Abstract
Two distinct cell surface endothelin receptors were identified, namely a 73-kDa protein referred to as ET-R1 and a 60-kDa protein named ET-R2. ET-R1 was expressed as the sole endothelin receptor on rat A10 vascular smooth muscle cells and C6 glial cells. Binding of 125I-ET-1 to these cells was inhibited by 50-200 pM endothelin-1 and -2, whereas endothelin-3 did not compete for this receptor subtype. Binding of 125I-ET-1 to intact A10 and C6 cells was reversible, indicating that ET-R1 is located on the cell surface. Affinity labelling of a single 73-kDa band on sodium dodecyl sulfate-polyacrylamide gels by 125I-ET-1 in A10 and C6 cells was inhibited by endothelin-1 but not by endothelin-3. In A10 cells, endothelin-1 but not endothelin-3 elicited a concentration-dependent increase in intracellular inositol trisphosphate levels. ET-R1 was also expressed in cultured rat glomerular mesangial cells based on findings of a subset of receptors with an apparent molecular mass of 73 kDa that bound 125I-ET-1 displacable by endothelin-1 and endothelin-2 but not by endothelin-3. These cells also expressed the ET-R2 receptor subtype, based on findings of a 60-kDa binding site that could be labeled by both 125I-ET-1 and 125I-ET-3. Labeling of ET-R2 by the radioactive endothelins-1 and -3 was inhibited competitively by endothelins-1, -2, and -3. Furthermore, ET-R2 was shown to be a functional receptor, as endothelin-3 caused inositol trisphosphate levels to rise in mesangial cells. An endothelin binding site with high affinity for endothelin-3 was also identified on rat PC12 pheochromocytoma cells, although the apparent molecular mass of this receptor could not be verified by cross-linking studies. Since endothelin-1 or -3 failed to augment inositol trisphosphate levels in these cells, this binding site could represent a third endothelin receptor subtype. Thus, two distinct functional receptors for endothelins were identified on rat cells, namely the 73-kDa ET-R1 which has an exceedingly low affinity for endothelin-3 and the 60-kDa ET-R2 which binds endothelin-3 with high affinity. Whether an additional endothelin receptor subtype exists in PC12 cells remains to be shown with certainty.
Highlights
From the Renal Division and Department of Medicine, Brigham and Women’s Hospital, of Kidney Diseases, Harvard Medical School, Boston, Massachusetts 02115 and the Harvard
Previous studies in this laboratory have shown the presence of an endothelin binding protein (ET-BP) in crude membranes prepared from kidney glomeruli and papillae which binds the endothelins with high affinity and selectivity
Despite the selectivity of ET-BP for the endothelins, the binding protein was not enriched in plasma membrane fractions prepared from the crude glomerular and papillary membranes and displayed a pH optimum for ET binding in the acidic range [44]
Summary
Two distinct cell surface endothelin receptors were identified, namely a 73-kDa protein referred to as ET-. An endothelin binding site with high affinity for endothelin-3 was identified on rat PC12 pheochromocytoma cells, the apparent molecular mass of this receptor could not be verified by cross-linking studies. High affinity binding sites for lZ51ET-l have been described in vascular smooth muscle cells [29, 32,33,34,35,36], mesangial cells [37, 38], cardiac myocytes [39], adrenal zona glomerulosa cells (2O), and neural glial cells [40]. A number of cell lines derived from the rat were used: namely, A10 vascular smooth muscle cells, C6 glial cells, glomerular mesangial cells, and PC12 pheochromocytoma cells All of these cells have been shown by others either to respond to endothelin or to express endothelin binding sites. More than one cell line was chosen in order to assess the possibility of endothelin receptor heterogeneity among the different cell lines
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