The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes

1 Extracellular adenosine 5'-triphosphate (ATP) is an agonist for a P2Z receptor on human lymphocytes which mediates opening of a cation-selective ion channel, activation of phospholipase D and shedding of the adhesion molecule, L-selectin, from the cell surface. The isoquinolinesulphonamides, KN-62, (1-[N, O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl] 4-phenylpiperazine), a selective antagonist of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and KN-04, (N-[1-[N-methyl-p-(5 isoquinoline sulphonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquinolinesulphonamide) an inactive analogue, were used to investigate the possible role of CaMKII in these diverse effects of extracellular ATP. 2 KN-62 potently antagonized ATP-stimulated Ba2+ influx into fura-2 loaded human lymphocytes with an IC50 of 12.7+/-1.5 nM (n=3) and complete inhibition of the flux at a concentration of 500 nM. Similarly, KN-62 inhibited ATP-stimulated ethidium(+) uptake, measured by time resolved flow cytometry, with an IC50 of 13.1+/-2.6 nM (n=4) and complete inhibition of the flux at 500 nM. 3 KN-04 antagonized ATP-stimulated Ba2+ influx with an IC50 of 17.3+/-2.7 nM (n=3). Similarly, KN-04 inhibited ATP-stimulated ethidium(+) uptake with an IC50 of 37.2+/-8.9 nM (n=4). Both fluxes were completely inhibited at 500 nM KN-04. 4 ATP-stimulated phospholipase D activity, measured in [H-3]-oleic acid-labelled lymphocytes by the transphosphatidylation reaction, was antagonized by KN-62 and KN-04, with 50% inhibition at 5.9+/-1.2 and 9.7+/-2.8 nM (n=3), respectively. Both KN-62 and KN-04 inhibited ATP-stimulated shedding of L-selectin, measured by flow cytometric analysis of cell surface L-selectin, with IC50 values of 31.5+/-4.5 and 78.7+/-10.8 nM (n=3), respectively. Neither of the isoquinolinesulphonamides (500 nM) inhibited phorbol ester- or ionomycin-stimulated phospholipase D activity or phorbol ester-induced shedding of L-selectin. 5 The inhibitory effect of KN-62 or KN-04 on P2Z-mediated responses was slow in onset (5 min) and only partially reversed by washing the cells. 6 Both KN-62 and KN-04 (at 500 nM) had no effect on uridine 5'-triphosphate (UTP)-stimulated Ca2+ transients in fura-2 loaded human neutrophils, a response which is mediated by the P2Y(2) receptor. 7 Thus, KN-62 and KN-04 are potent antagonists of the P2Z receptor and at nanomolar concentrations inhibit all known responses mediated by the P2Z receptor of human lymphocytes. In contrast, KN-62 and KN-04 had no effect on responses mediated by the P2Y(2) receptor of neutrophils. Moreover, since KN-62 and KN-04 are almost equipotent, the P2Z-mediated responses do not involve CaMKII, but indicate that the isoquinolinesulphonamides are potent and direct inhibitors of the P2Z-receptor.