Accelerated resequestration of cytosolic calcium and suppression of the pro-inflammatory activities of human neutrophils by CGS 21680 in vitro

Abstract

We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).

Figure 1

FMLP-activated fura-2 fluorescence responses of control and CGS 21680 (1 μM)-treated neutrophils. FMLP (1 μM) was added as indicated (↓) when a stable base-line was obtained (±1 min). The traces shown are from three different experiments.

Figure 2

FMLP (1 μM)-activated fura-2 fluorescence responses of control and ZM 241385 (2.5 μM)-treated neutrophils. FMLP was added as indicated (↓) when a stable baseline was obtained (±1 min). This is a typical trace from 12 different experiments.

Figure 3

Kinetics of efflux of ⁴⁵Ca²⁺ out of unstimulated neutrophils and neutrophils activated with FMLP (1 μM) in the absence and presence of CGS 21680 (1 μM). The results of 11 different experiments are expressed as the mean amount of cell associated ⁴⁵Ca²⁺ (pmol 10⁷ cells⁻¹) and vertical lines show s.e.mean. ^*P<0.05 for comparison with the FMLP-activated, CGS 21680-free control system.

Figure 4

Kinetics of influx of ⁴⁵Ca²⁺ into unstimulated neutrophils activated with FMLP (1 μM) in the absence and presence of CGS 21680 (1 μM). The results of eight different experiments are expressed as the mean amount of cell-associated ⁴⁵Ca²⁺ (pmol 10⁷ cells⁻¹) and vertical lines show s.e.mean. ^*P<0.05 for comparison with the FMLP-activated, CGS 21680-free control system.

Figure 5

The effects of varying concentrations of CGS 21680 (0.01–1 μM) on superoxide production by FMLP (1 μM)-activated neutrophils and on elastase release from FMLP/CB-activated neutrophils. The results of eight (superoxide), measured by lucigenin-enhanced chemiluminescence (LECL) and ten (elastase) different experiments are presented as the mean percentages of the drug-free control systems and vertical lines show s.e.mean. In the case of superoxide production, the absolute values for resting and FMLP-activated neutrophils were 256±23 and 911±84 mV s⁻¹ respectively. The corresponding values for elastase release were 40±2 and 598±70 milliunits enzyme per 10⁷ cells respectively.