Human basophil/mast cell releasability. VII. Heterogeneity of the effect of adenosine on mediator secretion

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than N6-(R-phenyl-isopropyl)-adenosine (R-PIA) inhibited in vitro anti-IgE-induced histamine and peptide leukotriene C4 (LTC4) release from human basophils in a concentration-dependent fashion. Micromolar concentrations of adenosine, NECA and R-PIA potentiated the anti-IgE-stimulated release of histamine and LTC4 from human lung parenchymal mast cells. Submillimolar concentrations of adenosine, NECA and R-PIA inhibited in a concentration dependent manner the release of histamine and prostaglandin D2 (PGD2) from skin mast cells challenged with anti-IgE. These results demonstrate marked heterogeneity of the modulatory effect exerted by adenosine on mediator release from human basophils and mast cells.     

Evidence for adenylate cyclase-coupled A1-adenosine receptors on ventricular cardiomyocytes from adult rat and dog heart

Isolated metabolically stable cardiomyocytes from adult rats and mongrel dogs were used to characterize the mechanism underlying the antiadrenergic effect of adenosine. In a system not affected by cellular heterogeneity, isoproterenol (3 x 10(-9) M - 10(-5) M) in the presence of adenosine deaminase (5U/ml) dose dependently increased cellular cAMP (5-80 pmol/mg). The effect of isoproterenol (0.1 microM) was inhibited by various adenosine derivatives, the rank order of potency being in the rat: (-)-N6-(R-phenyl-isopropyl)-adenosine (R-PIA) greater than 5'-N-ethylcarboxamidoadenosine (NECA) greater than S-PIA, and in the dog NECA greater than R-PIA greater than S-PIA. The cAMP increase induced by forskolin (1 microM) was attenuated in the rat by R-PIA. 8-phenyltheophylline (3 microM) antagonized the effect of R-PIA on isoproterenol-stimulated cAMP formation. Basal cAMP content was not influenced by R-PIA or NECA. Omission of adenosine deaminase from the incubation medium attenuated the isoproterenol-induced cAMP increase in the rat by about 30%. Our findings provide evidence for the presence of adenylate cyclase-coupled A1-adenosine receptors on cardiomyocytes which may mediate the antiadrenergic effect of adenosine in the heart.     

Adenosine receptor on human basophils: modulation of histamine release.

Adenosine, at physiologic concentrations, inhibits in vitro IgE-mediated human basophil histamine release in a dose-dependent fashion. The inhibition dose-response curve is paralleled by an adenosine-induced increase in cAMP levels of human leukocyte preparations. Further evidence that the adenosine effect is related to changes in cAMP levels is that the nucleoside inhibits only in the first stage of antigen-induced histamine release and fails to inhibit the release caused by ionophore A23187. A poorly metabolized derivative of adenosine, 2-chloroadenosine inhibits as effectively as adenosine; dipyridamole, which blocks adenosine uptake, does not impair the inhibition caused by adenosine. Finally, theophylline, which is a competitive antagonist of adenosine in human lymphocytes also blocks the inhibition of release caused by adenosine. These data suggest that adenosine acts via a specific cell-surface receptor linked to adenylate cyclase. It appears that the human basophil has a specific receptor for adenosine and that this nucleoside may modulate the in vivo release of the mediators of immediate hypersensitivity reactions.     

Pertussis toxin pretreatment reveals differential effects of adenosine analogs on IgE-dependent histamine and peptidoleukotriene release from RBL-2H3 cells

The effects of adenosine (A) and the nonmetabolizable adenosine analogs, N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (L-PIA), D-PIA and 2-chloroadenosine (2CHA) were examined on the IgE-dependent mediator release from RBL-2H3 cells, a model for mast-cell function. Adenosine and the adenosine analogs failed to influence mediator release from cells, previously sensitized with monoclonal anti-TNP mouse immunoglobulin E (anti-TNP IgE), when added alone. When added prior to conjugated trinitrophenol-ovalbumin (TNP-OVA), adenosine and the adenosine analogs (10(-8)-10(-4) M) significantly potentiated the release of both histamine (marker for degranulation) and peptidoleukotrienes (LT) (marker for de novo synthesized mediators). The effects were concentration-dependent with the potency order being L-PIA greater than NECA greater than A greater than D-PIA, 2CHA. The stimulatory effect on both histamine and LT release were reversed by prior treatment of the cells with pertussis toxin but not by the purinoceptor antagonists, theophylline and 8-phenyltheophylline, nor adenosine uptake blockers. At higher concentrations (above 10(-5) M), adenosine and adenosine analogs were also inhibitory on LT but not on histamine release. This inhibition was more evident on pertussis-toxin-treated cells in which there was no effect of adenosine or adenosine analogs on histamine release, but a concentration-dependent inhibition of IgE-dependent LT release. These findings demonstrate that adenosine analogs have two distinct mechanisms on mediator release from RBL-2H3 cells; a stimulatory effect on both histamine and LT release, mediated via a pertussis-toxin-sensitive G protein and an inhibitory effect on LT release via a pertussis-toxin-insensitive pathway. An abstract of this work has been published.     

The inhibitory effects of some adenosine analogues on transmitter release at the mammalian neuromuscular junction

An electrophysiological study was made of the effects of four adenosine analogues, 2-chloroadenosine (2-CIA), 5'-N-ethylcarboxamidoadenosine (NECA), L-N6-phenylisopropyladenosine (L-PIA), and 2-(p-methoxyphenyl)-adenosine (CV-1674) on neurotransmitter release in the mouse phrenic nerve - hemidiaphragm preparation. All four drugs decreased miniature end-plate potential frequency in a dose-dependent manner. Evoked transmitter release in the cut diaphragm preparation was depressed by 2-CIA and CV-1674 to a similar extent. The ability of theophylline to antagonize the inhibitory effect of CV-1674 on spontaneous transmitter release was also established. On the basis of these results, the rank order of potencies was: L-PIA greater than NECA greater than 2-CIA greater than CV-1674. A clear classification of receptor type could not be made, since the ratio of potencies of L-PIA and NECA was narrow. Different slopes of the concentration-effect curves for 2-CIA and CV-1674 compared with L-PIA and NECA suggest an additional component to simple agonist action in their overall effects.     

Adenosine as substrate and receptor agonist in the ovary

In the present study the possible dual effects of adenosine as substrate and adenosine receptor agonist in rat granulosa cells, cumulus-oocyte complexes, luteal cells and ovarian membranes are discussed. Adenosine is an indispensable compound in cell energy metabolism, as precursor to cofactors, second messenger and nucleic acids. Adenosine is also an agonist to adenosine receptors. The adenosine receptor can either inhibit (A1) or stimulate (A2) adenylate cyclase. Alternatively, in some cells adenosine receptor activation is linked to other cellular events like inhibition of Ca2+ fluxes. Adenosine is taken up by isolated preovulatory granulosa and luteal cells from pregnant mare serum gonadotropin-treated immature rats, but follicle stimulating hormone (FSH) decreases the uptake by granulosa cells. Adenosine, but not the non-metabolizable adenosine analogs 5'-(N-ethyl)carboxamide-adenosine (NECA), 2-chloro-adenosine (2-Clado), N6-(R-phenyl-isopropyl)-adenosine (R-PLA) and N6-(S-phenyl-isopropyl)-adenosine (S-PLA), increase granulosa cell ATP levels. FSH and luteinizing hormone (LH) decrease granulosa cell ATP levels in the presence or absence of adenosine. It has previously been shown that FSH and LH decrease oxygen consumption by cumulus-oocyte complexes and increase their lactate production. These effects have been suggested to be due to a competition of cofactors (e.g. ADP) common to glycolysis and the respiratory chain. The fact that adenosine reverse the gonadotropin-induced effects on oxygen consumption and lactate production support this theory. Adenosine and its analogs increase cAMP accumulation in luteal and granulosa cells only in the presence of gonadotropins, and this effect is antagonized by the adenosine receptor antagonist 8-phenyl-theophylline (8-PHT). Furthermore, adenylate cyclase is stimulated by adenosine analogs in membranes from non-luteinized and luteinized ovarian membranes and in luteal cell homogenates. The effect of NECA is antagonized by 8-PHT. In the membranes, the rank order of potency was NECA greater than 2-Clado greater than R-PLA greater than S-PLA, suggesting adenosine A2 receptors. In summary, it is suggested that adenosine can act both as a substrate to intracellular metabolism and as an adenosine A2 receptor agonist in granulosa and luteal cells. A paracrine short loop positive feedback model is proposed where extracellular adenosine, derived from a gonadotropin-induced extracellular increase in cAMP and a decrease in cellular ATP, enhances gonadotropin stimulation in granulosa and luteal cells.     

5'-N-ethylcarboxamide[3H]adenosine binding sites of mouse mastocytoma P815 cell membranes: characterization and solubilization

Mouse mastocytoma P815 cell membranes were found to possess adenosine binding sites as assessed by using the adenosine agonist [3H]5'-N-ethylcarboxamideadenosine (NECA). The Kd and Bmax for the [3H]NECA binding at 0 degrees C were 380 nM and 17 pmol/mg of protein, respectively. The rank order of potency for inhibition of [3H]NECA binding was NECA greater than 5'-N-cyclopropylcarboxamideadenosine greater than 2-chloroadenosine greater than 2',5'-dideoxyadenosine greater than isobutylmethylxanthine greater than theophylline greater than N6-[(R)-1-methyl-2-phenylethyl]adenosine = N6-[(S)-1-methyl-2- phenylethyl]adenosine. Thermodynamic analyses of the adenosine receptor agonist and antagonist binding showed that all such ligands displayed negative values of both enthalpy and entropy which suggested that the driving force for the binding was enthalpic. [3H]NECA binding sites of P815 cell membranes were solubilized with sodium cholate and retaining the same ligand-binding characteristics as those of the membrane-bound form. By gel filtration on a Sepharose CL-6B column, the adenosine binding site was estimated to have a Stokes radius of approximately 6.7 nm.     

Evidence for A1 and A2 adenosine receptors in guinea pig trachea

The adenosine analogs [5'-N-ethylcarboxamideadenosine (NECA), 2-Chloro-adenosine (2-ClA), R-phenylisopropyladenosine (R-PIA), N6-cyclohexyl adenosine (CHA), and N6-cyclopentyladenosine (CPA)] produced both relaxation and contraction responses in isolated guinea-pig trachea. A concentration-related relaxation response was observed in trachea which were precontracted with either histamine or KC1. This response followed an order of analog potency that was indicative of the A2 receptor subtype (NECA greater than 2-ClA greater than R-PIA greater than CPA greater than CHA). Theophylline, an adenosine-receptor antagonist, blocked this relaxation response. In addition, a concentration-related contractile response was produced with adenosine analogs in those trachea that were not previously contracted. In contrast, the contractile response followed an analog potency indicative of the A1 receptor subtype (R-PIA greater than 2-ClA = CPA = CHA). This contractile response was not mediated by cholinergic, adrenergic or histaminergic receptors. 2-ClA induced a biphasic response, while NECA only relaxed these tissue under basal tone. Unlike the relaxation response, these contractile responses were not attenuated by theophylline, but were blocked by 1,3 dipropyl-8-(2 amino-4-chlorophenyl)xanthine (PACPX). These findings confirm the existence of two subpopulations of adenosine receptors in guinea pig trachealis muscle.     

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.     

Effect of synthetic cyclic nucleotides on immunologic histamine release from calf granulocytes.

Sensitized bovine granulocytes release histamine when exposed to specific antigens. A unique modulation of histamine release by adrenergic agents has been shown in the bovine; beta-adrenergic agonists enhance and alpha-adrenergic agonists inhibit histamine release. This is an opposite response to that reported in other species. The present study was undertaken to determine the possible relationship between cyclic nucleotides and adrenergic agents in this species. Dibutyryl cAMP enhanced antigen-induced histamine release over the complete concentration range tested (10(-6)--10(-3)M); it also overcame, in a dose-dependent manner, the inhibition of antigen-induced histamine release produced by 10(-4) M phenylephrine. The 8-bromo cGMP AND 0-MONOBUTYRYL CGMP had no significant effect on antigen-induced histamine release nor did 8-bromo-cGMP have any significant effect on the enhancement of histamine release produced by 10(-4) M dibutyryl cAMP. These findings suggest that only cAMP has a role in the modulation of antigen-induced histamine release from bovine granulocytes.     

Studies on a modulatory role for adenosine in antigen and arachidonic acid induced contractions of guinea pig airways

The modulatory effects of adenosine and selected derivatives were examined on antigen and arachidonic acid (AA) induced contractions of indomethacin-treated tracheal spirals and lung parenchymal strips from actively sensitized guinea pigs. Adenosine (up to 2 X 10(-4) M) had no effect on antigen-induced contractions, but inhibited AA-induced contractions by 30-40% if added 30 min prior to challenge. The weak effect of adenosine suggests that endogenous adenosine may only have a limited modulatory role in allergic bronchospasm. 2-Chloroadenosine (10(-6)-10(-4) M) dose-dependently inhibited antigen- and AA-induced contractions of trachea, but was considerably less effective on parenchyma. The substituted adenosine derivatives, R-phenylisopropyladenosine (R-PIA) and 5'-(N-ethylcarboxamido)-adenosine (NECA), and the adenosine transport inhibitor, 6-[p-nitrobenzyl)thio]-9-beta-D-ribofuranosyl purine, were also active as modulators, but their activity was relatively weak and varied with the stimulus and the tissue. An order of potency for R-PIA, NECA, and 2-chloroadenosine could not be determined and 8-phenyltheophylline (10(-5) M) was not an effective inhibitor of the effects of adenosine or the adenosine derivatives. This suggests that adenosine and its derivatives may modulate cells through mechanisms other than activation of conventional A1 and A2 receptors. A lack of specificity for the adenosine derivatives must also be considered.     

Characterization of Adenosine Receptors in the PC 12 Pheochromocytoma Cell Line Using Radioligand Binding: Evidence for A-2 Selectivity

Examination of the binding characteristics of the adenosine agonist radioligands [3H]N6-cyclohexyladenosine [( 3H]CHA), [3H]cyclopentyladenosine [( 3H]CPA), and [3H]5'-N-ethylcarboxamido adenosine [( 3H]NECA) to membranes prepared from PC12 cells showed that the A-1-selective ligands (CHA and CPA) had minimal binding, which was not amenable to analysis using curve-fitting programs. However, [3H]NECA, a nonselective A-1/A-2 agonist, gave reproducible binding, which was enhanced by removal of endogenous adenosine, using the catabolic enzyme adenosine deaminase. This binding was of high affinity (KD = 4.7 nM) with limited capacity (263 fmol/mg of protein). Specific binding of [3H]NECA was unaffected by the presence of either CPA (50 nM) or MgCl2 (10 mM) but was sensitive to guanylylimidodiphosphate (100 microM), a finding suggesting involvement of an N-protein mechanism in the coupling of the adenosine receptor labeled by [3H]NECA to other components of the receptor complex. Binding of [3H]NECA to PC12 cell membranes was stereo-selective, with the R isomer of N6-phenylisopropyladenosine (PIA) being approximately 12 times more active than S-PIA. The A-1-selective agonist CPA was a weak inhibitor of [3H]NECA binding (Ki = 251 nM). The rank order of activity of adenosine agonists in displacing specific [3H]NECA binding was NECA greater than or equal to 2-chloroadenosine greater than CHA greater than or equal to 5'-N-methylcarboxamido adenosine greater than or equal to R-PIA greater than CPA greater than S-PIA. Binding was also displaced by the marine adenosine agonist 1-methylisoguanosine and by a series of xanthine antagonists with the activity order being 1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 8-phenyltheophylline greater than 8-p-sulfophenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS-1 cells: the possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.     

Regulation of Cyclic AMP Formation in Cultures of Human Foetal Astrocytes by β2-Adrenergic and Adenosine Receptors

Two cell cultures, NEP2 and NEM2, isolated from human foetal brain have been maintained through several passages and found to express some properties of astrocytes. Both cell cultures contain adenylate cyclase stimulated by catecholamines with a potency order of isoprenaline greater than adrenaline greater than salbutamol much greater than noradrenaline, which is consistent with the presence of beta 2-adrenergic receptors. This study reports that the beta 2-adrenergic-selective antagonist ICI 118,551 is approximately 1,000 times more potent at inhibiting isoprenaline stimulation of cyclic AMP (cAMP) formation in both NEP2 and NEM2 than the beta 1-adrenergic-selective antagonist practolol. This observation confirms the presence of beta 2-adrenergic receptors in these cell cultures. The formation of cAMP in NEP2 is also stimulated by 5'-(N-ethylcarboxamido)adenosine (NECA) more potently than by either adenosine or N6-(L-phenylisopropyl)adenosine (L-PIA), which suggests that this foetal astrocyte expresses adenosine A2 receptors. Furthermore, L-PIA and NECA inhibit isoprenaline stimulation of cAMP formation, a result suggesting the presence of adenosine A1 receptors on NEP2. The presence of A1 receptors is confirmed by the observation that the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine reverses the inhibition of isoprenaline stimulation of cAMP formation by L-PIA and NECA. Additional evidence that NEP2 expresses adenosine receptors linked to the adenylate cyclase-inhibitory GTP-binding protein is provided by the finding that pretreatment of these cells with pertussis toxin reverses the adenosine inhibition of cAMP formation stimulated by either isoprenaline or forskolin.     

An Investigation of the Low Intrinsic Activity of Adenosine and Its Analogs at Low Affinity (A2) Adenosine Receptors in Rat Cerebral Cortex

The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5'-N-Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2-fold). 2-Chloroadenosine (2-CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 microM dipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50 value for R-(-)-N6(2-phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to the KI value for inhibiting the response to NECA. The EC50 value for adenosine was substantially higher than the KI value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8-phenyltheophylline, 1,3-diethyl-8-phenylxanthine, and 8-p-sulfophenyltheophylline, with KI values from 1 to 10 microM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low-affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or changing the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5'-(N-ethylcarboxamido)adenosine (NECA, 10 microM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 +/- 0.02 (S.E.) to 1.80 +/- 0.02 (p less than 0.001) while inosine (10 microM), a poor adenosine receptor agonist, had no effect (1.73 +/- 0.04, p = n.s. vs. control, p less than 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA greater than adenosine = N6-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 microM) did not significantly change the number or affinity of [3H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

A low-affinity adenosine binding protein has recently been distinguished from the adenosine A2 receptor and purified from human placental membranes. Soluble human placental extracts contain an adenosine binding activity that has properties similar to those of the membrane low-affinity adenosine binding protein. The binding protein was purified from soluble human placental extracts 134-fold to 89% purity with a Bmax of 2.5 nmol/mg. It comprises 0.7-0.9% of the soluble protein. The major purified soluble protein has a subunit molecular mass of 98 kDa and a Stokes radius identical with that of the membrane-bound adenosine binding protein. Competition analysis of the soluble protein revealed similar affinities and an identical potency order for displacement of 5'-(N-ethylcarbamoyl)[2,8-3H]adenosine ([3H]NECA) as follows: NECA greater than 2-chloroadenosine greater than adenosine greater than (R)-N6-(2-phenylisopropyl)adenosine. The soluble binding protein was more acidic than the membrane binding protein as revealed by a comparison of the elution properties during ion exchange chromatography. A second form of soluble adenosine binding activity comprised 17% of the major form and had a charge similar to that of the membrane binding protein, a smaller Stokes radius, and a subunit molecular mass of 74 kDa. Carbohydrate composition analysis revealed that the major soluble form has 4.3% carbohydrate by weight as compared to the membrane-associated form, which has 5.5% carbohydrate by weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological profile of adenosine A2 receptor in PC12 cells

The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-(-)-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6-Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8-phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1-methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors.     

Antagonism by theophylline of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine at the guinea pig ileum

The inhibitory effect of the putative adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on acetylcholine release from the stimulated guinea pig ileum preparation and the nature of its antagonism by theophylline were investigated. NECA was shown to inhibit the response of the ileum preparation in a dose-dependent fashion, and an EC50 value of 1.62 X 10(-8) M was determined. This value was comparable with that determined for the A1 receptor agonist N6-R-phenylisopropyladenosine (R-PIA) (2.57 X 10(-8) M) using the same preparation. Competitive antagonism of the inhibitory effect of NECA by theophylline was quantitated and a pA2 value of 5.04 for the methylxanthine was obtained. This value was similar to those obtained previously for R-PIA and adenosine itself and suggests that these nucleosides may be interacting with the same receptor site on myenteric nerve endings. These findings do not permit the designation of the receptor as an A1 or A2 subtype according to current criteria.     

Involvement of Bidirectional Adenosine Transporters in the Release of l-[3H]Adenosine from Rat Brain Synaptosomal Preparations

Abstract: Adenosine transport inhibitors as enhancers of extracellular levels of endogenous adenosine would, presumably, only be effective if, for example, (1) the inhibitors block influx to a greater degree than efflux (release) of intracellular adenosine or (2) the inhibitors block equally well the influx and efflux of adenosine, but significant amounts of adenosine are formed as a result of dephosphorylation of released adenine nucleotides. Limited information is available regarding the directional symmetry of adenosine transporters in neural cells. Using rat brain crude P2 synaptosomal preparations preloaded with l -[³H]adenosine, our objectives here were to determine (1) if l -[³H]adenosine, a substrate for adenosine transporters that is more metabolically stable than physiological d -adenosine, was being released from synaptosomal preparations, (2) the optimal conditions necessary to observe the release, and (3) the degree to which this release was mediated by efflux through bidirectional nucleoside transporters. l -[³H]Adenosine release was found to be concentration and time dependent, temperature sensitive, and linear with synaptosomal protein. l -[³H]Adenosine release was inhibited dose-dependently by dipyridamole, nitrobenzylthioinosine, and dilazep; at concentrations of 100 µM inhibition was at least 40% for dipyridamole, 52% for nitrobenzylthioinosine, and 49% for dilazep. After loading with l -[³H]adenosine alone or l -[³H]adenosine plus unlabeled l -adenosine, d -adenosine, or uridine, l -[³H]-adenosine release was inhibited 42% by l -adenosine, 69% by uridine, and 81% by d -adenosine. The inhibition of l -[³H]adenosine release from the synaptosomal preparations by substrates for or inhibitors of nucleoside transporters suggests that a portion of the release was mediated by nucleoside transporters. This experimental system may prove useful for evaluating the effects of pharmacological agents on bidirectional transport of adenosine.