The vasopressin precursor is not processed in the hypothalamus of Wolfram syndrome patients with diabetes insipidus: evidence for the involvement of PC2 and 7B2

Wolfram syndrome (WS) is characterized by optic atrophy, insulin-dependent diabetes mellitus, vasopressin (VP)-sensitive diabetes insipidus, and neurosensory hearing loss. Here we report a disturbance in VP precursor processing in the supraoptic and paraventricular nuclei of WS patients. In these patients with diabetes insipidus we could hardly detect any cellular immunoreactivity for processed VP in the supraoptic and paraventricular nuclei. On the other hand, in the paraventricular nucleus a considerable number of cells immunoreactive for the VP precursor were present. In addition, the proprotein convertase PC2 and the molecular chaperone 7B2 were absent. As expression of PC2 and 7B2 was detected in the nearby nucleus basalis of Meynert of one WS patient and in the anterior lobe of the other WS patient, the absence of the two proteins in the paraventricular nucleus was not due to mutations in their genes. These results indicate that in WS patients with diabetes insipidus, not only does VP neuron loss occur in the supraoptic nucleus, but there is also a defect in VP precursor processing.     

Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids

The prohormone convertases (PCs) are processing enzymes that activate proproteins via cleavage at specific single or pairs of basic residues. The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) are primary sites of biosynthesis of several neuroendocrine hormone precursors, including provasopressin (pro-AVP), pro-oxytocin (pro-OT), and procorticotrophin-releasing hormone (pro-CRH), which require post-translational processing to yield active products. Using in situ hybridization, we observed PC1 and PC5 mRNAs in PVN and SON magnocellular neurons, while PC2 mRNA was observed in both magnocellular and parvocellular PVN neurons as well as magnocellular SON neurons. Similar to furin, PC7 mRNA was expressed throughout the PVN and SON, whereas PACE4 mRNA levels were undetectable. Both immunohistochemical and Western blot studies were performed to demonstrate the presence of PC proteins and forms in the PVN and SON. Using double-labeling in situ hybridization, we examined the cellular colocalization of each PC mRNA with pro-AVP, pro-OT, and pro-CRH mRNAs in PVN and SON. PC1 mRNA was colocalized with both AVP and OT mRNA in PVN and SON magnocellular neurons. All AVP, OT, and CRH neurons expressed PC2. In contrast, PC5 mRNA was colocalized only with OT mRNA. We examined the effects of adrenalectomy (ADX) on PVN PC mRNA levels. PC1 mRNA levels were increased selectively within CRH/AVP parvocellular neurons but were unchanged in PVN magnocellular AVP or OT neurons. These results established the anatomical organization of each convertase and proneuropeptide substrates in the PVN and SON and suggested potential roles for each enzyme under resting and stimulated conditions.     

Angiotensin-(1-7) immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat

The distribution of angiotensin-(1-7) immunoreactive neurons was compared to those of vasopressin-(VP) and oxytocin-(OT) immunoreactive (IR) neurons in the hypothalamus of adult (mRen-2d)27 transgenic hypertensive and Sprague-Dawley rats. In both strains, angiotensin (Ang)-(1-7)-IR cells were found in the supraoptic nucleus (SON), and in the anterior (ap-), medial (mp-), and lateral (lp-) parvocellular, and posterior magnocellular (pm-) subdivisions of the paraventricular (PVN) nucleus. Three-dimensional reconstructions showed that cells immunoreactive to Ang-(1-7) and VP were specifically co-distributed in the SON and in the pmPVN. Double-labeling neurons for both peptides revealed that both Ang-(1-7) and VP were colocalized in a subpopulation of neurons in the pmPVN and SON. In combination with previous studies, our results suggest that Ang-(1-7) and VP are colocalized, co-released and may have a combined action at a common target. In addition, the introduction of the mouse submandibular renin (mRen-2d) transgene into Sprague-Dawley rats does not appear to have altered the fundamental organization of hypothalamic peptide systems involved in fluid homeostasis.     

Somatomedin C (insulin-like growth factor I) levels in patients with chronic fatigue syndrome

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.     

Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus

The adipose tissue-derived hormone leptin regulates body weight homeostasis by decreasing food intake and increasing energy expenditure. The weight-reducing action of leptin is thought to be mediated primarily by signal transduction through the leptin receptor (LR) in the hypothalamus. We have used immunohistochemistry to localize LR-immunoreactive (LR-IR) cells in the rat brain using an antiserum against a portion of the intracellular domain of LR that is common to all LR isoforms. The antiserum recognized the short and long isoforms of LR in transfected hematopoietic BaF3 cells. To examine the chemical nature of target cells for leptin, direct double-labeling immunofluorescence histochemistry was applied. The results show extensive distribution of LR-like immunoreactivity (LR-LI) in the brain with positively stained cells present, e.g., in the choroid plexus, cerebral cortex, hippocampus, thalamus, and hypothalamus. In the hypothalamus, strongly LR-IR neurons were present in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), periventricular nucleus, arcuate nucleus, and lateral hypothalamus. Weaker LR-IR neurons were also demonstrated in the lateral and medial preoptic nuclei, suprachiasmatic nucleus, ventromedial and dorsomedial nuclei, and tuberomammillary nucleus. Confocal laser scanning microscopy showed LR-LI in the periphery of individual cells. In magnocellular neurons of the SON and PVN, LR-LI was demonstrated in vasopressin- and oxytocin-containing neurons. In parvocellular neurons of the PVN, LR-LI was demonstrated in many corticotropin-releasing hormone-containing neurons. LR-IR neurons were mainly seen in the ventromedial aspect of the arcuate nucleus, where LR-LI co-localized with neuropeptide Y. In the ventrolateral part of the arcuate nucleus, LR-LI was present in many large adrenocorticotropic hormone-IR proopiomelanocortin-containing neurons and in a few galanin-, neurotensin-, and growth hormone-releasing hormone-containing neurons. In the dorsomedial arcuate nucleus, few tyrosine hydroxylase (dopamine)-containing neurons were seen to have LR-LI. Melanin-concentrating hormone-containing neurons in the lateral hypothalamus had LR-LI. Based on the immunohistochemical results, possible interactions of leptin with brain mechanisms are discussed.     

Expression of estrogen receptor-beta messenger ribonucleic acid in oxytocin and vasopressin neurons of the rat supraoptic and paraventricular nuclei

The regulatory actions of estrogen on magnocellular oxytocin (OT) and vasopressin (VP) neurons of the paraventricular (PVN) and supraoptic (SON) nuclei are well documented. To date it is still debated whether the effect of estrogens is exerted directly or mediated by estrogen-sensitive interneurons. Previous immunocytochemical (ICC) and in situ hybridization (ISH) studies detected either low levels or absence of the classical estrogen receptor (ER-alpha) in the PVN and the SON of the rat. The present experiments using a combined ICC and ISH method were undertaken to examine the expression of the recently cloned beta form of ER (ER-beta) in OT- and VP-immunoreactive (IR) neuronal systems of the rat hypothalamus. The results demonstrate that the highest cellular levels of ER-beta messenger RNA (mRNA) in OT-IR neurons can be visualized in the caudal portion of the PVN and in an area ventro-medial to the central core of VP-IR cells. These neurons were previously shown to project caudally to the brain stem and the spinal cord to regulate autonomic functions. In addition, the whole rostro-caudal extent of the PVN and the SON contained OT-IR neurons that coexpressed variable levels of ER-beta mRNA. Similarly, the presence of ER-beta mRNA was seen in a large population of VP-IR paraventricular and supraoptic neurons. In the SON, somewhat stronger hybridization signal was detected in VP-IR neurons as compared with OT-IR neurons. Together, these findings provide strong support for the concept that the functions of OT- and VP-IR neurons in the PVN and the SON are regulated directly by estrogen and that the genomic effects of estrogens are mediated by ER-beta.     

Kindled seizures increase metabotropic glutamate receptor expression and function in the rat supraoptic nucleus

The spread of experimentally kindled seizures in rats results in sustained increases in plasma vasopressin (VP) and VP mRNA in the supraoptic nucleus (SON). These increases provide an excellent example of the pathological plasticity that can develop in normal cells exposed to recurrent seizure activity. To test whether this plasticity might be due in part to changes in metabotropic glutamate receptors (mGluRs), we examined mGluR mRNA expression in the SON 1 month after stage 5 amygdala kindling. Three mGluR subtypes were detected by in situ hybridization in the SON in the following relative levels: mGluR3 > mGluR1 > mGluR7. Both mGluR1 and mGluR3 mRNAs were significantly increased in the SON (+28-61%) and cortex (+27-42%) after kindling. Immunoreactivity for mGluR1 but not mGluR2/3 was significantly increased in vivo in the SON. Receptor protein expression and intracellular calcium accumulation in response to the mGluR agonist, 1S,3R ACPD, were evaluated after in vitro "kindling" of neuroendocrine cells by Mg2+ deprivation. Increased immunoreactivity for mGluR1 and mGluR2/3 was seen in all cultures 3 days after a brief exposure to Mg2+-free medium. 1S,3R 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) induced rapid peak responses and gradual accumulations of intracellular Ca2+ in neurons. Both responses were increased in the "kindled" cells. Increases in the expression of functional mGluR1 and perhaps mGluR3 receptors may contribute to the development of long-lasting plastic changes associated with seizure activity.     

Mutual interactions between HIV-1 and cytokines in adherent cells during acute infection

Intracerebroventricular (i.c.v.) infusions of angiotensin II (AII) reliably induced c-fos expression in the supraoptic (SON) and paraventricular (PVN) nuclei, as well as other areas of the basal forebrain including the OVLT, subfornical organ (SFO), and bed nucleus (BNST). Double-labelling showed that AII-induced c-fos was observed in both vasopressin (AVP-) and oxytocin (OXY)-containing neurons of the SON and PVN in male rats. Allowing rats to drink water after AII infusions suppressed c-fos expression both AVP- and OXY-stained magnocellular neurons. Intragastric infusions of water were also effective, showing that oro-pharyngeal stimuli were not critical. Maximal suppression occurred in rats in whom water had been infused intragastrically about 5 min before i.c.v. AII infusions, suggesting that changes in osmolarity were responsible. i.c.v. AII also induced c-fos expression in a number of brainstem structures, including the solitary nucleus (NTS), lateral parabrachial nucleus (LPBN), locus coeruleus (LC), and the area postrema (AP). These results indicate that AVP and OXY-containing neurons in the magnocellular parts of the SON and PVN alter their immediate-early gene response to AII after water intake, and that this does not depend upon oro-pharyngeal factors. Furthermore, AII can induce c-fos expression in a number of brainstem nuclei associated with autonomic function, and these do not respond to water intake.     

Differential colocalization of estrogen receptor beta (ERbeta) with oxytocin and vasopressin in the paraventricular and supraoptic nuclei of the female rat brain: an immunocytochemical study

Evidence exists for the localization of the newly identified estrogen receptor beta (ERbeta) within the rat paraventricular nucleus (PVN) and supraoptic nucleus (SON), regions which lack ERalpha. Presently, we investigate whether ERbeta-like-immunoreactivity (-ir) is found within cells of several major neuropeptide systems of these regions. Young adult Sprague-Dawley rats were ovariectomized (OVX), and 1 week later half of the animals received estradiol-17beta (E). Dual-label immunocytochemistry was performed on adjacent sections by using an ERbeta antibody, followed by an antibody to either oxytocin (OT), arginine-vasopressin (AVP), or corticotropin releasing hormone. Nuclear ERbeta-ir was identified within SON and retrochiasmatic SON, and in specific PVN subnuclei: medial parvicellular part, ventral and dorsal zones, dorsal and lateral parvicellular parts, and in the posterior magnocellular part, medial and lateral zones. However, the ERbeta-ir within magnocellular areas was noticeably less intense. OT-/ERbeta-ir colocalization was confirmed in neurons of the parvicellular subnuclei, in both OVX and OVX+E brains ( approximately 50% of OT and 25% of ERbeta-labeled cells between bregma -1.78 and -2.00). In contrast, few PVN parvicellular neurons contained both AVP- and ERbeta-ir. As well, very little overlap was observed in the distribution of cells containing corticotropin releasing hormone- or ERbeta-ir. In the SON, most nuclear ERbeta-ir colocalized with AVP-ir, whereas few OT-/ERbeta-ir dual-labeled cells were observed. These findings suggest that estrogen can directly modulate specific OT and AVP systems through an ERbeta-mediated mechanism, in a tissue-specific manner.     

Activation and degeneration during aging: a morphometric study of the human hypothalamus

During the course of aging both activation and degenerative changes are found in the human hypothalamus. Degeneration may start around middle-age in some neurotransmitter- or neuromodulator-containing neurons. For instance, a decreased number of vasoactive intestinal polypeptide (VIP) neurons was observed in the suprachiasmatic nucleus (SCN) of middle-aged males. The normal circadian fluctuations seen in the number of vasopressin (AVP) neurons in the SCN of young subjects diminished in subjects older than 50 years. Moreover, a sharp decline in cell number was found in the sexually dimorphic nucleus (SDN) after 50 years in males. On the other hand, many hypothalamic systems remain perfectly intact during aging like the oxytocin (OXT) neurons in the paraventricular nucleus (PVN). The AVP neurons in the PVN are activated during aging as appears from their increasing cell number. Also the corticotrophin-releasing hormone (CRH) neurons of the PVN are activated in the course of aging, as indicated by their increased number and their increased AVP coexpression. Part of the infundibular nucleus, the subventricular nucleus, contains hypertrophic neurokinin B neurons in postmenopausal women. It can be concluded that a multitude of changes in the various hypothalamic nuclei may be the biological basis for many functional changes in aging, i.e., both endocrine and central alterations, and that only a minority of the possible human hypothalamic changes have so far been studied.     

Diurnal fluctuations in mating-induced oxytocinergic activity within the paraventricular and supraoptic nuclei do not influence prolactin secretion

Previous studies have implicated oxytocin (OT) in the control of surge-type PRL secretion in the pregnant and pseudopregnant rat. The present studies examined the relationship between mating-induced activation of OT neurons in the paraventricular (PVN), supraoptic (SON), and anterior commissural (ACN) nuclei and PRL secretion. Activity within OTergic neurons, as measured by increased c-fos expression, was examined immediately and 5 days following mating in ovariectomized, estrogen-plus-progesterone-treated rats at the time when nocturnal PRL surges are expressed (0600 h) and at an intersurge time (2400 h). Females received fifteen intromissions (15I), 15 mounts-without-intromission (MO), or no stimulation (homecage, HC) from a sexually experienced male. Receipt of 15I at 0600 h induced significantly higher numbers of OT immunoreactive (OT-IR) cells and FOS/OT-IR double-labeled cells in the parvocellular division of the PVN (PVNparv) and in the SON than did 15I at 2400 h. Numbers of OT-IR and FOS/OT-IR cells in the ACN and in the magnocellular compartment of the PVN (PVNmag) were not influenced by mating at either time. In contrast, acute PRL secretion induced within 5-30 min by 15I was not influenced by whether mating occurred at 1800 h (diurnal surge), 2400 h, or 0600 h, nor were plasma OT levels elevated during the 1 h following 15I or MO at these times. Examination of FOS-IR cells throughout the hypothalamus across the two times of day revealed previously unreported differences between 15I and control MO treatments in the PVN, SON, and the ventrolateral part of the arcuate nucleus (ARCvl). On day 5 post mating, numbers of OT-IR and FOS/OT-IR cells in the PVN, SON, and ACN were very low and were similar between 0600 h and 2400 h and between females that showed (15I) or did not show (MO) mating-induced PRL surges characteristic of pregnancy. The results of these studies demonstrate that intromissive but not mounts-only stimulation from males induces a rapid increase in OT-IR staining and OT neuron activation in the PVNparv and the SON. These mating-induced responses in OT neurons occurred within 1 h after mating only at 0600 h, suggesting a diurnal fluctuation in sensitivity to intromissive stimulation. Changes in OTergic function were not seen in response to mating at other times of day, nor at the time of the nocturnal PRL surge 5 days after mating. We conclude that OT activity induced by mating does not act to stimulate PRL secretion directly, but may be involved in the process(es) by which genitosensory stimulation initiates surge-type PRL secretion.     

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC 7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF.     

Morphological and electrophysiological classification of hypothalamic supraoptic neurons

In mammals, the magnocellular neurons of the supraoptic nucleus (SON) have been classified into vasopressin- (VP) and oxytocin- (OT) producing subtypes. The degree to which these neurons have distinguishable characteristics is considered in the present review. Most of the cytoarchitectonic diversity observed in some Golgi studies has yet to be attributed to differences between OT and VP neurons. The predominant SON cell type is a large bipolar neuron with relatively short and simply branching dendrites. Based on intracellular filling, large multipolar neurons probably represent a small subset of neurosecretory cells. Parvicellular multipolar and bipolar neurons may represent interneurons or subsets of neurosecretory cells. Suggestive evidence that axonal origin and spine density may differ between OT and VP neurons remains to be confirmed in rat. Different fiber systems are thought to preferentially innervate VP or OT subgroups, but only rarely have inputs to OT and VP neurons been compared at the ultrastructural level. Potentially selective inputs to OT somata may derive from the raphe system and the nucleus of the solitary tract, whereas the apparent preferential innervation of VP neurons (e.g. from the A1 region of the ventrolateral medulla) is less certain because of the overlapping dendritic fields of OT and VP neurons. Electrophysiologically, OT and VP neurons are best distinguished in vivo by their reaction to gastric, cardiovascular and suckling stimuli. The firing patterns of activated OT and VP neurons often differ, but can transiently appear indistinguishable in vivo and especially in vitro. Classification in vitro without immunochemical labelling may be aided by the presence of phasic bursting (mostly in VP neurons) and by the differential response of these neurons to certain neurochemicals or to stimulation of certain inputs. The membrane properties of OT and VP neurons are generally similar in vitro, but the range of tests has not been extensive. The depolarizing afterpotential is more often exhibited by, but is not exclusive to, VP neurons.     

Age-induced hypertrophy of astrocytes in rat supraoptic nucleus: a cytological, morphometric, and immunocytochemical study

BACKGROUND: In the adult rat, neuron-astroglia interactions in the supraoptic nucleus (SON) are characterized by the structural and functional plasticity of astrocytes in response to several physiological and experimental conditions. This study has analyzed the plasticity of the supraoptic nucleus astrocytes in response to the age-induced changes in neuronal activity. METHODS: The study was performed in 5-, 12-, 18- and 24-month-old rats. The cytology and organization of astrocytes in the SON were examined using glial fibrillary acidic and vimentin immunocytochemistry and ultrastructural and morphometric analysis. RESULTS: No significant age-related variations in the total number of neurons and astrocytes in the SON were detected, although a few degenerating neurons were found in old rats. An age-dependent increase in GFAP immunoreactivity was observed at the ventral glial lamina, perivascularly and between neuronal perikarya. Vimentin overexpression was also detected in ventral lamina astrocytes with advancing age. At the cell nucleus level, we observed an age-associated increase in nuclear size and in the number of coiled bodies, nuclear bodies, and "cleared" nucleoplasmic areas, as well as changes in the nucleolar organization. At the cytoplasmic level, characteristic ultrastructural features in astrocytes of old rats were the hypertrophy of intermediate filament bundles and the formation of an extensive network of Golgi stacks interlinked by tubulovesicular elements. Glial filaments were often associated with the nuclear envelope and polyribosomes. CONCLUSIONS: The increased GFAP and vimentin immunoreactivity and the morphometric and cytological changes in rat SON astrocytes may reflect a sustained upregulation of cellular activity with age, resulting in hypertrophy of glial perikarya and cell processes. Several factors that are known to influence the expression of the astrocytic phenotype, such as signals produced by degenerating neurons and activated microglia, as well as variations in neuronal activity are considered possible causes of the age-associated changes in SON astrocytes.     

Galanin-R1 receptor in anterior and mid-hypothalamus: distribution and regulation

The distribution and regulation of galanin-R1 receptor (GAL-R1-R) mRNA has been studied in the anterior and mid-diencephalon by using in situ hybridization. Moreover, possible colocalization of GAL-R1-R mRNA and prepro-galanin or vasopressin mRNAs has been analyzed at the cellular level using double in situ hybridization methodology. Many nuclei in the hypothalamus expressed GAL-R1-R mRNA, including the paraventricular nucleus (PVN) and the supraoptic nucleus (SON). Strong expression was also seen in the same sections in various areas outside of the diencephalon. The distribution patterns are similar to those described in earlier studies. Double labeling experiments showed GAL-R1-R mRNA in vasopressin neurons in the PVN and SON. Moreover, GAL-R1-R mRNA and prepro-galanin mRNA were colocalized in several hypothalamic nuclei. GAL-R1-R mRNA levels showed a high degree of plasticity. Thus, salt loading resulted in a marked increase in GAL-R1-R mRNA levels in the PVN and SON and a moderate decrease was seen during lactation. In contrast, hypophysectomy caused a decrease in GAL-R1-R mRNA levels. Differential effects of colchicine were recorded with a decrease of GAL-R1-R mRNA in the magnocellular hypothalamic neurons. After salt loading or during lactation, GAL-R1-R mRNA and prepro-galanin mRNA were regulated in parallel, whereas their levels changed in opposite directions after hypophysectomy and colchicine injection. In conclusion, GAL-R1-Rs are present in several hypothalamic nuclei, partly in neurons synthesizing galanin. The receptors are regulated in a specific fashion in the various nuclei, depending on the stimulus applied. The results suggest that the effect of galanin in the hypothalamus partly depends on the state of receptor expression.     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

Vasopressin in the forebrain of common marmosets (Callithrix jacchus): studies with in situ hybridization, immunocytochemistry and receptor autoradiography

The distribution of vasopressin (AVP) producing cells, their projections and AVP receptors was examined in the brain of common marmosets (Callithrix jacchus) using in situ hybridization, immunocytochemistry and receptor autoradiography. Clusters of cells labeled for AVP mRNA or stained for AVP immunoreactivity (AVP-ir) were found in the paraventricular (PVN), supraoptic (SON) and suprachiasmatic nuclei (SCN) of the hypothalamus. Scattered AVP producing cells were also found in the lateral hypothalamus and the bed nucleus of the stria terminalis (BST). Neither AVP mRNA-labeled nor AVP-ir cells were detected in the amygdala. Although AVP-ir fibers were evident outside of the hypothalamic-neurohypophyseal tract, a plexus of fibers in the lateral septum, as observed in the rat brain, was not detected. Receptor autoradiography using 125I-linear-AVP revealed specific binding for AVP receptors in the nucleus accumbens, diagonal band, lateral septum, the BST, SCN, PVN, amygdala, anterodorsal and ventromedial nucleus of the hypothalamus, indicating sites for central AVP action in the marmoset brain. Together, these data provide a comprehensive picture of AVP pathways in the marmoset brain, demonstrating differences from rodents in the distribution of cell bodies, fibers and receptors.