A(1) adenosine receptors in human neutrophils: Direct binding and electronmicroscope visualization

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical st udies have shown that Al adenosine receptors modulate chemotaxis in respons e to chemotactic peptides. Until now, the characteristics of the specific a gonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [H-3 ] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [H-3] CHA bound A(1) adeno sine receptors with selectivity and specificity, although with a low bindin g capacity. Scatchard analysis showed a Kd Value of 1.4 +/- 0.08 nM and a m aximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The go od affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine r eceptors allowed us to visualize them, after conjugation with colloidal gol d-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a recept or-mediated pathway. The kinetics of the intracellular trafficking was fast , taking less than 5 min. These data suggest that the CHA-biotin XX-strepta vidin-gold complex is a useful marker for the specific labelling of A(1) bi nding sites and to follow the intracellular trafficking of these receptors. J. Cell. Biochem.75:235-244, 1999. (C) 1999 Wiley-Liss, Inc.