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Endothelins (ETs) are 21-amino acid peptides that bind to membrane receptors to initiate pathophysiological effects. This report characterizes ET receptors in benign prostatic hyperplasia-1 (BPH-1) cells, a prostate cell line isolated from a specimen of a 60-yr-old man with benign prostatic hyperplasia. [(125)I]ET-1 or -3 binding was of high affinity, with B(max) and K(d) values of 48 fmol/1 x 10(6) cells and 0.16 nM for ET-1, and 2.9 fmol/1 x 10(6) cells and 0.033 nM for ET-3, respectively. ET-1, ET-3, FR139317, Ro 46-2005, and IRL1620 inhibited [(125)I]ET-1 binding to these cells with IC50 values of 0.22, 186, 0.20, 52.8, and 772.3 nM, respectively. Reverse transcription-polymerase chain reaction confirmed that BPH-1 cells expressed more ET(A) than ET(B) receptors. ET-1 did not have any effect on arachidonic acid release, but caused a modest stimulation of phosphatidylinositol hydrolysis, and induced a prominent, sustained elevation in intracellular Ca2+ concentrations. The functional effects of ET-1 were completely inhibited by the ET(A)-selective antagonists FR139317 and A-127722, suggesting that the effects were mediated by the ET(A) receptor. These results suggest that ET may play functional roles in benign prostatic hyperplasia.

The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.

Endothelin-1 (ET-1) binding to human astrocytoma U138MG cells was time-dependent, and bound [125I]ET-1 was difficult to dissociate. The B(max) and Kd values of [125I]ET-1 binding were 70 fmol/mg and 0.07 nM, respectively. Interestingly, different from other astrocytoma cells and astrocytes, the U138MG cells expressed predominantly ETA receptor as shown by RT-PCR results and binding studies. ET-1, FR139317, BQ123, PD142893 and Ro46-2005 inhibited specific [125I]ET-1 binding with Ki values of 0.10, 0.53, 4.3, 22, and 320 nM, respectively. ETB selective ligands ET-3 and IRL1620 were much less potent. The inhibitory effects of antagonists BQ123 and PD142893 on [125I]ET-1 binding diminished following the incubation time. ET-1 binding caused a modest stimulation in phosphatidylinositol hydrolysis with an EC50 value of 24 nM. In comparison to the human U373MG cells, ET-1-induced receptor internalization in U138MG cells was less efficient with 42% of bound ET-1 internalized after 30 min of incubation. These results imply that human astrocytoma cells/astrocytes are able to express either ETA or ETB receptor under different pathophysiological conditions.

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of 16 metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, basic fibroblast growth factor, and epidermal growth factor in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated growth, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer growth. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.

Endothelin (ET) receptor in human astrocytoma U373MG cells was characterized. ET-1, ET-3, sarafotoxin S6C, IRL1620, BQ788, Ro46-2005 and PD142893 inhibited specific [125I]ET-1 binding with Ki values of 0.03 0.06, 0.74, 5.01, 4.45, 2275 and 157 nM, respectively. ETA selective antagonists BQ123 and FR139317 at 1 microM did not block [125I]ET-1 binding. Reverse transcription-polymerase chain reaction confirmed the results from competition studies that U373 cells expressed predominantly ETB receptor. The Bmax and KD values of [125I]ET-1 binding were 0.15 pmol/1 x 10(6) cells and 0.23 nM. The molecular mass for the receptor was 45 kDa. ET-1 binding did not stimulate Ca+2 mobilization, phosphatidylinositol hydrolysis or arachidonic acid release, nor did it affect the intracellular cAMP or cGMP level. Interestingly, a majority of ET (> 80%) bound to the receptor was rapidly internalized, consistent with emerging evidence that a major function of ETB receptor is to clear ET. [125I]ET-1 binding was time-dependent and bound [125I]ET-1 was difficult to dissociate. In contrast, bound antagonists were much easier to dissociate. The results suggest that agonists and antagonists of the ET receptor exhibited different dissociation characteristics, with antagonist binding more reversible than agonist binding.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Recently the identification of endothelin (ET) receptors and ET in the pituitary gland has induced much interest in studying the potential role of ET in neuroendocrine regulation. MMQ, isolated from rat pituitary, is a prolactin-secreting cell line. Similar to primary pituitary cells, the secretory response in MMQ cells is regulated by calcium and cAMP. In this report, by combining radioligand binding, cross-linking, and reverse transcription-polymerase chain reaction (RT-PCR) techniques, we characterized the properties of ET receptors in MMQ cells. 125I-ET-1 bound to membranes prepared from MMQ cells in a time-dependent manner, reaching a plateau at 150 min at 25 degrees. 125I-ET-1 binding was inhibited by ET-1 with an IC50 value of 0.17 nM but was only partially (approximately 60%) inhibited by 1 microM ET-3. BQ123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]) and FR139317 (cC6N-L-Leu-D-Trp-Me-D-2Pya-OH), two antagonists that are selective for the ETA receptor, inhibited 125I-ET-1 binding with IC50 values of 5 nM and 0.9 nM, respectively. RT-PCR detected mRNA for the ETA receptor but not the ETB receptor. RT-PCR detected mRNA for both ETA and ETB receptors in control experiments using rat kidney RNA. 125I-ET-1 binding was saturable, reaching a plateau at 0.1 nM. Scatchard analysis of the data from saturation studies yielded a straight line, with Bmax and Kd values of 0.11 pmol/mg and 0.038 nM, respectively. The number of receptors was 6.6 x 10(10) sites/mg of protein or 13,200 sites/cell. Cross-linking studies using bis(sulfosuccinimidyl)suberate revealed an apparent molecular mass of 65 kDa for the ET receptor. Labeling of the 65-kDa protein was abolished by ET-1, BQ123, or FR139317 at 0.1 microM. ET-1 stimulated the formation of total inositol phosphates in a dose-dependent manner, with an EC50 of 0.1 nM. The phosphatidylinositol hydrolysis response was also inhibited by BQ123 and FR139317. We conclude that MMQ cells express the ETA receptor, which is coupled to phosphatidylinositol hydrolysis. MMQ cells may be useful for elucidating the mechanisms through which ET exerts its regulatory effects on pituitary cells.