A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.
By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] 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. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-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 receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.
Falleni
A
A
Dipartimento di Morfologia Umana e Biologia Applicata, Università di Pisa, Pisa, Italy.
Trincavelli
M L
ML
Macchia
M
M
Salvetti
F
F
Hamdan
M
M
Calvani
F
F
Gremigni
V
V
Lucacchini
A
A
Martini
C
C
D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000648:ultrastructure; D017979:Gold Colloid / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D009504:Neutrophils / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure*; D011485:Protein Binding; D018047:Receptors, Purinergic P1 / Q000378:metabolism / Q000648:ultrastructure*; D015398:Signal Transduction
D017979:Gold Colloid; D018047:Receptors, Purinergic P1; C027513:N(6)-cyclohexyladenosine; D001710:Biotin; D000241:Adenosine