Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

In Vitro Studies on the Putative Function of N-acetylaspartate as an Osmoregulator

Efflux and tissue content of N-acetylaspartate (NAA) and amino acids were evaluated from cultured and acutely prepared hippocampal slices in response to changes in osmolarity. The osmoregulator taurine, but not NAA, was lost from both types of slices after moderate reductions in extracellular osmolarity (−60 mOsm) for 10–48 h. Hypoosmotic shock (−166 mOsm) for 5 min resulted in unselective efflux of several amino acids from acutely prepared slices. Notably, the efflux of taurine, but not NAA, was prominent also after the shock. Efflux of NAA was markedly enhanced by NMDA and high K⁺, in particular after the stimulation period. The high K⁺-mediated efflux was decreased by high extracellular osmolarity and a NMDA-receptor antagonist. The results indicate that NAA efflux can be induced by a sudden non-physiological decrease in extracellular osmolarity but not by prolonged more moderate changes in osmolarity. The mechanisms behind the efflux of NAA by high K⁺ are complex and may involve both swelling and activation of NMDA-receptors.     

Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

Reduced and carboxymethylated-κ-casein (RCM-κ-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Aβ), which is associated with Alzheimer’s disease. In this study, a series of flavonoids, many known to be inhibitors of Aβ fibril formation, were screened for their ability to inhibit RCM-κ-CN fibrilisation, and the results were compared with literature data on Aβ inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-κ-CN fibril formation. IC₅₀ values were between 10- and 200-fold higher with RCM-κ-CN than literature results for Aβ fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-κ-CN assay make it an economic alternative first screen for Aβ inhibitory activity, especially for use with large compound libraries.     

Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels

Omission of extracellular Ca(2+) for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X(7) receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca(2+) omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca(2+) omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca(2+) and Mg(2+) were omitted from the medium. Omission of Ca(2+) for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca(2+) stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.