Biochemical and Pharmacological Profile of a Potent and Selective

Home , Endothelin receptor

Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4892-4896, May 1994 Medical Sciences Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788 (receptor subtype) KIYOFUMI ISHIKAWA*, MASAKI IHARA, KAZUHITO NOGUCHI, TOSHIAKI MASE, NOBUYUKI MINO, ToSHIHIKO SAEKI, TAKAHIRO FUKURODA, TAKEHIRO FUKAMI, SATOSHI OZAKI, TOSHIO NAGASE, MASARU NISHIKIBE, AND MITSUO YANO New Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-33, Japan Communicated by Edward M. Scolnick, January 28, 1994

ABSTRACT We describe the characteristics of a potent mediated by ETB receptors (20-22). A recent in vivo study, and selective endothelin (ET) B-receptor antagonist, BQ-788 using an ETA antagonist, BQ-123 [cyclo(-D-Trp-D-Asp-Pro- [N-cis-2,6-dimethylpiperidinocarbonyl-L--methylleucyl-D-1- D-Val-Leu-] (23, 24), and an ETB agonist, [Ala'.3,11315]ET-1 methoxycarbonyltryptophanyl-D-norleucinel. In vitro, this (25), has demonstrated that bronchoconstrictor response as compound potently and competitively Inhibits 12SI-labeled en- well as transient pressor response to ET-1 or ET-3 in guinea dothelin 1 (ET-1) binding to ETB receptors on human Girardi pigs is mediated by ETB receptors, while sustained pressor heart cells (IC",, 1.2 nM) but only poorly inhibits the binding response to ET-1 is mediated by ETA receptors (26). As to ETA receptors on human neuroblastoma cell line SK-N-MC mentioned above, several subtype-selective ET ligands are cells (IC50, 1300 nM). In isolated rabbit pulmonary arteries, now available and undoubtedly useful in elucidating the BQ-788 shows no agonist activity up to 10 pM and competi- physiological and pathological roles of the ETs and their tively antagonizes the vasoconstriction induced by an ETB- receptor subtypes. Of these selective ligands, only one, IRL selective agonist, BQ-3020 (pA2, 8.4). In rat, an ETA-selective 1038, has been reported as an ETB antagonist (21). a onist, BQ-123 (1 mg/kg, i.v.), does not affect trnsient We previously reported on a series of potent tripeptide depressor response to ET-1 (0.3 nmol/kg, i.v.) but potently ETA antagonists, which were identified through systematic inhibits following s d pressor response; vice versa, BQ- and extensive modification of a new lead tripeptide, N-iso- 788 (1 mg/kg, i.v.) abolishes the depressor response, resulting varelyl-L-leucyl-D-tryptophanyl-(3-alanine, designed on the in a rapid onset of apparently enhanced pressor response. basis of a structure-activity relationship on cyclic pentapep- Thus, being a potent and selective ETB receptor antgonit, tide ETA antagonists (27). In the course of these extensive BQ-788 may be considered as a powerful tool for investigting modifications, it was also found that certain structural alter- the role of ET in physiological and pathological processes. ations intensified ETB affinity of some of these analogues. Further modification has resulted in the development of a potent and selective ETB antagonist, BQ-788 [N-cis-2,6- Endothelin 1 (ET-1) was first isolated from the culture dimethylpiperidinocarbonyl-L- -methylleucyl-D-1-methoxy- supernatant of porcine aortic endothelial cells (1). The pep- carbonyltryptophanyl-D-norleucine] (Fig. 1). We herein de- tide is some 10-fold more potent than angiotensin II as a scribe in vitro and in vivo the biological profile of the vasoconstrictor and has extremely long-lasting pressor ef- compound. fects (2). Subsequent studies, including human genomic analysis, identified two additional structurally and functionally related isopeptides named ET-2 and ET-3 (3). MATERIALS AND METHODS ET receptors are widely distributed not only in vascular Materials. Compounds 1-5 (Table 1) were synthesized as but also in nonvascular tissues (4, 5) and are classified into a free acid except for compound 5 (BQ-788) using conven- two subtypes termed ETA (6-8) and ETB (9-13) according to tional liquid-phase peptide synthesis in our chemistry labo- their distinctions in affinity for the ET isopeptides-i.e., the ratory. BQ-788 was prepared as a sodium salt by treatment of ETA receptor has high affinity for ET-1 and ET-2 and the corresponding free acid with NaHCO3. All of these relatively low affinity for ET-3, while the ETB receptor has compounds were characterized by consistent 1H NMR and high affinity equally for all ET isopeptides. high-resolution fast-atom-bombardment mass spectra. BQ- ETA receptors are predominantly found in peripheral tis- 123 as a sodium salt and an ETB-selective agonist, BQ-3020, sues, especially in vascular smooth muscle tissues to mediate were also prepared in house according to the previously vasoconstriction, though they are also present in certain described methods (refs. 23 and 28, respectively). ET-1 and regions ofthe brain (6, 7). On the other hand, ETB receptors ET-3 were purchased from Peptide Institute (Osaka). '25I- have been thought to be exclusively localized to the endo- ET-1 (2000 Ci/mmol; 1 Ci = 37 GBq) was purchased from thelium and nonvascular tissues such as the liver, kidney, and Amersham. brain (14). Endothelial ETB receptors are functionally linked Membranes from porcine cerebellum were prepared as to vasodilation possibly through the release of endothelium- described (16). pCASM cells were obtained by the previously derived relaxing factor (15). However, it has now been described explant method with minor modifications (29). confirmed that ETB receptors are also located in certain hGH cells and human neuroblastoma cell line SK-N-MC cells vascular smooth muscle tissues, mediating vasoconstriction were purchased from Dainippon Seiyaku (Osaka). pCASM (16-19). Thus, the function ofETB receptors is yet to be fully cells and SK-N-MC cells were cultured in Dulbecco's mod- elucidated. In certain nonvascular tissues such as guinea pig bronchus and trachea, ET-1-induced constriction is also Abbreviations: ET, endothelin; 1251-ET-1, 1251-labeled ET-1; pCASM, porcine coronary artery smooth muscle; hGH, human The publication costs of this article were defrayed in part by page charge Girardi heart; FCS, fetal calf serum; BSA, bovine serum albumin; payment. This article must therefore be hereby marked "advertisement" [Ca2W+, intracellular free calcium ion concentration. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4892 Downloaded by guest on September 28, 2021 Medical Sciences: Ishikawa et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4893

at 370C for 30 min. The cell suspensions were diluted with a 10-fold volume of DMEM/Hepes/BSA and again incubated at 3TC for 20 min. The fura-2-loaded cells were centrifuged and resuspended in Krebs-Henseleit Hepes buffer containing 0.1% BSA (pH 7.4) in a cuvette and the resultant suspension was continuously stirred at 370C throughout the following measurement process. Each test compound or vehicle (final 1% dimethyl sulfoxide) was given 5 min before the addition of 100 nM ET-1, fluorescence intensity at emission wave- length of 510 nm and excitation wavelengths of 340 and 380 FIG. 1. Chemical structure of BQ-788. nm was monitored with a JASCO CAF-100 spectrofluorom- eter (Tokyo), and [Ca2]1i values were calculated according to ified Eagle medium (DMEM) containing 10% fetal calf serum the reported method (30). (FCS), 100 units of penicillin per ml, and 100 ug of strepto- Vasoconstriction Studies. Vasoconstriction studies were mycin per ml at 37°C under an atmosphere of 95% air/5% performed as described (17). Briefly, porcine coronary and C02, while hGH cells were grown in minimum essential rabbit pulmonary arteries (each 1.0-2.0 mm in diameter) medium with Earle's salt (MEM) containing 10%6i FCS and the were isolated. The arteries were cleaned and cut into spiral above-mentioned antibiotics under the same conditions of strips. Each tissue was denuded of endothelium and then temperature and atmosphere as above. suspended with an initial tension of 1 g in an organ bath Bining Assay. Porcine cerebellar membranes were incu- containing Krebs-Henseleit solution bubbled with 95% bated at 250C with 125I-ET-1 (12 pM) in the presence of each 02/5% CO2 at 37C. ET-1 or an ETB-selective agonist, test compound or vehicle (1% dimethyl sulfoxide) in 50 mM BQ-3020, was cumulatively added to the strips. BQ-788 or Tris-HCI (pH 7.4) containing 0.1 mM phenylmethanesulfonyl vehicle (0.1% dimethyl sulfoxide) was added 10 min before fluoride, 1 pM pepstatin, 2 pM leupeptin, 1 mM 1,10- the addition of ET-1 or BQ-3020. Constrictions were ex- phenanthroline, 1 mM EDTA, 10 AM CaCk2, 10 PM MgCl2, pressed as percentages where the response to 50 mM KCl and 0.1% bovine serum albumin (BSA) in a total volume of was 100%. 0.4 ml. After 4 hr of incubation, cold 5 mM Hepes/Tris (pH Measurement of Pulmonary Ination Pressure in Guinea 7.4) containing 0.3% BSA (buffer A) was added to the Pis. Bronchoconstrictor response was measured as de- mixture. Free and bound 125I-ET-1 were separated by filtra- scribed (26). Briefly, male Hartley guinea pigs (380-400 g) tion using Whatman GF/C glass fiber filters. After the were anesthetized with urethane (750 mg/kg, i.p.) and a-chlo- filtration, the filters were washed with buffer A, and radio- ralose (37.5 mg/kg, i.p.). A catheter was inserted into the left activity was measured in a y counter (COBRA 5002, Pack- jugular vein for drug administration. The animals were con- ard). pCASM cells, SK-N-MC cells, or hGH cells that formed nected to a respirator (model 7025, Ugo Basile, Vorese) a confluent monolayer were incubated with 125I-ET-1 (12 pM through a tracheal cannula and ventilated at 10 ml of air per except 5-500 pM for Scatchard analysis) in Hanks' balanced kg and 60 strokes/min. Spontaneous breathing was sup- salt solution (pH 7.4) contaning 0.1% glucose and 0.3% BSA pressed with succinylcholine (5 mg/kg, s.c.). Pulmonary (HBSS/BSA) in a total volume of 0.2 ml at 3TC under an inflation pressure was recorded on a bronchospasm trans- atmosphere of 95% air/5% CO2 for 4 hr. Cells were washed ducer (model 7020, Ugo Basile, Vorese) connected to a side three times with ice-cold HBSS/BSA. Cell-bound radioac- arm of the tracheal cannula. Bronchoconstrictor responses tivity was then measured in a Packard COBRA 5002 y were expressed as percentages, where a maximum pulmo counter. Specific IzI-ET-1 binding was defined as the dif- nary inflation pressure reading obtained by clamping the ference between total binding and nonspecific binding in the trachea at the end ofeach experiment was 100%. One percent presence of 200 nM ET-1. IC50 values were determined by polyoxyethylene (60) hydrogenated castor oil (HCO 60; Nik- regression analysis of displacement curves. ko Chemicals, Tokyo) in saline was used as vehicle. BQ-788 Measurement of Incelar Free Calcium Ion Concentra- or vehicle was i.v. administered 5 min before an i.v. bolus tiOn ([C(2+j). [Ca2i]i concentrations were fluorometrically injection of ET-1 (0.5 nmol/kg). measured using a Ca2+-sensitive fluorescent dye, fura-2 (30). Measurement of Blood Presure in Rats. Male Sprague- pCASM cells or hGH cells were harvested using 0.25% Dawley rats were used at the age of8-9 weeks. Under sodium trypsin and 0.02% EDTA. The cells were washed once with pentobarbital anesthesia (50 mg/kg, i.p.), a tracheostomy DMEM containing 20 mM Hepes and 0.3% BSA (pH 7.4) was performed and right and left femoral veins were cannu- (DMEM/Hepes/BSA). Then the cells were suspended in lated for a supplementary infusion of sodium pentobarbital DMEM/Hepes/BSA and incubated with 2 pM (for hGH (20 mg/kg per hr) and for injection of drug (BQ-788 or cells) or 4 pM (for pCASM cells) fura-2 acetoxymethylester BQ-123) or vehicle (0.1% HCO 60 in saline for BQ-788, or Table 1. Inhibition of 125I-labeled ET-1 (125I-ET-1) binding to ET receptors by tripeptide ET antagonists Compound Structure ETA* ETBt 1 Boc-Leu-D-Trp-3-Ala-OH 210 + 24 >105 2 Boc-Leu-D-Trp-D-Nle*-OH 580 ± 47 19,000 ± 1300 3 Boc-Leu-D-Trp(1-CO2CH3)-D-Nle-OH 820 ± 19 140 ± 17 4 Dmpc§-Leu-D-Trp(l-CO2CH3)-D-Nle-OH 130 ± 7.9 2.0 ± 0.3 5 (BQ-788) Dmpc--MeLeul-D-Trp(1-CO2CH3)-D-Nle-OH 280 ± 24 1.2 ± 0.2 *IC5o in nM (mean + SEM, n = 3) for inhibition of 125I-ET-1 binding to ETA receptors on porcine coronary artery smooth muscle (pCASM) cells. tIC5o in nM (mean + SEM, n = 3) for inhibition of125I-ET-1 binding to ETB receptors on human Girardi heart (hGH) cells. *o-Norleucyl. §cis-2,6-Dimethylpiperidinocarbonyl. ly-Methylleucyl. Downloaded by guest on September 28, 2021 4894 Medical Sciences: Ishikawa et al. Proc. Nad. Acad Sci. USA 91 (1994)

saline for BQ-123), respectively. Mean arterial blood pres- those on BQ-123; the compound selectively inhibited ET sure was monitored via a catheter inserted in the left femoral binding to ETA receptors (Table 2). The specificity ofBQ-788 artery with a pressure transducer (DX-360, Nihon Kohden, for the ET receptor relative to other peptide hormone recep- Tokyo) connected to a blood pressure amplifier (AP-641G, tors was assessed by using three additional radioligand bind- Nihon Kohden) and recorded on a polygraph (RM-6100, ing assays; as the result, it was found that BQ-788 (10 AM) did Nihon Kohden). Throughout the experiment, rectal temper- not significantly inhibit nor facilitate the specific binding of ature of the rats was maintained at 37 ± 0.50C by a homeo- 125I-labeled angiotensin II to bovine cerebellar membranes, thermic blanket with a rectal probe (KN-474, Natsume, that of 125I-labeled calcitonin gene-related peptide to rat Tokyo). After a 60-min postsurgical equilibration period, hepatic membranes, or that of '25I-labeled peptide YY to BQ-788 or vehicle was injected as an i.v. bolus in a volume porcine hippocampal membranes, indicating high specificity of 1 ml/kg, 5 min prior to an i.v. bolus injection of ET-1 (0.3 of the compound for the ET receptor (data not shown). nmol/kg). The response ofblood pressure was observed for To determine whether BQ-788 interacts competitively or 60 min after the injection of ET-1. In this experiment, ET-1 noncompetitively with ETB receptors, 125I-ET-1 binding to was injected into each animal only once to avoid a possible hGH cells in the presence or absence of 1.0 nM of the tachyphylaxic effect of ET-1. compound was analyzed according to Scatchard (33). The data are not shown here, but the linearity of the control Scatchard plot indicates that the cells have a single popula- RESULTS AND DISCUSSION tion of specific and saturable, high-affinity binding sites for In the course of a structure-activity relationship study aimed 12I-ET-1 with a maximum number ofreceptors (B..) of 12.3 toward the identification of potent and selective tripeptide ± 1.2 fmol per 106 cells (mean ± SEM, n = 3) and an apparent ETA antagonists, we observed that some structural alter- dissociation constant (Kd) of 32.2 ± 7.6 pM. BQ-788 reduced ations intensified an ETB rather than ETA affinity of some of the slope but not the x intercept of the Scatchard plot, our analogues. The replacement of the C-terminal f-alanyl indicating a change in Kd of125I-ET-1 for the receptor (to 56.3 residue in compound 1 with a D-norleucyl residue (compound ± 7.9 pM) without a significant change in B.2. These data 2) considerably increased ETB affinity but, at the same time, suggest that BQ-788 interacts competitively with ETB recep- decreased ETA affinity (Table 1). The introduction of a tors on hGH cells. methoxycarbonyl group onto the indole nitrogen of the Functional in vitro studies using hGH and pCASM cells D-tryptophanyl residue in compound 2 resulted in an ana- were performed to further characterize the nature of the logue with a markedly increased ETB affinity (compound 3). interaction of BQ-788 with ETB receptors as well as ETA Subsequent modification of compound 3 revealed that the receptors. Neither BQ-788 nor BQ-123 alone (each 100 pLM) replacement of the N-terminal tert-butoxycarbonyl (Boc) increased [Ca2+]i in either cells, indicating a lack of any group with a cis-2,6-dimethylpiperidinocarbonyl group (com- agonist activity (data not shown). However, BQ-788 inhibited pound 4) further improved ETB affinity. The replacement of an increase in [Ca2+i induced by ET-1 (100 nM) in hGH cells, the leucyl residue in compound 4 with a 'y-methylleucyl as a function of concentration, with an IC(o value of 0.54 ± residue finally yielded compound 5 (BQ-788), a potent and 0.02 nM (mean ± SEM, n = 3) (Fig. 2). The inhibition by selective inhibitor of ET binding to ETB receptors (IC50, 1.2 BQ-788 was far more potent than that by BQ-123 (I(C5, >1(X 0.2 nM; mean ± SEM, n = 3). LM). Furthermore, BQ-788 (100 pMA) did not significanty It is known that pCASM cells (16) and SK-N-MC cells (31) affect an increase in [(aW+]i induced by other agonists such express ETA receptors, while porcine cerebellar membranes as neuropeptide Y, bradykinin, argunine vasopressin, throm- (16) and hGH cells (32) exclusively possess ETB receptors. bin, histamine, or acetylcholine, again indicating high spec- Indeed, as shown in Table 2, 'l2I-ET-1 binding to pCASM ificity of the compound for the ET receptor even on a cell cells as well as SK-N-MC cells was far more potently function level (data not shown). On the other hand, in inhibited by ET-1 than by ET-3, clearly indicating that pCASM cells, BQ-788 inhibited an ET-1-induced increase in specific ET binding sites on these two types ofcells are ofthe [C(a2]i almost three orders of magnitude less potently (1C5o, ETA subtype. On the other hand, 125I-ET-1 binding to porcine 2500 ± 290 nM; mean ± SEM, n = 3) than BQ-123 (IC5o, 2.9 cerebellar membranes or hGH cells was almost equipotently ± 0.6 nM; mean ± SEM, n = 3) (Fig. 2). On a cell function inhibited byboth ET-1 and ET 3, indicating that ET receptors level, BQ-788 was thus identified as a potent, specific, and in these tissues are ofthe ETB subtype. BQ-788 together with selective ETB receptor antagonist with no agonist activity. the representative ETA antagonist BQ-123 (24) was further We next investigated the antagonist properties of BQ-788 characterized using the above-mentioned assay systems. As in vascular tissues. In isolated porcine coronary arteries, the result, it was confirmed that BQ-788 potently inhibited BQ-123 concentration-dependently displaced the concentra- ET binding to ETB receptors both in porcine cerebellar tion-contractile response curve to ET-1 rightward (pA2, 7.4) membranes and on hGH cells, with IC50 values of0.9 nM and without affecting the maximal response or basal tension, as 1.2 nM, respectively, but that the compound only weakly previously reported (34). In contrast to the result, BQ-788, inhibited the binding to ETA receptors on pCASM and which also showed no effect on basal tension, did not SK-N-MC cells, with ICo values of 280 nM and 1300 nM, significantly affect the concentration-response curve to ET-1 respectively. These data on BQ-788 strikingly contrast with in this tissue (pD) for ET-1 in the absence and presence of 1.0 Table 2. IC5o values in nM on specific binding of 1251-ET-1 in various tissues ETA receptor ETB receptor Compound pCASM cells SK-N-MC cells* pCMt hGH cells BQ-788 280 ± 24 1300 ± 200 0.9 + 0.1 1.2 ± 0.2 BQ-123 8.9 ± 1.4 24 ± 3 18,000 ± 2000 9700 ± 2100 ET-1 0.12 ± 0.03 0.42 ± 0.16 0.11 ± 0.02 0.02 ± 0.002 ET-3 71 ± 13 >100 0.09 ± 0.002 0.03 ± 0.005 Values are presented as mean ± SEM (n - 3). *Human neuroblastoma cell line SK-N-MC cells. tPorcine cerebellar membranes. Downloaded by guest on September 28, 2021 Medical Sciences: Ishikawa, et al. Proc. Natl. Acad. Sci. USA 91 (1994) 4895

O 120 1001 0

I -Ir- -I 80 r. ._ 0 8 80 0 '0 0 + Ne'0 60 0. _ .6 0 40 40 gs 20 a g 20 .0 0 4 -8 -6 -4 log[Drug, M] 0 0.005 0.05 0.5 BQ-788 (mg/kg) FIG. 2. Effect of BQ-788 (circles) or BQ-123 (squares) on ET-1- induced increase in [Ca2+]i in pCASM cells (open symbols) or hGH FIG. 4. of on cells (closed symbols). Fura-2-loaded cells were pretreated with Effect BQ-788 ET-1-induced bronchoconstriction in guinea pigs. One percent HCO 60/saline was used as vehicle. various concentrations of BQ-788 or BQ-123 and then challenged BQ-788 or vehicle was i.v. administered 5 an i.v. with 10-7 M ET-1. Each point with vertical bar represents the mean min before bolus injection ofET-1. Bronchoconstrictor responses are expressed as percentages ± SEM (n = 3). where a maximum pulmonary inflation pressure reading obtained by clamping the trachea at the end of each experiment is 100%. Each HM BQ-788, 10.13 ± 0.13 and 10.05 ± 0.10, respectively; column with error bar represents the mean + SEM of five animals. mean ± SEM, n = 4). On the other hand, in isolated rabbit pulmonary arteries rich in ETB receptors, BQ-788 potently, to utilize this compound in vivo as a tool for investigating the and as a function ofconcentration, antagonized the constric- roles of ET receptor subtypes. tions produced by an ETB-selective agonist, BQ-3020 (28) We previously reported that, in guinea pigs, transient [N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His- bronchoconstrictor response to ET-1 (0.5 nmol/kg, i.v.) was Leu-Asp-Ile-Ile-Trp], with a pA2 value of 8.4 (Fig. 3), and never affected by a 1 mg/kg dose of the ETA antagonist BQ-788 alone (10 pM) again showed no effect on basal BQ-123 i.v. given 5 min prior to ET-1 injection, though the tension ofthe tissue. The antagonism was characterized by a dose was enough to significantly suppress ET-1-induced parallel and rightward shift of the BQ-3020 concentration- sustained.pressor response (26). In the same model, BQ-788 response curves without significant alteration in the maximal potently (ED50, 0.11 mg/kg, i.v.) suppressed the maximal bronchoconstrictor response in a dose-dependent manner contractile response; this result is consistent with competi- (Fig. and again alone about tive inhibition of ET binding to ETB receptors by BQ-788. In 4), BQ-788 (2 mg/kg, i.v.) brought no significant change in pulmonary inflation pressure (data this tissue, BQ-788 (10 I.M) exhibited no significant effects on not shown). These results demonstrate that ETB receptors concentration-response curves to norepinephrine, serotonin, are responsible for in vivo ET-1-elicited bronchoconstriction not histamine, and KC1 (data shown). in guinea pigs. It has thus been confirmed that BQ-788 is a potent and On the other hand, a bolus i.v. injection of ET-1 (0.3 highly specific ET receptor antagonist with no agonist activ- nmol/kg) to rats produced a rapid and transient decrease ity in vitro and that the compound is selective to one of the followed by a sustained increase in blood pressure, as shown receptor subtypes-namely, ETB. This finding prompted us in Fig. 5. BQ-123 (1 mg/kg) i.v. administered 5 min before

150 60

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0 -60 -11 -10 -9 -8 -7 -6 -5 -5 0 10 20 30 40 50 60 Time after ET-1 administration (min) log[BQ-3020, M] FIG. 5. Effect of BQ-788 or BQ-123 on ET-1-induced changes in FIG. 3. Concentration-contractile response curve to BQ-3020 in mean arterial blood pressure (MBP) in anesthetized rats. One-tenth the presence of 0.1% dimethyl sulfoxide (vehicle, o) or 0.1 ELM (0) percent HCO 60/saline and saline were used as vehicles for BQ-788 or 1 ,&M (m) BQ-788 in the isolated rabbit pulmonary artery. Re- and BQ-123, respectively. Vehicle (o, n = 4 x 2), BQ-788 (1 mg/kg; sponses are expressed as percentages where the response to 50 mM A, n = 3), or BQ-123 (1 mg/kg; e, n = 3) was i.v. administered 5 min KCI is 100%. Each point with vertical bar represents the mean before an i.v. bolus injection of ET-1. Each point with vertical bar SEM (n = 3). represents the mean + SEM. Downloaded by guest on September 28, 2021 4896 Medical Sciences: Ishikawa et al. Proc. Nad. Acad Sci. USA 91 (1994) ET-1 injection potently inhibited the pressor response but did 12. Saito, Y., Mizuno, T., Itakura, M., Suzuki, Y., Itoh, T., not affect the transient depressor response. In contrast, Hagiwara, H. & Hirose, S. (1991) J. Biol. Chem. 266, 23433- BQ-788 (1 mg/kg, i.v.) completely abolished the depressor 23437. response, resulting in a rapid onset of apparently enhanced 13. Elshourbagy, N. A., Lee, J. A., Korman, D. R., Nuthalaganti, pressor response. It had also been confirmed as a premise P., Sylvester, D. R., Dilella, A. G., Sutiphong, J. A. & Kumar, C. S. (1992) Mol. Pharmacol. 41, 465-473. that neither BQ-788 (3 mg/kg, i.v.) nor BQ-123 (10 mg/kg, 14. Gomezsanchez, C. E., Cozza, E. N., Foecking, M. F., Chiou, i.v.) alone produced any substantial change in blood pressure S. & Ferris, M. W. (1990) Hypertension 15, 744-747. in rats (data not shown). These results indicate that the 15. Takayanagi, R., Kitazumi, K., Takasaki, C., Ohnaka, K., transient depressor response to ET-1 in rats is mediated by Aimoto, S., Tasaka, K., Ohashi, M. & Nawata, H. (1991) FEBS ETB receptors but that the sustained pressor response is Lett. 282, 103-106. mediated by ETA receptors. 16. Ihara, M., Saeki, T., Funabashi, K., Nakamichi, K., Yano, M., Fukuroda, T., Miyaji, M., Nishikibe, M. & Ikemoto, F. (1991) J. Cardiovasc. Pharmacol. 17, S119-S121. CONCLUSION 17. Fukuroda, T., Nishikibe, M., Ohta, Y., Ihara, M., Yano, M., BQ-788 has been identified as a potent and selective inhibitor Ishikawa, K., Fukami, T. & Ikemoto, F. (1992) Life Sci. 50, of ET binding to ETB receptors through chemical modifica- PL107-PL112. tion. Further in vitro characterization has made it clear that 18. Panek, R. L., Major, T. C., Hingorani, G. P., Doherty, A. M., Taylor, D. G. & Rapundalo, S. T. (1992) Biochem. Biophys. the compound is a potent, competitive, ET-specific, and Res. Commun. 183, 566-571. 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