Abstract
This work compares the pharmacological and biochemical properties of A3 adenosine receptors in human polymorphonuclear neutrophil granulocytes (PMNs) and promyelocytic HL60 cells. The gene expression of A3 receptors was examined by reverse transcription-polymerase chain reaction experiments, whereas the amount of A3 subtype on the plasma membrane was quantified by using the high-affinity and selective A3 antagonist [3H]5N-(4-methoxyphenyl-carbamoyl)amino-8-propyl-2-(2-furyl)pyrazolo-[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine ([3H]MRE 3008F20). Saturation experiments reveal a single high-affinity binding site with K Dvalues of 2.3 ± 0.3, 2.6 ± 0.4 nM, andB 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 A3 subtype. EC50 values ofN 6-(3-iodo-benzyl)-2-chloro-adenosine-5′-N-methyluronamide (Cl-IB-MECA) for inhibition of cAMP levels via A3 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 Ca2+ mobilization, respectively, are not completely consistent with the involvement of an A3receptor. Furthermore, an important finding of this work is that the inhibition of PMNs oxidative burst is predominantly A2A-mediated, even though an effect of A3subtype 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 A3 receptors similar to those in PMNs, thus providing a useful model for investigation of biochemical pathways leading to A3 receptor activation.
- The American Society for Pharmacology and Experimental Therapeutics
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