Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

Abstract

N-Methyl-D-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A2 inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A2 activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 microM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.

Fig. 1

Effect of 15-min incubation of organotypic hippocampal slice cultures with 10 μM NMDA/10 μM glycine on the efflux rates of glutathione, PEA, taurine, and glutamate (n = 19). *P <.05 comparing NMDA-mediated efflux rates with the efflux rates before NMDA introduction (45 – 60 min).

Fig. 2

Glutathione efflux rate from organotypic hippocampal slice cultures in the presence (o) or absence (▴) of 5 mM BSO (n = 5). BSO was present during preincubation and throughout the experiment. The black bar indicates the incubation period with NMDA/glycine. *P <.05 comparing efflux rate with and without BSO. # P <.05 comparing efflux rate with that during the first incubation period (0–15 min).

Fig. 3

Effect of indicated drugs on NMDA-mediated efflux rates of glutathione, PEA, taurine, and glutamate. Values are expressed as percent of basal efflux rate, comparing the efflux rates before introduction of NMDA (45–60 min) with the efflux rates during NMDA/glycine incubation (gray bars, 60–75 min), and with the efflux rates after NMDA incubation (black bars, 75–90 min). All drugs were introduced 30 min before NMDA introduction and were present throughout the experiment except in experiments denoted 500 Quin*, in which quinacrine was introduced simultaneously with NMDA. Used concentrations: PMXB, 10 μM; 500 Quin, 500 μ; staurosporine, 1 μM; W7, 100 μM; H9, 100 μM; DNDS, 1 mM; AA, 30 μM; 50 Quin, 50 μM; dantrolene, 40 μM; PMA, 1 μM. *P <.05 comparing efflux rates with and without the indicated treatments (n ≥4).

Fig. 4

Glutathione, PEA, taurine, and glutamate efflux rates in the presence (▴) or absence (o) of calcium in the medium (n = 5). Calcium-free medium was introduced 30 min before NMDA/glycine introduction. The black bar indicates the incubation period with NMDA/glycine. * P <.05 comparing efflux rates with and without calcium.