Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio.

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

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.

Evidence for prolactin feedback actions on hypothalamic oxytocin, vasoactive intestinal peptide and dopamine secretion.

Ovariectomized rats, when transplanted with 4 anterior pituitaries (APs) to the kidney capsule for 2-3 weeks, had elevated plasma prolactin (PRL) levels (3.8-fold) and showed decreased in situ AP weights (0.62-fold) and PRL concentrations (0.63-fold). The concentrations of dopamine (DA) and oxytocin (OT) in pituitary portal plasma of hyperprolactinemic rats were increased 1.7- and 1.9-fold, respectively. However, the levels of vasoactive intestinal peptide (VIP) in pituitary portal plasma of these rats were decreased 0.31-fold. The secretion of DA, dihydroxyphenylalanine (DOPA) and OT from fetal hypothalamic cells in primary culture was increased, whereas VIP secretion from these cells was reduced in a dose-dependent fashion following PRL treatment. These data are the first in vivo and in vitro demonstration of a stimulatory action of PRL on OT release and an inhibitory action of PRL on VIP release. Furthermore, these data suggest that a subtle imbalance between the secretion of the PRL-inhibiting factor (DA) and the PRL-releasing factors (VIP and OT) during elevated systemic levels of PRL is responsible for decreased lactotrophic function.

Hypothyroidism increases substance P concentrations in the heterotopic anterior pituitary.

The regulatory effects of thyroid hormone on adenohypophysial substance P (SP) were studied in heterotopically implanted anterior pituitaries. Three or four anterior pituitaries from 21-day-old rat pups were implanted under the renal capsule in 175- to 200-g adult rats. The donor and recipient animals were sex matched. One week after implantation, animals were thyroidectomized or sham operated. A separate group of animals received daily T4 treatment (1.5 g/100 g, ip). After 2 weeks, the native and heterotopic pituitaries were assayed for SP, TSH, PRL, and LH. Thyroidectomy resulted in a 3- to 10-fold increase in the SP concentration in both the heterotopic and native pituitaries compared to euthyroid values. T4 treatment suppressed the SP levels in the heterotopic pituitaries of the thyroidectomized rats. In contrast to the reduction of TSH concentrations in native pituitaries in thyroidectomized animals vs. controls, TSH concentrations in the heterotopic pituitaries of thyroidectomized rats were approximately 10 times greater than those in euthyroid animals. PRL concentrations were unaffected by hypothyroidism in native and heterotopic pituitaries. Thyroidectomy resulted in a decrease in LH concentrations in the native anterior pituitary, without affecting LH concentrations in the implanted pituitary. These findings indicate that a direct link from the hypothalamus to the anterior pituitary is not required for the pituitary SP response to hypothyroidism.

Effect of experimentally induced hyperprolactinemia on growth hormone secretion.

In order to study the existence of possible interrelation-ships between prolactin (PRL) and growth hormone (GH) secretions, adult male rats bearing an anterior pituitary graft under the kidney capsule since day 90 of life and their sham-operated controls were submitted to a single i.p. administration of L-dopa (50 mg/kg weight) or saline 30 days after the operation. Plasma PRL and GH levels were measured by using specific RIA methods. Dopamine (DA) and norepinephrine (NE) contents in the hypothalamus and in the in situ anterior pituitary gland were measured by using a specific radioenzymatic assay. An increase in plasma PRL levels and a decrease in plasma GH levels were shown in grafted rats. Hypothalamic contents of DA and NE were increased in these animals, while the anterior pituitary content of DA was not modified as compared to controls. The administration of a single injection of L-dopa led to decreases of plasma PRL and GH levels in both grafted and control rats, but while marked increases in hypothalamic and anterior pituitary contents of DA were shown in both groups, the hypothalamic content of NE was only increased in control animals. These data suggest that PRL and GH secretions were closely related. Dopamine could be mediating the action of PRL on GH, while NE would be less involved.

Hyperprolactinemia disrupts neuroendocrine responses of male rats to female conspecifics.

An increase in plasma luteinizing hormone levels which occurs in the male rat in response to the presence of a receptive female was associated with increased norepinephrine metabolism in several regions of the hypothalamus and reduced norepinephrine metabolism in preoptic area and olfactory bulbs. Hyperprolactinemia induced by grafting three anterior pituitary glands beneath the kidney capsule blocked female-induced changes in norepinephrine metabolism and attenuated the increase in luteinizing hormone release. These results suggest that endocrine and behavioral responses of male rats to the presence of a female are mediated by changes in catecholamine metabolism in several brain regions. The ability of hyperprolactinemia to induce deficits in male sexual behavior may be due to the blockade of these CNS responses to exteroceptive stimuli originating from the female.

Direct inhibitory effect of long term estradiol treatment on dopamine synthesis in tuberoinfundibular dopaminergic neurons: in vitro studies using hypothalamic slices.

The mechanism of the inhibitory effect of long term treatment with estradiol on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons was studied by using hypothalamic slices from ovariectomized rats. Treatment with 2 mg estradiol valerate (EV) at a 3-week interval increased the weight of the anterior pituitary gland and the concentration of serum PRL. In vivo and in vitro dopamine synthesis in TIDA neurons were estimated in EV-treated animals by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after injections of 3-hydroxybenzylhydrazine (NSD 1015), a DOPA decarboxylase inhibitor, and after incubation of hypothalamic slices with NSD 1015, respectively. In vivo DOPA accumulation in the median eminence was less in EV-treated rats than in control rats. The basal rate of in vitro DOPA accumulation in the median eminence of hypothalamic slices from EV-treated rats was lower than that in control rats. Ca2+-dependent DOPA accumulation in the median eminence, determined by incubation in medium containing depolarization agents such as 50 mM K+ and veratridine, was decreased in EV-treated rats. Furthermore, cAMP-dependent DOPA accumulation, determined by incubation with Bu2cAMP or forskolin, was also suppressed in EV-treated rats. The decreased depolarization-induced DOPA accumulation in the median eminence recovered after cessation of EV treatment. Hyperprolactinemia lasting for 6 weeks, achieved by transplantation of anterior pituitaries under the kidney capsule, increased the rate of depolarization-induced DOPA accumulation in the median eminence. On the other hand, EV treatment was effective in inhibiting depolarization-induced DOPA accumulation in hypophysectomized rats regardless of the presence of anterior pituitary transplants. These results suggest that chronically administered estradiol inhibits dopamine synthesis in TIDA neurons via a direct action on the hypothalamus and overcomes the facilitatory action of PRL on dopamine synthesis; and estradiol inhibits all three distinct systems that regulate basal, Ca2+-dependent, and cAMP-dependent dopamine synthesis in TIDA neurons.

Angioarchitecture of the CNS, pituitary gland, and intracerebral grafts revealed with peroxidase cytochemistry.

Blood vessels of the fetal, neonatal, and adult subprimate and primate CNS, including circumventricular organs (e.g., median eminence, pituitary gland, etc.), and of solid CNS and nonneural (anterior pituitary gland) allografts placed within brains of adult mammalian hosts were visualized with peroxidase cytochemistry applied in three ways: to tissues from animals injected systemically with native horseradish peroxidase (HRP) or peroxidase conjugated to the lectin wheat germ agglutinin (WGA) prior to perfusion fixation; to tissues from animals infused with native HRP into the aorta subsequent to perfusion fixation; and to tissues from animals fixed by immersion and incubated for endogenous peroxidase activity in red cells retained within blood vessels. In neonatal and adult animals receiving native HRP intravascularly, non-fenestrated vessels contributing to a blood-brain barrier were outlined with HRP reaction product when tetramethylbenzidine (TMB) as opposed to diaminobenzidine (DAB) was used as the chromogen; fenestrated vessels of circumventricular organs were not discernible due to the density of extravascular reaction product. Fenestrated and non-fenestrated cerebral and extracerebral blood vessels exposed to bloodborne WGA-HRP were visible when incubated in TMB and DAB solutions. Native HRP infused into the aorta of fixed animals likewise labeled non- fenestrated vessels throughout the brain upon exposure to TMB or DAB but obscured fenestrated vessels of the circumventricular organs. Endogenous peroxidase activity of red cells, seen equally well with TMB and DAB, outlined blood vessels throughout the cerebral gray and white matter and all circumventricular organs in fetal, neonatal, and adult animals. Application of the three peroxidase cytochemical approaches to study the development or absence of a blood-brain barrier in intracerebral allografts demonstrated that the vascularization of day 16-19 fetal/1 day neonatal CNS allografts is not well defined prior to 7 days following intracerebral placement of the grafts. CNS allografts secured from donor sites expected to possess a blood-brain barrier exhibited blood vessels that were not leaky to HRP injected intravenously in the host. Fenestrated blood vessels associated with anterior pituitary allografts were evident prior to 3 days posttransplantation within the host brain and permitted blood-borne HRP in the host to enter the graft and surrounding host brain parenchyma.

Rathke's pouch grafts in adult brain sites.

Donor tissue containing Rathke's pouch (RP) with its associated mesenchyme and neural lobe was isolated from 15-day fetal rats and stereotaxically transplanted either to hypothalamic hypophysiotropic sites or to cerebral cortex of adult females for 30 days. Hosts either were intact or had been hypophysectomized 2-4 weeks prior to transplantation of Rathke's pouch. Grafts in the hypothalamus of either intact or hypophysectomized hosts were pleomorphic and large, often as wide as 1-2 mm, and occasionally larger. Grafts in the cortex of either intact or hypophysectomized hosts were nodular and occasionally projected upward in association with the meninges (cortex/meninges grafts). Certain features were characteristic of the grafts in all experimental groups, i.e., development of histotypic pars distalis with cell cords and fenestrated capillaries. In all experimental groups gonadotrophs and somatotrophs, when present, were localized at the graft margin adjacent to the connective-tissue interface; mammotrophs, when present, were distributed throughout the graft. Features specific to each experimental group also were apparent. Grafts in the hypothalamus of both intact and hypophysectomized hosts typically were encapsulated by a labyrinthine meshwork of cell processes, whereas cortex/meninges grafts directly abutted dense connective tissue or neural tissue. In hypothalamic grafts in intact hosts, moderately differentiated mammotrophs, gonadotrophs, and somatotrophs could be identified by their cytological features and immunopositivity for prolactin, luteinizing hormone, and growth hormone, respectively. In hypothalamic grafts in hypophysectomized hosts, mammotrophs were absent, and gonadotrophs and somatotrophs were poorly granulated and not abundant. Grafts in the cortex of intact hosts contained numerous, well-differentiated mammotrophs, gonadotrophs, and somatotrophs. Many of the mammotrophs in these grafts were hypertrophied, and profiles of exocytosis were common. In grafts in the cortex of hypophysectomized hosts, mammotrophs were either absent or very few, whereas gonadotrophs and somatotrophs were numerous. Gonadotrophs in these grafts were dramatically hypertrophied, although exocytosis was rare. The results indicate that development of histotypic pars distalis may occur in hypophysiotropic and non-hypophysiotropic brain sites and that the hormonal state of the host as well as implantation site modulate cytodifferentiation of specific pars distalis cell types.

The inhibition of prolactin secretion due to intrahypothalamic pituitary grafts is reversed by estradiol benzoate but not by progesterone.

Anterior pituitary (AP) tissue grafted into the hypothalamus inhibited the luteotrophic response to mating and prevented pseudopregnancy (PSP) and pregnancy. All normal rats given 10 micrograms estradiol benzoate (EB) on estrus became PSP (15 days) while the same treatment caused 10-day PSPs in 20/21 grafted rats. Doses of 30 micrograms EB or 10 micrograms EB plus reserpine (1 mg/kg) resulted in 15-day PSP in grafted rats. By contrast progesterone (P; 10 mg on estrus) did not prolong cycles in rats with hypothalamic grafts though it did in 50% of normals. Earlier studies showed that PSP or pregnancy was restored in the grafted rats by blockade of dopamine (DA) secretion. The results above show that EB was similarly effective in restoring PSP while P was not, suggesting that EB both raised prolactin and lowered DA while P was unable to lower DA in rats with AP grafts in the hypothalamus.

Thyroxine 5'-deiodinase activity in anterior pituitary glands transplanted under the renal capsule in the rat.

The conversion of T4 to T3 by the anterior pituitary gland appears to be of considerable physiological importance in the control of pituitary function. To determine a possible role of hypothalamic factors in controlling this enzymatic process, iodothyronine 5'-deiodinase (I5'D) activity was studied in rats 6 weeks after homologous transplantation of pituitary (implanted animals) or muscle tissue (sham animals) under the renal capsule. Intrasellar pituitaries remained intact, and serum T3, T4, and TSH levels were similar in both groups. I5'D activity was determined by quantifying T3 production rates in tissue homogenates at T4 concentrations of 0.002-4 microM, and with 20 mM dithiothreitol. Sellar pituitaries from sham and implanted animals displayed similar nonlinear reaction kinetics, suggesting the presence of two enzymatic processes having approximate Michaelis-Menten constant (Km) values of 2 nM and 0.3 microM. Maximum velocity (Vmax) was 51.3 +/- (SE) 4.0 fmol T3/min X mg protein (units) and 40 +/- 6 U for the low and high Km components, respectively. In transplanted pituitary tissue, I5'D activity was markedly altered; the low Km activity was significantly decreased (Km, 6 nM; Vmax, 13.0 +/- 1.1 U; P less than 0.001 compared to sellar pituitaries), whereas the high Km activity was increased 15-fold (Km, 5 microM; Vmax, 620 U). The in vitro addition of 6-n-propyl-2-thiouracil (0.1 mM) inhibited high Km I5'D activity in homogenates of both transplanted pituitary and renal tissue by approximately 50% (P less than 0.001), but had no effect on low Km I5'D activity in either sellar or transplanted pituitaries. In sham and implanted animals rendered hypothyroid by the inclusion of 1 g/dl NaClO4 in their drinking water for 6 weeks, low Km I5'D activity was increased approximately 3-fold in sellar and transplanted pituitary tissue. The levels of activity reached in transplanted tissue, however, were only 20-30% of those noted in sellar pituitary homogenates (P less than 0.001). High Km I5'D activity was estimated to be decreased 55% in transplanted tissue from hypothyroid animals. These studies demonstrate that transplantation of the anterior pituitary gland under the renal capsule in the rat results in marked alterations in two distinct components of pituitary I5'D activity. This suggests that neuroendocrine factors are important in the control of pituitary T4 to T3 conversion. Furthermore, it provides evidence for a unique mechanism whereby the hypothalamus, by modulating local thyroid hormone metabolism, may influence pituitary function.

Dopamine blockade reverses the inhibition of prolactin which results from intrahypothalamic pituitary grafts.

When mated with fertile bucks, rats with anterior pituitary (AP) tissue grafted into the hypothalamus did not exhibit prolongation of the diestrous cycle. Treatment of these rats with alpha-methyl-p-tyrosine, reserpine, or haloperidol for 1 or 2 days after mating increased the interestrous interval by a few days in all rats and to more than 8 days (leading to pseudopregnancy or pregnancy) in 20% of the cases. The same treatment in unmated normals resulted in 80% becoming pseudopregnant. To get more than 70% of rats with hypothalamic AP grafts pregnant or pseudopregnant required dopamine-blocking drugs for 3 or 4 consecutive days. Pregnancy was prolonged in 50% and lactation was impaired in 78% of the grafted rats which littered. Both impairments, like the original failure of the luteotrophic response, are attributed to the effects of PRL autofeedback from the hypothalamic AP grafts. These experiments provide further evidence that the mechanism whereby PRL in the hypothalamus inhibits PRL secretion involves elevation of dopamine.

Comparison of plasma thyroxine levels following short exposure to cold and TRH administration in intact and pituitary autografted quail (Coturnix coturnix japonica).

The thyrotropic functional abilities of ectopically transplanted anterior pituitaries were tested by subjecting quail bearing their adenohypophysis in juxtarenal position either to a short cold exposure or to an intravenous injection of TRH. Thyroxine was determined in plasma samples collected from 20 to 120 min after treatment. Intact birds exhibited increasing T4 levels up to a peak at 40 min, then decreasing slowly within 2 hr after either cold or TRH stimulation. Autografted birds exhibited significant although lower and delayed increase of plasma thyroxine following both stimuli.

Effect of adrenalectomy or castration on the inhibition of gonadotrophin secretion induced by hyperprolactinaemia in the adult male rat.

To investigate the role of adrenal and gonadal steroids in the long-term suppression of gonadotrophin secretion induced by prolactin the effects of adrenalectomy or castration on the serum and pituitary levels of LH, FSH and prolactin and the hypothalamic content of LH releasing hormone (LH-RH) have been studied in adult male rats with hyperprolactinaemia produced by the transplantation of pituitary glands under the kidney capsule. Levels of LH and FSH in serum were significantly suppressed in al intact pituitary-grafted rats. Adrenalectomy on the day of pituitary implantation or 20 days later did not affect the suppression. However, castration on days 0, 28 or 49 after pituitary grafting resulted in a rise in levels of FSH in serum indistinguishable from that in control rats. While the rise in levels of LH after castration on day 0 was the same as the controls, this increase was significantly reduced 2 days after castration on days 28 and 49 after pituitary grafting. Castration resulted in an increase in the pituitary content of LH and a reduction in the hypothalamic content of LH-RH but no change in the pituitary content of FSH. Hyperprolactinaemia did not appear to affect these response. The present results showed clearly that the gonad but not the adrenal must be present for prolactin to exert an inhibitory effect on gonadotrophin secretion.

Evidence that the hypothalamus mediates endotoxin stimulation of adrenocorticotropic hormone secretion.

The site of action of Escherichia coli endotoxin in inducing ACTH secretion was studied in vivo and in vitro. Hypophysectomized rats, bearing two to three transplanted pituitaries under the kidney capsule and "primed" with exogenous ACTH, responded to 2.0-7.5 microgram/100 g BW ip or iv endotoxin with a several-fold increase of plasma corticosterone. This response was markedly reduced by hypothalamic lesions and completely abolished by removing the entire forebrain. Endotoxin added directly to cultured rat adenohypophyseal cells in a concentration up to 10 microgram/ml did not induce significant ACTH secretion. We conclude that endotoxin-induced ACTH secretion from heterotopically transplanted pituitaries is mediated primarily by the hypothalamus, presumably through hypothalamic CRF that reaches the transplanted pituitaries via the systemic circulation.

Effect of the isolated removal of the median eminence (ME) and pituitary stalk (PS) on the immunohistology and hormone release of the anterior pituitary gland grafted into the hypophysiotrophic area (HTA) and/or of the in situ pituitary gland.

Isolated removal of the median eminence (ME) and pituitary stalk (PS) of female rats was performed under visual control, using a new instrument to open up the 3rd ventricle. Atrophy of the uterus, the follicles and the intersitial tissue in the ovaries was accompanied by persistent corpora lutea and persistent diestrous vaginal smears in rats which had undergone a successful removal of ME and PS. No change was, however, detected in the weight of the thyroid and adrenal glands at the end of the six weeks experimental period. An adenohypophysis implanted in the place of the ME at the time of the surgery, could not prevent these changes. In animals, in which the removal of the ME was not complete, the changes of the gonadal system were less pronounced. Immunocytology of the pituitary LH-, FSH, TSH- and prolactin-cells in animals with completely removed ME and PS showed inactive LH- and FSH-cells both in the grafted and in situ pituitaries, while the TSH- and prolactin-cells appeared to be in a stimulated state. In animals with ME remnant, LH-RH axon terminals were localized only on the blood vessels of the remnant. The part of the pituitary graft in contact with these blood vessels, as well as some areas of the in situ pituitary gland, contained active LH cells as judged from their size and immunohistological appearance. Since in the absence of the ME, the hypophysiotrophic area is not able to exert its regulatory effect on the gonadotrophs of the pituitary implant in this area, the authors suggest that this effect is mediated by the blood circulation of the ME which is rich in releasing hormones and is drained toward both the anterior pituitary and the medial basal hypothalamus.