Using Receptor Antagonists in Binding Studies to Characterize a Mammalian Endothelin Receptor

Wu-Wong, Jinshyun R.

doi:10.1385/1-59259-289-9:071

Cited by 3 publications

(2 citation statements)

References 26 publications

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“…33 To measure ET-1 and vascular endothelial growth factor-A (VEGF) in conditioned medium, we utilized an enzyme-linked immunosorbent assay (ELISA). Medium (2 mL) was collected after 48 h exposure to 2.5 × 10 5 cells plated in six-well dishes.…”

Section: Methodsmentioning

confidence: 99%

Endothelin-1 inhibits prostate cancer growth in vivo through vasoconstriction of tumor-feeding arterioles

Weydert

Esser

Mejia

et al. 2009

Cancer Biology & Therapy

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The vasoactive peptide endothelin-1 (ET-1) has been implicated in promoting the progression of prostate and other cancers though its precise mechanism(s)-of-action remain unclear. To better define the role of ET-1 in prostate cancer progression, we generated prostate cancer cell lines (PC-3 and 22Rv1) that express elevated levels of ET-1. As anticipated, increased ET-1 lead to modest autocrine growth stimulation of PC-3 cells in monolayer culture and increased colony formation in soft agar by both cell lines. Unexpectedly, however, metastatic colonization of 22Rv1 cells expressing elevated levels of ET-1 was reduced, as was the size of subcutaneous tumors produced by both 22Rv1- and PC-3 cells. Based on these data, we hypothesized that high levels of ET-1 may negatively impact the tumor microenvironment. We found that increased ET-1 expression did not consistently inhibit angiogenesis, indicating that this was not the cause of poor tumor growth. As an alternative explanation, we examined whether elevated ET-1 results in local vasoconstriction and thus reduces the blood supply available to the tumor. Consistent with this hypothesis, treatment of mice bearing PC-3 xenografts with a vasodilator increased tumor perfusion and partially restored tumor growth. Moreover, analysis of tumor vascular casts indicated vasoconstriction of tumor-feeding arterioles. Taken together, our data suggest that the local concentration of the ET-1 peptide is critical for determining a balance between its previously unrecognized tumor growth-suppressing activity (vasoconstriction) and known growth-promoting (mitogenesis, survival and angiogenesis) activities. These findings may have implications for the modification of current prostate cancer therapies involving ET-1.

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Section: Methodsmentioning

confidence: 99%

Endothelin-1 inhibits prostate cancer growth in vivo through vasoconstriction of tumor-feeding arterioles

Weydert

Esser

Mejia

et al. 2009

Cancer Biology & Therapy

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“…[ 125 I] Endothelin-1 binding assay [ 125 I]-Endothelin-1 Ligand Binding experiments were carried out as described by Wu-Wong (2002). Confluent cells in 24-well plates were preincubated in ice-cold binding buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, 25 mM HEPES, and 0.1% BSA, pH 7.4) for 15 min.…”

Section: [ 3 H]bq123 Binding Assaymentioning

confidence: 99%

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Taylor

Wilson

et al. 2012

Journal Cellular Physiology

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Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca 2+ influx in response to endothelin-1 (ET-1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phosphoantibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET-1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC while it continues in IPAH. Importantly the data suggest that caution must be taken when applying ET-1 receptor antagonist therapy to PAH patients.

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Signalling and regulation of collagen I synthesis by ET‐1 and TGF‐β1

Horstmeyer¹,

Licht

Scherr³

et al. 2005

The FEBS Journal

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Endothelin‐1 (ET‐1) plays an important role in tissue remodelling and fibrogenesis by inducing synthesis of collagen I via protein kinase C (PKC). ET‐1 signals are transduced by two receptor subtypes, the ETA‐ and ETB‐receptors which activate different Gα proteins. Here, we investigated the expression of both ET‐receptor subtypes in human primary dermal fibroblasts and demonstrated that the ETA‐receptor is the major ET‐receptor subtype expressed. To determine further signalling intermediates, we inhibited Gαi and three phospholipases. Pharmacologic inhibition of Gαi, phosphatidylcholine‐phospholipase C (PC‐PLC) and phospholipase D (PLD), but not of phospholipase Cβ, abolished the increase in collagen I by ET‐1. Inhibition of all phospholipases revealed similar effects on TGF‐β1 induced collagen I synthesis, demonstrating involvement of PC‐PLC and PLD in the signalling pathways elicited by ET‐1 and TGF‐β1. ET‐1 and TGF‐β1 each stimulated collagen I production and in an additive manner. ET‐1 further induced connective tissue growth factor (CTGF), as did TGF‐β1, however, to lower levels. While rapid and sustained CTGF induction was seen following TGF‐β1 treatment, ET‐1 increased CTGF in a biphasic manner with lower induction at 3 h and a delayed and higher induction after 5 days of permanent ET‐1 treatment. Coincidentally at 5 days of permanent ET‐1 stimulation, a switch in ET‐receptor subtype expression to the ETB‐receptor was observed. We conclude that the signalling pathways induced by ET‐1 and TGF‐β1 leading to augmented collagen I production by fibroblasts converge on a similar signalling pathway. Thereby, long‐time stimulation by ET‐1 resulted in a changed ET‐receptor subtype ratio and in a biphasic CTGF induction.

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Using Receptor Antagonists in Binding Studies to Characterize a Mammalian Endothelin Receptor

Cited by 3 publications

References 26 publications

Endothelin-1 inhibits prostate cancer growth in vivo through vasoconstriction of tumor-feeding arterioles

Endothelin-1 inhibits prostate cancer growth in vivo through vasoconstriction of tumor-feeding arterioles

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Signalling and regulation of collagen I synthesis by ET‐1 and TGF‐β1

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