NMDA preconditioning protects against seizures and hippocampal neurotoxicity induced by quinolinic acid in mice

PURPOSE: N-methyl D-aspartate (NMDA) preconditioning has been used to prevent cellular death induced by glutamate or NMDA in cultured neurons. Quinolinic acid (QA)-induced seizures are used to average NMDA receptors-evoked neurotoxicity in animal models. The purpose of this study was to investigate the potential neuroprotective effects of NMDA preconditioning against QA-induced seizures and hippocampal damage in vivo. METHODS: Mice were pretreated with nonconvulsant doses of NMDA for different times before i.c.v. QA infusion and observed for the occurrence of seizures. Hippocampal slices from mice were assayed to measure cellular viability. RESULTS: NMDA preconditioning presented 53% protection against QA-induced seizures, as well as QA-induced cellular death in the hippocampus. The NMDA receptor antagonist, MK-801, prevented the protection evoked by NMDA preconditioning. The adenosine A1 receptor antagonist, CPT, prevented the protection evoked by NMDA preconditioning against QA-induced seizures, but not against QA-induced hippocampal cellular damage. The adenosine A1 receptor agonist, CPA, did not mimic the NMDA preconditioning-evoked protective effects. CONCLUSIONS: These results suggest that in vivo preconditioning with subtoxic doses of NMDA protected mice against seizures and cellular hippocampal death elicited by QA, probably through mechanisms involving NMDA receptors operating with adenosine A1 receptors.     

NMDA preconditioning protects against quinolinic acid-induced seizures via PKA, PI3K and MAPK/ERK signaling pathways

Preconditioning by N-methyl-d-aspartate (NMDA) may be promoted in vivo by the administration of a sub-convulsing dose of NMDA, with a neuroprotective effect against seizures and neuronal death induced by the infusion of quinolinic acid (QA) in mice. This study aimed to evaluate the participation of protein kinase C (PKC), cyclic AMP-dependent protein kinase (PKA), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), Ca(2+)/calmodulin dependent protein kinase II (CaMKII) and phosphatidilinositol-3 kinase (PI3K) signaling pathways in this neuroprotection model. Adult Swiss male mice were preconditioned with NMDA 24 h before the infusion of QA, and were treated with inhibitors of the aforementioned signaling pathways either 15 min before the preconditioning or infusion of QA. Inhibition of the PKA and PI3K pathways abolished the protection evoked by NMDA, and inhibition of the MEK pathway significantly diminished this protection. Treatment with PKC and CaMKII inhibitors did not alter the protection rate. Inhibition of the MEK and PKC pathways resulted in an increased mortality rate when followed by the infusion of QA, or NMDA preconditioning and QA infusion, respectively. These results suggest that the PKA, PI3K and MEK pathways have a crucial role in the achievement of a neuroprotective state following preconditioning.     

NMDA Preconditioning Attenuates Cortical and Hippocampal Seizures Induced by Intracerebroventricular Quinolinic Acid Infusion

Searching for new therapeutic strategies through modulation of glutamatergic transmission using effective neuroprotective agents is essential. Glutamatergic excitotoxicity is a common factor to neurodegenerative diseases and acute events such as cerebral ischemia, traumatic brain injury, and epilepsy. This study aimed to evaluate behavioral and electroencephalographic (EEG) responses of mice cerebral cortex and hippocampus to subconvulsant and convulsant application of NMDA and quinolinic acid (QA), respectively. Moreover, it aimed to evaluate if EEG responses may be related to the neuroprotective effects of NMDA. Mice were preconditioned with NMDA (75 mg/kg, i.p.) and EEG recordings were performed for 30 min. One day later, QA was injected (36.8 nmol/site) and EEG recordings were performed during 10 min. EEG analysis demonstrated NMDA preconditioning promotes spike-wave discharges (SWDs), but it does not display behavioral manifestation of seizures. Animals that were protected by NMDA preconditioning against QA-induced behavioral seizures, presented higher number of SWD after NMDA administration, in comparison to animals preconditioned with NMDA that did display behavioral seizures after QA infusion. No differences were observed in latency for the first seizure or duration of seizures. EEG recordings after QA infusion demonstrated there were no differences in the number of SWD, latency for the first seizure or duration of seizures in animals pretreated with saline or in animals preconditioned by NMDA that received QA. A negative correlation was identified between the number of NMDA-induced SWD and QA-induced seizures severity. These results suggest a higher activation during NMDA preconditioning diminishes mice probability to display behavioral seizures after QA infusion.     

Regulation of extracellular signal-regulated kinase phosphorylation in cultured rat striatal neurons

Recent studies demonstrate that activation of Ca²⁺-permeable N-methyl-D-aspartate (NMDA) receptors upregulates phosphorylation of mitogen-activated protein kinases (MAPKs) in heterologous cells and neurons. In cultured rat striatal neurons, the present work systematically evaluated the role of a number of protein kinases in forming a signaling cascade transducing NMDA receptor signals to MAPKs. It was found that a brief NMDA application consistently induced rapid and transient phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), a best characterized subclass of MAPKs. This ERK1/2 phosphorylation was resistant to the inhibition of protein kinase C, p38 MAPK, cyclin-dependent kinase 5, receptor tyrosine kinase (epidermal growth factor receptors), or non-receptor tyrosine kinases (including Src) by their selective inhibitors. However, the increase in ERK1/2 phosphorylation was partially blocked by a protein kinase A (PKA) inhibitor. The inhibitors for Ca²⁺/calmodulin-dependent protein kinase (CaMK) or phosphatidylinositol 3-kinase (PI3-kinase) completely blocked the NMDA-stimulated ERK1/2 phosphorylation. In an attempt to characterize the sequential role of CaMK and PI3-kinase, we found that NMDA increased PI3-kinase phosphorylation on Tyr⁵⁰⁸, which kinetically corresponded to the ERK1/2 phosphorylation and was blocked by the CaMK inhibitor. These results indicate that the protein kinases are differentially involved in linking NMDA receptors to ERK1/2 in striatal neurons.     

Atorvastatin Prevents Hippocampal Cell Death due to Quinolinic Acid-Induced Seizures in Mice by Increasing Akt Phosphorylation and Glutamate Uptake

Statins are cholesterol-lowering agents due to the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Recent studies have shown statins possess pleiotropic effects, which appear to be independent from its cholesterol-lowering action. In this study, we investigated whether atorvastatin would have protective effects against hippocampal cell death promoted by quinolinic acid (QA)-induced seizures in mice. Mice were pretreated with Atorvastatin (1 or 10 mg/kg) or vehicle (saline, 0.9%), orally, once a day for 7 days before the intracerebroventricular (i.c.v.) QA infusion (36.8 nmol/site). Atorvastatin treatment with 1 mg/kg/day did not significantly prevent QA-induced seizures (13.34%). However, administration of atorvastatin 10 mg/kg/day prevented the clonic and/or tonic seizures induced by QA in 29.41% of the mice. Additionally, administration of atorvastatin 10 mg/kg/day significantly prevented QA-induced cell death in the hippocampus. Atorvastatin treatment promoted an increased Akt phosphorylation, which was sustained after QA infusion in both convulsed and non-convulsed mice. Moreover, atorvastatin pretreatment prevented the reduction in glutamate uptake into hippocampal slices induced by QA i.c.v. infusion. These results show that atorvastatin attenuated QA-induced hippocampal cellular death involving the Akt pathway and glutamate transport modulation. Therefore, atorvastatin treatment might be a useful strategy in the prevention of brain injury caused by the exacerbation of glutamatergic toxicity in neurological diseases such as epilepsy.     

Brain-derived neurotrophic factor enhances the expression of the monocarboxylate transporter 2 through translational activation in mouse cultured cortical neurons

MCT2 is the predominant neuronal monocarboxylate transporter allowing lactate use as an alternative energy substrate. It is suggested that MCT2 is upregulated to meet enhanced energy demands after modifications in synaptic transmission. Brain-derived neurotrophic factor (BDNF), a promoter of synaptic plasticity, significantly increased MCT2 protein expression in cultured cortical neurons (as shown by immunocytochemistry and western blot) through a translational regulation at the synaptic level. Brain-derived neurotrophic factor can cause translational activation through different signaling pathways. Western blot analyses showed that p44/p42 mitogen-activated protein kinase (MAPK), Akt, and S6 were strongly phosphorylated on BDNF treatment. To determine by which signal transduction pathway(s) BDNF mediates its upregulation of MCT2 protein expression, the effect of specific inhibitors for p38 MAPK, phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), p44/p42 MAPK (ERK), and Janus kinase 2 (JAK2) was evaluated. It could be observed that the BDNF-induced increase in MCT2 protein expression was almost completely blocked by all inhibitors, except for JAK2. These data indicate that BDNF induces an increase in neuronal MCT2 protein expression by a mechanism involving a concomitant stimulation of PI3K/Akt/mTOR/S6, p38 MAPK, and p44/p42 MAPK. Moreover, our observations suggest that changes in MCT2 expression could participate in the process of synaptic plasticity induced by BDNF.     

Involvement of extracellular regulated kinase and p38 kinase in hippocampal seizure tolerance

The mechanisms underlying brain seizure tolerance, a phenomenon in which brief periods of seizures protect brain against the lethal effects of subsequent sustained seizures, are poorly understood. Because brain seizure tolerance and brain ischemia tolerance likely share certain common mechanisms, the recent evidence that activation of extracellular regulated kinase (ERK) and p38 kinase pathways plays a critical role in ischemic preconditioning suggests that a similar mechanism may underlie brain seizure tolerance. We investigated the hypothesis in a rat kainic acid preparation of seizure preconditioning and tolerance, which was established by induction of one episode of priming epileptic status lasting for 20 min on the first day and another episode of sustained epileptic status lasting for 2 hr on the second day. We observed that acute seizures lead to a rapid activation of ERK and p38 in the hippocampal CA3 area, the brain region most susceptible to the lethal effects of epileptic status. Pretreatment with the ERK inhibitor PD98059 and the p38 inhibitor SB203580 selectively reduces seizure-elicited activation of ERK and p38, respectively, and significantly reduces priming seizure-induced protection of CA3 neurons. These findings indicate that, similar to brain ischemia tolerance, brain seizure tolerance also involves the ERK and p38 signaling pathways.     

Activation of MAP kinases, Akt and PDGF receptors in injured peripheral nerves

A number of receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein (MAP) kinase signaling pathways have been critically involved in peripheral nerve regeneration. Here, we examined the activation of PI3K/Akt and MAP kinase pathways, and platelet-derived growth factor receptors (PDGFRs) in the distal segments of crushed rat sciatic nerve from 3 to 28 days after injury. In Western blot analyses, the phosphorylated forms of extracellular signal-regulated protein kinase (ERK) and c-Jun NH₂-terminal kinases (JNKs) were highly augmented on days 3 and 7 and on days 7 and 14 after injury, respectively. Phosphorylated Akt and p38 consistently increased from 3 to 28 days after injury. Phosphorylated PDGFR-α and -β were also increased from 3 to 14 days. In the immunohistological analyses, phosphorylated ERK and PDGFR-α were co-localized in many activated Schwann cells and regrowing axons 3 days after injury, while PDGFR-β was localized in a few spindle-shaped cells. The detected temporal profile of RTK signaling appears to be crucial for the regulation of Schwann cell proliferation and following redifferentiation. Furthermore, the immunohistological studies suggested a role of ERK and PDGFR-α in axon regeneration as well.     

Adaptor Protein APPL1 Couples Synaptic NMDA Receptor with Neuronal Prosurvival Phosphatidylinositol 3-Kinase/Akt Pathway

It is well known that NMDA receptors (NMDARs) can both induce neurotoxicity and promote neuronal survival under different circumstances. Recent studies show that such paradoxical responses are related to the receptor location: the former to the extrasynaptic and the latter to the synaptic. The phosphoinositide 3-kinase (PI3K)/Akt kinase cascade is a key pathway responsible for the synaptic NMDAR-dependent neuroprotection. However, it is still unknown how synaptic NMDARs are coupled with the PI3K/Akt pathway. Here, we explored the role of an adaptor protein-adaptor protein containing pH domain, PTB domain, and leucine zipper motif (APPL1)-in this signal coupling using rat cortical neurons. We found that APPL1 existed in postsynaptic densities and associated with the NMDAR complex through binding to PSD95 at its C-terminal PDZ-binding motif. NMDARs, APPL1, and the PI3K/Akt cascade formed a complex in rat cortical neurons. Synaptic NMDAR activity increased the association of this complex, induced activation of the PI3K/Akt pathway, and consequently protected neurons against starvation-induced apoptosis. Perturbing APPL1 interaction with PSD95 by a peptide comprising the APPL1 C-terminal PDZ-binding motif dissociated the PI3K/Akt pathway from NMDARs. Either the peptide or lentiviral knockdown of APPL1 blocked synaptic NMDAR-dependent recruitment and activation of PI3K/Akt pathway, and consequently blocked synaptic NMDAR-dependent neuroprotection. These results suggest that APPL1 contributes to connecting synaptic NMDARs with the intracellular PI3K/Akt cascade and the downstream prosurvival signaling pathway in rat cortical neurons.     

MK801 prevents quinolinic acid-induced behavioral deficits and neurotoxicity in the striatum

Bilateral intrastriatal injections of quinolinic acid (QA) (180 nmoles) induced weight loss and neurologic and behavioral deficits including convulsions, decreased catalepsy response to haloperidol, increased nocturnal locomotor activity, and abnormal feeding behavior in adult male Sprague-Dawley rats. Pretreatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK801 (4 mg/kg IP) 30 min prior to stereotaxic surgery prevented the appearance of all QA-induced behavioral abnormalities and prevented weight loss. Twelve weeks after surgery the QA-lesioned animals recovered to sham levels on feeding behavior and nocturnal locomotor activity, but showed persistent reductions in haloperidol-induced catalepsy. Histological examination of the QA-lesioned brains showed extensive lesions of the dorsolateral striatum and frontoparietal cortex. MK801 pretreatment protected against these lesions. These results confirm that MK801 treatment prevents the appearance of neuropathological damage after QA neurotoxicity, and further show that neuronal protection with MK801 is correlated with the absence of QA-induced behavioral deficits.     

Induction of Akt by endogenous neurosteroids and calcium sequestration in P19 derived neurons

Neuronal cell death caused by pathophysiological over-activation of glutamate receptors and the subsequent CaII overloading, has been implicated in neurodegeneration after stroke, cerebral trauma and epileptic seizures. Recent findings suggest that certain progesterone metabolites (neurosteroids) such as allopregnanolone and dehydroepiandrosterone can protect neuronal cells from such insults. In the present study, murine P19 cells were induced to differentiate into postmitotic neurons expressing specific neuronal markers, including GABA(A) and NMDA receptors. Activation of NMDA receptors in P19-N neurons resulted in excitotoxic cell death, which involved suppression of the phosphorylation of the survival kinase PKB/Akt. Allopregnanolone and DHEA induced a rapid and prolonged phosphorylation of the Akt kinase and they were able to reverse the NMDA-induced suppression of the PI3-K/Akt pathway. The specificity of the neuroprotective effects of these neurosteroids was confirmed by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin, as well as by the GABA(A) receptor antagonist, bicuculline. The neurotoxic effect of NMDA on P19-N neurons was directly correlated with increased CaII entry, since the addition of EGTA or BAPTA-AM, significantly suppressed the NMDA-induced decrease of phospho-Akt and subsequent neuronal death. These results suggest that neurosteroids are able to act as survival factors on P19-N neurons, promoting the activation of the PI3-K/Akt pathway through a calcium-entry dependent mechanism.     

Olanzapine produces trophic effects in vitro and stimulates phosphorylation of Akt/PKB, ERK1/2, and the mitogen-activated protein kinase p38

Olanzapine has previously been shown to stimulate the growth of neuronal cells in culture. A major goal of the present studies was to determine if olanzapine also provided neuroprotection to pheochromocytoma (PC12) cells, SH-SY5Y neuroblastoma cells, and primary cultures of rat cortical neurons. Olanzapine was mitogenic and enhanced the survival of PC12 cells, SH-SY5Y cells and 3T3 preadipocytes, but not L6 myoblasts or myeloma cells. It protected neuronal cells from death induced by serum and glutamine deprivation, amyloid beta peptide (25-35), and fluphenazine. Molecular mechanisms of the neuroprotection by olanzapine were explored, specifically the activation of various protein kinase signaling pathways including Akt/protein kinase B (PKB), extracellular-regulated kinase (ERK), ERK1/2, and mitogen-activated protein kinase (MAPK), p38. Olanzapine treatment led to rapid phosphorylation of kinases from all three pathways in PC12 cells. Phosphorylation of Akt was blocked with selective inhibitors (wortmannin and LY294002), which implicates phosphoinositide 3-kinase (PI3K) in the signaling cascade. Short-term mitogenic effects of olanzapine were abolished with a selective inhibitor of Akt, but not by inhibition of the ERK pathway. Other antipsychotic drugs stimulated phosphorylation of a subset of the kinase panel, but not all three kinases. The present findings demonstrate that olanzapine has both mitogenic and neuroprotective effects in neuronal cells.     

Neuroprotection by estrogen via extracellular signal-regulated kinase against quinolinic acid-induced cell death in the rat hippocampus

Extracellular signal-regulated kinase (ERK) belongs to the family of mitogen-activated protein kinases (MAPKs), which are serine-threonine kinases activated by phosphorylation in response to a variety of mitogenic signals. We previously reported that 17 beta-estradiol rapidly activates ERK in the rat hippocampus. However, the physiological role of this rapid activation of ERK by estrogen in vivo has not yet been elucidated. This study investigated whether ERK may participate in mediating the neuroprotective effects of estrogen against quinolinic acid (QA) toxicity in the rat hippocampus in vivo. Injection of QA into the hippocampi of male rats produced a loss of Nissl-stained neurons in the CA1 after 24 h. Prior administration of 17 beta-estradiol (50 pmol/animal) to the ventricles prevented the QA-induced decrease in Nissl-stained neurons. Pretreatment with U0126, an inhibitor of MAPK/ERK kinase, inhibited the rapid activation of ERK by 17 beta-estradiol in the rat hippocampus. Moreover, the neuroprotective effects of 17beta-estradiol against QA toxicity were blocked by the pretreatment with U0126. U0126 alone did not produce a loss of neurons. These results indicate that ERK mediates estrogen neuroprotection after QA toxicity in the rat hippocampus.     

Erythropoietin exerts cell protective effect by activating PI3K/Akt and MAPK pathways in C6 Cells

Abstract

Even though erythropoietin (EPO) is a neurotropic cytokine that is recognized widely for its role in the development, maintenance, protection, and repair of the nervous system, there are few reports concerning EPO-mediated influences on the glial cells in the central nervous system. In this study, we investigated anti-inflammatory and anti-apoptotic effects of EPO on C6 glioma cells (C6 cells). Erythropoietin did not attenuate inflammatory response, such as nitrite production, iNOS gene expression, and pro-inflammatory cytokines when LPS/TNF-alpha mixture was treated. However, EPO increased C6 cell viability by exerting cell protective effect against staurosporine stimulation. Erythropoietin increased the transient Akt expression at 30 minutes and induced the gradual elevation of ERK1/2 and p38 expression as time progressed. The cell protective effect of EPO was also significantly attenuated with pretreatment of specific PI3K, pERK1/2, or pP38 inhibitor. In summary, these results suggest that EPO may exert its cell protective functions via the direct cell protective activity rather than via its anti-inflammatory effect. Moreover, the PI3K/Akt and mitogen activated protein kinase (MAPK) pathways may be responsible for cell survival against cytotoxicity.     

MEK/ERKs signaling is essential for lithium-induced neurite outgrowth in N2a cells

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.     

Acute intrastriatal administration of quinolinic acid provokes hyperphosphorylation of cytoskeletal intermediate filament proteins in astrocytes and neurons of rats

In the present study we investigated the effect of in vivo intrastriatal injection of quinolinic acid (QA) on cytoskeletal proteins in astrocytes and neurons of young rats at early stage (30 min) after infusion. QA (150nmoles/0.5 µL) significantly increased the in vitro phosphorylation of the low molecular weight neurofilament subunit (NFL) and the glial fibrillary acidic protein (GFAP) of neurons and astrocytes, respectively. This effect was mediated by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II (PKCaMII). In contrast, mitogen activated protein kinases were not activated by QA infusion. Furthermore, the specific N-methyl-D-aspartate (NMDA) antagonist MK-801 (0.25 mg/kg i.p), the antioxidant L-NAME (60 mg\kg\day), and diphenyldisselenide (PheSe)₂ (0.625 mg\kg\day) injected prior to QA infusion totally prevented QA-induced cytoskeletal hyperphosphorylation. We also observed that QA-induced hyperphosphorylation was targeted at the Ser55 phosphorylating site on NFL head domain, described as a regulatory site for NF assembly in vivo. This effect was fully prevented by MK801, by the PKA inhibitor H89 and by (PheSe)₂, whereas staurosporine (PKC inhibitor) only partially prevented Ser55 phosphorylation. The PKCaMII inhibitor (KN93) and the antioxidant L-NAME failed to prevent the hyperphosphorylation of Ser55 by QA infusion. Therefore, we presume that QA-elicited hyperphosphorylation of the neural cytoskeleton, and specially of NFLSer55, achieved by intrastriatal QA injection could represent an early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum. Our observations also indicate that NMDA-mediated Ca²⁺ events and oxidative stress may be related to the altered protein cytoskeleton hyperphosphorylation observed with important implications for brain function.     

Cyclic nucleotides inhibit MAP kinase activity in low-dose collagen-stimulated platelets

Collagen-induced platelet activation is a complex process involving multiple signaling pathways. The role(s) of MAP kinases (ERKs and p38^MAPK) are unclear, although at high, but not low, collagen concentrations p38^MAPK is involved in cPLA₂-mediated arachidonic acid release, prior to thromboxane generation. Cyclic nucleotides are conventionally regarded as mediators of platelet inhibition. However recent studies suggested a role for cGMP early in a MAP kinase pathway in platelet activation. In the current study the roles and relationships of MAP kinases, cyclic nucleotides and cPLA₂ in platelet activation by low-dose collagen and a thromboxane analogue (U46619) have been evaluated. Stimulants of neither adenylate cyclase (PGI₂) nor guanylate cyclase (NaNP) alone had any effect on the basal phosphorylation of either MAP kinase. PGI₂ inhibited ERK/p38^MAPK phosphorylation in response to both agonists which was unaffected by a cPLA₂ inhibitor (AACOCF₃). NaNP inhibited collagen-induced ERK/p38^MAPK phosphorylation, which was enhanced by AACOCF₃ and reversed by a guanylate cyclase inhibitor (ODQ). However NaNP had no effect on U46619-induced p38^MAPK phosphorylation. Thus adenylate cyclase activation inhibits low-dose collagen-induced MAP kinase phosphorylation both prior, and distal, to thromboxane release. The study also supports an inhibitory, rather than stimulatory, role for guanylate cyclase in platelet signaling.     

Tobacco cembranoids protect the function of acute hippocampal slices against NMDA by a mechanism mediated by alpha4beta2 nicotinic receptors

Nicotine has been reported to be neuroprotective in experimental and epidemiological studies. In addition to nicotine, tobacco and cigarette smoke contain cembranoids, which are antagonists of neuronal nicotinic receptors (nAChR). Exposure of hippocampal slices to N-methyl-D-aspartate (NMDA) decreases the population spikes (PS). This parameter has been used as a measure of excitotoxicity. Surprisingly, both nicotine and tobacco cembranoids protected against NMDA and this neuroprotection was not blocked by methyllycaconitine (MLA), an antagonist of alpha7 nAChR. On the contrary, MLA had a neuroprotective effect of its own. We examined the effect of the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) on the neuroprotection against NMDA. DHbetaE, a selective antagonist of alpha4beta2 nAChR, inhibited the neuroprotection by nicotine, 4R, and MLA, suggesting the involvement of alpha4beta2 nAChRs in the neuroprotection. The cell-signaling pathways underlying the neuroprotection by 4R and by nicotine are different. The activity of phosphatidylinositol-3 kinase (PI3K) was required in both cases; however, 4R required the activity of L-type calcium channels and CAM kinase, whereas nicotine required the extracellular signal regulated kinase-1,2 (ERK) and protein kinase C (PKC). In addition, 4R did not enhance total phospho-ERK-1/2 but increased the amount of total Akt/PKB phosphorylated on the activation site and of glycogen synthase kinase 3-beta phosphorylated on the inhibitory site. Total levels of phosphoenzymes are presented instead of the ratio of phospho- over total enzyme because in preliminary experiments total ERK-1/2 levels were slightly increased by 4R. In conclusion, these findings demonstrate that there are two different nicotinic neuroprotective mechanisms mediated by alpha4beta2.     

Glutamate receptors and phosphoinositide metabolism: stimulation via quisqualate receptors is inhibited by N-methyl-D-aspartate receptor activation

Excitatory amino acid receptors in the neonatal rat hippocampus have opposing actions on phosphoinositide (PI) metabolism. Quisqualic acid (QA), but not the QA receptor agonist AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), potently stimulates inositol phosphate (IP) formation. Activation of NMDA (N-methyl-D-aspartate) receptors inhibits the QA-induced stimulation by 70% by a mechanism which is dependent on extracellular calcium.