There is accumulating evidence that endothelial cells express a non- isopeptide-selective endothelin (ET) receptor, ET(B), which may be responsible for ET-1-induced transient vasorelaxation. The purpose of the present study was to seek direct evidence for ET(B) receptor expression in human umbilical vein endothelial cells (HUVEC) and to characterize its functional role in HUVEC and in Chinese hamster ovary cells stably transfected with ET(B) receptor cDNA (CHO-ET(B)). Reverse polymerase chain reaction using HUVEC total RNA and ET(B) receptor-specific oligonucleotide primers firmly demonstrated the presence of an endogenous transcript of the appropriate molecular size. Next, a biotinylated ligand specifically recognizing the ET(B) receptor, IRL-1620, was synthesized, and immunocytochemical mapping of binding sites was performed in CHO-ET(B) cells. Specific binding of biotinylated IRL-1620 was evident in CHO-ET(B) cells, confirming appropriate cell surface receptor expression. Continuous nitric oxide (NO) monitoring with NO-selective electrode revealed a dose-dependent ET-1 stimulation of NO production by HUVEC. Stable transfection of CHO-ET(B) cells with endothelial nitric oxide synthase (NOS), but not mock- transfection, imparted responsiveness to ET-1 similar to that for HUVEC and was characterized by the immediate release of NO. Protein tyrosine kinase- dependent and calcium-calmodulin-dependent pathways were involved in ET-1- induced activation of the constitutive NOS in CHO-ET(B)/NOS cells, but coupling of the receptor to the enzyme in HUVEC appeared to be predominantly protein tyrosine kinase-dependent. Although sufficient, calcium/calmodulin system was not an obligatory prerequisite for the ET-1-induced activation of NOS in HUVEC. In conclusion, using two cell systems, we demonstrated that the ET(B) receptor is functionally coupled to NOS and coordinates the generation of NO via a tyrosine kinase-dependent and a calcium/calmodulin-dependent pathway.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology