Increased exocytosis of secretory granules in contrast to reduced serum hormone levels in pituitary adenomas of humans and rats treated with dopamine agonist

The release of secretory granules outside the cells increased in frequency, in inverse proportion to the marked decrease in serum prolactin (PRL) levels in human prolactin-secreting adenomas (PRLomas) treated with bromocriptine (CB), a dopamine agonist. Rat pituitary adenomas induced by diethylstilbestrol also showed an increase in exocytosis of the granules despite a reduction in serum PRL levels after CB treatment. To elucidate this curious phenomenon, which is contrary to current general knowledge that serum hormone levels are sustained by the exocytosis of the secretory granules, we combined morphometric analysis with analyses using a cell fractionation technique and immunocytochemistry. The results indicated that the proteins in secretory granules consisted of 90% nonhormonal proteins and 10% PRL. CB treatment did not change the levels of the former, while the levels of the latter were markedly reduced. Immunocytochemistry, using a protein-A gold method, revealed that the secretory granules in rats treated with CB contained fewer PRL molecules than the controls. Serum PRL elevation caused by the exocytoses, estimated by counting the exocytotic granules, were markedly lower (less than 10%) in CB-treated rats than in the controls. It was suggested that, after CB treatment, the composition of secretory granules altered, or that nonhormonal constituents in the granules disintegrated more slowly, and that the granules became detectable at the site of release for a longer period. Consequently, although granules that appear ultrastructurally similar to those in untreated adenomas are observed to be more frequently released in treated adenomas, it seems that they no longer contribute to raising the serum PRL levels.     

Decreased tuberoinfundibular and pituitary dopamine and dihydroxyphenylacetic acid concentrations in rats with estrogen-induced pituitary tumors

The majority of pituitary adenomas are prolactin (PRL)-secreting, but it is still uncertain whether their pathogenesis results from a central nervous system (CNS) disturbance or autonomous lactotroph growth and function. We have measured dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) concentrations in rats bearing estradiol-induced PRL-secreting pituitary tumors. Female rats, injected at 3-week intervals with 2 mg estradiol valerate (EV), had increased plasma prolactin concentrations, up to 3 micrograms/ml, at 31 weeks. Inversely, there was a reduction of DA and DOPAC in the median eminence and arcuate nucleus as well as a decreased DOPAC/DA ratio in the arcuate nucleus. DA-containing nuclei in the other parts of the brain were not affected. Anterior pituitary weight increased while its DA concentration decreased during estradiol treatment. In the neurohypophysis, DA concentrations were unchanged while DOPAC and the ratio DOPAC/DA decreased following the estrogen treatment. Our data suggest that rats with primary estrogen-induced PRL-secreting tumors have a defect in the CNS-DA neurotransmission.     

Formation of secretory granules in the Golgi apparatus of prolactin cells in the rat pituitary gland: a stereoscopic study.

The mode of secretory granule formation in prolactin cells was analyzed in thin or thick sections of pituitary glands from non-lactating or lactating female as well as from male rats. In all these animals, the Golgi apparatus of prolacting cells consists of a continuous twisted ribbon-like structure that branches and anastomoses to form a hollow sphere located in the juxtanuclear area. The early signs of secretory granule formation are observed along the trans-aspect of the Golgi ribbon where progranules appear as focal distensions simultaneously occurring anywhere in the last trans thiamine pyrophosphatase (TPPase)-containing Golgi element. In the transmost Golgi saccule, such dilatations usually contain several nodular masses of electron opaque material which are separated from each other and from the saccular membrane by a less intensely stained material. While this transmost saccule becomes more fenestrated, its focal polynodular distensions seemingly yield polynodular tubular progranules which are initially closely apposed and usually parallel to the trans face of the Golgi ribbon. Subsequently, these progranules, which frequently show small membranous tubules or tubular networks attached to them, are seen some distance from the Golgi stacks and progressively transform into the more compact polymorphous granules characteristic of prolactin cells. These observations suggest that the polynodular tubular progranules arise by fragmentation of portions of the trans-Golgi elements rather than by fusion of small uninodular granules budding from the edges of a trans-Golgi saccule. Once the progranules have been liberated, the rest of the transmost Golgi element appears to break down into small residual networks, tubules, and vesicles. Thus, in prolactin cells as in other glandular cells, the whole transmost Golgi element would fragment during formation of prosecretory granules.     

The morphogenesis of motor rituals in rats treated chronically with the dopamine agonist quinpirole

Rats injected repeatedly with the dopamine agonist quinpirole develop motor rituals that evolve through a cascade of 4 behavioral processes. The 1st involves increased activity. The 2nd involves increased path stereotypy, reflected in traveling repeatedly along the same few paths. The 3rd is an increase in the frequency of stopping in a few places, along with a decrease in stopping in other places. The 4th is a decrease in the repetition of movements performed in the specific stopping places. Altogether, these processes culminate in stereotypy, a typical short set of movements composed of a single performance of each movement type. Thus, stereotypy arises from changes in the temporal and spatial organization, but not the content, of behavioral patterns. These results provide a model for the development of motor rituals and their linkage to normal behavior and to the physical properties of the environment.     

Ultrastructural localization of prolactin in estrogen-induced prolactinoma of the rat pituitary. Experimental models for the human prolactinomas and the effects of bromocriptine

In order to establish the experimental model for human prolactinomas and its bromocriptine treatment, estrogen-induced prolactinoma was produced in the female Wistar rats, in which daily bromocriptine treatment was given for a week, 3 days or 1 day (24 hours). The prolactinoma was produced by giving 5 mg estrogen depot every four weeks. The rats were killed at 4, 12, 20, and 24 weeks after the initial injection of the estrogen. By immunoelectron microscopy, in the estrogen induced prolactinoma, PRL was localized in well developed lamellar or whorling rough endoplasmic reticulum (RER) and Golgi saccules. In the rats treated with bromocriptine for a week, the serum PRL levels were lowered and the atrophied cytoplasm was filled with secretory granules containing PRL. These data were comparable to those of the human prolactinomas and the present experiment will serve as an animal model for studying the morphofunctional changes of human prolactinomas induced by bromocriptine treatment. The experiment with bromocriptine treatment for 24 hours supported the proposed mechanism that bromocriptine evokes inhibition of exocytosis followed by continuous granule formation in Golgi complexes and subsequently lowers synthesis of PRL.     

Glycoprotein hormone alpha-subunit-producing pituitary adenomas in rats treated for one year with calcitonin.

Calcitonin, a calcium-lowering hormone, has been associated with an increased incidence of nonfunctioning pituitary tumors in rats. In this study, rats were treated with calcitonin (80 IU/kg/d) for 52 weeks. After treatment with calcitonin, immunohistochemistry and in situ hybridization analyses demonstrated that most pituitary tumors expressed the glycoprotein hormone alpha-subunit. Expression of the alpha-subunit was identified rarely in hyperplastic lesions of control animals. Serum levels of GH, PRL, ACTH, LH, and FSH were unchanged in calcitonin-treated rats relative to controls. However, TSH levels were increased 2.1 fold after chronic treatment with calcitonin in both male and female rats (P less than 0.001). The level of glycoprotein hormone alpha-subunit was markedly increased (20-fold) in male rats with smaller elevations in female rats. Time course studies demonstrated that increases in serum alpha-subunit levels could be detected by 24 weeks of treatment and that elevations in alpha-subunit were present in the majority of animals by 40 weeks of treatment with calcitonin. The authors conclude that high doses of calcitonin, administered to rats for 6 months or longer, increases the incidence of alpha-subunit-producing pituitary tumors.     

Plasma luteinizing hormone bioactivity and immunoactivity in ovariectomized rats treated with a long-acting agonist of luteinizing hormone releasing hormone.

The dose response of a luteinizing hormone releasing hormone (LHRH) agonist in rats with high endogenous gonadotrophin levels was determined. Female rats were ovariectomized and injected with 0.3 microgram (group B), 3.2 micrograms (group C), 32 micrograms (group D) and 320 micrograms (group E) of a slow-releasing microcapsule preparation of the LHRH agonist D-Trp 6-LHRH. Control ovariectomized rats (group A) remained untreated. Plasma luteinizing hormone (LH) concentrations were measured by radioimmunoassay (RIA) before the LHRH agonist injection as well as 5, 15 and 30 days thereafter. Furthermore, LH bioactivity was determined by an in vitro rat LH bioassay in order to evaluate changes in bioactivity after administration of LHRH agonist. In control rats, plasma LH concentrations increased to 4.8 +/- 1.3 ng/ml on day 5, reaching peak levels of 9.9 +/- 1 ng/ml on day 30. In contrast to the control group, those rats which received 320 micrograms of LHRH agonist did not show any increase. Rats which received intermediate doses (groups C and D) tended to maintain levels of LH halfway between group A and group E during the first 15 days of treatment. Thereafter LH concentrations were similar to the untreated control group. The course of the LH concentrations during treatment measured by bioassay (BA) showed a similar pattern to the LH concentrations measured by RIA. The BA/RIA ratio was similar in all groups.     

Decreased level of the cell cycle regulator p27 and increased level of its ubiquitin ligase Skp2 in endometrial carcinoma but not in normal secretory or in hyperstimulated endometrium

p27 is a cyclin-dependent kinase (CDK) inhibitor whose specific late G(1) destruction allows progression of the cell across the G(1)/S boundary. The protein is ubiquitinated by S-phase kinase-interacting protein-2 (Skp2) following its specific phosphorylation, and is subsequently degraded by the 26s proteasome. There is a direct relationship between low level of p27 and rapid proliferation occurring in several benign states and in many malignancies. In the glandular cells of the normal endometrium, the level of p27 is exceedingly low during the proliferative phase, whereas it is markedly increased during the secretory phase. The expression of p27 in endometrial carcinoma is very low but has been found to increase following treatment with progesterone. However, estrogen exposure is considered as a major risk factor in developing endometrial cancer. The implications of the high dose of estrogen and progesterone induced during IVF treatment are still unknown. We have examined the expression of p27 and Skp2 as well as of Ki67 proliferation marker by using endometrial extracts and cells from normal endometrium, from ovarian hyperstimulated patients, and from endometrial carcinoma patients. The expression of p27, Skp2 and Ki67 was found to be similar in both normal secretory endometrium and endometrium from ovarian hyperstimulated patients. In striking contrast, p27 is significantly lower while Skp2 and Ki67 are significantly higher in the endometrial carcinoma and in endometrium from the proliferative phase compared with their normal secretory counterpart tissue.     

Glutathione homeostasis in rats chronically treated with ethanol. Evidence for an increased hepatic GSH export in vivo.

The influence of chronic ethanol feeding to rats on the hepatic glutathione (GSH and GSSG) system (synthesis, catabolism, export) and on the GSH and GSSG concentrations in extrahepatic tissues was investigated. Histological examination of livers from ethanol pretreated rats revealed a minor dilatation of the hepatic sinusoids. After ethanol administration the distribution pattern of gamma-glutamyltranspeptidase (enzymehistochemistry) was nearly unchanged, but the hepatic activity of this enzyme was increased. The ethanol pretreatment led to a decrease in hepatic GSH content. The hepatic activity of the GSSG-reductase were increased after ethanol treatment whereas the activities of the GSH synthesizing enzymes (gamma-glutamyl-cysteinyl-synthetase and GSH-synthetase) were not affected. A strong increase in sinusoidal GSH export was found in the ethanol-pretreated rats. The GSH- and GSSG concentrations of brain, lung, kidney and skeletal muscle were unchanged. It can be concluded that the ethanol-induced alteration of the hepatic GSH metabolism is caused mainly by changes of the sinusoidal membrane of the hepatocytes (direct effect of ethanol on the sinusoidal GSH carrier) leading to an increased GSH export into plasma. This effect should not due to an increased extrahepatic requirement for GSH.     

Sustainability of forestomach hyperplasia in rats treated with ethyl acrylate for 13 weeks and regression after cessation of dosing.

In a chronic study conducted by the National Toxicology Program (NTP), gavage administration of 100 or 200 mg ethyl acrylate (EA)/kg/day, 5 days/week, to F344 rats and B6C3F1 mice resulted in a significant dose-dependent increase in the incidence of squamous cell papillomas and carcinomas of the forestomach of both sexes of rats and mice. No increase in the incidence of tumors was observed at any other site in these rats. Chemically-induced cell proliferation is currently thought to play a role in the development and progression of chemically-induced neoplasia. Therefore, a stop-study was initiated where 100 or 200 mg EA/kg (in corn oil) was administered daily, 5 days/week, for 13 weeks. Rats sacrificed at the end of the treatment regimen had severe epithelial hyperplasia of the forestomach. No lesions were observed in the glandular stomach or liver of EA-treated rats. Forestomach hyperplasia induced by EA included upward and downward cell proliferation. However, forestomachs of rats treated for 13 weeks and sacrificed 8 weeks after the last EA dose exhibited a significant decline in the incidence and severity of forestomach mucosal hyperplasia. Histopathologic evaluation of forestomachs of EA-treated rats (13 weeks) which were allowed a 19-month-recovery (with no exposure to EA) showed further decline in the incidence and severity of mucosal cell hyperplasia. These results indicate that gavage administration of EA to rats results in extensive and sustained forestomach mucosal hyperplasia. The sustainability of forestomach hyperplasia is apparently dependent on the continued exposure of rats to ethyl acrylate, and regressed after cessation of dosing. Furthermore, although enough post-treatment time was allowed for tumors to develop after cessation of EA administration, forestomachs exhibited a nearly complete recovery with no increased incidence of papillomas or carcinomas. It, therefore, remains to be determined what duration of exposure or other factors are critical for reversibility or progression of EA-induced forestomach mucosal hyperplasia to neoplasia.     

Missing secretory granules, dilated endoplasmic reticulum, and nuclear dislocation in the thyroid gland of rdw rats with hereditary dwarfism

Previous studies on the rdw rat have suggested that its dwarfism is caused primarily by dysfunction of the thyroid gland. In this study, rat thyroid glands were analyzed endocrinologically and morphologically to clarify the primary cause of dwarfism in the rdw rat. The rdw rat showed lowered thyroid hormone (T4 and T3) levels but elevated TSH in serum. The rdw thyroid gland was almost proportional in size and it was not goiter in gross inspection. Our histological investigation produced three results that may lend important evidence in understanding the problem in the thyroid gland of rdw rats. First of all, secretory granules could not be detected in the follicular epithelial cells of the rdw. Secondly, thyroglobulin was found at very low levels in the follicular lumen by immunohistochemical analysis. In contrast, it could be detected in a substantial quantity inside the dilated rER and in the huge vacuoles that are formed by swelling of the rough endoplasmic reticulum (rER) at the basal side of the follicular epithelial cells. Additionally, the nucleus of the follicular epithelial cells was pressed to the luminal side by the enlarged rER. These morphological changes would indicate that the transport of thyroglobulin is stopped at or before the formation of the secretory granules and thyroglobulin is not secreted into the follicular lumen. The rdw characterization strongly supports that rdw dwarfism is induced by hypothyroidism due to some defect(s) in the thyroid gland.     

In vivo dopamine release and uptake impairments in rats treated with 3-nitropropionic acid

Recent evidence has suggested that mitochondrial dysfunction may lead to impaired neurotransmitter exocytosis in transgenic Huntington's disease (HD) model mice. To gain insight into the impact of mitochondrial impairment on striatal dopamine release in vivo, we used fast-scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes to measure dopamine release and uptake kinetics in anesthetized Lewis rats continuously treated for 5 days with 3-nitropropionic acid (3NP). Our results indicate that, even though striatal dopamine content was unchanged, remotely stimulated dopamine release evoked per electrical stimulus pulse ([DA]_p) is decreased in 3NP-treated rats (33% of that observed in sham control rats) and that this decrease is uniform throughout all stereotaxic depths tested. Nevertheless, unlike data collected previously from transgenic HD model rodents, the maximum rate of dopamine uptake (V_max) in 3NP-treated rats is diminished (30% of controls) while K_m is unchanged. Treatment with 3NP also resulted in a corresponding decrease in locomotor activity, presumably due in part to the impaired dopamine release. These results indicate that dopamine release is degraded in this HD model, as is observed in transgenic HD model rodents; however, the results also imply that there are fundamental differences in dopamine uptake between 3NP-treated animals and transgenic animals.     

Multifocal ductal cell hyperplasia in the submandibular salivary glands of Wistar rats chronically treated with a novel steroidal compound

A high incidence of multifocal ductal hyperplasia was observed in the submandibular salivary gland of rats treated for 26 weeks with a high dose of a novel synthetic steroid with combined estrogenic and progestagenic properties. Hyperplastic foci consisted of microcystic duct-like structures lined by a single or multilayered epithelium, sometimes showing a tendency towards a cribiform growth pattern. The hyperplastic ducts wereembedded in a collagen-rich stroma and surrounded by numerous myoepithelial cells. Immunohistochemical methods used for the detection of estrogen- and progesterone receptors revealed that progesterone receptors were abundantly present in the nucleus of epithelial cells within the lesions, exclusively. Estrogen receptors could not be detected in both the normal tissue and hyperplasic foci. The morphological, ultrastructural, and immunohistochemical characteristics strongly suggest that these hyperplastic lesions originated from the intercalated ducts. The rodent-specific granular duct cell was not involved in the pathogenesis as was clearly demonstrated by the lack of immunoreactive epidermal growth factor within the lesions. Lesions were not observed in studies with progestagens and estrogens alone or with other combined estrogen/progestagen compounds, suggesting that the specific ratio of estrogenic and progestagenic activity of the present steroid had played an important role in the development of ductal hyperplasia in this study. Lesions of the intercalated ducts, as described in this study, have not been reported before in the literature.     

Studies on prolactin-secreting cells in aging rats of different strains. II. Selected morphological and immunocytochemical features of pituitary tumors correlated with serum prolactin levels

Sixty pituitary tumors from 151 aging rats of the BN/BiRij strain, the WAG/Rij strain and their F1 hybrid were studied light microscopically. The lowest tumor incidence (18%) was found in the BN/BiRij strain, the highest incidence (71%) in the WAG/Rij strain. In the F1 hybrid the incidence (45%) was intermediate between those of the parental strains. With a hematoxylin-phloxin-saffron (HPS) staining two tumor types, i.e. hemorrhagic and solid could be distinguished. Immunocytochemical staining with anti-r-PRL antiserum revealed a positive reaction in the hemorrhagic and a negative reaction in the solid type, whereas staining with anti-sh-GH and anti-ACTH generally were negative. In animals with hemorrhagic as well as with solid tumors elevated serum PRL levels were found, although some differences were noticed. Serum levels in females of the WAG/Rij strain with hemorrhagic adenomas were significantly higher than the levels in females with solid adenomas. In the same strain, age-related differences in PRL levels were found. Significantly higher serum PRL levels were found at the age of 26-32 months than in older animals. The same tendencies were noticed in both other strains.