Thyroid hormone receptor expression in the human hypothalamus and anterior pituitary

In the present study, we describe for the first time the distribution of thyroid hormone receptor (TR) isoforms in the human postmortem hypothalamus and anterior pituitary using immunocytochemistry. We used a set of polyclonal antisera raised against the specific isoforms of the human TR. The distribution of TR alpha 1, alpha 2, beta 1, and beta 2 was studied in consecutive sections of six hypothalami and pituitaries. Staining intensity showed strong interindividual variation but was consistently present in the infundibular nucleus, paraventricular nucleus, and supraoptic nucleus. In addition, strong TR immunoreactivity was observed in the anterior pituitary. Neuropeptide Y and proopiomelanocortin mRNA-positive cells in the infundibular nucleus, which were studied in three other hypothalami, appeared not to express TRs, and thus, the neurons expressing TRs in the human mediobasal hypothalamus remain to be characterized.     

Immunohistochemical analysis of human growth hormone-releasing hormone gene expression in transgenic mice

The tissue-specific expression of a fusion gene encoding the mouse metallothionein-1 promoter and the coding region of the human GH-releasing hormone (hGRH) gene was studied in transgenic mice by immunohistochemistry using an anti-hGRH serum that does not recognize endogenous mouse GRH. hGRH immunoreactivity (GRH-IR) was detected in specific cells of the pituitary, pancreas, kidney, duodenum, lung, testis, ovary, adrenal, heart, and brain. In the pituitary, using double immunofluorescent staining, GRH-IR was found in some, but not all, somatotrophs, gonadotrophs, thyrotrophs, and mammotrophs. GRH-IR was found in both pancreatic exocrine cells and endocrine islets. Within the islet, GRH-IR was colocalized in A and D cells with glucagon and somatostatin, respectively. Immunopositive cells in other tissues were localized in kidney proximal convoluted tubules, duodenal submucosal glands of Brunner, the smooth muscles of pulmonary arterioles, testicular Leydig cells, oocytes, adrenal medullary chromaffin cells, and cardiac atria. In the brain, GRH-IR was seen in the external layer of the median eminence and in perikarya and fibers of the hypothalamic arcuate nucleus, the parvocellular region of the paraventricular nucleus, the supraoptic nucleus, and the amygdala. Somatostatin-immunoreactive cell bodies and fibers in transgenic and control mouse hypothalamus were not appreciably different. In summary, hGRH expression in transgenic mice occurs in a cell-specific manner in the hypothalamus as well as in numerous other tissues, many of which have secretory functions.     

Localization of growth hormone-releasing hormone in the human hypothalamus and pituitary stalk

The localization of the recently identified GH-releasing hormone (GHRH) in the human hypothalamus and pituitary stalk was determined by microdissection techniques and a specific RIA for GHRH. The highest concentrations of GHRH immunoreactivity (IR-GHRH) in the hypothalamus were found in the area of the infundibular nucleus (83 +/- 4 ng/mg protein; average +/- range). Lower quantities were found in other hypothalamic regions. Very high concentrations of IR-GHRH were present in the upper portion of the pituitary stalk (1454 +/- 48 ng/mg protein), and they decreased gradually toward the distal end of the stalk (21 +/- 3 ng/mg). This concentration gradient suggests that the peptide reaches the anterior pituitary mainly by way of the long portal vessels. Somatostatin, the second neuropeptide involved in the regulation of GH secretion from the anterior pituitary, had a pattern of distribution along the pituitary stalk very similar to that of IR-GHRH.     

Gonadotropin-releasing hormone receptor gene expression in rat anterior pituitary

Gonadotropin-releasing hormone (GnRH) effects on the lactotroph function have been widely studied, but they probably result from paracrine interactions. No visual data about GnRH receptor in the pituitary are available. In order to identify the GnRH target cells in the pituitary of adult rats, the cellular distribution of rat GnRH receptor mRNA was investigated by electron microscopy, usingin situ hybridization on ultrathin pituitary frozen sections.In situ hybridization was performed using a digoxigenin-labeled oligonucleotide probe revealed by an indirect immunogold reaction. Gonadotropin-releasing hormone receptor mRNA was found in the cytoplasmic matrix, apposed to the endoplasmic reticulum and the nucleus of the gonadotrophs, which were identified by their ultrastructural characteristics, and by the presence of luteinizing hormone (LH) immunoreactivity. It was also found in the lactotrophs, which were revealed by the immunocytological detection of prolactin. No GnRH receptor mRNA was detected in corticotrophs, somatotrophs, thyrotrophs or hepatocytes. This result, without excluding paracrine effects, clearly showed that in addition to the gonadotrophs, the lactototrophs are likely to be direct target cells for the hypothalamic GnRH.     

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.     

Luteinizing hormone-releasing hormone neurons in human preoptic/hypothalamus: differential intraneuronal localization of immunoreactive forms

Luteinizing hormone-releasing hormone (LRH) may be synthesized as part of a larger prohormone, as are several other neuropeptides. In this study, we sought not only to define the distribution and morphological characteristics of LRH neurons within the human preoptic area and hypothalamus, but also to identify sites of initial synthesis, posttranslational conversion to the decapeptide, and storage of LRH in these neurons. Immunoreactive molecular forms were differentiated using a series of antisera with distinct specificities in the peroxidase-antiperoxidase technique. These antisera were capable of detecting the fully processed hormone as well as extended decapeptide sequences. Immunopositive LRH neurons were more abundant in the infundibular area of the hypothalamus than in the preoptic area. Numbers of immunopositive perikarya and subcellular distribution of reaction product varied with binding requirements of the antisera. After treatment with an antiserum that requires the fully processed decapeptide for binding, the reaction product was associated almost entirely with granules in perikarya and processes, while very little was associated with either rough endoplasmic reticulum (RER) or Golgi apparatus. In contrast, with an antiserum capable of detecting extended forms of the decapeptide, the RER and Golgi were labeled in addition to granules. From these data, we infer that in humans, mature decapeptide is present in granules within LRH neuronal perikarya and processes. Furthermore, the molecular forms associated with RER and Golgi may be precursors in which the decapeptide sequence is extended.     

Galanin within the normal and hyperplastic anterior pituitary gland: localization, secretion, and functional analysis in normal and human growth hormone-releasing hormone transgenic mice

Studies evaluating estrogen-induced anterior pituitary tumors revealed a strong direct correlation between expression of the peptide galanin and tumor growth. To evaluate further the potential roles of galanin in the hyperplastic pituitary, we used a model of estrogen-independent anterior pituitary tumor formation, the male human GH-releasing hormone (hGHRH) transgenic mouse. Pituitaries of hGHRH transgenic mice are characterized by a hyperplasia of somatotrophs and contain markedly elevated levels of galanin. We examined the population of galanin-producing pituitary cells in 4- to 6-month-old male hGHRH transgenic mice and their nontransgenic siblings. The percentage of galanin-containing pituitary cells was significantly increased within the anterior pituitaries of hGHRH transgenic mice. By using the cell immunoblot assay we found that the basal secretion of galanin and GH from individual pituitary cells of hGHRH transgenic mice was significantly greater than that from pituitary cells of nontransgenic mice. By modifying the cell immunoblot assay, we determined that somatotrophs from both hGHRH transgenic and normal mice that were positive for galanin immunoreactivity secreted significantly greater amounts of GH than those somatotrophs devoid of galanin immunoreactivity. Moreover, immunoneutralization of galanin significantly decreased GH secretion from pituitary cells obtained from hGHRH transgenic mice. Thus, we now show that the increased levels of galanin peptide within the hyperplastic pituitaries of hGHRH transgenic mice are due to an increase in the population of cells containing galanin, and that galanin participates in the augmented secretion of GH from hyperplastic proliferating pituitary cells.     

Biosynthesis of human growth hormone-releasing hormone (hGRH) in the pituitary of hGRH transgenic mice

We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(1-44)-NH2 and hGRH(1-40)-OH, and its carboxyl-terminal peptide (hGCTP) in pituitary cells from transgenic mice bearing a metallothionein-hGRH fusion gene after incubation with [35S]methionine. After separation on HPLC, 35S-labeled and unlabeled hGRH in medium and cell extracts were characterized by RIA and immunoprecipitation with antisera against hGRH and against hGCTP. After a 4-h pulse, unlabeled and [35S]pro-hGRH, hGRH(1-44)-NH2, and hGRH(1-40)-OH were identified in medium and cell extracts by both RIA and immunoprecipitation with anti-hGRH serum. In cell extracts, after a 0.5-h pulse, [35S]pro-hGRH and hGRH(1-44)-NH2 but not [35S]hGRH(1-40)-OH were detectable. After a 0.5-h chase, however, 35S-labeled hGRH(1-40)-OH, pro-hGRH, and [35S]hGRH(1-44)-NH2 were all measurable. After a 4-h chase, comparable levels of [35S]hGRH(1-44)-NH2 and hGRH(1-40)-OH were present, and very little intracellular 35S-pro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(1-40)-OH to hGRH(1-44)-NH2 was observed in the two chase periods. In medium, [35S]hGRH(1-44)-NH2 was detectable at all times, whereas only minimal amounts of [35S]hGRH(1-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH (less than 0.02) suggests a more rapid turnover rate of hGCTP than of hGRH. These results demonstrate the processing of hGRH prohormone to both mature forms of hGRH and provide evidence that hGRH(1-40)-OH is derived from hGRH(1-44)-NH2.     

The expression of the pituitary growth hormone-releasing hormone receptor and its splice variants in normal and neoplastic human tissues

Various attempts to detect human pituitary growth hormone-releasing hormone receptor (pGHRH-R) in neoplastic extrapituitary tissues have thus far failed. Recently, four splice variants (SVs) of GHRH-R have been described, of which SV1 has the highest structural homology to pGHRH-R and likely plays a role in tumor growth. The aim of this study was to reinvestigate whether human tumors and normal human extrapituitary tissues express the pGHRH-R and to corroborate our previous findings on its SVs. Thus, we developed a real-time PCR method for the detection of the mRNA for the pGHRH-R, its SVs, and the GHRH peptide. Using real-time PCR, Western blotting, and radioligand-binding assays, we detected the mRNA for pGHRH-R and pGHRH-R protein in various human cancer cell lines grown in nude mice and in surgical specimens of human lung cancers. The expression of mRNA for SVs of pGHRH-R and GHRH was likewise found in xenografts of human non-Hodgkin's lymphomas, pancreatic cancer, glioblastoma, small-cell lung carcinomas, and in human nonmalignant prostate, liver, lung, kidney, and pituitary. Western blots showed that these normal and malignant human tissues contain SV1 protein and immunoreactive GHRH. Our results demonstrate that some normal human tissues and tumors express mRNA and protein for the pGHRH-R and its splice variants. These findings confirm and extend the concept that GHRH and its receptors play an important role in the pathophysiology of human cancers.     

Detection of a recurring mutation in the human growth hormone-releasing hormone receptor gene

OBJECTIVE: Mutations in the gene encoding for the GH-releasing hormone receptor (GHRHR) have been recently described in patients with familial isolated GH deficiency (IGHD) type IB. To date, all reported mutations have been found in kindreds sharing common ancestors. The only exception is a T to A transversion which causes a substitution of histidine for leucine in codon 144 (L144H) and creates a DraIII restriction site. This mutation was described in two families with different ethnic background residing in two different continents (Europe and North America). DESIGN: We searched for GHRHR mutations in a new family with IGHD from a third continent (South America) and found the affected individuals to be homozygous for the same L144H change. We performed linkage analysis with intra- and para-genic polymorphisms to determine if the three families carrying the L144H allele are related. RESULTS: Linkage analysis studies demonstrated that one of the three families does not share the same para- and intragenic GHRHR polymorphisms with the other two. CONCLUSIONS: The L144H mutation has arisen at least twice and should be considered for initial genetic analysis in patients with familial IGHD in whom the a GHRHR mutation is suspected.     

Age-related decrease of growth hormone and prolactin gene expression in the mouse pituitary

The effects of aging on pituitary GH, PRL, and alpha-tubulin messenger RNA (mRNA) levels were measured in 3-, 12-, and 27-month-old male C57BL/6J mice by dot-blot hybridization. The amount of GH and PRL mRNA in the pituitary deceased dramatically with age. However, total poly(A+) RNA (mRNA), as measured by hybridization with radioactively labeled oligo-(dT), was not altered during aging. In addition, there were no age-related changes in the level of alpha-tubulin mRNA. Thus, the effects of aging on GH and PRL mRNA levels are specific; the levels of the majority of cellular mRNAs are not altered with age. GH and mRNA levels decreased 35% between 3 and 12 months (P less than 0.05) and a total of 75% after 27 months (P less than 0.01). PRL mRNA levels decreased 65% between 12 and 27 months (P less than 0.01), although there was no significant decrease before 12 months. Whereas T3 is the most potent regulator of GH gene expression, we did not detect any significant age-related change in serum T3 levels. These results suggest that factors other than T3 play a role in the age-related decline in GH and PRL gene expression.     

Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells

In terms of regulation of gene expression, gonadotropin-releasing hormone receptor (GnRHR) found in extrapituitary tissues has been suggested to be different from that in the pituitary. In the present study, we examined the molecular basis of this difference using the pituitary alphaT3-1 and ovarian carcinoma OVCAR-3 cells. As a first step, the different expression levels of GnRHR mRNA in the pituitary and ovarian cells were investigated using semi-quantitative RT-PCR. Quantitative analysis showed that the expression level of hGnRHR is a nine-fold higher in primary pituitary tissues than the primary culture of ovarian carcinomas (PCO). In pituitary alphaT3-1 cells, the expression level of hGnRHR was ten-fold higher than ovarian carcinoma OVCAR-3 cells. The possibility of the differential use of various cell-specific promoters in different cells was addressed by transiently transfecting cells with 3'-deletion clones of human GnRHR promoter. Sequential deletion of the 5'-flanking region of the gene revealed the use of two putative promoters, designated PR1 (-771 to -557) and PR2 (-1351 to -1022), and a negative control region (-1022 to -771), in the pituitary and ovarian cells. The same promoters appeared to be utilized for driving the basal promoter activities in both alphaT3-1 and OVCAR-3 cells, suggesting that there is no cell-specific promoter usage for the human GnRHR gene. Alternatively, the involvement of different regulatory protein factors was investigated using electrophoretic gel mobility shift assays. When end-labeled PR1 was used as a probe, two unique shifted complexes were identified in OVCAR-3 cells when compared to alphaT3-1 cells. One unique protein-DNA complex was observed in alphaT3-1 cells compared to OVCAR-3 cells when incubated with end-labeled PR2 as a probe. These DNA-protein complexes appeared to be specific, as they competed with excess amount of unlabelled competitor PR1 and PR2, respectively. In summary, it is unlikely that the use of a cell-specific promoter contributes to the different characteristics of ovarian GnRHR. Our study demonstrates that one mechanism by which cell-specific expression of human GnRHR is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5'-flanking region of the gene.     

Restoration of growth hormone-releasing hormone (GHRH) responsiveness in pituitary GH3 cells by adenovirus-directed expression of the human GHRH receptor

GH-secreting GH3 cells lack GH-releasing hormone (GHRH) receptors. In this study we used adenoviral vectors to transfer the human GHRH receptor to GH3 cells in an effort to restore GHRH responsiveness. A replication-deficient recombinant adenovirus (AdGHRH-R) was designed to allow cytomegalovirus promoter-driven expression of the GHRH receptor messenger RNA. COS-7 cells and GH-producing GH3 cells infected with AdGHRH-R showed GHRH receptor expression on their membranes and exhibited specific GHRH binding. The addition of GHRH to GH3 cells infected with AdGHRH-R increased cAMP levels, induced cAMP response element-binding protein phosphorylation and restored GH secretory responsiveness. GHRH treatment also caused activation of mitogen-activated-protein kinase, induction of c-fos, stimulation of GH promotor activity, and increased cellular proliferation. These findings indicate that adenoviral vectors carrying human GHRH receptor are useful for in vitro studies of GHRH receptor biology and represent a first step toward the development of gene therapy for dwarfism caused by GHRH receptor mutations.     

Sexual dimorphism of growth hormone-releasing hormone and somatostatin gene expression in the hypothalamus of the rat during development.

The secretory pattern of GH secretion is markedly sexually dimorphic in the adult rat. The patterning of GH secretion is determined by the coordinated activity of somatostatin (SS)- and GH-releasing hormone (GHRH)-containing neurosecretory cells located in the hypothalamus. In this study we examined whether there is sexual dimorphism in the expression of the SS and GHRH genes and, if so, at what developmental stage this becomes evident. To address these questions, we measured SS messenger RNA (mRNA) levels in neurons of the periventricular nucleus and GHRH mRNA levels in the arcuate nucleus and ventromedial nucleus of the hypothalamus in male and female rats at 10, 25, 35, and 75 days of age. Using in situ hybridization and a computerized image analysis system, we measured SS mRNA and GHRH mRNA signal levels in individual neurons and compared these levels among the different age groups. We found that male animals had significantly higher levels of SS mRNA than females at every age. Similarly, males had higher GHRH mRNA levels than females; however, this difference was statistically significant only at 10 and 75 days of age. Developmental changes in GHRH mRNA levels were similar for both sexes, with GHRH message levels increasing gradually over the course of maturation. SS mRNA signal levels also changed over the course of development in both male and female animals. In the male rat, SS mRNA levels increased significantly between 10 and 25 days of age and declined significantly between 35 and 75 days of age. In the female rat, SS mRNA levels increased gradually between 10 and 35 days of age, then, as in the male, declined significantly between days 35 and 75. We conclude that sex differences and age-dependent changes in the expression of the SS and GHRH genes may subserve the sexual dimorphism and developmental alterations in the pattern of GH secretion in the rat.     

Prognosis in human glioblastoma based on expression of ligand growth hormone-releasing hormone,pituitary-type growth hormone-releasing hormone receptor,its splicing variant receptors,EGF receptor and PTEN genes

Purpose

Glioblastoma (GB) is the most frequent brain tumor. Despite recent improvement in therapeutic strategies, the prognosis of GB remains poor. Growth hormone-releasing hormone (GHRH) may act as a growth factor; antagonists of GHRH have been successfully applied for experimental treatment of different types of tumors. The expression profile of GHRH receptor, its main splice variant SV1 and GHRH have not been investigated in human GB tissue samples.

Methods

We examined the expression of GHRH, full-length pituitary-type GHRH receptor (pGHRHR), its functional splice variant SV1 and non-functional SV2 by RT-PCR in 23 human GB specimens. Epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog gene (PTEN) expression levels were also evaluated by quantitative RT-PCR. Correlations between clinico-pathological parameters and gene expressions were analyzed.

Results

Expression of GHRH was found to be positive in 61.9 % of samples. pGHRH receptor was not expressed in our sample set, while SV1 could be detected in 17.4 % and SV2 in 8.6 % of the GB tissues. In 65.2 and 78.3 % of samples, significant EGFR over-expression or PTEN under-representation could be detected, respectively. In 47.8 % of cases, EGFR up-regulation and PTEN down-regulation occurred together. Survival was significantly poorer in tumors lacking GHRH expression. This worse prognosis in GHRH negative group remained significant even if SV1 was also expressed.

Conclusion

Our study shows that GHRH and SV1 genes expressed in human GB samples and their expression patterns are associated with poorer prognosis.     

Hypothalamic somatostatin and growth hormone-releasing hormone mRNA expression depend upon GABA(A) receptor expression in the developing mouse

Gonadal steroids exert an important regulatory influence upon the biosynthetic and secretory activity of the somatostatin and growth hormone-releasing hormone (GHRH) neurons controlling the release of growth hormone. It is hypothesized that some of these effects occur in an indirect transsynaptic manner through the steroid regulation of GAGAergic inputs to these cells. Using GABA(A) receptor gamma(2) subunit knockout mice (gamma(2)(-/-)), which exhibit marked deficiencies in GABA(A) receptor functioning, we have examined here whether signaling through the GABA(A) receptor has any role in maintaining normal levels of somatostatin and GHRH mRNA expression in vivo. In situ hybridization experiments using (35)S-labeled oligonucleotide probes revealed that cellular levels of somatostatin mRNA in the periventricular nucleus were significantly (p < 0.01) reduced by 16% in newborn gamma(2)(-/-) mice compared with wild-type litter mates (gamma(2)(+/+)). Somatostatin mRNA expression in the striatum was not changed. Cellular levels of GHRH mRNA expression in the arcuate nucleus were significantly (p < 0.05) reduced by 30% in gamma(2)(-/-) compared with gamma(2)(+/+) mice. These results demonstrate that deletion of the gamma(2) subunit of the GABA(A) receptor reduces somatostatin and GHRH mRNA expression within the hypothalamopituitary axis and indicate that GABA exerts a tonic stimulatory influence upon both somatostatin and GHRH biosynthesis in vivo in the neonatal mouse.     

Transgenic mice expressing the human growth hormone gene provide a model system to study human growth hormone synthesis and secretion in non-tumor-derived pituitary cells: differential effects of dexamethasone and thyroid hormone

Growth hormone (GH) is regulated by pituitary and hypothalamic factors as well as peripheral endocrine factors including glucocorticoids and thyroid hormone. Studies on human GH are limited largely to the assessment of plasma levels in endocrine disorders. Thus, insight into the regulation of synthesis versus secretion has come mainly from studies done on non-human GH and/or pituitary tumor cells. However, primate and non-primate GH gene loci have differences in their structure and, by extension, regulation. We generated transgenic (171hGH/CS-TG) mice containing the intact hGH1 gene and locus control region, including sequences required for integration-independent and preferential pituitary expression. Here, we show hGH co-localizes with mouse (m) GH in somatotrophs in situ and in primary pituitary cells. Dexamethasone treatment increased hGH and mGH, as well as GH releasing hormone (GHRH) receptor RNA levels, and hGH release was stimulated by GHRH treatment. By contrast, triiodothyronine decreased or had no effect on hGH and mGH production, respectively, and the negative effect on hGH was also seen in the presence of dexamethasone. Thus, 171hGH/CS-TG mouse pituitary cultures represent a model system to investigate hormonal control of hGH synthesis and secretion.     

In vivo protective effect of melatonin on cadmium-induced changes in redox balance and gene expression in rat hypothalamus and anterior pituitary

Cadmium (Cd) is widely used in industrial applications and is an important side contaminant of agricultural products. As an endocrine disruptor, Cd modifies pituitary hormone release. It has been shown that this metal causes oxidative stress in primary cultures of anterior pituitary cells. To examine whether Cd induces redox damage in the hypothalamic-pituitary axis in vivo and to evaluate the efficacy of the antioxidant molecule melatonin to prevent Cd activity, rats were exposed to Cd (5 p.p.m. in drinking water) with or without melatonin (3 microg/mL drinking water) for 1 month. In the anterior pituitary, Cd increased lipid peroxidation and mRNA levels for heme oxygenase-1 (HO-1) at both time intervals tested (09:00 and 01:00 hr, beginning of rest span and middle of activity span, respectively). Melatonin administration prevented the Cd-induced increase in both parameters. In the hypothalamus, Cd affected the levels of mRNA for HO-1 by decreasing it in the evening. Melatonin reduced hypothalamic HO-1 gene expression. Cd treatment augmented gene expression of nitric oxide synthase (NOS)1 and NOS2 in the pituitary whereas melatonin decreased it, impairing the activity of Cd. Exposure to Cd increased the levels of hypothalamic NOS1 mRNA at 09:00 hr and decreased the levels of NOS2 mRNA at 01:00 hr, with melatonin treatment preventing Cd effects. Cd treatment decreased plasma thyroid-stimulating hormone levels at both examined times, while melatonin reversed the effect of Cd at 09:00 hr and partially counteracted the effect at 01:00 hr. There were important variations between day and night in the expression of all the genes tested in both tissues. Melatonin treatment was effective reducing all examined effects of Cd, documenting its effectiveness to protect the rat hypothalamic-pituitary axis from the toxic metal effects.