Two endothelin receptor antagonists (ERAs), bosentan and ambrisentan, are 
currently approved for the treatment of pulmonary arterial hypertension (PAH), a 
devastating disease involving an activated endothelin system and aberrant 
contraction and proliferation of pulmonary arterial smooth muscle cells (PASMC). 
The novel ERA macitentan has recently concluded testing in a Phase III 
morbidity/mortality clinical trial in PAH patients. Since the association and 
dissociation rates of G protein-coupled receptor antagonists can influence their 
pharmacological activity in vivo, we used human PASMC to characterize inhibitory 
potency and receptor inhibition kinetics of macitentan, ambrisentan and bosentan 
using calcium release and inositol-1-phosphate (IP(1)) assays. In calcium 
release assays macitentan, ambrisentan and bosentan were highly potent ERAs with 
K(b) values of 0.14 nM, 0.12 nM and 1.1 nM, respectively. Macitentan, but not 
ambrisentan and bosentan, displayed slow apparent receptor association kinetics 
as evidenced by increased antagonistic potency upon prolongation of antagonist 
pre-incubation times. In compound washout experiments, macitentan displayed a 
significantly lower receptor dissociation rate and longer receptor occupancy 
half-life (ROt(1/2)) compared to bosentan and ambrisentan (ROt(1/2):17 minutes 
versus 70 seconds and 40 seconds, respectively). Because of its lower 
dissociation rate macitentan behaved as an insurmountable antagonist in calcium 
release and IP(1) assays, and unlike bosentan and ambrisentan it blocked 
endothelin receptor activation across a wide range of endothelin-1 (ET-1) 
concentrations. However, prolongation of the ET-1 stimulation time beyond 
ROt(1/2) rendered macitentan a surmountable antagonist, revealing its 
competitive binding mode. Bosentan and ambrisentan behaved as surmountable 
antagonists irrespective of the assay duration and they lacked inhibitory 
activity at high ET-1 concentrations. Thus, macitentan is a competitive ERA with 
significantly slower receptor dissociation kinetics than the currently approved 
ERAs. Slow dissociation caused insurmountable antagonism in functional 
PASMC-based assays and this could contribute to an enhanced pharmacological 
activity of macitentan in ET-1-dependent pathologies.