2-Chloroadenosine stimulates granule exocytosis from mouse natural killer cells: Evidence for signal transduction through a novel extracellular receptor

The effect of 2-chloroadenosine (2CA), an adenosine receptor agonist, on the activation status of mouse natural killer (NK) cells was determined. Splenic lymphocytes incubated with 2CA exocytosed an NK cell-associated granzyme with Nα-CBZ-L-lysine thiobenzyl ester (BLT) esterase activity in a dose- and time-dependent manner. Selective depletion of NK cells by anti- asialoGM1 antibody plus complement pretreatment confirmed that NK cells were the source of the BLT esterase activity, 2CA-induced granule exocytosis was not reduced in the presence of the nucleoside uptake blockers NBTI, dilazep, or dipyridamole, indicating the involvement of an extracellular receptor. However, adenosine or other A₁, A₂, or A₃ cell-surface adenosine receptor agonists failed to trigger the exocytotic process. Furthermore, the nonselective adenosine receptor antagonist theophylline, as well as the selective A₁ receptor antagonist DPCPX and the selective A₂ receptor antagonist DMPX, did not interfere with 2CA-induced BLT esterase secretion. These data suggest that 2CA acts on NK cells via a novel (non-A₁/A₂/A₃) cell-surface receptor. Genistein, a protein tyrosine kinase inhibitor, and calphostin C, a protein kinase C inhibitor, both interfered with 2CA-induced granule exocytosis. Pertussis toxin, an ADP-ribosylating toxin to which certain GTP-binding proteins are sensitive, also inhibited 2CA-stimulated BLT esterase release. In addition, 2CA-induced granule exocytosis was reduced in the presence of cyclosporin A, an inhibitor of Ca²⁺-dependent signaling pathways, and the Ca²⁺-chelating agent EGTA. We conclude that 2CA, acting through a novel extracellular receptor on mouse NK cells, triggers granule exocytosis via a Ca²⁺-dependent signal transduction pathway that is coupled to GTP-binding proteins and involves protein tyrosine kinase and protein kinase C activation.