• Animal cells and systems
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• 제3권3호
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• pp.311-317
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• 1999

Prolactin (PRL) was reported to be locally synthesized in many brain areas including the hypothalamus, thalamus (TH) and hippocampus (HIP). In the pituitary lactotrophs, PRL synthesis is dependent upon a pituitary-specific transcription factor, Pit-1. In the present study, we attempted to identify Pit-1 or Pit-1-like protein in brain areas known as the synthetic sites of PRL. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis showed the same Pit-1 transcripts in brain areas such as the medial basal hypothalamus (MBH), preoptic area (POA), TH, and HIP with the Pit-1 transcripts in the anterior pituitary (AP). Electrophoretic mobility shift assay (EMSA) was run with nuclear protein extracts from brain tissues using a double strand oligomer probe containing a putative Pit-1 binding domain. Shifted bands were found in EMSA results with nuclear proteins from MBH, POA, TH and HIP. Specific binding of the Pit-1-like protein was further confirmed by competition with an unlabeled cold probe. Antisense Pit-1 oligodeoxynucleotide (Pit-1 ODN), which was designed to bind to the Pit-1 translation initiation site and block Pit-1 biosynthesis, was used to test Pit-1 dependent brain PRL transcription. Two nmol of Pit-1 ODN was introduced into the lateral ventricle of a 60-day old male rat brain. RNA blot hybridization and in situ hybridization indicated a decrease of PRL mRNA signals by the treatment of Pit-1 ODN. Taken together, the present study suggests that Pit-1 may play an important role in the transcriptional regulation of local PRL synthesis in the brain.

• BMB Reports
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• 제40권6호
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• pp.979-985
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• 2007

Induction of growth hormone (GH) by Glycyrrhizae Radix (GR), one of the most popular herbal medicine, and its major ingredients were studied in rat pituitary cells in vitro and in vivo assay. The MeOH extract and the n-hexane (HX) fraction of GR induced rat GH (rGH) release up to 1.89 times ($0.34{\pm}0.04 nM$) and 4.59 times ($0.83{\pm}0.03 nM$), compared to the basal level (p < 0.05). Among many ingredients isolated and purified from GR both glycyrrhetinic acid and glycyrrhizin induced significantly rGH release compared to the control (p < 0.05). After an intravenous injection of rat growth hormone releasing hormone (rGHRH) ($10{\mu}g$/kg) as positive control, in SD rats, $T_{max}$ of plasma rGH level was 10 min, $C_{max}$ was $3.84{\pm}0.01 nM$ (n = 3), and enhanced plasma rGH level returned to the baseline in 90 min. Both $AUC_{0-90}$ (area under the curve) of plasma rGH level after HX fraction and that after rGHRH administration were increased significantly from the basal level, respectively (p < 0.01). In conclusions, HX fraction is the most active fraction of MeOH extract of GR in rGH induction.

• Toxicological Research
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• 제22권4호
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• pp.307-313
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• 2006

The development of in vitro assays has been recommended to screening and testing the potential endocrine disruptors (EDs). These assay systems focus only on identifying the estrogenic or antiestrogenic activity of EDs, whereas a few studies have been carried out to screen the thyroid hormone (TH) disruptors. The aim of this study was to evaluate a test system to detect TH disruptors using rat pituitary tumor $GH_3$ cells. The test system is based on the TH-dependent increase in growth rate. As expected, L-3,5,3-triiodothyronine ($(T_3)$ markedly induced a morphological change in $GH_3$ cells from flattened fibroblastic types to rounded or spindle-shaped types. $T_3$ stimulated $GH_3$ cell growth in a dose-dependent manner with the maximum growth-stimulating effect being observed at a concentration $1{\times}10^9M$. In addition, $T_3$ increased the release of growth hormone and prolactin into the medium of the $GH_3$ cells culture. Using this assay system, the TH-disrupting activities of bisphenol A (BPA) and its related compounds were examined. BPA, dimethy/bisphenol A (DMBPA), and TCI-EP significantly enhanced the growth of $GH_3$ cells in the range of $1{\times}10^{-5}M\;to\;1{\times}10^{-6}M$ concentrations. In conclusion, this in vitro assay system might be useful for identifying potential TH disruptors. However, this method will require further evaluation and standardization before it can be used as a broad-based screening tool.

• The Korean Journal of Physiology and Pharmacology
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• 제5권2호
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• pp.139-146
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• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.

• Molecules and Cells
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• 제23권2호
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• pp.215-219
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• 2007

In mammals light input resets the central clock of the suprachiasmatic nucleus by inducing secretion of pituitary adenylate cyclase-activating polypeptide (PACAP) from retinal ganglion cells (RGCs). We previously showed that thyroid transcription factor 1 (TTF-1), a homeodomain-containing transcription factor, specifically regulates PACAP gene expression in the rat hypothalamus. In the present study we examined the expression of TTF-1 in PACAP-synthesizing retinal cells. Fluorescence in situ hybridization (FISH) showed that it is abundantly expressed in RGCs of the superior region of the retina, but in only a small subset of RGCs in the inferior region. Double FISH experiments revealed that TTF-1 is exclusively expressed in PACAP-producing RGCs. These results suggest that TTF-1 plays a regulatory role in PACAP-expressing retinal ganglion cells.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1999년도 학술발표회 진행표 및 논문초록
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• pp.58-58
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• 1999

The paraventricular nucleus (PVN) is a complex structure comprised of several different populations of cells divided into two main groups, the magnocellular (type I) neurons which secrete vasopressin and oxytocin and the parvocellular (type II) neurons which regulate hormone secretion from the anterior pituitary.(omitted)

• 대한약리학회지
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• 제32권3호
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• pp.347-355
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• 1996

흰쥐 뇌하수체 전엽배양세포에서 PKC나 cAMP에 의한 LH 분비와 $LH{\beta}$subunit mRNA 증가과정에서 세포외 $Ca^{++}$의 역할을 검증하기 위하여 phorbol ester와 forskolin에 의해 촉진된 LH 분비와 $LH{\beta}$ subunit mRNA 수준에 미치는 EGTA와 verapamil의 영향을 조사하였다. Forskolin에 의해 촉진된 LH 분비와 $LH{\beta}$ subunit mRNA 수준은 $Ca^{++}$ chelator인 EGTA나 $Ca^{++}$ 채널차단제인 verapamil의 처리로 세포외부로부터 $Ca^{++}$의 이동을 억제시켰을때 모두 감소하였다. PMA에 의해 유도된 LH 분비는 EGTA와 verapamil 처리에 의해 영향을 받지 않았으나 PMA에 의해 촉진된 $LH{\beta}$ subunit mRNA는 억제되었다. 따라서 본 연구에 의하면 PKC 활성화나 세포내 cAMP 농도 증가는 $LH{\beta}$ subunit mRNA 수준을 증가시키며 이러한 과정은 세포외 $Ca^{++}$ 의존적인 것으로 생각된다. 그러나 PKC 활성화나 cAMP 증가에 의한 세포외 $Ca^{++}$의 역할이 서로 다른 양상을 보인다. PKC 활성화에 의한 LH 분비는 세포외 $Ca^{++}$에 비의존적이나 cAMP에 의해 촉진된 LH 분비는 세포외 $Ca^{++}$ 유입의 영향을 받는 것으로 사료된다.

• 생명과학회지
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• 제10권1호
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• pp.22-36
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• 2000

The mammary gland contains a subpopulation of epithelial cells with large proliferative potentials which are the likely targets for carcinogens. These clonogenic cells can proliferate and differentiate into functional glandular structures. Multicellular secretory alveolar units (AU) develop from these clonogens in grafts of monodispersed rat mammary epithelial cells (RMEC) in gland-free mammary fat pads in intact recipient F344 rats co-grafted with mammotropic hormone-secreting pituitary tumors (MtT F4). Multicellular nonsecretory ductal units (DU) develop in grafts of monodispersed RMEC in gland-free fat pads in adrenalectomized recipient WF rats co-grafted with MtT W10. However, this effect were reversed by hydrocortisone replacement therapy. RMEC were isolated from appropriate donor rats as monodispersed mixed cells or, alternatively, RNA＋ cells were sorted by flow cytometry of mixed RMEC stained with FITC-RNA and PE-anti-Thy-1.1 monoclonal antibody. We grafted mixed or sorted PNA+ cells in gland-free mammary fat pads in recipient rats that were endocrinologically manipulated to induce AU or DU. Cells were also isolated from these AU or DU as mixed or sorted RNA＋ cells and sub-transplanted in recipient rats treated appropriately to induce AU or DU, respectively. Cells obtained from AU in grafts gave rise to clonal AU and from DU in grafts to DU on sub-transplantation in appropriate recipients. When adrenalectomized recipient WF rats co-grafted with MtT W10 received daily subcutaneous injections of hydrocortisone for periods of 21 days following the PHA＋ cell transplantation, AU, instead of DU, were developed. The histologies of these secondary AU and DU were not different from those of the primary AU and DU. Casein and laminin proteins were demonstrated by immunocytochemical staining of primary and secondary AU. Electron micrographs also demonstrated that AU were composed of secretory cells with milk protein in the cytoplasm. DU were composed of little or non-secretory ductal epithelial cells. These AU and DU also secreted large amounts of lipids. Clonogenic cells were more common in DU than in AU. Thus, AU and DU contain persistent subpopulations of clonogenic stem-like cells.

• The Korean Journal of Physiology and Pharmacology
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• 제3권3호
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• pp.339-350
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• 1999

The present study was attempted to examine the effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on catecholamine (CA) secretion evoked by cholinergic stimulation, membrane depolarization and calcium mobilization from the isolated perfused rat adrenal gland. The perfusion of PACAP (10 nM) into an adrenal vein for 60 min produced a great inhibition in CA secretion evoked by ACh $(5.32{\times}10^{-3}\;M),$ high $K^+\;(5.6{\times}10^{-2}\;M),$ DMPP $(10^{-4}\;M\;for\;2\;min),$ McN-A-343 $(10^{-4}\;M\;for\;2\;min),$ cyclopiazonic acid $(10^{-5}\;M\;for\;4\;min)$ and Bay-K-8644 $(10^{-5}\;M\;for\;4\;min).$ Also, in the presence of neuropeptide (NPY), which is known to be co-localized with norepinephrine in peripheral sympathetic nerves, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with PACAP (10 nM) under the presence of VIP antagonist $[(Lys^1,\;Pro^{2.5},\;Arg^{3.4},\;Tyr^6)-VIP\;(3\;{\mu}M)]$ for 20 min, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were not altered greatly in comparison to the case of PACAP-treatment only. Taken together, these results suggest that PACAP causes the marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization, indicating that this effect may be mediated by inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells.

• The Korean Journal of Physiology and Pharmacology
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• 제7권2호
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• pp.79-84
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• 2003

We have previously reported that expression of the somatostatin receptor subtypes, sst1-5, is differentially regulated by growth hormone (GH)-releasing hormone (GHRH) and forskolin (FSK), in vitro. GHRH binds to membrane receptors selectively located on pituitary somatotropes, activates adenylyl cyclase (AC) and increases sst1 and sst2 and decreases sst5 mRNA levels, without significantly altering the expression of sst3 and sst4. In contrast FSK directly activates AC in all pituitary cell types and increases sst1 and sst2 mRNA levels and decreases sst3, sst4 and sst5 expression. Two explanations could account for these differential effects: 1) GHRH inhibits sst3 and sst4 expression in somatotropes, but this inhibitory effect is masked by expression of these receptors in unresponsive pituitary cell types, and 2) FSK inhibits sst3 and sst4 expression levels in pituitary cell types other than somatotropes. To differentiate between these two possibilities, somatotropes were sequentially labeled with monkey anti-rat GH antiserum, biotinylated goat anti-human IgG, and streptavidin-PE and subsequently purified by fluorescent-activated cell sorting (FACS). The resultant cell population consisted of 95% somatotropes, as determined by GH immunohistochemistry using a primary GH antiserum different from that used for FACS sorting. Purified somatotropes were cultured for 3 days and treated for 4 h with vehicle, GHRH (10 nM) or FSK ($10{\mu}M$). Total RNA was isolated by column extraction and specific receptor mRNA levels were determined by semi-quantitative multiplex RT-PCR. Under basal conditions, the relative expression levels of the various somatostatin receptor subtypes were sst2＞sst5＞sst3=sst1＞ sst4. GHRH treatment increased sst1 and sst2 mRNA levels and decreased sst3, sst4 and sst5 mRNA levels in purified somatotropes, comparable to the effects of FSK on purified somatotropes and mixed pituitary cell cultures. Taken together, these results demonstrate that GHRH acutely modulates the expression of all somatostatin receptor subtypes within GH-producing cells and its actions are likely mediated by activation of AC.