Prolactin secretory rhythm of mated rats induced by a single injection of oxytocin

Mating or vaginocervical stimulation [copulatory stimulus (CS)] induces two daily surges of the hormone prolactin (PRL) in rats. This unique secretory pattern of PRL surges is characteristic for the first half of pregnancy and is also present in ovariectomized (OVX) rats. Studies have shown that CS additionally provokes an acute release of the hormone oxytocin (OT). In this study, we tested whether a single injection of OT (iv) is sufficient to initiate the PRL secretion pattern of OVX/CS rats. Furthermore, we measured the 24-h profile of dopamine (DA) content in the anterior lobe of the pituitary gland, because DA is the major inhibitory factor of PRL secretion. The results indicated that a single injection of OT induces a PRL secretory rhythm and a DA release pattern similar to that initiated by CS. Immunocytochemical investigation showed that particular OTergic neurons in the hypothalamus express receptors for PRL, as well as for vasoactive intestinal polypeptide, which indicates an involvement in generating the PRL rhythm and entraining it to the ambient photoperiod. On the basis of this study, we suggest that the PRL-DA inhibitory feedback loop between lactotrophs and DAergic neurons plays a crucial role in generating the oscillatory PRL secretion pattern in CS rats. A timing signal, likely provided by the hypothalamic suprachiasmatic nucleus, entrains the autonomous PRL oscillation to a particular time of day. Mathematical modeling was used to illustrate the proposed network function. The experimental results further suggest an additional feedback mechanism in which certain hypothalamic OTergic neurons are influenced by PRL.     

A mathematical model for the mating-induced prolactin rhythm of female rats

For the first 10 days of pregnancy and the first 12 days of pseudopregnancy, the secretion of prolactin (PRL) from pituitary lactotrophs is rhythmic, with two surges/day. This rhythm can also be triggered by bolus injection of oxytocin (OT). We describe a mathematical model for the initiation, maintenance, and termination of the OT-induced PRL rhythm. In our model, the mechanism for this circadian rhythm is mutual interaction between lactotrophs and neuroendocrine dopamine (DA) neurons. This rhythm is, under normal lighting conditions, entrained by the suprachiasmatic nucleus (SCN) but persists in the absence of input from the SCN. We postulate that OT injection triggers the rhythm by activating a population of bistable hypothalamic neurons that innervate and inhibit DA neurons. The bistable nature of these neurons allows them to act as a memory device, maintaining the rhythm long after OT has been cleared from the blood. The mechanism for this memory device and the arguments supporting it are detailed with computer simulations. Finally, we consider potential targets for a rhythm-terminating factor and make predictions that may be used to determine which mechanism is operational in terminating the OT- or mating-induced PRL rhythm.     

Oxidative stress induces vascular heme oxygenase-1 expression in ovariectomized rats

Heme oxygenase-1 (HO-1), an inducible stress protein, has been implicated in cytoprotection against oxidative stress in vitro and in vivo. Estrogens also have antioxidant effects. This study investigated the time course of HO-1 and inducible nitric oxide synthase (iNOS) expression in the aortas of ovariectomized rats, and the regulatory relationship between the NO/NOS and the carbon monoxide/HO systems. HO-1 and iNOS protein expression was induced by ovariectomy (Ovx) and was extremely high 2-6 weeks after Ovx compared with the sham-operated group. Expression of the constitutive enzymes HO-2 and endothelial NOS did not differ significantly between sham-operated and Ovx rats. 17beta-Estradiol (E(2)) replacement reversed these changes in rats after Ovx. Long-term treatment with the antioxidant tempol significantly inhibited HO-1 and iNOS expression. The iNOS inhibitor aminoguanidine significantly suppressed the induction of HO-1. Oxidized glutathione in the hearts of Ovx rats increased gradually, with significant elevation at 3-6 weeks after Ovx compared with the sham-operated group, whereas plasma levels of NO metabolites were significantly reduced 4-6 weeks after Ovx. Treatment with the HO inhibitor zinc protoporphyrin IX blocked HO-1 induction, but significantly increased the plasma levels of NO metabolites. In conclusion, HO-1 is induced by oxidative stress resulting from E(2) depletion. The NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause.     

Circadian rhythm of circulating corticosterone and prolactin levels in spontaneously hypertensive rats

Circulating levels of corticosterone and prolactin were measured by radioimmunoassay at hourly intervals during a 24 h period to establish the diurnal rhythm of these hormones in spontaneously hypertensive rats (SHR). Corticosterone levels exhibited a distinct circadian variation with concentrations reaching a zenith at 2000 h and a nadir at 1200 h in male and female SHR. Corticosterone levels in females were consistently greater than males. Circulating prolactin levels were greater during the light h than dark in the female; the opposite occurred in males. Measurement of pituitary prolactin content tended to be low when serum prolactin levels were high and vice versa. The circadian rhythm of circulating corticosterone and prolactin in the hypertensive SHR was found to be similar to the day-night patterns established for normotensive rats. However, these measurements were made under quiescent conditions. It is suggested that because SHR are hyper-responsive to stress and because corticosterone and prolactin have synchronous effects on stress-induced adrenal steroidogenesis, further investigation of prolactin and corticosterone may reveal a participatory role of these hormones in the pathogenesis of the genetically-programmed hypertension of SHR.     

Effect of exercise training on cardiac oxytocin and natriuretic peptide systems in ovariectomized rats

Exercise training results in cardiovascular and metabolic adaptations that may be beneficial in menopausal women by reducing blood pressure, insulin resistance, and cholesterol level. The adaptation of the cardiac hormonal systems oxytocin (OT), natriuretic peptides (NPs), and nitric oxide synthase (NOS) in response to exercise training was investigated in intact and ovariectomized (OVX) rats. Ovariectomy significantly augmented body weight (BW), left ventricle (LV) mass, and intra-abdominal fat pad weight and decreased the expression of oxytocin receptor (OTR), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and guanylyl cyclase-A (GC-A), in the right atrium (RA) and LV, indicating estrogenic control of these genes. These effects of ovariectomy were counteracted by 8-wk-long exercise training which decreased fat pad weight (33.4 +/- 2.3 to 23.4 +/- 3.1 g, n = 8, P < 0.05), plasma free fatty acids (0.124 +/- 0.033 to 0.057 +/- 0.010 mM, n = 8, P < 0.01), and plasma triacylglycerol (0.978 +/- 0.174 to 0.588 +/- 0.115 mM, n = 8, P < 0.05). Chronic exercise tended to decrease BW and stimulated ANP (4- to 5-fold) and OTR gene expression in the LV and RA and BNP and inducible NOS (iNOS) mRNA in the LV. In sham-operated rats, exercise augmented ANP expression in the RA, downregulated GC-A mRNA in the LV and RA, but increased its expression threefold in the RA of OVX animals. Endothelial NOS and iNOS expression was enhanced in the left atrium of sham-operated rats. Altogether, these data indicate that in OVX animals, chronic exercise significantly enhances cardiac OT, NPs, and NOS, thus implicating all three hormonal systems in the beneficial effects of exercise training.     

Limited proteolysis of prolactin in secretory granules from the pituitary gland of estrogenized rats

The LTH-converting proteolytic activity in LTH granules isolated from estrogenized rat hypophysis was studied. Suspensions of granules were incubated at different values of pH for 4 hours at 37 degrees C. The reaction was controlled by SDS electrophoresis. Intensive proteolysis of LTH was observed at pH 6.0 and 3.9, which was accompanied by the formation of fragments with Mr 10, 12 and 17 kD and probably of smaller peptides. An inhibitory analysis revealed that the formation of the 17 kD fragment at pH 3.9 was partly and selectively inhibited by chloroquine, phenanthroline and phenylmethylsulfonyl fluoride. Pepstatin A fully inhibited the proteolysis, whereas leupeptin had no inhibiting influence. The data obtained testify to the presence in the granular fraction of the endopeptidase LTH-converting activity which is sensitive to pepstatin A, an aspartyl proteinase inhibitor as well as to chelators and a serine proteinase inhibitor.     

Alpha-synuclein targets the plasma membrane via the secretory pathway and induces toxicity in yeast

A pathological feature of Parkinson's disease is the presence of Lewy bodies within selectively vulnerable neurons. These are ubiquitinated cytoplasmic inclusions containing alpha-synuclein, an abundant protein normally associated with presynaptic terminals. Point mutations in the alpha-synuclein gene (A30P and A53T), as well as triplication of the wild-type (WT) locus, have been linked to autosomal dominant Parkinson's. How these alterations might contribute to disease progression is unclear. Using the genetically tractable yeast Saccharomyces cerevisiae as a model system, we find that both the WT and the A53T isoforms of alpha-synuclein initially localize to the plasma membrane, to which they are delivered via the classical secretory pathway. In contrast, the A30P mutant protein disperses within the cytoplasm and does not associate with the plasma membrane, and its intracellular distribution is unaffected by mutations in the secretory pathway. When their expression is elevated, WT and A53T, but not A30P, are toxic to cells. At moderate levels of expression, WT and A53T induce the cellular stress (heat-shock) response and are toxic to cells bearing mutations in the 20S proteasome. Our results reveal a link between plasma membrane targeting of alpha-synuclein and its toxicity in yeast and suggest a role for the quality control (QC) system in the cell's effort to deal with this natively unfolded protein.     

The possible role of prolactin in the circadian rhythm of leptin secretion in male rats

In humans there is a circadian rhythm of leptin concentrations in plasma with a minimum in the early morning and a maximum in the middle of the night. By taking blood samples from adult male rats every 3 hr for 24 hr, we determined that a circadian rhythm of plasma leptin concentrations also occurs in the rat with a peak at 0130h and a minimum at 0730h. To determine if this rhythm is controlled by nocturnally released hormones, we evaluated the effect of hormones known to be released at night in humans, some of which are also known to be released at night in rats. In humans, prolactin (PRL), growth hormone (GH), and melatonin are known to be released at night, and adrenocorticotropic hormone (ACTH) release is inhibited. In these experiments, conscious rats were injected intravenously with 0.5 ml diluent or the substance to be evaluated just after removal of the first blood sample (0.3 ml), and additional blood samples (0.3 ml) were drawn every 10 min thereafter for 2 hr. The injection of highly purified sheep PRL (500 microg) produced a rapid increase in plasma leptin that persisted for the duration of the experiment. Lower doses were ineffective. To determine the effect of blockade of PRL secretion on leptin secretion, alpha bromoergocryptine (1.5 mg), a dopamine-2-receptor agonist that rapidly inhibits PRL release, was injected. It produced a rapid decline in plasma leptin within 10 min, and the decline persisted for 120 min. The minimal effective dose of GH to lower plasma leptin was 1 mg/rat. Insulin-like growth factor (IGF-1) (10 microg), but not IGF-2 (10 microg), also significantly decreased plasma leptin. Melatonin, known to be nocturnally released in humans and rats, was injected at a dose of 1 mg/rat during daytime (1100h) or nighttime (2300h). It did not alter leptin release significantly. Dexamethasone (DEX), a potent glucocorticoid, was ineffective at a 0. 1-mg dose but produced a delayed, significant increase in leptin, manifest 100-120 min after injection of a 1 mg dose. Since glucocorticoids decrease at night in humans at the time of the maximum plasma concentrations of leptin, we hypothesize that this increase in leptin from a relatively high dose of DEX would mimic the response to the release of corticosterone following stress in the rat and that glucocorticoids are not responsible for the circadian rhythm of leptin concentration. Therefore, we conclude that an increase in PRL secretion during the night may be responsible, at least in part, for the nocturnal elevation of leptin concentrations observed in rats and humans.     

The influence of long-term estrogen treatments on daily plasma prolactin levels in ovariectomized rats

Prolactin levels were determined in the plasma of female rats from 4 to 54 days after ovariectomy or ovariectomy and treatment with a long acting polymer of estradiol, polyestradiol phosphate (PEP), or Silastic implants of crystalline estradiol-17β. Blood samples were withdrawn from indwelling aortic catheters in the morning (0900–1100) and afternoon (1500–1700). Both methods of estrogen delivery elevated plasma prolactin in the morning and afternoon compared to ovariectomized controls. However, the increases in the afternoon were significantly greater than those in the morning. The difference from ovariectomized controls and the morning-afternoon differences were maintained for 25–26 days in the polyestradiol phosphate-treated group whereas those differences in the group receiving the implants of estradiol were significant for the entire length of the experiment (54 days). In addition, there were periodic fluctuations in the morning and afternoon levels of prolactin in the estradiol implanted animals. It is suggested that the plasma prolactin response to estrogen varies with time of day, time after administration of estrogen and with the method of estrogen delivery.     

Evidence for a rapid in vivo effect on estradiol-17 beta on prolactin secretion in ovariectomized rats.

Repeated intraarterial injections of synthetic thryrotropin releasing hormone (TRH, 1 microgram/rat) increased plasma prolactin levels 4 hours after a single subcutaneous injection of 10 micrograms estradiol-17 beta (E2-17 beta) in rats ovariectomized 1, 2 or 4 weeks and at 2 hours after E2-17 beta injection in rats ovariectomized for 6 weeks. The effect of TRH was still present at 24 but not 48 hours after estradiol treatment. TRH-induced increases in plasma prolactin were similar in groups of rats treated with 10 micrograms E2-17 beta (s.c.) or implanted with 0.5 cm Silastic capsules of crystalline E2-17 beta (s.c.) whereas smaller, yet significant, TRH-induced increases in plasma prolactin were observed in rats injected s.c. with 1.0 microgram E2-17 beta. Single intraarterial injections of TRH at 4 or 8 hours after E2-17 beta treatment induced increases in plasma prolactin similar in magnitude to those observed at the same times after E2-17 beta in rats given repeated TRH injections. No effect of TRH was observed in ovariectomized rats given sesame oil and E2-17 beta treatment did not influence plasma prolactin in rats given saline instead of TRH. Intraarterial administration of serotonin creatinine sulfate (5-HT, 10 mg/kg body weight) induced marked increases in plasma prolactin in rats ovariectomized for 4 weeks which were potentiated at 2 and 6 hours after E2-17 beta (10 micrograms) treatment. The data show that estradiol has a fairly rapid stimulatory effect on plasma levels of prolactin induced by two different secretagogues but the exact site and mechanism of action remain unresolved.     

Relative and combined effects of estradiol and prolactin on corticosterone secretion in ovariectomized rats

In vivo and in vitro experiments were designed to assess the relationship of the estradiol (E2) and prolactin (PRL) on glucocorticoid secretion in ovariectomized (Ovx) rats. Female rats were Ovx for two weeks and then subcutaneously injected with oil or estradiol benzoate (EB) for 3 days before experimentation. Venous blood samples were collected from right jugular vein at 0, 30, 60, 90, and 120 min after challenge with adrenocorticotropin (ACTH). Adrenal zona fasciculata-reticularis (ZFR) cells from Ovx rats were isolated and incubated with E2 or PRL. In the morning and afternoon, EB enhanced the basal and ACTH-stimulated concentrations of plasma corticosterone (CORT) and PRL. Administration of E2 in vitro increased the basal and ACTH-stimulated release of CORT and production of adenosine 3', 5'-cyclic monophosphate (cAMP) in ZFR cells. E2 enhanced the forskolin-stimulated release of CORT by ZFR cells. However, the 3-isobutyl-l-methylxanthine (IBMX)- or 8-Br-cAMP-stimulated release of CORT was not affected by E2. E2 augmented the lower doses of PRL-stimulated release of CORT and cAMP accumulation as compared with the PRL-treated group alone. Incubation of higher doses of PRL increased the production of cAMP. Administration of nifedipine and R(+) BK8644 (classic L-type Ca2+ channel blocker) significantly attenuated the PRL-stimulated release of CORT. Taken together, these data indicate that E2- and PRL-related increase of CORT in Ovx rats is associated with the increase of cAMP accumulation and calcium influx in ZFR cells. In conclusion, E2 and PRL play a stimulatory role in the co-regulation of CORT secretion.     

Dopaminergic mediation of Y-aminobutyric acid in the control of prolactin release: Plasma prolactin and brain tyrosine hydroxylase levels in ovariectomized conscious rats

The present experiments were carried out to further elucidate the mechanism by which dopamine mediates the actions of Y-aminobutyric acid on prolactin release from anterior pituitary following its intraventricular injection in overiectomized conscious rats, Y-Aminobutyric acid significantly suppressed the prolactin levels at 0.1 Μmol concentration while at 4 Μmol dose, the level was elevated. The activity of tyrosine hydroxylase was increased significantly in the anterior pituitary at the lower dose while the higher concentration of Y-aminobutyric acid did not bring about any change in the activity both in the hypothalamus and the anterior pituitary. The results presented suggest that intracellular dopamine in the anterior pituitary may directly inhibit prolactin release; the plasma prolactin level is elevated by Y-aminobutyric acid, by way of either inhibiting dopaminergic tone or possible stimulation of a physiological prolactin releasin g hormone.     

Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons

Magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) show considerable plasticity during pregnancy and lactation. Prolactin receptors (PRL-R) have been identified in both these nuclei. The aim of this study was to investigate the cell type(s) expressing mRNA for the long form of prolactin receptor (PRL-R(L)) and to determine whether patterns of expression change during pregnancy and lactation. In addition, we examined effects of prolactin on excitability of oxytocin and vasopressin neurons. Sections from brains of nonpregnant, pregnant, and lactating rats were hybridized with an 35S-labeled probe to label PRL-R(L) mRNA together with digoxigenin-labeled probes to detect either oxytocin or vasopressin mRNA. In the SON, PRL-R(L) mRNA was predominantly colocalized with oxytocin mRNA, with over 80% of oxytocin neurons positive for PRL-R(L) mRNA. Very few (<10%) vasopressin neurons expressed PRL-R(L) mRNA. In the PVN, PRL-R(L) mRNA was also predominantly found in oxytocin neurons, and the proportion of PRL-R(L)-positive oxytocin neurons increased significantly during pregnancy and lactation. As in the SON, relatively few vasopressin cells contained PRL-R(L) mRNA. For in vivo electrophysiology, nonpregnant rats were anesthetized, and then extracellular single neuron activity was recorded in identified oxytocin and vasopressin neurons. After a period of baseline recording, the effect of prolactin (1 microg i.c.v.) on firing rate was examined. Prolactin treatment of nonpregnant rats induced a significant decrease in firing rates of oxytocin neurons. There was no effect of prolactin on the activity of vasopressin neurons. Together, these data provide strong evidence that prolactin directly and specifically regulates activity of oxytocin neurons.     

Effects of arginine vasotocin on levels of plasma gonadotropins and prolactin in ovariectomized conscious rats

Arginine vasotocin was injected into the third ventricle or intravenously in conscious, ovariectomized rats and its effect on gonadotropin and prolactin release evaluated. The peptide lowered plasma levels of both LH and prolactin in doses of 40 or 100 ng given intraventricularly. The higher dose was slightly more effective than the lower dose. Intravenous injection of a 1-microgram dose of vasotocin failed to alter plasma LH in the ovariectomized animals; however, a 5-micrograms dose induced a slight depression apparent at only 60 min following injection. Intravenous injection of 1 microgram produced a significant lowering of plasma prolactin, whereas a dramatic lowering followed the injection of the higher dose. Plasma FSH was unaffected in these experiments. Incubation of dispersed anterior pituitary cells from ovariectomized rats with various doses of vasotocin revealed no effect of the peptide on the release of FSH, LH, or prolactin. It also did not alter the response to LHRH, but it partially blocked the action of dopamine to inhibit prolactin release. The data indicate that quite low doses of arginine vasotocin act within the brain to inhibit LH and prolactin secretion in ovariectomized, conscious animals.     

Long-term prolactin exposure differentially stimulated the transcellular and solvent drag-induced calcium transport in the duodenum of ovariectomized rats

Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P < 0.001) and 9.78 +/- 0.72 to 15.99 +/- 1.75 (P < 0.001) nmol.hr(-1).cm(-2), respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 +/- 10.12 to 47.77 +/- 5.42 nmol.hr(-1).cm(-2) (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.     

High-calcium diet modulates effects of long-term prolactin exposure on the cortical bone calcium content in ovariectomized rats

High physiological prolactin induced positive calcium balance by stimulating intestinal calcium absorption, reducing renal calcium excretion, and increasing bone calcium deposition in female rats. Although prolactin-induced increase in trabecular bone calcium deposition was absent after ovariectomy, its effects on cortical bones were still controversial. The present investigation, therefore, aimed to study the effect of in vivo long-term high physiological prolactin induced by either anterior pituitary (AP) transplantation or 2.5 mg/kg prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of normal adult rats has not been reported, we first determined mRNA expression of both short- and long-form PRLRs at the cortical sites (tibia and femur) and trabecular sites (calvaria and vertebrae) by using the RT-PCR. Our results showed the mRNA expression of both PRLR isoforms with predominant long form at all sites. However, high prolactin levels induced by AP transplantation in normal rats did not have any effect on the femoral bone mineral density or bone mineral content. By using (45)Ca kinetic study, 2.5 mg/kg prolactin did not alter bone formation, bone resorption, calcium deposition, and total calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical total calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that 2.0% wt/wt high-calcium diet significantly increased the tibial total calcium content in 9-wk-old young AP-grafted ovariectomized rats but decreased the tibial total calcium content in 22-wk-old adult rats. As for the vertebrae, the total calcium contents in both young and adult rats were not changed by high-calcium diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.