Involvement of the secretory pathway for AMPA receptors in NMDA-induced potentiation in hippocampus.

A chemical form of synaptic potentiation was produced with a brief bath application of NMDA to rat hippocampal slices. Two methods were used to assess changes in membrane-bound AMPA receptors. Traditional subcellular fractionation was used to isolate synaptic membranes; alternatively, membrane receptors were cross-linked with the membrane-impermeable reagent bis(sulfosuccinimidyl) suberate, and levels of nonmembrane receptors were determined. In both cases, Western blots were used to determine the content of receptor subunits in various subcellular fractions. NMDA-induced potentiation was associated with increased levels of glutamate receptor 1 (GluR1) and GluR2/3 subunits of AMPA receptors in synaptic membrane preparations, whereas no change was observed in whole homogenates. Both KN-62, an inhibitor of calcium/calmodulin kinase, and calpain inhibitor III, a calpain inhibitor, inhibited NMDA-induced potentiation and changes in GluR1 and GluR2/3 subunits of AMPA receptors. Brefeldin A (BFA) inhibits protein trafficking between the Golgi apparatus and cell membranes. Pretreatment of hippocampal slices with BFA significantly decreased NMDA-induced potentiation and completely prevented an NMDA-induced increase in GluR1 levels in membrane fractions. Thus, the levels of GluR1 and GluR2/3 subunits of AMPA receptors are rapidly upregulated in synaptic membranes under conditions associated with potentiation of synaptic responses, and this upregulation requires a functional secretory pathway.

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus.

Estrogen replacement therapy in women is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. The present study indicates that estrogen is neuroprotective against N-methyl-d-aspartate (NMDA)- and kainate-mediated neurotoxicity, an effect mediated by tyrosine kinase/mitogen-activated protein kinase (MAPK) pathways. Estrogen also stimulates tyrosine phosphorylation of NMDA receptors via an src tyrosine kinase/MAPK pathway. Finally, estrogen-mediated enhancement of long-term potentiation in hippocampal slices is mediated by activation of an src tyrosine kinase pathway. Thus, estrogen, by activating an src tyrosine kinase and the extracellular signal-related protein kinase/MAPK signaling pathway, both enhances NMDA receptor function and long-term potentiation and retains neuroprotective properties against excitotoxicity. These findings warrant further evaluation of the usefulness of estrogenic compounds for the treatment of Alzheimer's disease and other neurodegenerative diseases.

Antisense knockdown of glutamate transporters alters the subfield selectivity of kainate-induced cell death in rat hippocampal slice cultures.

Organotypic rat hippocampal slice cultures were used to study the role of excitatory amino acid transporters (EAATs) in kainate-induced cell death. Expression of the neuronal (EAAT3) or glial (EAAT2) transporters was inhibited with antisense phosphothioate oligonucleotides, and cytotoxicity was assessed with propidium iodide uptake. In control cultures, a concentration of 10 microM kainate was more cytotoxic in CA3 than in CA1. Treatment for 24 h with EAAT3 antisense oligonucleotide decreased kainate toxicity in CA1 but had an opposite effect in CA3. Neither antisense oligonucleotide to EAAT2 nor mismatch oligonucleotide to EAAT3 decreased kainate toxicity in CA1. Immunoblotting with affinity-purified antibodies showed that EAAT3 antisense oligonucleotide decreased selectively EAAT3 but not EAAT2 protein levels, and vice versa. NMDA was more cytotoxic in CA1 than in CA3, and antisense oligonucleotides to either EAAT3 or EAAT2 did not decrease the NMDA effect in CA1 or CA3. Dihydrokainate and DL-threo-beta-hydroxyaspartic acid were more cytotoxic in CA1 than in CA3, suggesting that the higher vulnerability of CA3 to kainate was not the result of its activity as transporter blocker. We conclude that glutamate transporters differentially regulate excitotoxicity in different hippocampal subfields.

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity.

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.

Regulation of electrolyte transport with IL-1beta in rabbit distal colon.

Interletrkin-1beta levels are elevated in inflammatory bowel disease. In this study the mechanism by which interleukin-1beta affects electrolyte transport in the rabbit distal colon, was investigated. Interleukin-1beta caused a delayed increase in short-circuit current (I(sc)) which was attributed to protein synthesis since the effect was inhibited by cycloheximide. The interleukin-1beta induced increase in I(sc) was not affected by amiloride treatment but was completely inhibited by bumetanide or in chloride-free buffer and by indomethacin. Prostaglandin E(2) levels increased in tissue treated with interleukin-1beta, but this increase was reversed by cycloheximide. These data suggest that interleukin-1beta causes its effect via a yet to be identified second messenger, by increasing chloride secretion through a prostaglandin E(2) mediated mechanism.

Regulation of ileal electrolyte transport by K+ channel activators.

Pinacidil (N"-cyano-N-4-pyridyl-N'-1,2,2-trimethylpropylguanidine monohydrate) and BRL 38227, a benzopyran derivative, two K+ channel activators, were found to decrease short-circuit current (ISC), a measure for ion movement across the intestinal tissue. This decrease in ISC was correlated with an increase in NaCl absorption. These results suggest the possibility of new forms of drug therapy for diarrheal diseases. The effects of pinacidil were compared to galanin which also increased NaCl absorption. Galanin increased potassium currents in whole-cell patch clamp studies. The effects of galanin and pinacidil on Isc were not additive suggesting a common pathway in their mechanism of action.