Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

Endothelin 1 protects HN33 cells from serum deprivation-induced neuronal apoptosis through Ca2+-PKCalpha-ERK pathway

Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.

Expression of Nitric Oxide Synthase and Endothelin-1 in Human Uterine Artery from Full-Term Pregnancies

The aim of this study was to determine the roles of ET-1 and NO on uterine blood flow in pregnancy. Uterine arteries were isolated from 17 nonpregnant and 12 pregnant women. Nonpregnant group included patients with median age of 48.6+/-2.3 years who underwent hysterectomy, because of myoma. Pregnant group included patients with median age of 31.3+/-1.4 years undergoing cesarean delivery. ET-1 and ET-2 induced concentration-dependent contraction in isolated nonpregnant and pregnant uterine arteries. The contractile response and maximal contraction were increased in pregnant uterine arteries. In nonpregnant uterine arteries, there was no contraction in response to ET-3, whereas pregnancy induced concentration-dependent contraction by ET-3. Tissue nitrite/nitrate level and immunohistochemical staining of eNOS and iNOS were increased in pregnant uterine arteries, compared with nonpregnant uterine arteries. In addition, the expressions of eNOS and iNOS mRNA were significantly increased in pregnancy. Moreover, contractions by ET isopeptides, including ET-1, were enhanced, and immunohistochemical staining of ET-1 and ET-1 mRNA expression was increased in pregnant uterine arteries. These results suggest that NO production by increased NOS activity, especially eNOS activity, is related to placental and uterine blood flow. Furthermore, ET-1 appears to play a pathophysiological role in pregnant complications such as hypertension.

Endothelin-1, Endothelin-2 and Endothelin-3 Induced Expression of Monocyte Chemoattractant Protein-1 in Rat Mesangial Cells / 대한신장학회잡지

BACKGROUND: Monocyte chemoattractant protein- 1 (MCP-1) is an important mediator for monocyte/ macrophage infiltration in various inflammatory renal diseases and is produced by renal cells. In the process of renal diseases, endothelin-1 (ET-1) is known to play an active role in cell growth, inflammation and fibrosis. The aim of this study was to investigate whether three isoforms of endothelin regulate MCP-1 expression in cultured mesangial cells. METHODS: Mesangial cells were incubated with or without various doses of ET-1, ET-2 or ET-3. To determine the monocyte chemotactic activity, chemotaxis assay was performed in modified Boyden chambers using freshly isolated human monocytes. MCP-1 mRNA expression in mesangial cells was measured by Northern blot analysis. RESULTS: ET-1, ET-2 and ET-3 stimulated monocyte chemotactic activity released from mesangial cells in a dose-dependent manner. ET-1, ET-2 and ET-3 also stimulated MCP-1 mRNA expression in a time-dependent manner, which was seen as early as 4 hours and was maintained up to 24 hours. CONCLUSION: These data suggest that ET-1, ET- 2 and ET-3 stimulate MCP-1 expression in mesangial cells and may contribute to the monocyte/ macrophage infiltration in inflammatory renal diseases.

Analysis of HPV-16 E6-related gene expression using cDNA microarray / 대한산부인과학회잡지

OBJECTIVE: To examine the effect of HPV-16 E6 expression on the transcription of cellular genes, we used cDNA microarray in HPV-16 E6 transfected stable cancer cell lines. METHODS: Using cDNA microarray consisting of 1,024 genes, we have performed a systematic characterization of gene expression in A549E6 human lung adenocarcinoma and RC10.1 human colon adenocarcinoma cell lines stably expressing HPV-16 E6 gene. The up-regulated and down-regulated genes were classified into the different functional categories; oncogenes, apoptosis, cell cycle, signal transduction, gene regulation, immune response, cell adhesion, protein transport, metabolism, redox control and angiogenesis. RESULTS: Among 1,024 known genes and ESTs (expressed sequence tags) tested, we found 27 up- regulated and 43 down-regulated genes in A549E6 (HPV-16 E6) compared to A549. The major up-regulated genes were as follows. GTPase-activating protein Rho 4, transcription factor D2, IKAROS, integrin-alpha 6, cadherin 11, ephrin-beta 2, RAN binding protein 2, branched-chain amino transferase 2. The major down-regulated genes were as follows. K-ras 2, CDC (cell division cycle) 37, CDC16, CDC7L1, IRF3, interferon-gamma-inducible protein 30, cadherin 6, desmoglein 1, desmocollin 2, endothelin 2. Also, we found 48 up-regulated and 34 down-regulated genes in RC10.1 (HPV-16 E6) compared to RKO. The major up-regulated genes were as follows. Colon cancer familial nonpolyposis type 1 (COCA 1), Bcl 2, jagged 1, MAP2K6, E2F1, ephrin receptor-beta 2, ephrin-beta 2, desmoglein 1, transforming growth factor-beta 3. The major down-regulated genes were as follows. KIT, Rad51C, Bcl 2 antagonist killer 1, STAT 4, epidermal growth factor receptor, high mobility group protein 2, cadherin 11, cadherin 12, cadherin 3, integrin-alpha 1, intergrin-alpha 8, chromosome segregation 1-like. CONCLUSION: Various expression patterns of cellular genes by HPV-16 E6 could be wholy grasped and classified into different functional groups using both cell line system stably expressed HPV-16 E6 and cDNA microarray analysis. These analysis methods must be helpful to understand multiple effects of a specific gene on cellular genes in a short period.

Endothelial-dependent and independent vasodiatation; an index for endothelial dysfunction in women with previous gestational diabetes mellitus

Aim: This work was intended to assess whether healthy women with a history of gestational diabetes mellitus [GDM] may have abnormalities in endothelial function at a very early stage before glucose intolerance occurs. Subjects and the work included 45 subjects classified into 3 groups; I] included 15 obese women with previous GDM, II] included 15 nonobese women with previous GDM and III] included 15 nonobese healthy women as controls. All women were subjected to the following: thorough history taking, full clinical examination, laboratory investigations including serum uric acid, glycosylated haemoglobin, plasma endothelin-1 and complete lipid profile "total cholesterol, triglycerides, HDL-C and LDL-C". Oral glucose tolerance test [OGTT] was done using 75g glucose. Insulin sensitivity index and relative resistance for insulin were estimated during OGTT. The vasodilatory responses of the brachial artery during reactive hyperemia [endothelium-dependent Vasodilatation], and after nitroglycerine administration [endothelium-independent Vasodilatation] were measured using high-resolution echo-Doppler ultrasound 3-6 months after the last delivery. Flow mediated dilatation [FMD] was significantly and equally decreased in both groups of women with previous GDM, compared with control subjects [1.6 +/- 3.7% in the nonobese GDM group and 1.6 +/- 2.5% in the obese GDM group versus 10.3 +/- 4.4% in control subjects, P <0.05]. FMD correlated inversely and significantly with serum uric acid levels, BMI, serum total cholesterol, plasma endothelin-1 and relative resistance for insulin. Nitrate-induced dilatation [NID] was significantly decreased only in the obese GDM group compared with controls [21.4 +/- 5.1% versus 27.9 +/- 9.5%, P < 0.05]. OGTT was within normal range in all groups, although glucose concentrations at 30 and 60 min were significantly higher in both GDM groups, and glucose at fasting time, 90 and 120 min were significantly higher only in obese GDM women. Insulin levels at fasting, 30, 60, 90 and 120 min during the OGTT were significantly higher in obese GDM group. The relative resistance for insulin was significantly higher in the obese GDM group when compared with both the normal and nonobese GDM groups. Glycosylated haemoglobin levels were similar in all groups. Serum uric acid and plasma endothelin-1 were significantly higher in both obese and nonobese GDM women. Total cholesterol, triglycerides and LDL-cholesterol were significantly higher in obese GDM women. Conclusions: Our results suggest that endothelial dysfunction, which is considered as a very early index of atherogenesis, is already present in both obese and nonobese women with a history of GDM, even when they have normal glucose tolerance

Alteration in Endothelin Receptor-Mediated Responses in Femoral Arteries from Hyperlipidemic Rabbits / 대한정형외과학회잡지

OBJECTIVE: To investigate the roles of ET in the regulation of peripheral vascular tone, we studied the effect of hyperlipidemia on vascular responsiveness in femoral arteries from rabbits with control groups of rabbits and test groups receiving a hyperlipidemic diet. MATERIALS AND METHODS: New Zealand Whites were anesthetized with pentobabital and killed by exsanguination from the femoral arteries. Arteries which were suspended on muscle chambers at their optimal length for contractile properties, were examined. RESULTS: 1. After 14-16 weeks of cholesterol-rich diet, plasma cholestrol and HDL levels were significantly higher in the hyperlipidemic rabbits than in the control rabbits. There was no significant difference in the triglyceride levels between the two groups. 2. The contractions caused by 60 mM KCI in the femoral arterial strips were significantly augmented (P<0.01). The contractile responses to phenylephrine or angiotensin II were also augmented, whereas 5-hydroxytryptamine or U46619- induced contraction was not affected by the hyperlipidemic diet. 3. In control rabbits, ET-1 and ET- 2 contracted femoral arteries in a concentraction-dependent manner, whereas sarafotoxin S6c and IRL 1620 had no effect. 4. Contractions caused by ET-1 and ET-2 were significantly diminished by hyperlipidemia. 5. ET-1-induced concentration-response curves were inhibited by BQ-610, but not affected by BQ-788 in the femoral arterial strips from control and hyperlipidemic rabbits. CONCLUSIONS: These results suggest that ET is involved in the regulation of vascular tone in peripheral arteries and ETA receptor subtypes are mainly present in rabbit femoral arteries. Further more, ET-induced contraction is attenuated in hyperlipidemic rabbit, and the attenuated responses might be caused at least in part by the alteration of ET receptors (e.g. desensitization).

Contractile Responses to Endothelins in Isolated Arteries from Human Uterus / 대한산부인과학회잡지

OBJECTIVE: We examined the vasoconstricting poperties of endothelin (ET) on isolated arteries from pregnant as well as non-pregnant uterus. METHODS: Arteries of the uterus were obtained from both hysterectomized uterus and during pregnany hysterectomy for control group and cesarean section for pregnant group. Rings of uterine artery were suspended on muscle chambers at their optimal length for generating tension and contractile properties were examined. RESULTS: ET-1 and ET-2 induced concentration-dependent constriction of both isolated arterial strips from non-pregnant and pregnant uterus. The contraction to ET-1 and ET-2 were more enhanced in full-term pregnancy. Furthermore, in pregnant group, sarafotoxin S6c and IRL 1620, ET. agonists, induced a dose-dependent contraction, which was not shown in those from non-pregnant human. Pretreatment of human uterine arterial strips from pregnant uterus with BQ610, an ET. antagonist, for 10 min resulted in a dose-related rightward shift of ET-1 response curve with diminution of maximal response. Schild plot analysis yielded a pA value of 7.29 with a slope of 0.98. However, BQ788, an ET antagonist, did not produce any rightward shift. The contraction to lower concentration (10-8~3*10-7 M) of sarafotoxin S6c was not affected by BQ788, whereas that to higher concentration (10-s-8*10-7 M) was marked diminished. However, BQ610 did not exnt any efFect on sarafotoxin S6c-induced contraction in arterial staips from pregnant uterus. When the bath solution was replaced with Ca-free physiological salt solution (PSS) containing 1 mM EGTA for 10 min prior to adding sarafotoxin S6c, sarafotoxin S6c-induced contraction was completely abolished. Sarafotoxin S6c (10 nM)-induced contraction was prefetentially blocked by a protein kinase C antagonist, H-7, whereas it was less sensitive to a calmodulin antagonist, calmidazolium, CONCLUSION: Based on above results, we concluded that ET plays an important role in regulating uterine blood flow through the activation of ETa and ETB receptors. Furthermote, ETB receptors may predominantly contribute to the modulation of human uterine circulation in full-term pregnancy.

Changes in Plasma and Urine Endothelin Levels During Acute Exacerbation of Asthma / 결핵

BACKGROUND: Recent studies have documented increased release of endothelin(ET) during acute attack of asthma. The purpose of this study is to observe the link between plasma level and urinary excretion of each and changes during acute exacerbation. METHOD: Plasma and 24 hour urine were collected from sixteen asthmatics during acute exacerbation, twice ; first day of symptomatic exacerbation and two weeks after treatment. Controls were ten healthy normal subjects. All patients were treated with corticosteroid and beta-2 adrenergic agonist on admission. ET was determined by radioimmmunoassay and had 100% cross reactivity with ET-1, 67% with ET-2, 84% with ET-3, and 8% with Big-ET. RESULTS: Plasma ETs were significantly elevated during acute attack of asthma compared with those in remission and controls. However, there was no significant changes in urine ET concentrations or total ET amounts in 24 hour urine during exacerbation upto two weeks. Those levels of urine ET in asthmatics were still higher than controls. ET concentrations in plasma or urine were not correlated with pulmonary functional parameters and hypoxemia. CONCLUSION: The findings suggests that increased plasma ETs are related with exaggerated release during acute asthma. Urinary ET excretion is increased in asthma. However, urine ET changes during exacerbation should be observed in a larger and longer scale.

Endothelin increases intracellular free calcium in isolated rat nephron

In the freshly isolated rat nephron, the effect of endothelin-1, -2 and -3 (ET-1, -2 and -3) on cytosolic free calcium concentration ((Ca2+)i) was determined using the fluorescent indicator Fura-2/AM. (Ca2+)i increase was investigated in 9 parts of the single nephron including glomerulus (Glm), S1, S2, S3, Cortical and medullary thick ascending limb and cortical (CCT) and outer medullary collecting tubule (OMCT). Endothelins increased (Ca2+)i in Glm (ET-1; 127+/-17%, ET-2; 93+/-5%, ET-3; 169+/-17%), CCT (ET-1; 30+/-6%, ET-2; 38+/- 19%, ET-3; 158+/-18%) and OMCT (ET-1; 197+/- 11%, ET-2; 195+/- 11%, ET-3; 215+ 37%) at 10(-7) M. In OMCT, ET-1 and ET-2 increased (Ca2+)i in a dose-dependent manner (10(-10) ~ 10(-6) M). To the contrary, ET-3-induced (Ca2+)i rise was begun from 10(-12) M. BQ-123Na, an antagonist of ETA receptor, at 10(-4) M inhibited about 30% of (Ca2+)i rise induced by ET-1 and -3. Binding experiments using (125I)ET-3 showed the existence of ETB receptor in OMCT. This binding was replaced by ET-1, ET-2 or ET-3 by the almost same degree but not by angiotensin II or vasopressin.