• P2Z receptor;
  • KN-62;
  • KN-04;
  • lymphocytes (human leukaemic);
  • isoquinolinesulphonamide;
  • extracellular ATP receptor;
  • antagonist;
  • cation channel;
  • lymphocyte;
  • phospholipase D;
  • P2Y2 receptor;
  • L-selectin
  • 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.

  • 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.

  • 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.

  • ATP-stimulated phospholipase D activity, measured in [3H]-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.

  • 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.

  • 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 P2Y2 receptor.

  • 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 P2Y2 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.

British Journal of Pharmacology (1997) 120, 1483–1490; doi:10.1038/sj.bjp.0701081