Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase

Hirokazu Tsukahara, Howard Ende, Harold I. Magazine, Wadie F. Bahou, Michael S. Goligorsky

Research output: Contribution to journalArticle

231 Citations (Scopus)

Abstract

There is accumulating evidence that endothelial cells express a non-isopeptide-selective endothelin (ET) receptor, ETB, which may be responsible for ET-1-induced transient vasorelaxation. The purpose of the present study was to seek direct evidence for ETB 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 ETB receptor cDNA (CHO-ETB). Reverse polymerase chain reaction using HUVEC total RNA and ETB receptor-specific oligonucleotide primers firmly demonstrated the presence of an endogenous transcript of the appropriate molecular size. Next, a biotinylated ligand specifically recognizing the ETB receptor, IRL-1620, was synthesized, and immunocytochemical mapping of binding sites was performed in CHO-ETB cells. Specific binding of biotinylated IRL-1620 was evident in CHO-ETB 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-ETB 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-ETB/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 ETB receptor is functionally coupled to NOS and coordinates the generation of NO via a tyrosine kinase-dependent and a calcium/calmodulin-dependent pathway.

Original languageEnglish
Pages (from-to)21778-21785
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number34
Publication statusPublished - Aug 26 1994
Externally publishedYes

Fingerprint

Endothelial cells
Human Umbilical Vein Endothelial Cells
Nitric Oxide Synthase
Endothelial Cells
Endothelin-1
Nitric Oxide
CHO Cells
Calmodulin
Protein-Tyrosine Kinases
Transfection
Focal Adhesion Kinase 2
Calcium
Chemical activation
Endothelin Receptors
DNA Primers
Nitric Oxide Synthase Type III
Cell Surface Receptors
Cricetulus
Polymerase chain reaction
Vasodilation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular and functional characterization of the non-isopeptide-selective ETB receptor in endothelial cells. Receptor coupling to nitric oxide synthase. / Tsukahara, Hirokazu; Ende, Howard; Magazine, Harold I.; Bahou, Wadie F.; Goligorsky, Michael S.

In: Journal of Biological Chemistry, Vol. 269, No. 34, 26.08.1994, p. 21778-21785.

Research output: Contribution to journalArticle

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