The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes
Version of Record online: 11 FEB 2009
1997 British Pharmacological Society
British Journal of Pharmacology
Volume 120, Issue 8, pages 1483–1490, April 1997
How to Cite
Gargett, C. E. and Wiley, J. S. (1997), The isoquinoline derivative KN-62 a potent antagonist of the P2Z-receptor of human lymphocytes. British Journal of Pharmacology, 120: 1483–1490. doi: 10.1038/sj.bjp.0701081
- Issue online: 11 FEB 2009
- Version of Record online: 11 FEB 2009
- (Received October 24, 1996, Revised January 9, 1997, Accepted January 17, 1997)
- P2Z receptor;
- lymphocytes (human leukaemic);
- extracellular ATP receptor;
- cation channel;
- phospholipase D;
- P2Y2 receptor;
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