[Inhibitory effects of melatonin on the development of 17-beta-estradiol induced prolactinoma in relation to plasma prolactin and peroxidative lipid contents].

In the present study, we have examined inhibitory effects of melatonin on the development of pituitary prolactin-producing tumors (prolactinoma) induced by 17-beta-estradiol (E(2)) in vivo. The prolactinomas were established by implanting E(2)-laden silastic capsules subcutaneously in Sprague-Dawley male rats weighing 80 approximately 100 g. Melatonin (0.05, 0.25, 0.50, 1.00, and 2.00 mg/0.1 ml/rat) was administrated subcutaneously at l8:00 h for 90 days, beginning from d 7 prior to tumor induction. Controls were given equal volumes of 4 percent; alcohol in 0.9 percent; saline. Our results showed: (1) In control group and groups given respectively 0.05, 0.25, 0.50, 1.00 and 2.00 mg melatonin, the weight of prolactinoma was 115.0+/-71.0, 85.2+/-41.0, 58.9+/-24.1, 72.7+/-23.6, 79.3+/-56.1, 74.5+/-46.8 mg respectively; the plasma prolactin (PRL) content was 493.46+/-33.3, 373.78+/-26.5, 125.13+/-13.3, 201.79+/-11.2, 418.88+/-41.3, 281.94+/-36.4 ng/ml respectively; the plasma peroxidative lipid content was 1.21+/-0.23, 0.89+/-0.32, 0.92+/-0.27, 0.64+/-0.24, 0.41+/-0.14 and 0.43+/-0.21 delta233/ml respectively. (2) The correlation coefficients between tumor weight and plasma PRL content, tumor weight and plasma peroxidative lipid content, and plasma PRL content and plasma peroxidative lipid content were 0.8738, 0.5550 and 0.2141 respectively. These results indicate: (1) The dosages of 0.25 (P<0.01) and 0.50 (P<0.05) mg, but not 0.05 (P>0.05), 1.00 (P>0.05) and 2.00 (P>0.05) mg, melatonin significantly inhibited the development of the E(2)-induced prolactinoma and the secretion of PRL in comparison with the matched control. (2) The levels of 0.05 approximately 2.00 (P<0.05 approximately 0.00l) mg melatonin showed a dose-dependent antioxidative action. (3) There are positive correlation between tumor weight and plasma PRL content (P<0.05), but no correlation between tumor weight and plasma peroxidative lipid content (P>0.05), and plasma PRL content and plasma peroxidative lipid content (P>0.05). Therefore, our experiments demonstrate that the inhibition of the development of E(2)-induced prolactinoma by adequate dosage of melatonin may be related to the inhibitory effects of MLT on the secretion of PRL, but not to the antioxidative action of MLT.

[Inhibitory effect of melatonin on the development of pituitary prolactin-producing tumors induced by 17-beta-estradiol].

OBJECTIVE: To examine the inhibitory effect of melatonin (MLT) on the development of pituitary prolactin-producing tumors (prolactinoma) induced by 17-beta-estradiol (E2), in vivo, and explore MLT's oncostatic mechanisms. METHODS: The prolactinomas were established by implanting E2-laden silastic capsules subcutaneously in Sprague-Dawley male rats. MLT doses 0.05, 0.25, 0.50, 1.00, and 2.00 mg/rat were administrated separately to 5 groups subcutaneously starting seven days prior to tumor induction for 97 days. The matched controls were given equal volumes of 4% alcohol in saline. RESULTS: (1) The prolactinoma weights in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 25.91% (P > 0.05), 48.78% (P < 0.01), 36.78% (P < 0.05), 31.04% (P > 0.05) and 35.22% (P > 0.05) respectively which were lower than that of control group; (2) The PRL mRNA levels of prolactinoma in 0.05, 0.25, and 0.50 mg MLT dose groups were 33.67% (P < 0.05), 25.51% (P < 0.05) and 41.84% (P < 0.01) respectively which were lower than that of control group as estimated by Northern Blot, and the in situ hybridization studies; (3) The DNA contents of prolactinoma in 0.05, 0.25 and 0.50 mg MLT dose groups were 40.73% (P < 0.001), 51.15% (P < 0.001) and 60.23% (P < 0.001) respectively which were lower than that of control group by laser scanning confocal microscopy; (4) Plasma peroxidative lipid contents in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 26.45% (P < 0.05), 23.97% (P < 0.05), 47.11% (P < 0.001), 66.12%(P < 0.001) and 64.46% (P < 0.001) respectively which were lower than that of control group. The correlation coefficient between MLT doses and plasma peroxidative lipid contents was -0.8257 (P < 0.05). CONCLUSIONS: MLT in suitable doses is able to inhibit the development of E2-induced prolactinoma by inhibiting the expression of PRL gene and the DNA synthesis. The link between MLT antioxidative action and its inhibitory effect on development of prolactinoma should be further investigated.

Pituitary prolactin-secreting tumor formation: recent developments.

Prolactinoma is the most common type of primary pituitary tumors. It occurs more frequently in women than in men. Dopaminergic agonists are effective in the shrinkage of prolactin-secreting pituitary tumor and are preferred in some patients. However, pituitary radiotherapy may enable the long-term removal of prolactin-secreting tumor cells. Recent evidence suggests that prolactinoma is a heterogeneous disorder with complicated and multifactorial etiology and pathogenesis. Apparently, a thorough understanding of prolactinoma tumorigenesis would be important. To facilitate investigations on tumorigenesis of prolactinoma, animal models for prolactinomas have been developed. These models have expedited our progress in the recent years. Many researchers consider the F(344) rat to be the most sensitive strain of rats to estrogen (E(2))-induced prolactinoma formation. Nonetheless, E(2) treatment for 60 days also induces the formation of pituitary prolactin-secreting adenoma in male Sprague-Dawley (SD) rats. Evidently, the SD rat is also a good animal for prolactinoma investigations. Following E(2) implantation, prolactinomas developed in the eutopic adenohypophysis in situ and/or ectopic pituitary grafted under the renal capsule in SD rats. These observations favor the hypothesis that prolactinoma growth is the result of pathological changes in the adenohypophysis and/or hypothalamus. In the latter case, abnormal release of hypothalamic dopamine, GABA, or brain-gut peptides (such as cholecystokinin, vasoactive intestinal polypeptide, galanin, angiotensin, opioid peptide, gastrin, gastrin-releasing peptide, pancreatic polypeptide, and adrenocorticotropic hormone) results in some of the pathological changes that may lead to hyperprolactinemia and/or prolactinoma development. Dysregulation of prolactin synthesis and secretion may be the result of prolactin gene modulation. In E(2)-induced rat prolactinomas, prolactin mRNA contents and the expression of some proto-oncogenes, e.g. c-myc and c-ras, TGFalpha and TGFbeta1 mRNA were significantly changed. The above findings are consistent with results in human prolactinoma development. In addition, in rats abnormal expression of the prolactin gene was correlated with hypomethylated status of CpG sites in exons 1, 2 and 4 of the prolactin gene, as well as the increase in hypersensitive sites to DNase 1 in the encoding region of the prolactin gene. In E(2)-treated rats, a point mutation with a base substitution from cytidine (C) to adenine (A) was found at the -36-bp site of the proximal promoter of the prolactin gene in eutopic pituitary prolactinomas, but no change was observed in the same sequence of the prolactin gene in ectopic prolactinoma. The association of a base substitution with the hyperexpression of the prolactin gene in eutopic prolactinomas suggests that different mechanisms may mediate the formation of eutopic and ectopic prolactin-secreting tumors. Melatonin decreases the expression of the prolactin gene in vitro suggesting that this pineal hormone may be a potential anticarcinogen in vivo. It has also been shown that MT(2) (Mel(1b)) melatonin receptors are expressed in anterior pituitary cells. The use of melatonin as a preventive or therapeutic drug for prolactinomas should be further investigated. In summary, improved knowledge on tumorigenesis of prolactinomas, especially in the rat model, was noted. These E(2)-induced rat prolactinoma models would facilitate future investigations, and expected results shall be fruitful and exciting for the development of future drug designs for the prevention and/or treatment of prolactin-secreting pituitary tumors.

[Expression of prolactin, TGF alpha and TGF beta 1 genes in estrogen-induced eutopic and ectopic pituitary prolactin-secreting tumors of rats].

The Sprague-Dawley (SD) rat bearing a heteroplasted pituitary underneath renal capsule was used to observe differential expression of prolactin (PRL), transforming growth factor alpha (TGF alpha) and transforming growth factor beta 1 (TGF beta 1) genes during the formation of pituitary prolactin-secreting tumor (prolactinoma) induced by 17 beta-estradiol (E2). Our results indicated that in both eutopic and ectopic pituitaries disconnected from hypothalamus formed simultaneously PRL-secreting tumors after the rats treated with E2 for 120 days in vivo, which was accompanied by overexpression of PRL gene (P < 0.05-0.01). The PRL mRNA level was higher in eutopic prolactinoma than that in ectopic prolactinoma (P < 0.05). Overexpression of TGF alpha and TGF beta 1 genes were also detected in eutopic prolactinoma. However, the expression of TGF alpha and TGF beta 1 genes in ectopic prolactinoma was similar to that in normal pituitary. It is suggested that TGF alpha and TGF beta 1 may be involved in prolactinoma tumorigenesis of eutopic pituitary. However, the mechanism mediating eutopic and ectopic prolactin-secreting tumor formation seems different.

Neuroendocrinology of melatonin in reproduction: recent developments.

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

[The effects of gastrin-releasing peptide (GRP) and vasoactive intestinal polypeptide (VIP) on the prolactin (PRL) gene transcription of pitutitary PRL releasing tumor of rat].

The effects of gastrin-releasing peptide (GRP) and vasoactive intestinal polypeptide (VIP) on the prolactin gene transcription of cultured pituitary of male Sprague-Dawley (SD) rats PRL releasing tumor (PPRT) (induced by estradiol) cells were studied. The PRL mRNA levels were determined by in situ hybridization of cytoplasmic RNA with a DIG-labeled PRL cDNA probe. PRL mRNA levels didn't change when the PPRT cells were incubated with 10(-8) mol/L or 10(-7) mol/L GRP for 24 h, but decreased by 20% when GRP was increased to 10(-6) mol/L (P < 0.05). The PRL mRNA level increased to 1.60, 2.10, 2.21 times of the control group when the PPRT cells were respectively incubated with 10(-8), 10(-7), 10(-6) mol/L VIP for 24 h (P < 0.05). The PRL mRNA level didn't change when the PPRT tumor cells were incubated with 10(-8) mol/L E2 for 48 h, but did increase to 2.80 and 2.92 times of the control group respectively when 10(-7) mol/L and 10(-6) mol/L E2 were used. The results above indicated that GRP and VIP exert an inhibitory and a stimulatory effect on RPL gene transcription respectively, while the stimulatory action of E2 on PRL secretion is a direct one.

[Regulatory role of galanin on prolactin and beta-endorphin release from anterior pituitary lobe of rat].

The role of brain-gut peptide galanin in the regulation of prolactin (PRL) and beta-endorphin (beta-EP) release from anterior pituitary lobe was studied in vivo in conscious male rats and in vitro with cultured anterior pituitary cells of the rat. Galanin (1 microgram or 3 micrograms/rat) injected into the third cerebral ventricle of rats produced highly significant, dose-related increases of PRL resting secretion, but did not alter resting secretion of beta-EP and restraint stress-induced release of PRL and beta-EP. However, galanin (0.05, 0.5 and 1.0 micrograms/5 x 10(5) cells.ml-1) induced highly significant, dose-related increase of beta-EP secretion from dispersed anterior pituitary cells, but failed to alter significantly PRL secretion from the cultured cells. These results indicate that central galanin has a stimulatory role in pituitary PRL resting secretion via the hypothalamus, whereas peripheral galanin stimulates beta-EP secretion only via direct action of this peptide in anterior pituitary cells.

[Pancreatic polypeptide in the control of beta-endorphin and prolactin release from rat anterior pituitary in vivo and in vitro].

This study was designed to examine the possible effects of pancreatic polypeptide (PP) on beta-endorphin (beta-EP) and prolactin (PRL) release from rat anterior pituitary in vivo and in vitro. Injection of 0.5 microgram or 2.0 micrograms PP into the third ventricle of the brain (3rd v.i.) produced a significant decrease of the beta-EP and PRL resting secretion. 0.5 microgram PP (3rd v.i.) did not affect restraint stress-induced release of beta-EP, but partially lowered stress induced release of PRL. 2.0 micrograms PP (3rd v.i.) partially reduced restraint stress-induced release of beta-EP and completely suppressed stress-induced release of PRL. In order to investigate a possible direct action of PP on beta-EP and PRL secretion from the anterior pituitary gland, we incubated dispersed anterior pituitary cells with synthetic PP (0.05, 0.625 and 1.00 micrograms) for 1 n, the secretion of beta-EP was not affected at any dosage tested, but 0.625 and 1.00 micrograms PP significantly decreased the PRL secretion. These data indicate that PP may have an inhibitory role in the control of beta-EP secretion at the level of the hypothalamus, and an inhibitory role in the control of PRL secretion at the level of either hypothalamus or anterior pituitary.

[Role of central endogenous dynorphin in control of ACTH and prolactin release during resting and stress].

To determine the role of central endogenous dynorphin (Dyn) in resting and stress-induced release of ACTH and prolactin (PRL), Dyn (1-17) antiserum (AS) or normal rabbit serum (NRS) were microinjected into the 3rd ventricle of freely-moving male rats. Plasma ACTH and PRL levels were measured by radioimmunoassay (RIA) using RIA kits supplied by NIADDK. Our results clearly indicate that Dyn (1-17) AS and NRS had no effect on resting secretion of ACTH and PRL, and restraint stress-induced release of ACTH and PRL were simultaneously lowered significantly in the Dyn (1-17) AS-treated group as compared with the NRS-treated group. The results suggest that central endogenous dynorphin plays an important role in stimulating the concomitant release of ACTH and PRL under certain stresses.

Role of substance P in suppressing growth hormone release in the rat.

To evaluate a possible physiological role of endogenous substance P (SP) in the control of growth hormone (GH; somatotropin) secretion, a specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third cerebral ventricle) in unanesthetized unrestrained normal male rats. Control rats received an equivalent volume of normal rabbit serum (NRS). Intraventricular injection of the NRS lowered plasma GH concentrations significantly. The lowering was detected on first measurement at 10 min after injection and was maximal at 30 min. This was followed by a return toward the initial levels. Third ventricular injection of antiserum significantly increased plasma GH in comparison with control animals injected with NRS. The effect was observed within 10-20 min, and levels remained elevated for the 120-min duration of the experiment. To confirm the possible inhibitory role of endogenous SP on GH release, 3 microliters of 0.9% NaCl (saline) alone or saline containing a specific antagonist of SP, [D-Pro2,D-Trp7,9]SP, was injected into the third ventricle of normal male rats. The antagonist also increased plasma GH significantly (P less than 0.005) within 5 min compared with values in the saline-injected control group. Levels remained elevated for 30 min but had returned toward control values 60 min after injection. In contrast, synthetic SP significantly decreased plasma GH when injected intravenously or intraventricularly compared with plasma GH in the control saline-injected group. To investigate a possible direct action of SP on GH release from the anterior pituitary gland, we incubated synthetic SP with dispersed anterior pituitary cells for 1 hr. The release of GH from incubated anterior pituitary cells was not affected at any dose of SP (10(-9) to 10(-6) M) tested. These data strongly indicate that endogenous SP has a physiological inhibitory role in the control of GH secretion at the level of the hypothalamus in the male rat.

[Effects of kappa-opiate receptor antagonist MR-2266-BS on ACTH and prolactin release].

The effect of intravenous injection of different doses of MR-2266-BS, a selective antagonist of kappa-opiate receptor, on plasma adrenocorticotropin (ACTH) and prolactin (PRL) in conscious male rats bearing an intrajugular cannulae was assessed. The results revealed that the MR-2266-BS of 3 mg/kg completely blocked the restraint stress-induced increase in plasma ACTH levels, and further elevated plasma PRL levels in these animals, while there were no effects on the resting levels of ACTH and PRL. MR-2266-BS of 6 mg/kg significantly increased the resting levels of plasma ACTH and also further elevated the restraint stress-induced increase of plasma ACTH and PRL. The present data suggest that kappa-opiate receptor and its endogenous ligand may be involved in the regulation of the resting and restraint stress-induced release of ACTH, and their action appears to be both stimulatory and inhibitory. Furthermore, kappa-opiate receptor and its endogenous ligand may only inhibit the stress-induced release of PRL.

Differential effects of naloxone on basal and stress-induced release of ACTH and prolactin in the male rat.

The effect of i.v. injection of various doses of naloxone (NAL) on plasma adrenocorticotropin (ACTH) and prolactin (Prl) in conscious animals bearing an indwelling intrajugular catheter was assessed. The effects were evaluated in animals which were left undisturbed and in others subjected to either restraint or ether stress. The results revealed that the dose of 3 mg/kg of NAL significantly reduced basal Prl levels, whereas a dose of 6 mg/kg of NAL was required to block completely either ether or restraint stress-induced release of Prl. The behavior of ACTH contrasted with that of Prl. There was no effect whatsoever of the 3 mg/kg dose of NAL on either resting or stress-induced ACTH levels, whereas a 6 mg/kg or 12 mg/kg dose of NAL elevated resting ACTH levels and only partially attenuated the further elevation induced by stress in these animals. The results clearly indicate a NAL sensitive step in the control of resting and stress-induced Prl release but indicate that the control of resting and stress-induced release of ACTH is different in that the predominantly millimicron receptor blocker, NAL, can elevate ACTH at high doses and can only partially block the response to stress. In contrast to Prl where opioid peptide control is solely stimulatory, this control of ACTH secretion appears to have both stimulatory and inhibitory features.