Modulation of muscarinic cholinoceptor-stimulated inositol 1,4,5-trisphosphate accumulation by [formula omitted] in neonatal rat cerebral cortex

Abstract

The mechanisms by which N- methyl- d-aspartate (NMDA) receptor activation can modulate muscarinic receptor-stimulated phosphoinositide turnover have been studied in neonatal rat cerebral cortex slices. A maximally effective concentration of carbachol (1 mM) caused a large stimulation of both total [ 3H]inositol phosphate ([ 3H]InsP x) accumulation (30–40-fold over basal levels after 15 min in the presence of 5mM LiCl) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3] mass accumulation (consisting of a rapid peak increase of about 8–10-fold within 15 sec followed by a sustained plateau rise of 4–5-fold which persisted for > 10 min). Low concentrations of NMDA enhanced carbachol-stimulated [ 3H]InsP x and Ins(1,4,5)P 3 accumulations with a maximal effect being observed at 10 μM NMDA. However, at higher concentrations of NMDA (30–300 μM) a dramatic inhibition of these indices of phosphoinositide turnover was observed. Time-course studies demonstrated that NMDA (100 μM) caused a significant enhancement of the initial increases in [ 3H]InsP x and Ins(1,4,5)P 3 accumulations stimulated by carbachol, with the profound inhibitory effects becoming evident at longer incubation times. The modulatory effects of NMDA were antagonized by D-2-amino-5-phosphonopentanoate and MK-801. Reducing extracellular calcium concentration ([Ca 2+] e) to the low micromolar range decreased basal Ins(1,4,5)P 3 accumulation and attenuated the response to carbachol. Under these conditions NMDA (10–100 μM) caused only a potentiation of agonist-stimulated Ins(1,4,5)P 3 accumulation. Under control conditions ([Ca 2+] e = 1.3 mM), addition of MK-801 (1 μM) 10 min after carbachol + 100 μM NMDA challenge failed to reverse the inhibitory effect of NMDA on carbachol-stimulated [ 3H]InsP x accumulation. Furthermore, pre-incubation of cerebral cortex slices with 100 μM NMDA for 15 min (followed by extensive washing of slices to remove NMDA) dramatically decreased [ 3H]inositol incorporation into the cellular inositol phospholipid fraction and decreased basal and carbachol-stimulated Ins(1,4,5)P 3 mass accumulations. We conclude that the enhancement of agonist-stimulated phosphoinositide turnover seen at concentrations of NMDA up to 10 μM may be due to Ca 2+ entry and Ca 2+ facilitation of phosphoinositide-specific phospholipase C activity. In contrast, the inhibitory effect of high concentrations of NMDA on agonist-stimulated phosphoinositide turnover may be due to progressive, irreversible and, at least in part, Ca 2+-dependent damage to the cell populations in the slice preparation responding to muscarinic-receptor stimulation.