We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.
The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.
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