Identification and characterization of type A endothelin receptors in MMQ cells
Wu-Wong, J.R.; Chiou, W.; Magnuson, S.R.; Witte, D.G.; Lin, C.W.
Molecular Pharmacology 44(2): 285-291
1993
ISSN/ISBN: 0026-895X PMID: 8355666 Document Number: 411483
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 degree . 125I-ET-1 binding was inhibited by ET-1 with an IC-50 value of 0.17 nM but was only partially ( apprx 60%) inhibited by 1 mu-M 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 IC-50 values of 5 nM and 0.9 nM, respectively. RT-PCR detected mRNA for the ET-A receptor but not the ET-B receptor. RT-PCR detected mRNA for both ETA and ET-B 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 B-max and K-d values of 0.11 pmol/mg and 0.038 nm, respectively. The number of receptors was 6.6 times 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 mu-M. ET-1 stimulated the formation of total inositol phosphates in a dose-dependent manner, with an EC-50 of 0.1 nm. The phosphatidylinositol hydrolysis response was also inhibited by BQ123 and FR139317. We conclude that MMQ cells express the ET-A 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.