1 Although extracellular adenosine 5'-triphosphate (ATP) is the natura
l ligand for the P2Z receptor of human lymphocytes it is less potent t
han 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) in opening the associated ion
channel, which conducts a range of permeants including Ba2+ and ethidi
um(+). We have quantified the influx of ethidium(+) into lymphocytes p
roduced by BzATP, ATP, 2-methylthio-ATP (2MeSATP) and ATP gamma S, stu
died competition between ATP and BzATP and investigated the effects of
KN-62, a new and potent inhibitor of the P2Z receptor. 2 BzATP and AT
P stimulated ethidium(+) influx with EC50 values of 15.4 +/- 1.4 mu M
(n=5) and 85.6 +/- 8.8 mu M (n=5), respectively. The maximal response
to ATP was only 69.8 +/- 1.9% of that for BzATP. Hill analysis gave n(
H) of 3.17 +/- 0.24 (n=3) and 2.09 +/- 0.45 (n=4) for BzATP and ATP, s
uggesting greater positive cooperativity for BzATP than for ATP in ope
ning the P2Z receptor-operated ion channel. 3 A rank order of agonist
potency of BzATP>ATP = 2MeSATP>ATP gamma S was observed for agonist-st
imulated ethidium(+) influx, while maximal influxes followed a rank or
der of BzATP>ATP>2MeSATP>ATP gamma S. 4 Preincubation with 30-50 mu M
oxidized ATP (ox-ATP), an irreversible P2Z inhibitor, reduced the maxi
mal response but did not change the steepness of the Ba2+ influx-respo
nse curve produced by BzATP (n(H) 3.2 and 2.9 for 30 and 50 mu M ox-AT
P, respectively (n=2)). 5 ATP (300-1000 mu M) added simultaneously wit
h 30 mu M BzATP (EC90) inhibited both ethidium(+) and Ba2+ fluxes to a
maximum of 30-40% relative to the values observed with BzATP alone. M
oreover, ATP (300 mu M) shifted the concentration-response curve to th
e right for BzATP-stimulated Ba2+ influx, confirming competition betwe
en ATP and BzATP. 6. KN-62, a new and powerful inhibitor of the lympho
cyte P2Z receptor, showed less potency in antagonizing BzATP-mediated
fluxes than ATP-induced fluxes when maximal concentrations of both ago
nists (BzATP, 50 mu M; ATP, 500 mu M) were used. 7 These data suggest
that the natural ligand, ATP, is a partial agonist for the P2Z recepto
r while BzATP is a more efficacious agonist. Moreover the competitive
studies show that only a single class of P2-receptor (P2Z class) is ex
pressed on human leukaemic lymphocytes.