A(3) adenosine receptors in human neutrophils and promyelocytic HL60 cells: a pharmacological and biochemical study.
This work compares the pharmacological and biochemical properties of A(3) adenosine receptors in human polymorphonuclear neutrophil granulocytes (PMNs) and promyelocytic HL60 cells. The gene expression of A(3) receptors was examined by reverse transcription-polymerase chain reaction experiments, whereas the amount of A(3) subtype on the plasma membrane was quantified by using the high-affinity and selective A(3) antagonist [(3)H]5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]MRE 3008F20). Saturation experiments reveal a single high-affinity binding site with K(D) values of 2.3 +/- 0.3, 2.6 +/- 0.4 nM, and B(max) values of 430 +/- 35, 345 +/- 31 fmol/mg of protein for PMNs and HL60 cells, respectively. Competition of radioligand binding by adenosine ligands displays a rank order of potency typical of the A(3) subtype. EC(50) values of N(6)-(3-iodo-benzyl)-2-chloro-adenosine-5'-N-methyluronamide (Cl-IB-MECA) for inhibition of cAMP levels via A(3) receptors are in good agreement with the binding data; furthermore, the response is potently inhibited by MRE 3008F20. In contrast, the high micromolar concentrations of Cl-IB-MECA and MRE 3008F20 in stimulating and blocking Ca(2+) mobilization, respectively, are not completely consistent with the involvement of an A(3) receptor. Furthermore, an important finding of this work is that the inhibition of PMNs oxidative burst is predominantly A(2A)-mediated, even though an effect of A(3) subtype could not be excluded. This conclusion is based on potent blockade of Cl-IB-MECA-mediated inhibition of oxidative burst by SCH 58261 and a minor but significant blockade by MRE 3008F20. In conclusion, HL60 cells express A(3) receptors similar to those in PMNs, thus providing a useful model for investigation of biochemical pathways leading to A(3) receptor activation.
Gessi
Stefania
S
Department of Clinical and Experimental Medicine, University of Ferrara, Italy.
Varani
Katia
K
Merighi
Stefania
S
Cattabriga
Elena
E
Iannotta
Valeria
V
Leung
Edward
E
Baraldi
Pier Giovanni
PG
Borea
Pier Andrea
PA
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001667:Binding, Competitive; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010671:Phenylurea Compounds / Q000494:pharmacology*; D058915:Purinergic P1 Receptor Antagonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000037:antagonists & inhibitors; D014230:Triazoles / Q000494:pharmacology*
C405605:MRE 3008-F20; D010671:Phenylurea Compounds; D058915:Purinergic P1 Receptor Antagonists; D043684:Receptor, Adenosine A3; D018047:Receptors, Purinergic P1; D014230:Triazoles; D013481:Superoxides; C084956:N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine; D000242:Cyclic AMP; D000241:Adenosine; D002118:Calcium