The C-terminal glycopeptide of propressophysin potentiates excitatory transmission in the rat lateral septum.

Effects of peptides synthesized from the same precursor as vasopressin, i.e. the C-terminal 39-amino acid long glycopeptide and neurophysin II, were investigated for biological activities in electrophysiological experiments in brain slices of the rat lateral septum. These slices contained the glycopeptide as the predominant form and a fragment of it, amino acid sequence 22-39, as a minor form (8% of the glycopeptide 1-39), as shown by high performance liquid chromatography of extracts and by radioimmunoassay. None of the peptides, neurophysin II, the glycopeptide 1-39 and the fragment 22-39, tested in a concentration of 10(-12) M, had measurable effects on the resting membrane potential of the neurons. The glycopeptide and the fragment 22-39, however, increased, in some cells, for tens of minutes the excitatory postsynaptic potentials evoked in these neurons by stimulation of the fimbria fibers. The increase in input resistance, seen in many septal neurons treated with either of the peptides was not correlated with the excitatory postsynaptic potential increase. Neurophysin II affected neither the excitatory postsynaptic potentials nor the input resistance of the neurons. It is concluded that the glycopeptide 1-39 and the fragment 22-39 possess biological activities amongst which the facilitation of excitatory amino acid transmission on lateral septum neurons. Therefore, these peptides derived from the vasopressin precursor may act in concert with vasopressin to establish facilitation of excitatory transmission in the brain.

Stimulation of pro-opiomelanocortin gene expression by glucocorticoids in the denervated rat intermediate pituitary gland.

The effect of frontal deafferentation of the medial basal hypothalamus on pro-opiomelanocortin (POMC) gene expression was studied in the intermediate lobe (IL) and anterior lobe (AL) of the pituitary gland in the absence and presence of corticosterone (CORT). The lesion in the basal hypothalamus removed neural inputs to the IL and induced the glucocorticoid receptor (GR) in this tissue. The GR was visualized in the denervated IL by immunocytochemistry. Induction of the GR had a slow onset and was detectable at 3 weeks after lesion, but not at one week after the lesion. In order to study the effect of IL denervation on pro-opiomelanocortin (POMC) gene expression, the level of messenger RNA specifically encoding POMC was measured 1 and 3 weeks after lesion, in the IL and AL. In adrenalectomized (ADX) animals, the changes in POMC mRNA levels were not significant 1 week after lesion in the IL. Three weeks after denervation there was a 3-fold decrease in POMC mRNA in the IL in ADX rats which was blocked by chronic CORT replacement via subcutaneously implanted pellets. In the AL, CORT reduced the level of POMC gene expression in both the lesioned and control animals. It is concluded that (1) removal of neural input induces GR in the denervated IL cells; (2) with the appearance of the GR, POMC gene expression in the IL becomes sensitive to circulating glucocorticoids; (3) under these conditions, CORT may stimulate POMC gene expression in the IL as opposed to its inhibitory effect in the AL.

Biosynthesis of pro-opiomelanocortin is increased in the rat intermediate pituitary following denervation.

In order to study the effect of pituitary intermediate lobe denervation on pro-opiomelanocortin (POMC) biosynthesis and processing, radioactive amino acids were incorporated in vitro into whole neurointermediate lobe (NIL) explants obtained from hypothalamic lesioned rats and control animals. The lesion in the basal hypothalamus removed the neural input to the intermediate pituitary and cut the neurohypophyseal neurons. One week after the lesion, approximately a 3-fold increase in the rate of synthesis of POMC peptides was found in the NIL. The content of POMC peptides was decreased. The results imply that denervation increases the rate of POMC synthesis and release, without altering the pattern of proteolytic processing.

The presence and in vivo biosynthesis of fragments of CPP (the C-terminal glycopeptide of the rat vasopressin precursor) in the hypothalamo-neurohypophyseal system.

The existence and rate of formation of fragments of the 39-residue C-terminal glycopeptide of propressophysin (CPP1-39) was investigated in the hypothalamo-neurohypophyseal system. Newly-prepared antisera to CPP were used to confirm the existence of smaller C-terminal fragments derived from CPP1-39. Radiolabelled fucose was injected into rats in vivo into the area of the supraoptic nucleus, and the labelled peptides formed in the neurohypophysis were examined at various time intervals up to five weeks after the injection. The products derived from the neurohypophyseal hormone precursors were separated by high-performance liquid chromatography. The level of the major immunoreactive C-terminal fragment (CPP22-39) was constant and represented about 5% of the intact CPP1-39 in the neurohypophysis. The appearance of newly-synthesized N-terminal fragment of CPP1-39 occurred only after 3 or 4 days. This fucose labelled fragment increased slowly thereafter until it reached the same level as the CPP C-terminal fragment immunoreactivity between 21 and 28 days after injection. The results suggest that CPP1-39 is extremely stable in the hypothalamo-neurohypophyseal neurons, and that the cleavage at Arg21-Leu22 is a delayed proteolytic event in the magnocellular neurons of the SON.

Structure and bioactivity of the amino-terminal fragment of pro-opiomelanocortin.

The primary sequence of the amino-terminal or 16 K fragment (16 K) of pro-opiomelanocortin (POMC) is highly conserved throughout the mammals. This suggests an important biological role for this peptide. We have performed studies to determine the structure, biosynthetic origin and bioactivity of this pituitary peptide. A comprehensive study of all the biosynthetic derivatives of POMC in the neurointermediate lobe of the rat and mouse pituitary was undertaken. Inspection of the amino acid composition of these peptides indicated that cleavage at all available dibasic processing sites within POMC is essentially complete except for Arg49-Lys50 within the 1-74 16 K fragment (16 K1-74). Only about 50% of 16 K1-74 was found to be processed to give rise to the extreme amino-terminal 1 to 49 sequence (16 K1-49) and the carboxyl-terminal 50 to 74 sequence (Lys1 gamma 3 melanotropin). Sufficient 16 K1-77 and 16 K1-49 was purified from bovine posterior pituitaries in order to determine if there are any structural features controlling the limited degree of processing of 16 K within the intermediate lobe. Both bovine 16 K1-77 and 16 K1-49 were found to have cystine bridges linking cystine residues 2 and 24 and linking cystine residues 8 and 20. While 16 K1-77 was found to be O-glycosylated at threonine45 and N-glycosylated as asparagine65, 16 K1-49 was found to have no carbohydrate content. Thus the presence of O-glycosylation at threonine45 apparently inhibits cleavage at -Arg49-Lys50-. Lys1 gamma 3 MSH 16 K1-74 and 16 K1-49 purified from rat neurointermediate pituitaries were tested for their ability to potentiate the action of corticotropin (ACTH) in an isolated rat adrenal cell bioassay. None of the 16 K-related peptides showed any intrinsic steroidogenic activity. Experiments were performed in which dispersed adrenal cells were incubated with serial dilutions of ACTH. Constant amounts of test peptides were added in concentrations ranging from 10 pM to 5 nM. Lys1 gamma 3 MSH potentiated the steroidogenic activity of ACTH by up to 2-fold with an ED50 of approx 0.5 nM. 16 K1-49 showed no ability to potentiate the action of ACTH. In contrast the most highly glycosylated form of 16 K1-74 potentiated the action of ACTH by up to 6-fold.

Human phosphoserine 31 corticotropin1-39. Isolation and characterization.

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.