During the past decade it became
During the past decade, it became increasingly clear that the affinity and efficacy of small agonists acting on the orthosteric binding site of a GPCR can be modulated by ligands that bind to a topographically distinct (allosteric) binding site on the same GPCR molecule 33, 39, 40, 41, 42. This allosteric modulation involves cooperativity between the affinity and efficacy of the distinct ligands acting on their distinct binding sites. In our opinion, a large agonist molecule such as ET1 that consists of two parts and that acts on two orthosteric binding domains of a 4E1RCat engages similar cooperativity between the orthosteric binding domains (Figure 2); in other words, endothelins might be considered as endogenous bitopic agonists  of at least ETA.
Long-lasting agonism Conditions of dynamic equilibrium govern most classical and modern theories of molecular pharmacology . These conditions do not easily apply to ETA in view of the slow dissociation of the agonist–receptor complex 4, 31, 45, 46. Hence, reported affinity measures (e.g. the ‘equilibrium’ dissociation constant Kd) must be regarded as approximations. When the dissociation rate constant of the agonist–receptor complex (k–1) is considerably less than its association rate constant (k+1), very low concentrations of agonist can act locally with high potency because Kd=k–1/k+1. This is an effective mechanism for a paracrine mediator. However, conditions of dynamic equilibrium become hard to establish in routine experimental settings such as ligand-binding and concentration–response studies. Not only is the agonist concentration a crucial factor, but the duration and history of agonist exposure also become determining factors that influence binding and effects 4, 16. Figure 3 illustrates this for ET1–ETA-induced contractile responses in isolated arteries. Over time, the concentration–response curve shifts to the left; the agonist seems to become more potent. Moreover, the response persists if the organ chamber content is flushed and free (unbound) agonist is no longer available. The half-life of the response is >20min (compared to < 1min for the classical vasoconstrictor agonist noradrenaline) . In theory, both the tightness of agonist–receptor complexes (R*-ET and R-ET in Figure 1) and the slow reversibility of receptor activation (slow conversion of R* to R in Figure 1) can contribute to such long-lasting agonism. However, using fluorescently labeled ET1 in the same functional assay system, we established that the contractile response and binding of the agonist to the cell membrane of smooth muscle cells in the intact arterial wall were maintained for >20min in the absence of the free ligand . Moreover, intravenous bolus administration of ET1 in vivo causes a long-lasting increase in blood pressure 2, 48 despite the short half-life of the peptide in circulation (T1/2 <1.5min) as a result of its scavenging by the lungs and its elimination by the kidneys 12, 13. These in vitro arterial contractile responses to ET1 and the in vivo vasopressor effects of ET1 can be prevented by prior application of a selective ETA antagonist (ERA). In summary, ET1 binds tightly to ETA and consequently causes long-lasting cardiovascular effects not only in vitro, but also in vivo.
ERAs Antagonists are still frequently seen as agents that can occupy an orthosteric binding site without altering the activation of the receptor , in other words, as neutral competitive antagonists. According to the International Union of Basic and Clinical Pharmacology (IUPHAR), however, ‘an antagonist is a drug that reduces the action of another drug, generally an agonist’ . An antagonist can thus be a chemical antagonist, a functional antagonist, a physiological antagonist, a neutral competitive antagonist, an inverse agonist and a negative allosteric modulator. In most ligand-binding and functional experiments, the latter three cannot be discriminated unless the allosteric inhibitory effect is only moderate .