Distortion of KBestimates of endothelin‐1 ETAand ETBreceptor antagonists in pulmonary arteries: Possible role of an endothelin‐1 clearance mechanism

Figures

• 

  Figure 1. Average single exposure concentration‐contraction curves to endothelin‐1 in rat (A) mesenteric artery (n = 15) and (B) pulmonary artery (n = 15), pretreated with L‐NAME 100 μmol L−1, in the absence Control, (0 μmol L−1) or presence of bosentan 1, 10 or 100 μmol L−1. Data are expressed as % KPSS maximum contraction (y axis). (C) Clark plot display for the relationship in the rat mesenteric artery between the endothelin‐1 pEC 50 values (y axis; −log M) and −log(B + KB) where B is concentration of bosentan (0, 1, or 10 μmol L−1) and KB is the global‐fitted dissociation constant. The error bars are ± 2 SEM of the difference between the nonlinear regression‐fitted pEC 50 values for endothelin‐1 and the pEC 50 values fitted for the individual artery for each concentration of bosentan (B). (D) The pEC 50 values for the endothelin‐1 curves in (A) and (B) are plotted on the y axis against the bosentan concentration (x axis) for each artery type. Vertical error bars in (A, B, and D) are ± 1 SEM (those not shown are contained within the symbol). Horizontal error bars (A‐B) represent the EC 50 ± 1 SEM. n, number of arteries isolated from separate animals. *P < .05, pEC 50 values compared…

• 

  Figure 2. Average single exposure concentration‐contraction curves to sarafotoxin S6c in rat pulmonary artery in the (A) absence or (B) presence of L‐NAME 100 μmol L−1 and of bosentan 0 (Control), 1, 10, or 100 μmol L−1. Data are expressed as % KPSS maximum contraction (y axis). Vertical error bars are ± 1 SEM (those not shown are contained within the symbol). Horizontal error bars represent the EC 50 ± 1 SEM. (C‐D) Clark plot displays for the corresponding figure panel above for the relationship between the sarafotoxin S6c pEC 50 values (y axis; −log M) and −log(B + KB) values (see legend for Figure 1C) in the absence (C) or presence (D) of L‐NAME. n, number of arteries isolated from separate animals. *P < .05, pEC 50 values compared with respective control (0 μmol L−1) pEC 50 values. Variations in n are due to violation of a predetermined criterion: arteries that contracted to KPSS with <1 mN force

• 

  Figure 3. Average single exposure concentration‐contraction curves to (A‐C) endothelin‐1 or (D) sarafotoxin S6c in rat pulmonary artery in the (A) absence of endothelium or (B‐D) presence of endothelium plus L‐NAME 100 μmol L−1. (A, B, and D) Curves were completed in the absence (Control, 0 μmol L−1) or presence of BQ788 0.3, 1, or 3 μmol L−1. (C) Curves were completed in the presence of BQ788 3 μmol L−1 plus bosentan 0, 1, or 10 μmol L−1. Data are expressed as % KPSS maximum contraction (y axis). (E‐F) The pEC 50 values from (A‐B) are plotted on the y axis against the BQ788 concentration (x axis). Vertical error bars (A‐F) are ± 1 SEM (those not shown are contained within the symbol). Horizontal error bars (A‐D) represent the EC 50 ± 1 SEM. (G‐H) Clark plot displays for the corresponding left figure panel for the relationship between the (G) endothelin‐1 or (H) sarafotoxin S6c pEC 50 values (y axis; −log M) and −log(B + KB) values (see legend for Figure 1C) in the presence of L‐NAME. *P < .05, pEC 50 values compared with respective control (0 μmol L−1) pEC 50 values. n, number of arteries isolated from separate animals. Variations in n are due to violation of a predetermined criterion:…

• 

  Figure 4. Average single exposure concentration‐contraction curves to endothelin‐1 in Swiss mouse isolated (A) main pulmonary, (B) mesenteric, and (C) tail arteries in the absence and presence of bosentan (0 (Control), 0.3, 1, 3, or 10 μmol L−1). Data are expressed as % KPSS maximum contraction (y axis). Vertical error bars are ± 1 SEM (those not shown are contained within the symbol). Horizontal error bars represent the EC 50 ± 1 SEM. (D‐F) Clark plot displays for the corresponding figure panel above for the relationship between the endothelin‐1 pEC 50 values (y axis; −log M) and −log(B + KB) values (see legend for Figure 1C). n, number of arteries isolated from separate animals. *P < .05, pEC 50 values compared with respective control (0 μmol L−1) pEC 50 values. Variations in n are due to violation of predetermined criteria: pulmonary arteries that contracted to KPSS with <1 mN force; mesenteric arteries that contracted to KPSS with <3 mN force; or tail arteries that contracted to KPSS with <20 mN force

• 

  Figure 5. Average single exposure concentration‐contraction curves to endothelin‐1 in rat (A) and (C) mesenteric artery (n = 13‐15) or (B) and (D) pulmonary artery (n = 12‐14) in the absence (Control, 0 μmol L−1) or presence of (A‐B) macitentan 0.3, 1, or 10 μmol L−1 or (C‐D) ambrisentan 1, 3, or 10 μmol L−1. Data are expressed as % KPSS maximum contraction (y axis). Vertical error bars are ± 1 SEM (those not shown are contained within the symbol). Horizontal error bars represent the EC 50 ± 1 SEM. *P < .05, pEC 50 values compared with respective control (0 μmol L−1) pEC 50 values. Variations in n are due to violation of predetermined criteria: mesenteric arteries that contracted to KPSS with <3 mN force or pulmonary arteries that contracted to KPSS with <1 mN force

• 

  Figure 6. Average single concentration‐contraction curves to (A) sarafotoxin S6c or (B) endothelin‐1 in rat isolated trachea with intact epithelium in the absence (Control, 0 μmol L−1) or presence of bosentan 3, 10, or 30 μmol L−1. (C‐D) Corresponding agonist curves in trachea with epithelium denuded. Data are expressed as % KPSS maximum contraction (y axis). Error bars in (A‐D) are ± 1 SEM (those not shown are contained within the symbol). (E‐F) Clark plot displays for the relationship in the rat trachea between the (E) sarafotoxin S6c or (F) endothelin‐1 pEC 50 values (y axis; −log M) and −log(B + KB) where B is concentration of bosentan (0, 3, 10, or 30 μmol L−1) and KB is the global‐fitted dissociation constant (see legend for Figure 1C). n, number of tracheal rings isolated from separate animals. Variations in n are due to violation of a predetermined criterion: tracheae that contracted to KPSS with <1 g force

• 

  Figure 7. (A) Hypothetical relationship between the pEC 50 values for endothelin‐1 or sarafotoxin S6c concentration‐contraction curves and the concentration of a theoretical dual ETA and ETB receptor antagonist with a pKB of 8.5 at both receptors. For simplicity, the control (0 antagonist) pEC 50 for sarafotoxin S6c () was set 1 log unit higher (10‐fold more potent) than for endothelin‐1 (). In the presence of ETB receptors and the clearance (C) mechanism for endothelin‐1, the maximum clearance was set at 10‐fold (1 log unit) so that the pEC 50 in the presence of no antagonist (0) rises 1 log unit (● or ▲). As the ETB antagonism starts to block the endothelin‐1 clearance, so the pEC 50 rises (●) but just as does the ETB and ETA antagonism so that the resultant shows the actual pEC 50 is not altered. (B) The Schild plot for endothelin‐1 (or sarafotoxin S6c) as the agonist and the dual ETA and ETB antagonist with pKB of 8.5 is shown. Separate theoretical lines are shown for ETA (; eg, rat aorta) and ETB (; eg, trachea). In the presence of ETB‐mediated clearance (C) that removes endothelin‐1, as in pulmonary artery, the Schild plot points (▲) move parallel 1 log unit to decrease the potency…

• 

  Figure 8. (A) Schild plot for a theoretical endothelin‐1 antagonist that is 30‐fold more selective at ETA vs ETB receptors (pKB: ETA 8.5 and ETB 7.0). Note that in the presence of ETB‐mediated clearance, the plasma concentration of the dual antagonist must rise to 10 μmol L−1 to give a 10‐fold antagonism at ETB constrictor receptors and 3000‐fold antagonism at ETA constrictor receptors. (B) Schild plot for a theoretical endothelin‐1 antagonist that is 10‐fold more selective at ETB vs ETA receptors (pKB: ETB 8.5 and ETA 7.5). In the presence of ETB‐mediated clearance, the plasma concentration of the dual antagonist must rise to 0.3 μmol L−1 to give a 10‐fold antagonism at both ETB and ETA receptors. (C) Schild plot for endothelin‐1 antagonist A‐182086 that is threefold more selective for ETA vs ETB receptors (pKB: ETA 8.5 and ETB 8.0; see Table 1). In the presence of ETB‐mediated clearance, the plasma concentration of the dual antagonist must rise to 1 μmol L−1 to give a 10‐fold antagonism at ETB constrictor receptors and 300‐fold antagonism at ETA constrictor receptors. (D) Schild plot for endothelin‐1 antagonist Ro 46‐8443 that is 25‐fold more selective at ETB vs ETA receptors (pKB: ETB…

• 

  Figure 9. Schematic diagram of the location and function of ETA and ETB receptors in 3 tissue assays. (A) ETA receptors located on vascular smooth muscle mediate contraction. ETA receptors are internalized and recycled slowly through endosomes.36 (B) ETB receptors located on smooth muscle cells mediate contraction and are rapidly removed by phosphorylation.36 (C) ETB receptors located on smooth muscle cells bind endothelin‐1 and clear endothelin‐1 from the environment through lysosomal metabolism. The remaining endothelin‐1 binds to ETA and ETB receptors on smooth muscle to mediate contraction before being recycled by endosomes or destroyed by phosphorylation, respectively. In (A) and (C), ETB receptors on the endothelium mediate release of NO that transiently relaxes smooth muscle. Examples of the species and tissues assumed to have these particular receptor profiles are given below each panel. ET‐1, endothelin‐1. NO, nitric oxide