS 15943 or 100 microM theophylline). In conclusion, adenosine receptors of coronary arteries have been detected for the first time by radioligand binding and photoaffinity labeling. This ligand appears to label porcine A4 binding sites that may correspond to A2a receptors assayed at 4 degrees C.

Adenosine↗

PubMed1996

Characterization of 8-(N-methylisopropyl)amino-N6-(5'-endohydroxy- endonorbornyl)-9-methyladenine (WRC-0571), a highly potent and selective, non-xanthine antagonist of A1 adenosine receptors.

Previous studies in our laboratory identified N6-endonorbornyl-9-methyladenine (N-0861) as a highly selective (100-fold) A1-adenosine receptor antagonist (KB = 500 nM). However, its moderate potency limits the degree of A1-receptor blockade that can be achieved by systemically administered N-0861. Structure activity studies were undertaken to invent a compound that had greater affinity for the A1-adenosine receptors than N-0861. C8-N-methylisopropylamino-N6-5'-endohydroxy-N-0861 (WRC-0571) inhibited [3H]-N6-cyclohexyladenosine (CHA) binding to guinea pig A1-receptors with a Ki value of 1.1 nM. WRC-0571 was 200-fold less potent at inhibiting [3H]-5'-N-ethylcarboxamidoadenosine binding to bovine A2a receptors (Ki = 234 nM). WRC-0571 also inhibited the binding of radioligands to cloned human A1, A2a and A3 adenosine receptors with affinities of 1.7, 105 and 7940 nM, respectively. Thus in human adenosine receptors, WRC-0571 is 62-fold selective for the A1 vs. A2a and 4670-fold selective for the A1 vs. A3 receptors; WRC-0571 is therefore the most A1 vs. A3 selective compound yet described. In guinea pig isolated atria, WRC-0571 antagonized the A1-mediated negative inotropic responses to 5'-N-ethylcarboxamidoadenosine (NECA) with a KB of 3.4 nM. WRC-0571 was more than 2500-fold less potent at antagonizing NECA-induced A2b-mediated relaxation in guinea pig aorta. In anesthetized rats WRC-0571 antagonized adenosine-induced bradycardia at concentrations as low as 1 nmol/kg but failed to antagonize A2-mediated hindquarter vasodilation at concentrations up to 10,000 nmol/kg. WRC-0571 is orally active at concentrations as low as 0.3 mumol/kg. WRC-0571 is therefore a highly potent, highly selective antagonist of A1-adenosine receptors both in vitro and in vivo.

Adenine↗

PubMed1996

Effects of long-term treatment with the allosteric enhancer, PD81,723, on Chinese hamster ovary cells expressing recombinant human A1 adenosine receptors.

In this study, desensitization and down-regulation of A1 adenosine receptors (A1AR) by the allosteric enhancer PD81,723 (PD) and by N6-cyclopentyladenosine (CPA) were investigated after 24-hr pretreatment of CHO-K1 cells stably expressing recombinant human A1AR. Pretreatment with 20 microM PD and 10 microM CPA caused a 1.5- and 4.0-fold, respectively, desensitization (reduced potency of CPA to lower cAMP). Pretreatment with PD and/or CPA did not modify the acute effect of PD to increase (5-fold) the potency of CPA. Radioligand binding was used to measure receptor down-regulation in cell membranes and in intact cells. Pretreatment of cells with PD had no effect on the number of membrane binding sites for the agonist [125I] N6-(3-iodo-4-aminobenzyl) adenosine or for the antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine, but the binding of these radioligands to intact cells was modestly reduced (20-37%), possibly reflecting an effect of pretreatment on receptor subcellular distribution. Pretreatment of cells with CPA produced large ( > 40%) reductions in the binding of radioligands to both membranes and intact cells. Pretreatment of cells with CPA also increased the number of presumed internalized receptors measured as [3H]8-cyclopentyl-1,3-dipropylxanthine binding sites in intact cells insensitive to blockade by the charged antagonist 8-sulfophenyltheophylline. The relatively small degree of functional desensitization and down-regulation of A1AR caused by long term exposure of cells to PD is considered to be encouraging in terms of the therapeutic potential of the allosteric enhancer class of compounds.

Adenosine↗

PubMed1996

Covalent modification of transmembrane span III of the A1 adenosine receptor with an antagonist photoaffinity probe.

Structure-based design of subtype-selective ligands for the A1 adenosine receptor will require a reliable model of the ligand-binding pocket. It should be possible to develop a reliable model based on the results of affinity labeling experiments that provide atomic coordinates for the ligand in relation to predicted receptor helices. A high affinity, A1-selective xanthine antagonist photoaffinity probe, 125l-3-(4-azidophenethyl)-1-propyl-B-cyclopentylxanthine, was used to covalently modify the A1 receptor. Chemical or enzymatic fragmentation experiments were performed to localize the region or regions of incorporation within the receptor. The fragmentation profiles for radiolabeled A1 receptor obtained with endoproteinase Glu-C, endoproteinase Lys-C, cyanogen bromide, and hydroxylamine were consistent with the interpretation that the covalent linkage was within the first four predicted transmembrane regions. This interpretation was confirmed by the demonstration that the radioactive endoproteinase Glu-C fragment derived from an A1 receptor that contains an amino-terminal FLAG epitope was recognized by an anti-FLAG monoclonal antibody. Sequential digestion with endoproteinase Glu-C/endoproteinase Lys-C limited the possible labeling to the first three predicted transmembrane spans, and endoproteinase Glu-C/trypsin digestion refined this prediction to include only transmembrane spans III and IV. Taken together, our findings suggest that the adenosine antagonist 125l-3-(4-azidophenethyl)-1-propyl-8-cyclopentyl-xanthine covalently modifies transmembrane III of the A1 receptor because this was the only receptor region common to all radiolabeled fragments.

Affinity Labels↗

PubMed1996

Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.

We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.

Animals↗

PubMed1995

Modulation of TCDD-induced wasting syndrome by portocaval anastomosis and vagotomy in Long-Evans and Han/Wistar rats.

Portocaval anastomosis and vagotomy operations were performed in Long-Evans (L-E) and Han/Wistar (H/W) rats to elucidate the mechanism of anorexia induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). TCDD-sensitive L-E rats were given a sublethal (5 micrograms/kg) or a lethal dose (20 micrograms/kg) by gavage 5-8 weeks after portocaval anastomosis. TCDD-resistant H/W rats were given a nonlethal dose (500 or 7200 micrograms/kg). The shunt operation did not reduce lethality from TCDD. The effect on wasting of the marginally toxic dose of 5 micrograms/kg in L-E rats was potentiated by the portocaval operation, and the lethal dose was effective in both shunted and sham-operated L-E rats. TCDD failed to decrease food intake and body weight in shunted rats of H/W strain at either dose level though it did so in sham-operated controls. The lack of effect may be due to the already reduced weight of shunted rats at the time of TCDD dosing. TCDD anorexia was not explained by changes in histamine or serotonin (5-HT) turnover in the brain. Vagotomy did not influence lethality after TCDD, although reduction in food intake was somewhat blunted in H/W rats. The results seem to indicate that the anorectic effect of TCDD is modified when portal blood bypasses the liver. The mechanisms remain to be elucidated in detail, but the results do not favor the role of liver as the only or the major initiator of TCDD anorexia. Little evidence was found to support a crucial role of vagal afferent input.

Animals↗

PubMed1995

The allosteric enhancer, PD 81,723, stabilizes human A1 adenosine receptor coupling to G proteins.

2-Amino-3-benzoylthiophenes such as PD 81,273 (PD) have been shown to increase agonist, but not antagonist, radioligand binding and to enhance functional effects of A1 adenosine receptor (A1AR) activation in tissues derived from rats and guinea-pigs. The mechanism by which PD produces this allosteric enhancement, and its effect on human A1ARs was not known. In this study, we demonstrate that PD modifies recombinant human A1AR binding and function in stably transfected CHO cells. In membranes, PD (20 microM) causes: (i) a 3-fold increase in the fraction of receptors found in a high affinity G-protein coupled conformation as assessed by the binding of [125I]N6-(3-iodo-4-aminobenzyladenosine) (125I-ABA), an A1AR agonist; (ii) a 2.44-fold increase in the potency of the agonist R-N6-phenylisopropyladenosine (R-PIA) to compete for binding with the antagonist radioligand, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX); (iii) a 1.5-fold increase in the t1/2 of 125I-ABA to dissociate from A1AR; and (i.v.) a 2.2-fold increase in the concentration of guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) required to half-maximally uncouple receptor-G-protein complexes. In intact CHO cells expressing A1AR, PD increases the potency of R-PIA to decrease forskolin-stimulated cAMP accumulation by 3.3-fold. We speculate that PD binds to A1AR at a site distinct from the agonist binding site and stabilizes agonist-R-G complexes.

Allosteric Regulation↗

PubMed1995

Photoaffinity labeling with 2(-)[2-(4-azido-3(-)[125I]- iodophenyl)ethylamino]adenosine and autoradiography with 2(-)[2-(4-amino-3(-)[125I]iodophenyl)ethylamino]adenosine of A2a adenosine receptors in rat brain.

The A2a adenosine receptor agonist 2(-)[2-(4-amino-3- iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125I]APE, discriminates between high- and low-affinity conformations of A2a adenosine receptors. In this study, [125I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [125I]APE binding is consistent with that expected of an A2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125I]APE binding to these brain regions is abolished by 1 microM 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 nM 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [125I]APE to the corresponding arylazide results in [125I]AzPE. The rank-order potency of compounds to compete for [125I]AzPE binding in the dark is CGS-21680 > D-(R)-N6-phenylisopropyladenosine > N6- cyclopentyladenosine, indicating that it also is an A2a-selective ligand. Specific photoaffinity labeling by [125I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 +/- 4% by 100 microM guanosine 5'-O-(3-thiotriphosphate) and 91 +/- 1.3% by 100 nM CGS-21680. [125I]APE and [125I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.

Adenosine↗

PubMed1995

Evidence for regulated coupling of A1 adenosine receptors by phosphorylation in Zucker rats.

Studies were designed to find the molecular basis for previous observations that lipolysis is less active and A1 adenosine receptor signaling is more active in adipocytes from obese than from lean Zucker rats. With quantitative immunoblot procedures for detection, Gi alpha 1 and Gs alpha 45 levels were found anomalously low in obese compared with lean membranes (50 and 30%, respectively), but other G alpha subunit levels were normal. However, the sensitivity of the receptor-Gi protein to GTP was about 5- to 10-fold higher in obese compared with lean membranes when assessed from 1) the ability of GTP to inhibit forskolin-stimulated adenylyl cyclase in the presence of an adenosine receptor agonist and 2) the ability of a nonhydrolyzable guanine nucleotide analogue to alter A1 adenosine receptor agonist binding. Alkaline phosphatase treatment of isolated adipocyte membranes from obese but not lean animals decreased guanine nucleotide sensitivity of agonist binding. Surprisingly, solubilized adipocyte A1 adenosine receptors from all animals exhibited the same high sensitivity to guanine nucleotides as that of intact obese membranes, and this high sensitivity could be decreased 20-fold by treatment with alkaline phosphatase. These data suggest that protein phosphorylation may regulate coupling of the A1 adenosine receptor in rat adipocyte membranes.

Adenosine↗

PubMed1995

Ontogeny of rat myocardial A1 adenosine receptors.

Adenosine functions as a counterregulatory hormone in the myocardium by decreasing work and thereby protecting the myocardium against ischemia. Functional adenosine A1 receptors could serve as an important regulatory system in the developing preinnervated heart by balancing the humoral sympathetic input to the heart. The aims of this study were to determine if A1 adenosine receptors were functionally coupled to their Gi protein in the immature preinnervated heart and to determine if A1 adenosine receptors were present in greater numbers in the immature heart. One- to 3-day-old rat ventricular cardiomyocyte cultures were exposed to (1) control conditions; (2) isoproterenol, a beta-receptor agonist, (3) R-PIA, an A1 agonist, or (4) isoproterenol and R-PIA, cAMP levels were determined by RIA in each group. Adenosine A1 receptor density and the equilibrium dissociation constant were determined by binding of an adenosine A1 receptor antagonist in newborn, 1-week-old, 2-week-old, and adult rat hearts. A1 stimulation decreased the isoproterenol-induced increase in cAMP by 30%, demonstrating functional A1 receptors in immature preinnervated myocytes. The A1 receptor density in the newborn age group was twice the adult and 2-week-old level. We conclude that A1 receptors in the immature heart are functionally coupled to their effector and that A1 receptors are present in greater numbers in the immature heart.

Adrenergic beta-Agonists↗

PubMed1995

Comparison of A4 and A2a binding sites in striatum and COS cells transfected with adenosine A2a receptors.

A putative A4 adenosine receptor is characterized by a distinct structure activity profile of compounds in competition for [3H]2-phenylaminoadenosine ([3H]CV 1808) binding sites on rat brain membranes assayed at 4 degrees C. We now confirm that A4 binding sites can be demonstrated on ice-cold membranes of rat striatum and demonstrate a similar binding site on COS cells transfected with rat A2a adenosine receptors (COS/A2a). The characteristic A4 potency order is: CV 1808 > [1R-(1 alpha, 2 alpha, 3 beta, 5 beta)]-3-(2,6-diamino-N2-(3-carbethoxyphenyl)-9 H-purin-9-yl)-5'-(N-ethylcarbamoyl)-1,2-cyclopentanediol (CGS 22988) >> 5'-N-ethylcarboxamidoadenosine (NECA) > or = 2-[4-(2- carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680); 9-chloro-2-(2-furyl)[1,2,4]-triaolo[1,5-c]-quinazolin-5-a min e (CGS 15943) only partially inhibits binding at 1 microM. If [3H]CGS 21680 is used for ice-cold assays, or if either [3H]CV 1808 or [3H]CGS 21680 are used for assays at 21 degrees C, the potency order of competing compounds changes markedly and becomes characteristic of A2a adenosine receptor binding sites; CGS 15943 > or = CGS 21680 congruent to NECA > CGS 22988 > or = CV 1808. Binding of [3H]CGS 21680, but not [3H]CV 1808, is significantly enhanced by the pore-forming antibiotic, alamethicin. Guanosine 5'-O-(3-thiotriphosphate) decreases the binding of both radioligands to striatal membranes at 21 degrees C significantly more than to membranes on ice.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1995

Characterization of two affinity states of adenosine A2a receptors with a new radioligand, 2-[2-(4-amino-3-[125I]iodophenyl)ethylamino]adenosine.

Adenosine analogs substituted in the 2-position with arylamino groups have been found to have high affinity and selectivity for A2a adenosine receptors. Two such compounds, 2-[2-(4-aminophenyl)ethylamino]adenosine and 2-[2-4-amino-3-iodophenyl)ethylamino]adenosine (I-APE), were synthesized and found to be potent coronary vasodilators (ED50 < 3 nm). These compounds bind weakly to A1 adenosine receptors of rat cortex (Ki > 150 nM). 125I-APE was synthesized and the new radioligand was found to bind to two affinity states of rat striatal A2a adenosine receptors (Kd = 1.3 +/- 0.1 nM and 19 +/- 4.5 nM). The high affinity site represents a previously unrecognized small (15-20%) fraction of A2a adenosine receptors coupled to G proteins. Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) reduces specific binding of 125I-APE half-maximally at a concentration of 45 +/- 2 nM. [3H]CGS21680 also binds to two affinity states of A2a receptors on striatal membranes (Kd = 3.9 +/- 0.9 and 51 +/- 5.5 nM), although in previous studies single Kd values ranging from 5 to 15 nM have been reported. This high affinity site is substantiated by the finding that the IC50 of CGS21680 in competition with 125I-APE binding to striatal membranes is shifted leftward in membranes diluted for 4 min before filtration, to selectively dissociate radioligand from low affinity receptors. Assuming that agonist radioligands bind to both coupled and uncoupled forms of striatal A2a adenosine receptors, we could simulate with the computer the finding that the decrease in specific binding induced by GTP gamma S (100 microM) is variable and depends on radioligand concentration, ranging from 20 to 90%. Unlike 125I-APE, [3H]CGS21680 is charged at physiological pH, and treatment of membranes with the pore-forming antibiotic alamethicin uncovers cryptic [3H]CGS21680 but not 125I-APE binding sites. We conclude that the GTP gamma S-sensitive high affinity form of the A2a adenosine receptor can be preferentially labeled by 125I-APE, due to both its high specific activity and its physicochemical properties. Possible functional manifestations of poor coupling of A2a adenosine receptors to G proteins are discussed.

Adenosine↗

PubMed1994

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively influences the binding of the ribose-substituted adenosine analog, 5'-N-ethylcarboxamidoadenosine to a variable extent, depending on the nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.

Adenosine↗

PubMed1994

Hemodynamic and metabolic correlates of dipyridamole-induced myocardial thallium-201 perfusion abnormalities in multivessel coronary artery disease.

The mechanisms responsible for the development of reversible thallium-201 (TI-201) defects with dipyridamole stress in patients with coronary artery disease (CAD) is not well understood. Previous experimental animal studies have demonstrated coronary steal characterized by an absolute decrease in subendocardial flow distal to a stenosis in response to dipyridamole infusion. Accordingly, the purpose of this study was to determine if reversible TI-201 defects in response to dipyridamole infusion are reflective of myocardial ischemia or secondary to regional differences in flow reserve. Dipyridamole (0.56 mg/kg) TI-201 imaging was performed in 23 patients in whom serial electrocardiographic, hemodynamic, aortic and coronary sinus lactate, and coronary sinus adenosine measurements were obtained. All patients with CAD had TI-201 redistribution (3.8 +/- 2.0 defects/patient), and all patients without CAD had normal scans. Mean aortic pressure was similar in both groups and did not change in response to dipyridamole (non-CAD 103 +/- 11 vs CAD 99 +/- 15 mm Hg, p = NS). Pulmonary capillary wedge pressure was similar at baseline (non-CAD 11 +/- 4 vs CAD 13 +/- 5 mm Hg, p = NS) and did not change in response to the drug (non-CAD 14 +/- 3 vs CAD 15 +/- 7 mm Hg, p = NS). Lactate extraction fraction was similar at baseline (non-CAD 0.22 +/- 0.09 vs CAD 0.17 +/- 0.14, p = NS) and decreased similarly in both groups (non-CAD 0.08 +/- 0.06 vs CAD 0.05 +/- 0.12, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1994

Purification and characterization of an acetyl esterase from Aspergillus niger.

Optimized acetyl esterase enzyme production conditions using Aspergillus niger ATCC 10864 in 14-L fermentation jars were determined to be 33 degrees C, 1.5 vvm aeration, and 300 rpm agitation without pH control. The acetyl esterase was purified by precipitation in 60-80% saturation in ammonium sulfate. The pellet was applied directly to a Pharmacia high-load Phenyl Sepharose column for hydrophobic interaction chromatography and purified to homogeneity in two steps. Stability and kinetic characteristics of the acetyl esterase were determined over a pH range of 4.0-7.5 and from 4 to 45 degrees C. At temperatures > 25 degrees C, stability was superior at pH values < 5.0. The temperature activity optimum was 35 degrees C, and the pH optimum was 7.0. The Vmax was determined to be 46,700 U/mg protein, and the Km was 0.023M p-nitrophenyl acetate at pH 6.5 in 0.2M phosphate buffer at 35 degrees C. The mol wt of the enzyme was 35,000 dalton by size-exclusion chromatography and SDS gel electrophoresis. The N-terminal amino acid sequence and the glycosylation composition were also determined.

Acetylesterase↗

PubMed1994

Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions.

In this review, Joel Linden summarizes what is known about a new and intriguing member of the adenosine receptor family, the A3 receptor. This receptor exhibits unusually large differences in structure, tissue distribution and pharmacological properties between species. Rat A3 receptors are resistant to blockade by xanthine antagonists, but human and sheep A3 receptors can be potently blocked by certain xanthines, notably acidic 8-phenylxanthines. One function of the receptor is to facilitate degranulation of mast cells, and a role for mast cells and A3 receptors in mediating myocardial preconditioning has been proposed. Therefore, selective antagonists of A3 receptors have potential for the treatment of allergic, inflammatory and possibly ischaemic disorders.

Amino Acid Sequence↗