Characteristics of adenosine binding sites in atrial sarcolemmal membranes

The studies reported here involve an exploration of the sites on atrial myocyte membranes with which adenosine interacts to produce its potent physiological effects in atrial muscle. Specific, high affinity binding of the stable adenosine analogs 2-chloro[3H]adenosine (2-ClAdo) and [3H]adenosine 5'-N-ethylcarboxamide (NECA) to atrial sarcolemmal membranes was measured in kinetic and equilibrium studies at 4 degrees C and 35 degrees C. Analysis of the [3H]2-ClAdo binding isotherm indicated the presence of two classes of binding site with equilibrium Kassoc values estimated to be 5.7 X 10(7) M-1 and 2.7 X 10(6) M-1. Displacement of bound [3H]2-ClAdo by adenosine 5'-N-cyclopropylcarboxamide (NCPCA) and by several N6-substituted adenosine analogs confirmed the presence of two classes of binding site. Analysis of the [3H]NECA binding also revealed the presence of two types of binding site for this ligand. The methylxanthines isobutylmethylxanthine and theophylline displaced bound [3H]2-ClAdo whereas adenosine uptake inhibitors and several other purines showed little activity. These atrial membrane binding sites exhibit many of the characteristics of the physiological adenosine receptors studied in intact atria. Furthermore, the [3H]2-ClAdo binding sites were sensitive to treatment with proteolytic enzymes, suggesting that these sites exist on sarcolemmal membrane proteins.     

Adenosine receptors mediate pigment dispersion in leucophores of the medaka, Oryzias latipes

Using the medaka, Oryzias latipes (orange-red variety), the mechanisms of action on leucophores of adenosine and adenine nucleotides, including cyclic AMP, were studied. All these substances were found to be very effective in dispersing leucosomes. Their pigment-dispersing action was antagonized by methylxanthines. These substances did not aggregate leucosomes. It was concluded that leucophores possess specific binding sites for adenosine, i.e. adenosine receptors, on the cell membrane, which mediate leucosome dispersion. Further, it was shown that even the action of an established intracellular second messenger, cyclic AMP, is primarily manifested through the receptors.     

Adenosine deaminase activity of insect-derived growth factor is essential for its growth factor activity

Insect-derived growth factor (IDGF) was originally isolated from conditioned medium of NIH-Sape-4 cells derived from flesh fly embryos. Here we demonstrated that IDGF has adenosine deaminase activity. The substrate specificity of IDGF was similar to that of the mammalian cytoplasmic adenosine deaminase. The adenosine deaminase activity of IDGF was shown to be indispensable for its growth factor activity toward NIH-Sape-4 cells. We found that there are specific binding sites for IDGF on the surface of NIH-Sape-4 cells and that it binds to these sites with a K(d) value of 2.4 x 10(-10) m. We propose that the cell surface binding sites for IDGF are specific receptors modified with an adenosine moiety. When IDGF binds to these receptors, it may deaminate the adenosine moiety, and this process may be prerequisite for the signal transduction via this receptor.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

Effects of chronic caffeine on brain adenosine receptors: Regional and ontogenetic studies

The effect of chronic caffeine treatment on three different binding sites in five brain areas of mice is characterized. The sites studied were the adenosine receptor, using [³H] diethylphenylxanthine, the benzodiazepine receptor, using [³H] diazepam and the adenosine uptake site, using [³H] nitrobenzylthioinosine. Significant increases were only observed in adenosine receptors with the greatest degree of change seen in the cerebellum and brain stem at both 16 and 23 days of caffeine treatment. The lack of significant effects of chronic caffeine on benzodiazepine receptors and adenosine uptake sites indicates that the caffeine effect is specific. The effect of chronic caffeine treatment on the ontogency of adenosine receptors was also studied with the result showing a significantly accelerated development of the receptor in the caffeine treated animals. The adult adenosine receptor levels were 20–30% higher than those observed in control animals. The observed alterations in adenosine receptor number which occur as a consequence of caffeine consumption may underlie some of the behavioral effects of this cortical stimulant as well as provide insights concerning the mechanisms of tolerance to and dependence on caffeine.     

Phylogenetic distribution of [3H]cyclohexyladenosine binding sites in nervous tissue

The specific binding of the A1 adenosine receptor ligand, [3H]CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific [3H]CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, [3H]CHA binding sites were not detectable in nervous tissue of the invertebrate species studied. The densities of [3H]CHA binding sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by [3H]CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A1 adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation.     

Anomalous binding of MgADP to myosin of skeletal muscle

Binding of adenosine diphosphate to skeletal muscle myosin was studied using a range of concentrations from 0 to 2 mM. Up to 0.2 mM adenosine diphosphate two equivalent and independent nucleotide binding sites were detected, characterized by the single association constant of 5 x 10(4)M(-1). At greater adenosine diphosphate concentrations a decreasing binding capacity was noticed, bound nucleotide being essentially approximately 0.1 mol/mol at a 1-2mM adenosine diphosphate concentration. We tentatively propose that nucleotides act indirectly on myosin by promoting the perturbation of the solvent, which is supported by the fact that polyphosphates are known powerful kosmotropes.     

Adenosine binding sites of rat pheochromocytoma PC 12 cell membranes: partial characterization and solubilization

Rat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine [( 3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0 degrees C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30 degrees C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0 degrees C was NECA greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine much greater than phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membrane-bound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7 nm for these adenosine binding sites/detergent complexes.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

Sites of methylation of purified transfer ribonucleic acid preparations by enzymes from normal tissues and from tumours induced by dimethylnitrosamine and 1,2-dimethylhydrazine

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNA(Asp) from yeast and on tRNA(Glu) (2), tRNA(fMet) and tRNA(Val) (1) from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNA(Glu) (2) was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5'-end of the molecule; tRNA(Asp) was methylated at the guanosine residue at position 26 from the 5'-end of the molecule; tRNA(fMet) was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5'-end; tRNA(Val) (1) was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5'-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.     

Kinetic studies on rat liver and beef heart mitochondrial adenosine triphosphatase: the effects of the chromium complexes of adenosine triposphate and adenosine diphosphate on the kinetic properties.

The kinetics of isolated rat liver and beef heart mitochondrial adenosine triphosphatase (ATPase) were studied by using the chromium ATP and ADP complexes as substrate analogs. It was found that both chromium ATP (CrATP) and chromium ADP (CrADP) are competitive inhibitors of ATP hydrolysis. The presence or absence of ATPase-activating anions, e.g., bisulfite, had little effect on the type or potency of the inhibition by these chromium complexes. Both CrADP and CrATP were noncompetitive inhibitors of the hydrolysis of ITP with both the heart and liver-derived enzymes. It was also found that CrADP was a consistently more effective inhibitor than the ATP complex with the beef heart enzyme. These results are consistent with the existence of two types of nucleotide binding sites on mitochondrial ATPases: One site is regulatory and is rather specific for adenosine polyphosphates, while the other site is relatively nonspecific and serves as the hydrolytic site.     

A Computer Generated Model of Adenosine Receptors Rationalising Binding and Selectivity of Receptor Ligands

Abstract

Computer graphic analyses on a broad spectrum of adenosine receptor ligands has shown that both the A1 and A2 adenosine receptors have three binding sites. The spatial relationship of these three binding sites has been defined. Adenosine orientation at A1 and A2 is different.     

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.     

Sensitization by chronic diazepam treatment of A2A adenosine receptor-mediated relaxation in rat pulmonary artery

The effects of a 10-day i.p. treatment of rats with diazepam on responses to subtype selective adenosine receptor agonists were studied 3 h, 2 and 8 days after termination of diazepam treatment in isolated cardiovascular tissues possessing distinct adenosine receptors. After long-lasting diazepam exposure, the relaxation elicited by the specific A2A receptor agonist CGS 21680 was enhanced in rat main pulmonary arteries (a tissue containing A2A adenosine receptors). The increased sensitivity of A2A receptors observed 3 h and 2 days after withdrawal of diazepam was completely restored by the 8th day of the wash-out period. N6-cyclopentyladenosine (CPA)-induced suppression in mechanical activity of electrically stimulated rat atrial myocardium (a tissue containing A1 adenosine receptors) was not altered following diazepam treatment. In order to reveal the possible role of inhibition of membrane adenosine transport in the effects of diazepam (a moderate inhibitor of membrane adenosine transport), the action of a 10-day treatment with dipyridamole or S-(p-nitrobenzyl)-6-thioinosine (NBTI; prototypic adenosine uptake inhibitors) was also studied. Dipyridamole or NBTI treatment, like diazepam, increased the responsiveness of rat pulmonary artery to CGS 21680, but did not influence the cardiodepressive effect of CPA in electrically driven left atrial myocardium. The CGS 21680-induced relaxations were significantly antagonized by 10 nM ZM 241385 (a selective A2A adenosine receptor antagonist) in vessels of diazepam-treated rats. The relaxation responses to verapamil were unaltered in pulmonary arteries obtained from animals chronically treated with diazepam, dipyridamole or NBTI. These results suggest that chronic diazepam treatment is able to enhance the A2A adenosine receptor-mediated vascular functions, but does not modify the responses mediated via A1 receptors of rat myocardium, where nucleoside transport inhibitory sites of membrane are of a very low density. It is possible that sensitization of A2A adenosine receptor-mediated vasorelaxation is due to a long-lasting inhibition of membrane adenosine transporter during diazepam treatment.     

Stoichiometric binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate to bovine heart cytochrome c oxidase.

The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.     

Two mechanisms for inhibition of ADP-induced platelet shape change by 5'-p-fluorosulfonylbenzoyladenosine. Conversion to adenosine, and covalent modification at an ADP binding site distinct from that which inhibits adenylate cyclase

The interaction of ADP with platelets leads to shape change, exposure of fibrinogen binding sites, and aggregation, all of which have been shown to be inhibited by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), an alkylating analogue of adenine nucleotides which binds covalently to a 100-kDa polypeptide in intact platelet membranes (Figures, W. R., Niewiarowski, S., Morinelli, T., Colman, R. F., and Colman, R. W. (1981) J. Biol. Chem. 256, 7789-7795). In plasma, FSBA can break down to adenosine which stimulates adenylate cyclase. To distinguish between direct effects of FSBA and the actions of adenosine, we have used washed platelet suspensions and adenosine deaminase. We studied the effects of FSBA on shape change and cyclic AMP metabolism, and on the binding of 2-methylthio-ADP, which mimics the effects of ADP on cyclic AMP metabolism at concentrations too low to activate platelets. Inhibition of ADP-induced shape change of platelets incubated with FSBA for 2 min in platelet-rich plasma was greatly reduced by adenosine deaminase. In the presence of a phosphodiesterase inhibitor, 100 microM FSBA increased platelet cyclic AMP to the same extent as did 10 microM adenosine. These effects were inhibited by theophylline, an adenosine receptor antagonist, and by adenosine deaminase. Incubation of washed platelets for 60 min with FSBA and adenosine deaminase caused a concentration-dependent inhibition of ADP-induced shape change. Inhibition closely paralleled the covalent incorporation of 3H from tritiated FSBA into platelet membranes. Under these conditions, FSBA did not block inhibition of cyclic AMP accumulation by ADP, nor did it block the binding of 2-methylthio-ADP. We conclude that part of the inhibition of shape change caused by brief exposure to FSBA is due to adenosine, but at longer times shape change is inhibited in association with covalent incorporation of sulfonylbenzoyladenosine. This effect of FSBA is independent of adenosine and occurs at a site distinct from that at which ADP inhibits adenylate cyclase.     

Inhibition of hexose transport by adenosine derivatives in human erythrocytes

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.     

Inhibition of tryptophanyl-tRNA synthetase by modifying ATP analogs

The interaction between tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef pancreas and the ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of ATP was studied as an approach to investigate the structure of the enzyme active center. Some of the compounds under study were shown to irreversibly inhibit the enzyme activity; the presence of ATP in the most cases protects the enzyme against inactivation. The kinetic constants Ki and k2 of interaction of the irreversible inhibitors with the enzyme were determined. It was found that the Ki values for a number of irreversible competitive inhibitors are by 1-2 orders of magnitude less than the Km value for ATP; the k2 values were found equal to 0.02-0.04 min-1. this suggests that the compounds may be used as affinity reagents, the most efficient ones being adenosine 5'-(beta-chloroethyl phosphate) and mixed AMP-mesithylene carbonic acid anhydride. The absence of a protective effect of ATP in the case of adenosine 5'-(beta-bromoethane phosphonate) and non-competitive type of reversible inhibition inhibition of the enzyme by adenosine 5'-chloromethane phosphonate indicate that the molecule of tryptophanyl-tRNA synthetase contains sites interacting with adenine nucleotides, other than the ATP binding sites of the active center.