A1 Affinity Research Articles

1030-13 Alterations in Endothelial Cell Adenosine Receptors Mediate Endothelium-Dependent Vasoconstriction in Experimental Vein Grafts

Adenosine is a potent endogenous vasodilator which mediates its action through A1 and A2 receptor subtypes located on both vascular endothelial and vascular smooth muscle cells (SMC). Adenosine-mediated relaxation in vein grafts has not been examined. This study assesses the in vitro isometric tension responses to specific adenosine receptor agonists (A1: R-phenyl-isopropyl-adenosine and A2: CSG-21680; 10–9 to 10–4 M) of common carotid external jugular vein bypass grafts (VG) in New Zealand White rabbits. These responses were compared to those obtained in the extemal jugular vein (JV) and in the common carotid artery (CA). All vessels were precontracted with prostaglandin F2a (10–5 M). Endothelialized and de-endothelialized vessels were examined. The A1 mediated relaxation in JV was endothelium-independent, whereas A2 mediated relaxation was endothelium-dependent. In CA, A1 mediated relaxation was partially endothelium-dependent, while A2 mediated relaxation was endotheliumindependent. In VG, Al activation induced endothelium-dependent vasoconstriction. Endothelial denudation restored Al mediated relaxation, but reduced compared to that of JV (max. 19 ± 9%). A2 relaxation in VG was endothelium-independent (max. 39 ± 4%). Primary cultures of arterial and vein graft SMC expressed both A1 and A2 receptors on northern blot analysis. There was, however, a marked reduction in the A1 affinity of the SMC from the VG compared to the arterial SMC (40.9 ± 1.3% arterial binding). This study, therefore, demonstrates that there is a substantial change in the adenosine mediated vasoreactivity in VG compared to JV, due to a change in endothelial A1 receptor mediated response from relaxation to constriction, coupled with a decreased affinity of the A1 receptors on the VG SMC. The adenosine responses of the VG are also different from CA. Therefore, the endothelial celis of VG appear to be unique, in that functionally they neither maintain a venous phenotype nor acquire an arterial phenotype in response to adenosine.

Physiological red cell, 2,3-diphosphoglycerate increase by the sixth hour after birth

As the available hemoglobin A1 at birth ranges from 20 to 30% a possible mechanism to favor oxygen release to the tissues could be a decrease of hemoglobin A1 affinity to oxygen. This may be accomplished by an increase in blood pH soon after birth and by an elevation in red cell 2,3-diphosphoglycerate (2,3-DPG). This hypothesis is supported by Valleri and Hirsch, who described a rapid 2,3-DPG recovery of transfused depleted 2,3-DPG red cells. That being so, we carried out this current study by assaying the 2,3-DPG of cord blood from 22 newborns and at 6, 24 and 72 hours after birth, as well as those enzymes assumed to be envolved in the 2,3-DPG levels regulation. 2,3-DPG (nmoles g-1 Hb) demonstrated the following values: cord blood: 9,770 +/- 1,026; 6h: 12,773 +/- 1,726; 72 h: 11,990 +/- 728, unveiling a distinct behavior of a sharp increase of 30% by the sixth hour. This confirmed our hypothesis. Regarding the metabolic mechanisms which can account for the 2,3-DPG increase, besides the rise of blood pH, we detected a significant decrease of the 2,3-DPG phosphatase activity, which might diminish the 2,3-DPG breakdown.

Both RNA-Binding Domains in Heterogeneous Nuclear Ribonucleoprotein A1 Contribute Toward Single-Stranded-RNA Binding

Heterogenous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A1 are tightly associated with heterogenous nuclear RNAs (hnRNAs) within eukaryotic nuclei and are thought to be involved in hnRNA processing and splice site selection. The NH2-terminal two-thirds of hnRNP A1 contains two 92-amino acid RNA binding domains (RBDs) that are arranged in tandem and are more than 30% homologous with each other. Following this region is a flexible glycine-rich COOH-terminal domain. We have studied the nucleic acid binding properties of the two isolated RBDs (residues 1-92 and 93-184, respectively) and of A1 fragments corresponding to residues 1-184 and 1-196 (i.e., the latter fragment is called UP1) in order to evaluate their relative contributions to A1 binding. We have determined that the individual RBDs of A1 bind poly[r(epsilon A)], a fluorescent single-stranded RNA (ssRNA), with a surprisingly low apparent association constant of only 1.5 x 10(4) M-1 (1-92) and 4.5 x 10(4) M-1 (93-184), respectively. We hypothesize that this low affinity represents a basal level of binding that is common to most RBD-containing proteins. Oligonucleotide binding studies suggest the interaction site size for the 93-184 fragment is approximately 4 nucleotides or less and salt sensitivity studies indicate that only about 27% of the free energy of binding of this RBD derives from ionic interactions. Since the affinity of the 1-184 fragment is at least 10-fold above that of either of its component RBDs, both must contribute to binding. This conclusion is further supported by the increased occluded site size of 1-184 (n = 14 +/- 2), as compared to its 93-184 RBD (n = 6 +/- 1), and by the biphasic binding that was observed for the UP1:poly(U) interaction at pH 6.0. Our finding that the affinity of the 1-184 fragment is 1000-fold less than the product of the affinities of its 1-92 and 93-184 RBDs is consistent with these domains being joined by a flexible linker. By comparing the affinities of the 1-184 fragment with that for A1, we conclude that together the two RBDs in A1 account for only 53% of the free energy of A1 binding. Comparative binding studies with UP1 demonstrate that the short region spanning residues 185-->195 represents an important determinant of the binding affinity of A1 and, since this region contains a site of dimethylation, it may provide a mechanism for regulating the affinity of A1 for specific nucleic acid targets.

AMINOPHYLLINE AND CAFFEINE HAS DIFFERENT EFFECTS ON CBF

Background Studies using Doppler ultrasonography have been unable to identify a direct effect of aminophylline and caffeine on Cerebral Blood Flow(CBF) ot neonates. In one study using Xe-clearance, CBF decreased following intravenous administration of aminophylline. Design As part of a double-blind, randomised, clinical study of preterm neonates relieving either caffeine or aminophylline, CBF and Left Ventricular Output(LVO) were measured in 35 spontaneously breathing infants (GA median 28 wks, BW mean 1129 gr). Measurements were done at a mean postnatal age of 137 min., two hours after an intravenous loading dose (aminophylline 5mg/kg., caffeine 10mg/kg). Methods CBF was measured using tho 133Xe clearanee method. LVO was estimated using M-mode echocardiography and Doppler ultrasound velocimetry. Results Values are mean(SD). No differences in GA, BW or MABP, HR, pCO2, pO2 and a/A-ratio at the time of measurements were found between the groups. Conclusion These results apparently reflect differences in the ability of aminophylline and caffeino to block adenosine-induced vasodilation with the doses uaed. Aminophylline and caffeine may have different affinity for A1- and A2- adenosine receptors or subtypes of receptors.

Nucleosides and nucleotides. 112. 2-(1-Hexyn-1-yl)adenosine-5'-uronamides: a new entry of selective A2 adenosine receptor agonists with potent antihypertensive activity.

Chemical modifications of the potent A2 adenosine receptor agonist 2-(1-hexyn-1-yl)adenosine (7, 2-HA) at the 5'-position have been carried out to find more potent and selective A2 agonists. These analogues were evaluated for adenosine A1 and A2 receptor binding affinity in rat brain tissues and antihypertensive effects in spontaneously hypertensive rats (SHR). Among the series of compounds, 2-(1-hexyn-1-yl)adenosine-5'-N-cyclopropyluronamide (16d) had the most potent affinity to the A2 receptor with a Ki of 2.6 nM, which is essentially the same as that of the parent agonist, 2-HA. However, the most selective agonist for the A2 receptor was 2-(1-hexyn-1-yl)adenosine-5'-N-methyluronamide (16b) with a Ki of 11 nM and a 162-fold selectivity. The N-alkyl substituents of 5'-uronamide derivatives did not seem to potentiate the A2 binding affinity but drastically reduced the A1 affinity compared with the parent 2-HA. Therefore, the A1/A2 selectivity was consequently increased. Other 5'-deoxy-5'-substituted derivatives of 2-HA such as the chloro (20), carboxamide (27, 28), sulfonamide (29), urea (30), and thiourea (22) analogues were also prepared. Among these nucleosides, no active compounds with potent or selective affinities to both receptors were found except 20. Although glycosyl conformations and sugar-puckering of these nucleosides were studied by 1H NMR spectroscopy, there were no positive correlations between active and inactive agonists. 2-(1-Hexyn-1-yl)adenosine-5'-uronamide (16a) and 16d had a potent hypotensive effect at ED30 values of 0.18 and 0.17 micrograms/kg, respectively, upon iv administration to anesthetized SHR.

2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamide as selective agonists at A2 adenosine receptors.

In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A2 receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cyclase, and platelet aggregation studies. Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited by compounds 2a-d with Ki values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affinity binding at solubilized A2 receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [3H]DPCPX and for the agonist [3H]CCPA gave Ki values in the nanomolar range, and the compounds showed moderate A2 selectivity. In order to improve this selectivity, the corresponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. As expected, the 5'-N-ethyluronamide derivatives retained the A2 affinity whereas the A1 affinity was attenuated, resulting in an up to 10-fold increase in A2 selectivity. A similar pattern was observed in adenylate cyclase assays and in platelet aggregation studies. A 30- to 45-fold selectivity for platelet A2 receptors compared to A1 receptors was found for compounds 8a-c in adenylate cyclase studies.

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1Receptor in the Rat Cortex

Abstract The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [3H]XCC and [3H]XAC2, and [3H]CPX3) which bind with high and selective affinity to the adenosine A1 receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 82165, represents the first reported non-xanthine structure that potently blocks adenosine receptors6. CGS 15943 has nanomolar affinity for both A1 and A2 receptor subtypes6. However, in contrast to many of th...

Functional correlation between subclasses of brain adenosine receptor affinities and ethanol-induced motor incoordination in mice

To further investigate if the modulation of ethanol-induced motor incoordination is by brain adenosine A1 and/or A2 receptor, adenosine analogs with wide variability in their affinity for A1 and A2 subtypes were administered ICV and their effect on ethanol-induced (IP) motor incoordination was evaluated by rotorod. A dose-dependent marked accentuation of ethanol-induced motor incoordination by adenosine agonists (CHA, NECA, CPA, DCCA) tested, with nearly no effect on normal motor coordination in the absence of ethanol, was observed. There was a positive correlation between A2 affinity, A2/A1 affinity ratio but a negative correlation between A1 affinity and the potency (ED50) of adenosine agonists to accentuate ethanol-induced motor incoordination. However, with the high potency of CHA and NECA, both having significant affinity for A1 and A2 receptors, together with the well known membrane perturbation by ethanol, it seems difficult to rule out until more information becomes available the contribution of A1 receptor activation to adenosine modulation of ethanol-induced motor incoordination. The high density of high affinity A2 (A2a) in the striatum and of A1 in the cerebellum and several brain areas and the known importance of these two brain areas in the motor control, indirectly supports or at least provides a circumstantial evidence for a functional correlation between ethanol-induced motor incoordination and brain adenosine receptors.

7-Deaza-2-phenyladenines: structure-activity relationships of potent A1 selective adenosine receptor antagonists

A series of derivatives of 7-deazapurines with varying substituents in the 2-, 6-, and 9-position was synthesized in an attempt to improve the adenosine receptor affinity and A1 or A2 selectivity. The adenosine receptor affinities were assessed by measuring the inhibition of [3H]-(R)-N6-(phenylisopropyl) adenosine (R-PIA) binding to rat brain A1 and inhibition of [3H]-5'-(N-ethylcarboxamido)adenosine (NECA) binding to rat striatum A2 adenosine receptors. A selected set of compounds representing the main structural variations was further examined in adenosine receptor coupled adenylate cyclase assays. All tested compounds antagonized the inhibition of adenylate cyclase elicited by interaction of R-PIA with A1 receptors in rat fat cell membranes and the activation of adenylate cyclase elicited by interaction of NECA with A2 receptors of pheochromocytoma PC12 cell membranes. The results indicate that 7-deazahypoxanthines have a potential for A2 selectivity, while all 7-deazaadenines are A1 selective. Introduction of a phenyl residue in the 2-position of 7-deazaadenines increases A1 activity tremendously. 2-(p-Chlorophenyl)-7,8-dimethyl-9-phenyl-7-deazaadenine (29) is potent and specific for the A1 receptors of rat brain (Ki = 122 nM), having no affinity for the A2 receptors of rat striatum. The compound has low activity at the A2 receptors of rat PC12 cell membranes where it appears to act as a noncompetitive inhibitor. A 1-phenylethyl substituent at the 9-position was found to be superior to a phenyl residue in terms of A1 affinity. The most potent A1 antagonist in the present series is the highly A1 selective (790-fold) (R)-7,8-dimethyl-2-phenyl-9-(1-phenylethyl)-7-deazaadenine (31, Ki = 4.7 nM), which is 30-35 times more potent at A1 receptors than its S enantiomer. The solubility of six of the potent 7-deaza-2-phenyladenines was determined by means of an A1 binding assay. Chloro substitution of the 2-phenyl ring appeared to improve the solubility as well as the solubility over A1 affinity ratio of 9-phenyl- and 9-(1-phenylethyl)-substituted 7-deazadenines.

4-Amino[1,2,4]triazolo[4,3-a]quinoxalines. A novel class of potent adenosine receptor antagonists and potential rapid-onset antidepressants

A series of 4-amino[1,2,4]triazolo[4,3-a]quinoxalines has been prepared. Many compounds from this class reduce immobility in Porsolt's behavioral despair model in rats upon acute administration and may therefore have therapeutic potential as novel and rapid acting antidepressant agents. Optimal activity in this test is associated with hydrogen, CF3, or small alkyl groups in the 1-position, with NH2, NH-acetyl, or amines substituted with small alkyl groups in the 4-position, and with hydrogen or 8-halogen substituents in the aromatic ring. Furthermore, many of these 4-amino[1,2,4]triazolo[4,3-a]quinoxalines bind avidly, and in some cases very selectively, to adenosine A1 and A2 receptors. A1 affinity of these compounds was measured by their inhibition of tritiated CHA (N6-cyclohexyladenosine) binding in rat cerebral cortex membranes and A2 affinity by their inhibition of tritiated NECA (5'-(N-ethylcarbamoyl)adenosine) binding to rat striatal homogenate in the presence of cold N6-cyclopentyladenosine. Structure-activity relationship (SAR) studies show that best A1 affinity is associated with ethyl, CF3, or C2F5 in the 1-position, NH-iPr or NH-cycloalkyl in the 4-position, and with an 8-chloro substituent. Affinity at the A2 receptor is mostly dependent on the presence of an NH2 group in the 4-position and is enhanced by phenyl, CF3, or ethyl in the 1-position. The most selective A1 ligand by a factor of greater than 3000 is 121 (CP-68,247; 8-chloro-4-(cyclohexyl-amino)-1- (trifluoromethyl)[1,2,4]triazolo[4,3-a]quinoxaline) with an IC50 of 28 nM at the A1 receptor. The most potent A2 ligand is 128 (CP-66,713; 4-amino-8-chloro-1- phenyl[1,2,4]triazolo[4,3-a]quinoxaline) with an IC50 of 21 nM at the A2 receptor and a 13-fold selectivity for this receptor. Representatives from this series appear to act as antagonists at both A1 and A2 receptors since they antagonize the inhibiting action of CHA on norepinephrine-stimulated cAMP formation in fat cells and they decrease cAMP accumulation induced by adenosine in limbic forebrain slices. Thus certain members of this 4-amino[1,2,4]triazolo[4,3-a]quinoxaline series are among the most potent and A1 or A2 selective non-xanthine adenosine antagonists known.

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation

Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results, it was concluded that chitinase A1 is the key enzyme in the chitinase system of this bacterium.

Possible presence of the difference peptide in alkali light chain 1 of fish fast skeletal myosin

1. 1. Presence of N-terminal peptide (“difference peptide”) in alkali light chain 1 (A1) of fish fast skeletal myosin was examined by comparing two kinds of light chain-based myosin subfragment 1 (S1) isozymes from the yellowtail Seriola quinqueradiata. 2. 2. On tryptic digestion, A1 was cleaved to a smaller fragment (mol. wt decrement by 2000) along with the cleavage of S1 heavy chain, while A2 was resistant to trypsin. Two-dimensional gel electrophoresis showed that A1 released a basic peptide by tryptic digestion. 3. 3. Both S1 isozymes showed clear kinetic differences in actin-activated Mg-ATPase activity, suggesting a higher affinity of A1 for actin. Affinity of A2 for heavy chain was also estimated to be about 2-fold higher than that of A1, as judged by the model experiments in which rabbit S1 isozymes were hybridized with heterologous alkali light chains.

The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re‐uptake site

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

Comparison of the behavioral effects of adenosine agonists and dopamine antagonists in mice

The adenosine agonists 5'-N-ethylcarboxamideadenosine (NECA), 2-chloroadenosine (2-CLA), N6-cyclohexyladenosine (CHA), N6-cyclopentyladenosine (CPA), 2-(phenylamino)adenosine (CV-1808) and R and S isomers of N6-phenylisopropyladenosine (R-PIA and S-PIA) decreased spontaneous locomotor activity in mice and, except for CPA, did so at doses that did not impair motor coordination, a profile shared by dopamine antagonists. CV-1808, the only agent with higher affinity for A2 as compared with A1 adenosine receptors, displayed the largest separation between locomotor inhibitory and ataxic potency. Like dopamine antagonists, NECA and CV-1808 also decreased hyperactivity caused by d--amphetamine at doses that did not cause ataxia whereas A1-selective adenosine agonists reduced amphetamine's effects only at ataxic doses. Unlike dopamine antagonists, adenosine agonists inhibited apomorphine-induced cage climbing only at doses that caused ataxia. Involvement of central adenosine receptors in these effects was suggested by the significant correlation obtained between potency for locomotor inhibition after IP and ICV administration. Affinity for A1 but not A2 adenosine receptors was significantly correlated with potency for inducing ataxia. These results suggest that the behavioral profile of adenosine agonists in mice is related to their affinity for A1 and A2 adenosine receptors and indicate that adenosine agonists produce certain behavioral effects that are similar to those seen with dopamine antagonists.

ChemInform Abstract: Benzo(1,2‐c:5,4‐c′)dipyrazoles: Non‐Xanthine Adenosine Antagonists.

AbstractThe diacetoxybenzenes (I) undergo Fries rearrangements in the presence of a Lewis acid, yielding the diacetyldihydroxybenzenes (II).

Benzo[1,2-c:5,4-c']dipyrazoles: non-xanthine adenosine antagonists.

3,5-Dimethylbenzo[1,2-c:5,4-c']dipyrazoles, optionally substituted in the 1-, 7-, and 8-positions, were synthesized from resorcinols. These compounds display affinity for adenosine A1 (rat brain) and A2 (human platelet) receptors. In addition, these compounds reverse contractions of guinea pig tracheal cylindrical segments induced by potassium chloride, histamine, acetylcholine, and 5-hydroxytryptamine, as well as reverse bronchospasm induced by aerosolized histamine in the conscious guinea pig.

1,2,4]Triazolo[4,3-a]quinoxalin-4-amines: a new class of A1 receptor selective adenosine antagonists

Several [1,2,4]triazolo[4,3-a]quinoxalines that were reported as antidepressants in the patent literature were found to possess moderate affinity for the adenosine A1 and A2 receptors. On the basis of structural parallels with adenine and adenosine, the N-cyclopentyl derivative was synthesized and found to have improved affinity and selectivity for the A1 receptor. In the N-cyclopentyl series, affinity was optimal with trifluoromethyl substitution at the 1-position, resulting in a compound (9) with 7.3 nM A1 affinity and 138-fold selectivity for the A1 receptor.

Characterization of the human T lymphocyte adenosine receptor: comparison of normal and systemic lupus erythematosus cells

To determine whether a defect in the T cell response to adenosine exists at the level of the adenosine receptor in systemic lupus erythematosus (SLE) we measured the binding affinity and maximum binding of T cell membranes from both normal and SLE T cells by utilizing radiolabeled adenosine ligands. Normal T lymphocyte membranes possess a single class of [3H]5-N-ethylcarboxamide adenosine binding sites with a Kd of 0.61 microM, a Bmax of 23.5 pmol/mg protein, and a Hill coefficient of 0.98, which indicates the presence of noncooperative sites. In contrast, T cell membranes do not bind significant amounts of either [3H]cyclohexyladenosine or [3H]phenylisopropyladenosine. These data indicate that T lymphocyte membranes have only A2, and not A1, adenosine receptors. Similarly, T cells from both active and inactive SLE subjects also express only A2 receptors with a Kd of 0.93 microM, a Bmax of 20.4 pmol/mg protein, and a Hill coefficient of 0.85, which is consistent with the presence of noncooperative sites. There is no difference in the on-rate, affinity, or density of T cell A2 receptors from active SLE patients, inactive SLE patients, or healthy controls. We conclude that T lymphocytes from both healthy and SLE subjects express A2, but not A1, receptors. Thus, the inability of SLE T cells to respond to adenosine does not reflect a decreased density of A2 (stimulatory) receptors, diminished A2 receptor binding, or an increased affinity or number of A1 (inhibitory) adenosine receptors. These observations support the conclusion that the defect in the T cell cAMP-dependent pathway may occur at a point distal to the adenosine receptor.

Autoradiographic evidence for multiple CNS binding sites for adenosine derivatives

The binding of the adenosine analogues, 5′-N-ethylcarboxamido[ 3H]adenosine and N6-cyclohexyl[ 3H]adenosine, to the rat brain was examined utilizing light-microscopic autoradiographic techniques. While associated with many of the same structures, binding sites for these compounds showed distinct differences in both their patterns of distribution and their capacities to be inhibited by the adenosine analogue, R-phenylisopropyladenosine. Previous studies have shown that, at nanomolar concentrations, cyclohexyladenosine and R-phenylisopropyladenosine bind rather exclusively to the A1 type of adenosine receptors. In contrast, 5′-N-ethylcarboxamidoadenosine has almost equal affinity for A1 and A2 sites. Taking advantage of these characteristics, non-A1 binding sites were resolved by examining 5′-N-ethylcarboxamidoadenosine binding in the presence of micromolar concentrations of unlabeled R-phenylisopropyladenosine. The autoradiographically demonstrated distribution of R-phenylisopropyladenosine-insensitive 5′-N-ethylcarboxamidoadenosine binding sites differed significantly from that of the cyclohexyladenosine binding sites. Such non-A1 binding sites were concentrated in the striatum, nucleus accumbens, medial genicuiate, olfactory tubercle, amygdala and certain thalamic nuclei. In contrast to the distribution of A1 adenosine receptor sites, R-phenylisopropyladenosine-insensitive 5′-N-ethylcarboxamidoadenosine binding was only low to moderate in the hippocampus, cerebellum and superior colliculus, regions which are strongly positive for cyclohexyladenosine binding. The present study provides the first autoradiographic evidence for multiple adenosine binding sites in the brain by demonstrating that the adenosine analogue 5′-N-ethylcarboxamidoadenosine can bind to a site or sites distinct from the A1 adenosine receptor site. The 5′-N-ethylcarboxamidoadenosine binding site which is not displaced by low concentrations of R-phenylisopropyladenosine may correspond to an A2 adenosine receptor site and/or an as yet uncharacterized type of adenosine receptor.