Differential Trafficking and Desensitization of Human ET A and ET B Receptors Expressed in HEK 293 Cells

Abstract

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ET_A and ET_B receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ET_A and ET_B receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ET_A receptors, 83 ± 2% of these receptors colocalized with pan cadherin. In ET_B receptor–transfected cells, 54 ± 2% of the receptor colocalized with pan cadherin. When ET_A and ET_B receptors were cotransfected, 97 ± 1% of ET_B receptors colocalized with ET_A receptors and 84 ± 2% of ET_B receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ET_B receptor agonist) increased [Ca²⁺]_i in cells transfected with ET_A or ET_B receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ET_B receptors desensitized faster (t_1/2 = 21 ± 1 sec) than ET_A receptors (t_1/2 = 48 ± 1 sec). S6c-induced increases in [Ca²⁺]_i desensitized in cells expressing ET_B receptors only (t_1/2 = 17 ± 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ET_A receptors localize to the cell membrane, whereas ET_B receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ET_B receptors desensitize faster than ET_A receptors, but receptor coexpression eliminates desensitization. Finally, ET_A and ET_B receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.