Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an A1 adenosine receptor agonist

Adenosine, via interaction with A1 adenosine receptors, increases insulin sensitivity and inhibits lipolysis in adipocytes. To investigate regulation of this system, adipocytes were incubated for up to 72 h with the nonmetabolizable adenosine receptor agonist, N6-phenylisopropyl adenosine (PIA). Adenosine receptors were measured by the binding of 125I-hydroxyphenylisopropyl adenosine to membranes. PIA down-regulated adenosine receptors, decreasing the number of binding sites with no change in affinity. Adipocytes were incubated for 48 h without or with 100 nM PIA to down-regulate the A1 receptors by approximately 60%. The cells were washed, and lipolysis and glucose transport were assessed. The ability of PIA to inhibit lipolysis was markedly attenuated in the down-regulated cells. Furthermore, the EC50 of insulin was increased approximately 3-fold in the PIA-treated cells. 125I-Insulin binding to the PIA-treated cells was unchanged, demonstrating that the decreased insulin sensitivity is not due to decreased insulin receptor binding. Pertussis toxin catalyzed ADP-ribosylation of a 41-kDa protein thought to be the alpha-subunit of Gi. This 41-kDa protein was decreased in membranes from cells treated with PIA, with a maximal 50% loss. This suggests that Gi is down-regulated and that loss of both the A1 adenosine receptor and Gi are involved in the metabolic changes observed after PIA treatment.     

Differential Binding Properties of Adenosine Receptor Agonists and Antagonists in Brain

The binding properties of N6-cyclohexyl [3H]adenosine ( [3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ( [3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD = 0.4 +/- 0.05 nM and 4.2 +/- 0.3 nM; Bmax = 159 +/- 17 and 326 +/- 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD = 13.9 +/- 1.1 nM; Bmax = 634 +/- 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 microM, 250 microM, and 70 microM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50 = 350 microM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with L-N-phenylisopropyladenosine (PIA) being 50-fold more potent than D-PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.     

Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems

Specific high-affinity binding sites for 125I-alpha-bungarotoxin and (-)-[3H]nicotine have been measured in rat brain and locust (Schistocerca gregaria) ganglia. The binding sites for 125I-alpha-bungarotoxin had similar Kd values of 1.5 x 10(-9) and 0.8 x 10(-9) M for rat and locust preparations, respectively; the corresponding values for the (-)-[3H]nicotine-binding site were 9.3 x 10(-9) and 1.7 x 10(-7) M. Methyllycaconitine (MLA) potently inhibited 125I-alpha-bungarotoxin binding in both rat and locust. MLA was a less effective inhibitor of (-)-[3H]nicotine binding whereas (+)-anatoxin-a was a very potent inhibitor at this site in the rat but not in the locust. These data suggest that (+)-anatoxin-a is a useful probe for the high-affinity nicotine-binding receptor in vertebrate brain, whereas MLA is a preferential probe for the subclass of receptor that binds alpha-bungarotoxin.     

A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes.

In contrast to endothelin-1 (ET-1) and several of its analogues, sarafotoxin S6c (S6c) was a much more potent inhibitor of [125I]-ET-1 binding in rat hippocampus and cerebellum (Ki approximately 20 pM) than in rat atria and aorta (Ki approximately 4500 nM), suggesting the existence of ET-1 receptor subtypes (aorta/atria, ETA; hippocampus/cerebellum, ETB). S6c was a potent activator of PI turnover in hippocampus (EC50 approximately 10 nM) but not atria (EC50 greater than 1 microM), unlike ET-1 which was active in both tissues. S6c, therefore, is a highly selective ETB agonist. Furthermore, S6c was a potent pressor agent in the pithed rat (ED25 mm Hg approximately 0.1 nmoles/kg, i.v.), suggesting that the ETB receptor subtype may be important in cardiovascular function.     

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.     

Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Study of stereoisomers of N6-phenylisopropyladenosine on the secretion of pancreatic glucagon

This work was designed to characterize the adenosine receptor (A1 or A2) involved in glucagon secretion. The most potent adenosine analogues on A1 receptors are the N6 substituted compounds, among them N6-phenylisopropyladenosine (PIA); furthermore L-PIA is 50 to 100 times more potent than D-PIA on the A1 receptor, whereas it is 3 to 5 times more potent on the A2 receptor; thus the A1 receptor shows a much higher stereoselectivity. The effects of L-PIA and D-PIA were studied on glucagon secretion from the isolated perfused rat pancreas. 1) L-PIA at 1.65 microM induced a transient glucagon secretion which was not greater than that induced by the same concentration of adenosine. 2) D-PIA at a 3 fold higher concentration (4.95 microM) elicited a secretion of glucagon comparable to that induced by L-PIA 1.65 microM; thus the involved receptor does not present a high stereoselectivity for L-PIA. These results support the fact that the receptor involved in glucagon secretion is not of the A1 type.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate.

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.     

Barbiturates Are Selective Antagonists at A1 Adenosine Receptors

Barbiturates in pharmacologically relevant concentrations inhibit binding of (R)-N6-phenylisopropyl[3H]adenosine ([3H]PIA) to solubilized A1 adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. Ki values are similar to those obtained for membrane-bound receptors and are 31 microM for (+/-)-5-(1,3-dimethyl)-5-ethylbarbituric acid [(+/-)-DMBB] and 89 microM for (+/-)-pentobarbital. Kinetic experiments demonstrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-N6-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The stimulation of adenylate cyclase via A2 adenosine receptors in membranes from N1E 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. It is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A1 adenosine receptor antagonism may convey excitatory properties to barbiturates.     

Inhibition of IMP-specific cytosolic 5''-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5''-deoxy-5''-isobutylthio derivatives of adenosine and inosine.

1. The partially purified IMP-specific cytosolic 5'-nucleotidases from rat liver, polymorphonuclear leucocytes and heart were inhibited by 50% by 2-6 mM-5'-deoxy-5'-isobutylthioadenosine (IBTA) or 7-10 mM-5'-deoxy-5'-isobutylthioinosine (IBTI). IBTA and IBTI inhibited the rat liver and polymorphonuclear-leucocyte enzymes non-competitively. IBTA, but not IBTI, also inhibited the ecto-5'-nucleotidase of polymorphonuclear leucocytes. IBTI was, by contrast, a more potent inhibitor than IBTA of the AMP-specific soluble 5'-nucleotidase from pigeon heart. 2. During 2-deoxyglucose-induced ATP-catabolism in rat polymorphonuclear leucocytes, adenosine formation was inhibited by approx. 80% by 3 mM-IBTA and by approx. 70% by 7 mM-IBTI. 3. The results show that 5'-modified nucleosides are inhibitors of cytosolic 5'-nucleotidases and that they penetrate to inhibit their target enzymes in intact cells. Such inhibitors may be useful to clarify the mechanisms of adenosine formation and to prevent mononucleotide hydrolysis during ATP breakdown.     

Adenosine Receptors Mediating Cyclic AMP Productioin the Rat Hippocampus

In the transversely cut rat hippocampus, adenosine caused a dose-dependent increase in the accumulation of [3H]cyclic AMP from [3H]ATP. Adenosine breakdown products were inactive. AMP was somewhat less effective than adenosine, and its effect could be partially, but not completely, abolished by alpha, beta-methylene-ADP and GMP, which inhibited its metabolism by 5'-nucleotidase. The effect of adenosine was unaffected by inhibitors of adenosine deaminase, but enhanced by several inhibitors of adenosine uptake. Some analogues of adenosine, including N6-phenylisopropyladenosine (PIA), 2-chloroadenosine and adenosine 5'-ethylcarboxamide (NECA), were more active than adenosine, whereas others such as 2-deoxyadenosine and 9-(tetrahydro-2-furyl)adenine (SQ 22536) actually inhibited the response. The effect of PIA was highly stereospecific. The action of adenosine was inhibited by several alkylxanthines, the most potent of which was 8-phenyltheophylline. [3H]Cyclohexyladenosine (CHA) bound specifically to cell membranes from the rat hippocampus. The extent of binding was similar to that found in other cortical areas. The relative potency of some adenosine analogues and alkylxanthines to displace labelled CHA was essentially similar to their potency as effectors of the cyclic AMP system. Adenosine contributed to the cyclic AMP-elevating effect of alpha-adrenoceptor-stimulating drugs and several amino acids, but not to that seen with isoprenaline. The cyclic AMP increase seen following depolarization was only partially adenosine-dependent. The present results demonstrate that the rat hippocampus contains adenosine receptors mediating cyclic AMP accumulation and that these receptors have similar characteristics to those mediating pyramidal cell depression. Adenosine-induced cyclic AMP accumulation may be used as a biochemical correlate to electrophysiology and as a convenient parameter to assess the influence of drugs on adenosine mechanisms in the rat hippocampus.     

Functionalized congeners of 1,4-dihydropyridines as antagonist molecular probes for A3 adenosine receptors.

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA [N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyl-adenosine] in the submicromolar range. In this study, functionalized congeners of 1,4-dihydropyridines were designed as chemically reactive adenosine A3 antagonists, for the purpose of synthesizing molecular probes for this receptor subtype. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined in radioligand binding in comparison to binding at rat brain A1 and A2A receptors. Benzyl ester groups at the 3- and/or 5-positions and phenyl groups at the 2- and/or 6-positions were introduced as potential sites for chain attachment. Structure-activity analysis at A3 adenosine receptors indicated that 3,5-dibenzyl esters, but not 2,6-diphenyl groups, are tolerated in binding. Ring substitution of the 5-benzyl ester with a 4-fluorosulfonyl group provided enhanced A3 receptor affinity resulting in a Ki value of 2.42 nM; however, a long-chain derivative containing terminal amine functionalization at the 4-position of the 5-benzyl ester showed only moderate affinity. This sulfonyl fluoride derivative appeared to bind irreversibly to the human A3 receptor (1 h incubation at 100 nM resulting in the loss of 56% of the specific radioligand binding sites), while the binding of other potent dihydropyridines and other antagonists was generally reversible. At the 3-position of the dihydropyridine ring, an amine-functionalized chain attached at the 4-position of a benzyl ester provided higher A3 receptor affinity than the corresponding 5-position isomer. This amine congener was also used as an intermediate in the synthesis of a biotin conjugate, which bound to A3 receptors with a Ki value of 0.60 microM.     

Adenosine inhibits divalent cation influx across human neutrophil plasma membrane via surface adenosine A2 receptors.

Adenosine and its analogues inhibited increases in divalent cation influx stimulated by platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (FMLP) in a dose-dependent fashion. This effect was antagonized by theophylline, an adenosine receptor antagonist. When extracellular adenosine was removed by adenosine deaminase, the effect of adenosine was completely abolished. Two adenosine analogues with different affinities for adenosine receptor subtypes, 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (PIA), also inhibited divalent cation influx, NECA being more potent than PIA. These results suggest that adenosine and its analogues inhibit divalent cation influx across neutrophil plasma membranes via surface adenosine A2 receptors. Adenosine had little effect on the initial peaks of intracellular free calcium rises induced by chemoattractants, but it inhibited the subsequent rise in free calcium. Since calcium influx through the divalent cation channels or neutrophil plasma membranes is responsible for maintaining free calcium concentration following the initial peaks, we suggest that adenosine modulates neutrophil function by interfering with this calcium influx.     

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.