Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists

Abstract

Novel 2-propynylcyclohexyl-5'-N:-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-(3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl] -prop-2-ynyl)-cyclohexylmethyl ester (ATL193) were >50 x more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-N:-ethylcarboxamidoadenosine (CGS21680) for human A(2A) AR binding. Human A(2A) AR affinity for substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A(2A) AR agonist stimulation of human neutrophil [cyclic AMP](i), and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were approximately equipotent agonists of human A(3) ARs. We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP](i) and the effect of PKA inhibition on A(2A) AR agonist activity. ATL193-stimulated neutrophil [cyclic AMP](i) was blocked by antagonists with the potency order: ZM241385 (A(2A)-selective)>MRS1220 (A(3)-selective)>>N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A(2B)-selective) approximately 8-(N-methylisopropyl)amino-N(6)-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A(1)-selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89. The data indicate that activation of A(2A)ARs inhibits neutrophil oxidative activity by activating [cyclic AMP](i)/PKA.

Figure 1

Molecular structures of some A_2A AR agonists discussed in the study.

Figure 2

Competition by A_2A AR agonists for binding of ¹²⁵I-ZM241385 to recombinant human A_2A ARs. (A) ATL146e, ATL146a and CGS21680. (B) ATL193 and ATL2037. Membranes (2 μg protein tube⁻¹) prepared from HEK-293 cells stably transfected with human A_2A ARs were incubated with ¹²⁵I-ZM241385 (0.5 nM) and various concentrations of competing compounds. Specific binding was >80% of total binding. Each point is the mean±s.e.mean of triplicate determinations. Similar results were obtained in two or more additional experiments. K_i values are reported in Table 1.

Figure 3

Inhibition of the neutrophil oxidative burst and stimulation of neutrophil [cyclic AMP]_i by agonists of A_2A ARs. (A) ATL146e, ATL146a, and CGS21680. (B) ATL193 and ATL2037. Luminol-enhanced chemiluminescence was assayed in human neutrophils (1×10⁶ ml⁻¹) primed with TNFα (10 u ml⁻¹; 30 min; 37°C) and then stimulated with fMLP (1 μM; 10 min; 37°C)±A_2A AR agonists+ADA (1 u ml⁻¹). The results are reported as peak chemiluminescence as per cent of a TNF-primed control. Each point is the mean±s.e.mean, n=4−6 experiments. The EC₅₀s for decreasing oxidative activity are: ATL146e=0.3 nM; ATL193= 0.8 nM; ATL146a=16.7 nM; ATL2037=4.4 nM; and CGS21680= 9.2 nM. Inset: Human neutrophils (8×10⁶ ml⁻¹) were incubated (15 min; 37°C; with 1 μM rolipram and 1 u ml⁻¹ ADA)±A_2A AR agonists. Neutrophil [cyclic AMP]_i was measured by radio immunoassay. Each point (cyclic AMP pmol ml⁻¹) is the mean±s.e.mean (n=6). The EC₅₀ values for increased neutrophil [cyclic AMP]_i are ATL146e=7.1 nM; ATL193=5.6 nM; ATL146a= 19.7 nM; ATL2037=8.6 nM and CGS21680=17.1 nM.

Figure 4

Competitive antagonism of ATL193-stimulated neutrophil cyclic AMP accumulation. (A) ZM241385 (100 nM). (B) ATL193 (1 μM), WRC0571, MR1754, MRS1220, and ZM241385 (10–1000 nM). Human neutrophils (8×10⁶ ml⁻¹) were incubated (15 min; 37°C) with 1 μM rolipram and 1 u ml⁻¹ ADA)±ATL193 (0.01–100 nM) and±adenosine receptor antagonists (WRC0571, MRS1754; MRS1220 or ZM241385; 10–1000 nM). Neutrophil [cyclic AMP]_i was measured by radioimmunoassay. Each point (cyclic AMP pmol ml⁻¹) is the mean±s.e.mean (n=6).

Figure 5

Effect of ZM241385 on ATL193-inhibited neutrophil oxidative activity. Luminol-enhanced chemiluminescence was assayed in neutrophils (1×10⁶ ml⁻¹) primed with TNFα (10 u ml⁻¹; 30 min; 37°C) and then stimulated with fMLP (1 μM; 10 min; 37°C)±ATL193 (0.01 nM–100 nM)±ZM241385+ADA (1 u ml⁻¹). The results are reported as percent TNF-primed control. Each point is the mean±s.e.mean (n=6). Inset: Schild analysis, pA₂=8.86.

Figure 6

Synergistic effects of rolipram and ATL193 on the oxidative burst in human neutrophils. Luminol-enhanced chemiluminescence was measured in human neutrophils (1×10⁶ ml⁻¹) primed with TNFα (10 u ml⁻¹; 30 min; 37°C) and then stimulated with fMLP (1 μM 10 min; 37°C)±ATL193 (0.01 nM–100 nM)±rolipram (100 nM)+ADA (1 u ml⁻¹). The results are reported as per cent of TNF-primed control. Each point is the mean±s.e.mean (n=4–5).

Figure 7

H-89 counteracts ATL193-inhibited neutrophil oxidative activity. Luminol-enhanced chemiluminescence was measured in human neutrophils (1×10⁶ ml⁻¹) primed with TNFα (10 u ml⁻¹; 30 min; 37°C) and then stimulated with fMLP (1 μM; 10 min; 37°C)±ATL193 (10 nM)±H-89 (100 nM–10 μM)+ADA (1 u ml⁻¹). The results are reported as percent of a TNF-primed control. Each point is the mean±s.e.mean (n=6).

Figure 8

Kinetics of declines in the bioactivity of ATL146e and ATL193 in human plasma. (A) ATL146e. (B) ATL193. ATL193 and ATL146e (15 μl of 1 mM) were added to 1.5 ml aliquots of human plasma and incubated (37°C; 5% CO₂) for 0–240 min. Aliquots (200 μl) were transferred to 1.8 ml of cold acetone at each time point. Following removal of precipitated proteins and rapid evaporation bioactivity was assayed by competitive radioligand binding to A_2A ARs. Each point is the mean±s.e.mean (n=3–4).