Neurohypophyseal peptides in aging and Alzheimer's disease.

The neurohypophyseal hormones arginine-vasopressin (AVP) and oxytocin (OT) are produced in the neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nucleus and in the much smaller cells of the suprachiasmatic (SCN) nucleus. The SON is the main source of plasma AVP. Part of the AVP and OT neurons of the PVN join the hypothalamo-neurohypophyseal tract, whereas others send projections to the median eminence or various brain areas, where AVP and OT are involved in a number of central functions as neurotransmitters/neuromodulators. AVP and OT from the PVN can also regulate via the autonomous innervation endocrine glands and fat tissue. OT is produced for a major part in the PVN but some OT neurons are present in the SON. Moreover, both AVP and OT containing neurons are observed in the "accessory nuclei", i.e. islands situated between the SON and PVN. The SCN is the biological clock, and the number of AVP expressing neurons in the SCN shows both diurnal and seasonal rhythms. In addition to these hypothalamic areas, AVP and OT may be found to a lesser extent in some other brain areas, such as the bed nucleus of the stria terminalis, diagonal band of Broca, nucleus basalis of Meynert, lateral septal nucleus, globus pallidus and the anterior amygdaloid nucleus, as well as in the peripheral tissues. The AVP and OT containing neurons should not be considered as one system. Prominent functional differences exist between the different nuclei. The heterogeneity also becomes clear from the marked differences in the neurohypophyseal peptides containing neurons of the SON, PVN and SCN during aging, and in the most prevalent age-related neurodegenerative diseases, i.e. Alzheimer's disease (AD). For those reasons, we will discuss the SON, PVN and SCN separately.

The hypothalamo-hypophyseal system of the white seabream Diplodus sargus: immunocytochemical identification of arginine-vasotocin, isotocin, melanin-concentrating hormone and corticotropin-releasing factor.

The distribution of the neurosecretory hormones vasotocin, isotocin and melanin-concentrating hormone and the hypophysiotropic hormone corticotropin-releasing factor was studied in the hypothalamo-hypophyseal system of the white seabream (Diplodus sargus) using immunocytochemical techniques. Magnocellular and parvocellular perikarya immunoreactive for arginine-vasotocin and isotocin were present in the nucleus preopticus. Perikarya immunoreactive for arginine-vasotocin extended more caudally with respect to isotocin-immunoreactive perikarya. Parvocellular perikarya were located at rostroventral levels and magnocellular perikarya in the dorsocaudal portion of the nucleus. Arginine-vasotocin and isotocin did not coexist in the same neuron. Fibres immunoreactive for arginine-vasotocin and isotocin innervated all areas of neurohypophysis and terminate close to corticotropic and melanotropic cells. Perikarya immunoreactive for melanin-concentrating hormone and corticotropin-releasing factor were observed in the nucleus lateralis tuberis, with a few neurons in the nucleus periventricularis posterior. In addition, melanin-concentrating hormone immunoreactive perikarya were detected in the nucleus recessus lateralis. The preoptic nucleus did not show immunoreactivity for these antisera. Fibres showing melanin-concentrating hormone and corticotropin-releasing factor immunoreactivity ended close to the melanotropic and somatolactotrophic cells of the pars intermedia, and close to the corticotrophic cells of the rostral pars distalis.

Identification of neurohypophysial peptides in the ovaries of several mammalian and nonmammalian species.

Ovarian tissue from a variety of mammalian and nonmammalian species were extracted in acid. All extracts contained both oxytocin- and vasopressin-like immunoreactivites as determined by radioimmunoassay. Analysis by high performance liquid chromatography revealed the presence of oxytocin in all ovarian extracts examined. This was in contrast to the corresponding posterior pituitary gland which other workers have shown do not necessarily contain the oxytocin peptide. It is suggested that oxytocin may play an important role in ovarian function in species of differing phylogeny.

Neurohypophysial hormones of the 1-month-old bovine fetus: absence of vasotocin during mammal development.

The neurohypophysial hormones of the 1-month-old bovine fetus have been identified by their positions in ion-exchange chromatography and their retention times in high-pressure reverse-phase partition chromatography. Arginine vasopressin and oxytocin have been recognized. The molar ratio vasopressin/oxytocin in neurohypophysis is about 6 in the 1-month-old fetus compared with 4 in the 3-month-old fetus, 2.7 in the 7-month-old fetus and 1 in the adult. Vasotocin is virtually absent even in the early fetus (less than 0.1% of arginine vasopressin). The occurrence of a vasotocin gene expressed in the fetus but silent in the adult appears unlikely.

Immunohistochemical studies on the distribution of endogenous digitalis-like substance (EDLS)-containing neurons in the rat hypothalamus, with special consideration on the possibility of their coexistence with posterior lobe hormones.

The distribution of the perikarya of endogenous digitalis-like substance (EDLS)-containing neurons in the rat hypothalamus was studied by immunohistochemistry using digoxin and digitoxin antiserum. In addition, the possible coexistence of EDLS and posterior lobe hormones was examined using an immunohistochemical double-staining technique. Digoxin-like immunoreactive neurons were demonstrated in the paraventricular and supraoptic nuclei and other hypothalamic areas. However, digitoxin-like immunoreactive neurons were not detected in the hypothalamus. A portion of the digoxin-like immunoreactive neurons showed immunoreactivity for vasopressin or oxytocin. From these results, it is suggested that these digoxin-like immunoreactive neurons correspond to "EDLS-producing neurons," and that EDLS seems to act not only as a natriuretic substance but also as a neurohormone or neurotransmitter.

The neurohypophysis: recent developments.

The hormones of the neurohypophysis, vasopressin and oxytocin, have now been shown to be synthesized as part of a prohormone complex that includes a vasopressin-neurophysin and oxytocin-neurophysin, respectively. In addition, for vasopressin, there is a glycopeptide as part of the prohormone. For each hormone the prohormone is packaged into neurosecretory granules and transported via axons to the posterior pituitary gland. In addition to this "classic" system, axons containing neurohypophyseal hormones project to the median eminence for release into portal vessels, and to other areas of the brain and spinal cord where the peptides may function as neurotransmitters rather than as hormones. As neurotransmitters, the neurohypophyseal hormones may be involved in the regulation of certain autonomic functions. Vasopressin and oxytocin are secreted into the cerebrospinal fluid where there is a diurnal rhythmic secretion of the peptides in several animal species (some species have a predominant rhythm of vasopressin and others a rhythm of oxytocin). Neurohypophyseal peptides are synthesized in some non-neuronal tissues where the function is unknown, and recently a novel peptide with similarities to oxytocin and vasotocin has been identified. The relationship of this novel peptide to the neurohypophyseal peptides is unknown. These new developments are likely to elucidate many new functions for the hormones of the neurohypophysis.

[Phylogeny of neurohypophyseal hormones in birds: microidentification of mesotocin and vasotocin in the ostrich (Struthio camelus)]. / Phylogénie des hormones neurohypophysaires des Oiseaux: microidentification de la mésotocine et de la vasotocine chez l'Autruche (Struthio camelus).

Ostrich (Struthio camelus) neurohypophysial hormones have been isolated from 5 freeze-dried posterior pituitary glands. Purification has involved three steps: a first molecular sieving on Sephadex G-75 for eliminating proteins, a second molecular sieving on Bio-Gel P4 for separating the two active principles and a high pressure reverse-phase liquid chromatography (HPLC) on Nova-Pak C 18 with an 10 mM acetate-acetonitrile gradient for isolating each hormone. The active peptides have been identified by their retention time in HPLC and their amino acid composition. Mesotocin and vasotocin have thus been characterized. Although the phylogeny of Ratites is disputed, in particular their possible common origin with Carinates, which include most of the living birds, species of the first sub-class seem to have the same neurohypophysial hormones as those of the second.

The nonmammalian-mammalian transition through neurohypophysial peptides.

Neurohypophysial hormones are particularly proper evolutionary tracers. Whereas Eutherian mammals have oxytocin and virtually always arginine vasopressin, nonmammalian tetrapods possess instead mesotocin and vasotocin. The transitions of mesotocin-oxytocin and vasotocin-vasopressin involved in the passage of reptiles-mammals seem to have occurred independently. Australian marsupials are endowed with mesotocin but American marsupials have either oxytocin (South-American opossums) or both oxytocin and mesotocin (North-American opossum). These results suggest that Australian Metatherians have preserved reptilian mesotocin and used it for milk-ejecting function whereas the change mesotocin-oxytocin appeared only in the American line. All marsupials have either arginine vasopressin or lysipressin and phenypressin (Australians) or lysipressin and arginine vasopressin (Americans). It is assumed that the change of vasotocin into arginine vasopressin occurred very early, perhaps in mammal-like reptiles, and duplication of the gene with subsequent mutations has led to the presence of two vasopressin-like peptides in most Metatherians.

Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules.

A method is described for the quantitative determination of the content of subcellular organelles such as secretory granules. Purified subcellular fractions of the organelle are prepared and aliquots are assayed for hormones, for example. To determine the number of organelles per fraction, known numbers of latex particles of a size similar to the organelle are added to other aliquots of the subcellular fractions. Latex particles and organelles are then pelleted together by centrifugation. The ratio between latex particles and organelles can be determined by morphometric analysis of ultrathin sections taken through the full thickness of the pellet. The number of organelles and hence their content of the substance assayed can then be calculated. We have applied this technique to posterior pituitary neurosecretory granules, the content of which has already been estimated by a different method. Newly formed neurosecretory granules from oxen and rats were found to have a content of approximately equal to 85,000 molecules of hormone and neurophysin. Aged neurosecretory granules from the same neural lobes appeared to contain less hormone and neurophysin, but this was shown to be the result of loss of material from the granules during isolation in media of 360 mosM. Such loss could be prevented by isolation in hypertonic (660 mosM) media.

Effect of adrenalectomy and administration of hypertonic saline on the content of aldehyde fuchsin-positive neurosecretory material and posterior lobe hormones in the median eminence and the neural lobe of rats.

The influence of adrenalectomy and administration of hypertonic saline on the amount of vasopressin, oxytocin, and neurophysin contained in the median eminence and the neural lobe of rats was studied by means of the following methods: (i) morphometric and microphotometric analyses of aldehyde fuchsin-stained histological sections of the neurohypophysis; (ii) immunohistochemical demonstration of vasopressin, oxytocin, and neurophysin in the neurohypophysis, and (iii) radioimmunological measurement of vasopressin and oxytocin in extracts of the median eminence and the neural lobe. Adrenalectomy increases the amount of vasopressin and neurophysin in the external layer of the median eminence but does not change the content of oxytocin. It has no influence on the amount of vasopressin, oxytocin, and neurophysin demonstrable in the inner layer of the median eminence and in the neural lobe two weeks after the operation. Hypertonic saline markedly diminishes the vasopressin, oxytocin, and neurophysin content of the inner layer of the median eminence and the neural lobe but reduces only slightly, if at all, the amount of vasopressin and neurophysin in the outer layer of the median eminence. The findings support the concept that osmotic stress reduces only the vasopressin and oxytocin content of the hypothalamus-neural lobe system and has no or only little influence on the vasopressin content of the outer layer of the median eminence.

Reversed phase high pressure liquid chromatography for the identification and purification of neuropeptides.

Reversed phase HPLC has wide applications in studies on neuropeptides. It provides a fast and effective technique for assessing the purity of synthetic peptides and for purifying mg amounts of synthetic peptides (examples: angiotensins II and III and analogues; neurohypophysial hormones). Due to the very small quantities of peptides which can usually be safely recovered after HPLC, the method is also useful in the isolation, purification and sequencing of peptides from biological sources (examples: urotensin I and II), and in the identification of neuropeptides in tissues when coupled with radioligand-binding displacement assays (example: [arginine 8]vasotocin in the anterior ganglia of Aplysia californica.

Protein binding of coherin and other small molecules by thin-layer gel chromatography.

The protein binding stoichiometry of small molecules is here determined on a nanomole scale by a simplified procedure utilizing chromatography on thin layers of cross-linked dextran gels. New data are presented on the thin layer chromatographic properties of representative ligands, including a-amino acids, peptides, dyes and fluorigenic reagents, in relation to their molecular weights, polar characteristics, gel water regain values and denaturants, providing criteria for the general application of this method to studies of ligand binding with large as well as small molecules. By this procedure coherin peptides, A1 and B1--4, respectively, bind to coherin C in the molar ratio, 2:1, with a binding constant of about 10(5) M-1. Coherin C is believed to act as a carrier peptide.

Development pharmacokinetics of the posterior pituitary hormones.

Arginine vasotocin, arginine vasopressin, and oxytocin play a critical role in the stimulation of labor and delivery and in salt and water homeostasis in the newborn infant. The authors present information on their chemistry, secretion, and metabolism, and discuss the clinical effects upon target organs of their presence or absence.