DISSOCIATION CHARACTERISTICS OF ENDOTHELIN RECEPTOR AGONISTS AND ANTAGONISTS IN CLONED HUMAN TYPE-B ENDOTHELIN RECEPTOR

The human type-B endothelin receptor (h-ETB) was cloned from human lun g poly A(+)RNA and stably expressed in CHO cells. Endothelin (ET) rece ptor binding and stimulation of PI hydrolysis demonstrated that the cl oned h-ETB receptor is functional and linked to intracellular signal t ransduction pathways in CHO cells. The molecular mass of the h-ETB rec eptor was determined to be 65 KDa, and B-max and K-d were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligan ds revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, w hereas the natural agonists (ET-1 and ET-3) were not. When competing w ith ET-1 in the h-ETB receptor binding, the ICS, increased from 1.2 nM to 8.2 nM for IRL1620, 0.058 mu M to 1.9 mu M for PD142893, and 0.76 mu M to 12.7 mu M for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differenc es in the dissociation characteristics between the artificial ligands and the natural ligands.