Biomedical subjects

M E Vrontakis

Publications and source records attributed to M E Vrontakis.

13 recordsLinked to original sources

PubMed2003

Transgenic mice over-expressing galanin exhibit pituitary adenomas and increased secretion of galanin, prolactin and growth hormone.

Galanin is a biologically active 29 amino acid peptide, widely distributed in the central and peripheral nervous system, and most abundantly in the hypothalamus where it may serve in the regulation of anterior pituitary hormones. We herein report that mice carrying the rat preprogalanin cDNA specifically targeted to the somatomammotroph cell lineage, under the control of the rat GH promoter, over-express and over-secrete galanin. Galanin peptide is localised within the GH and prolactin secretory granules. GH and prolactin release is increased as well, predominantly in males, while older transgenic animals develop pituitary hyperplasia and adenoma. In both male and female transgenic mice there is a significant increase in serum galanin (P<0.00003 and P<0.001 respectively) and prolactin (P<0.002 and P<0.05 respectively) levels, while only in male transgenic mice is there a significant increase in the serum levels of GH. Furthermore, in male transgenic mice serum prolactin levels are significantly correlated with the serum galanin levels (P<0.03). We conclude that galanin plays a key role in the process of pituitary hyperplasia, acting as a growth factor to promote pituitary cell proliferation, and participates in pituitary adenoma formation not necessarily dependent on oestrogens. Targeted over-expression and over-secretion of galanin in the somatomammotroph cell lineage stimulates predominantly hyperprolactinaemia in an oestrogen-independent manner.

Adenoma↗

PubMed1995

Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats.

We describe a sensitive technique combining dual-label immunocytochemistry (ICC) with isotopic in situ hybridization histochemistry (ISHH). We developed this technique to characterize the receptor and/or peptide content of pheno-typically identified neurons that express cell markers of neuronal activity (immediate early gene products) after physiological or pharmacological perturbation. Tissue was fixed by perfusion with 4% paraformaldehyde in PBS, sucrose-infiltrated, and cryosectioned. Sections were stored in cryoprotectant or immediately hybridized. After stringent hybridization wash procedures, Fos and luteinizing hormone-releasing hormone (LHRH) neurons were visualized sequentially using immunocytochemistry. Finally, galanin mRNA was detected autoradiographically. We applied the technique to study of subpopulations of LHRH-containing neurons. Results of this study indicate that a majority of the LHRH neurons activated during the luteinzing hormone (LH) surge (as indicated by presence of nuclear Fos staining) also express mRNA encoding galanin. However, there is not a complete overlap between the subpopulation of LHRH neurons that express Fos and that which expresses galanin mRNA.

Animals↗

PubMed1993

Growth hormone expression in human Burkitt lymphoma serum-free Ramos cell line.

A human nonpituitary cell line grown under serum-free (sf) conditions (sfRamos Burkitt lymphoma cell line) has been reported to secrete a 29K PRL-like peptide which acts as an autocrine growth factor. Conditioned medium from these cells was examined for lactogenic activity using the Nb2 bioassay and RIAs specific for human GH (hGH) and hPRL. SfRamos conditioned medium stimulated the growth of Nb2 cells. Anti-hGH monoclonal antibodies but not anti-hPRL inhibited the mitogenic effect of sfRamos conditioned medium on Nb2 cells. Immunoreactive hGH but not hPRL was detected by RIA. Immunoprecipitation with anti-hGH polyclonal antibody followed by Western blot analysis with anti-hGH monoclonal antibody revealed a specific 22K band with the same mobility as pituitary hGH. Northern blot analysis with an hGH complementary DNA (cDNA) probe revealed a 1.0-kilobase transcript migrating coincident with pituitary hGH messenger RNA. A less abundant, 1.6-kilobase transcript was also observed. Reverse transcriptase-polymerase chain reaction using specific primers for the hGH cDNA generated the predicted 248-base pair band. Polymerase chain reaction sequencing of this fragment revealed sequence identity to the hGH-N cDNA, demonstrating conclusively the expression of the hGH-N gene in the sfRamos cell line.

Antibodies↗

PubMed1992

Expression and secretion of galanin during pregnancy in the rat.

The expression of galanin messenger RNA (mRNA) in the pituitary and conceptus of pregnant rats has been studied at various stages of gestation. Using Northern blot analysis and in situ hybridization we have found that high levels of mRNA coding for galanin were detected in the conceptus during early pregnancy. The level of expression in conceptuses increased until day 11-12 after which the levels decreased rapidly. In contrast, in the pituitary galanin mRNA continued to increase throughout pregnancy, especially in the latter half of pregnancy as serum estradiol levels has been reported to be increased. The size of the galanin transcript was the same in the conceptus and pituitary (0.9 kilobase). Expression of this mRNA was confined to the decidua and was first seen at day 5 of pregnancy at a time when implantation swellings were first observed. Galanin antisense probe hybridized strongly to the decidual cells that surround the implantation site. At day 11 of pregnancy the galanin mRNA was found in both the antimesometrial and the mesometrial tissue with the highest concentration in the region lateral to the antimesometrial cells and continued toward the mesometrial cells. Serum galanin levels measured by an RIA using synthetic rat galanin as standard and antisera raised against porcine galanin, exhibited a temporal pattern similar to the pattern of mRNA expression in decidua, with a 7.1-fold increase at day 12 of pregnancy followed by a decline. In summary, decidual cells may differentiate into an endocrine cell during pregnancy. The galanin secreted by these cells, in addition to acting locally in a paracrine/autocrine fashion, may function as a placental hormone with systemic effects on distal target tissues.

Animals↗

PubMed1992

Tissue-specific regulation of vasoactive intestinal peptide messenger ribonucleic acid levels by estrogen in the rat.

The early and chronic effects of 17 beta-estradiol on vasoactive intestinal peptide (VIP) gene expression in rats were examined. Total RNA of four VIP-producing tissues were subjected to Northern blot analysis 15 and 30 min, and 1, 3, 6, and 24 h after a single injection of 17 beta-estradiol (100 micrograms/kg ip). Pituitary, hypothalamus, brain, and ileum VIP messenger RNA (mRNA) levels rose in a time-dependent manner after estrogen treatment. In the pituitary, the increase was maximal at 30-60 min, whereas in the hypothalamus, the increase reached significance only at 3 h but then persisted until at least 24 h. In the brain, a transient increase in VIP mRNA was observed at 30 min, whereas VIP mRNA levels in the ileum responded in a biphasic pattern; the initial early increase was followed by a second elevation occurring at 6 h. A smaller 1-kilobase VIP-related transcript particularly abundant in the pituitary was regulated in parallel with the 1.7-kilobase mature VIP mRNA species. Continuous estrogen stimulation for 7 weeks dramatically increased both mRNA species in the pituitary but did not affect VIP mRNA levels in the other tissues. These data suggest that the regulation of VIP gene expression by transient increases in estrogen levels is rapid and that the pattern of induction is tissue specific.

Animals↗

PubMed1992

Steroids and tissue-specific modulation of galanin gene expression in the male rat reproductive system.

Galanin is a neuropeptide widely distributed throughout the vertebrate neural and endocrine system. Galanin can influence pituitary hormone secretion, intestinal motility, and other biological activities. The precise physiological role of galanin is unknown. We studied the control of galanin gene expression in peripheral organs in the male rat using Northern blot and in situ hybridization techniques. In the adrenals and prostate, galanin mRNA was undetectable in the controls and did not change after the administration of dexamethasone (0.0001-10.0 mg/kg, ip) and diethylstilbestrol (0.1 mg/kg, ip). In the testis, thymus, seminal vesicles, medial basal hypothalamus, and colon, galanin message was detectable, but was not influenced by steroids. On the other hand, dexamethasone (0.5-10.0 mg/kg) was very effective in enhancing galanin expression in the vas deferens and epididymis (4- to 7-fold in the vas deferens), with a peak 6-9 h after the treatment. Diethylstilbestrol (0.1 mg/kg) stimulated galanin mRNA transcription only in the vas deferens (2- to 3-fold), with a peak 1-3 h after the treatment. Dihydrotestosterone treatment (0.2-0.4 mg/kg) was ineffective in all tissues examined. In the vas deferens and seminal vesicles, galanin mRNA has been localized at a cellular level by in situ hybridization. In these tissues only fibroblast-like cells contained the message. These data demonstrate that galanin is expressed in the male rat reproductive system and that steroid hormones participate in the control of galanin gene expression in a tissue- and hormone-specific fashion.

Adrenal Glands↗

PubMed1991

Galanin.

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Animals↗

PubMed1991

Galanin immunoreactivity in neuroendocrine tumors.

We investigated immunoreactivity for galanin, a 29-amino acid peptide, in formalin-fixed, paraffin-embedded sections of 123 neuroendocrine tumors. Galanin-immunoreactive cells were found in one of 12 hypothalamic gangliocytomas, nine of 18 adrenal pheochromocytomas, nine of 14 pituitary corticotroph adenomas, and one of two thymic endocrine tumors. In pheochromocytomas, galanin-immunoreactive cells were seen either singly or in clusters. In corticotroph adenomas, many tumor cells were positive for galanin, indicating colocalization of corticotropin and galanin in the same tumor cells. No galanin-immunoreactive cells were noted in four extra-adrenal paragangliomas; 10 medullary carcinomas of the thyroid; 35 endocrine tumors arising in the lung, pancreas, and gastrointestinal tract; and 28 pituitary adenomas composed of cells other than corticotrophs. In nontumorous counterparts of these neuroendocrine tumors, galanin immunoreactivity was observed in nerve cells of the hypothalamus, nerve fibers of the duodenum, and adenohypophyseal cells corresponding to corticotrophs. These findings indicate that galanin expression in neuroendocrine tumors is uncommon and restricted to some tumor types.

Endocrine Gland Neoplasms↗

PubMed1990

Presence of galanin-like immunoreactivity in nontumorous corticotrophs and corticotroph adenomas of the human pituitary.

Galanin is a 29-amino acid neuropeptide widely distributed in the central nervous system. Galanin-like immunoreactivity (galanin-LI) was investigated in 23 nontumorous human adenohypophyses and 27 pituitary adenomas. Galanin-LI was demonstrated by immunohistochemistry on formalin-fixed paraffin-embedded sections, using the avidin-biotin-peroxidase complex method. All 23 nontumorous pituitaries showed the presence of galanin-positive cells which were similar to the corticotrophs in shape, size, histological features, and distribution. Immunoreactivity based on the study of mirror sections was found to be present in the cytoplasm of corticotrophs, indicating that galanin-LI colocalizes with ACTH. Crooke's cells and basophilic cells spreading to the posterior lobe (basophil invasion) were also positive for galanin. No galanin-LI was seen in lactotrophs of the human pituitaries, including 4 estrogen-treated men and 4 pregnant women. Varying numbers of tumor cells in 13 of 18 corticotroph adenomas were positive for galanin. Galanin-LI and ACTH were coexpressed in the cytoplasm of the same adenoma cells. In 19 pituitary adenomas composed of cells other than corticotrophs, no galanin-LI was noted. This study is the first report providing evidence of the presence of galanin-LI in corticotrophs of the human pituitary.

Adenoma↗

PubMed1989

Estrogen induction of galanin synthesis in the rat anterior pituitary gland demonstrated by in situ hybridization and immunohistochemistry.

The distribution of galanin messenger (mRNA) and galanin-like immunoreactivity (Gal-LI) in the anterior and posterior pituitaries of control and estrogen-implanted female rats was determined by in situ hybridization and immunohistochemical methods. In control ovariectomized animals galanin mRNA was undetectable in the posterior, intermediate and anterior pituitary. However, 4 days after implantation with 10 mg of diethylstilbestrol, galanin mRNA was clearly present in the anterior pituitary, but not in the posterior or intermediate lobe. By immunohistochemistry Gal-LI was readily visualized and detected in the posterior lobe, but clearly undetectable in cells of the intermediate and anterior pituitary lobes of control animals. After estrogen administration numerous cells exhibiting intense Gal-LI were evident in the anterior lobe, while Gal-LI remained unchanged in the intermediate and posterior lobes. These results indicate that in control animals galanin is stored, but not synthesized, in the posterior pituitary and that after estrogen administration galanin production is substantially increased in the anterior pituitary. We conclude that the expression of galanin in the anterior pituitary is regulated by estrogen and suggest that galanin may be a pituitary hormone.

Animals↗

PubMed1987

Isolation and characterization of a complementary DNA (galanin) clone from estrogen-induced pituitary tumor messenger RNA.

The administration of high levels of estrogen is a well established method for producing prolactin-secreting pituitary tumors in rodents but the mechanism of tumor induction is not clear. In this paper we describe a cDNA clone (pEIC) which has been isolated from an estrogen-induced pituitary tumor cDNA library. The mRNA transcript corresponding to the pEIC clone is 0.9 kilobase in length and is not detectable in normal pituitaries but is expressed as early as 3 h after estrogen stimulation. Nucleotide sequence analysis of two 700-base pair recombinant clones shows that they encode a 124-amino acid protein which is 70% identical to the porcine galanin precursor. The sequence of 29 amino acid residues coded for by the pEIC cDNA clone is 88% identical with porcine galanin with only three amino acid substitutions near the C terminus. This extensive homology suggests that the pEIC cDNA clone codes for rat galanin or a protein belonging to the galanin gene family. These results provide the first evidence of a physiological regulator (estrogen) of the expression of the galanin gene. They also imply that galanin is secreted by prolactin-secreting tumors. Because intracerebroventricular injection of galanin can stimulate prolactin secretion and galanin inhibits hypothalamic dopamine release, it is conceivable that galanin may play a role in the induction of prolactin-secreting tumors.

Amino Acid Sequence↗

PubMed1987

Influence of bromocriptine and oestrogen on prolactin synthesis, secretion and tumour growth in vivo in rats.

The effects of diethylstilboestrol implants and bromocriptine administration on serum prolactin concentrations, prolactin messenger RNA (mRNA) and pituitary tumour weight were examined. Intact female Fischer 344 rats were implanted s.c. with 10 mg diethylstilboestrol (DES) under light anaesthesia. All animals except the control group carried the implant for 7 weeks at which time the rats were subdivided into five groups: A, control; B, DES for 7 weeks; C, DES for 7 weeks followed by withdrawal of DES for 1 week; D, DES for 7 weeks followed by withdrawal of DES and administration of bromocriptine for 1 week; E, DES for 8 weeks with concurrent administration of bromocriptine during the last week. Serum concentrations of prolactin were measured by radioimmunoassay, prolactin mRNA concentrations were measured by dot-blot hybridization and sodium dodecylsulphate-polyacrylamide gel electrophoresis of the in-vitro translated mRNA. Pituitary growth was estimated by changes in pituitary weight and assessed by light and electron microscopic examination. Treatment with DES dramatically increased serum prolactin concentrations and prolactin mRNA and induced pituitary tumour formation as shown by histological changes. Withdrawal of DES for 1 week did not lead to a decrease in pituitary tumour weight but was accompanied by a decrease in serum prolactin concentrations and prolactin mRNA from peak concentrations although they remained significantly increased above controls. Treatment with bromocriptine after DES implants were removed led to a significant reduction in pituitary tumour weight and a decrease in serum prolactin concentrations and prolactin mRNA. Histology of the pituitary tumour after the bromocriptine treatment showed pituitary cells similar to those from normal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Hormonal regulation of thyrotropin alpha and beta subunit mRNAs.

We have examined the effects of 3,5 3'-triiodo-L-thyronine (T3), dexamethasone, bromocriptine, thyrotropin releasing hormone (TRH) and estrogen on the levels of pituitary alpha and TSH-beta protein and mRNA levels in hypothyroid mice. After 3 days of treatment with T3 (0.5 micrograms/100 g body weight) serum TSH, alpha and TSH-beta levels were 77%, 79% and 44% of control, respectively. Pituitary alpha and TSH-beta mRNA content was estimated by dot blot hybridization of total RNA with 32P-labelled alpha and TSH-beta plasmid probes. There was no change in alpha mRNA after 3 days of T3 treatment but TSH-beta mRNA had decreased to 60% of control. With T3 at 2 micrograms/100 g body weight for 3 days, TSH protein was 27% of control and TSH-beta was undetectable, but there was no change in alpha. TSH-beta mRNA was decreased to 40% of control at 1 day and was barely detectable at 3 days, whereas alpha mRNA was 70% of control at 1 day and 42% at 3 days. Dexamethasone and bromocriptine caused no consistent change in pituitary levels of alpha and TSH-beta mRNA. Treatment with TRH caused small increases in serum TSH and in both alpha and TSH-beta mRNA levels. Estrogen treatment increased serum TSH and subunit levels and TSH-beta mRNA, but not alpha. We conclude that thyroid hormones decrease alpha and beta subunit mRNA levels discordantly in both the hypothyroid pituitary and in thyrotropic tumors and that the suppressive effect of thyroid hormone is the major regulator of TSH.

Animals↗