Activin A is a member of the transforming growth factor-β family and has known roles in the adrenal cortex, from which activin A is secreted. We aimed to find whether activin A induces secretion of catecholamines from chromaffin cells of the adrenal medulla, which neighbours the adrenal cortex in vivo. Using carbon fibre amperometry, we were able to measure catecholamine secretion in real-time from single chromaffin cells dissociated from the rat adrenal medulla. Activin A stimulated catecholamine secretion in a rapid and dose-dependent manner from chromaffin cells. This effect was fully reversible upon washout of activin A. The minimum dose at which activin A had a maximal effect was 2 nM, with an EC50 of 1.1 nM. The degree of secretion induced by activin A (2 nM) was smaller than that due to membrane depolarization caused by an increase in the external K+ concentration from 5 to 70 mM. No response to activin A was seen when Ca2+ channels were blocked by Cd2+ (200 μM). We conclude from these findings that activin A is capable of stimulating a robust level of catecholamine secretion from adrenal chromaffin cells in a concentration-dependent manner. This occurs via the opening of voltage-gated Ca2+ channels, causing Ca2+ entry, thereby triggering exocytosis. These findings illustrate a new physiological role of activin A and a new mechanism in the control of catecholamine secretion from the adrenal medulla.
Damien J Keating and Chen Chen
Yu-Feng Zhao, Jianming Pei and Chen Chen
ATP-sensitive potassium channels (KATP channels) determine the excitability of pancreatic β-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to β-cells, but the effects of exogenous FFAs on KATP channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and KATP channels were observed in primary cultured rat pancreatic β-cells. LA (20 μM) induced hyperpolarization of MP and opening of KATP channels, which was totally reversed and inhibited by tolbutamide, a KATP channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 μM), partially inhibited LA-induced opening of KATP channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 μM), induced an opening of KATP channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of KATP channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on KATP channels. The present study demonstrates that LA induces hyperpolarization of MP by activating KATP channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and β-cell dysfunction induced by FFAs.
SK Peirce, WY Chen and WY Chen
Human prolactin (hPRL) has been reported to be involved in breast and prostate cancer development. The hPRL receptor (hPRLR) is expressed in a wide variety of tissues in at least three isoforms. In this study, a one-step real time reverse transcription PCR technique was used to determine relative expression levels of hPRLR mRNA in eleven human breast cancer cell lines, HeLa cells, three prostate cancer cell lines and nine normal human tissues. The housekeeping gene beta-actin was used for internal normalization. We demonstrate that hPRLR mRNA is up-regulated in six of the eleven breast cancer cell lines tested when compared with normal breast tissue. Of the cancer cell lines tested, we found that T-47D cells have the highest level of hPRLR mRNA, followed by MDA-MB-134, BT-483, BT-474, MCF-7 and MDA-MB-453 cells. In two breast cancer cell lines (MDA-MB-468 and BT-549), the hPRLR levels were found to be comparable to that of normal breast tissue. Three breast cancer cell lines (MDA-MB-436, MDA-MB-157 and MDA-MB-231) expressed hPRLR mRNA at levels lower than that of normal tissue. In contrast, in all three commonly used prostate cancer cell lines (LNCaP, PC-3 and DU 145), the levels of hPRLR mRNA were found to be down-regulated relative to that of normal prostate tissue. Of nine normal human tissues tested, we found that the uterus and the breast have the highest levels of hPRLR mRNA, followed by the kidney, the liver, the prostate and the ovary. The levels of hPRLR mRNA were the lowest among the trachea, the brain and the lung.
H. J. Chen
Exposure of male golden hamsters to short photoperiods of 6 h light: 18 h darkness led to testicular and accessory sex organ atrophy in 5 weeks. Short photoperiods also significantly depressed serum levels of LH, FSH, prolactin and testosterone in samples obtained by decapitation, but not in samples collected on the preceding day under ether anaesthesia. Injections of luteinizing hormone releasing hormone (LH-RH) at 09.00 h (lights on) or at 15.00 h (lights off) prevented testicular regression when compared with hamsters receiving injection vehicle only. However, the hamsters receiving LH-RH injections at lights on had significantly greater testicular weight and accessory sex organ (seminal vesicles and coagulating glands) weight and testosterone concentration than those receiving LH-RH at lights off. No increase in testicular weight was observed in hypophysectomized male hamsters given the same LH-RH injections and the same lighting regimen.
These results indicate that LH-RH alone can prevent, at least partially, testicular and sex organ atrophy and increase serum testosterone concentration by stimulating release of LH and FSH in hamsters exposed to short photoperiods, involving temporal difference of LH-RH action. Further implications of the results are discussed.
Bo Chen, Yanrong Lu, Younan Chen and Jingqiu Cheng
Endothelial dysfunction is an important risk factor for cardiovascular disease, and it represents the initial step in the pathogenesis of atherosclerosis. Failure to protect against oxidative stress-induced cellular damage accounts for endothelial dysfunction in the majority of pathophysiological conditions. Numerous antioxidant pathways are involved in cellular redox homeostasis, among which the nuclear factor-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1)–antioxidant response element (ARE) signaling pathway is perhaps the most prominent. Nrf2, a transcription factor with a high sensitivity to oxidative stress, binds to AREs in the nucleus and promotes the transcription of a wide variety of antioxidant genes. Nrf2 is located in the cytoskeleton, adjacent to Keap1. Keap1 acts as an adapter for cullin 3/ring-box 1-mediated ubiquitination and degradation of Nrf2, which decreases the activity of Nrf2 under physiological conditions. Oxidative stress causes Nrf2 to dissociate from Keap1 and to subsequently translocate into the nucleus, which results in its binding to ARE and the transcription of downstream target genes. Experimental evidence has established that Nrf2-driven free radical detoxification pathways are important endogenous homeostatic mechanisms that are associated with vasoprotection in the setting of aging, atherosclerosis, hypertension, ischemia, and cardiovascular diseases. The aim of the present review is to briefly summarize the mechanisms that regulate the Nrf2/Keap1–ARE signaling pathway and the latest advances in understanding how Nrf2 protects against oxidative stress-induced endothelial injuries. Further studies regarding the precise mechanisms by which Nrf2-regulated endothelial protection occurs are necessary for determining whether Nrf2 can serve as a therapeutic target in the treatment of cardiovascular diseases.
Y Chen and H J Arnqvist
The present study was undertaken to investigate the metabolic regulation of insulin-like growth factor binding proteins (IGFBPs) gene expression in muscles from diabetic or fasted rat. The messenger RNA (mRNA) levels for IGFBP-2 and -4 were analysed by solution hybridization in heart, skeletal and smooth muscle and liver from fasted (3 days) and refed (6, 12, 24, 72 h) rats and rats made diabetic with streptozotocin. In aortic intima-media, the mRNA levels for IGFBP-2 and -4 were decreased by diabetes or fasting and were restored gradually by refeeding. The response of IGFBP-4 mRNA to diabetes appeared two days after injection of streptozotocin, while a significant decrease of IGFBP-2 mRNA was found after a diabetes duration of two weeks. Both diabetes and fasting decreased IGFBP-4 mRNA levels in heart muscle and skeletal muscle and refeeding restored mRNA for IGFBP-4 to normal level. IGFBP-2 mRNA was undetectable in heart muscle and skeletal muscle. In liver IGFBP-4 mRNA was abundantly expressed. It was slightly but significantly decreased by fasting and approached normality with refeeding, while no change was found in diabetic liver. In contrast, liver IGFBP-2 mRNA was much lower in amount than IGF-I mRNA and IGFBP-4 mRNA and was sharply elevated by fasting, and decreased by refeeding. In conclusion, 1) both IGFBP-2 and -4 mRNA in various tissues are regulated by diabetes or fasting; 2) the mRNA for IGFBP-2 is metabolically regulated in a discordant, organ-specific manner.
Journal of Endocrinology (1994) 143, 235–242
LOUISE CHEN, H. R. LINDNER and M. LANCET
Dispersed human endometrial and myometrial cells grown for up to 4 months as monolayer cultures responded to addition of oestradiol-17β (1 × 10−9 mol/l) to the medium by increased [3H]thymidine incorporation into DNA, and an increased rate of cell division. Cultures derived from uterine leiomyomata showed a less consistent mitotic response to the hormone.
Normal endometrial and myometrial cells grown in the oestradiolenriched medium showed a significantly higher efficacy of colony formation (increases of about 60 and 90%, respectively) than cells grown in control medium throughout the experimental period. Exposure to the hormone for 3–4 days was sufficient to induce this effect, which suggests that it does not depend on selection of hormone-sensitive cells. The mitogenic effect of oestradiol, as expressed by increased cloning efficacy, persisted for several days after transfer of the cells to a hormone-free medium. Cultures of foetal rat skeletal muscle failed to respond to oestradiol-17β in the cloning test, and oestradiol-17α was without effect on uterine cells.
It is concluded that oestradiol-17β exerts a direct mitogenic action on some cellular components of the human endometrium and myometrium in vitro.
ST Chen, JD Lin and KH Lin
The expression of TSH receptor (TSHR) gene is frequently lost in thyroid cancers during the process of dedifferentiation that involves perturbation of several nuclear transcription factors. We have established that thyroid hormone receptor beta1 (TRbeta1) is associated with the loss of TSHR gene expression in an anaplastic human thyroid cancer cell line, ARO. To demonstrate that TRbeta1 regulates TSHR gene expression, we performed electrophoresis mobility shift and 3,5,3'-triiodothyronine (T3) transactivation assays. As expected, TRbeta1 bound the synthesized oligomer containing TSHR promoter sequence by heterodimerizing with retinoid X receptor. When a chimeric reporter pTRCAT5'-146 enclosing the minimal TSHR promoter was applied for T3 transactivation assay, two TRbeta1-overexpressing transfectants of ARO cells (ARO1 and ARO2) demonstrated higher basal activity than their parental cells. Consequentially, T3 suppressed the reporter gene activity only in ARO1 and ARO2, but not in ARO cells. A point mutation creating a cAMP response element (CRE) in the reporter pTRCAT5'-146 CRE led to T3-induced suppression of the reporter gene in ARO cells without changing the basal or T3-induced activities in ARO1 and ARO2 cells. We conclude that the regulatory effect of T3 on TSHR gene expression is TR- and promoter DNA sequence-determined.
Y Chen, ML Nagpal and T Lin
Basal and LH/human chorionic gonadotropin (hCG)-stimulated testosterone formation by Leydig cells is dependent on ambient glucose levels. Inhibition of glucose uptake is associated with decreased testosterone formation. Recently, glucose transporter 8 (GLUT8) has been shown to be highly expressed in the testis. In the present study, we have investigated the expression and regulation of the GLUT8 gene in rat Leydig cells. Primers were designed by using sequences that are not conserved in GLUT1 to GLUT5 and that contain the glycosylation region of GLUT8. This yielded an amplicon of 186 bp. The tIssue-specific expression experiments in adult rat (55- to 65-day-old) tIssues revealed that GLUT8 is expressed predominantly in the testis, in smaller amounts in heart and kidney, and in negligible amounts in liver and spleen. Furthermore, GLUT8 mRNA was found to be highly expressed in crude interstitial cells, Leydig cells and testicular and epididymal germ cells. In prepubertal rat (20-day-old) tIssues, GLUT8 expression was comparatively much lower than in the adult rat tIssues. By comparative RT-PCR, hCG caused dose- and time-dependent increases of GLUT8 mRNA levels. hCG and IGF-I had synergistic effects on GLUT8 mRNA and protein expression. GLUT1 and GLUT3 were also found to be expressed in Leydig cells. However, neither GLUT1 nor GLUT3 were affected by treatments with hCG, IGF-I or hCG and IGF-I combined. The addition of murine interleukin-1alpha (mIL-1alpha; 10 ng/ml), murine tumor necrosis factor-alpha (mTNF-alpha; 10 ng/ml), murine interferon-gamma (mIFN-gamma; 500 U/ml) separately or in combination decreased hCG-induced GLUT8 mRNA levels significantly. In conclusion, GLUT8 mRNA in Leydig cells was positively regulated by hCG and IGF-I and down-regulated by cytokines, mIL-1alpha, mTNF-alpha and mIFN-gamma. These results indicate that hCG, growth factors and cytokines affect Leydig cell steroidogenesis by modulating GLUT8 expression.
HL Huang, ST Chu and YH Chen
We examined 24p3 expression in the mouse uterus at various stages of the natural estrous cycle and during the preimplantation period. The level of 24p3 mRNA appeared intensively in proestrus and estrus, then declined sharply from metestrus to diestrus. Consistent with this observation, 24p3 protein was abundant in proestrus, decreased from estrus to metestrus and declined to a very low level in diestrus. The uterine 24p3 expression closely overlapped with the estradiol (E2) surge in proestrus and estrus but it was suppressed when progesterone (P4) rose to a high level during the reproductive cycle. Neither the protein nor its message was detected in the uteri of immature mice or ovariectomized adult animals. While an injection of P4 to these animals was unable to initiate uterine 24p3 expression, administration of estrogenic steroids to these animals markedly stimulated the gene expression. Treatment of these animals with E2 together with P4, on the other hand, did not stimulate the gene expression. In pregnant animals (day 1 (D1)=day of vaginal plug), 24p3 mRNA remained at a high level on D1 and D2 but dropped to an almost undetectable level on D3 and D4. This was accompanied by a decrease in 24p3 protein from D1 to D2 and a decline in the protein to undetectable levels from D3 to D4. The staining patterns of both the immunohistochemical localization of 24p3 protein and in situ hybridization for the detection of 24p3 mRNA in the uterine sections showed that 24p3 expression took place mainly in the luminal and glandular epithelial cells of the endometrium. This together with our previous observation that 24p3 protein is found in uterine luminal fluid indicates that the protein is secreted primarily from these cells to their respective luminal surfaces during proestrus and estrus.