Neurophysin biosynthesis in normal rats and in rats with hereditary diabetes insipidus.

Abstract

When [35S]cysteine was injected adjacent to the supraoptic nucleus (SON) in rats, it was rapidly incorporated into proteins in the SON. The [35S]cysteine-labeled proteins extracted from the SON were separated by isoelectric focusing on polyacrylamide gels. Twenty minutes after the injection of [I Scysteine, two major labeled peaks (pI = 5.4 and 6.1) were found in the SON of normal rats; Brattleboro rats had only one major labeled peak (pI = 5.4). One hour after the injection, four major radioactive peaks were found in the SON of normal animals (pI = 5.1,5.4,5.6, and 6.1). Animals with diabetes insipidus had only two major labeled proteins (pl = 5.1 and 5.4). Twenty-four hours after normal rats were injected with l35S-cysteine, all of the labeled peaks described above, except for the one with pI = 5. Neurons in the supraoptic nucleus (SON) and paraventricular nucleus synthesize two peptide hormones (oxytocin and vasopressin) as well as their "carrier" proteins (neurophysins) (1-3). The oxytocin, vasopressin, and neurophysins are transported, in axons of the neurons that synthesize them, to the posterior pituitary via the median eminence. Sachs and his colleagues (4-8) have hypothesized that vasopressin and its neurophysin come from a common precursor protein that is synthesized in the hypothalamus by a ribosomal mechanism. We have already presented some evidence consistent with this hypothesis (9, 10).We have found that, within 1 hr, [a3S]cysteine injected adjacent to the SON is incorporated into two proteins with molecular weights (Mr) of about 20,000 and pI of 5.4 and 6.1 (9, 10). It appears that, in time, these two proteins give rise to the two neurophysins (Mr 12,000). sopressin and its associated neurophysin (11, 12) and hence have diabetes insipidus-to determine which of the putative precursors, intermediates, and neurophysins are related to vasopressin and which to oxytocin. MATERIALS AND METHODS Animals and Operative Procedures. Female OsborneMendel (225-250 g) and Brattleboro (130-150 g) rats were used in these studies. Some of the normal Osborne-Mendel rats were given 2% (wt/vol) saline to drink for 1 week prior to use in order to stimulate vasopressin and neurophysin synthesis; others were given water ad lib as were the Brattleboro animals.The animals were anesthetized with ether, their heads were fixed in a stereotaxic device (50, nose down), and a flap of bone was removed from their skulls. The 30-gauge stainless steel needles were positioned 7 mm rostral to the interauricular line (6.8 mm for Brattleboro rats), and 2.5 mm on each side of the midline (2 mm for Brattleboro animals). The needles were lowered 8.6 mm (8.2 mm for Brattleboro animals) beneath the dural surface, and 1 ,ul of a solution containing 20,Ci of I35S]cysteine (50 Ci/mmol; New England Nuclear) in 0.9% NaCl and 10 mM dithiothreitol was injected through each needle over a 4-min period. After the injection, the needles were left in place for 10 min and then removed. At various times after the injections the rats were killed by...

Immunological and biochemical characterization of distinct high molecular weight forms of neurophysin and somatostatin in mouse hypothalamus extracts

“…The above-reported observations reinforce and extend the previous findings from pulse-chase analysis by others [7,8,15] that neurophysin biosynthesis may occur via higher molecular weight precursors. In addition to the '17 k' form, we are able also to detect a =;> '20 k' species which seems to be relatively labile and could preferably be isolated as a product of in vitro translation of reticulum-bound polysomes from hypothalamic neurons.…”

“…These larger forms, elaborated on the rough-surfaced endoplasmic reticulum [5,6], undergo several transformations, named processing, which generally lead to the unmasking of biologically active 'final' compounds. Biosynthetic evidence [7,8] for such a general scheme, derived from in vivo pulse chase experiments, seems to apply to the biogenesis of the neurophysins, neurohypophyseal hormone-associated proteins, synthesized by neurosecretory neurons of the magnocellular system in the supraopticus and paraventricular nuclei of the hypothalamus. In the course of our biochemical approach to the characterization of such putative pro-neurophysins we were able to detect, using a radioimmunological assay, neurophysin-like materials with molecular weight '17 k' and ~ '20 k'.…”

Nature of the immunoreactive neurophysins in ectopic vasopressin-producing oat cell carcinomas of the lung. Demonstration of a putative common precursor to vasopressin and neurophysin.

“…Recently, bovine hypothalamic messenger (m)RNA has been shown to direct the synthesis of possible neurophysin biosynthetic precursors in a cell-free system (16)(17)(18). Similarly, it has been reported that labeled cysteine injected adjacent to the supraoptic nucleus of the rat was rapidly incorporated into a 20,000-dalton protein that, in time, was converted to a 12,000-dalton protein (19,20). Along with the findings that the 20,000-dalton protein reacts with antineurophysin serum (21) and that trypsin liberates neurophysin and vasopressin-like peptide from the protein (22), these authors suggested that this 20,000-dalton protein may be a precursor to neurophysin and vasopressin in the rat.…”

The Emerging Concept of Relaxn as a Centrally Actng Peptide Hormone with Hemodynamic Actions

“…Relaxin also causes a pressor and tachycardic response in Brattleboro rats (41); animals which do not sjnthesize and secrete a functional vasopressin molecule (43). The mechanism of this pressor response is not established but Parry showed that exogenous relaxin causes a substantial release of oxytocin in Brattleboro rats (41 ) and this oxytocin may be responsible for the cardiovascular actions.…”