Opiate binding differentially associated with oxytocin and vasopressin nerve endings from porcine neurohypophyses

Summary We examined opioid binding in fractions with disconnected nerve endings (secretosomes) which were prepared from porcine neurohypophyses by centrifugation in a discontinuous Percoll gradient. Specific (= displaceable) binding was observed with ³H-etorphine and with ³H-diprenorphine, two ligands with low selectivity for distinct opiate receptor sub-classes. No displaceable binding was found with the prototypic mu- and delta-ligands ³H-dihydromorphine and ³H-(D-Ala, D-Leu) enkephalin. Displacement of ³H-diprenorphine binding was almost absent with the selective mu- and delta-ligands morphiceptin and ICI-174864. Partial displacement occurred with the selective kappa-ligand U-50488 and with dynorphin (1–8). Binding of ³H-etorphine was stereo-specific. ³H-diprenorphine binding was saturable with a K_D between 2 and 4 nM. Maximum of opiate binding activity was detected in the fractions with accumulated secretosomes. By autoradiography specific ³H-diprenorphine binding is shown to be mainly associated with secretosomes. In imunocytochemical preparations an oxytocin antibody was immunoreactive in 85% of the secretosomes in the fraction with highest opiate binding. These fractions in radioimmunoassays exhibited the largest contents in oxytocin and low vasopressin levels. The data therefore suggest that in the porcine neurohypophysis opioid binding sites of the kappa-type occur in secretory endings presumably of the oxytocin type.     

Opioid binding in a rat neurohypophysial fraction enriched in oxytocin and vasopressin nerve endings

Binding of the opiate antagonists [3H]diprenorphine and [3H]naloxone and of the opioid agonists [3H]Met-enkephalin and [3H]dynorphin(1-8) was studied in a fraction of the rat neurohypophysis containing disconnected oxytocin and vasopressin nerve endings ('neurosecretosomes'). There was specific binding of [3H]diprenorphine in the fraction enriched with neurosecretosomes. This binding was only partially displaceable by naloxone; naloxone binding was stereospecific. Intact and unoxidized [3H]Met-enkephalin was found in the neurosecretosome pellet; binding of the analogue D-Ala-D-Leu-enkephalin was very low. Our data favour the assumption of a direct action of endogenous opioids at the neurosecretory nerve endings.     

The hypothalamic neurosecretory pathways for the release of oxytocin and vasopressin in the cat

1. The neurones of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) were stimulated electrically in lactating cats under chloralose anaesthesia. Milk-ejection responses were used to monitor the release of oxytocin and vasopressin and both hormones were assayed in samples of blood collected during stimulation. The position of the tip of the stimulating electrode was confirmed from brain sections stained selectively for cystine-rich neurosecretory material.2. A previous finding that stimulation of the SON in the cat releases vasopressin without oxytocin was confirmed.3. Stimulation of the PVN caused both hormones to be released. The ratio of their concentrations in blood was variable; this suggests release from separate neurones.4. Both hormones were also released on stimulation of the median eminence but not of the zone lying vertically between this structure and the PVN. No neurosecretory material was detected in this zone. These findings argue against the existence of a direct or medial paraventriculo-hypophysial pathway running downwards along the wall of the third ventricle.5. Study of sections from unstimulated brains confirmed that the tractus paraventricularis cinereus of Greving which runs ventro-laterally from the PVN towards the SON, represents the principal efferent pathway for neurosecretory fibres from the PVN.6. The results are discussed in relation to the problem of the independent release of oxytocin and vasopressin in response to physiological stimulation of the neurohypophysis.     

Chloride and magnesium dependence of vasopressin release from rat permeabilized neurohypophysial nerve endings

The role of Cl- and Mg+ ions has been studied on the secretory mechanism leading to the release of vasopressin from digitonin permeabilized nerve endings isolated from the rat neurohypophysis. Secretion was triggered by challenging the permeabilized nerve endings with 1.1 microM free Ca2+. Magnesium enhances secretion and its maximal effect occurred at a concentration of about 2 mM. Further increase of this divalent cation concentration however led to an inhibition of secretion. Chloride ions are necessary for the final steps in exocytosis and this effect of Cl- was inhibited by the chloride channel antagonist N144. It is concluded that in neurosecretory nerve endings magnesium and chloride ions are crucial components for exocytosis to occur.     

The role of the actin cytoskeleton in oxytocin and vasopressin release from rat supraoptic nucleus neurons

Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K⁺, 50 m m ) or TG (200 n m ) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 μ m ) or depolymerizing agents (latrunculin B, 5 μ m ) to treat SON and neural lobe (NL) explants in vitro and measure high K⁺-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K⁺-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K⁺-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.     

Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland

The magnocellular neurosecretory system of mammalia, which produces oxytocin and vasopressin and releases these hormones at a neurohaemal contact area in the neural lobe of the pituitary gland, provides a useful model for the study of release of granule-packaged material from both neurones and endocrine cells. Isolated neural lobes from rats were stimulated to release hormone by incubation for 15min in a depolarizing sodium-free medium containing 56 mM potassium ions. Controls were incubated in a sodium-free medium containing 5.6 mM potassium ions. The amounts of vasopressin and oxytocin released by depolarization together comprised ~83 mU. At the end of the stimulation the glands were fixed for electron microscopy and stereological analysis. Loss of neurosecretory granules from stimulated glands was restricted to the nerve endings. From knowledge of the amount of hormone stored in a single neurosecretory granule, the number of granules lost from the gland could be calculated to contain approximately the amount of hormone released. After stimulation, the nerve endings were unaltered in size or membrane area. There was also no change in the total microvesicle population of the endings, though the microvesicles were redistributed towards the basement membrane contact zone. The vacuole population of the endings was, however, increased three-fold after stimulation.We conclude that, after acute stimulation of hormone release from the neural lobe, neurosecretory granules are lost specifically from the nerve endings and that the excess membrane that results from their exocytosis is to be found primarily in vacuoles.     

3H]glutamate binding sites in the rat pituitary

A significant activity of [3H]glutamate binding was detected in homogenate particulate preparations obtained from the rat pituitary, in addition to central structures including the cerebral cortex. In contrast to the cerebral binding, the pituitary binding was significantly inhibited by Na+ ions. It was also found that neurohypophysis possessed more than a two-fold higher binding activity than that found in adenohypophysis. These results suggest a possible significance of glutamate in rodent pituitary.     

Distribution of vasopressin and oxytocin binding sites in the brain and upper spinal cord of the common marmoset

The aim of this study was to label selectively and to map central vasopressin (AVP) and oxytocin (OT) binding sites in the common marmoset. [¹²⁵I]VPA, a compound selective in rodents and human for the AVP V_1a receptor, yielded the same labeling pattern as [³H]AVP, thus suggesting that most AVP receptors present in the marmoset brain are of the V_1a subtype. Numerous areas exhibited AVP binding sites, among which the olfactory bulb, the accumbens nucleus, the bed nucleus of the stria terminalis, the hypothalamic suprachiasmatic, arcuate and ventromedial nuclei, the medial amygdaloid nucleus, the nucleus of the solitary tract and the cerebral cortex. Binding sites for [¹²⁵I]OTA, a selective OT receptor antagonist in rat and human, were markedly less abundant than [¹²⁵I]VPA ones, and, to a few exceptions, expressed in different areas. Neither AVP, nor OT binding sites were detected in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei identified by neurophysin immunoreactivity. Marked species-related differences were observed in the distribution of both AVP and OT binding sites. Altogether, our data provide a morphological basis to investigate the function of central AVP and OT in the marmoset.     

Ultrastructural demonstration of oxytocin and vasopressin release sites in the neural lobe and median eminence of the rat by tannic acid and immunogold methods

We have investigated the localization of oxytocin (OXT) and vasopressin (AVP) release sites in the neural lobe and median eminence (ME) of the rat. Ultrastructural and post-embedding immunocytochemical studies with protein A-gold as a marker were used for the identification of OXT and AVP neurosecretory granules (NSG). Release of the contents of these OXT and AVP NSG by exocytosis has been clearly demonstrated with the tannic acid-Ringer incubation method in axon terminals of the neural lobe and in varicose fibres with OXT and AVP NSG in the internal zone of the ME. As these fibers lack synaptic specializations, and are not located in the direct vicinity of capillaries of the primary portal plexus, these observations suggest that OXT and AVP are released in a paracrine way in the ME of the rat.     

Independent release of oxytocin and vasopressin during parturition in the rabbit

1. Oxytocin and vasopressin were assayed in samples of blood collected from seven conscious rabbits during parturition.2. Oxytocin was detected in the blood in ten out of fourteen samples collected during the expulsion of one or more foetuses. Four samples contained 6-100 muu./ml., three 100-200 muu./ml. and three 200-500 muu./ml.3. Vasopressin was detected in six blood samples collected during the delivery of foetuses but in only one experiment did the amount exceed that found in the corresponding control sample collected before or after delivery.4. When both hormones were detected in the same blood sample, the ratio of oxytocin to vasopressin varied from 5:1 to at least 26:1.5. It is concluded that, while oxytocin may not be essential for parturition in the rabbit, stretching of the birth canal during the expulsion of foetuses normally acts as a stimulus for the reflex release of oxytocin from the neurohypophysis and that oxytocin is released independently of vasopressin.     

Inhibitory effect of gaseous neuromodulators in vasopressin and oxytocin release induced by endotoxin in rats

Nitric oxide (NO) and carbon monoxide (CO) are endogenously synthesized gaseous molecules that act as neurotransmitters in central nervous system. In this study we investigated the modulatory role of NO and CO in lipopolysaccharide (LPS)-induced vasopressin and oxytocin secretion. Intracerebroventricular (i.c.v.) injection of N omega-L-nitro-arginine methyl ester (L-NAME), 3-morpholino-sydnonimine (SIN-1), zinc deuteroporphyrin 2,4-bis glicol (ZnDPBG) or hemin did not change the basal vasopressin and oxytocin plasma levels. After endovenous LPS administration, plasma vasopressin and oxytocin increased, reaching a peak at 60 min, and returning to basal levels afterwards. LPS administration induced a higher vasopressin and oxytocin plasma levels in rats previously treated with L-NAME and ZnDPBG (P<0.05) compared to rats pre-treated with vehicle. On the other hand, in rats previously treated with SIN-1 or hemin, there was a significant reduction in the vasopressin and oxytocin secretion. These findings confirm the inhibitory role of NO and CO in the LPS-induced vasopressin and oxytocin secretion.     

Sexually dimorphic expression of oxytocin binding sites in forebrain and spinal cord of the rat

The central actions of oxytocin on reproduction-related functions and behaviors are strongly steroid-dependent and gender specific. This study characterizes sexual differences in the oxytocin binding site expression in forebrain and spinal cord of the rat. Using film autoradiography, we quantified the density of oxytocin binding sites in the ventromedial hypothalamic nucleus, the medial and central nuclei of the amygdala, the medial bed nucleus of the stria terminalis and the spinal cord dorsal horns both in adult male and female rats, and during development. In addition, neonatal castrated males and intact neonatal females treated with a single injection of testosterone (1 mg) were examined. Data showed a sexual dimorphism in the expression of oxytocin binding sites in the spinal cord dorsal horns and in restricted areas of the forebrain that are sensitive to gonadal steroids such as the ventromedial hypothalamic nucleus, but not in gonadal steroid insensitive sites such as the central nucleus of the amygdala. Adult males had higher oxytocin binding site densities in the ventromedial hypothalamic nucleus and dorsal horns than females. In the forebrain, but not in the dorsal horn, this sexual difference required a perinatal exposure to testosterone. Neonatal castration only abolished the sexual difference in the ventromedial hypothalamic nucleus of adults, but not in the dorsal horn. Furthermore, females that received a single injection of testosterone 1 day after birth showed significant increases in the density of oxytocin binding sites in the ventromedial hypothalamic nucleus, medial nucleus of the amygdala and medial bed nucleus of the stria terminalis. In addition, the findings suggest that the sexual difference in the ventromedial hypothalamic nucleus also requires gonadal hormones in adulthood. Our data support the hypothesis that sexually dimorphic oxytocin binding sites may contribute to the regulatory central actions of oxytocin in gender specific functions and behaviors such as nociception and reproduction.     

Ontogeny of vasopressin and oxytocin binding sites in the brain of Wistar and Brattleboro rats as demonstrated by lightmicroscopical autoradiography

Vasopressin (VP) and oxytocin (OT) binding sites were localized and quantified in the developing brain of the Wistar, heterozygous (Het) and homozygous (Hom) VP-deficient Brattleboro rat using an autoradiographical technique. VP binding sites could be demonstrated from prenatal day 20 onwards in the septum and in the lateral reticular nucleus. Between this and postnatal day 15, VP binding sites appeared in all other brain areas known to contain VP binding sites in adulthood. In the caudate putamen the regional distribution of VP binding changed during development, while in some areas, for instance, the dorsal hippocampus and post cingulate cortex, the concentration of binding sites increased early but decreased with age. Comparison of VP binding between Het and Hom rats showed significant differences in the lateral reticular nucleus during development. Moreover, at postnatal day 15 there was more VP binding in the anterior commissural and suprachiasmatic nucleus and less in the central amygdala, dorsal hippocampus and post cingulate cortex of the Hom rat. This study shows, for the first time, OT binding sites in the developing rat brain. There is a considerable overlap with VP binding in the brain, sometimes with the same developmental pattern, e.g. in the anterior olfactory nucleus and caudate putamen and sometimes with a later appearance, e.g. in the central amygdala and thalamic nuclei. However most areas with VP binding sites did not show OT binding. In some areas only OT binding sites were present, for instance in the islands of Calleja and ventromedial hypothalamus. Similar to some areas with VP binding, OT binding decreased between postnatal day 5 and 15 in the dorsal hippocampus and even completely disappeared in the parietal cortex. The existence of VP binding sites in the Hom rat, together with the only occasional relationship between the previously described ontogeny of VP and OT innervation of the brain and the presently described developmental course of binding sites, indicates that the early expression of binding sites is not initiated by endogenous ligand. However, the setting of the number of VP binding sites has probably been affected by the VP deficiency of the Hom Brattleboro rat.     

The role of steroid hormones in the regulation of vasopressin and oxytocin release and mRNA expression in hypothalamo neurohypophysial explants from the rat

Vasopressin and oxytocin release from the neural lobe, and the vasopressin and oxytocin mRNA contents of the supraoptic and paraventricular nuclei are increased by hypertonicity of the extracellular fluid. The factors regulating these parameters can be conveniently studied in perifused explants of the hypothalamo-neurohypophysial system that include the supraoptic nucleus (but not the paraventricular nucleus) with its axonal projections to the neural lobe. Vasopressin and oxytocin release and the mRNA content of these explants respond appropriately to increases in the osmolality of the perifusate. This requires synaptic input from the region of the organum vasculosum of the lamina terminalis. Glutamate is a likely candidate for transmitting osmotic information from the organum vasculosum of the lamina terminalis to the magnocellular neurones, because agonists for excitatory amino acid receptors stimulate vasopressin and oxytocin release, and because increased vasopressin release and mRNA content induced in hypothalamo-neurohypophysial explants by a ramp increase in osmolality are blocked by antagonists of both NMDA ( N -methyl-D-aspartate) and non-NMDA glutamate receptors. Osmotically stimulated vasopressin release is also blocked by testosterone, dihydrotestosterone, oestradiol and corticosterone. Both oestrogen and dihydrotestosterone block NMDA stimulation of vasopressin release, and in preliminary studies oestradiol blocked AMPA stimulation of vasopressin release. Thus, steroid inhibition of osmotically stimulated vasopressin secretion may reflect inhibition of mechanisms mediated by excitatory amino acids. Recent studies have demonstrated numerous mechanisms by which steroid hormones may impact upon neuronal function. Therefore, additional work is warranted to understand these effects of the steroid hormones on vasopressin and oxytocin secretion and to elucidate the potential contribution of these mechanisms to regulation of hormone release in vivo.     

Restraint stress has no effect on morphine-induced inhibition of gastrointestinal transit in the rat

Stress in the rat has been reported to enhance the analgesic and thermic effects of opioids, drug effects that are mediated centrally. We examined whether this stress-induced enhancement of response to opioids could also be demonstrated for a drug effect mediated largely in the periphery, morphine-induced inhibition of gastrointestinal transit. Restrained (stressed) and unrestrained (unstressed) rats were injected with saline or morphine and then administered orally a charcoal suspension; after sacrifice, the distance the charcoal traveled through the intestine was determined. After the administration of saline, restrained rats had significantly lower gastrointestinal transit than did unstressed rats; however, both groups were comparably sensitive to inhibition of gastrointestinal transit by morphine.