Eliprodil prevents expression of the 70 kDa heat shock protein in MK801-injured neurones.

The present study examined whether eliprodil (SL 82.0715), an N-methyl-D-aspartate (NMDA) receptor antagonist acting on the polyamine sites induced expression of the 70 kDa heat shock protein (HSP70) in the rat brain. Whereas the NMDA channel blocker MK801 consistently induced HSP70 in posterior cingulate and retrosplenial cortices, eliprodil had no such effects even at the highest dose (50 mg/kg, intraperitoneally), supporting the idea that injury to the cerebrocortical neurones by NMDA receptor antagonists is probably related to specific sites of the receptor. Furthermore, eliprodil, given immediately after injection of MK801, blocked the effects of MK801 on HSP70. The result is discussed in terms of high affinity of eliprodil for the sigma receptor.

Expression of N-CAM in fertilized pre- and periimplantation and parthenogenetically activated mouse embryos.

The expression of the cell adhesion molecule of the immunoglobulin family, neural cell adhesion molecule (N-CAM), in the pre- and periimplantation embryo was examined by immunocytochemistry. N-CAM is expressed on unfertilized ovulated oocytes, fertilized preimplantation embryos at all stages of development and parthenogenetically activated eggs and embryos. In fertilized embryos, expression from the 4-cell stage can be partially inhibited by blocking embryonic transcription before 38 h post human chorionic gonadotropin (hCG). Expression of N-CAM was reduced on the trophoblast of day 6 blastocysts in culture, weak on the trophoblast of embryonic outgrowths and disappears from invading trophoblast in utero. An antibody against alpha(2-8) linked polysialic acid, mAb2-2B, reacted with embryos from the 8-cell stage, and staining was similarly reduced on the trophoblast of blastocysts at the time of implantation. These results suggest a role for N-CAM in the interactions of cells of the preimplantation mammalian embryo which requires further investigation.

Differential expression of neural cell adhesion molecule and cadherins in pancreatic islets, glucagonomas, and insulinomas.

The endocrine cells of the pancreas develop from the endoderm and yet display several characteristics of a neuronal phenotype. During embryonic life, ductal epithelial cells give rise to first the glugagon-producing cells (alpha-cells) and then cells that express insulin (beta-cells), somatostatin (delta-cells), and pancreatic polypeptide (PP-cells) in a sequential order. The endocrine cells are believed to arise from a stem cell with neuronal traits. The developmental lineage from a common neuron-like progenitor is evidenced by: transient coexpression of more than one cell type-specific hormone in immature cells, expression of neuronal markers during islet cell development, and the pluripotentiality of clones of insulinoma cells to develop into cells expressing other islet cell hormones. The four mature endocrine cell types assume a particular organization within the islets of Langerhans in a process where cell adhesion molecules are involved. In this study we have analyzed the expression of neural cell adhesion molecule (NCAM) and cadherin molecules in neonatal, young, and adult rat islet cells as well as in glucagonomas and insulinomas derived from a pluripotent rat islet cell tumor. Whereas primary islet cells at all ages express unsialylated NCAM and E-cadherin, as do insulinomas, the glucagonomas express the polysialylated NCAM, which is characteristic for developing neurons. The glucagonomas also lose E-cadherin expression and instead express a cadherin which is similar to N-cadherin in brain. Insulinoma cells express E-cadherin but differ from primary islet cells by expressing a second cadherin molecule, which is similar to N-cadherin. The expression of NCAM and cadherin isoforms in the glucagonoma suggest that this transformed alpha-cell type has converted to an immature phenotype with strong neuronal traits, reflecting the early palce of glucagon-producing cells in the islet cell lineage. In contrast, insulinoma cells are more islet-like in their phenotype and show less neuronal traits.

Neuronal and glial marker proteins in the evaluation of the protective action of MK 801.

A quantitative dot immunobinding procedure was used to quantify glial [the S-100 protein and the glial fibrillary acidic (GFA) protein] and neuronal (the 68- and 200-kDa neurofilament polypeptides, neuron-specific enolase, and neuronal cell adhesion molecule) markers. A single intraperitoneal administration of 10 mg/kg of MK 801 blocked the increase of glial parameters and the decrease in content of neuronal marker proteins that occurred as the response to an N-methyl-D-aspartate (NMDA) lesion in the rat hippocampus. The degradation products of GFA protein and the 68-kDa neurofilament polypeptide that were induced by the NMDA lesion did not appear after MK 801 treatment. This study shows that brain-specific proteins are a set of precise tools for the evaluation of neuroprotective effects of antagonists to excitatory amino acids.

NCAM in developing mouse gonads and ducts.

The expression of the Neural Cell Adhesion Molecule, NCAM, in mouse gonads and ducts was studied from fetal life to maturity. The methods used were immunocytochemical staining and Western blotting. The immunocytochemical studies showed that the only structures that remain NCAM-positive throughout life were the mesonephric-derived rete ovarii and rete testis. Also in the fetal gonads some somatic cell lining the groups of differentiating germ cells were stained. In the immature as well as in the mature ovary the granulosa cells and oocytes of growing and large follicles--but not of small follicles--were stained. A particularly strong staining of the cytoplasm of the oocyte, healthy as well as atretic, was seen. All cells of the testis remained negative except for weakly stained residual bodies and late spermatids. At all ages the male ducts showed only weak staining, whereas in the female Müllerian duct the epithelium became strongly positive at puberty. The stroma of the Müllerian duct was positive during a transitory period around day 16 of fetal life in both sexes. One-dimensional gel immunoblotting of total protein from gonads, rete and ducts from immature and mature mice showed that only the two largest isoforms of NCAM (NCAM-A and NCAM-B) were present. The gonads and the rete of both sexes and the adult uterus expressed only NCAM-B, whereas NCAM-A was also detected in the adult epididymis. The present findings suggest that NCAM may be involved in the normal development and formation of both the gonads and ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of N-CAM polypeptides in neurons.

N-CAM from rat brain consists of three polypeptides: 190,000 Mr (A), 140,000 Mr (B) and 120,000 Mr (C). It has been reported that cultured neurons express only A and B, whereas glial cultures synthesize mainly B and C. During postnatal development the relative biosynthesis of C increases. This could possibly reflect differentiation of neurons or an increased biosynthetic contribution of glial cells. We have investigated neuronal expression of N-CAM with the aim of determining whether neurons were able to synthesize the C-polypeptide. Biosynthetic labelling of explant cultures of peripheral ganglia and of chromaffin cells from adrenal medulla showed that cultured neurons synthesized not only A and B, but also C. However, the biosynthetic capacity for C production was low. Cell-free translation of microsomes from neuronal cell cultures showed that they contained a messenger RNA coding for C. Finally, retinal ganglion neurons expressed C when located in their natural environment as determined by biosynthetic labelling performed in living rats. Thus, both neurons and glial cells may be involved in the developmentally regulated change in C expression that occurs during postnatal life.

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C.

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.