Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

In cultured cerebrocortical neurons, mild excitotoxic insults or staurosporine result in apoptosis. We show here that N-methyl-D-aspartate (NMDA) receptor-mediated, but not staurosporinemediated, apoptosis is preceded by depolarization of the mitochondrial membrane potential (⌬m) and ATP loss. Both insults, however, release cytochrome c (Cyt c) into the cytoplasm. What prompts mitochondria to release Cyt c and the mechanism of release are as yet unknown. We examined the effect of inhibition of the adenine nucleotide translocator (ANT), a putative component of the mitochondrial permeability transition pore. Inhibition of the mitochondrial ANT with bongkrekic acid (BA) prevented NMDA receptor-mediated apoptosis of cerebrocortical neurons. Concomitantly, BA prevented ⌬ m depolarization, promoted recovery of cellular ATP content, and blocked caspase-3 activation. However, in the presence of BA, Cyt c was still released. Because BA prevented NMDA-induced caspase-3 activation and apoptosis, the presence of Cyt c in the neuronal cytoplasm is not sufficient for the induction of caspase activity or apoptosis. In contrast to these findings, BA was ineffective in preventing staurosporine-induced activation of caspases or apoptosis. Additionally, staurosporineinduced, but not NMDA-induced, apoptosis was associated with activation of caspase-8. These results indicate that, in cerebrocortical cultures, excessive NMDA receptor activation precipitates neuronal apoptosis by means of mitochondrial dysfunction, whereas staurosporine utilizes a distinct pathway. Apoptosis is an important mechanism in both the development and degeneration of the nervous system. Evidence suggests that the loss of neurons in many neurologic disorders occurs by apoptosis (1-4). The overstimulation of glutamate receptors can precipitate the death of neurons by either necrosis or apoptosis depending on the severity of the insult (5, 6). Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, in particular, results in an increase in the intracellular free calcium concentration ([Ca 2ϩ ] i ) to levels above the buffering capacity of neurons (7). This increase in [Ca 2ϩ ] i leads to the activation of toxic events, including oxidative and nitrosative stress (8). Mitochondria, which undergo harmful Ca 2ϩ -loading after NMDA receptor activation (9, 10), also have an important signaling function in apoptosis (11-13).Apoptotic cell death is often mediated by a caspase cascade. Although many stimuli exist, the final phases of apoptosis are executed by a few common effector caspases. Mitochondria appear to provide a link between the initiator caspases and the downstream effector caspases. In nonneuronal cells, mitochondria have been shown to accelerate activation of caspases by releasing proapoptotic molecules, such as cytochrome c (Cyt c) (11, 12) and the apoptosis-inducing factor (13). The release of these molecules can be stimulated by some caspases and by Bid and Bax (14-16), whereas Bcl2 prevents their release (11-13).The mechan...

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p38 is a member of the mitogen-activated protein (MAP) kinase superfamily and mediates intracellular signal transduction. Recent studies suggest that p38 is involved in apoptotic signaling in several cell types, including neurons. In the mammalian retina, approximately 50% of the retinal ganglion cells (RGCs) die by apoptosis during development. Additionally, transection of the optic nerve close to the eye bulb causes apoptotic cell death of RGCs in adulthood. We investigated the role of p38 in axotomy-induced apoptosis of RGCs. One day after axotomy, activated (phosphorylated) p38 was visualized by immunocytochemistry in the nuclei of RGCs, but not in control retinas. Phosphorylated p38 was first detected on immunoblots 12 hr after axotomy, reached a maximum at 1 d, and then decreased. To investigate possible roles of p38 in RGC death, a p38 MAP kinase inhibitor, SB203580, was administered intravitreally at the time of axotomy and repeated at 5 and 10 d. Assayed 14 d after axotomy, SB203580 increased the number of surviving RGCs in a dose-dependent manner (the minimum effective concentration was 1.6 micrometer). Furthermore, MK801, a selective inhibitor of NMDA receptors, not only showed protective effects against RGC apoptosis but also attenuated p38 MAP kinase activation in a dose-dependent manner. Our findings imply that p38 is in the signaling pathway to RGC apoptosis mediated by glutamate neurotoxicity through NMDA receptors after damage to the optic nerve. p38 inhibitors could be potentially useful for the treatment of optic nerve trauma and neurodegenerative diseases that affect RGCs, such as glaucoma.

Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N‐methyl‐d‐aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin‐1β‐converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA‐induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V‐ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z‐VAD·fmk) that inhibits caspase activity. Pretreatment with either V‐ICEinh or Z‐VAD·fmk protects cerebrocortical neurons from NMDA‐induced apoptosis, suggesting a role for caspases in NMDA‐induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA‐induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V‐ICEinh or Z‐VAD·fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V‐ICEinh and Z‐VAD·fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18 -24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.

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Trans-synaptic regulation of muscarinic, peptidergic, and purinergic responses after denervation has been reported previously in rat parotid acinar cells (McMillian, M. K., Soltoff, S. P., Cantley, L. C., Rudel, R., and Talamo, B. R. (1993) Br. J. Pharmacol. 108, 453-461). Characteristics of the ATP-mediated responses and the effects of parasympathetic denervation were further analyzed through assay of Ca 2؉ influx, using fluorescence ratio imaging methods, and by analysis of P 2x receptor expression. ATP activates both a high affinity and a low affinity response with properties corresponding to the recently described P 2x4 and the P 2z (P 2x7 )-type purinoceptors, respectively. Reverse transcription-polymerase chain reaction analysis reveals mRNA for P 2x4 as well as P 2x7 subtypes but not P 2x1 , P 2x2 , P 2x3 , P 2x5 , or P 2x6 . P 2x4 protein also is detected by Western blotting. Distribution of the two types of ATP receptor responses on individual cells was stochastic, with both high and low affinity responses on some cells, and only a single type of response on others. Sensitivity to P 2x4 -type activation also varied even among cells responsive to low concentrations of ATP. Parasympathetic denervation greatly enhanced responses, tripling the proportion of acinar cells with a P 2x4 -type response and increasing the fraction of highly sensitive cells by 7-fold. Moreover, P 2x4 mRNA is significantly increased following parasympathetic denervation. These data indicate that sensitivity to ATP is modulated by neurotransmission at parasympathetic synapses, at least in part through increased expression of P 2x4 mRNA, and suggest that similar regulation may occur at other sites in the nervous system where P 2x4 receptors are widely expressed.Extracellular ATP acts as a signaling molecule through the interaction of ATP with ligand-gated ion channels (P 2x ) as well as metabotropic receptors (P 2y ) (for reviews see Refs. 2-4). These receptors are distributed throughout the body, including synapses where ATP seems to function as a neurotransmitter (5, 6). The pharmacology and physiology of ATP-activated ion fluxes indicate the existence of multiple subtypes of P 2x receptors, confirmed by molecular cloning of seven genes for P 2x receptor subunits (3,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).Relatively little is known about the physiological regulation of P 2x receptors. We have shown that removal of the autonomic innervation alters ATP responses in parotid acinar cells, which therefore provide a good model for investigating trans-synaptic regulation of receptor-mediated signaling. We previously described two ATP responses in rat parotid acinar cells, a P 2z/x7 type and a high affinity ATP receptor with distinctly different P 2x -like pharmacology (1).Expression of the P 2x -type proteins in heterologous systems shows that each can form functional, homomeric, ligand-gated ion channels with a significant permeability to Ca 2ϩ ions as well as other inorganic cations, including Na ϩ and K ϩ . Properties of P 2x7 recept...