, and PP2B did not prevent the inhibitory effect of NMDA. In the presence of tetrodotoxin, NMDA produced a bell-shaped dose-response curve with stimulation of phospho-ERK2 at 10, 25, and 50 M NMDA and reduced stimulation at 100 M NMDA. NMDA (50 M) stimulation of phospho-ERK2 was completely blocked by pertussis toxin and inhibitors of phosphatidylinositol 3-kinase and was partially blocked by a calcium/calmodulin-dependent kinase II inhibitor. These results suggests that NMDA receptors can bidirectionally control ERK signaling.Mitogen-activated protein kinases constitute a family of serine/threonine kinases, the best understood of which are the extracellular signal-regulated kinases (ERKs 1 ; Ref. 1). Ras proteins belong to a superfamily of small GTPases that cycle between inactive GDP-bound states and active GTP-bound states, and represent a point of convergence for the transduction and integration of many extracellular signals that activate mitogen-activated protein kinases (2). Ras-GTP initiates a sequential cascade of events involving recruitment to the membrane and activation of Raf-1, activation of the dual-specificity kinases termed mitogen-activated protein kinase/ERK kinases (MEKs), and finally activation of ERK. Activated ERKs phosphorylate cellular substrates and translocate to the nucleus, where they play an important role in regulating gene transcription (3, 4). In mitotic cells, ERKs constitute a primary effector pathway in controlling cellular proliferation, differentiation, cell cycle regulation, and survival. In brain, recent studies indicate that ERKs play an important role in synaptic plasticity and memory formation (5). Glutamate is the major excitatory neurotransmitter in the vertebrate brain, and the NMDA subtype of glutamate receptors are among the most widely distributed and abundant receptor-operated ion channels in the central nervous system. In addition to mediating the slow component of glutamate-dependent excitatory postsynaptic currents, NMDA receptors play a vital role in a variety of processes, including neuronal development, synaptic plasticity, learning and memory, and neuronal survival and death (6). NMDA receptor-mediated increases in intracellular calcium have been shown to stimulate ERK signaling, and evidence suggests that NMDA receptormediated ERK activation may play an important role in neurotransmission and synaptic plasticity (7,8). NMDA-dependent hippocampal long-term potentiation is associated with activation of ERK and is blocked by compounds that inhibit the ability of MEK to activate ERK (9, 10). ERK activation has also been shown to be required for hippocampal-dependent associative learning in rats (11), and mice lacking Ras-guanine nucleotide-releasing factor (Ras-GRF) display impaired amygdaladependent memory consolidation (12).

The serine/threonine protein kinase B (PKB)/Akt is a phosphoinositide 3-kinase (PI3K) effector that is thought to play an important roll in a wide variety of cellular events. The present study examined whether PKB activation in cortical neuronal cultures is coupled with synaptic activity. A 1-h incubation of neuronal cultures with tetrodotoxin (TTX), the PI3K inhibitor wortmannin, the NMDA receptor antagonist MK-801 or removal of extracellular calcium significantly reduced basal levels of phospho(Ser473)-PKB, indicating that activitydependent glutamate release maintains PKB activation through an NMDA receptor-PI3K pathway. A 5-min exposure to NMDA (50 lM) in the presence of TTX increased phospho-PKB back to levels observed in the absence of TTX. NMDA stimulation of phospho-PKB was blocked by wortmannin, the CaMKII inhibitor KN-93, MK-801, and removal of extracellular calcium. We have previously shown that NMDA receptors can bi-directionally regulate activation of extracellular-signal regulated kinase (ERK), and NMDA receptor stimulation of PKB in the present study appeared to mirror activation of ERK. These results suggest that in cultured cortical neurons, PKB activity is dynamically regulated by synaptic activity and is coupled to NMDA receptor activation. In addition, NMDA receptor activation of ERK and PKB may occur through overlapping signaling pathways that bifurcate at the level of Ras.

SUMMARYThe effects of prolonged ethanol exposure on excitatory amino acid receptor stimulated nitric oxide (NO) formation were examined in primary rat cortical neuronal cultures. Chronic ethanol (4 days, 100 mM) potentiated N-methyl-D-aspartate (NMDA)-stimulated NO formation as determined by measuring the conversion of [ 3 H]arginine to [ 3 H]citrulline. In contrast, chronic ethanol had no effect on NO formation stimulated by kainate, ␣-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, or the calcium ionophore ionomycin. Potassium chloridestimulated NO formation was also enhanced by chronic ethanol treatment, but this effect was not seen in the presence of the ionotropic glutamate receptor antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione. Immunoblot analysis of expression of NR1, NR2A, and NR2B receptor subunits showed no difference between control and chronic ethanoltreated cultures. In support of this apparent lack of change in receptor density, there was no difference in the specific binding of 125 I-MK-801 between control and chronic ethanol-treated groups. These results demonstrate that prolonged ethanol exposure selectively enhanced NMDA receptor-stimulated NO formation, which may play an important role in alcohol dependence, withdrawal, and alcohol-associated brain damage. These results also suggest that chronic ethanol-induced increases in NMDA receptor function may not be due to a simple increase in the number of NMDA receptors or change in NMDA receptor subunit composition but may instead reflect more complicated and subtle changes.