1 We have investigated the e ects of the selective phosphodiesterase (PDE) type 4 inhibitor, rolipram (0.01 ± 1 mM) on cytosolic Ca 2+¯u xes in FMLP-activated human neutrophils, as well as on superoxide production by, and release of elastase from, these cells. 2 Cytosolic Ca 2+¯u xes were measured by use of fura-2 spectro¯uorimetry in combination with a radiometric procedure that enables distinction between net e ux and in¯ux of the cation. Superoxide production and elastase release were measured by lucigenin-enhanced chemiluminescence and a colorimetric procedure, respectively. 3 Pretreatment of neutrophils with rolipram did not a ect the FMLP-activated release of Ca 2+ from intracellular stores, but was associated with dose-related acceleration of the rate of decline in fura-2 uorescence and with decreased e ux, as well as store-operated in¯ux of 45 Ca 2+ , indicative of enhancement of resequestration of the cation by the endo-membrane Ca 2+ -ATPase. 4 Inhibition of superoxide production and elastase release was observed at concentrations of rolipram which accelerated the clearance of Ca 2+ from the cytosol of FMLP-activated neutrophils.5 These e ects of rolipram on FMLP-activated Ca 2+¯u xes, superoxide generation and elastase release were mimicked by pretreatment of neutrophils with dibutyryl cyclic AMP (0.5 ± 4 mM), while theophylline (10 ± 150 mM), a non-speci®c PDE inhibitor, as well as the b 2 -agonist, salbutamol, were less e ective. 6 We conclude that rolipram deactivates FMLP-stimulated human neutrophils by enhancement of cyclic AMP-dependent resequestration of cytosolic Ca 2+ .

No abstract

1 We have investigated the effects of salmeterol (0.3-50 yM) on several pro-inflammatory activities of human neutrophils in vitro. 2 Oxidant production by FMLP-and calcium ionophore (A23187)-activated neutrophils was particularly sensitive to inhibition by low concentrations (0.3-3 IIM) of salmeterol, while the responses of phorbol myristate acetate-and opsonised zymosan-stimulated cells were affected only by higher concentrations (3-50 yM) of the drug. At these concentrations salmeterol is not cytotoxic, nor does it act as a scavenger of superoxide. 3 These anti-oxidative interactions of salmeterol with neutrophils were insensitive to propranolol but could be eliminated by washing the cells, or by pretreatment with low concentrations (1-2 uM) of the pro-oxidative, membrane-destabilizing phospholipids, lysophosphatidylcholine (LPC), platelet activating factor (PAF) and lysoPAF (LPAF). 4 At concentrations of 6.25-50 yM salmeterol interfered with several other activities of stimulated neutrophils, including intracellular calcium fluxes, phospholipase A2 activity and synthesis of PAF. 5 In an assay of membrane-stabilizing activity, salmeterol (25 and 50 Mm) neutralized the haemolytic action of LPC, PAF and LPAF. 6 Of the other commonly used /32-adrenoceptor agonists, fenoterol, and formoterol, but not salbutamol, caused moderate inhibition of neutrophil oxidant generation by a superoxide-scavenging mechanism. However, unlike salmeterol, these agents possessed only weak membrane stabilizing properties. 7 We conclude that salmeterol antagonizes the pro-inflammatory, pro-oxidative activity of several bioactive lipids implicated in the pathogenesis of bronchial asthma, by a mechanism related to the membrane-stabilizing, rather than to the f2-agonist properties of this agent.

1 We have investigated the e ects of the adenosine A 2A receptor agonist CGS 21680 (0.01 ± 1 mM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca 2+¯u xes and intracellular concentrations of cyclic AMP. 2 Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca 2+¯u xes were measured by fura-2 spectro¯uorimetry in combination with radiometric procedures which distinguish between net e ux and in¯ux of the cation. 3 Treatment of neutrophils with CGS 21680 did not a ect the FMLP-activated release of Ca 2+ from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 uorescence, as well as decreases in both e ux and store-operated in¯ux of Ca 2+ , compatible with enhancement of resequestration of the cation by the endo-membrane Ca 2+ -ATPase. These e ects on neutrophil Ca 2+ handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. 4 In contrast, treatment of neutrophils with the selective A 2A receptor antagonist, ZM 241385 (2.5 mM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca 2+ during store-operated in¯ux. 5 The CGS 21680-mediated reduction of Ca 2+ e ux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 mM), as well as by thapsigargin (1 mM), an inhibitor of the endo-membrane Ca 2+ -ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-in¯ammatory interactions of CGS 21680 with neutrophils.

No abstract