Identification of neurohypophysial hormones with their antisera.

Identifying posterior pituitary hormones in body fluids or neurohypophysial extracts was heretofore partially achieved by using pharmacologic potency ratios or semispecific inactivation by thioglycolate or enzymes. Production of antisera against oxytocin and lysine-vasopressin has prompted us to test their specificity against lysine-vasopressin, arginine-vasopressin, arginine-vasotocin, and oxytocin. In ethanol anesthetized rats, antidiuretic and milk-ejection activities were assayed for each peptide-antiserum combination after 0, 30, 60, and 90 min of incubation. Results indicate that (a) oxytocin antiserum inactivates oxytocin, but not arginine-vasopressin, lysine-vasopressin, or arginine-vasotocin; vasopressin antiserum inactivates arginine-vasopressin and lysine-vasopressin, but neither oxytocin nor arginine-vasotocin; (b) an identifiable antigenic site exists for each hormone; (c) relatively specific identifications of natural neurohypophysial peptides are possible using antisera and bioassays; (d) this method is promising for identifying neurohypophysial peptides in body fluids and pituitary extracts; and (e) active and passive immunization against oxytocin and vasopressin may increase our understanding of their physiologic functions.

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.

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.

Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.

Hormone storage in individual neurosecretory granules of the pituitary gland: A quantitative ultrastructural approach to hormone storage in the neural lobe.

A quantitative ultrastructural study of the neurosecretory granules in the neural lobe of the rat was performed in order to calculate the number of such granules in the neural lobe and thus, provided all hormone is intragranular, to derive the hormone content of individual granules. The results suggest that the gland contains 1 - 44 X 10(10) granules and that an individual granule (mean diameter 160 nm) contains about 84000 hormone molecules. The errors involved in the many measurements made are analysed, and the calculated value for the amount of space available for each hormone molecule within the granule is shown to agree well with independently reported data for the expected size of a molecule of protein-hormone complex, and for the size of subunits visualized in freeze-etched material. These results are compatible with exclusive intragranular hormone storage as a 'solid' core, and the physical form that this core might take is discussed. The data are used to express what is known of hormone turnover and release in terms of the numbers of granules involved. Comparison of different parts of the neural lobe suggests that, apart from the most anterior part of the gland, there is little regional variation in quantitative aspects of granule storage in the neural lobe.

Distribution of neurosecretory granules among the anatomical compartments of the neurosecretory processes of the pituitary gland: a quantitative ultrastructural approach to hormone storage in the neural lobe.

The distribution of neurosecretory granules in various anatomical compartments of neurosecretory axons of the neural lobe of the rat pituitary has been studied. Apart from the most anterior tip of the gland, where granules are largely restricted to undilated axons and a few 'swellings', the proportional compartmental storage of granules is essentially homogeneous for the rest of the gland: 13% of granules are found in undilated axons, 31% in axonal 'endings' (which contain microvesicles and abut the basement membrane) and 55% in axonal 'swellings' (which are devoid of significant numbers of microvesicles). These values indicate that the 'endings' contain a much greater proportion of the total number of granules stored in the neural lobe than would be predicted if the readily releasable pool of hormone were composed of all the granules in the 'endings'. Some further constraint on granule release either physiological or anatomical (e.g. the position of the granule in relation to the plasmalemma of the 'ending') must be operative.

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.

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.

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.

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.