Further pharmacological characterization of the adenosine receptor subtype mediating inhibition of oxidative burst in human isolated neutrophils

Abstract

The aims of this study were to characterize the adenosine receptor subtype mediating inhibition of superoxide anion generation induced by N-formyl-methionyl-leucylphenylalanine (fMLP) in human neutrophils and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cAMP) is the second messenger mediating such inhibition. Superoxide anion generation induced by a submaximal concentration of fMLP (1 μM) was inhibited in a concentration-dependent manner by adenosine receptor agonists with a rank order of potency of N-ethylcarboxamidoadenosine (NECA) > 2(4-[(2-carboxyethyl)phenyl]ethylamino)-5'-N-ethylcarboxamido adenosine (CGS 21680) > (R)-N6-phenyl-2-propyladenosine ((R)-P1A) > 2-Chloro-N 6 -(3-iodobenzyl)9-[5-methylcarbamoyl)-β-D-ribofuranosyl] adenine (2-Cl-IB-MECA) > N 6 -cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine ((S)-PIA) N6-(4-amino-3-iodobenzyl) adenosine-5 -N-methyluronamide (AB-MECA); this order of potency is consistent with the activation of A 2A adenosine receptors. The nonselective A 1 , A 2A , and A 2B receptor antagonist 8-p-(sulphophenyl) theophylline (8-SPT; 10 μM) produced blockade of each of the agonists (pK B values 4.79-5.68). The selective A 1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM) and the selective A 3 adenosine receptor antagonist 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl- -propylxanthine (I-ABOPX; 200 nM) also produced blockade of all the agonists (PK B values 7.37-7.61 and 6.75-7.52, respectively); however, the concentrations required were higher than those which are considered selective for the A 1 or A 3 receptors. The selective A 2A receptor antagonist, 4-(2-[7-amino-2-(2-furyl [1,2,4] triazolo [2,3-a] [1,3,5] triazin-5-yl amino]ethyl) phenol (ZM 241385; 100 nM), powerfully suppressed the inhibition of the oxidative burst induced by each of the agonists. A Schild analysis of the effects of ZM 241385, 1-100 nM, against NECA and CGS 21680 was carried out. ZM 241385 produced concentration-dependent, parallel shifts of the concentration-effect curves to both NECA and CGS 21680, with pA 2 values of 9.62 and 9.59, respectively. Together, these data establish that inhibition of the oxidative burst in human isolated neutrophils, induced by adenosine receptor agonists, is mediated by the A 2A receptor. NECA (0.01-10 μM) induced a concentration-dependent increase in the intracellular cAMP content of neutrophils. This effect was inhibited in a dose-dependent manner by ZM 241385 (0.001-10 μM), consistent with activation of A 2A adenosine receptors. The results clearly demonstrate that in human neutrophils inhibition of the fMLP-induced oxidative burst by adenosine receptor agonists is mediated via activation of A 2A adenosine receptors linked positively to cAMP. No evidence of A 1 , A 2B , or A 3 adenosine receptor-mediated modulation of oxidative burst was found.