Studies on the vasopressin and oxytocin storage in the hypothalamus and neurohypophysis

The effects of modified adrenergic transmission on the bioassayed storage of vasopressin and oxytocin in the hypothalamus and neurohypophysis under conditions of stress (cold or immobilization), disturbed water balance and pinealectomy are reviewed. Alpha-adrenergic mechanisms seem to be included in the response of vasopressinergic and oxytocinergic neurones to stress; on the other hand, impulses of osmoreceptor origin are of importance in regulatory processes affecting the functional response of these neurones to altered alpha-adrenergic transmission and also to melatonin. The beta-adrenergic (and, to some extent, also the alpha-adrenergic) transmission is probably involved in the neural mechanisms of the pineal-neurohypophysial relationship. Furthermore, a possible regulatory role of cholecystokinin in water metabolism and release of neurohypophysial hormones is suggested.     

Differential regulation of vasopressin gene expression in the hypothalamus of endotoxin-treated 14-day-old rat.

The E. coli endotoxin 0111 B4, a lipopolysaccharide (LPS), in a dose of 200 ng/kg body weight/50 microl artificial cerebrospinal fluid (CSF) was given intracisternally to 14-day-old rats. Four hours later CSF, blood and urine were sampled, and consecutive brain sections from the hypothalamic area of the brain were prepared for in situ hybridization. The LPS treatment resulted in a significant (p<0.001) pleocytosis and an elevation of the protein content of the CSF. There were no changes observed in the chemical parameters of the CSF, plasma, blood or urine, i.e. vasopressin (VP) levels, osmolality, Na+ and K+ concentrations, glucose level, pH, bicarbonate or PaCO2, PaO2 values. LPS injection, however, resulted in a significantly (p<0.01) increased VP mRNA level (121% of the control value) in the supraoptic nuclei (SON), but not in the paraventricular nuclei (PVN), as compared to controls. Our findings suggest an early effect of LPS on VP gene expression selectively in the SON of 14-days-old rats. This animal model might be suitable for studying the regulation of VP gene expression and the role of this peptide in the pathogenesis of bacterial meningitis in pediatric patients.     

Administration of oxytocin affects vasopressin V2 receptor and aquaporin-2 gene expression in the rat.

Oxytocin (OT) binds to the vasopressin V2 receptor (V2R) because of its structural similarity to arginine vasopressin (AVP). Though the affinity of OT for V2R is low, it is known that OT causes antidiuresis. To clarify the effect of OT as an agonist of V2R, we investigated the influence of acute elevation of plasma OT levels on the rat mRNA expression of V2R and aquaporin-2 (AQP2), the water channel regulated by V2R. The plasma OT level increased from 11.1+/-1.6 pg/ml to 331.0+/-67.9 pg/ml by 1 h after subcutaneousinjection of 20 microg OT. V2R mRNA expression decreased to 68.3+/-4.1% of the control at 3 h, and AQP2 mRNA expression increased to 239.3+/-26.8% of the control at 6 h. The plasma AVP level did not change significantly during the experiment. The influence of a subcutaneous injection of 20 microg OT on V2R and AQP2 mRNA expression is comparable to that of 10 microg AVP that we documented in the previous study. In conclusion, OT can downregulate V2R mRNA expression and upregulate AQP2 mRNA expression in the collecting duct as an agonist of the V2R like AVP.     

Distribution of vasopressin and oxytocin in rat brain

Arginine-vasopressin and oxytocin in various portions of rat brain were determined by radioimmunoassays. The hormones were extracted from tissue samples into 0.1 N HCl and then purified partially with acetone-petroleum ether extraction. The non-equilibration method was used for the assays. In this method recovery rates of arginine-vasopressin and oxytocin were 73.0 +/- 4.4% and 75.0 +/- 3.8%, respectively. Sensitivities of the assays were 1 pg of arginine-vasopressin and 0.75 pg (0.3 microU) of oxytocin per assay tube. The higher concentrations of arginine-vasopressin and oxytocin were confirmed in the hypothalamo-neurohypophyseal system, where these hormones are synthesized, transported and stored. Relatively high concentrations of these hormones, especially oxytocin, were detected in spinal cord. Amygdala, hippocampus, limbic forebrain and pineal body contained a certain amount of arginine-vasopressin (2-20 pg/mg protein). Oxytocin (1-7 pg/mg protein) was also detected in amygdala, pons and medulla oblongata, pineal body and midbrain. The low concentrations of these hormones were also found in cerebral cortex and cerebellum.     

Upregulation of CRH gene expression and downregulation of arginine vasopressin gene expression in the hypothalamus of bilateral nephrectomized rats

The expression of the corticotropin-releasing hormone (CRH) gene and the arginine vasopressin (AVP) gene in the hypothalamus examined in bilateral nephrectomized rats by in situ hybridization histochemistry. The expression of the CRH gene was significantly increased in the parvocellular part of the paraventricular nucleus (PVN) 12 and 20 h after bilateral nephrectomy in comparison with that after sham operation. The plasma concentration of adrenocorticotropic hormone (ACTH) in nephrectomized rats was significantly higher than that in sham operated rats 20 h after surgery. In contrast, the expression of the AVP gene in both the parvocellular and magnocellular parts of the PVN and throughout the supraoptic nucleus (SON) was significantly decreased 20 h after bilateral nephrectomy in comparison with that after sham operation. These results suggest that nephrectomy-induced upregulation of the CRH gene with elevation of plasma ACTH may be due to the activation of the hypothalamo-pituitary adrenal (HPA) axis.     

Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat

Summary Perfusion of rat brain followed by immersion fixation with 2.5% glutaraldehyde-1% paraformaldehyde, purification of the first antisera and application of the unlabelled antibody enzyme method were used to specifically identify vasopressin and oxytocin containing cells and fibres. The conventional sites of production of these hormones were confirmed as follows: supraoptic and paraventricular nuclei, suprachiasmatic nucleus (only vasopressin), and other cells and cell groups of the hypothalamus. Fibres from the suprachiasmatic nucleus spread out in various directions, and probably project to the nucleus praeopticus periventricularis, organum vasculosum laminae terminalis and in the direction of the supraoptic nucleus. Oxytocin and vasopressin containing pathways could be traced from the paraventricular nucleus to the lateral ventricle, the stria terminalis and the stria medullaris. Some of the oxytocin and vasopressin containing tracts appear to continue onto the septum. The possible importance of these morphological findings for the behavioural effects of vasopressin and oxytocin is discussed.The authors wish to thank Dr. L. Sternberger for his generous gift of peroxidase-antiperoxidase complex, and Miss M.M. Smidt, Mr. A. Potjer and Mr. P. Wolters for their assistance. This work was supported in part by the Foundation for Medical Research FUNGO     

Simultaneous and independent release of vasopressin and oxytocin in the rat

The relative dependence or independence of the secretion of the neurohypophysial hormones, arginine vasopressin and oxytocin, was investigated using a wide variety of stimuli reported to cause the secretion of one or the other hormone. Differences in species, animal preparations, sampling techniques, assays, and other factors make comparison of many previous studies difficult. The aim of this study was to overcome these problems by using the same methodology, animal species, and assays to compare vasopressin and oxytocin release. To further strengthen the analysis, determinations of vasopressin and oxytocin were done in the same blood samples. The results demonstrated that during simultaneous release of both hormones, vasopressin is released in greater proportion following restraint stress, hemorrhage, isotonic hypovolemia, and nicotine, whereas oxytocin is released in greater proportion following endotoxin or hypertonic saline. Vasopressin was released without oxytocin following diethylstilbestrol. Oxytocin was released without concomitant vasopressin release following exercise, hypothermia, hyperthermia, labour, and lactation. Neither oxytocin nor vasopressin release was observed following thyroid-releasing hormone or insulin-induced hypoglycemia. These data illustrate the marked flexibility of the hypothalamo-neurohypophysial system that regulates secretion of vasopressin and oxytocin.     

Changes in oxytocin and vasopressin content in posterior pituitary and hypothalamus following pantethine treatment

Pantethine, a cysteamine precursor, depletes somatostatin in the cerebral cortex and hypothalamus and prolactin in the anterior pituitary and hypothalamus. This study investigated the effect of pantethine on oxytocin and arginine vasopressin content in the posterior pituitary and hypothalamus. Male Long-Evans rats were injected intraperitoneally with escalating doses of pantethine (i.e., 146.7 mg, 293.4 mg and 586.6 mg/100 gm body weight). Hormone content was determined by radioimmunoassay. Three hours after pantethine treatment, the oxytocin content in the posterior pituitary and the hypothalamus was markedly reduced with all doses of the drug. Vasopressin content in the posterior pituitary and hypothalamus was decreased but to a lesser extent than oxytocin and only with the highest dose of pantethine. Pantethine may act to reduce oxytocin and vasopressin content through intracellular conversion to cysteamine. The exact mechanism of action of pantethine on oxytocin and vasopressin remains to be elucidated.     

Expression of corticosteroid-binding protein in the human hypothalamus, co-localization with oxytocin and vasopressin.

Corticosteroid-binding globulin, a specific steroid carrier in serum with high binding affinity for glucocorticoids, is expressed in various tissues. In the present study, we describe the immunocytochemical distribution of this protein in neurons and nerve fibers in the human hypothalamus. CBG immunoreactive perikarya and fibers were observed in the paraventricular, supraoptic, and sexual dimorphic nuclei in the perifornical region, as well as in the lateral hypothalamic and medial preoptic areas, the region of the diagonal band, suprachiasmatic and ventromedial nuclei, bed nucleus of the stria terminalis and some epithelial cells from the choroid plexus and ependymal cells. Stained fibers occurred in the median eminence and infundibulum. Double immunostaining revealed a partial co-localization of corticosteroid-binding globulin with oxytocin and, to a lesser extent, with vasopressin in the paraventricular and the supraoptic nuclei. Double immunofluorescence staining showed coexistence of these substances in axonal varicosities in the median eminence. We conclude that neurons of the human hypothalamus are capable of expressing corticosteroid-binding globulin, in part co-localized with the classical neurohypophyseal hormones. The distribution of CBG immunoreactive neurons, which is widespread but limited to specific nuclei, indicates that CBG has many physiological functions that may include neuroendocrine regulation and stress response.     

Chromosomal and comparative mapping of rat oxytocin, oxytocin receptor and vasopressin genes

Oxytocin and its receptor are potentially important for cardiovascular functions. In the present paper, we report their chromosome locations in the rat and their comparative mapping with the mouse and human. They are located in chromosome regions previously known to contain quantitative trait loci for blood pressure in various genetic crosses. Thus, they have become valid candidate genes for genetic hypertension.     

Demonstration of a different localization of perikarya immunoreactive to oxytocin and vasopressin in the cat hypothalamus

Using immunohistochemical techniques, we demonstrated oxytocin (OT) and vasopressin (AVP) neurons in the cat hypothalamus. The OT immunoreactive neurons were found mainly in the paraventricular nucleus, supraoptic nucleus and dorsal accessory group located lateral to the fornix. In addition to these hypothalamic structures, the AVP immunoreactive neurons were observed in the suprachiasmatic nucleus, ventral accessory group located in the retrochiasmatic area and lateral accessory group, dorsal to the supraoptic nucleus caudally, and ventral to the medial part of the internal capsule rostrally. We further demonstrated a different localization of the OT and AVP immunoreactive neurons in the paraventricular and supraoptic nuclei.     

Exposure to chronic isolation modulates receptors mRNAs for oxytocin and vasopressin in the hypothalamus and heart

The goal of our study was to explore the effect of social isolation stress of varying durations on the plasma oxytocin (OT), messenger ribonucleic acid (mRNA) for oxytocin receptor (OTR), plasma arginine vasopressin (AVP) and mRNA for V_1a receptor of AVP (V_1aR) expression in the hypothalamus and heart of socially monogamous female and male prairie voles (Microtus ochrogaster). Continuous isolation for 4 weeks (chronic isolation) increased plasma OT level in females, but not in males. One hour of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma AVP level. Chronic isolation, but not repeated isolation, significantly decreased OTR mRNA in the hypothalamus and heart in both sexes. Chronic isolation significantly decreased cardiac V_1aR mRNA, but no effect on hypothalamic V_1aR mRNA expression. We did not find a gender difference within repeated social isolation groups. The results of the present study reveal that although chronic social isolation can down-regulate gene expression for the OTR in both sexes, the release of the OT peptide was increased after chronic isolation only in females, possibly somewhat protecting females from the negative consequences of isolation. In both sexes repeated, but not chronic, isolation increased plasma AVP, which could be permissive for mobilization and thus adaptive in response to a repeated stressor. The differential effects of isolation on OT and AVP systems may help in understanding mechanisms through social interactions can be protective against emotional and cardiovascular disorders.     

The vasopressin and oxytocin content in the hypothalamus and neurohypophysis as influenced by reserpine treatment during long-term dehydration in the white rat.

In dehydrated rats both neurohypophysial hormones diminished in hypothalamus as well as in the neurohypophysis. Oxytocin disappearef from the hypothalamus and neurohypophysis at a more rapid rate than vasopressin did. The minimal content of vasopressin and oxytocin in the hypothalamus was observed during 3rd--4th day, but even in extreme dehydration it was found to be relatively high: 65 per cent of vasopressin and 27 per cent of oxytocin as compared with intact animals. At that time the neurohypophysial vasopressin and oxytocin content were almost fully exhausted. In dehydrated and additionally reserpinized animals (10 mg/kg intraperitoneally, then each 48 hr 5 mg/kg of initial body weight) the vasopressin and and oxytocin hypothalamus and neurohypophysis changed in a similar manner. In some experimental groups the decrease of neurohormones in both sites was more marked under reserpine treatment. The drug seems therefore rather to potentiate the effects of physiological stimulation of osmodetectors. So the existence of monoaminergic stimulatory synapses, directly involved in the neural pathway between the osmodetector and the neurosecretory cell, appears to be hardly probable.     

Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat

Summary An immunoelectronmicroscopic method for the specific localization of neurohypophyseal hormones was developed in neurohypophyses of Wistar and Brattleboro rats, the latter strain being homozygous for diabetes insipidus. If the proper precautions were omitted, a marked cross reactivity between antivasopressin and antioxytocin preparations was found. Cross reaction of an antivasopressin plasma with oxytocin, at a dilution of less than 11600, resulted in electron density of all granules within neurosecretory fibres of the Brattleboro and Wistar neurohypophyses. However, this cross reactivity could be eliminated either by sufficient dilution of the antiplasma, or by its purification. Purification of the antibodies was performed by absorption to agarose beads coated with the cross reacting component. Upon incubation with antivasopressin (diluted unpurified 11600 or purified 180) and unpurified antioxytocin (1400) plasma, sections of a Wistar neurohypophysis revealed two types of neurosecretory fibres, containing either electron dense or lucent granules. Oxytocin and vasopressin containing neurosecretory fibres were found as clusters in the neurohypophysis. The specificity of both unpurified antivasopressin (11600) and antioxytocin (1400) plasma was confirmed on serial sections of a Wistar neurohypophysis, alternately incubated with the solutions of the two antibodies.These data prove that the one-cell-one-hormone hypothesis holds true for the hypothalamic-neurohypophyseal system.The authors wish to thank Dr. L.A. Sternberger (Edgewood Arsenal, Md., U.S.A.) for the peroxidase-anti-peroxidase complex, Dr. J.G. Streefkerk (Free University, Amsterdam) and the members of our project group on neuroendocrinology for their suggestions and critical remarks, and Mrs. M. Mud, Mr. P. Wolters and Mrs. A. van der Velden for their skilful assistance     

Interleukin-6 and nitric oxide synthase expression in the vasopressin and corticotrophin-releasing factor systems of the rat hypothalamus.

Nitric oxide synthase (NOS) and interleukin-6 (IL-6) are constitutively expressed in hypothalamic cells. However, phenotypic and functional aspects of these cells remain unknown. We have studied the expression pattern of these two molecules in hypothalamic cells expressing corticotropin-releasing factor (CRF) and arginin-vasopressin (AVP), two major regulatory peptides in the hypothalamus-pituitary system, using immunofluorescence, intracerebroventricular injection of colchicine, and the study in parallel of the labeling pattern of axons in the median eminence. Within AVP cells, we distinguished two different populations: large, intensely stained AVP cells coexpressing IL-6; and large, intensely stained AVP cells coexpressing IL-6 and NOS. Within the CRF cells, we distinguished three different populations: large, intensely stained CRF cells immunonegative for AVP, NOS, and IL-6; large cells weakly stained for CRF and AVP, immunopositive for NOS and immunonegative for IL-6; and small cells intensely stained for CRF and AVP and immunonegative for IL-6 and NOS. In addition, we also found AVP cells containing IL-6 in the suprachiasmatic nucleus. These results suggest that neuronal NOS and IL-6 may be involved in different modulatory processes in hypophysiotropic and non-hypophysiotropic cells.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.     

Co-localization of putative vasopressin receptors and vasopressinergic neurons in rat hypothalamus

Summary Vasopressin and oxytocin are synthesized by neurons in the paraventricular and supraoptic nuclei of hypothalamus. Dense concentrations of vasopressin binding sites have also been localized in these nuclei. Using a vasopressin anti-idiotypic antiserum, a dual immunocytochemical labeling procedure has been employed to elucidate the distribution of putative vasopressin receptors in anatomical relation to vasopressin and oxytocin immunoreactive cells in rat brain. Putative vasopressin receptors are observed in relation to magnocellular neurons in hypothalamus that are vasopressin immunoreactive. They do not appear to be associated with parvocellular vasopressinergic cells or oxytocin immunoreactive neurons. The presence of these presumed autoreceptors would support evidence that vasopressin may autoregulate the activity of magnocellular vasopressinergic neurons in hypothalamus.