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

Abstract

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, , a selective antagonist of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), and -(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 e ects of extracellular ATP. 2 KN-62 potently antagonized ATP-stimulated Ba 2+ in¯ux into fura-2 loaded human lymphocytes with an IC 50 of 12.7+1.5 nM (n=3) and complete inhibition of the¯ux at a concentration of 500 nM. Similarly, KN-62 inhibited ATP-stimulated ethidium + uptake, measured by time resolved¯ow cytometry, with an IC 50 of 13.1+2.6 nM (n=4) and complete inhibition of the¯ux at 500 nM. 3 KN-04 antagonized ATP-stimulated Ba 2+ in¯ux with an IC 50 of 17.3+2.7 nM (n=3). Similarly, KN-04 inhibited ATP-stimulated ethidium + uptake with an IC 50 of 37.2+8.9 nM (n=4). Both¯uxes were completely inhibited at 500 nM KN-04. 4 ATP-stimulated phospholipase D activity, measured in [ 3 H]-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¯ow cytometric analysis of cell surface L-selectin, with IC 50 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 e ect 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 e ect on uridine 5'-triphosphate (UTP)-stimulated Ca 2+ 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 e ect 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.

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

“…The isoquinoline derivative KN62 is widely used as a potent and selective antagonist of both human and mouse P2X 7 receptors [30,31]. As seen in A similar response was observed (Fig.…”

“…The P2X 7 receptor shows a wide distribution including cells of the immune and hemopoietic system [27,34,45] and is distinguished by its low affinity for extracellular ATP and by its ability to trigger pore formation. The involvement of P2X 7 is supported by the finding that the increase in [Ca 2+ ] i is inhibited by KN62 [30,31] and oxATP [32], two selective antagonist of the human P2X 7 receptor, and by a blocking anti-P2X 7 mAb which has been reported to be highly selective for human P2X 7 receptors but does not recognize human P2X 1 and P2X 4 receptors [33]. When exposing monocytes to similar concentrations of ATP, the inhibitory effects of KN62, oxATP, and anti-P2X 7 mAb were also observed, confirming the participation of P2X 7 receptors.…”

The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro

“…One possible explanation for the lack of effect with A-438079 is that it is a competitive and reversible antagonist [34]. Oxidised ATP is an irreversible P2X7R inhibitor [35,36], whilst KN62 and the cyclic imide group of antagonists are non-competitive allosteric inhibitors [37,38]. Given that these cultures were performed over a three week period and we have measured a long-term response, it could be possible that A-438079 has been competed off the receptor during this time, thus reducing its efficacy.…”

Roles of extracellular nucleotides and P2 receptors in ectodomain shedding

“…Adenosine, ADP and UTP did not cause CD62L shedding, while oxidised ATP inhibited both ATP-and BzATP-induced CD62L shedding from these cells [55]. Furthermore, KN-62 inhibited ATP-induced CD62L shedding from CLL lymphocytes [34,56], while BzATP-induced CD62L shedding was impaired in CLL cells expressing non-functional P2X7 [57]. Collectively, confirming a role for P2X7 in this process.…”