PRL, placental lactogen, and GH induce NA(+)/taurocholate-cotransporting polypeptide gene expression by activating signal transducer and activator of transcription-5 in liver cells.

We investigated the transcriptional regulation of the Na(+)/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na(+)/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na(+)/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na(+)/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na(+)/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na(+)/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.

The effects of co-therapy with recombinant human insulin-like growth factor I and insulin on serum leptin levels in adolescents with type 1 diabetes mellitus.

We previously demonstrated that in patients with type 1 diabetes mellitus (DM), co-therapy with subcutaneous (s.c.) recombinant human insulin-like growth factor I (rhIGF-I) and insulin improves glycemic control and reduces daily insulin requirements without inducing a significant change in body weight. However, it has been postulated that treatment with IGF-I may promote beneficial changes in body composition. Consequently, we assayed serum leptin, a peptide highly correlated with total fat mass, before and during chronic rhIGF-I administration. We studied 14 adolescents with type 1 DM (age range 12-19 yr). All patients were treated for 12 weeks with twice daily (BID) sc rhIGF-I in combination with standard BID split-mix insulin. At baseline, leptin concentrations were positively correlated with body mass index (BMI) (r(2) = 0.52, p = 0.004), as previously described for non-diabetic individuals. Leptin levels in diabetic females were higher than in diabetic males, and more than two times higher than in non-diabetic female controls. Baseline leptin levels did not correlate with patient age, duration of DM or hemoglobin A1c (HbA1c) measurements. The relationship between leptin concentrations and gender was maintained throughout treatment; however, average leptin levels did not change during 12 weeks of IGF-I + insulin co-therapy. These data suggest that despite treatment-induced improvements in HbA1c and serum IGF-I levels, serum leptin concentrations are unchanged by co-therapy with IGF-I + insulin. Moreover, these results suggest that improved metabolic control with IGF-I therapy is not obtained at the expense of increasing adiposity, a complication seen frequently with intensive insulin therapy.

Estradiol attenuates the forskolin-induced increase in hypothalamic tyrosine hydroxylase activity.

The purpose of this study was to evaluate interactions between estradiol and the 3',5' cyclic adenosine monophosphate (cAMP) signaling pathway to regulate tyrosine hydroxylase (TH) activity in hypothalamic dopaminergic neurons. The first experiment examined the ability of forskolin to activate TH in the tuberoinfundibular dopaminergic neurons of adult ovariectomized rats with or without estradiol treatment. Estradiol treatment reduced both basal and forskolin-stimulated TH activity in the median eminence. The second group of experiments examined the effect of estradiol on the forskolin-induced activation of TH in fetal hypothalamic cells cultures. Estradiol decreased basal TH activity in the hypothalamic cell cultures to 80% of control levels. Forskolin treatment for 1 h increased TH activity in a concentration-dependent manner in control and estradiol-treated cells, but estradiol attenuated the stimulatory response to 0.01-10 microM forskolin. The suppressive effect of estradiol on cAMP-dependent activation of TH was evident with 1-12 h of forskolin treatment. The responses to other activators of the cAMP- protein kinase A pathway, including dibutyryl cAMP and 8-bromo-cAMP, and to a depolarizing stimulus were blunted in estradiol-treated cultures. Forskolin treatment for 1 h increased radiolabeled phosphate incorporation into TH protein in control but not estradiol-treated cells, suggesting that estradiol interferes with the ability of the cAMP pathway to phosphorylate TH. Forskolin caused a time-dependent increase in TH mRNA signal levels in control cultures. The magnitude of the forskolin-induced increase in TH mRNA levels was less in the estradiol-treated cells after 6 h of forskolin treatment, indicating that estradiol hinders cAMP-regulated TH gene expression. These data indicate that estradiol attenuates the ability of hypothalamic dopaminergic neurons to respond to cAMP-dependent stimulation by interfering with phosphorylation mechanisms in the short term and control of TH mRNA levels in the long term.

Horizontal basal cell proliferation in the olfactory epithelium of transforming growth factor-alpha transgenic mice.

Transgenic mice in which overexpression of the transforming growth factor alpha (TGF-alpha) gene was directed by the keratin-14 promoter were used to study the regulation of cell cycle progression and proliferation in vivo in the olfactory epithelium. The level of TGF-alpha protein was 73% greater in the nasal-olfactory epithelium of the transgenic mice than in that of nontransgenic littermate controls. Increased levels of TGF-alpha protein were accompanied by a 5.8-fold selective increase in the proliferation of phenotypically characterized horizontal basal cells in the transgenics compared with nontransgenics; in contrast, globose basal cells exhibited a similar low level of proliferation in both transgenics and nontransgenics. The level of expression of epidermal growth factor receptor protein, the receptor for TGF-alpha, was also upregulated in the transgenics, indicating a role for the ErbB tyrosine kinase receptor family in the response to TGF-alpha in the olfactory epithelium. TGF-alpha overexpression was also associated with increased expression of several early cell-cycle-associated proteins, including the growth factor sensor cyclin D1, retinoblastoma, E2F-1 transcription factor, and cyclin E, indicating the progression of relatively quiescent progenitor cells in the G1 phase of the cell cycle toward the G1/S restriction point, after which the cells become refractive to mitogens. These results demonstrate a role for the growth factor TGF-alpha in the in vivo regulation of cell cycle progression and proliferation in the mitotically active olfactory epithelium in these transgenic mice.

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.

Targeted overexpression of galanin in lactotrophs of transgenic mice induces hyperprolactinemia and pituitary hyperplasia.

We generated transgenic mice that carry 4.6 kb of the mouse galanin gene fused to 2.5 kb of the rat PRL promoter. In all transgenic lines that carried and transmitted the transgene, there were significant increases in galanin messenger RNA and peptide levels in the anterior pituitary in both male and female transgenic mice, and the elevation of galanin was restricted to the anterior lobe. Furthermore, galanin release from pituitary cells in vitro of both male and female transgenic mice was dramatically increased compared with that in control mice. At 2-4 months of age, pituitary PRL contents in female transgenic mice were increased compared with those in normal controls. Moreover, PRL messenger RNA levels were increased in female transgenic mice. However, plasma levels of PRL in female transgenic mice were not significantly higher until 6 months of age. By 11 months of age, cell numbers in the anterior pituitary were increased in female, but not male, transgenic mice. The percentage of lactotrophs in female transgenic mice as well as PRL gene expression per cell were significantly higher. No differences were detected in PRL content, gene expression, or release between normal and transgenic male mice. Six weeks of estrogen treatment significantly increased anterior pituitary weights and PRL secretion in male transgenic mice compared with that in normal male mice. In addition, anterior pituitary weights and PRL secretion were decreased in female transgenic mice compared with controls 6 weeks after ovariectomy. We conclude that overexpression of galanin in lactotrophs stimulates PRL synthesis and secretion and acts as a growth factor resulting in the formation of pituitary hyperplasia and hyperprolactinemia. Furthermore, estrogen appears critical for these galanin-mediated events.

The human growth hormone-releasing hormone transgenic mouse as a model of modest obesity: differential changes in leptin receptor (OBR) gene expression in the anterior pituitary and hypothalamus after fasting and OBR localization in somatotrophs.

We reported previously an increase in leptin receptor (OBR) gene expression in the anterior pituitary of human GH-releasing hormone (hGHRH) transgenic mice. The primary goal of this study was to investigate the possible mechanisms regulating OBR expression in these mice. Compared with normal sibling controls, hGHRH transgenic mice had significantly greater amounts of abdominal fat, higher levels of leptin messenger RNA (mRNA), and a 2-fold increase in plasma leptin concentrations. Despite normal plasma glucose levels, hGHRH transgenic mice had 4.5-fold elevated levels of plasma insulin. Using a ribonuclease protection assay, we measured the mRNA levels of the OBR long form (OBR(L)) in the anterior pituitary and hypothalamus after 48 h of fasting. In the anterior pituitary, food deprivation induced dramatic increases in OBR(L) mRNA levels in both normal and transgenic mice. In contrast, in the hypothalamus, fasting resulted in a significant decrease in OBR(L) gene expression in normal mice, and no changes were detected in hGHRH transgenic mice. Using dual in situ hybridization, OBR(L) mRNA was detected in somatotrophs. Moreover, the number of OBR(L)-positive pituitary cells as well as the percentage of OBR(L)-positive cells that express GH mRNA were increased in transgenic mice. In conclusion, 1) the modest obesity in hGHRH transgenic mice is associated with increases in leptin synthesis and secretion as well as insulin secretion; 2) GH and/or GHRH as well as leptin and insulin may differentially contribute to the changes in OBR(L) gene expression in the anterior pituitary and the hypothalamus; 3) the response of OBR(L) gene expression in the hypothalamus to fasting is absent in the modestly obese hGHRH transgenic mice; and 4) somatotrophs are target cells for leptin, and the increase in OBR(L) gene expression in the pituitary of hGHRH transgenic mice is due at least in part to the increase in the number of cells expressing OBR(L).

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.

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Function of galanin in the anterior pituitary of estrogen-treated Fischer 344 rats: autocrine and paracrine regulation of prolactin secretion.

Estrogen is a robust stimulator of galanin synthesis and secretion in the anterior pituitary. Galanin is colocalized in lactotrophs in the estrogen-treated anterior pituitary, and its roles in lactotroph function are still being elucidated. In the present studies, we quantified the phenotypes of estrogen-treated Fischer 344 rat anterior pituitary cells expressing the galanin gene by dual in situ hybridization. The total population of galanin-positive pituitary cells increased from undetectable levels to 16% of all cells after 2 weeks of estrogen treatment. More than 90% of the galanin-positive cells coexpressed PRL messenger RNA, and one-third of the lactotrophs expressed galanin messenger RNA. We hypothesized that galanin in the anterior pituitary may contribute to the heterogeneous secretion of PRL, and that one of the functions of galanin is to regulate PRL secretion in an autocrine/paracrine manner. To test this hypothesis, we performed the reverse hemolytic plaque assay combined with in situ hybridization to measure PRL secretion and galanin gene expression within the same individual cells. PRL secretion from galanin-positive lactotrophs was significantly greater than that from galanin-negative lactotrophs. Moreover, treatment with galanin antiserum significantly attenuated PRL secretion from galanin-positive cells, and treatment with galanin significantly enhanced PRL secretion from galanin-negative lactotrophs. In conclusion, these data provide direct evidence that galanin derived from the estrogen-treated anterior pituitary stimulates PRL secretion in both autocrine and paracrine manners.

Upregulation of leptin receptor gene expression in the anterior pituitary of human growth hormone-releasing hormone transgenic mice.

The recently cloned leptin receptor (OB-R) is expressed in many tissues, including the anterior pituitary. It is not known whether OB-R gene expression is regulated by pituitary hormones. In the present study, we detected the long isoform of OB-R (OB-R(L)) in the anterior pituitary of normal mice using RT-PCR, but were unable to detect the short isoform (OB-R(S)). In human growth hormone-releasing hormone (hGHRH) transgenic mice, we discovered a significant increase in OB-R(L) mRNA levels in the anterior pituitary as compared to controls, and OB-R(S) gene expression was detectable. In contrast to the pituitary, there were no significant changes in OB-R gene expression for either isoform in the hypothalamus of hGHRH mice. The dramatic increase in the gene expression of OB-R(L) in the anterior pituitary of hGHRH transgenic mice was confirmed by RNase protection assay. This is the first study to demonstrate that OB-R gene expression in the anterior pituitary gland is increased by GH and/or GHRH.

Galanin in normal and hyperplastic anterior pituitary cells. From pituitary tumor cell lines to transgenic mice.

Studies on the regulation of galanin expression in the epithelial cells of the anterior pituitary gland have provided a wealth of insight into the cellular and molecular biology of this unique peptide. Galanin is localized within subpopulations of specific pituitary cell types, and hypothalamic as well as gonadal factors including dopamine, somatostatin, thyrotropin-releasing hormone, growth hormone-releasing hormone (GHRH), estrogen, and progesterone dynamically regulate its expression and release. Galanin gene expression and peptide secretion are markedly increased in estrogen-induced prolactinomas, wherein galanin serves as both an autocrine and paracrine hormone regulating prolactin secretion. Galanin mRNA and peptide levels are also dramatically elevated in somatotroph adenomas of human GHRH transgenic mice. Moreover, galanin secretion is increased from the hyperplastic somatotrophs of hGHRH transgenic mice. However, not all pituitary adenomas are associated with increased galanin gene expression; galanin synthesis is repressed in 131I-induced thyrotroph adenomas. Thus, galanin acts locally to regulate pituitary hormone secretion and appears to act as a mitogenic factor to increase the proliferation of pituitary cells in a cell-type specific manner.

An estrogen receptor binding site within the human galanin gene.

Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.

Regulation of the rat liver sodium-dependent bile acid cotransporter gene by prolactin. Mediation of transcriptional activation by Stat5.

The intracellular mechanism(s) underlying the upregulation of the hepatic Na+/taurocholate cotransporting polypeptide (ntcp) by prolactin (PRL) are unknown. In this report, we demonstrate a time-dependent increase in nuclear translocation of phosphorylated liver Stat5 (a member of the ignal ransducers and ctivators of ranscription family) that correlated with suckling-induced increases in serum PRL levels. In electrophoretic mobility gel shift assays, nuclear Stat5 exhibited specific DNA-binding ability towards IFN-gamma-activated sequence (GAS)-like elements (GLEs; 5'TTC/A-PyNPu-G/TAA-3') located in the -937 to -904 bp region of the ntcp promoter. Transient cotransfections in HepG2 cells revealed that PRL inducibility (2.5-3-fold) required coexpression of the long form of the PRL receptor (PRLRL) and Stat5. Deletion analysis mapped the PRLinducible region to -1237 to -758 bp of the ntcp promoter. Linking this 0.5-kb region to a heterologous thymidine kinase (tk) promoter, or linking multimerized ntcp GLEs either upstream of the ntcp minimal promoter (-158 to +47 bp) or the heterologous promoter conferred dose-dependent PRL responsiveness. The short form of the PRL receptor failed to transactivate ntcp GLEs. These results indicate that PRL acts via the PRLRL to facilitate Stat5 binding to ntcp-GLEs and to transcriptionally regulate ntcp.

An interactive physiological role of neuropeptide Y and galanin in pulsatile pituitary luteinizing hormone secretion.

Both neuropeptide Y (NPY) and galanin (GAL) systems have been implicated in the excitatory regulation of pulsatile LH secretion in the ovariectomized rat. The present studies were designed to examine the possible interaction of these two neuropeptides in controlling episodic LH release by testing the effects of central infusion of antibodies (Ab) to NPY and GAL, alone or in combination; additional studies tested the effects of central administration of an antisense oligodeoxynucleotide (ODN) to NPY and GAL messenger RNA. Rats were ovariectomized, implanted with a cannula in the third ventricle, and used in experiments 2 weeks later. Central infusion, via Alzet osmotic minipumps, of IgG purified from an NPY Ab produced a dose-related suppression of pulsatile LH secretion. Although an Ab dilution of 1:10 was ineffective, a maximal inhibitory effect was obtained using an NPY Ab dilution of 1:1, which decreased the mean levels, pulse frequency, and pulse amplitude of LH. These parameters of episodic LH secretion were also significantly reduced by central injection of antisense NPY ODN compared to those in vehicle- or missense ODN-treated controls. Similar dose-related inhibitory effects on the parameters of LH secretion were seen after central infusion of GAL Ab. Furthermore, infusion of a combination of NPY Ab and GAL Ab, each at the ineffective dilution of 1:10, resulted in a profound inhibition of LH secretion equivalent to the pattern seen with the maximally effective 1:1 Ab dilution. These results strengthen the idea of a physiological role for both NPY and GAL systems in the mechanism underlying the LHRH pulse generator activity and further suggest that these two excitatory neuropeptides act in concert to generate pulsatile LHRH release.

Galanin gene expression in radiothyroidectomy-induced thyrotroph adenomas.

Galanin gene expression is markedly increased in the anterior pituitary glands of estrogen-treated rats (lactotroph hyperplasia) as well as human growth hormone-releasing hormone transgenic mice (somatotroph hyperplasia). The objective of this study was to examine galanin in a mouse model of thyrotroph adenoma formation. Male mice were radiothyroidectomized by use of iodine-131 (131I), and galanin peptide levels were assessed in the hypothalamic-pituitary axis. Immunoreactive galanin concentrations in the anterior pituitaries of 131I-treated mice were decreased 80% at 3, 6, 9, and 12 mo after radiothyroidectomy. Galanin peptide levels in the hypothalamus were decreased 20-25% at these times. Treatment with either estradiol or 3,3',5-triiodo-L-thyronine increased galanin peptide concentrations in the anterior pituitaries of 131I-treated mice, but neither treatment restored galanin concentrations. Galanin mRNA levels were decreased > 80% 1 yr after radiothyroidectomy. We conclude that, unlike animal models of lactotroph and somatotroph hyperplasia, galanin gene expression is suppressed throughout the development of thyrotroph adenomas, suggesting that galanin does not have a stimulatory role in the proliferation of thyrotrophs. Moreover, these data show that thyroid hormones are important positive regulators of galanin gene expression in the mouse and that estrogen may stimulate galanin gene expression in the absence of thyroid hormones.

Vasoactive intestinal polypeptide mRNA and peptide levels are decreased in the anterior pituitary of the human growth hormone-releasing hormone transgenic mouse.

We recently reported that galanin gene expression is markedly increased in the hyperplastic anterior pituitary gland of the human growth hormone-releasing hormone (hGHRH) transgenic mouse. To determine if another pituitary peptide hormone with putative growth-promoting activity is similarly affected, or if this effect is specific to the peptide galanin, we examined vasoactive intestinal polypeptide (VIP) gene expression in the hypothalamic-pituitary axis of male hGHRH transgenic and non-transgenic mice. The objectives were to: 1) assess VIP peptide concentrations, 2) estimate relative differences in VIP mRNA levels, 3) determine the effects of acute treatment with 17beta-estradiol on VIP peptide and mRNA levels, and 4) quantify the density of immunoreactive VIP pituitary cells by immunohistochemistry. Four to five month old male hGHRH transgenic mice and their non-transgenic siblings were identified by PCR. Immunoreactive VIP concentrations were decreased by 50% in the anterior pituitary glands of hGHRH transgenic mice as compared to non-transgenic siblings. In contrast, no differences in immunoreactive VIP concentrations were observed in the hypothalamus or frontal cerebral cortex of transgenic and non-transgenic mice. Treatment with 17beta-estradiol significantly increased VIP concentrations in the anterior pituitary gland of both transgenic and non-transgenic mice; however, VIP peptide concentrations in the anterior pituitary glands of hGHRH transgenic mice remained 50% lower. Relative differences in VIP mRNA levels were estimated by RT-PCR, and were found to be 2.5-fold higher in the anterior pituitary glands of non-transgenic mice. In contrast, no differences in VIP mRNA levels in the cerebral cortex were detected between transgenic and non-transgenic mice. Treatment with 17beta-estradiol increased VIP mRNA levels in the anterior pituitary, but not in the cerebral cortex. In concert with the changes in VIP peptide and mRNA, the density of immunoreactive VIP pituitary cells was decreased approximately 50% in hGHRH transgenic mice. In conclusion, unlike galanin gene expression, VIP peptide and mRNA levels are significantly decreased in the anterior pituitary gland of hGHRH transgenic mice. Moreover, these changes appear to be tissue-specific and are likely due, in part, to the decrease in the density of VIP-containing pituitary cells in the hyperplastic pituitary. Although the pituitary cell type(s) synthesizing VIP remains unclear, these data suggest that VIP in the anterior pituitary is not stimulating pituitary tumor development in hGHRH transgenic mice.

Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport.

We have shown that prolactin (Prl) increases the transhepatic transport of taurocholate (TC) in postpartum rats and following treatment of ovariectomized (Ovx) rats with ovine Prl (oPrl). The present studies were designed to determine if treatment of Ovx rats with oPrl (100, 300, or 600 micrograms/day, 7 days iv) 1) increases Na(+)-TC cotransport in basolateral plasma membrane vesicles (bLPM), 2) induces a corresponding increase in the steady-state levels of Na(+)-TC cotransport polypeptide (Ntcp mRNA), and 3) if the oPrl-mediated increase in Na(+)-TC cotransport activity is blocked by cycloheximide, an inhibitor of protein synthesis. oPrl (300 micrograms/day) induced a twofold increase in the maximal velocity for Na(+)-TC cotransport in both hepatocytes and bLPM vesicles with little change in the Michaelis constant. Infusion of oPrl at a dose of 100, 300, or 600 micrograms/day increased steady-state Ntcp mRNA four-, ten-, and twofold, respectively. Finally, cycloheximide blocked the oPrl-mediated increase in Na(+)-TC cotransport but did not affect basal activity. These data support the hypothesis that an increase in Ntcp mRNA followed by increased synthesis and incorporation of Ntcp in the plasma membrane is responsible for the oPrl-mediated increase in Na(+)-TC cotransport in the basolateral plasma membrane domain of the hepatocyte.

Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity.

Bilateral changes in striatal dopamine metabolism after unilateral intracarotid and intrastriatal administration of apomorphine.

Following cannulation of the common carotid artery of female Sprague-Dawley rats, 3 microCi (10 micrograms) of [3H]apomorphine were infused. At various time intervals, drug concentrations were determined in the right and left striata, anterior forebrains, posterior forebrains and cerebella. One minute following intracarotid infusion of apomorphine, approximately a 65-fold right/left difference in apomorphine concentrations was attained in all forebrain structures, and this difference steadily diminished with time as a result of declining drug levels in the infused hemisphere. The concentrations of dopamine and its metabolites (DOPAC, HVA and 3-MT) were quantified by gas chromatography-mass spectrometry in the right and left striata at 5 and 15 min after unilateral intracarotid infusion of 1 microgram apomorphine. At both time intervals and regardless of the side infused, the metabolites of dopamine increased ipsilateral to the side of infusion. Moreover, 3-MT levels were significantly decreased in the contralateral striatum. After direct intrastriatal injection of either 0.1 or 1.0 microgram apomorphine into the right striatum, the levels of dopamine metabolites were again increased in the ipsilateral striatum. 3-MT levels were also decreased significantly in the left striatum. In contrast to the effects observed after systemic administration of apomorphine, these results demonstrate that dopamine release in the striatum is increased by selectively delivering higher concentrations of apomorphine to the nerve terminals of the nigrostriatal neurons. The effects of unilateral apomorphine on dopamine metabolism in the contralateral striatum are most likely the effect of interhemispheric communication.