Data supporting a new physiological role for brain apelin in the regulation of hypothalamic oxytocin neurons in lactating rats

Apelin is a bioactive peptide identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ in 1998. The present data show that apelin modulates the activity of magnocellular and parvocellular oxytocin (OXY) neurons in the lactating rat. A combination of in situ hybridization and immunohistochemistry demonstrated the presence of apelin receptor mRNA in hypothalamic OXY neurons. Double immunofluorescence labeling then revealed the colocalization of apelin with OXY in about 20% of the hypothalamic OXY-positive neurons. Intracerebroventricular apelin administration inhibited the activity of magnocellular and parvocellular OXY neurons, as shown by measuring the c-fos expression in OXY neurons or by direct electrophysiological measurements of the electrical activity of these neurons. This effect was correlated with a decrease in the amount of milk ejected. Thus, apelin inhibits the activity of OXY neurons through a direct action on apelin receptors expressed by these neurons in an autocrine and paracrine manner. In conclusion, these findings highlight the inhibitory role of apelin as an autocrine/paracrine peptide acting on OXY neurons during breastfeeding.     

Dopaminergic control of oxytocin release in lactating rats.

During suckling, anaesthetized lactating rats release regular (about every 7 min) but brief pulses of oxytocin (0.5--1.0 mu.) which produce single transient increases in intramammary pressure. Drugs which selectively impair synaptic transmission were used to determine the role of dopamine and noradrenaline in regulating this natural reflex. Diethyldithiocarbamate (100--200 mg/kg, i.v.) and alpha-methylparatyrosine (100--400 mg/kg, i.v.) which inhibit the synthesis of catecholamines both blocked the suckling-induced release of oxytocin. The milk-ejection reflex was also inhibited in a dose-dependent manner by the intravenous administration of the dopamine antagonists, fluphenazine (0.7 mg/kg), pimozide (1.4 mg/kg), cis-dupenthixol (4.5 mg/kg) and metoclopramide (6.0 mg/kg), and caused a significant inhibition P less than 0.01) of the reflex in 50% of the rats tested. The alpha-adrenoceptor antagonist phenoxybenzamine (1.4 mg/kg) was similarly effective. Dopamine (40 micrograms), bromocriptine (10 micrograms), apomorphine (100 micrograms), noradrenaline (10 micrograms) and phenylephrine (2 micrograms) injected into the cerebral ventricles evoked a sustained release of oxytocin which produced multiple increases in intramammary pressure; isoprenaline (4 micrograms) was ineffective. The release of oxytocin evoked by dopamine and noradrenaline was prevented by cis-flupenthixol and phenoxygenzamine respectively. None of the drugs used affected the mammary sensitivity to exogenous oxytocin nor were their actions modified by pretreatment with propranolol (1 mg/kg). The results suggest that the neural pathway for the reflex release of oxytocin during suckling in the rat contains both dopaminergic and noradrenergic synapses, the latter acting through alpha-adrenoceptors and being distal in the pathway to the dopaminergic component.     

Activation of central D-1 dopamine receptors stimulates oxytocin release in the lactating rat: evidence for involvement of the hypothalamic paraventricular and supraoptic nuclei.

The stimulatory effect of dopamine (DA) on the release of oxytocin (OT) in lactating rats is exerted at the D-1 DA receptor subtype. Because the neural loci mediating this effect have not been identified, the objective of the present studies was to test whether OT release in the lactating rat would be elevated after central administration of a D-1 DA receptor agonist into the third ventricle (3V) or directly into either the rostral paraventricular/anterior commissural nucleus area (PVN/ACN), the central paraventricular nucleus area, or the supraoptic nucleus (SON), all of which contain OT neurosecretory cells. Lactating rats were implanted with a stainless steel cannula directed into one of the above areas or into the arcuate-ventromedial region of the medial basal hypothalamus (MBH), or sites dorsal to the PVN/ACN or SON, which served as anatomical controls. After 6-7 days of recovery, each animal received an intra-atrial cannula for sequential blood sampling, and was used in experiments 24 h later. Animals were separated from their litters, and following a period of basal blood sampling, received central microinjections of either vehicle, the D-1 DA receptor agonist SKF-38393, or the D-2 DA receptor agonist quinpirole, and blood samples were removed periodically for 60 min. An injection of angiotensin II (Ang II, 100 ng) was made into each site as a positive control for OT release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of eicosanoids in release of oxytocin and vasopressin from the neural lobe of the rat pituitary

Arachidonic acid (AA) is oxidized via three pathways which result in several series of distinct metabolites. Cyclooxygenase produces prostaglandins (PGs), prostacyclins, and thromboxanes. Lipoxygenase produces hydroperoxy/hydroxyeicosatetraenoic acids (HPETE/HETEs) and leukotrienes. Epoxygenase, a recently uncovered pathway, results in epoxyeicosatrienoic acids (EETs). Based on reverse phase HPLC product analysis, this study establishes that all three pathways of AA metabolism are present in microsomal incubates of the neural lobe of the pituitary gland. Addition of PGE2 to incubated fragments of neural lobes of the rat pituitary stimulates secretion of both arginine vasopressin (AVP) and oxytocin in vitro. Inclusion of 5-HETE and 12-HETE in the incubation medium stimulates marginal release of AVP and oxytocin by 12-HETE only. The magnitude of AVP and oxytocin secretion stimulated by the epoxygenase metabolites 8,9-, 11,12-, and 14,15-EET is equal to that caused by PGE2. Maximal stimulation of secretion (3- to 4-fold) requires an EET concentration 10-15 times greater than that of PGE2. In contrast, 5,6-EET is inactive. These data suggest that oxygenated products of AA play a role in AVP and oxytocin secretion. Although PGs appear to be the dominant arachidonate metabolites involved in the release of AVP and oxytocin, the EETs probably have a contributing role.     

Evidence for a physiological role for oxytocin in the control of prolactin secretion

The presence of oxytocin (OT) in neuronal elements of the external layer of the median eminence and in hypophysial portal plasma suggests a role for the peptide in the control of anterior pituitary function. We have reported previously that OT stimulates PRL release in vitro; therefore, we attempted to establish evidence for a physiological PRL-releasing role for OT. Plasma OT levels rose significantly just before the PRL surges occurring during a suckling stimulus in lactating rats (10 min after pup reinstatement vs. 15 min for PRL) and 48 h after estrogen injection in ovariectomized (OVX) rats (at 1200 h vs. 1300 h). Dispersed anterior pituitary cells harvested from lactating female rats and OVX estrogen-primed rats released PRL in a specific, significant, and dose-related fashion when perifused in vitro with incubation medium containing 10(-7)-10(-9) M OT, doses similar to levels found previously in hypophysial portal plasma. Infusion of antiserum specific for OT into lactating females before pup reinstatement and into estrogen-primed OVX rats 2 h before the expected release of endogenous OT delayed and significantly reduced subsequent PRL surges compared to levels in saline-or normal rabbit serum-infused rats; however, PRL release was not completely abolished. These data indicate that OT plays a physiological role in the hypothalamic control of PRL secretion and further suggest the importance of multiple factors in coordinated regulation of PRL release.     

Prolactin selectively stimulates ornithine decarboxylase in the lateral lobe of the rat prostate

In androgenized-hypophysectomized rats, ovine prolactin stimulated the activity of the ornithine decarboxylase (ODC) of the lateral lobes, but not the ventral and dorsal lobes of the prostate glands in a time- and dose-dependent fashion. High degrees of enzyme stimulation were associated with significant elevations in the endogenous levels of its product, putrescine. The relative response to prolactin over basal activities was relatively unaffected by indomethacin but decreased with cycloheximide, suggesting that prostaglandins do not mediate the effects of the hormone, but that a high rate of protein synthesis is a prerequisite for its expression. Indomethacin alone significantly increased the basal activity of the enzyme above control levels, suggesting that prostaglandins may normally exert a degree of inhibition on the ODC. The selective activation of the lateral lobe ODC supports previous reports of a differential response of the various prostatic lobes to prolactin, and also provides a convenient biochemical response for examining details of prolactin action on this organ.     

Inhibitory action of exogenous oxytocin on plasma cortisol in normal human subjects: evidence of action at the adrenal level

In previous studies we demonstrated that exogenous oxytocin induces a decrease of basal and stimulated cortisol and ACTH plasma levels in the normal male. To rule out the possibility that oxytocin could also act at the adrenal level, we tested, in the present work, the influence of exogenous oxytocin on the cortisol increase secondary to the intravenous injection of a supraphysiological dose (0.25 mg) of synthetic ACTH1-24 (Synachten) in 9 normal male volunteers. We demonstrate that oxytocin infusion does not modify the ACTH plasma levels but, however, induces a decreased cortisol response to ACTH1-24. These results support the hypothesis that, beside its hypophysial inhibitory action on ACTH release, oxytocin acts also at the adrenal gland level to decrease cortisol release and/or synthesis in normal human subjects.     

Evidence for stimulatory noradrenergic and inhibitory dopaminergic regulation of oxytocin release in the lactating rat

The present experiments tested the involvement of central catecholaminergic systems in the suckling-induced release of oxytocin (OT) during lactation in the rat. In the first experiment, female rats in midlactation were separated from their offspring for 4 h and then allowed to suckle their litters for 30 or 60 min or to remain nonsuckled. The turnover rates of norepinephrine (NE) and dopamine (DA) were calculated from the rate of decline after synthesis inhibition. Suckling decreased the turnover rate of DA in the median eminence and in the neurointermediate lobe of the pituitary gland. Suckling increased the turnover rate of NE in the rostral paraventricular and supraoptic nuclei, areas that contain most of the OT cells that project to the neural lobe of the pituitary, and in the interstitial nucleus of the stria terminalis, but not in the arcuate or caudal paraventricular nuclei, median eminence, or neurointermediate lobe. In a second experiment, midlactating females received intracerebral microinjections of the catecholamine neurotoxin 6-hydroxydopamine or of vehicle into the vicinity of the paraventricular and supraoptic nuclei 1 week before a suckling test. The release of OT was completely prevented in 6-hydroxydopamine-treated animals, and NE was significantly decreased in the paraventricular, supraoptic, and arcuate nuclei. In a third study, the increase in plasma OT in response to suckling was prevented by stimulation of DA receptors with bromocriptine, while blockade of DA receptors with domperidone significantly increased plasma OT levels in nonsuckled lactating rats. These results suggest that suckling stimulation activates the noradrenergic innervation to the rostral paraventricular nucleus and to the supraoptic nucleus, which exerts an excitatory influence on the release of OT and decreases activity of the tuberohypophyseal DA system, which provides a tonic inhibitory influence over the secretion of OT.     

The hypothalamic paraventricular nucleus has a pivotal role in regulation of prolactin release in lactating rats

The affect of paraventricular nucleus (PVN) lesions on PRL secretory response to suckling was studied in adult female rats. Basal levels of PRL were similar in the control and lesioned groups. Substantial decreases in PRL levels occurred after separation of pups from their mothers in the control as well as lesioned animals. When mothers and pups were reunited, the circulating PRL concentrations of the control groups rose immediately from basal values of 50-100 micrograms/liter to reach peaks of 450-550 micrograms/liter. PVN lesions significantly decreased the suckling-induced rise of PRL levels. Furthermore, PVN lesions abolished the high amplitude, episodic pattern of PRL release in continuously lactating rats. These findings are consistent with the view that PVN neurons produce PRL releasing factor(s), which is (are) required for normal secretory patterns of PRL in lactating rats.     

Relationship between the suckling-induced release of oxytocin and prolactin in the urethane-anaesthetized lactating rat

The effects of changes in the intensity of the suckling stimulus on the reflex release of oxytocin and prolactin were compared in urethane-anaesthetized lactating rats. Mothers which had previously suckled 12 pups (Group 1) showed a graded increase in the amount of oxytocin released during a 3 h suckling test when the number of pups applied to the nipples was increased from six to eight or ten. Mothers which had suckled six pups during their lactation (Group 2) appeared to show a maximum frequency of milk ejection whether six, eight or ten pups were applied to the nipples. The release of prolactin was not elicited from either Group 1 or Group 2 mothers when six pups were applied to their nipples. With eight pups suckling, the Group 1 mothers again showed no evidence of prolactin release. In contrast, the Group 2 mothers showed a significant increase in the level of prolactin in the plasma during the 3 h suckling test. With ten pups suckling the release of prolactin was evident in both groups of mothers, although the response was earlier and more pronounced in Group 2 than Group 1. These results suggest that in the urethane-anaesthetized rat, the threshold for the suckling-induced reflex release of oxytocin is distinct from the threshold for the release of prolactin and that these thresholds are, at least in part, set by the preceding suckling experience of the mothers. In those animals which showed both reflex milk ejection and prolactin release there was a linear relationship between the magnitude of the two endocrine responses.     

Obestatin inhibits vasopressin secretion: evidence for a physiological action in the control of fluid homeostasis

Obestatin, a product of post-translational processing of the ghrelin prohormone, has been reported to act in the brain to inhibit thirst. We extended our initial studies on water drinking by examining the effects of obestatin on hypovolemia-induced water and saline drinking and vasopressin release in male rats. Intracerebroventricular administration of obestatin significantly inhibited water, but not saline (0.3 M NaCl) drinking in response to a hypovolemic challenge. Obestatin also inhibited, in a dose-related fashion, dehydration-induced vasopressin secretion without affecting plasma oxytocin levels. Vasopressin release induced by central angiotensin II administration was attenuated significantly by prior administration of obestatin. Finally, central administration of an antiserum specific to obestatin resulted in an exaggerated basal vasopressin release and an increased vasopressin response to overnight water deprivation. Antiserum treatment also resulted in significantly increased ad libitum water drinking and drinking in response to dehydration. We conclude that this product of post-translational processing of the ghrelin prohormone may be an important contributor to the physiologic regulation of fluid and electrolyte homeostasis.     

Insulin stimulates cord blood erythroid progenitor growth: evidence for an aetiological role in neonatal polycythaemia

Polycythaemia in the neonate is a serious pathologic entity which occurs particularly in infants of diabetic mothers (IDM) and small-for-gestational age (SGA) infants. Both of these conditions are associated with fetal hyperinsulinaemia. Cultures of cord blood mononuclear cells from polycythaemic IDM showed increased growth of late erythroid progenitor colonies, compared to cord blood mononuclear cells from non-polycythaemic infants, reflecting a possible expansion of this progenitor population in the polycythaemic fetus. No changes were observed in early erythroid progenitor populations. Biosynthetic human insulin at physiological levels characteristic of IDM stimulated growth in culture of late erythroid progenitors in cord blood from premature, term and IDM infants. Three out of five polycythaemic infants had elevated cord blood plasma levels of insulin C-peptide at birth, whereas no infant with a haematocrit of less than 65% had high insulin C-peptide measurements. These data suggest that the polycythaemia noted in infants of diabetic mothers may be secondary, in large part, to a stimulatory effect on erythroid progenitor growth by the hyperinsulinaemic environment in which they develop in utero.     

Central inhibition of oxytocin release in the rabbit: role of the midbrain

Transection of the ventral central grey and surrounding midbrain tegmentum in anaesthetized lactating rabbits caused repeated milk ejections which, on comparison with the effects of i.v. infusions of synthetic oxytocin, synthetic arginine-vasopressin or a mixture of the two, were attributed to continuous release of 1.25-2.5 mu. oxytocin/min, although it is not known whether lesser amounts of vasopressin might also have been released. It is suggested that the ascending midbrain reticular formation, which is known to project rostrally through this region, controls the central inhibition of oxytocin release via the previously described septo-hippocampo-subicular route to the hypothalamus.     

Effects of porcine relaxin on oxytocin release from the neurohypophysis in the anaesthetized lactating rat

The effect of relaxin on electrically evoked release of oxytocin from the posterior pituitary was examined by monitoring changes in intramammary pressure in the anaesthetized lactating rat. The amount of oxytocin released by electrical stimulation of the neurohypophysis in vivo was dramatically reduced following i.v. injection of highly purified porcine relaxin (2.5-10 micrograms/rat). Relaxin inhibited oxytocin release in a dose-dependent manner and the onset of inhibition occurred within 6-10 min and lasted for 10-60 min. No effect on the sensitivity of the mammary gland to exogenous oxytocin was observed after relaxin treatment. During the period of inhibition, i.v. injection of the opioid antagonist naloxone chloride (1 mg/kg) completely and immediately restored electrically evoked oxytocin release. The neurohypophysis is known to contain endogenous opioid peptides, therefore the effect of relaxin on electrically stimulated release of oxytocin from the rat isolated neural lobe in vitro was examined. Relaxin (500-2000 ng/ml) failed to inhibit oxytocin release in vitro. The results suggest that relaxin can inhibit the release of oxytocin from terminals in the neurohypophysis, but by an indirect mechanism. This action appears to be mediated through endogenous opioid peptides whose source is not clear. They are unlikely to be of neurohypophysial origin and may probably come from the adrenal medulla, since acute adrenalectomy negated the inhibitory effect of relaxin on oxytocin release.     

The sweeter side of ACE2: physiological evidence for a role in diabetes

Diabetes mellitus is a growing problem in all parts of the world. Both clinical trials and animal models of type I and type II diabetes have shown that hyperactivity of angiotensin-II (Ang-II) signaling pathways contribute to the development of diabetes and diabetic complications. Of clinical relevance, blockade of the renin-angiotensin system prevents new-onset diabetes and reduces the risk of diabetic complications. Angiotensin-converting enzyme (ACE) 2 is a recently discovered mono-carboxypeptidase and the first homolog of ACE. It is thought to inhibit Ang-II signaling cascades mostly by cleaving Ang-II to generate Ang-(1-7), which effects oppose Ang-II and are mediated by the Mas receptor. The enzyme is present in the kidney, liver, adipose tissue and pancreas. Its expression is elevated in the endocrine pancreas in diabetes and in the early phase during diabetic nephropathy. ACE2 is hypothesized to act in a compensatory manner in both diabetes and diabetic nephropathy. Recently, we have shown the presence of the Mas receptor in the mouse pancreas and observed a reduction in Mas receptor immuno-reactivity as well as higher fasting blood glucose levels in ACE2 knockout mice, indicating that these mice may be a new model to study the role of ACE2 in diabetes. In this review we will examine the role of the renin-angiotensin system in the physiopathology and treatment of diabetes and highlight the potential benefits of the ACE2/Ang-(1-7)/Mas receptor axis, focusing on recent data about ACE2.