[ super(3)H]-SCH 58261 labelling of functional A sub(2A) adenosine receptors in human neutrophil membranes

The present study describes the direct labelling of A sub(2A) adenosine receptors in human neutrophil membranes with the potent and selective antagonist radioligand, [ super(3)H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1 ,2,4 triazolo[1,5-c]pyrimidine, ([ super(3)H]-SCH 58261). In addition, both receptor affinity and potency of a number of adenosine receptor agonists and antagonists were determined in binding, adenylyl cyclase and superoxide anion production assays. Saturation experiments revealed a single class of binding sites with K sub(d) and B sub(max) values of 1.34 nM and 75 fmol mg super(-1) protein, respectively. Adenosine receptor ligands competed for the binding of 1 nM [ super(3)H]-SCH 58261 to human neutrophil membranes, with a rank order of potency consistent with that typically found for interactions with the A sub(2A) adenosine receptors. In the adenylyl cyclase and in the superoxide anion production assays the same compounds exhibited a rank order of potency identical to that observed in binding experiments. Thermodynamic data indicated that [ super(3)H]-SCH 58261 binding to human neutrophils is entropy and enthalpy-driven. This finding is in agreement with the thermodynamic behaviour of antagonists binding to rat striatal A sub(2A) adenosine receptors. It was concluded that in human neutrophil membranes, [ super(3)H]-SCH 58261 directly labels binding sites with pharmacological properties similar to those of A sub(2A) adenosine receptors of other tissues. The receptors labelled by [ super(3)H]-SCH 58261 mediated the effects of adenosine and adenosine receptor agonists to stimulate cyclic AMP accumulation and inhibition of superoxide anion production in human neutrophils.