Isolation of neurosecretory granules containing vasotocin, mesotocin, MSEL- and VLDV-neurophysins from goose neurohypophysis

Neurosecretory granules have been isolated from goose posterior pituitaries and their contents have been analyzed by reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis. Vasotocin and mesotocin have been identified by their biological activities and their retention times compared with those of synthetic peptides. MSEL- and VLDV-neurophysins have been characterized by their N-terminal sequences, their electrophoretic migrations and their retention times, compared with those of purified goose neurophysins. In contrast to the two-step processing of mammalian provasopressin, processing of the vasotocin - MSEL-neurophysin precursor appears to involve only one cleavage giving the hormone and a "big" MSEL-neurophysin homologous to mammalian MSEL-neurophysin extended by copeptin.     

Cytosol androgen receptors in guinea pig brain and pituitary

Cytosol androgen receptors were assayed in guinea pig brain and pituitary tissues, using [3H]R1881 as ligand. These receptors had an apparent Kd of 0.04 nM and were androgen-specific (R1881 greater than dihydrotestosterone greater than testosterone = estradiol greater than progesterone). Concentrations of cytosol androgen receptors in castrated adult male guinea pigs were 12.2, 11.6, 6.9, 2.6 and 1.3 fmol per mg protein in anterior pituitary, hypothalamus, medial preoptic area, amygdala and cortex, respectively. No significant differences in receptor levels were observed between castrated adult males and females. The concentration of androgen receptors was significantly lower in the hypothalamus, medial preoptic area and anterior pituitary of castrated neonatal males than in castrated adult male guinea pigs. The systemic injection of the alpha 1-adrenergic antagonist, prazosin, had no significant influence on androgen receptor levels in castrated males in any brain area.     

Acetylcholinesterase containing nerve fibres in guinea pig ovary

Summary The distribution of acetylcholine esterase (AChE) in the ovary of normal as well as of sympathectomized guinea pigs was studied. In normal animals AChE-positive nerve fibres were found organized in a perivascular plexus. Some nerve fibres reach the corpus luteum and the follicular wall. Chemical sympathectomy performed with the neurotoxin 6-hydroxydopamine caused an almost complete disappearance of AChE positive nerve fibres suggesting that most of the AChE activity of ovarian nerves is localized in adrenergic nerve fibres. Enzyme activity was also present in smooth muscle cells of the vascular tree.     

Target cells for dihydrotestosterone in the guinea pig pituitary gland

Adult male guinea pigs were castrated and 24 h later injected with [3H]dihydrotestosterone. Their pituitary glands were then processed for autoradiographic and immunocytochemical analyses. Three percent of the parenchymal cells in the pars distalis were radiolabelled. These cells were primarily gonadotrophs; a small population of thyrotrophs were also found to retain the [3H]androgen. Labelling occurred in cells of the pars intermedia (3.2%) as well as in 9.6% of the pituicytes. These data indicate that in addition to gonadotrophs, the pars nervosa is a major target for dihydrotestosterone in the guinea pig pituitary gland.     

A sexually dimorphic vasopressin innervation of auditory pathways in the guinea pig brain

Vasopressin-like immunoreactivity was detected in the auditory brainstem of female guinea pigs. Stained cell bodies and fibres were found in the inferior colliculus and in the ventral trapezoid body, and immunoreactive fibres in the dorsal cochlear nucleus. No vasopressin immunoreactivity was detected in the auditory brainstem of male guinea pigs. Using oxytocin antisera we found neither immunoreactive perikarya nor fibres in the auditory pathways of guinea pigs of both sexes.     

Isolation and partial amino acid sequence analysis of nerve growth factor from the guinea pig prostate

Nerve growth factor (NGF) has been purified from the guinea pig prostate using a modification of the Bocchini-Angeletti method for isolating 2.5S NGF from mouse submaxillary glands. As with the mouse preparation, guinea pig prostate NGF appears to migrate as a high molecular weight entity at physiological pH. Following dissociation, NGF, active in neurite proliferation assays and similar in size to mouse 2.5S NGF, can be isolated by chromatography on a column of carboxymethyl-cellulose at pH 4.8. Based on gel filtration, SDS-polyacrylamide gel analysis, and amino-terminal sequence studies, this material consists of two, noncovalently linked, identical polypeptide chains each with a molecular weight of about 13,000. The amino-terminal third of the polypeptide chain is at least 90% identical to the corresponding region of the murine molecule, confirming the homology of the guinea pig prostate protein to NGFs obtained from different tissues in other species. However, in contrast to the mouse preparation, the putative high molecular weight form of guinea pig NGF does not contain a subunit with arginine esteropeptidase activity. Although there is an abundance of this enzymatic activity in the homogenate, it does not appear to be associated with the fractions containing NGF. This apparent difference in the mouse and guinea pig material is of interest because the mouse γ subunit, possessing the arginine esteropeptidase activity, has been alleged to participate in the processing of a precursor of the β NGF polypeptide chain.     

The hormone-binding site of neurophysins: Binding of vasopressin to the N-terminal sub-domain dissected from human MSEL-neurophysin through endopeptidase Lys-C

Human MSEL-neurophysin has been dissected into two halves by endopeptidase Lys-C, taking advantage of a peculiar Lys59-Ala60 bond. Two sub-domains, N-terminal (1–59) and C-terminal (60–93), have been separated. These sub-domains have been purified by reverse-phase high pressure liquid chromatography and identified by their N-terminal sequences. The N-terminal fragment comprises two chains 1–18 and 19–59, because of the presence of a second lysine residue in position 18, whereas the C-terminal fragment (60–93) is a single chain. Hormone-binding experiments have been carried out using vasopressin or vasopressinyl-Gly-Lys-Arg and testing the ability of the hormone-neurophysin complex to precipitate at pH 3.9 with 10% NaCl. The N-terminal sub-domain precipitates in presence of vasopressin in the same way as native neurophysin whereas the C-terminal sub-domain does not. It can be concluded that the hormone-binding site is located in the 1–59 region of neurophysin.     

Microheterogeneous neurophysins in newly formed and aged neurosecretory granules

To find out, whether microheterogeneity of neurophysins is due to aging of neurosecretory granules we investigated neurophysin proteins of porcine origin from newly formed and aged granules, as well as from two different extraction methods from whole posterior pituitary glands by high performance liquid chromatography. In newly formed as well as in aged granules all microheterogeneous forms of the neurophysins were present in nearly identical relation suggesting that microheterogeneity is not due to maturation or post matural degradation. In comparison, the crude material obtained by the method of Uttenthal and Hope showed a comparable content of the different neurophysins while Chauvet's method resulted in a different HPLC pattern. In summary, neurophysin heterogeneity is obviously not due to aging processes.     

Choline acetyltransferase activity in the neurointermediate lobe of the rat pituitary

Summary CAT activity was determined in the neurointermediate lobe of the rat's pituitary gland. The level of enzyme activity in the adult rat is of 386.2±39.1 picomoles/hour/neurointermediate lobe. CAT activity is present in the pituitary gland of the newborn animal and increases with age. In organotypic cultured neurointermediates lobes CAT decreases progressively and practically disappears after 10 days.     

Localization and characterization of binding sites for vasopressin and oxytocin in the brain of the guinea pig.

Using autoradiography on film, specific binding sites for arginine-vasopressin (AVP) and for oxytocin (OT) were localized in various areas of the brain of adult male guinea pigs. Vasopressin binding sites were detected with [3H]AVP or with [125I]VPA, a recently synthetized linear vasopressin antagonist radiolabeled with 125I. [125I]VPA and [3H]AVP yielded similar results, thus suggesting that AVP binding sites present in the guinea pig brain are V1 type receptors. Supporting evidence on this was obtained in competing studies using structural analogues allowing to discriminate V1 receptors from V2 and from OT receptors. Oxytocin binding sites were labeled with [3H]OT or with the iodinated OT antagonist [125I]OTA; both ligands yielded similar results. The localization in the guinea pig brain of AVP binding sites differed from that of OT binding sites. AVP binding sites were mainly detected in the olfactory bulb and throughout the cerebral cortex. Oxytocin binding sites were most noticeable in the hypothalamic ventromedial nucleus, in the amygdaloid complex and in restricted areas of the cerebral cortex. A comparison of the present data with those previously described in the rat, the mouse, the human and the hamster brain suggests that similar binding sites are present in these species, but that their anatomical distribution differs markedly. These data are discussed in relation to immunocytochemical and electrophysiological data which suggest that binding sites detected by autoradiography may represent, at least in part, functional neuronal receptors.     

Choline acetyltrasferase activity in the neurointermediate lobe of the rat pituitary

CAT activity was determined in the neurointermediate lobe of the rat's pituitary gland. The level of enzyme activity in the adult rat is of 386.2 +/- 39.1 picomoles/hour/neurointermediate lobe. CAT activity is present in the pituitary gland of the newborn animal and increases with age. In organo-typic cultured neurointermediates lobes CAT decreases progressively and practically disappears after 10 days.     

Mu and delta opioid receptor regulation of pro-opiomelanocortin peptide secretion from the rat neurointermediate pituitary in vitro

We investigated the ability of selective opioid agonists and antagonists to influence pro-opiomelanocortin peptide secretion from the rat neurointermediate lobe in vitro. The mu-opioid agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin) significantly stimulated beta-endorphin and alpha-melanocyte-stimulating hormone release relative to controls early (30 min) in the incubation period. Similar effects on beta-endorphin secretion were observed with the selective mu-opioid agonist dermorphin. The delta-opioid receptor agonist DPDPE ([D-Pen(2,5)]enkephalin) weakly inhibited beta-endorphin secretion relative to controls while the kappa-opioid receptor agonist U50488 had no effect. The mu-opioid selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)) inhibited basal beta-endorphin secretion while kappa- and delta-opioid receptor antagonists had no effect. Our data support a role for local mu-opioid receptor control of intermediate lobe pro-opiomelanocortin peptide secretion. Peptide secretion from melanotropes appears to be tonically stimulated by activation of mu-opioid receptors in the absence of intact neuronal innervation to the intermediate lobe.     

Angiotensin I-generating acid endopeptidase activity in neurosecretory vesicles isolated from bovine pituitary

Secretory vesicles purified from the neural and intermediate lobes of the bovine pituitary contain acidic endopeptidases which are capable of converting renin tetradecapeptide (RTD) substrate to Angiotensin I (AI). Preliminary characterization of the neurosecretory vesicle (NSV) endopeptidase showed that it had a pH optimum of 4.0, and unlike renin was inactive at pHs greater than 6.0. It is inhibited by 10(-6) M pepstatin A, but not by PMSF, leupeptin, PMBS, or the specific renin inhibitor H-142. This NSV endopeptidase differed from cathepsin D in that it was unable to degrade alpha-casein, but was quite active in generating AI from RTD (Vmax = 5 moles/g protein/hour). No enzyme activity that could convert AI to Angiotensin II could be detected in the NSVs suggesting that the acidic endopeptidase is involved in processing neurosecretory vesicle proteins other than those associated with the renin angiotensin system in the brain.     

Ultrastructural immunolocalization of vasopressin and neurophysin in neurosecretory cells of dehydrated rats

It has been proposed that neurosecretory material may be transported in a non-vesicular compartment in magnocellular neurons in the hypothalamus of osmotically stimulated rats. We have reexamined this issue using postembedding electron microscopic immunoperoxidase labeling of the secreted peptides (vasopressin and neurophysin) found in these neurons. Reaction product was found exclusively over neurosecretory vesicles in cell bodies, and in axons in the median eminence and neurohypophysis. The findings are consistent with an exclusive secretory vesicle location of the neuropeptides in the hypothalamo-neurohypophysial system of both normal and dehydrated rats.     

Corticotropin-releasing factor (CRF) and neurotransmitters modulate melanotropic peptide release from rat neurointermediate pituitary in vitro

Isolated pituitary neurointermediate lobes from adult male Sprague-Dawley rats were incubated for up to 90 minutes in media containing stimulatory or inhibitory agents, to study the potential effects on secretion of alpha-melanocyte stimulating hormone (alpha-MSH) from the pars intermedia. Dopamine (DA), as expected, inhibited the release of alpha-MSH, as measured by radioimmunoassay (RIA), while corticotropin-releasing factor (CRF) and serotonin (5-HT) stimulated peptide release to varying degrees over the incubation period. A combination of DA with CRF produced an inhibition of peptide release, suggesting that DA suppresses the stimulatory effects of the hypothalamic peptide. Immune staining of pituitary tissue for beta endorphin or alpha-MSH, and quantified for staining intensity, supported the specific effects of stimulation or inhibition of the respective treatments. In addition, electron microscopy of incubated tissues demonstrated alterations in secretory vesicles and membranous organelles which were consistent with peptide secretion patterns. Our in vitro findings suggest that neurotransmitters and hypothalamic peptide, which are present together in vivo in the pituitary intermediate lobe, interact at the level of the endocrine cells to modulate pro-opiomelanocortin peptide secretion.     

A vasopressin and oxytocin containing nucleus in the pig hypothalamus that shows neuronal changes during puberty

A vasopressin and oxytocin containing nucleus is described for the first time in the pig hypothalamus. It is located near the third ventricle, just dorsal to the suprachiasmatic nucleus, and consists of magnocellular neurons, similar to those of the supraoptic nucleus and paraventricular nucleus. Morphometric analysis of neuronal number, size, density, and volume was performed at four different ages: 1 day, 7 weeks, 16 weeks, and 30 weeks postnatally. No sex difference in these parameters was observed. In this period the volume of the nucleus increased gradually from 6.6 x 10(-3) to 54.2 x 10(-3) mm3. One day after birth 1,215 +/- 191 (mean +/- SEM) neurons were present in the vasopressin and oxytocin containing nucleus, followed by a decrease to 771 +/- 80 neurons at 7 weeks and 697 +/- 116 at 16 weeks. Between 16 and 30 weeks (puberty) there was a dramatic increase in neuron number up to 1,765 +/- 214 neurons. This increase in the number of vasopressin and oxytocin containing neurons in the pig hypothalamus is much later in development than has ever been reported so far.     

Locus coeruleus effects on baroreceptor responsiveness and activity of neurosecretory vasopressin cells

The locus coeruleus (LC) has previously been implicated in the regulation of vasopressin secretion. To further investigate this issue experiments were done in which extracellular recordings were obtained from functionally identified neurosecretory vasopressin (VP) cells of the rat supraoptic nucleus. Electrolytic lesions of the ipsilateral LC reduced the proportion of VP cells inhibited by carotid baroreceptor activation from 93% to 35%; the inhibitory effect of aortic depressor nerve stimulation was unchanged. Electrical stimulation of the LC altered the discharge probability of 20% of VP cells tested, the predominant effect being excitation. In contrast to the effects of electrolytic lesions and electrical stimulation, neither chemical inhibition nor stimulation of the LC, by local injection of neuroactive amino acids, altered VP cell baroreceptor responsiveness or spontaneous discharge. These data indicate that while fibres of passage in the LC region can influence VP cell excitability, particularly responses to carotid baroreceptor activation, LC cells do not regulate VP cell function or, by implication, the secretion of this vasoactive and antidiuretic hormone.     

Effects of neurointermediate pituitary lobectomy on humoral and cell-mediated immune responses in the rat

OBJECTIVE: Chronic stress is characterized by an increased activity of the hypothalamic-pituitary-adrenocortical axis and decreased humoral and cell-mediated immune responses. In the rat, corticosterone is the principal natural immune suppressor. Neurointermediate pituitary lobectomy (NIL) in rats induces diabetes insipidus and protracted increases in basal adrenocorticotropin and corticosterone plasma levels, a situation that resembles chronic stress. In this paper, we evaluated the effects of NIL on humoral (hemagglutinin titers and footpad swelling to sheep red blood cells--SRBC) and cell-mediated immune responses (contact hypersensitivity to dinitrochlorobenzene). METHODS: The studies were conducted on NIL Wistar rats (body weight 150-200 g) 3 weeks after surgery. For comparisons, nonoperated control rats were used. RESULTS: NIL resulted in an increased water intake. Body weight gain and adrenal, thymus, and spleen weights were within the range of nonoperated controls. Eight days after SRBC immunizations a second SRBC injection into the footpad resulted in a decreased swelling response in NIL rats. The hemagglutinin titers were also reduced in the NIL rats. CONCLUSIONS: These results indicate that: (1) NIL reduces humoral immune responses and decreases the cell-mediated immune response; (2) the immune alterations are most likely due to the increased activity of the hypothalamic-pituitary-adrenocortical axis induced by NIL, and (3) NIL animals constitute a valuable paradigm to study hypothalamic-pituitary-immune interactions.