We have previously described the isolation of a cDNA clone corresponding to an mRNA rapidly induced to high levels in PC12 cells by treatment with NGF. We report here the complete amino acid sequence of the protein (named VGF8a) as deduced by nucleotide sequencing of overlapping cDNA clones. VGF8a is particularly rich in proline residues and has a conspicuous number of short stretches of basic amino acid residues which may represent potential targets for proteolytic cleavage. Antibodies directed against recombinant VGF8a‐beta‐galactosidase fusion proteins were used for immunofluorescent staining of the protein in PC12 cells as well as for its localization, by Western blot analysis, in subfractions of cell homogenates. We demonstrate that in PC12 cells, VGF8a protein is stored in secretory vesicles and is released in response to a variety of stimuli that are known to induce the regulated secretion of neurotransmitters.
Several polypeptide neurotoxins affect presynaptic functions by interfering with chemical neurotransmission. This group of toxins includes botulinum toxin, tetanus toxin, beta-bungaro-toxin and black widow spider toxin (BWSTx). While the effect of the first three toxins is mainly a rapid and severe block of neurotransmitter release, BWSTx affects transmission by a massive stimulation of mediator release. Despite various hypotheses put forward to explain the action of BWSTx at the level of nerve terminals, there is still a considerable degree of uncertainty as to the cation dependence of venom action. Study of the toxin mode of action at the biochamical level has been hampered by the complexity and cellular heterogeneity of the preparations used, neuromuscular junction or synaptosomes. PC12 cell line, derived from a rat phaeochromocytoma, seems to be an excellent model in view of its property of synthesising and storing noradrenaline, dopamine and acetylcholine, and releasing them in depolarising conditions. We have recently shown that highly purified BWSTx stimulates secretion from PC12 cells of previously taken up radioactive dopamine (DA) and noradrenaline (NA) (ref. 14 and manuscript in preparation). We report here that the earliest detectable event after toxin treatment of such cells is a massive increase of cytosolic calcium.
The circadian rhythm of plasma proopiocortin-related peptides was studied in 15 heroin addicts and in 6 sex-and age-matched controls. ACTH, \g=b\-l i potrophin, (\g=b\-LPH), \g=b\-endorphin(\g=b\-EP) and cortisol were measured by RIA either directly (cortisol), or after plasma extraction (ACTH) and Sephadex G-75 gel chromatography (\g=b\-LPH and \g=b\-EP) every 4 h from 8 a.m. to 8 p.m. and again at 8 a. m. the next morning.The means of the two 8 a.m. measurements of \g=b\-LPH (2.67 \ m=+-\ 0.34 fmol/ml, mean \ m=+-\ SE), ACTH (2.74 \ m=+-\ 0.71) and cortisol (218 \ m=+-\ 31 pmol/ml) levels in heroin addicts were significantly lower than those in controls (6.28 \ m=+-\ 0.61, 10.1 \ m=+-\ 0.74 and 364 \ m=+-\ 27, respectively, P < 0.01) while \g=b\-EP concentrations in heroin addicts (5.1 \ m=+-\ 0.6) were similar to those of healthy volunteers (6.44 \m=+-\ 0.56).In controls, all three peptides and cortisol show a circadian rhythm of secretion, the lowest values being in the evening and the highest ones in the morning. Heroin addicts partially lack this phenomenon showing constant levels of the three proopiocortin-related peptides throughout the day, with a slight but significant decrease of plasma cortisol. In the 7 subjects who took heroin throughout the study, no systematic changes were observed in the three proopiocortin-related peptides, while it seems that this group of addicts shows a cortisol decrease in the evening to a lesser extent than subjects receiving methadone maintenance only.These data show a decreased basal \g=b\-LPH, ACTH and cortisol secretion in heroin addicts, despite normal \g=b\-EP levels. This would indicate that anterior pituitary proopiocortin synthesis and/or secretion is affected in these subjects, and that there is also some impairment in the structures controlling the circadian rhythmicity of ACTH and related peptides.Partly supported by CNR grant 82.02145.04 and CNR PF 'MPR' SP 7.Since the discovery of opiate receptors in human brain, a great deal of attention has been paid to the possible relationship between their natural ligands, the endogenous opioid peptides and the onset of morphine or heroin addiction. In this context, it has been proposed that a genetic deficiency of endogenous opioid peptides may be a cause of drug-seeking behaviour (Goldstein 1978), as has also been suggested for alcohol abuse (Blum 1980). The link between exogenous and endogenous opioids is supported by the observation that chro¬ nic morphine treatment causes a decrease in the ß-endorphin-like immunoactivity (ß-ELI) content of the anterior and intermediate posterior pituitarylobes of rats (Volker et al. 1978) while plasma concentrations of these substances were found to be depressed in heroin addicts (Ho et al. 1980). However, the antisera employed in the above stud¬ ies recognize all the peptides or fragments con¬ tained in the C-terminal portion of ß-lipotrophin (ß-LPH), so even though the results are suggestive, they do not establish which of the peptides whithin the endorphin family are depressed in...
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