March 1970
ANALOGSOF AXGIOTENSIS11. I1
181
Table 11; amino acid ratios oil an acid hydrolysate: .hp, of Et3S. This heptapeptide polynier wa.. prepared as de0.96; .4rg, 0.98; J-al>1.00; Tyr, 0.92; Ile, 0.95; His, 1.02; Pro, scribed above. The reaction mixture wap ,haken in an ice 1.05; Phe, 1.00: on an enzymatic hydrolysate: Asp, 1.00; Arg, bath for 2 hr, then at room temperature overnight. The result1.01; Val, 1.08; Ile, 0.98; His, 0.51; Pro, 0.58; (O?\Ie)Tyr, 1.07; ing protected peptide polymer was collected by filtration, Phe, 1.00. -4nal. ( C ~ ~ H , S , ~ ~ ~ Z . C H I CC, O H, O HX. ) washed [DP\.IF(3 times with 50 ml), absolute EtOH ( 3 times with Aspartylarginylvalyl-O-methyltyrosylvalylhistidylprolylphenyl- -50 ml)], and finally dried over PtO, in vacuo t o yield 2.33 g. alanine ([4-(OMe)Tyr,5-Val] -angiotensinII).-The free octapepThe protected peptide polymer was suspended in 30 ml of antide was prepared as described above to give 56% yield of the hydrous FICC02H and a slow stream of HBr was passed through desired peptide; see Table 11; amino acid ratios on an acid with occasional shaking for 30 min under anhydrous condihydrolysate: Asp, 0.92; Arg, 1.06; Val, 2.13; Tyr, 0.98; His, tions. The suspension was filtered and the polymer was washed 1.08; Pro, 1.00; Phe, 1.00; on an enzymatic hydrolysate, Asp, (3 times with 8 ml) wit,h anhydrous F~CCOIH. The combined 1.00; Arg, 1.01; Val, 2.12; His, 0.58; Pro, 0.61; Phe, 1.00; filtrate was concentrated in oacuo at 20" and peptide was pre(OAIe)Tyr, 1.00. Anal. (C50H;1Nl~O~z.CH~COOH) C , H, S . cipitated by addition of anhydrous EtlO. The solid was reAsparaginylarginylvalyl-O-methyltyrosylvalylhistidylprolylphe- moved by filtration and washed with aiihydroiis Et?O. This nylalanine ( [l-Asp(NH2),4-(OMe)Tyr,5-Val] -angiotensin II).part,ially protected octapeptide was dissolved in lIeOH-AcOHWoodward's Reagent K24 (0.633 g, 2.5 mmol) was dissolved in HzO (10: 1: 1) and hydrogenated over Pd black for 48 hr. The 23 ml of D l I F with vigorous stirring. At O " , 0.67 g ( 2 . 5 mmol) peptide was isolated in the usual manner to yield 329 mg of solid. of carbobenzoxyasparagine and 0.35 ml ( 2 . 5 mmol) of Et3N This was purified by chromatography on a Sephadex G-25 (coarse) dissolved in 25 ml of D l I F were added. Stirring was continued column by elution with BAW solvent. The peptide emerged until the soln cleared (about 3 hr). This was then added to a mainly as one fraction, was precipitated from 50% .IcOH with suspension of 2 . 5 g of heptapeptide polymer initroarginylvaly1-0Et20-AIezC0 ( l : l ) , and dried over P,Oj, S a O H , and paraffin methyltyrosylvalyl-.~~-beiiz~lhiutidylprolylphenylalaninepolyin V ~ C U Oto yield 490 mg (50% yield based 011 0.84 mmol of Xmer) siispended in 25 ml of D l I F containing 0.35 ml (2.5 mmol) terminal nitroarginyl heptapeptide with polymer I : see Table 11; rat,ios on an enzymatic hydrolysate: ALpiYHJ, 0.95; - h g , 1.05; Val, 1.92: His, 0.51; Pro, 0.58; Phe, 1.00; (OlIe)Tyr, 0.98. Anal. CsoH,.SlrOll .PCHICOOH .2H?O 11200.67): C, 124) (a) R. B. Woodward and R . A . Olofson, J . Smer. Chem. Soc., 83, 53.97; H, 7.05; S , 16.33. Found: C, 53.42: H, 6.30; N, 1007 (1961). (h) R B. Woodnard. R . h. Olofson and H. Maser, zbid., 1010 16.09. (196 1).
Analogs of Angiotensin 11. 11. Mechanism of Receptor Interaction' P. A.
ICHrlIRALLAH,
A. TOTH,A K D F. 31. B U I I P C S
Research Dioision, Cleveland Clinic Foundation, Cleveland, Ohio 44106 Recewed September 15, 1969 [.i-Ilel-aiigiotensiu I1 has two sites of action on guinea pig ileum. I t directly interacts with receptor siteh on umooth muscle, leading to contraction, and also indirectly contracts miiscle by interacting with receptor sites on the parasympathetic innervation of the ileum, releasing acet,ylcholine. An attempt has been made to study the-e two receptors, using responsiveness of the tissue to analogs of angiotensin 11. Substit,utions in positions 1-7 showed close correlations between pressor activit,y, smooth muscle activity, and release of acetylcholine. Substitiitions ill position 8, however, indicated that [5-Ile,8-(031e)Tyr]-angiotensin I1 is about, three times more active on guinea pig ileum than on blood pressure, while [5-Ile,&Tyr]-angiotensin I1 has roughly the hame activity in both. The release of acetylcholine by both peptides was the same; the smooth muscle respoilbe t o the latter peptide was the same as the parent compound, while response to the former peptide was less than half that of the parent compound. On the other hand [5-Ile,8-Ala]-angiotensin I1 produced no response on guinea pig ileum similar to its effect on blood pressure, but it, inhibited subsequent response to the parent compound. I t is assumed that the analog binds to recept,or sites producing no excitation but preventing other analogs from interacting. These result,s have been interpreted by a speculative scheme concerning conformations of muscle and nerve receptors.
The octapeptide, angiotensin 11, is known to have a multiplicity of actions.2 Until recently, angiotensin I1 analogs were usually assayed either by their pressor responses in ganglion-blocked or nephrectomized rat's, or by their musculot'ropic effect,s on isolated rat uteri. In general, biological activity in these assay systems was roughly equivalent and this led to an assumption that angiotensin acted only on the smooth muscle cells in these t'wo assay syst'ems, and that the receptor sit'es on these cells were, a t least grossly, similar. Recently, Peach, Bumpus, and Khairallah3 reported that angiotensin I1 inhibited uptake of norepinephrine into sympathetic nerve endings in rabbit heart, a t dose levels of less than 50 pg/ml. Angiotensin I1 analogs (1) This investigation was supported b y U. S. Public Health Serrice Research Grant HE-6835f r o m t h e National Heart Institute. (2) I. H. Page and J. W.McCuhbin, Ed., "Renal Hypertension," Year Book Medical Publishers, Inc., Chicago, Ill., Chap. 5 and 9 (1968). (3) 11.J. Peach, F. M.Bumpus, and P. A . Khairallah, J . Pharmacol. Exp. Ther., 167, 2 9 1 (1969).
substituted in positions 1-7 showed a close correlation between pressor/musculotropic activity and inhibition of uptake. Substitutions in position 8. however, indicated that the benzene ring of phenylalanine was not needed for inhibition of uptake but n - a ~required for pressor activity. Substituting phenylalanine by aminophenylbutyric acid or aminophenylisobutyric acid also dissociates inhibition of uptake from the pressor response. The free C-terminal C02H was required for both activities. This led to the conclurion that angiotensin receptor sites on sympathetic nerve endings were very similar to those on smooth muscle cells. except that the latter required an aromatic ring structure in position 8, while the former did not. To study receptor sites on other tiqsueu. guinea pig terminal ileum was used. Response of ileum to angiotensin is biphasic, a rapid component due to release of ACh from the intrinsic parasympathetic nerve ganglia of Meissner and Auerbach or postganglionic nerve end-
Results Overall bio1ogic:d activity of :iiigiotensiii aiialog:,. recorded :is isotonic contraction., :ire listed in Table I.
general, it caii be heell that pressor and contractile. re-poii-e- TI ere roughly parallel with the exception of [ .i-Ile.S- (ONe)Tyr ]-angiotensin 11. With ibometric contraction., there also was a rough p:tralleli\m between prebsor rebponses and both t h e direct mu-culotropic and indirect acetylcholine mediated reipori-es. The major exception was observed \I hcii u-ing peptides -ubstituted in position 8. When ~.i-Ilc,S-,lla]-axigiote~isin I1 TKLS given in doses over
111
1'. .I. Khairallah and I. €I. Page, A m ? . J . Phiisid., 200, 51 (1961). 1'. .\. Robertson and D. Rubin. Brit. J . Pharmueol., 19, 5 (1962). r6) T. Godfraind. .I.Krtba, and P. Polster, ibid.,28, 9 3 (1966). (71 S . C. Chaturredi, \I7. IC. Park, R . R. Smebg, a n d F. hI. Rumpus. 11)
. I . .lfr.d. Chtm.. 1 3 , 177 110701.
(8) 3'. Ciodfrainii. ,\. Kalm, and P. l'dster, .trch. I n t e r n . Phurnimvi!:i, . , 163, 227 (1966;. (9) C. Chamyyaiid E. Gley, C o m p t . 12f~cd.Soc. B i d . , 11, I59 1,1911J . (10) I
