Species and response dependent differences in the effects of MAPK inhibitors on P2X₇ receptor function

Abstract

Background: Recent studies have implicated the mitogen activated protein kinase (MAPK) in cellular permeability changes following P2X 7 receptor activation in native tissues. In this study we have further studied the effect of MAPK inhibitors on recombinant and native P2X 7 receptors. Experimental Approach: The MAPK inhibitors SB-203580, SB-202190 and SB-242235 were examined in HEK293 cells expressing recombinant P2X 7 receptors and in THP-1 cells expressing native human P2X 7 receptors using a range of experimental approaches. Key results: At human recombinant P2X 7 receptors, SB-203580 and SB-202190 were weak, non-competitive inhibitors (pIC 50 ¼ 4.8 -6.4) of ethidium accumulation stimulated by 2'-& 3'-O-(4benzoylbenzoyl)-ATP (BzATP) but SB-242235 (0.1-10mM) had no effect. SB-203580 and SB-202190 had no effect on rat or mouse recombinant P2X 7 receptors and studies with chimeric P2X 7 receptors suggested that SB-203580 was only effective in chimeras containing the N-terminal 255aa of the human P2X 7 receptor. SB-203580 did not consistently affect BzATP-mediated increases in cell calcium levels and, in electrophysiological studies, it slightly decreased responses to 30mM BzATP but potentiated responses to 100mM BzATP. In THP1 cells, SB-203580 modestly inhibited BzATP-stimulated ethidium accumulation (pIC 50 5.7 -o5) but SB-202190 had no effect. Finally, SB-203580 did not block BzATP-stimulated interleukin-1b release in THP-1 cells. Conclusions: This study confirms that high concentrations of SB-203580 and SB-202190 can block human P2X 7 receptormediated increases in cellular ethidium accumulation but suggest this is not related to MAPK inhibition. Overall, the data cast doubt on a general role of MAPK in mediating P2X 7 receptor mediated changes in cellular permeability.

Structure-based identification and characterisation of structurally novel human P2X7 receptor antagonists

“…1D–G). Such striking species specificity has been repeatedly reported for known hP2X7R antagonists such as KN62 [19], AZ11645373 [40] and SB203580 [31]. Examination of ligand-receptor interactions has led us to hypothesise that the species specificity of C23, C40 and C60 arises from structural differences in the ATP-binding pocket such as Tyr288.…”

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

“…Of relevance to the current study, the p38 MAPK inhibitors SB202190 or SB203580 can impair P2X7-induced pore formation in human THP-1 myeloid cells, murine 2BH4 thymic epithelial cells and primary murine macrophages [33,34]. Although others have demonstrated that these compounds impair ATPinduced pore formation mediated by human, but not rat or murine, recombinant P2X7 [61]. This latter finding is consistent with the absence of an inhibitory effect of SB202190 or SB203580 on P2X7-induced pore formation in MEL cells.…”

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

“…Whether this second permeability state is attributed to intrinsic channel dilation [3], the pannexin-1 channel [4] or an alternate but unknown uptake pathway [5][6][7] remains controversial. Moreover, our understanding of this permeability state is further complicated with some [8][9][10] but not other [11][12][13] studies showing that P2X7-induced dye uptake involves the p38 mitogen-activated protein kinase. Regardless of the true identity of the P2X7 pore and the mechanism by which it opens, P2X7 activation stimulates several intracellular signalling pathways to induce various cellular events including inflammatory mediator release, reactive oxygen and nitrogen species formation, and cell proliferation or death [14,15].…”