Proportions Of Molecular Forms Research Articles

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis.

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.

Effects of Neonatal Administration of Monosodium Glutamate on Four Neuropeptide Concentrations in the Rat Brain

The present study was designed to measure concentrations of four neuropeptides in different brain regions in monosodium glutamate(MSG)-treated rats and to assess molecular forms of each peptide with gel and high performance liquid chromatography (HPLC). MSG(4mg/kg body weight) or 10% NaCl was injected subcutaneously on postnatal days 1, 3, 5, 7 and 9 to male littermates which were subsequently used on postnatal day 100. Rats were sacrificed by decapitation, and the brains were dissected into ten discrete regions. The brain extracts were subjected to measurement of four neuropeptides; somatostatin (SRIF), neuropeptide Y (NPY), atrial natriuretic polypeptide(ANP), and a novel pituitary polypeptide 7B2 by specific radioimmunoassays. Significant increase (p less than 0.01) in midbrain SRIF content was observed in MSG-treated rats, though there was no significant change in hypothalamic SRIF content. Significant reduction (p less than 0.05) in hypothalamic NPY content was also found in MSG-treated rats. Hypothalamic ANP content was similar in both MSG-treated and control rats. A significant increase of 7B2 content was found in substantia nigra/ventral tegmentum and hypothalamus (p less than 0.05 or p less than 0.01, respectively) in MSG-treated rats. These four immunoreactivities were further characterized by gel permeation or high pressure liquid chromatography (HPLC). Chromatographic analysis of SRIF immunoreactivity revealed that there were two distinctive peaks and smaller molecular weight component corresponding to SRIF. Fractionation of NPY or 7B2 immunoreactivity by gel permeation showed a single major peak which was identical to the synthetic NPY or 7B2 immunoreactivity from porcine pituitary extract. HPLC analysis for ANP immunoreactivity also showed that the major immunoreactive component corresponded to synthetic rat ANP. MSG treatment could not produce any major alterations in proportions of molecular forms studied. These results suggest that MSG treatment in neonates might produce the alterations in SRIF, NPY and 7B2 content in the discrete brain regions including the hypothalamus.

Heavy isotope-labeling study of the metabolism of monomeric and tetrameric acetylcholinesterase forms in the murine neuronal-like T 28 hybrid cell line.

We have used the method of heavy isotope labeling to study the metabolic turnover of acetylcholinesterase forms in the neuroblastoma-derived T 28 hybrid cells in their differentiated state. These cells contain mostly G1 and G4 forms, together with a small proportion of G2, and secrete all these forms into the culture medium. The cells maintained constant and equal levels of acetylcholinesterase, with the same proportions of molecular forms, in a medium containing heavy isotope-labeled amino acids and in a control light medium of similar composition. In addition, they secreted acetylcholinesterase at the same rate in both media. After transfer of the cells into the heavy medium, heavy isotope-labeled acetylcholinesterase molecules progressively replace preexisting light molecules. We analyzed heavy and light components of acetylcholinesterase for each of the two major G1 and G4 forms, by reconstructing the pattern obtained in sucrose gradient differential sedimentation, using combinations of weighted elementary distributions. Heavy molecules were detected in cellular extracts after about 30 min for G1 and 3 h for G4. Both heavy forms also appeared in the medium after a lag of about 3 h. The cellular complement of G1 was renewed much faster than that of G4, the levels of the light forms being reduced to 50% of the original level after 3.5 and 40 h, respectively. Each of these forms appeared to consist of several metabolic pools, and we present simplified models which describe their possible relationships.