Marc Lauber

Immunohistochemical localization and radioimmunoassay of corticotropin-releasing factor in the forebrain and hypophysis of the frog Rana ridibunda

The distribution of immunoreactive corticotropin-releasing hormone (CRF) in the forebrain and pituitary of the frog Rana ridibunda was studied by means of specific radioimmunoassay and immunohistochemistry using the indirect immunofluorescence and the peroxidase-antiperoxidase techniques. Relatively high concentrations of CRF-like material were found in both chiasmatic and infundibular regions of the hypothalamus (352 +/- 11 and 422 +/- 36 pg, respectively). Large amounts of CRF were also found in neurointermediate lobe extracts. Standard curves of synthetic CRF and the dilution curves for hypothalamic or neurointermediate lobe extracts were parallel. After Sephadex G-75 gel filtration, CRF-like immunoreactivity eluted in a single peak, in the same position as synthetic ovine CRF. Reversed-phase high-performance liquid chromatography of the material purified on Sephadex G-75 revealed 5 components with CRF-like immunoreactivity. The major peak had a retention time of 22 min as compared to 25.4 min for ovine CRF and 36 min for rat CRF. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the frogs. The great majority of the CRF-positive perikarya were seen in the ventral region of the preoptic nucleus. A few scattered perikarya were also observed in the dorsal preoptic nucleus and in the retrochiasmatic region. Immunoreactive fibers were found in the infundibular nucleus and in various extrahypothalamic zones. CRF-containing neurons were apparently distinct from mesotocinergic and vasotocinergic neurons. A large number of immunoreactive nerve fibers were observed in the median eminence in close contact with the capillaries of the pituitary portal plexus and in the neural lobe. A few CRF-positive fibers were detected in the intermediate lobe, whereas the distal lobe was totally negative. These results show that the diencephalon and pars intermedia-nervosa of the frog contain a peptide immunologically related to mammalian CRF.

Evidence for higher molecular weight immunoreactive forms of vasopressin in the mouse hypothalamus: Relationships with putative proneurophysins

The neurohypophyseal nonapeptide hormones, oxytocin and vasopressin are synthesized in specialized nuclei of the hypothalamus. They are translocated to the neurohypophysis, the storage and excretion organ, within membrane-limited secretory granules where they are found as non-covalent complexes with the 10 000 mol. wt proteins, the neurophysins (reviewed [l-4]). The biogenesis of secretory protein components has been found to occur in the producing cells via larger biosynthetic precursors which generate the active molecules by post-translational processing. This appears to be the case for some peptide hormones [5-l 11. As early as 1964, classical experiments of in vivo pulse followed by in vitro chase allowed Sachs and coworkers to obtain evidence that the appearance of radioactively labeled, and active, Arg’-vasopressin (AVP) occurred after a lag period. Since this process was not inhibited by puromycin added during the chase, it was proposed that AVP was produced, first, as a larger inactive precursor molecule [ 12 ,I 31. So far this hypothesis has not been demonstrated. Analogous

Selective processing of the 15 000 Mr prosomatostatin by mouse hypothalamic extracts releases the tetradecapeptide

Brain peptides seem to be synthetized in neuronal systems as larger Mr precursors. A 15 000 Mr form of the tetradecapeptide somatostatin was characterized in hypothalamic extracts [ 1,2] and its digestion by copurified enzymes led to lower M, somatostatin-like species [ 11. Pre-prosomatostatins with comparable size were identified by cell-free translation of mRNA extracted from the Brockmann organ of the Anglerfish [3,4]. The cloning of cDNA coding for somatostatin precursors from the pancreas was accomplished by 2 groups and indicated some divergence in the sequence of the predicted pre-prohormone [5,6]. These observations were interpreted [ 51 as suggesting the existence of several (at least 2) genes coding for 2 distinct somatostatin sequences: the normal tetradecapeptide (somatostatin I) and its derivative (Tyr’-Gly”; somatostatin II). In spite of obvious analogies of molecular size with the pancreatic precursor, little is known about the structure and the function(s) of the somatostatin precursor produced in the central nervous system; in particular, the peptide sequences recognized by the putative converting enzyme(s). Here we demonstrate that hypothalamic extracts contain protease capable of converting selectively the Mr 15 000 precursor into somatostatin I as evidenced by radioimmunological and high-pressure liquid chromatography (HPLC) criteria.

Immunological detection of pro-corticotropin releasing factor (CRF) in rat hypothalamus and pancreatic extracts: Evidence for in vitro conversion into CRF

Extracts of both rat hypothalamus and pancreas were analyzed for their corticotropin releasing factor (CRF)‐like immunoreactivity by radioimmunoassay (RIA). In the case of the hypothalamus, besides the rat CRF, further identified by high‐pressure liquid chromatography (HPLC), two peptide components, a 20‐kDa and a 10‐kDa species were detected. The 20‐kDa component was stable under acidic pH conditions and was further purified by reverse‐phase HPLC. When exposed to proteolytic activities coeluting with ‘high‐molecular‐mass CRF’ at pH 6, processing was observed and the CRF generated was identified both by RIA, molecular sieve filtration and HPLC under different experimental conditions. It is concluded that this 20‐kDa CRF may represent the CRF precursor and that hypothalamic extracts may contain processing enzymes involved in its selective post‐translational cleavage. In the pancreatic extract two immunoreactive forms of CRF were detected, the smaller coeluting with the rat CRF and the other corresponding to the intermediate 10‐kDa component detected in the hypothalamus. Pancreatic rat CRF, analyzed using RIA both by molecular sieve filtration and HPLC, was indistinguishable from the hypothalamic rat CRF.

BIOSYNTHETIC PRECURSORS OF NEUROHORMONES: HIGH MOLECULAR WEIGHT IMMUNOREACTIVE FORMS OF NEUROPHYSIN : VASOPRESSIN AND OF SOMATOSTATIN

Extracts of bovine neurohypophysis made from delipidified tissues, were fractionated by filtration on various molecular sieves and analyzed for their immunoreactivity toward antineurophysin (Np) and anti-Arg 8 vasopressin (AVP) antibodies. The pattern obtained indicates the presence of high Mr forms with apparent Mr 140,000, 60,000 and 25 to 30,000 daltons besides the 10,000 and 1,000 neurophysin and AVP respectively. The neurophysin- and AVP-like immunoreactivities contained in the 140,000 or 25–30,000 materials, were not aggregates of lower size immunoreactive products with high Mr molecules. They were found co-adsorbed on an (-antineurophysin-Sepharose 4B) conjugate and desorbed by a drastic pH change. The recovered 140,000 daltons material, in the contact of mild proteolytic activities, generated the intermediary size immunoreactive species (60,000, 25–30,000) and the “final” products, neurophysin and “vasopressin”, as identified by their chromatographical and immunological behaviour. Similar forms were detected in the mouse hypothalamus extracts as well as in lysates of purified bovine neurosecretory granules. Analysis of the somatostatin-like immunoreactivity detectable in these hypothalamic extracts showed the presence of 15,000, 10,000 and 6,000 daltons species besides the 1,500 somatostatin. The 15,000 daltons somatostatin-like form was purified by immunoadsorption on antisomatostatin antibodies immobilized on sepharose labeled with 125 I, and was shown to generate, by exposure to proteolytic activities co-eluted in the fractionation of the extracts, the 10,000, 6,000 and 1,500 daltons somatostatin-like forms.