Inhibition of NMDA‐gated ion channels by the P2 purinoceptor antagonists suramin and reactive blue 2 in mouse hippocampal neurones

Abstract

1 The action of suramin and reactive blue 2 on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture by use of whole-cell patch-clamp recording. 2 Suramin and reactive blue 2 inhibited steady-state current activated by 25 mM NMDA with IC 50 values of 68 and 11 mM, respectively. 3 Reactive blue 2 produced a gradual decline of NMDA-activated current to a steady-state, but this slow onset was not an indication of use-dependence, as it could be eliminated by exposure to reactive blue 2 before NMDA application. In addition, NMDA-activated current recovered completely from inhibition by reactive blue 2 in the absence of agonist. 4 The slow onset of inhibition by reactive blue 2 was not apparently due to an action at an intracellular site, as inclusion of 250 mM reactive blue 2 in the recording pipette did not alter inhibition by 25 mM reactive blue 2 applied externally. 5 Reactive blue 2 and suramin inhibited NMDA-gated channels in a voltage-independent manner. 6 Reactive blue 2, 25 mM, decreased the maximal response to NMDA from 1441 to 598 pA without changing its EC 50 . In contrast, 75 mM suramin increased the EC 50 for NMDA from 13 to 35 mM, and decreased the maximal response to NMDA from 1822 to 1498 pA. Schild analysis of suramin inhibition of NMDA-activated current yielded a nonlinear plot. 7 Both agents decreased the maximal response to glycine without altering its EC 50 . 8 Suramin and reactive blue 2 appear to inhibit NMDA receptor-channels in a manner that is noncompetitive with respect to both NMDA and glycine. However, inhibition by suramin di ered from that by reactive blue 2, in that suramin signi®cantly increased the EC 50 of NMDA.

Reactive Blue 2 Induces Calcium Oscillations in HeLa Cells.

“…RB has been used as a blocker of ATP receptors [9,10], but there are also reports that RB blocks some channels and receptors [11][12][13][14][15]. Besides these inhibitory actions of RB, we here demonstrated that RB had a stimulatory action on the PLC cascade.…”

“…RB, however, is a histochemical dye containing anthraquinone and has been reported to have other effects on cells and enzymes. RB inhibited GABA, glutamate [11], and NMDA [12] receptor channels; volume sensitive Cl Ϫ channels [13]; a thylakoid protein kinase [14]; and a certain dehydrogenase [15].…”

Role of P2X7 Receptors in Ischemic and Excitotoxic Brain Injury In Vivo

“…Previous studies in vitro have described neuroprotective actions of P2X receptor antagonists, particularly suramin and reactive basilin blue. These are both nonselective antagonists, however, and may exert additional actions at NMDA receptors (Peoples and Li, 1998). Selective antagonists of P2X 7 receptor function, GSK883 and GSK671 (provided by GlaxoSmithKline, Harlow, U.K.) (which have 50% inhibitor concentration effects of 735 nmol and 1 mol/L, respectively, in the P2X 7 YO-PRO assay [Michel et al, 1999] and confirmed by electrophysiologic methods [Chessell et al, 1998] without affecting P2X 4 ) also failed to reduce excitotoxic damage in vivo (data not shown).…”

Divergent Effects of the Purinoceptor Antagonists Suramin and Pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) (PPNDS) on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors

“…There are also reports that suramin inhibits glutamatergic synaptic transmission (Motin and Bennett, 1995;Gu et al, 1998) and whole-cell currents mediated by AMPA receptors (Nakazawa et al, 1995;Zona et al, 2000) and NMDA receptors (Ong et al, 1997;Peoples and Li, 1998). Suramin was observed to inhibit binding of radioligands to NMDA receptors (Balcar et al, 1995) but not kainate receptors (Ong et al, 1997), which suggested some selectivity among glutamate receptors.…”