Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

Identification of glutathione conjugates of 2,3-dibromopropene in male ICR mice

Hepatotoxic potential of 2, 3-dibromopropene (2, 3-DBPE) and its conjugation with glutathione (GSH) were investigated in male ICR mice. Treatment of mice with 20, 50, and 100 mg/kg of 2, 3-DBPE for 24 h caused elevation of serum alanine aminotransferase and aspartate aminotransferase activities. The hepatic content of GSH was not changed by 2, 3-DBPE. Meanwhile, the GSH content was slightly reduced when mice were treated with 2, 3-DBPE for 6 h and significantly increased 12 h after the treatment. Subsequently, a possible formation of GSH conjugate of 2, 3-DBPE was investigated in vivo. After the animals were treated orally with 20, 50, and 100 mg/kg of 2, 3-DBPE, the animals were subjected to necropsy 6, 12, and 24 h later. A conjugate of S-2-bromopropenyl GSH was identified in liver and serum treated with 100 mg/kg of 2, 3-DBPE by using liquid chromatography-electrospray ionization tandem mass spectrometry. The protonated molecular ions [M+H]+ of S-2-bromopropenyl GSH were observed at m/z 425.9 and 428.1 in the positive ESI spectrum with a retention time of 6.35 and 6.39 min, respectively. In a time-course study in livers following an oral treatment of mice with 100 mg/kg of 2, 3-DBPE for 6, 12, and 24 h, the 2, 3-DBPE GSH conjugate was detected maximally 6 h after the treatment. The present results suggested that 2, 3-DBPE-induced hepatotoxicity might be related with the production of its GSH conjugate.     

Analysis of biomarkers in rats and dogs exposed to polymeric methylenediphenyl diisocyanate (pMDI) and its glutathione adduct

Hemoglobin adducts (Hb-MDX) of monomeric methylenediphenyl diisocyanate (MDI) are often interpreted as indirect evidence of hydrolysis of the diisocyanate moiety to the respective amine (diphenylmethane-4,4'-diamine, 4,4'-MDA) which constitutes the rationale of using this biomarker as an internal dosimeter of exposure to putatively formed MDA. In contrast, more recently published data suggest that following inhalation the high concentration of glutathione (GSH) present in lungs favor an adduct formation with GSH and/or peptides/proteins rather than hydrolysis. The focus of this study was to test this alternate hypothesis, viz. whether Hb-MDX can also be formed by the GSH bis-adduct of monomeric MDI. The synthesized mMDI-GSH bis-adduct was administered to rats by single intratracheal instillation. Additional groups were dosed by gavage and intraperitoneal injection. Biomarkers of exposure were determined in blood (plasma protein and hemoglobin adducts) and urine after harsh alkaline and acid hydrolysis, respectively. Data from previous single inhalation exposure studies with aerosols of MDI and 4,4'-MDA in rats served as reference. As to whether N-acetylation plays any modifying role to yield these mMDI-specific biomarkers was addressed in similarly head-only exposed dogs, a species with no appreciable N-acetylation capacity whereas rats are strong N-acetylators. The results obtained suggest that biomarkers in blood from controlled exposures above current workplace standards of mMDI appear not to be suitable for reliable assessments of past exposures. The biomarkers typically used to assess past exposures to MDI were also identified following exposure to the MDI-GSH bis-adduct. Their yield was low but quite similar for MDI aerosol and the MDI-GSH bis-adduct, whilst that of MDA was distinctively higher. The findings of this study are supportive of a conceptual pathway that the MDI-derived biomarkers of exposure are formed through MDI-GSH adducts rather than MDA. Data from dogs support the findings from rats and show that N-acetylation does not appear to be an essential modifying factor. It is concluded that the yield of MDI-related markers of exposure is relatively low and dependent on the exposure dose (and route). MDA originating from hydrolyzed serum protein or hemoglobin appear to be confounded by false-positive background levels which are surmised to be associated with the method of hydrolysis. The determination of urinary biomarkers might be a useful tool to identify recent exposures (by any route). Due methodological uncertainties associated with the harsh hydrolysis of biological specimens may be reduced substantially when using incremental pre- to post-shift changes rather than relying solely on absolute data.     

N-acetylcysteine augmentation in serotonin reuptake inhibitor refractory obsessive-compulsive disorder

Rationale Dysfunction of glutamatergic neurotransmission has been implicated in the pathophysiology of obsessive-compulsive disorder (OCD) and recent clinical reports suggest that some glutamate modulating agents are efficacious in the treatment of this disorder. N-acetylcysteine (NAC) is a readily available amino acid compound that is thought to attenuate glutamatergic neurotransmission. NAC may be useful in treating psychiatric disorders involving glutamatergic dysfunction such as OCD. Objectives To examine the efficacy of augmentation with NAC in a patient with serotonin reuptake inhibitor (SRI)-refractory OCD. Methods A patient with SRI-refractory OCD was treated with an off-label use of NAC augmentation of fluvoxamine over several weeks. Results NAC augmentation of fluvoxamine resulted in a marked decrease in Yale-Brown Obsessive Compulsive Scale (Y-BBOCS) score and a clinically significant improvement in OCD symptoms. Conclusions NAC augmentation was effective in treating SRI-refractory OCD in this single case. Further research is warranted to investigate the use of NAC and other glutamate modulating agents in the treatment of OCD.     

An electron microscope study of the cortico-pretecto-olivary projection in the cat by a combined degeneration and horseradish peroxidase tracing technique

The anterior pretectal nucleus (PTA) of the cat was observed electron microscopically after motor cortical ablation and horseradish peroxidase (HRP) injection into the inferior olive of the same animal. Direct synaptic connections were found between degenerated cortico-pretectal axon terminals and dendrites of pretecto-olivary projection neurons retrogradely labeled with HRP in the ventrolateral part of the PTA. Therefore, this combined method revealed that the PTA is a relay station of the cortico-olivary projection.     

Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea

Olivocochlear fibers have been demonstrated to have acetylcholinesterase-positive staining both in brainstem and cochlea. Olivocochlear fibres in the cochlea have also been determined to contain enkephalin-like immunoreactivity. In this study, we first determined the source of olivocochlear fibers in the guinea-pig using horseradish peroxidase and wheat germ agglutinin in retrograde transport studies. These cells were then examined for enkephalin-like immunoreactivity followed by acetylcholinesterase staining on the same sections to determine which cells and fibers showed staining for both. It was found that cells in the guinea-pig lateral superior olive that project to the cochlea have both enkephalin-like immunoreactivity staining and acetylcholinesterase-positive staining. Cells in other areas giving rise to olivocochlear fibers showed only acetylcholinesterase staining. These results suggest that there is co-localization of enkephalin and acetylcholine in a population of olivocochlear cells and fibers.     

Mixed megakaryocytic-granulocytic differentiation during diffusion chamber culture of peripheral blast cells from the blast crisis of chronic myelocytic leukemia

Morphologically and cytochemically undifferentiated peripheral blast cells from three patients in blast crisis of Ph'-positive chronic myelocytic leukemia were analysed morphologically, immunologically and cytogenetically prior to and during in-vivo diffusion chamber culture (DC) to investigate their differentiation capacity. In two patients immunological markers characteristic of the megakaryocytic lineage were found on the original cells, but lineage-specific differentiation markers were absent on the blast cells of the third patient. During DC culture multilineage differentiation capacity could be demonstrated immunologically and morphologically in all three patients with expression of megakaryocytic and granulocytic markers as well as terminal differentiation along these lineages. Cytogenetic analysis prior to and during DC culture provided evidence that the cells differentiating in culture were derived from the blast crisis clones and that the event leading to blast crisis might have occurred in a pluripotent precursor cell which retained its differentiation capacity along several lineages.     

Location of single neurons containing HRP for electron microscopy

A technique is described for finding single neurons containing transported horseradish peroxidase (HRP) for study in the electron microscope. Tissue chopper sections which have been reacted to demonstrate HRP are cleared in glycerol and examined with darkfield light microscopy. The granular reaction product is clearly seen, enabling accurate drawings to be made for later dissection for ultramicrotomy. The neuron sought can then be found within a few microns of the cutting face of the block. While some distortion does occur, most of the cellular morphology remains intact, enabling recognition of specific organelles such as neurosecretory granules in cells which have transported HRP from the neurohypophysis.     

The mechanism of glutamate-induced degeneration of cultured Huntington's disease and control fibroblasts

Human cultured skin fibroblasts undergo rapid cellular degeneration and cell death when exposed to moderate levels of glutamate (10-30 mM). Huntington's disease (HD) skin fibroblast cultures are more sensitive to the toxic effects of glutamate (Gray et al. 1980) and elucidation of the toxic mechanism may bear on degenerative processes occurring in the HD brain. Glutamate toxicity was found to be inversely related to cystine content in the culture medium. Supplementing culture media with cystine effectively suppressed toxicity of L-Glu and that of a closely related but more potent analogue, L-homocysteic acid (L-HCA). Glutamate and L-HCA treatment provoked a rapid and marked decrease in cellular glutathione levels prior to cell death. Depletion of cellular glutathione with buthionine sulfoximine potentiated the toxicity of glutamate and L-HCA. Glutamate- or L-HCA-induced cell death also could be reduced or inhibited by co-treatment with antioxidants, thereby suggesting that free radical-induced peroxidative damage may ultimately be responsible for the loss of cell viability. While no site for the differential sensitivity of HD and normal fibroblasts to glutamate was clearly discerned, our results suggest that HD fibroblasts are more susceptible to peroxidative damage. The increased deposition of lipofuscin (Tellez-Nagel et al. 1974) and abnormal neuronal membrane ultrastructure seen in HD (Roizin et al. 1979) suggest similar degenerative processes may occur in HD brain.     

The diencephalo-olivary projection in the cat as studied with retrograde transport of horseradish peroxidase

Summary The projection from certain diencephalic regions (zona incerta, Forel's fields, the parafascicular and subparafascicular nuclei, the periventricular grey and the hypothalamus) to the inferior olive in the cat was studied by means of retrograde protein tracing. Small injections of horseradish peroxidase were made into various parts of the inferior olive from a ventral approach. The number of retrogradely labelled neural cells in the diencephalic nuclei of all cats is presented in Table 2. The majority of the labelled cells was found in the parafascicular and subparafascicular nuclei, especially within the medial part of the former. The connection is ipsilateral, and the caudal as well as the rostral part of the olivary complex appears to receive the descending afferents. The findings are discussed and related to recent observations concerning descending afferents to the olivary complex.