Continuous and selective measurement of oxytocin and vasopressin using boron-doped diamond electrodes.

The electrochemical detection of oxytocin using boron-doped diamond (BDD) electrodes was studied. Cyclic voltammetry of oxytocin in a phosphate buffer solution exhibits an oxidation peak at +0.7 V (vs. Ag/AgCl), which is attributable to oxidation of the phenolic group in the tyrosyl moiety. Furthermore, the linearity of the current peaks obtained in flow injection analysis (FIA) using BDD microelectrodes over the oxytocin concentration range from 0.1 to 10.0 µM with a detection limit of 50 nM (S/N = 3) was high (R(2) = 0.995). Although the voltammograms of oxytocin and vasopressin observed with an as-deposited BDD electrode, as well as with a cathodically-reduced BDD electrode, were similar, a clear distinction was observed with anodically-oxidized BDD electrodes due to the attractive interaction between vasopressin and the oxidized BDD surface. By means of this distinction, selective measurements using chronoamperometry combined with flow injection analysis at an optimized potential were demonstrated, indicating the possibility of making selective in situ or in vivo measurements of oxytocin.

Identification and expression of two oxytocin/vasopressin-related peptides in the cuttlefish Sepia officinalis.

Two novel members of the oxytocin/vasopressin superfamily have been identified in the cephalopod Sepia officinalis. Oxytocin/vasopressin gene sequences were cloned by Race PCR. The two precursors we identified exhibit the classical organization of OT/VP superfamily precursors: a signal peptide followed by a nonapeptide and a neurophysin domain. The neurophysin domain is entirely conserved for the cuttlefish precursors, but the nonapeptides and the signal peptides differ. The first nonapeptide, called sepiatocin, is highly homologous to Octopus vulgaris octopressin. The second nonapeptide, called pro-sepiatocin, shows sequence homologies with a Crustacean oxytocin/vasopressin-like peptide identified in Daphnia culex and with a novel form of oxytocin described in New World monkeys. The expression of pro-sepiatocin is restricted to the supraesophageal and subesophageal masses of the brain whereas sepiatocin is expressed in the entire central nervous system. Sepiatocin, as described for octopressin, modulates the contractile activity of several muscles such as penis, oviduct and vena cava muscles; this suggests its involvement in reproduction and blood circulation. Pro-sepiatocin is released in the hemolymph; it is a neurohormone able to target numerous peripheral organs.

A high recovery microsampling device based on a microdialysis probe for peptide sampling.

A high recovery microsampling probe based on microdialysis was devised. The new probe showed a high recovery (100%) of peptides in vitro at different perfusion flow rates (0.1-1.0 microl/min). At a high flow rate, 1.0 microl/min, a 10-fold increased in recovery of peptides compared to the conventional microdialysis probe was achieved. A probe made of a low molecular weight cutoff membrane is suitable for filtering off proteins. The new probe can be a useful tool for high recovery of peptides from living tissues.

Isolation, cloning, and expression mapping of a gene encoding an antidiuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus.

After a blood meal, Rhodnius prolixus undergoes a rapid diuresis to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas' disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresis in R. prolixus is under the neurohormonal control of serotonin and peptidergic diuretic hormones, and thus, diuretic hormones play an important role in the transmission of Chagas' disease. Although diuretic hormones may be degraded or excreted, resulting in the termination of diuresis, it would also seem appropriate, given the high rates of secretion, that a potent antidiuretic factor could be present and act to prevent excessive loss of water and salts after the postgorging diuresis. Despite the medical importance of R. prolixus, no genes for any neuropeptides have been cloned, including obviously, those that control diuresis. Here, using molecular biology in combination with matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry, we determined the sequence of the CAPA gene and CAPA-related peptides in R. prolixus, which includes a peptide with anti-diuretic activity. We have characterized the expression of mRNA encoding these peptides in various developmental stage and also examined the tissue-specific distribution in fifth-instars. The expression is localized to numerous bilaterally paired cell bodies within the central nervous system. In addition, our results show that RhoprCAPA gene expression is also associated with the testes, suggesting a novel role for this family of peptides in reproduction.

Ganglioneuroblastoma of the thymus: an adult case with the syndrome of inappropriate secretion of antidiuretic hormone.

A 61-year-old woman was admitted to the hospital because of general fatigue. Laboratory examinations showed hyponatremia, plasma hypo-osmolarity, and inappropriate increased concentration of the plasma antidiuretic hormone (ADH) in the presence of concentrated urine. Magnetic resonance imaging revealed a mass lesion in the anterior mediastinum. An extended thymectomy was performed under the diagnosis of thymoma with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Histologically the tumor was located in the thymic tissue and was diagnosed as ganglioneuroblastoma. Immunohistochemical studies showed the existence of ADH in the tumor cells. To the knowledge of the authors, this is the first case of ganglioneuroblastoma of the thymus with SIADH.

Localization of vasopressin mRNA and immunoreactivity in pituicytes of pituitary stalk-transected rats after osmotic stimulation.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.62-kb AVP mRNA was detected in the NL and was found to increase 10-fold with 7 days of continuous salt loading. Morphological analysis of the NL of 7-day salt-loaded rats revealed the presence of AVP mRNA in a significant number of GFAP-positive pituicytes in the NL and in areas most probably containing nerve fibers. Eight days after pituitary stalk transection the NL AVP mRNA diminished in animals given water to drink, whereas in those given 2% saline for 18 h followed by 6 h of water, a treatment repeated on 6 successive days beginning 2 days after surgery, the 0.62-kb AVP mRNA was present. The AVP mRNA in the pituitary stalk-transected, salt-loaded rats showed an exclusive cellular distribution in the NL, indicative of localization in pituicytes. Immunoelectron microscopy showed the presence of AVP immunoreactivity in a subpopulation of pituicytes 7 and 10 days after pituitary stalk transection in salt-loaded animals, when almost all AVP fibers had disappeared from the NL. These data show that a subset of pituicytes in the NL is activated to synthesize AVP mRNA and AVP in response to osmotic stimulation.

Synthesis, turnover, and release of peptides from the neurohypophysis of the Jerboa Jaculus orientalis.

Peptide contents of neural lobes from adult jerboas (Jaculus orientalis) under different states of hydration were determined by radioimmunoassay. The amounts of vasopressin, oxytocin, and their associated neurophysins in animals dehydrated for up to 4 weeks were not significantly different from those of controls. The different neurohypophyseal peptide were separated on two different types of gradient using reverse-phase high-performance liquid chromatography. The shape of the chromatograms suggests that, in contrast to the case of the rat, for which only three types of neurophysins have been shown, there are, in jerboa, many subspecies of neurophysins. This was also shown using two-dimensional electrophoresis. Injection of [35S]cysteine into the supraoptic nucleus followed by HPLC of extracts from the neural lobes from animals under different states of dehydration showed that the labeled material is not released any faster in dehydrated animals than in controls. Labeled vasopressin, oxytocin, and neurophysins could still be detected by HPLC 4 weeks after injection. Neural lobes from animals injected with [35S]cysteine were perfused in vitro and the release of neuropeptides was triggered by bursts of electrical pulses and also by K(+)-induced depolarization. The amplitude of the rate constant for release and the amounts of vasopressin and of radiolabeled material released were similar in animals dehydrated for up to 3 weeks and in controls. Under physiological conditions similar to those that would be expected to occur in their natural habitat, the jerboas appear to have a hypothalamoneurohypophyseal system which is down-regulated.

Single-step isolation and sequencing of vasopressin and oxytocin precursors.

The pre- and post-Golgi processing of preprovasopressin and prepro-oxytocin was evaluated by microsequencing for incorporated radiolabel. 35S-Cysteine and 3H-fucose were microinjected into rat supraoptic nuclei (SON), and proteins and peptides related to the biosynthesis of vasopressin (VP) and oxytocin (OT) were isolated at various times from the supraoptic nuclei and neural lobe by employing a one-step procedure of high performance liquid chromatography (HPLC). These proteins and peptides were recognized through their binding to specific antibodies against VP, OT, and rat neurophysins (RNPs), and by their binding to ConA-Sepharose. Two immunoreactive glycoproteins related to VP biosynthesis were recovered from the SON and both contained fucose and had a 35S-cysteine placement consistent with the location of the hormone sequence at the N-terminus. SDS-electrophoresis revealed the major protein form to be 21,000 daltons and the minor protein form to be 19,000 daltons. One nonglycosylated protein of 16,000 daltons related to oxytocin biosynthesis was recovered from the SON, and this protein also had a 35S-cysteine placement consistent with an N-terminal OT sequence. These data provide the first sequential evidence that prior to, or shortly after, packaging in the Golgi the preprohormones of VP and OT have lost their entire leader-peptide structures.

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.

The vasopressin precursor in the Brattleboro (di/di) rat.

The vasopressin precursor in the rat hypothalamus has been studied, using trypsin to release desglycinamide vasopressin and coupling it to glycinamide (T & G treatment). The resulting amidated nonapeptide was detected and measured with a radioimmunoassay for vasopressin. The "vasopressin" produced in this way had the full immunoreactivity of the authentic peptide but eluted from an hplc column 1 min earlier and appeared to have a larger molecular weight. It was found that T&G treatment generated vasopressin immunoreactivity in extracts of the supraoptic nucleus (SON) of the Brattleboro rat in just the same way as it did in normal animals. Furthermore, this procedure produced vasopressin immunoreactivity in those hplc fractions from Brattleboro SON extracts that corresponded with the elution time of vasopressin precursor. Similar amounts of "vasopressin" could be generated from Brattleboro and normal SONs. These results support the suggestion that the Brattleboro SON synthesizes an aberrant vasopressin precursor which is not processed by the cell.

Preparative high-performance liquid chromatography of peptides on a new reversed-phase packing material, Kromasil C18.

Preparative reversed-phase high-performance liquid chromatography has found wide use in the production of peptides for pharmaceutical formulations. Purity of the substance and overall economy of the chromatographic system are the most important criterias. In this sense optimized, silica particles and production process with capability to separately control parameters important to chromatography, are essential to high-performance chromatography. Kromasil C18 packing material was tested and evaluated in respect of its selectivity, flow and pressure properties, resolution, load capacity, recovery, adsorption effects, mechanical strength and chemical degradation.

Two vasopressin-like peptides in the pig testis?

Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR. High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP. The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms.

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.

[Evidence of apparent vasopressin and oxytocin peptides in the brain of the leech Rhynchobdelle Theromyzon tessulatum (O.F.M.)]. / Mise en évidence de peptides apparentés à la vasopressine et à l'ocytocine dans le cerveau de l'hirudinée Rhynchobdelle Theromyzon tessulatum (O.F.M.).

Vasopressin- and oxytocin-immunoreactive cells have been demonstrated in the brain of the leech Theromyzon tessulatum. A mapping of their localization in the different compartments of the brain has been undertaken. The cells immunohistochemically identified have been compared to previously described cell types defined by classical staining methods for neurosecretory material. Preliminary results obtained with high performance liquid chromatography confirm the presence in brain homogenates of substances with chromatographic properties similar to that of vertebrate nonapeptides. The possible role of these vasopressin- and oxytocin-like substances in osmoregulation is discussed.

HPLC separation of vasopressin-like hormones in chicken neurohypophysial extracts.

HPLC techniques were used to identify peptides that possess vasopressin-like immunoreactivity in the chicken neurohypophysis. The presence of arginine vasotocin (AVT) was confirmed together with arginine vasopressin (AVP). That the presence of AVP may be confined to the chicken, and not other avian species, was concluded from the absence of the hormone in the neurohypophysis of the duck and turkey. The chicken thus resembles some members of the suiformes and metatheria in possessing two vasopressin-like peptides.

Ontogeny of bovine neurohypophysial hormone precursors. II. Foetal copeptin, the third domain of the vasopressin precursor.

Vasopressin, MSEL-neurophysin and a glycopeptide, here referred to as copeptin, are three fragments of a common protein precursor processed during axonal transport from hypothalamus to neurohypophysis. Neurohormones and neurophysins purified from 7-9-month-old bovine foetuses have previously been shown to be identical with those found in the adult. Copeptin has now been isolated from 7-9-month and 3-month-old bovine foetuses and chemically characterized. It can be concluded from the nature of the three precursors that the same vasopressin gene is expressed in the adult and the 7-9-month-old foetus.

Human neuropeptide Y, somatostatin and vasopressin precursors identified in cell-free translations of hypothalamic mRNA.

Messenger mRNA has been prepared from post mortem human hypothalami and translated in a cell-free system. Using specific antibodies, biosynthetic precursors have been identified to neuropeptide Y (12 kDa), somatostatin (15 kDa) and vasopressin/neurophysin (19 kDa).