Oxytocinase in the female rat hypothalamus: a novel mechanism controlling oxytocin neurones during lactation.

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase; P-LAP) is expressed both peripherally and centrally. P-LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, although its role in the hypothalamus is unclear. We investigated P-LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, as well as its role in vivo during the milk-ejection reflex. P-LAP mRNA and protein were expressed in magnocellular neurones of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurones co-expressed P-LAP without strong subcellular co-localisation of oxytocin and P-LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P-LAP showed a redistribution of P-LAP to within 1 µm of the plasma membrane in the somata of oxytocin neurones during lactation. Both P-LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c.v. injection of amastatin in anaesthetised suckling mothers increased the frequency of reflex milk ejections. Because hypothalamic P-LAP expression and activity increase in lactation, and the prevention of its action mimics central oxytocin administration, we conclude that P-LAP regulates auto-excitatory oxytocin actions during the suckling-induced milk-ejection reflex.

Intranasal application of vasopressin fails to elicit changes in brain immediate early gene expression, neural activity and behavioural performance of rats.

Intranasal administration has been widely used to investigate the effects of the neuropeptides vasopressin and oxytocin on human behaviour and neurological disorders, although exactly what happens when these neuropeptides are administered intranasally is far from clear. In particular, it is not clear whether a physiological significant amount of peptide enters the brain to account for the observed effects. In the present study, we investigated whether the intranasal administration of vasopressin and oxytocin to rats induces the expression of the immediate-early gene product Fos in brain areas that are sensitive to centrally-administered peptide, whether it alters neuronal activity in the way that centrally-administered peptide does, and whether it affects behaviour in the ways that are expected from studies of centrally-administered peptide. We found that, whereas i.c.v. injection of very low doses of vasopressin or oxytocin increased Fos expression in several distinct brain regions, intranasal administration of large doses of the peptides had no significant effect. By contrast to the effects of vasopressin applied topically to the main olfactory bulb, we saw no changes in the electrical activity of olfactory bulb mitral cells after intranasal vasopressin administration. In addition, vasopressin given intranasally had no significant effects on social recognition or short-term recognition memory. Finally, intranasal infusions of vasopressin had no significant effects on the parameters monitored on the elevated plus maze, a rodent model of anxiety. Our data obtained in rats suggest that, after intranasal administration, significant amounts of vasopressin and oxytocin do not reach areas in the brain at levels sufficient to change immediate early gene expression, neural activity or behaviour in the ways described for central administration of the peptides.

Increased sensitivity of monoamine release in the supraoptic nucleus in late pregnancy: region- and stimulus-dependent responses.

Oxytocin neurone activation at birth depends upon noradrenaline-mediated signals from the uterus via a brainstem pathway, as well as on factors within the supraoptic nucleus (SON), including oxytocin itself, and the system adapts during pregnancy to optimise the delivery process. We determined whether noradrenaline release in the SON in response to stimuli activating brainstem inputs or antidromically activating magnocellular neurones is enhanced at term pregnancy. Noradrenaline, serotonin and dopamine concentrations were measured in microdialysis samples collected from the dorsal and ventral SON before, during and after either i.v. cholecystokinin (CCK) or neural stalk stimulation in virgin and late pregnant rats. Each stimulus transiently increased noradrenaline and serotonin but not dopamine concentration in the dorsal SON, and responses were increased on days 21 and 22 of pregnancy compared to day 20 pregnant and virgin rats. Neural stalk stimulation induced sensitisation to subsequent stalk stimulation and so the responses in the dorsal SON were doubled; on day 22 of pregnancy, the area under the curve of monoamine concentration was 3.4-fold greater than in virgins, suggesting that adaptations perinatally enhance responsiveness. In conclusion, there are enhanced responses of noradrenaline and serotonin release in the SON that can generate very high, transient extracellular concentrations at term. This may be a consequence of neuroendocrine adaptations in late pregnancy and probably contributes to optimal oxytocin neurone activation during parturition.

The actin filament and dendritic peptide release.

F-actin remodelling has been implicated in regulated secretion from many cell types, in particular secretion from neuron axon terminals and neuroendocrine cell types. Cortical F-actin has long been postulated to act as a barrier to vesicle movement and hence to inhibit secretion; however, more recent studies point to F-actin remodelling providing both supporting and restraining roles in secretion. Magnocellular neurons of the supraoptic nucleus secrete either oxytocin or vasopressin from their dendrites as well as their axon terminals; and peptide release from these two compartments can be differentially controlled to allow secretion from one compartment in isolation from the other. While oxytocin and vasopressin secretion can be provoked by F-actin depolymerization in both compartments, acutely stimulated secretion is dependent on F-actin remodelling in dendrites but not axon terminals, suggesting that F-actin plays a different role in regulating the readily releasable pool of secretory vesicles in the two compartments. In addition, activity-dependent secretion from the dendritic compartment can be primed by prior exposure to agents, including oxytocin, that stimulate release of Ca(2+) from intracellular stores. While remodelling of F-actin is involved, it is not solely responsible for priming secretory responses.

How important is stimulation of alpha-adrenoceptors for melatonin production in rat pineal glands?

The objective of this study was to determine the role of alpha-adrenoceptors in melatonin production by rat pineal gland. Pineal glands were isolated from adult male rats and maintained in organ baths. The perfusate was sampled every 5 min, stored, and later assayed for melatonin. Exposure to norepinephrine (10 microM) or the beta-adrenoceptor agonist orciprenaline (2-10 microM) increased the glands' production of melatonin. The time courses of melatonin production in response to these agonists were unaffected by the rats' pretreatment in vivo with the alpha-adrenoceptor antagonist prazosin (2 mg/kg i.p., three times). Rats that had had their superior cervical ganglia removed were primed with either orciprenaline (2 mg/kg i.p) or both orciprenaline and phenylephrine (1 mg/kg i.p) 1 hr before decapitation. Exposure of the pineal glands from these rats to orciprenaline evoked melatonin release that was similar in each group. These results lend weight to the suggestion that the marked potentiation by alpha-adrenoceptor agonists of the stimulation of cAMP and N-acetyltransferase (NAT) by beta-adrenoceptor agonists, demonstrated most readily in cultured glands or dispersed rat pinealocytes, does not carry over into significant augmentation of melatonin production in intact pineal glands.

The percentage of pituitary gonadotropes with immunoreactive oestradiol receptors increases in the follicular phase of the ovine oestrous cycle.

During the oestrous cycle, there is an alteration in gonadotrope responsiveness to gonadotropin releasing hormone (GnRH). One cellular mechanism that may be involved in these changes at the pituitary level is the hormonal regulation of oestrogen receptor (ER) expression. Using double-label immunohistochemistry, we examined the proportion of gonadotropes, lactotropes and somatotropes with immunoreactive (ir) oestrogen receptor alpha (ERalpha) in pituitary sections from ewes at three stages of the ovine oestrous cycle (n = 8 per group). The percentage of ERalpha positive cells that also stained positive for luteinizing hormone (LH) increased in the transition from the luteal phase to the follicular phase (n = 8), with no further increase at the time of oestrus (n = 8). In the pituitaries from the luteal phase sheep, only a small number (15%) of lactotropes and 4% of somatotropes were found to contain ir-ERalpha and there were no alterations across the oestrous cycle. When we examined pituitaries from ovariectomized (OVX) ewes treated (i.m.) with either oestradiol benzoate (50 microg) or oil vehicle for 2, 4, 6 or 16 h (n = 4 per group), there was no effect of treatment. In fact, the percentage of gonadotropes that were ERalpha-positive in OVX ewes was similar to that observed in the pituitaries from the follicular phase ewes, both of which display a high frequency of pulsatile GnRH secretion. We conclude that the number of gonadotropes that contain ir-ERalpha increases in the follicular phase of the oestrous cycle and this may enhance the responsiveness of these cells to oestrogen and GnRH. We suggest that this may be due to increased pulsatile GnRH input rather than rising oestrogen levels.

The regulation of gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs by testosterone is mediated by dihydrotestosterone.

The biological effects of testosterone (T) may be mediated directly by T or indirectly by its metabolites, dihydrotestosterone (DHT) and estradiol. The present study examined whether the metabolism of T is involved in the regulation of GnRH-induced Ca2+ signaling at the pituitary. In gonadotrophs from castrated rats, a significantly greater percentage of gonadotrophs demonstrated oscillatory Ca2+ responses to 100 nM GnRH than cells from intact rats (72% vs. 24%; P < 0.05). This increase was prevented by the administration of T propionate (0.1 mg/kg x day), DHT benzoate (2 mg/kg x day,), estradiol benzoate (EB; 5 microg/kg x day), or the combination of the above doses of DHT benzoate and EB. In all cases the proportion of gonadotrophs from the steroid-treated rats having oscillatory Ca2+ responses to 100 nM GnRH was between 21-25% (P > 0.05, compared with intact rats). To assess the importance of T metabolism, intact male rats were treated with the aromatase inhibitor letrozole (1 mg/kg x day), the 5alpha-reductase inhibitor finasteride (50 mg/kg x day), or their respective vehicles for 7 days. Letrozole had no effect on GnRH-induced Ca2+ signals, serum LH concentrations, or ventral prostate or testes weight. Finasteride treatment, however, mimicked the effects of castration, with significantly more gonadotrophs exhibiting Ca2+ oscillations in response to 100 nM GnRH than gonadotrophs from the vehicle-treated group (71% vs. 20% respectively; P < 0.05). Finasteride also caused a significant (P < 0.05) decrease in prostatic weight and DHT concentration, but had no significant effect on either prostatic T or serum LH concentrations. These findings suggest that in the intact male rat, the effects of T on GnRH-induced Ca2+ signaling are preferentially mediated via DHT. The results of this study also show that in the absence of androgens, estradiol may regulate GnRH-induced Ca2+ signaling in the male rat pituitary.

Testosterone acts directly at the pituitary to regulate gonadotropin-releasing hormone-induced calcium signals in male rat gonadotropes.

We have recently shown that castration alters GnRH-induced calcium (Ca2+) signaling in the gonadotropes of male rats. Instead of generating spike-plateau Ca2+ responses to high concentrations of GnRH (100 nM), the majority of gonadotropes from castrated rats have oscillatory Ca2+ responses, which are generally only seen with low concentrations of GnRH in the gonadotropes of intact rats. This change in the nature of GnRH-induced Ca2+ responses is prevented by in vivo testosterone treatment. The aims of the present study were, therefore, to determine if testosterone acts directly at the pituitary or via the regulation of hypothalamic GnRH secretion. Accordingly, castrated male rats were treated with a GnRH antagonist to ablate the effects of increased GnRH secretion at the pituitary gland. GnRH antagonist treatment (10 microg/100 g BW, twice daily for 7 days from the time of castration) decreased the concentration of LH in the serum of castrated rats (0.4 +/- 0.1 ng/ml vs. 11.2 +/- 0.4 ng/ml in untreated castrated rats, mean +/- SEM) but had no effect on the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH when compared with untreated castrated rats (63% in antagonist-treated castrated rats vs. 70% in untreated castrated rats). The GnRH antagonist treatment did not, however, interfere with the ability of in vivo testosterone treatment (100 microg/100 g body weight/day) to decrease the proportion of gonadotropes having oscillatory Ca2+ responses to 100 nM GnRH (26% in testosterone-treated rats vs. 25% in testosterone and antagonist-treated rats). These findings indicate that testosterone acts directly at the pituitary, and not by altered GnRH secretion, to modulate GnRH-induced Ca2+ signals. To confirm this suggestion, cultured gonadotropes of castrated male rats were treated in vitro with 10 nM testosterone. Testosterone treatment for twelve, but not 4 h, restored the proportion of gonadotropes having oscillatory Ca2+ responses to that seen in gonadotropes from intact rats. The in vitro effects of testosterone over 12 h were prevented by concomitant treatment with the protein synthesis inhibitor cycloheximide (10 microM), which, when given alone, had no effect on GnRH-induced Ca2+ signals in cells from castrate male rats. Taken together, these findings suggest that testosterone has a direct genomic action at the pituitary to regulate GnRH-induced Ca2+ signals, via a process that involves new protein synthesis.

Testosterone regulates gonadotropin-releasing hormone-induced calcium signals in male rat gonadotrophs.

As the GnRH-induced secretion of gonadotropins is critically dependent upon an increase in the intracellular calcium ion concentration ([Ca2+]i) and modulated by gonadal factors, the effects of gonadal steroids on the pattern of calcium mobilization in single gonadotrophs of the male rat were examined using the fluorescent Ca2+ indicator fura-2/AM. In cells from intact rats, low concentrations of GnRH induce repetitive oscillations in [Ca2+]i, whereas spike-plateau responses are observed at higher concentrations in single gonadotrophs. After castration, there was a significant change in the relationship between the GnRH concentration and the changes in [Ca2+]i. Increasing concentrations of GnRH (to 1 micron) generate fewer spike-plateau responses in gonadotrophs from castrate rats, with oscillatory responses predominating. This change develops with time after castration, with the proportion of cells oscillating in response to 100 nM GnRH peaking by 7 days. This effect of castration on GnRH-induced [Ca2+]i signals was reversed by treatment with testosterone propionate (100 microgram/100 g BW-day). Castration-induced decreases in serum testosterone, seminal vesicle, and prostate weights and increases in serum LH concentration were also corrected by testosterone propionate treatment. These findings demonstrate that testosterone regulates GnRH-stimulated Ca2+ signals in gonadotrophs and suggest that gonadal steroids exert a regulatory role in the secretion of gonadotropins at the level of Ca2+ mobilization.