Your SlideShare is downloading. ×
ENDOTHELIN RECEPTOR ANTAGONISTS EXHIBIT DIMINISHING POTENCY FOLLOWING INCUBATION WITH AGONIST
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

ENDOTHELIN RECEPTOR ANTAGONISTS EXHIBIT DIMINISHING POTENCY FOLLOWING INCUBATION WITH AGONIST

51
views

Published on

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible …

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [t25I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC5o values (0.08nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC5o values of 570 + 50 nM and 410 + 100 nM, respectively. Consistent with previous observations, bound [t25I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 laM inhibited 0.1 nM [125I]ET-1 binding by >80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to <10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding.


0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
51
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
1
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. Life Sciences, Vol. 54, No. 22, pp. 172%1734, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-32O5/94 $6.OO + .OO Pergamon 0024-3205-(94)E0025-M ENDOTHELIN RECEPTOR ANTAGONISTS EXHIBIT D I M I N I S H I N G POTENCY FOLLOWING INCUBATION WITH AGONIST Jinshyun R. Wu-Wong*, William J. Chiou, Kurt E. Naugles, Jr. and Terry J. Opgenorth Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064 (Received in final form March 16, 1994) Summary Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [t25I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC5o values (0.08 nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC5o values of 570 + 50 nM and 410 + 100 nM, respectively. Consistent with previous observations, bound [t25I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 laM inhibited 0.1 nM [125I]ET-1 binding by >80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to <10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding. Key Words: e n d o t h e l i n r e c e p t o r , e n d o t h e l i n r e c e p t o r antagonists, PD142893, Ro46-2005 Endothelin (ET), originally isolated from cultured porcine aortic endothelial cells, is a highly potent vasoconstricting peptide with 21-amino acid residues (1). Three distinct members of the ET family, namely, ET-1, ET-2 and ET-3, have been identified through cloning (2). The effects of ETs on mammalian organs and cells are initiated by their binding to high affinity Gprotein linked receptors. ET receptors are found in various tissues and cells, such as brain, lung, and mesangial cells (3-5). Two types of ET receptors, ETA and ETB, have been characterized, isolated (4, 6) and their cDNA cloned (7-9). ETA receptors are selective for ET-I and ET-2, while ETB receptors bind to ET-I, ET-2 and ET-3 with equal affinity. Several antagonists and agonists for ET receptors have been developed. Among them, BQ123 (10) and FR139317 (11) are selective antagonists for ETA receptors; Ro46-2005 and PD142893 *Corresponding Author
  • 2. 1728 ET Receptor Antagonist Binding Vol. 54, No. 22, 1994 antagonize both ETA and ETB receptors (12, 13), while IRL1620 (14) and BQ3020 (15) are selective agonists for ETB receptors. Previously we have reported that bound [125I]ET-1 is difficult to dissociate from membranes prepared from mouse 3T3 fibroblasts, rat cerebellum, and bovine atrium (16). This unique binding characteristic has also been observed in plasma membranes prepared from rat liver cells (17) and porcine lung (14). The development of ET receptor antagonists with clinical utility is being actively pursued. However, little is known about the dissociation characteristics of antagonists, or the effects of ET's unique, difficult-to-dissociate binding on the potency of antagonists. Because previously it has been reported that ET binding to ETa is even more difficult to dissociate than that to ETA (18), we have chosen to study ETa receptor in membranes prepared from porcine cerebellum in order to compare the dissociation characteristics of ET and antagonists. Our results indicate that antagonist binding is more reversible than ET binding and the antagonizing effects of Ro46-2005 and PD142893 diminish following incubation with ET-1. Materials and Methods Materials: [125I]ET-1 (2200 Ci/mmol) and [125I]ET-3 (2200 Ci/mmol) were obtained from Du Pont, NEN (Boston, MA). ET-1 and ET-3 were purchased from American Peptide Company, Sunnyvale, CA. Ro46-2005 (N-[6-(2-hydroxyethoxy)-5-(m-methoxyphenoxy)-4-pyrimidinyl]p-tert-butylbenzenesulphonamide) and PD142893 (Ac-D-diphenylalanine-Leu-Asp-Ile-Ile-Trp) were synthesized in house. Other reagents were of analytical grade. Preparation of membranes: Porcine cerebellum membranes were prepared as previously described (19). Briefly, cerebellum was homogenized in 25 volumes (w/v) of 10 mM Hepes (pH 7.4) containing 0.25 M sucrose and protease inhibitors (3 mM EDTA, 0.1 mM PMSF, and 5 lag/ml Pepstatin A) by 3-10 sec polytron at 13,500 rpm with 10 sec intervals. The mixture was centrifuged at 1000xg for 10 min. The supernatant was collected and centrifuged at 30,000xg for 30 min. The precipitate was resuspended in Buffer A (20 mM Tris, 100 mM NaC1, 10 mM MgC12, pH 7.4) containing the aforementioned protease inhibitors and centrifuged again. The final pellet was resuspended in Buffer A containing protease inhibitors and stored at -80°C until used. Protein content was determined by the Bio-Rad dye-binding protein assay. Radiolieand bindin~ to membranes: Binding assays were performed in 96-well microtiter plates pretreated with 0.1% BSA. Membranes were diluted -100 fold in Buffer B (20 mM Tris, 100 mM NaCI, 10 mM MgCI2, pH 7.4, with 0.2% BSA, 0.1 mM PMSF, 5 lag/ml Pepstatin A, 0.025% bacitracin, and 3 mM EDTA) to a final concentration of 0.2 mg/ml of protein. In time course studies, membranes (10 lag of protein) were incubated with 0.1 nM of [125I]ET in Buffer B (final volume: 0.2 ml) for different periods of time. Unlabeled test ligands were added either at time 0 or at indicated time. In competition studies, membranes were incubated with 0.1 nM of [125I]ET in Buffer B (final volume: 0.2 ml) in the presence of increasing concentrations of unlabeled test ligands for an indicated period of time at 25°C. After incubation, unbound ligands were separated from bound ligands by vacuum filtration using glass-fiber filter strips in PHD cell harvesters (Cambridge Technology, Inc., MA), followed by washing the filter strips with saline (1 ml) for three times. Nonspecific binding was determined in the presence of 1 laM ET. Bind-and-wash experiments; Membranes (200 lag) were incubated with or without test ligands at indicated concentrations in 1 ml of Wash Buffer (Buffer B without BSA) for 3 h at 25"C. After the incubation, 10 ml of Wash Buffer was added and the mixture was centrifuged at 30,000xg for 30 min. The pellet was resuspended into 25 ml of Wash Buffer and centrifuged again. The final pellet was resuspended into 0.5 ml of Wash Buffer and a small portion was used for the protein content determination. Protein loss during this wash procedure was approximately 30%. BSA was added to a final concentration of 0.2 % and then membranes (10
  • 3. Vol. 54, No. 22, 1994 ET Receptor Antagonist Binding 1729 lag per well) were assayed for [t25I]ET-1 binding. [t25I]ET-1 binding was performed as described above with a 3 h incubation period at 25°C. Results ET receptors in porcine cerebellum were first characterized by competition studies in which unlabeled ET-1 or -3 at various concentrations were used to compete against [t25I]ET-1 binding (Fig. 1A). Both ET-1 and ET-3 at 1 laM completely abolished specific [t25I]ET-1 binding. The IC5o value was 0.08 nM for ET-3 vs. 0.17 nM for ET-1. The fact that ET-1 and ET-3 exhibit nearly equal potency in inhibiting []25I]ET-1 binding is consistent with a previous observation that cerebellum expresses mainly ETa receptors (20). Fig. 1B shows that the IC50 values for non-selective antagonists Ro46-2005 and PD142893 were 570 +_ 50 nM and 410 + 100 nM, respectively. These results show that the potency of the ligands against ET binding is in the order of ET-1 >> PD 142893 > Ro46-2005. 120 120 100- 100- 80- (A) 80- i 00- 60- 40- 40 2000-1 --O--ET-3 5 10-13 ~L 10-11 10-9 Llgands, M 204 - ~ - , - P 0142893 - ~,,~ 0 ~ ~ 10-12 l ! 0-I1 0 F 10-11 ~ 10-6 Llgands, M 10-7 Fie. ~ (B) l T 10-4 1. Competition studies. Membranes (0.-01 mg) were incubated with (A) 0.1 nM [t25I]ET-1 or (B) []25I]ET-3 in the presence of increasing concentrations of unlabeled ligands for 3 h at 25*C. ET-1, O; ET-3, O; PD142893, A; Ro46-2005, 0 . Results are expressed as % of control (specific binding in the absence of unlabeled ligand). Nonspecific binding, determined in the presence of 1 laM of ET- 1 (A) or ET-3 (B), was subtracted from total binding to give specific binding. Each value represents the mean + S.D. of 3 determinations. The dissociation characteristic of bound ET in porcine cerebellum has not been reported previously. In Fig. 2A, [125I]ET-1 binding was time-dependent. Addition of 1 laM ET-1 at time zero blocked most of [125I]ET-1 binding (>90% after 24 h of incubation). However, when 1 I.tM ET- 1 was added at 2 h after the initiation of the incubation, it dissociated <20% of bound [t25I]ET-1 even after 22 h of incubation. These results suggest that bound [t25I]ET-1 is indeed difficult to dissociate in membranes prepared from porcine cerebellum, consistent with previous studies using membranes from mouse 3T3 fibroblasts, rat cerebellum, or bovine atrium (16). Although it has been shown that IRL1620 and BQ3020, two ETB selective agonists derived from ET-1, bind to ET receptors more reversibly in comparison to ET (14, 15), the dissociation characteristics of antagonists have not been studied. Lacking radiolabeled Ro462005 and PD142893, we designed and conducted a series of bind-and-wash experiments. As shown in Fig. 2B, specific [125I]ET-I binding to control membranes (washed with buffer in the absence of any ligand as described) was 176.4 + 2.5 frnol/mg. Ro46-2005 and PD142893 at 50 laM completely inhibited [125I]ET-1 binding, as expected. However, extensive washing of
  • 4. 1730 ET Receptor Antagonist Binding Vol. 54, No. 22, 1994 membranes pretreated with antagonists restored most of [I~I]ET-1 binding (72% for Ro462005 and 100% for PD142893). In contrast, membranes pre-incubated with 10 nM ET-1 followed by washing still exhibited >98% inhibition. These results show that bound Ro462005 and PD142893, but not bound ET-1, could be readily washed away, suggesting that the binding of Ro46-2005 and PD142893 is more reversible than that of ET-1. o) 20O 200 (A) o) ,~ 11;0- 1so $ ~100O m "- "~ 1 0 0 o m SO- "7 t-~ 121 I S 10 Time, ' 15 gO D 2 l0 2 Control Ro PD ET-1 h Dissociation studies. (A) ET-1: Membranes (0.01 mg) were incubated with 0.1 nM [125I]ET-1 for different periods of time at 25°C (O). At time 0 (ISl) or after 2 h of incubation (A), 1 laM ET-1 was added. The dotted line indicates the level of [125I]ET-1 bound at 2 h of incubation. (B) Bind-and-wash study: Membranes (0.2 mg) were incubated with or without ligands (50 I.tM Ro46-2005, or 50 I.tM PD142893, or 10 nM ET-1) for 3 h at 250C. Membranes were then washed and processed as described in "Materials and Methods" before assaying for [125I]ET-1 binding with or without test ligands for 3 h at 25*C. Solid bar: ligands were added in the first incubation period to membranes before washing. Hatched bar: ligands were added in the second incubation period with [125I]ET-1 to membranes after washing. Control membranes were also washed and processed without adding test ligand. Data shown are specific binding that have been corrected for nonspecific binding. Each value represents the mean + S.D. of 3 determinations. We further investigated if the difference in the dissociation characteristics between ET- 1 and antagonists has any effect on the potency of Ro46-2005 and PD142893. To best illustrate the ligand's potency on ET-1 binding, data were expressed as % inhibition by normalizing bound ET in the presence of test ligand to that in the absence of test ligand at each time point. In Fig. 3A, when 0.5 IIM of Ro46-2005 (5,000-fold greater than the concentration of [125I]ET-1) was added at time zero, 80 % of [t25I]ET-1 binding was inhibited after 15 min of incubation. However, after 3 h of incubation, only 50% of [125I]ET-1 binding was inhibited, and after 24 h the inhibitory effect of Ro46-2005 decreased to <20%. Similar results were obtained for PD142893 at 0.5 ~tM (Fig. 3A). When these antagonists were tested at 50 ~tM, the decrease in the antagonizing effect was still observed for PD142893 (97% at 3 h vs. 81% at 24 h). In contrast, unlabeled ET-1 at 0.3 nM inhibited 65% of [125I]ET-I binding after 15 min of incubation, and the effect increased, rather than decreased, with time, leveling off after 3 h (Fig. 3B). These results suggest that the potency of Ro46-2005 and PD142893, but not ET-1, is diminishing following the incubation with ETo 1. The above observation was further confirmed by the shift of IC5o values for Ro46-2005 and PD142893. Competition studies were performed as in Fig. IB except that three different
  • 5. Vol. 54, No. 22, 1994 ET Receptor Antagonist Binding 1731 incubation periods, 1 h, 6 h, and 24 h, were compared. Table I summarizes the IC5o values for Ro46-2005, PD142893 and ET-3 determined at the chosen incubation time. For both Ro462005 and PD142893, the IC5o values increased when the incubation time was extended. In contrast, the IC5o value for ET-3 did not show a significant change with time. To evaluate whether the decrease in the antagonists' potency following time resulted from the degradation of compound during the prolonged incubation period, compound integrity was assessed. Ro46-2005 at various concentrations was pre-incubated with membranes for either 3 h or 21 h at 25°C and then assayed against [t25I]ET-1 binding to determine the IC5o values. Fig. 4 shows that the IC5o values were identical (450 nM) for the two pre-incubation conditions, and were similar to that obtained without pre-incubation (570 nM from Fig. 1B). Similar results were obtained when PD142893 was tested (not shown). Therefore, the decrease in the potency of Ro46-2005 and PD142893 following time was not likely due to compound degradation. ¢: o m .,~ 1 2 0 0 .t.-, 1 2 0 rl r100- _=1oo- 80- 80- >, ¢1 IQ 110- ~ o 60" a. 40- ~. 40- 10 r" ¢1 Ot ..I 20- ~ 20¢1 I 0 I I 10 15 ,.1 I S 0 20 Time, (B) 0 t J I I S 0 2 10 15 20 h Time, Fi~. h 3. Potency determination. Membranes (0.01 mg) were incubated with 0.1 nM [t25I]ET-1 for different periods of time at 25°C. At each time point, [125I]ET-1 binding was determined + test ligand. Specific [125I]ET-1 binding with test ligands was normalized to control binding without test ligands of the corresponding time point to calculate % inhibition. Each value represents the mean + S.D. of 3 determinations. (A) Antagonists PD142893 at 50 laM (A) or 0.5 laM (A), Ro46-2005 at 50 l.tM (O) or 0.5 I.tM (0). (B) Agonist ET-1 at 100 nM (A) or 0.3 nM (0). The dotted line in (A) indicates the level of 100% inhibition. Table I IC50 values of antagonists at different incubation time. IC5o, n M a Incubation lime Ro46-2005 PD 142893 ET-3 lh 50 80 0.10 6h 230 340 0.07 24 h 1460 1700 a. IC5o values were determined as in Fig. 1B except that the incubation was allowed to continue for 1 h, 6 h, or 24 h at 25*C 0.06
  • 6. 1732 ET Receptor Antagonist Binding Vol. 54, No. 22, 1994 120 100: P o u Examination of compound integrity during incubation. Membranes (0.01 mg) were pre-incubated with increasing concentrations of Ro46-2005 for either 3 h ( 0 ) or 21 h (O) before assayed for [125I]ET-1 (0.1 nM) binding for 3 h. Results are expressed as % of control as in Fig. 1. ao60 40 2O 0 10 -10 1 0 -O 1 0 "11 Ro46-200S, 10 "7 1 0 -(; M Discussion Although radioligand binding studies have characterized ET receptors as ones that belong to the superfamily of heptahelical G-protein linked receptors (20, 21), ET receptors are unique in that ET binding appears to be of high affinity and bound ET is difficult to dissociate. For example, in membranes prepared from cultured Swiss 3T3 fibroblasts, addition of unlabeled ET-1 at time zero completely blocks [125I]ET-1 binding. However, once []25I]ET-1 is bound, adding 1 ~tM ET-1 plus 500 l.tM of guanylimidodiphosphate (GppNHp) dissociates bound [125I]ET-1 by <10% (16). Similar results were observed for membranes prepared from other tissues (14, 16-17). Although several antagonists have been developed for ET receptors, no studies have been done to investigate the dissociation characteristics of antagonists, and/or the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In this report, we demonstrate that, in membranes prepared from porcine cerebellum, the binding of Ro462005 and PD142893 is more reversible than ET, and the potency of antagonists decreases following incubation time even though the concentrations of the antagonists are 5000-fold higher than ET-1. The unique binding nature of ET may necessitate that special methods be used for the determination of binding parameters. Waggoner et al. (22) has suggested using a modification of a radioligand binding analysis program "Kinetic" (Biosoft, MO, U.S.A.) to calculate the kinetic data for ET binding. In their studies, they reported that the dissociation half-life (to, 05) for bound [12SI]ET-I was >30 h in membranes prepared from rat heart, rat lung, rat brain, and porcine vascular smooth muscle. By fitting the results from Fig. 2A into the method suggested by Waggoner et al., we observed a to, o5 in excess of 24 h for bound [125I]ET-I in porcine cerebellum membranes. It is important to note that the results obtained from the above kinetic analysis may be useful for comparison purposes only. Our results suggest that the diminishing in potency of Ro46-2005 and PD142893 is due to the difference in the dissociation characteristics between ET-1 and antagonists, but not due to compound degradation during the prolonged incubation period. It is clear that ET-1 binding to porcine cerebellum membranes is of extremely high affinity and that bound ET-1 is difficult to dissociate. In contrast, antagonists exhibit lower affinity, and bound antagonists can be readily washed away. These results suggest that antagonist binding to ET receptors does not form a stable receptor-ligand complex as does ET binding. This difference in receptor-ligand complex stability is likely to account for the observed time-related decrease in the inhibitory effect of antagonists on ET binding. It is important to note that [125I]ET-1 binding in the time course studies (Fig. 2A) starts to level off at around 4 h, and gradually decreases after 17 h of incubation. This observation was unexpected, inconsistent with the fact that bound ET is difficult to dissociate. Interestingly,
  • 7. Vol. 54, No. 22, 1994 ET Receptor Antagonist Binding 1733 binding studies on ET receptor in membranes from human pericardium smooth muscle cells showed that ET-1 binding does not reach a steady state after 24 h of incubation at 25°C (unpublished data). One possible explanation for the present observation is that proteins and receptors are being degraded in cerebellum membranes in spite of the presence of various protease inhibitors. We have observed in parallel experiments in the absence of protease inhibitors that the decrease in [125I]ET-1 binding occurred earlier (at 7 h), and was more severe than what was shown in Fig. 2A. Thus, the leveling off of [12SI]ET-1 binding is not the result of ET binding reaching a steady state but rather is the result of receptor degradation. Because antagonist binding and ET binding are compared under the same experimental conditions, and because very little antagonist or ET degradation occurs during the 24 h incubation period, receptor or ligand degradation is not likely the cause for the observed decrease in the potency of antagonists. Because live cells and tissues may process agonists and antagonists differently, one important question that is not addressed in this study but is currently being pursued by our laboratory is whether the potency of ET antagonists would be similarly curtailed when tested in functional assays. Our preliminary results suggest that in human vascular smooth muscle cells the functional effects of ET-1 are mediated mainly through the ETA receptor since ET-3 is not as effective as ET-I in stimulating arachidonic acid release, PI hydrolysis, or DNA synthesis. However, BQ123 and Ro46-2005 fail to completely inhibit the ET-1 induced DNA synthesis during a 24 h incubation period, although BQ123 effectively inhibits ET stimulated phosphatidylinositol hydrolysis and arachidonic acid release in a 30 - 60 min incubation period. Recently Zamora et al. (23) have also reported that BQI23 at 1 laM inhibits only 60% of ET-I induced DNA synthesis in human pulmonary artery smooth muscle cells. It is thus implicated that the potency of antagonists on the prolonged functional effects of ET will be similarly reduced. In conclusion, our results indicate that (1) bound ETn receptor antagonists can be readily washed away~ but bound agonists are difficult to dissociate, (2) antagonists, even at 5000-fold higher concentrations than ET- 1, exhibit reduced potency after prolonged incubation with ET-1, and (3) the decreased potency of Ro46-2005 and PD142893 is not the result of compound degradation during the prolonged incubation period. Whether the antagonizing effcct of Ro46-2005, PD 142893, or other ET receptor antagonists may diminish during chronic in vivo treatment of ET-induced pathological conditions awaits further studies. Acknowledements The authors would like to thank D47V chemists for making the antagonists and agonists used in this study. We are in debt to Drs. Chi-Ming Lee, and Chun Wel Lin for their valuable comments on this work. References 1. M. YANAGISAWA, H. KURIHARA, S. KIMURA, Y. TOMOBE, Y. KOBAYASHI, M. MITSUI, Y. YAZAKI, K. GOTO, and T. MASAKI, Nature (London) 332 411-415 (1988). 2. A. INOUE, M. YANAGISAWA, S. KIMURA, Y. KASUYA, T. MIYAUCHI, K. GOTO and T. MASAKI, Proc. Natl. Acad. Sci. USA 86 2863-2867 (1989). 3. I. SCHVARTZ, O. ITTOOP and E. HAZUM, Endocrinology 128 126-130 (1991). 4. M. KOZUKA, T. ITO, S. HIROSE, K. M. LODHI and H. HAGIWARA, J. Biol. Chem. 266 16892-16896 (1991). 5. E. BALDI and M. J. DUNN, J. Pharm. Exp. Ther. 256 581-586 (1991). 6. K. WADA, H. TABUCHI, R. OHBA, M. SATOH, Y. TACHIBANA, N. AKIYAMA, O. HIRAOKA, A. ASAKURA, C. MIYAMOTO and Y. FURUICHI, Biochem, Biophys. Res. Commun. 167 251-257 (1990).
  • 8. 1734 ET ReceptorAntagonist Binding Vol. 54, No. 22, 1994 7. H. ARAI, S. HORI, I. ARIMORI, H. OHKUBO and S. NAKANISHI, Namm (London) 348 730-732 (1990). 8. T. SAKURAI, M. YANAGISAWA, Y. TAKUWA, H. MIYAZAKI, S. KIMURA, K. GOTO and T. MASAKI, Nature (London) 348 732-735 (1990). 9. H. Y. LIN, E. H. KAJI, G. K. WINKEL, H. E. IVES and H. F. LODISH, Proc. Natl. Acad. Sci. USA 88 3185-3189 (1991). 10. M. IHARA, T. FUKURODA, T. SAEKI, M. NISHIKIBE, K. KOJIRI, H. SUDA and M. YANO, Biochem. Biophys. Rcs. Commun 178 132-137 (1991). 11. K. SOGABE, H. NIREI, M. SHOUBO, A. NOMOTO, S. AO, Y. NOTSU and T. ONO, J. Pharm. Exp. Ther. 254 1040-1046 (1993). 12. M. CLOZEL, V. BREU, K. BURRI, J.-M. CASSAL, W. FISCHLI, G. A. GRAY, G. HIRTH, B. M. LOFFLER, M. MULLER, W. NEIDHART and H. RAMUZ, Nature 365 759-761 (1993). 13. W. L. CODY, A. M. DOHERTY, J. X. HE, P. L. DEPUE, S. T. RAPUNDALO, G. A. HINGORANI, T. C. MAJOR, R. L. PANEK, D. T. DUDLEY, S. J. HALEEN, D. LADOUCEUR, K. E. HILL, M. A. FLYNN and E. E. REYNOLDS, J. Med. Chem. 35 3301-3303 (1992). 14. T. WATAKABE, Y. URADE, M. TAKAI, I. UMEMURA and T. OKADA, Biochem. Biophys. Res. Commun 185 867-873 (1992). 15. M. IHARA, T. SAEKI, T. FUKURODA, S. KIMURA, S. OZAKI, A. C. PATEL and M. YANO, Life Science 51 47-52 (1992). 16. J. R. WU-WONG, W. CHIOU and T. J. OPGENORTH, Mol. Pharmacol. 44 422-429 (1993). 17. B. HOCHER, C. RUBENS, J. HENSEN, P. GROSS and C. BAUER, Biochem. Biophys. Res. Commun. |34 498-503 (1992). 18. T. TAKASUKA, N. AKIYAMA, I. HORII, Y. FURUICHI and T. WATANABE, Biochem. J. I 11 748-753 (1992). 19. J. R. WU-WONG, G. P. BUDZIK, E. M. DEVINE and T. J. OPGENORTH, Biochem. Biophys. Res. Commun 171 1291-1296 (1990). 20. M. SOKOLOVSKY, J. Neurochem. 59 809-821 (1992). 21. W. G. HAYNES and D. J. WEBB, Clinical Science 84 485-500 0993). 22. W. G. WAGGONER, S. L., GENOVA, and V. A. RASH, Life Science 51 1869-1876 (1992) 23. M. A. ZAMORA, E. C. DEMPSEY, S. J. WALCHAK and T. J. STELZNER, Am. J. Respir. Cell Mol. Biol. 9 429-433 (1993).

×