Isolation and identification of vasopressin- and oxytocin-immunoreactive substances from bovine pineal gland. Presence of N alpha-acetyloxytocin.

Peptides with vasopressin- and oxytocin-immunoreactivity were purified from bovine pineal glands. Three immunoreactive peptides were purified by successive high performance liquid chromatography steps in sufficient quantities for identification by fast atom bombardment-mass spectrometry. Two peptides were characterized as authentic vasopressin and oxytocin.

Increased concentration of alpha- and gamma-endorphin in post mortem hypothalamic tissue of schizophrenic patients.

The concentrations of alpha-, beta- and gamma-endorphin were determined by radioimmunoassay in HPLC fractionated extracts of post mortem hypothalamic tissue obtained from schizophrenic patients and controls. The hypothalamic concentration of alpha- and gamma-endorphin was significantly higher in patients than in controls (+72.9% and +50.

Measurement of mRNA by solution hybridization with 32P-labelled single stranded cRNA probe ("SP6 test"). Comparison with a 32P-labelled single stranded cDNA as hybridization probe ("S1 test") for measurement of AVP mRNA.

Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA ("SP6 test"). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA ("S1 test"). Both tests had a comparable sensitivity of up to 0.

Leu-Phe cleaving endopeptidase activity, gamma-endorphin, and beta-endorphin in the rat pituitary gland and brain. Effect of adrenalectomy and corticosterone substitution.

The influence of adrenalectomy and corticosterone substitution was investigated on Leu-Phe cleaving endopeptidase activity and on the levels of gamma-endorphin and beta-endorphin in the pituitary gland and the brain. The enzyme activity was quantitated by a specific radiometric assay based on the cleavage of the Leu17-Phe18 bond in a NH2- and COOH-terminally protected synthetic substrate which was analogous to beta-endorphin-(15-19). This activity may mimick the formation of gamma-endorphin.

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.

Detection and high performance liquid chromatography identification of the summer rises of vasopressin and oxytocin immunoreactivity in the rat pineal gland.

The vasopressin (VP) and oxytocin (OT) contents of the rat pineal gland during the summer period were determined by RIAs. Both the levels of VP immunoreactivity and OT immunoreactivity rose markedly in August. The highest level of VP immunoreactivity occurred on August 6 [82 +/- 19 fmol/gland (+/- SEM)], compared to basal levels of 14 +/- 2 fmol/gland.

Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly(A) tails in hypothalamus.

The POMC gene is predominantly expressed in the pituitary gland; it is also expressed in various extrapituitary tissues. While POMC mRNAs of similar size (approximately equal to 1000 nucleotides) are present in the anterior and neurointermediate lobes of the pituitary, other POMC-expressing tissues contain POMC mRNAs of different sizes. Longer POMC mRNAs are observed in the hypothalamus.

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.

Morphometry, histochemistry, and contractility of dystrophic hamster diaphragm.

Differences in gross and microscopic morphology, fiber-size distribution, and fiber-type composition were present in the diaphragm of 35-, 130-, and 180-day-old dystrophic (Bio 14.6) compared with age-matched control (Bio F1B) hamsters. The dystrophic diaphragm was significantly thicker than the control at 130 and 180 days.

Ultrastructure of cardiocyte degeneration and myocardial calcification in the dystrophic hamster.

The myocardium of the Bio 14.6 cardiomyopathic hamster was examined with the electron microscope to identify cellular and organelle changes during the acute lesioning stage, a period typified by concomitant cardiocyte destruction and calcium elevation. Most cardiocytes retained their normal histologic and ultrastructural features, but scattered foci of altered and necrotic cells were observed in association with degenerative calcifying lesions.

Modified forms of vasopressin and oxytocin in a bovine pineal preparation.

A bovine pineal acid extract displays a vasotocin-like bioactivity in several bioassays, and is recognized by antibodies against the Pro-Arg-Gly-amide ending common to vasopressin and vasotocin. By using molecular sieve filtration and reversed-phase HPLC, a vasopressin- and oxytocin-like peptide was isolated from this pineal preparation, while no evidence for a vasotocin-like peptide was obtained. The isolated neuropeptides contain a modified amino acid at position 2.

In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups.

Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded 35S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.

Measurement and distribution of vasopressin-converting aminopeptidase activity in rat brain.

The aminopeptidase activity in the brain which converts vasopressin into centrally active metabolites, was quantitated on basis of the release of 3H-Phe from the substate [3H-Phe3]vasopressin and separation by hydrophobic interaction chromatography on mini-columns. After subcellular fractionation of whole rat brain homogenates the highest specific activity of the peptidase was recovered in membrane fractions, in particular microsomes and the P3 fraction, and the cytosol. The peptidase activity was present in all brain areas.

Structure activity relationship studies with C-terminal fragments of vasopressin and oxytocin on avoidance behaviors of rats.

The potency of various C-terminal fragments of the neurohypophyseal hormone [Arg8]vasopressin [AVP-(1-9)] was determined using different avoidance behavioral test procedures in rats. Passive avoidance behavior was facilitated by these peptides. The fragments [Cyt6]AVP-(5-8) and [Cyt6]AVP-(5-9) were the most potent peptides tested after postlearning injection (consolidation) and preretention treatment (retrieval), respectively, after s.

Oxytocin gene expression in discrete hypothalamic magnocellular cell groups is stimulated by prolonged salt loading.

The effect of prolonged osmotic stimulation on the oxytocin (OT) mRNA levels of OT-producing neurons was investigated in separate hypothalamic nuclei of the rat. After drinking 2% NaCl for 2 weeks, a 2-fold increase in the OT mRNA content was found by Northern blot analysis of microdissected supraoptic nucleus (SON) and paraventricular nucleus (PVN). The same samples showed a similar change in vasopressin (VP) mRNA levels.

Characterization of vasopressin and oxytocin immunoreactivity in the sheep and rat pineal gland: absence of vasotocin and detection of a vasopressin-like peptide.

The presence of arginine-vasopressin (VP), arginine-vasotocin (VT) and oxytocin (OT) were studied in sheep and rat pineal gland by the combination of HPLC and radioimmunoassays. Three immunoreactive substances, which had the same retention time as OT, VP and VT, were detected in sheep and rat pineal extracts after HPLC separation. Parallelism of dilution curve and standard curve demonstrated that two of them were identical to OT and VP respectively.

Quantitation of vasopressin mRNA and oxytocin mRNA in hypothalamic nuclei by solution hybridization assays.

Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magnocellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on recombinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA.

Effects of dystrophy and age on hamster tracheal smooth muscle function.

Pharmacological and morphometric evaluations of airway smooth muscle were performed on tracheal segments from control and dystrophic hamsters at 50, 145 and 315 days of age. An age-related change in muscle volume index (a measure of the total smooth muscle per tracheal segment) was not observed in the control group but decreased with age in the dystrophic strain. In the control strain, correlations were not observed between age and maximum pharmacological response to the drugs tested, but a change in sensitivity to carbachol and KCl was noted at 145 days.

Vasopressin gene expression is attenuated in the fetal Brattleboro rat.

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30.

Characterization and quantitation of vasopressin metabolites in the rat brain by specific radioimmunoassay and high-pressure liquid chromatography.

The content of endogenous vasopressin (AVP1-9) and its metabolites in rat's various brain areas and pituitary gland was investigated by the combination techniques of high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA). In addition to the AVP1-9, several compounds with C-terminal AVP immunoreactivity were detected in hypothalamus, hippocampus, amygdala, medulla oblongata and septum. The C-terminal AVP1-9 metabolites were virtually absent in the pituitary gland.

gamma-Endorphin and schizophrenia: amino acid composition of gamma-endorphin and nucleotide sequence of gamma-endorphin cDNA from pituitary glands of schizophrenic patients.

The possibility has been mentioned that a change in the structure is responsible for the deviant behavioral activity of gamma-endorphin in extracts of postmortem brain and pituitary gland samples of schizophrenic patients. This paper describes the investigation of this possibility by means of: amino acid composition analysis of alpha- and gamma-endorphin isolated from a pituitary gland of a schizophrenic patient; and nucleotide sequence analysis of the gamma-endorphin coding region of pro-opiomelanocortin (POMC) mRNA from two other pituitary glands, using the primer extension method. Both methods require no more than a single pituitary to obtain reliable results.

Isolation and primary structure of novel neurointermediate pituitary peptides derived from the C-terminal of the rat vasopressin-neurophysin precursor (propressophysin).

Four novel peptides were isolated from rat neurointermediate lobes by gel filtration and high-pressure liquid chromatography. Analyses of amino acid composition and sequence showed that all four peptides were derived from the C-terminal portion of propressophysin (CPP); they were identified as the glycopeptides CPP 1-19, CPP 1-20, CPP 22-37 and CPP 22-39. Processing of CPP 1-39 could thus produce the four isolated peptides by specific post-Arg or post-Leu cleavages.

Postnatal development of vasopressin mRNA content in supraoptic and paraventricular nucleus of the Wistar rat.

Levels of mRNA coding the vasopressin precursor (VP mRNA) were determined in the supraoptic and paraventricular nucleus during the first month of postnatal development of the Wistar rat by solution hybridization assay and Northern blot analysis. The supraoptic and paraventricular nuclei showed a marked increase in VP mRNA to adult levels during that period. These results indicate an upregulation of vasopressin gene expression in supraoptic and paraventricular nuclei after birth, a period in which vasopressin starts to become involved in kidney function.

Formation of metabolites of [Arg8]vasopressin (AVP) by brain peptidases. Conversion of the intermediate [Cyt6]AVP-(3-9).

[Cyt6]AVP-(3-9), an intermediate in the metabolism of AVP-(1-9) in vitro, was used to investigate the mechanism of formation of centrally active AVP metabolites. Exposure of [Cyt6]AVP-(3-9) to rat brain membranes resulted in formation of [Cyt6]AVP-(4-9), -(5-9), [pGlu4, Cyt6]AVP-(4-9) and AVP-(3-5), which were isolated and chemically identified. Products derived from cleavage of the C-terminus of the substrate were absent.

Vasopressin and oxytocin. Their presence in the central nervous system and their functional significance in brain processes related to behaviour and memory.

Vasopressin and oxytocin exert pronounced effects on behaviour by a direct action on the brain. A single injection of vasopressin results in a long-term inhibition of extinction of a conditioned avoidance response suggesting that vasopressin triggers a long-term effect on the maintenance of a learned response, probably by facilitation of memory processes. In addition vasopressin improves passive avoidance behaviour, delays extinction of appetitive discrimination tasks, affects approach behaviour to an imprinting stimulus in ducklings, improves copulation rewarded behaviour of male rats in a T-maze, prevents or reverses amnesia induced by electroconvulsive shock, CO2 inhalation, pentylenetetrazol or puromycin.