Pineal peptides in the regulation of milk-ejection reflex in lactating rats

The effects of pineal peptides (mol. mass 1-4 kDa) intranasal infusions on some parameters of milk-ejection reflex were investigated. Peptides were extracted from dairy-cattle pineal glands. Pineal peptides increase body weight, levels of water intake, plasma prolactin concentration and milk yield in rats when infusing daily in dose 1 microgram/kg from the third day of lactation. On 9th and 12th days of lactation during 1-hour nursing seance the significantly greater number of reflective milk ejections were found. When 1 hour before the peptides infusion rats were intraperitoneally injected by rabbit antiserum to oxytocin (200 microliters at dilution of 1:20) the effects of pineal peptides were significantly less expressive or were absent at all. Using enzymimmunoassay it was demonstrated that there were greater increasing of oxytocin content in pineal gland in suckling-induced oxytocin release from neurohypophysial system in the chronic pineal peptides-treated female rats compared with control. This effect was absent when rats were injected by oxytocin antibodies. These data suggest that pineal peptides can participate in forming of reflect oxytocin release pattern. This pattern is initiated by suckling and is limited by oxytocin content in blood.     

The pineal and oxytocin synthesis.

The aim of this study was to investigate the effect of pineal removal on oxytocin synthesis in the hypothalamus using the colchicine method. To this end, rats were injected intracerebroventricularly (i.c.v.) with colchicine solution (5 microg/5 microl) or normal saline and decapitated 20 h later. The animals were either pinealectomized or sham-operated two or eight weeks before i.c.v. injection. The oxytocin content in the hypothalamus was significantly higher in colchicine-treated rats whereas no significant differences were seen in the neurohypophysial hormone level between saline- or colchicine-injected animals. Thus, colchicine inhibited the hormonal transport but probably did not affect the function of the neurohypophysis. Two weeks after pinealectomy neither the oxytocin synthesis rate nor its neurohypophysial content were significantly different from control values. The oxytocin synthesis rate was increased markedly eight weeks after pineal removal. At that time, the neurohypophysial oxytocin content was reduced suggesting the increased secretion of the hormone. It is concluded that the pineal has an inhibitory impact on both oxytocin synthesis and release.     

Centrally administered galanin modifies vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of euhydrated and dehydrated rats.

Galanin (Gal) as a neuropeptide with widespread distribution in the central nervous system may be involved in the mechanisms of vasopressin (AVP) and oxytocin (OT) release from the hypothalamo-neurohypophysial system. Vasopressin and oxytocin content in the hypothalamus and neurohypophysis as well as plasma level of both neurohormones were studied after galanin treatment in euhydrated and dehydrated rats. In not dehydrated rats intracerebroventricular (i.c.v.) injections of Gal did not affect the hypothalamic and neurohypophysial OT content, however, distinctly increased plasma OT concentration. In the same animals Gal diminished the hypothalamic AVP content but was without the effect on neurohypophysial AVP storage; plasma AVP level then raised. Galanin, administered i.c.v. to rats deprived of water, distinctly inhibited AVP and OT release from the hypothalamo-neurohypophysial system. Simultaneously, plasma AVP and OT level was significantly diminished after Gal treatment in dehydrated rats. These results suggest that modulatory effect of galanin on vasopressin and oxytocin release depends on the actual state of water metabolism. Gal acts as an inhibitory neuromodulator of AVP and OT secretion under conditions of the dehydration but stimulates this process in the state of equilibrated water metabolism.     

The hypothalamic and neurohypophysial vasopressin and oxytocin content under various states of adrenergic transmission in dehydrated and subsequently rehydrated rats

Rats dehydrated for 8 days and subsequently rehydrated were given intracerebroventricularly (i.c.v.) methoxamine hydrochloride (MX) or dihydroergotamine methanosulphonate (DHE), each in a daily dose of 10 micrograms dissolved in 10 microliter of 0.9% sodium chloride. A single dose of MX injected to normally hydrated animals increased the release of hypothalamic and neurohypophysial vasopressin but did not affect significantly the oxytocic activity in the hypothalamus as well as in the neurohypophysis. Under conditions of dehydration MX did not influence the hypothalamic vasopressin content but it stimulated the neurohypophysial vasopressin depletion. On the contrary, MX distinctly inhibited the decrease of hypothalamic and neurohypophysial oxytocin content in dehydrated animals. In rehydrated animals MX restrained some what the renewal of hypothalamic vasopressin and oxytocin storage but intensified this process in the neurohypophysis. A single dose of DHE decreased the vasopressin content in the hypothalamus as well as the oxytocin content both in the hypothalamus and neurohypophysis. Under conditions of dehydration DHE stimulated the depletion of hypothalamic vasopressin and oxytocin. On the contrary, DHE strongly inhibited the depletion of oxytocin in the neurohypophysis of dehydrated rats. DHE restrained the renewal of hypothalamic vasopressin and oxytocin stores as well as intensified this process in the neurohypophysis of subsequently rehydrated rats.     

Neurophpophysial hormones and the emission of semen in rabbits.

To elucidate the importance of the neurohypophysial hormones for the emission of semen, several neurohypophysial peptides were tested in male rabbits and the sperm density in the ejaculates was determined. Besides oxytocin and vasopressin, vasotocin and one oxytocin analogue (de-amino1-oxytocin) were used. Only vasopressin, in a dose as low as 10 mU, increased the number of spermatozoa in the ejaculates. It is suggested that vasopressin is of physiological significance for the emission of semen, at least in rabbits.     

Multifaceted purinergic regulation of stimulus-secretion coupling in the neurohypophysis

The neurohypophysis is an original model of the CNS secretory system releasing vasopressin (AVP) and oxytocin (OXT), two neuropeptides hormones synthesized by the magnocellular neurons of the hypothalamus. Specific patterns of action potentials originating from cellular bodies of magnocellular neurons control the release of AVP and OT, but intra-neurohypophysis regulations do modulate the neuropeptides release. There is now good evidence for the effects of extracellular purines in the control of neurohypophysial secretion. This paper brings together evidence for the multiple, intricate actions of purines in the extracellular space of the neurohypophysis. It covers four main points. First, the activity-dependent release of endogenous ATP in the neurohypophysis. Second, the action of ATP on both neuronal and non-neuronal compartments of the neural lobe. Third, the termination of ATP positive feedback by ecto-nucleotidases. And finally the possible involvement of adenosine in the regulation of neurohypophysial secretion and glial plasticity. The data suggest that ATP and adenosine are physiological modulators of the release of neurohypophysial peptides by acting directly on nerve terminals and indirectly on neurohypophysial astrocytes. Since purinergic receptors are widespread in nervous and endocrine systems, the neurohypophysis appears as an useful model for studying the role of purines in the regulation of stimulus-secretion coupling and neuron-glia interactions. The feedback mechanisms found in the neurohypophysis could be ubiquitous, occurring throughout the central nervous system and in other secretory systems.     

Galanin affects vasopressin and oxytocin release from the hypothalamo-neurohypophysial system in haemorrahaged rats.

The effect of centrally administered galanin (Gal; 100 pM i.c.v.) on the hypothalamo-neurohypophysial storage as well as blood plasma level of vasopressin and oxytocin was estimated in haemorrhaged (1 ml per 100 g b.w.) male Wistar rats. Gal i.c.v. treatment did not alter vasopressin and oxytocin content both in the hypothalamus and neurohypophysis as well as their concentration in blood plasma of not haemorrhaged rats. Haemorrhage decreased the hypothalamic and neurohypophysial vasopressin and oxytocin storage but increased the neurohormones plasma level in animals injected with vehicle solution. During the haemorrhage, the increase in plasma vasopressin and oxytocin was inhibited in rats previously treated i.c.v. with galanin. The hypothalamic and neurohypophysial vasopressin as well as oxytocin content significantly increased in animals treated with galanin and subsequently haemorrhaged. These results suggest that galanin may have a regulatory role in the hypothalamo-neurohypophysial function especially under condition of hypovolemia.     

The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats

The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats. Acta physiol. pol., 1985, 36 (3): 193-200. The effects of phenoxybenzamine (PBA; an alpha-adrenergic blocker) on hypothalamic and neurohypophysial vasopressin and oxytocin were investigated in stressed rats. Immobilization resulted in a decrease of both vasopressor and oxytocic activities in the hypothalamus and neurohypophysis, whereas in rats, exposed to cold the vasopressin and oxytocin content in the hypothalamo-neurohypophysial system was increased. Under treatment with PBA the vasopressin and oxytocin content in the neurohypophysis was diminished in stressed (both immobilized and cold-exposed) rats when compared to respective groups of untreated animals subjected to appropriate kind of stress. The response of the vasopressinergic and oxytocinergic neurones seems, therefore, to be dependent on the type of stress. The alpha-adrenergic transmission is probably in some way involved in the mechanisms of modified neurohypophysial function in stressed animals.     

Effects of hormone-releasing stimuli on the area of the perivascular space in the neural lobe of the rat

Summary Neural lobes from rats subjected to neurohypophysial hormone-releasing stimuli were examined electron microscopically following fixation in 4 % tannic acid in 2.5 % glutaraldehyde. This fixation allowed the delineation of the perivascular space in the neural lobe tissue. Measurement of the area of the perivascular space showed that it was significantly increased in the rats subjected to vagal stimulation and intraarterial calcium ions compared to the control rats. The rats which had been subjected to haemorrhage as a hormonereleasing stimulus did not show any significant change in the area of the perivascular space. The significance of these findings in relation to hormone release is discussed.     

The vasopressin and oxytocin neurohypophysial content as influenced by bleeding or dehydration: effect of cholecystokinin octapeptide.

The effect of CCK-8 (50 ng, i.c.v.) on the neurohypophysial vasopressin and oxytocin storage was estimated in haemorrhaged (1 ml per 100 g b.w.) male Wistar rats. In another experimental series rats dehydrated for three days were given CCK-8 in a daily i.c.v. dose of 50 ng. The neurohypophysial vasopressin and oxytocin content was bioassayed by pressor effect following Dekański or milk-ejection activity in vitro following van Dongen and Hays, respectively. The decrease of neurohypophysial vasopressin and oxytocin content, brought about by dehydration, was significantly less marked in animals treated with CCK-8. The depletion of neurohypophysial vasopressin and oxytocin content in haemorrhaged animals could be completely inhibited by earlier i.c.v. administration of CCK-8. It is suggested that hypothalamic cholecystokinin may serve as a modulator of neurohypophysial function.     

Regulation of bioactive beta-endorphin processing in rat pars intermedia

Acid extracts of rat pituitary neuro-intermediate lobes have been shown by ion-exchange chromatography and radio-immunoassay to contain predominantly the inactive derivatives of beta-endorphin, alpha, N-acetyl beta-endorphin 1-27 and alpha, N-acetyl beta-endorphin 1-26; the biologically active form, beta-endorphin 1-31, is a minor component. In contrast, it was found that beta-endorphin generated in neuro-intermediate lobe cells in monolayer culture was less processed: the principal peptides related to bioactive beta-endorphin 1-31. When the cultured cells were incubated in the presence of 10(-5) M dopamine or 10(-6) M alpha-ergocryptine there was a marked increase in the degree of proteolysis and acetylation: the processing pattern reverted to that characteristic of the neuro-intermediate lobe in situ, with alpha-N-acetyl beta-endorphin 1-26 and alpha, N-acetyl beta-endorphin 1-27 as the prominent peptides. The results demonstrate that dopaminergic agents can influence the processing of beta-endorphin-related peptides in rat pars intermedia, indicating a new level at which the bioactivity may be regulated.     

Comparative Physiology of the Vasomotor Effects of Neurohypophysial Peptides in the Vertebrates

The neurohypophysial peptides are vasopressor or depressor inaction depending on the species. Isotocin, mesotocin and oxytocinconstrict the branchial vessels in fish and induce a reflexvasodilation in the systemic vasculature. The vasodilation haspersisted in some higher vertebrates and is particularly prominentin the snakes and birds where vasotocin and arginine vasopressinalso are vasodepressor but are much less potent than mesotocinand oxytocin. In other vertebrates including fish, vasotocinand vasopressin are pressor and exert their effects mainly onthe peripheral resistance. The newt, toads and soft-shell turtlegave pressor responses to all neurohypophysial peptides, withvasotocin showing the highest potency. The frogs, big-headedturtle and lizards were intermediate with vasotocin being pressor,mesotocin being pressor and oxytocin exhibiting a dual effect.     

Morphine, beta-endorphin and [D-Ala2] Met-enkephalin inhibit oxytocin release by acetylcholine and suckling

Morphine inhibits suckling-induced oxytocin (OT) release in lactating mice. Since beta-endorphin and enkephalins have several actions in common with morphine, the action of these opioid peptides on OT release was investigated. In anesthetized lactating rats, OT release was achieved by intraventricular injection of acetylcholine (ACh) or by the physiological stimulus of suckling. The amount of OT released was estimated by comparing milk-ejection responses to these stimuli to those following known amounts of intravenous (IV) OT. Both beta-endorphin and [D-Ala2]Met-enkephalin inhibited ACh-induced and suckling-induced OT release. Naloxone antagonized opiate inhibition in both cases.     

Co-transport of sex hormone-binding globulin/SHBG with oxytocin in transport vesicles of the hypothalamo-hypophyseal system.

The intrinsic expression of sex hormone binding globulin (SHBG) in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was recently described. This study is focused on the ultrastructural localization of SHBG in the hypothalamo-neurohypophyseal pathway in rats. Immunostaining for SHBG in the hypothalamic perikarya was increased by colchicine treatment, indicating that the steroid-binding globulin is subject to rapid axoplasmic transport along with the classical posterior lobe peptides. With immunoelectron-microscopic double labeling, we found co-localization of oxytocin and sex hormone binding globulin in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. Our observations show that SHBG is processed, transported and stored along with oxytocin suggesting that SHBG is released from nerve terminals in the posterior lobe, the median eminence and possibly the brain similarly to and in conjunction with oxytocin.     

Effects of acute opiate-peptide administration on pro-opiomelanocortin cells of the intermediate lobe of the rat pituitary

Summary The effects of acute injections of synthetic opiate peptides into the lateral cerebral ventricle of young adult male rats on cells of the intermediate lobe of the pituitary were studied. Met-enkephalin (100/g) injected into anesthetized rats, or 20 g beta-endorphin administered via a previously implanted cannula to unanesthetized animals, will lead to cell degranulation and often to expanded Golgi zones and prominent regions of rough endoplasmic reticulum in secretory cells when tissue is fixed 45–60 min after peptide administration. Treatment of animals with the opiate antagonist naloxone hydrochloride prior to enkephalin injection appeared to prevent the cellular changes elicited with peptide alone. Observations suggest that opiate peptides administered to the cerebrospinal fluid may stimulate release of pro-opiomelanocortin-peptide from pituitary cells.     

Atrial natriuretic peptide inhibits neurohypophysial hormones' release in the rat (in vitro and in vivo studies).

Intracerebroventricular hANP (50 nmol) inhibits release of vasopressin and oxytocin following dehydration as well as after haemorrhage. 10 nmol/L hANP markedly inhibits vasopressin and oxytocin release in vitro from the neurointermediate lobes both under basal condition as well as during stimulation with excess (56 mM) potassium. It is suggested that ANP may serve as a modulator of vasopressin and oxytocin release. The respective processes are localized, at least in part, at the neurohypophysial level.     

Isolated neurosecretory nerve endings as a tool for studying the mechanism of stimulus-secretion coupling

In the present paper we discuss the properties of a recently developed preparation of isolated neurosecretory nerve endings obtained from the rate neurohypophysis. These nerve terminals release two neurohormones, oxytocin and vasopressin, which are easily assayed by radioimmunoassay. Depolarization-induced secretion is dependent on the same parameters as those regulating release from the whole neural lobe. The isolated nerve endings can be permeabilized by means of digitonin; a treatment which gives direct access to the cytoplasm allowing the study of the minimal requirements for inducing neuropeptide release. Furthermore, some nerve endings are large enough to allow the use of the patch-clamp technique. In the present paper we present evidences which show that the isolated neurohypophysial nerve terminals represent a protent tool for studying the mechanism of stimulus-secretion.     

The vasopressor and oxytocic activities of the hypothalamus and neurohypophysis influenced by stimulated alpha-adrenergic transmission during dehydration and subsequent rehydration in the white rat

Under conditions of equilibrated water metabolism a single dose of methoxamine increased the content of vasopressin in the hypothalamus as well as that of oxytocin both in the hypothalamus and neurohypophysis. During dehydration the depletion of hypothalamic and neurohypophysial vasopressin was more marked in methoxamine-treated animals; this effect, however, was absent in the neurohypophysis on the 2nd day and in the hypothalamus on the 8th day of water deprivation. After two days of dehydration methoxamine inhibited the decrease of oxytocin content in the hypothalamus; simultaneously (2nd and 4th day of dehydration) it intensified this process in the neurohypophysis. During rehydration methoxamine impaired the renewal of vasopressin both in the hypothalamus and neurohypophysis; this effect was most marked on the 8th day of rehydration. On the contrary, it favoured somewhat the renewal of hypothalamic oxytocin in rehydrated rats (such an event was not found on the 8th day of rehydration). Moreover, methoxamine restrained initially (on the 2nd and 4th day of rehydration) the restoration of neurohypophysial oxytocin stores; following eight days of rehydration an opposite effect was here found. It is concluded that the response of the vasopressinergic and oxytocinergic neurons to alpha-adrenergic stimulation, brought about by using methoxamine as pharmacological tool, seems to be depended on the actual state of water metabolism. Impulses from the osmoreceptors may be therefore of some importance in modifying the change in vasopressin and oxytocin synthesis, transport and release resulting from stimulation of alpha-adrenergic transmission through neural chains including units susceptible to methoxamine.     

Endocrine actions of opioids

The widespread occurrence of opioid peptides and their receptors in brain and periphery correlates with a variety of actions elicited by opioid agonists and antagonists on hormone secretion. Opioid actions on pituitary and pancreatic peptides are summarized in Table 1. In rats opioids stimulate ACTH and corticosterone secretion while an inhibition of ACTH and cortisol levels was observed in man. In both species, naloxone, an opiate antagonist, stimulates the release of ACTH suggesting a tonic suppression by endogenous opioids. In rats, a different stimulatory pathway must be assumed through which opiates can stimulate secretion of ACTH. Both types of action are probably mediated within the hypothalamus. LH is decreased by opioid agonists in many adult species while opiate antagonists elicit stimulatory effects, both apparently by modulating LHRH release. A tonic, and in females, a cyclic opioid control appears to participate in the regulation of gonadotropin secretion. Exogenous opiates potently stimulate PRL and GH secretion in many species. Opiate antagonists did not affect PRL or GH levels indicating absence of opioid control under basal conditions, while a decrease of both hormones by antagonists was seen after stimulation in particular situations. In rats, opiate antagonists decreased basal and stress-induced secretion of PRL. Data regarding TSH are quite contradictory. Both inhibitory and stimulatory effects have been described. Oxytocin and vasopressin release were inhibited by opioids at the posterior pituitary level. There is good evidence for an opioid inhibition of suckling-induced oxytocin release. Opioids also seem to play a role in the regulation of vasopressin under some conditions of water balance. The pancreatic hormones insulin and glucagon are elevated by opioids apparently by an action at the islet cells. Somatostatin, on the contrary, was inhibited. An effect of naloxone on pancreatic hormone release was observed after meals which contain opiate active substance. Whether opioids play a physiologic role in glucose homeostasis remains to be elucidated.     

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.