Endogenous adenosine regulates neutrophil pro-inflammatory activities by cyclic AMP-dependent accelerated clearance of cytosolic calcium.
OBJECTIVE
To identify the involvement of adenosine in restoration of Ca2+ homeostasis to activated human neutrophils.


METHODS
Neutrophils were isolated from venous blood taken from healthy, adult, human volunteers.


METHODS
The cells were exposed to adenosine deaminase (ADA, 0.1-2 units/ml) for 10 min at 37 degrees C prior to activation with N-formyl-L-methionyl-L-leucyl-L-phenylala-nine (FMLP, 1 microM).


METHODS
Cytosolic Ca2+ concentrations and transmembrane fluxes of the cation in FMLP-activated neutrophils +/- ADA were measured using spectrofluorimetric and radiometric procedures respectively, while intracellular cAMP and inositol triphosphate were measured by radioassay, and superoxide production and elastase release by, chemiluminescence and colourimetric methods respectively. Levels of statistical significance were calculated using the Mann-Whitney U-test and ANOVA.


RESULTS
Although FMLP-activated generation of inositol triphosphate and mobilisation of Ca2+ from neutrophil internal stores, as well as the magnitude of the subsequent efflux and store-operated influx of the cation were unaffected by ADA, there was a prolonged elevation in cytosolic Ca2+ in the presence of the enzyme, which was associated with failure to activate adenylate cyclase and with increased production of superoxide and release of elastase. These effects of ADA were attenuated by dibutyryl cAMP (4 mM), CGS 21680 (1 microM) and rolipram (0.5 microM), as well as by EGTA (10 mM).


CONCLUSIONS
These results are compatible with a physiological role for adenosine in promoting deactivation of neutrophils, possibly by promoting cAMP-dependent clearance of Ca2+ from the cytosol of the cells by the endo-membrane Ca2+-ATPase.
Theron
A J
AJ
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, South Africa. atheron@medic.up.ac.za
Steel
H C
HC
Tintinger
G R
GR
Anderson
R
R
D000241:Adenosine / Q000502:physiology*; D000243:Adenosine Deaminase / Q000494:pharmacology; D000328:Adult; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002132:Calcium Radioisotopes; D020013:Calcium Signaling / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D007249:Inflammation / Q000503:physiopathology*; D007293:Inosine Triphosphate / Q000378:metabolism; D019272:Leukocyte Elastase / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D016877:Oxidants / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism
D002132:Calcium Radioisotopes; D005456:Fluorescent Dyes; D016877:Oxidants; D013481:Superoxides; D007293:Inosine Triphosphate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000242:Cyclic AMP; D019272:Leukocyte Elastase; D000243:Adenosine Deaminase; D000241:Adenosine; D002118:Calcium; D016257:Fura-2