Coexpression studies with endothelin receptor subtypes indicate the existence of intracellular cross-talk between...

Human Girardi heart cells expressing endothelin ET(B) receptors (GH(B) cells) were transfected with human ET(A) cDNA, and coexpression of ET(A) and ET(B) in the ratio of 4:6 was demonstrated by Scatchard analysis. [125I]Endothelin (ET)-1 binding to ET(A)-transfected GH cells (GH(AB) cells) was displaced by an ET(A) antagonist, BQ-123, in a biphasic manner. An ET(B) agonist, BQ-3020, and an ET(B) antagonist, BQ-788, inhibited [125I]ET-1 binding to GH(AB) cells in a monophasic manner with low affinities (IC50 = 2,800 and 890 nM, respectively); IC50 values for ET(B) receptors seemed to be as weak as those for ET(A) receptors. However, BQ-3020 and BQ-788 had a high affinity for ET(B) receptors in a binding experiment using [125I]ET-1 in the presence of 1 microM BQ-123, where ET(A) receptors are masked (IC50 = 0.49 and 0.89 nM, respectively). The ET(B)-mediated increase in intracellular calcium concentrations in GH(AB) cells was not affected by 0.1 microM BQ-788 alone but was inhibited significantly by the same concentration of BQ-788 in combination with 10 microM BQ-123. ET-1 suppressed forskolin-stimulated accumulation of cAMP through the activation of ET(A) and ET(B) in GH(AB) cells; 1 microM BQ-123 or BQ-788 inhibited the suppression by only 20%, whereas a mixture of BQ-123 and BQ-788 (1 microM each) completely inhibited the cAMP decrease. These findings suggest that the stimulation of ET(A) receptors with ET-1 results in a lowering of the affinity of BQ-3020 and BQ-788 for ET(B) receptors in GH(AB) cells. We conclude that there is intracellular cross-talk between ET(A) and ET(B) receptors in GH(AB) cells.