Paeoniflorin and liquiritin, two major constituents in Chinese herbal formulas used to treat hyperprolactinemia-associated disorders, inhibits prolactin secretion in prolactinoma cells by different mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin and liquiritin are major constituents in some Chinese herbal formulas, such as Yiru Tiaojing (YRTJ) Granule (a hospitalized preparation) and Peony-Glycyrrhiza Decoction, used for hyperprolactinemia-associated disorders. AIM OF THE STUDY: To investigate the effect of paeoniflorin and liquiritin on prolactin secretion. MATERIALS AND METHODS: The effect of YRTJ Granule on metoclopramide-induced hyperprolactinemia was tested in rats. Paeoniflorin and liquiritin in the YRTJ Granule extract were identified and quantified by HPLC. The effects of paeoniflorin and liquiritin on prolactin secretion were examined in prolactinoma cells that were identified morphologically and by Western blot. The concentration of prolactin was determined by ELISA. The gene expression was analyzed by Western blot. RESULTS: YRTJ Granule ameliorated metoclopramide-induced hyperprolactinemia in rats. The contents of paeoniflorin and liquiritin in YRTJ Granule were 7.43 and 2.05mg/g extract, respectively. Paeoniflorin, liquiritin and bromocriptine (a dopamine D2 receptor (D2R) agonist) decreased prolactin concentration in MMQ cells expressing D2R. However, the effect of liquiritin and bromocriptine was abolished in GH3 cells lacking D2R expression. Interestingly, paeoniflorin still decreased prolactin concentration in GH3 cells in the same manner. Furthermore, paeoniflorin suppressed prolactin protein expression, and was without effect on D2R protein expression in both MMQ and GH3 cells. CONCLUSIONS: The present results suggest that paeoniflorin and liquiritin play a role in YRTJ Granule-elicited improvement of hyperprolactinemia. While the effect of liquiritin is D2R-dependent, paeoniflorin D2R-independently inhibits prolactin secretion in prolactinoma cells that may especially benefit the hyperprolactinemic patients who are refractory to dopaminergic therapies.

Therapeutic effects of Schisandra chinensis on the hyperprolactinemia in rat.

Prolactin (PRL) is secreted from the pituitary gland in response to eating, mating, and ovulation. Increased serum concentration of PRL during pregnancy contributes to enlargement of the mammary glands of the breasts and prepares for production of milk. However, high PRL levels derived from prolactinoma and hyperprolactinemia induce physiological disorders such as infertility and early menopause. Natural compounds isolated from S. chinensis have been known to possess anti-oxidative, anti-inflammatory and anti-diabetic effects. In the present study, we examined the therapeutic effect of S. chinensis and its single compounds on hyperprolactinemia in the pituitary gland. In rat pituitary cells, PRL expression levels were examined using real-time PCR and western blot assay. Crude S. chinensis extract and its single compound, gomisin N, reduced mRNA and protein levels of PRL in GH3 cells. In addition, cell proliferation and PRL target gene expression in cells were modulated by S. chinensis. Similar to the in vitro experiments, crude S. chinensis extract and gomisin N reduced PRL levels in the pituitary and serum of immature female rats. These results show that S. chinensis and its single compound, gomisin N, are regulators of PRL production and may be candidates for treatment of hyperprolactinemia and prolactinoma.

Curcumin (diferuloylmethane) inhibits cell proliferation, induces apoptosis, and decreases hormone levels and secretion in pituitary tumor cells.

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

Multifunctional cells in human pituitary adenomas: implications for paradoxical secretion and tumorigenesis.

Pituitary adenomas are very common in humans. They are of monoclonal origin, very heterogeneous, and produce frequently paradoxical secretion. The normal anterior pituitary (AP) contains some unorthodox multifunctional cells able to store more than one AP hormone (polyhormonal) and/or to express multiple hypothalamic-releasing hormone receptors (multiresponsive). Multifunctional AP cells seem to be involved in plasticity processes such as transdifferentiation or paradoxical secretion. Here, we have characterized the single-cell phenotypes of 15 human pituitary tumors, including prolactinomas, nonfunctioning adenomas, and adenomas from multiple endocrine neoplasia type I (MEN-I) and pituitary Cushing's disease patients. Individual tumor cells were typed according to expression of AP hormones and hypothalamic-releasing hormone receptors by combination of calcium imaging and multiple sequential immunocytochemistry in the same cells. We found a large heterogeneity among the different tumors. In eight of the 15 tumors studied, more than 80% of the cells presented a multifunctional phenotype. This may explain the occurrence of paradoxical secretion. In addition, our results suggest that human pituitary adenomas might derive from multifunctional cells. This is consistent with the existence of a link between pituitary plasticity and tumorigenesis.

Age-associated changes in hypothalamic and pituitary neuroendocrine gene expression in the rat.

A cDNA membrane array displaying 1183 probes was used to detect hypothalamic and pituitary changes in gene expression accompanying ageing and age-associated pituitary macroadenomas. Four groups of male Sprague-Dawley rats (3-, 15-, 24-month-old and 24-month-old with prolactinoma) were compared in two independent hybridizations. cDNA array data were confirmed and completed by comparative reverse transcriptase-polymerase chain reaction on selected genes. The expression of 454 and 116 mRNAs was detected in hypothalamus and pituitary, respectively. Growth hormone (GH) mRNA alone represented 85% of total gene expression in the gland of young rats, and other pituitary hormone transcripts 2.8%, while melanin-concentrating hormone (MCH) mRNA, the most expressed neuropeptide transcript involved in neuroendocrine regulation, accounted for only 0.8% of total hypothalamic transcripts. The proportion of genes modified in the hypothalamus and pituitary was rather modest: 1.5% and 5.2%, respectively, for ageing per se, and 1.1% and 5.2% for age-associated macroprolactinomas. Among pituitary specific RNAs, GH mRNA expression was notably decreased with age. At the hypothalamic level, expression of genes directly involved in GH regulation, such as somatostatin and growth hormone-releasing hormone, was not altered, while neuropeptide transcripts involved in feeding behaviour [orexin/hypocretin, MCH, pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART)] were significantly altered. In addition, a few ubiquitous transcripts (hnRNP-K, PFKm, CCND 2, calponin and set) were differently affected in both tissues. Modifications in hypothalamic orexigenic (orexin, MCH) and anorexigenic (POMC, CART) gene expression are in keeping with an age-associated decrease in energy consumption but a higher one in the presence of macroprolactinomas.

Enhanced uptake of ifosfamide into GH3 prolactinomas with hypercapnic hyperoxic gases monitored in vivo by (31)P MRS.

Previously, (31)P magnetic resonance spectroscopy (MRS) has been used to detect ifosfamide (IF) in vivo and to show that breathing carbogen (5% CO(2)/95% O(2)) enhances the uptake and increases the efficacy of IF in rat GH3 prolactinomas [Rodrigues LM, Maxwell RJ, McSheehy PMJ, Pinkerton CR, Robinson SP, Stubbs M, and Griffiths JR (1997). In vivo detection of ifosfamide by (31)P MRS in rat tumours; increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas. Br J Cancer 75, 62-68]. We now show that other hypercapnic and/or hyperoxic (5% CO(2) in air, 2.5% CO(2) in O(2)) gas mixtures also increase the uptake of IF into tumors, measured by (31)P MRS. All gases caused an increased uptake (C(max)) of IF compared to air breathing, with carbogen inducing the largest increase (85% (P<.02) compared to 46% with 2.5% CO(2) in O(2) (P<.004) and 48% with 5% CO(2) in air (P<.004)). The T(max) (time of maximum concentration in tumor posintravenous injection of IF) was significantly (P<.04) later in the cohort that breathed 5% CO(2) in air. The increased uptake of IF with carbogen breathing was selective to tumor tissue and there were no significant increases in any of the normal tissues studied, suggesting that any host tissue toxicity would be minimal. Carbogen breathing by patients causes breathlessness. There was no significant difference in IF uptake between breathing carbogen and 2.5% CO(2) in O(2) and, therefore, the ability of 2.5% CO(2) in O(2) to also increase IF uptake may be clinically useful as it causes less patient discomfort.

Different sensitivity to sodium valproate in healthy, non-tumoral and tumoral hyperprolactinemic subjects.

GABAergic drugs affect PRL secretion in both rat and man. Sodium valproate (SV) inhibits GABA transaminase so increasing the endogenous GABAergic tone. The aim of this study was to evaluate the effects of SV at low and high doses on PRL release in healthy subjects and hyperprolactinemic patients. Fifteen patients with prolactinomas, 8 patients with non-tumoral hyperprolactinemia and 10 healthy subjects were studied: in non consecutive days, all subjects received placebo and SV at the dose of 400 and 800 mg po. Serum PRL levels were assessed 30, 15 and 5 min before and every 30 min for 4 hours after administration. SV at the dose of 400 mg induced a significant decrease of serum PRL in healthy subjects (p < 0.05), whereas no effect was noted in both tumoral and non-tumoral hyperprolactinemia. The administration of 800 mg SV induced a significant decrease of PRL levels in healthy subjects and in patients with non-tumoral hyperprolactinemia (p < 0.05). Conversely, in prolactinomas a paradoxical increase of serum PRL concentration (p < 0.05) was observed 120 min after the administration of the drug. These data confirm the inhibitory activity of SV on PRL release in healthy subjects, and suggest the existence of a partial resistance to GABA in non-tumoral hyperprolactinemia. In prolactinomas, the paradoxical PRL increase after high dose of SV suggests the existence of a complete pituitary resistance to GABA. This finding might be explained by the appearance of the stimulatory effect of GABA at hypothalamic level that could have been unmasked by the lack of pituitary GABA effects on adenomatous lactotrophs.

In vivo detection of ifosfamide by 31P-MRS in rat tumours: increased uptake and cytotoxicity induced by carbogen breathing in GH3 prolactinomas.

The direct detection and monitoring of anti-cancer drugs in vivo by magnetic resonance spectroscopy (MRS) may lead to improved anti-cancer strategies. 31P-MRS has been used to detect and quantify ifosfamide (IF) in vivo in GH3 prolactinomas and N-methyl-N-nitrosourea (MNU)-induced mammary tumours in rats. The average concentration of IF in the GH3 prolactinoma over the first 2 h following a dose of 250 mg kg-1 i.v. was calculated to be 0.42 micromol g-1 wet weight, with a half-life of elimination (t1/2) of 2-4 h. Carbogen (95% oxygen/5% carbon dioxide) breathing increased the amount of IF taken up by the GH3 prolactinoma by 50% (P<0.01) to 0.68 micromol g-1 wet weight, although t1/2 elimination rates were unchanged. IF was also detected in the liver in vivo, with a t1/2 of about 1 h. Carbogen breathing did not affect the maximum peak area (Cmax) or the t1/2 in the liver. Most importantly, the carbogen-induced increase in IF uptake by the tumour caused significant growth delay at all time points in the GH3 tumour growth between day 5 and day 12 (P< 0.01) compared with IF alone. These findings show that carbogen breathing has potential for increasing the efficacy of anti-cancer drugs. Isolated GH3 cells were sensitive to the parent drug (IF) in vitro (IC50 = 1.3 +/- 0.2 mM) suggesting that the GH3 cells may be either expressing P450 enzymes or are sensitive to the parent drug per se.

Modification of tumour perfusion and oxygenation monitored by gradient recalled echo MRI and 31P MRS.

Gradient recalled echo (GRE) 1H images can be used to monitor changes in blood oxygenation via the dephasing effects of paramagnetic deoxyhaemoglobin (Hb). We have modulated the blood flow/oxygenation of GH3 rat tumours by i.v. calcitonin gene-related peptide (CGRP) or carbogen (95% O2, 5% CO2) inhalation, and obtained GRE 1H images interleaved with 31P spectra before, during and after the insult. With CGRP the GRE image intensity decreased (6/10) by > 10% with a concomitant 40% decrease (4/4) in beta NTP/P1 and a small decrease in pH. Both the image intensity and 31P spectra returned to near their pre-CGRP levels after 50 min, consistent with a transient episode of hypoxia. Carbogen breathing (5/5) caused > 40% increases in average GRE image intensity, with no significant changes in the 31P spectra (4/4). Three-dimensional GRE images were obtained to confirm that a T2* increase, rather than just an 'in-flow' effect due to increased blood flow, was responsible for the GRE enhancement. Increases in average image intensity > 40% were observed for the three-dimensional GRE images (2/2), indicating a T2* increase. Using Hb as an endogenous contrast agent, the high sensitivity of the GRE technique may provide a method of monitoring heterogeneous tumour perfusion and oxygenation, both in the laboratory and the clinic.

Sexually dimorphic effects of aging on tuberoinfundibular dopaminergic neurons and lactotropes of rats.

The effect of aging on plasma prolactin (PRL) levels, hypothalamic tuberonifundibular dopaminergic (TIDA) neurons and pituitary lactotropes was evaluated in prolactinoma-free young (5-month-old) and old (23- to 24-month-old) Long-Evans rats of either sex. The young female rats were in diestrus, the old ones in recurrent pseudo-pregnancy. The tyrosine hydroxylase (TH)-labelled neurons in the arcuate nucleus (AN) and axons in the median eminence (ME) as well as the PRL-labelled lactotropes in the pituitary gland were studied by morphometry and densitometric immunohistochemistry. Further, we investigated the secretory function of isolated lactotropes by reverse hemolytic plaque assay (RHPA) and by cell culture in comparable animal groups. Compared with young animals, the plasma PRL levels of old rats of both sexes were similar or reduced. All morphometric and densitometric measurements of the AN neurons, ME axons (except number) and pituitary lactotropes were comparable in young and old female rats. In old male rats the AN and ME measurements were mostly decreased, while the lactotropes remained almost unchanged. The RHPA generally showed a reduced PRL release from lactotropes of old animals of both sexes. The PRL release from the cultured lactotropes, on the contrary, was greatly increased in old female rats and unchanged in old male rats. Our functional and morphological data suggest that the in vivo function of lactotropes in old prolactinoma-free female and male rats does not seem to be strongly influenced by the mildly reduced TIDA neuron activity, yet emphasize the differences of the aging process in the two sexes.

An assessment of artefacts in localized and non-localized 31P MRS studies of phosphate metabolites and pH in rat tumours.

UA hepatomas, GH3 prolactinomas and N-methyl-N-nitrosourea-induced mammary tumours, which were subcutaneously grown in rats, have been studied by 31P MRS using non-localized pulse-acquire, image selected in vivo spectroscopy (ISIS) and one-dimensional chemical shift imaging (1-D CSI) techniques. Comparisons have been made with measurements from acid extracts of these tumour types and surrounding tissues (i.e., muscle and skin). Since muscle containing high concentrations of phosphocreatine (PCr) is often found adjacent to the tumour, we have compared the ratio of the PCr to gamma-NTP peaks in the spectra with the same ratio calculated from the acid extract data, and have used deviations between the two sets of data to assess the discrimination of the MRS localization technique to signals from the tissue surrounding the tumour. Extract data showed an average NTP content of 1.25 mumol/g wet wt for all three tumour types. PCr (at 0.42 mumol/g wet wt), was significant only in the GH3 prolactinoma whereas it was negligible in the other tumour types (< 0.1 mumol/g wet wt). There was good agreement between the ISIS PCr/gamma-NTP ratio and the extract data for all tumours. However, the 1-D CSI data showed an unexpectedly large contamination of the tumour spectrum with PCr signals from the skin which was shown by subsequent phantom experiments to be due to the curved geometry of tumour and skin rather than Fourier bleed. In pH measurements by MRS it was found that biological variability was greater than the effects of artefacts (due to either the chemical shift artefact in the ISIS technique or partial volume effects) in the localization technique. An average pH of 7.2 was observed for all tumours. By initially comparing data from different localization schemes with that from chemical extracts potential sources of error have been highlighted and show that phantom studies alone are not sufficient to fully assess the accuracy of localized MRS data.

Tuberoinfundibular dopaminergic neurons and lactotropes in young and old female rats.

Aging in female rats is accompanied by several endocrine dysfunctions, such as reproductive decline associated with characteristic hyperprolactinemia, lactotrope hyperplasia, and functional impairment of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. The aim of this morphometrical, immunocytochemical, and densitometrical study was to gain a better anatomical knowledge of TIDA neurons and axons as well as of lactotropes in old female rats with (A) or without (NA) pituitary adenomas, compared with young animals. At the hypothalamic level, we found that tyrosine hydroxylase (TH)-labeled neurons in the arcuate nucleus were comparable in young and old NA yet their size and TH-content were increased in A animals. Also the TH-labeled median eminence axons did not differ significantly between young and old NA but were more numerous in the old A rats. Independently from adenomas, both number of prolactin (PRL)-labeled structures and content of immunoreactive PRL were increased in pituitaries of old rats, the plasma PRL levels, however, were high only in A. Our findings support the documented lactotrope hypertrophy and hyperplasia in old female rats and suggest that TIDA-neuron changes only occur in hyperprolactinemic animals carrier of adenomas.

Single cell levels of hypothalamic messenger ribonucleic acid encoding luteinizing hormone-releasing hormone in intact, castrated, and hyperprolactinemic male rats.

We have examined the changes that occur in neuronal expression of LHRH mRNA in response to castration and hyperprolactinemia in male rats. Single cell levels of LHRH mRNA were determined by quantitative in situ hybridization histochemistry using an 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Nine days postcastration, a 10.4-fold increase in mean plasma LH titers was observed which was associated with significantly increased LHRH mRNA in rostral hypothalamic neuronal cell bodies. Both increases were blocked in rats rendered hyperprolactinemic by the presence of the 7315a PRL-secreting pituitary tumor. The location and number of neurons expressing LHRH mRNA were unchanged, indicating that these differences were attributable to amounts of mRNA expressed per neuron. Experimental differences occurred in LHRH perikarya situated throughout the rostral hypothalamus from the organum vasculosum of the lamina terminalis to the caudal extent of the medial preoptic nucleus. These results suggest that gonadal steroids and PRL are involved, either directly or indirectly, in regulating the biosynthesis of LHRH in the rostral hypothalamus.

1H image-guided localized 31P MR spectroscopy of human brain: quantitative analysis of 31P MR spectra measured on volunteers and on intracranial tumor patients.

1H image-guided 31P MR spectra of normal human brain and of intracranial tumors have been analyzed quantitatively. Tumor types examined include prolactinoma, lymphoma, and various grade gliomas. The experimental signals were processed by means of a time-domain least-square fitting procedure, which yields the spectral parameters, as well as a prediction of the standard deviations. Significant spectral variations are observed within both populations of normal brain and of intracranial tumor 31P MR spectra. The metabolic ratios derived from the glioma 31P MR spectra and from corresponding uninfiltrated brain tissue do not differ significantly. Significant differences are, however, observed between the metabolic ratios of prolactinoma and uninfiltrated tissue 31P MR spectra. Alkaline pH values are found for the prolactinoma and the high-grade gliomas. Furthermore, spectral differences are observed between the patient's uninfiltrated tissue 31P MR spectra and those of an unmatched population of volunteers. This underscores the necessity for control measurements on the uninfiltrated tissue of the patient and for controls from a matched population of healthy individuals.

GABAergic biochemical parameters of the tuberoinfundibular neurons following chronic hyperprolactinemia.

The effect of chronic hyperprolactinemia was studied on (a) GABA concentration in the pituitary anterior lobe; (b) GABA biosynthesis enzyme, glutamate decarboxylase (GAD) activity in the hypothalamic median eminence, and (c) GABA degradation enzyme GABA-transaminase (GABA-T) activity at both levels. In male rats bearing the prolactin-secreting tumor MtTF4 for 1 month or treated for 5 days with estradiol benzoate, the plasma prolactin concentration was markedly increased (between 4- and 10-fold basal values). In both cases, GABA concentration was significantly increased (40-60%) in the anterior pituitary lobe. A slight reduction (20-30%) in GABA-T activity was observed in the anterior lobe while no change in GAD or GABA-T activity was measured in the median eminence. These results are discussed in relationship to a possible feedback input of prolactin on the tuberoinfundibular GABAergic system.