Evidence for Different Angiotensin II Receptors in Rat Adrenal Glomerulosa and Rabbit Vascular Smooth Muscle Cells

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1 Evidence fr Different Angitensin II Receptrs in Rat Adrenal Glmerulsa and Rabbit Vascular Smth Muscle Cells STUDIES WITH COMPETITIVE ANTAGONISTS By Grdn H. Williams, Lynne M. McDnnell, Marie C. Raux, and Nrman K. Hllenberg ABSTRACT Angitensin II (All) acts as bth an endgenus vascnstrictr agent and a specific trphic hrmne that cntrls aldsterne secretin. The characteristics f vascular and adrenal receptrs fr All were examined in vitr by cmparing stimulatin and blckade induced by tw structural analgues f AH. The adrenal respnse was assessed n the basis f crticsterne and aldsterne prductin by islated rat glmerulsa cells; islated rabbit artic strips prvided an index f the respnse f vascular smth muscle. Threshld sensitivity t AH in bth tissues was ng/ml with a peak respnse at abut 300 ng/ml. P113 (l-sar-8-ala-aii) was a cmpetitive antagnist in bth systems; 1-10 ng/ml f PI 13 prduced a significant parallel shift f the All dse-respnse curve {P < ). The maximum respnse was nt reduced; increasing dses f AH vercame the blckade, and the respnses t ther agnists were nt influenced. PI 13 did nt stimulate either tissue. P 4 T 8 (4-Phe-8-Tyr-AII) was a partial agnist in the rabbit arta; 100 ng/ml f P 4 T 8 induced cmpetitive blckade, and cntractin fllwed dses abve 1,000 ng/ ml. Cnversely, P 4 T 8 neither stimulated nr blcked the adrenal receptrs, despite very high cncentratins. Thus, the results f this study reveal functinal differences in the All receptrs in the tw systems and prvide an apprach t designing therapeutic agents that might blck nly ne f its tw majr bilgical effects. KEY WORDS aldsterne secretin vascular reactivity crticsterne l-sar-8-ala-angitensin II 4-Phe-8-Tyr-angitensin II angitensin analgues Dwnladed frm by n December 12, 2018 Althugh angitensin II (All) has a variety f pharmaclgic effects, the tw mst physilgically significant are the effect n vascular smth muscle and the effect n the adrenal glmerulsa cells. Little is knwn abut the cmparative characteristics f the All receptrs in these tw tissues. Classically, insight int receptrs has largely cme frm the assessment f the respnse t structural analgues. An especially useful apprach has been t examine the effect f cmpetitive inhibitrs. Fr example, epinephrine is the Frm the Endcrine-Metablic and the Cardirenal Units, Department f Medicine and Department f Radilgy, Peter Bent Brigham Hspital and Harvard Medical Schl, Bstn, Massachusetts This study was supprted in part by U. S. Public Health Service Grants HL14944, GM18674, and HE05832 frm the Natinal Institutes f Health, by the Jhn A. Hartfrd and the Smith, Kline, and French Fundatins, and by U. S. Army R & D Cmmand Cntract DA MD This study was presented in part at the 55th Annual Meeting f The Endcrine Sciety, Chicag, Illinis, June 20, Please address reprint requests t Dr. Grdn H. Williams, Peter Bent Brigham Hspital, 721 Huntingtn Avenue, Bstn, Massachusetts Received Nvember 26, Accepted fr publicatin January 2, mst ptent agnist fr bth alpha-and beta-receptr effects; yet, antagnists that selectively prduce alpha- r beta-receptr blckade have helped t dissciate these tw types f adrenergic receptrs. The develpment f All analgues that are cmpetitive inhibitrs prvides a similar tl fr cmparing the All receptrs in vascular smth muscle and glmerulsa cells. Such analgues have already been used t assess the rle f AH in the maintenance f bld pressure and t discriminate All receptrs in ther tissues (1-4). The present study used this apprach t demnstrate that the adrenal and the vascular receptrs fr All are functinally different. Methds Studies were perfrmed in vitr in the adrenal system (rat glmerulsa cells,) and the vascular system (rabbit artic strips). In each system a dse-respnse curve fr AH was established alng with the dse-respnse relatinship fr a nnangitensin agnist Crtrsyn fr the adrenal system and nrepinephrine fr the vascular system. Tw angitensin analgues were assessed ver a wide dse range fr their ability t stimulate each system, t blck the actins f All, and t influence the Circulatin Research, VL XXXIV, March 1974

2 ADRENAL AND VASCULAR ANGIOTEIN II RECEPTORS 385 Dwnladed frm by n December 12, 2018 respnses f each system t the nnangitensin stimulus. PREPARATION OF RAT GLOMERULOSA CELLS Nrmal female rats (200 g) (Charles River Labratries) were maintained n a nrmal sdium intake (Purina rat chw). The rats were decapitated, and their adrenal glands were remved and decapsulated as previusly described (5). A glmerulsa cell suspensin was then prepared accrding t the techniques f Haning et al. (6). In brief, this technique cnsisted f incubating the adrenal capsules fr 50 minutes at 37 C in a mdified Krebs-Ringer's bicarbnate slutin cntaining 200 mg glucse/100 ml, 4% bvine serum albumin, and 3.7 meq ptassium/liter t which 3.7 mg f crude cllagenase and 0.05 mg dexyribnuclease/ml were added. Fllwing incubatin, the cells were mechanically disrupted. Cells frm the capsules f eight glands (fur rats) were then reincubated in 2 ml f the mdified Krebs-Ringer's slutin fr 2 hurs at 37 C under a 95% O 2-5% CO 2 atmsphere. Crticsterne and aldsterne utput were measured by radiimmunassay techniques which have been previusly described (5). Cmputatins were perfrmed n a General Electric 635 cmputer. Statistical analysis f the data included a tw-way analysis f variance f the means. A (value fr the respnse f each dse level cmpared with its apprpriate cntrl was cmputed n a lg transfrmatin f the data. Hmgeneity f variances was assessed by Bartlett's test (7), and P values were btained frm Dunnett's tables (8). RABBIT AORTA PREPARATION The rabbit arta preparatin has been described in detail (9, 10). In brief, 4 strips cut frm each rabbit arta were munted with 4 g f tensin in muscle chambers with a 10-ml wrking vlume cntaining a mdified Krebs-Ringer's bicarbnate medium. The slutin was maintained at 37 ± 0.5 C and aerated cnstantly with a gas mixture cntaining 95% O 2-5% CO 2 - Istnic cntractins were mnitred with a frce transducer. Cumulative dse-respnse curves fr All r nrepinephrine were btained with an initial dse f 10"" g/ml and subsequent lgarithmic increments until a maximum cntractin was achieved; at least 60 minutes were allwed fr equilibratin prir t administratin f the agent. Only a single agnist (angitensin r nrepinephrine) was used n each strip because f the pssibility f desensitizatin. Alternate strips were expsed t antagnists (10~ 9-10~ 5 g/ml) fr 10 minutes prir t adding the agnist. A cntractile respnse ti PI 13 was never seen. Each cmbinatin was studied in at least 12 artic strips taken frm at least fur rabbits. REAGENTS Radiactive Sterids. l,2 3 H-crticsterne (specific activity apprximately 40 c/mmle), and 1,2 3 H- aldsterne-y-lactne (specific activity apprximately 40 c/mmle), and l,2 3 H-aldsterne (specific activity apprximately 50 c/mmle) were btained frm New Circulatin Research, Vl. XXXIV, March 1974 England Nuclear Crpratin. The purity f l,2 3 H-crticsterne and l,2 3 H-aldsterne were checked by paper chrmatgraphy (benzene-methanl-water slutin, 1500:400:400), and the purity f 1,2 3 H- aldsterne-y-lactne was checked by anther system (cyclhexane-benzene-methanl-water slutin 500:200:500:100). Other Reagents. The fllwing substances were used: Crtrsyn (csyntrpin), All (l-asp-5-ue-ah), P 4 T 8 (4-Phe-8-Tyr-AII derivative), PI 13 (l-sar-8-ala- AII derivative), AH (Hypertensin, Ciba), nrepinephrine bitartrate, bvine serum albumin (fractin V, Pentex Crpratin, lt n. 153), dextran, activated charcal, crude cllagenase (Wrthingtn Bichemical Crpratin, lt CLS 21B), and dexyribnuclease (Sigma DN-C, lt n. 112C-2720). Results GLOMERULOSA CELL AND AORTIC STRIP RESPOE TO ANGIOTEIN II Aldsterne prductin increased significantly (P < ) when 0.25 ng All/ml was added (Fig. 1). The cntrl level (1.53 ±0.11 ng/rat hur" 1 ) rse t 2.16 ± 0.14 ng/rat hur' 1 with 0.25 ng f All and peaked at 3.93 ± 0.27 ng/rat hur" 1 with 250 ng All/ml. Cntrl crticsterne prductin (37 ± 3 ng/rat hur" 1 ), hwever, did nt increase significantly until 2.5 ng All/ml was added. A peak respnse (72 ± 5 ng/rat hur" 1 ) was again btained with 250 ng All/ml. The rabbit arta shwed similar relatinships with a threshld respnse ccurring between 0.3 ng/ml and 1.0 ng/ml and a maximal respnse ccurring between 200 ng/ml and 300 ng/ml (Fig. 2). EFFECT OF P113 (1-SAR-8-ALA-ANGIOTEIN II) PI 13 (30 ng/ml) prduced a significant (P < ) parallel shift f the All dse-respnse curve (Fig. 3). Hwever, the maximum aldsterne prductin induced by All (3.85 ± 0.34 ng/rat hur" 1 ) was nt different frm that prduced in the presence f 30 ng P113/ml (3.98 ± 0.33 ng/rat hur" 1 ). Similarly, maximum crticsterne utput with (64 ± 7 ng/ rat hur" 1 ) r withut (68 ± 6 ng/rat hur' 1 ) PI 13 was nt different. Thus, the blckade was surmuntable. In six experiments, the effect f 1 ng P113/ml n the All dse-respnse curve was assessed. As anticipated, the effect n the aldsterne respnse was less nticeable, but a significant (P < 0.05) parallel shift f the curve was demnstrated. The effect f P113 n the All dserespnse curve in the artic strip preparatin was similar with a significant parallel shift f the curve after the additin f 10 ng P113/ml (Fig. 4). Again, the maximum abslute respnse induced by All

3 386 WILLIAMS, MCDONNELL, RAUX, HOLLENBERG,_ CORTICOSTERONE 90 _ CORTICOSTERONE => i ALDOSTERONE < LJ ^ ^ 9-2 ALDOSTERONE PM3<30ng/ml) z 111 LU Y r/ / A AH + PI 13 (30ng/ml Dwnladed frm by n December 12, 2018 = sigfrm cntrl p<o.oi NANOGRAMS ANGIOTEIN II / ML MEDIUM FIGURE 1 Aldsterne and crticsterne utput frm islated glmerulsa cells in respnse t increasing dses f angitensin II. Values are means ± SE f 20 experiments. (1.99 ± 0.33 g) was nt reduced when P113 was present (1.91 ± 0.34 g). This blckade was further accentuated with dses f 30 ng/ml, but there was n cnsistent effect with 1 ng/ml. There was n evidence f intrinsic activity, i.e., ^ 2 I I I J I 0.3 I ANGIOTEIN II (ng / ml) Tracing f a typical rabbit artic respnse t cumulative dses f angitensin i-*"" 20 i it NANOGRAMS ANGIOTEIN H / ML MEDIUM FIGURE 3 i m - sig frm cntrl p <O.OI Aldsterne and crticsterne utput pltted as percent f peak respnse t angitensin 11 with and withut 30 ng PI 131ml medium. Values are means ± SE f ten experiments. n increase in aldsterne and crticsterne prductin ccurred, when glmerulsa cells were incubated with increasing cncentratins f PI 13 (Fig. 5). In fact, a dse f 25 ng P113/ml significantly decreased aldsterne prductin t 71 ± 6% f cntrl. The crticsterne respnse mimicked that f aldsterne, but t a lesser degree. Similarly, PI 13 at dses as high as 3 fig P113/ml did nt induce cntractin f the artic strip. The specificity f the PI 13 blckade was assessed by its effect n Crtrsyn-stimulated aldsterne and crticsterne prductin and n nrepinephrine-stimulated smth muscle cntractin. PI 13 (30 ng/ml) had n effect n the Crtrsyn-stimulated utputs f aldsterne and crticsterne at dses f 0.16 munits/ml and 2.56 munits/ml. These dses increased aldsterne utput tw- and fivefld, respectively. Similarly, 10 ng P113/ml did nt alter the respnse f the arta t nrepinephrine ver a dse range f 1 ng/ml t 300 ng/ml. Circulatin Research, Vl. XXXIV, March 1974 i

4 ADRENAL AND VASCULAR ANGIOTEIN II RECEPTORS 387 Dwnladed frm by n December 12, id. u. a* 60 Id O 40 Q. (O Id cr 20 Cntrl P 113 (long/ml) I ANGIOTEIN H. (ng/ml) FIGURE 4 Respnse f arta t angitensin 11 as percent f peak respnse with and withut 10 ngp113/ml medium. Values are means ± SE f ten experiments. PI 13 did nt reduce the abslute maximum respnse achieved. I 20i n t z a. t u r = sig. frm cntrl p<0.0l = siq. frm cntrl p<0.05 Aldsterne EFFECT OF P4T8(4-PHE-8-TYR-ANGI0TEIN II) P 4 T 8 in dses as high as 2.5 fig/m\ did nt alter the All-stimulated aldsterne utput frm glmerulsa cells (Table 1). In cntrast, P 4 T 8 (100 ng/ml) blcked All-mediated cntractin f the arta (Fig. 6). Furthermre, the blckade was cmpetitive, since bth lines n the Lineweaver-Burke plt intercept the rdinate at the same pint, and the maximum respnse induced by All with PjT 8 (2.04 ± 0.43 g) was the same as the respnse induced withut it (1.99 ± 0.33 g). In three experiments, neither 25 ng/ml r 2,500 ng/ml f P 4 T 8 cnsistently charlged aldsterne r crticsterne prductin frm the cntrl levels. Hwever, in the smth muscle preparatin, dses f 1-30 /LAg/ml, i.e., levels 10- t 300-fld greater than that prducing blckade, prduced a cnsistent dse-related respnse (Fig. 7). Discussin Ultimately, the characterizatin f receptrs will demand their islatin and the definitin f their structure. Hwever, this apprach may nt be pssible if the receptr is an integral part f a membrane, since destructin f the membrane may alter the structure f the receptr. An alternative, albeit indirect, apprach is t assess the relative bilgic effect (intrinsic activity) f a parent cmpund and a variety f structural analgues. Thus, Crticsterne NANOGRAMS PII3/ ML MEDIUM FIGURE S Aldsterne and crticsterne utput as percent f cntrl with increasing dses f P113. Values are means ± SE f fur experiments. Circulatin Research, Vl. XXXIV, March 1974

5 388 WILLIAMS, MCDONNELL, RAUX, HOLLENBERG Dwnladed frm by n December 12, 2018 TABLE 1 Effect fp 4 T 8 n the Angitensin-Induced Increases in Aldsterne Output by Glmerulsa Cells Test substance Aldsterne utput (ng/rat hur- 1 ) the structural determinants f an agent's interactin with its receptr can be determined thrugh these structure-activity relatinships. This apprach has been used by several investigatrs (11-16) t define the structural aspects f AH that are essential fr activity. A secnd way t assess a receptr's characteristics is t determine the effect f varius structural analgues that may r may nt pssess intrinsic activity but are cmpetitive inhibitrs. Bth types f assessments assume a cmplementary relatinship between the parent cmpund, its analgues, and the receptr. Recent studies suggest that such a relatinship des exist fr All. In a preliminary reprt (17), bth All and its analgue PI 13 displaced radiactive angitensin frm adrenal binding sites, but nly All increased adenyl cyclase activity, implying an interactin with a cmmn receptr but the prductin f different bichemical events. Since Khairallah et al. (12) demnstrated that the 5-IIe-8-Ala-analgue f All blcked the actin f All n strips f guinea pig ileum, varius analgues that are cmpetitive inhibitrs f the smth muscle effect f All have been synthesized (1-4, 16, 18-21). Hwever, in nly a few instances has the effect f these analgues n the adrenal crtex been assessed (2,17, 22, 23), and nly rarely has the effect f analgues n different angitensin receptrs been specifically cmpared (1, 22). Peach et al. (1) and Khairallah (24), wh used varius structural analgues, have suggested that angitensin receptrs n sympathetic nerve endings are structurally different frm thse n heart muscle. A similar apprach in the present study demnstrated similarities and differences in the effect f All and tw f its analgues n the arta and islated glmerulsa cells. The angitensin sensitivity f the tw systems was similar ( ng/ml fr threshld and abut 300 ng/ml fr maximal respnses). The excellent sterid respnse prbably resulted frm the preparatin methd; the cells had been under the influence f adrencrtictrpin in viv but they had been incubated withut it in vitr. Likewise, the respnses f these tw tissues f PI 13 were essentially similar. In bth tissues, PI 13 antagnized the actin f All at dses f 1-10 ng/ml; the blckade was cmpetitive and specific, and intrinsic activity was nt demnstrated in either tissue, even at dses as high as /ug/ ml. The respnse f the artic strip t angitensin was blcked, althugh the cncentratin required was cnsiderably higher than that required fr PI 13 (100 ng/ml). In additin, at higher cncentratins, P 4 T 8 pssessed intrinsic activity and induced artic cntractin. Cnversely, P 4 T 8 neither stimulated nr blcked the glmerulsa cells, suggesting that the All receptrs are different in the tw tissues. It is pssible that higher dses f PI 13 might have stimulated smth muscle r that higher dses f P 4 T 8 might have increased adrenal sterid utput; hwever, even if these pssibilities are true the difference in the cncentratins required wuld still suggest a majr difference in the tw receptrs. This prpsal is als supprted by a recent preliminary reprt (22) in which a different analgue (l-sar-8-ile-aii) was used. Because changes in the 8 psitin significantly altered the interactin f the analgues with these tw receptrs, the critical difference between the vascular and the adrenal receptrs prbably is their relatinship with the carbxy end f the mlecule. The differences in the receptrs may als accunt fr Frm cntrl P Frm pair Cntrl All (0.25 ng/ml) AH + P 4 T 8 All (25 ng/ml) All + P 4 T 8 All (25 ng/ml) AH + P 4 T ± ± ± ± ± ± ± 0.6 All values are means ± SE. - nt significant. P 4 T g was given in a dse f 2.5 /ig/ml. Circulatin Research, VL XXXIV, March 1974

6 ADRENAL AND VASCULAR ANGIOTEIN II RECEPTORS r P 4 T 8 - RABBIT AORTA 0.50 I RESPOE 0.25, P 4 T 8 [O.I y/ml] Cntrl O.I DOSE ANGIOTEIN E FIGURE 6 Duble reciprcal (Lineweaver-Burke) plt f the respnse f the arta t angitensin II with and withut P 4 T 8 (100 ng/ml). The lines meet at rdinate, which is characteristic f cmpetitive kinetics. Each pint represents the mean f eight determinatins. 10 IOne gram 10 mins Dwnladed frm by n December 12, 2018 the ppsite effects that salt intake has n the respnses f the tw systems t All; restrictin f sdium intake blunts vascular respnses t angitensin, but it ptentiates the respnses f the adrenal glands (25). The difference in the respnse f the tw systems t P 4 T 8 dcumented in this paper pssibly reflects a species difference. The tissues were selected because it is imprtant t wrk in vitr; the rat prvides an excellent in vitr adrenal system, but islated vascular tissue frm the rat develps angitensin tachyphylaxis t rapidly (9) fr studies such as thse perfrmed in this investigatin. Hwever, in the rat, P 4 T 8 acts as a partial agnist bth in nnvascular islated smth muscle (26) and n pressr respnses t All (18) used as an index f the integrated vascular respnse. Mrever, PI 13 shws remarkably similar effects in bth systems, free f any apparent species influence in rat, rabbit, dg, and man (2, 3, 22). It seems unlikely, therefre, that the differences reprted in this paper can be attributed t species. Further studies are necessary t delineate the characteristics f adrenal and vascular All receptrs in varius species. The mst extensive studies with P 4 T 8 (18, 26, 27) have demnstrated specific inhibitin f All-induced respnses in islated uterine muscle and in the bld pressure f the intact rat. There have been n previus reprts n the effect f P 4 T 8 n Circulatin Research. Vl. XXXIV, March 1974 FIGURE 7 Tracings f the artic respnse t increasing dses f P 4 T g in fur strips derived frm a single arta. glmerulsa cells. PI 13 was first studied by Pals et al. (3) wh demnstrated specific cmpetitive inhibitin f All respnses f rabbit artic strips; these findings are reprduced in the present study. Brunner et al. (4), Jhnsn and Davis (2), and Mimran et al. (28) have als shwn that PI 13 is a ptent antagnist f the pressr effect f All in viv in the rat, the dg, and the rabbit. Furthermre, Jhnsn and Davis (2) have demnstrated that PI 13 decreases aldsterne secretin in the intact dg and Peach and Chin (23), using islated glmerulsa cells, have reprted that PI 13 is a cmpetitive inhibitr f AH. Their demnstratin f different structure-activity relatinships f adrenal stimulatin amng several angitensin analgues in the rabbit adrenal glands further supprts the cncept f functinally different receptrs and makes species differences less likely in this study. Thus, the present study cnfirms sme previus bservatins and demnstrates a previusly unrecgnized phenmenn: a striking difference in the actin f P 4 T 8 in the tw systems studied. This difference in the respnses f adrenal glands and vascular smth muscle suggests that there are functinal differences in their All receptrs; this

7 390 WILLIAMS, MCDONNELL, RAUX, HOLLENBERG Dwnladed frm by n December 12, 2018 characteristic may be useful in designing therapeutic agents t blck nly ne f the tw majr physilgical effects f All. Mrever, the difference in the receptrs may accunt fr the difference in their respnses t restrictin f sdium intake (25). Acknwledgment It is a pleasure t acknwledge the assistance prvided in varius prtins f this study by Ralph Tdesc, Randall Gaz, and Kathern Hinrichs. References 1. PEACH, M.J., BUMPUS. F.M., AND KHAIRALLAH, P.A.: Inhibitin f nrepinephrine uptake in hearts by angitensin II and analgs. ] Pharmacl Exp Ther 167: , JOHON, J.A., AND DAVIS, J.O.: Angitensin II: Imprtant rle in the maintenance f arterial bld pressure. Science 179: , PALS, D.T., MASUCCI, F.D., DENNING, G.S., JR., SIPOS, F., AND FESSLER, D.C.: Rle f the pressr actin f angitensin II in experimental hypertensin. Circ Res 29: , BRUNNER, H.R., KIRSHMAN, J.D., SEALEY, J.E., AND LARAGH, J.H.: Hypertensin f renal rigin: Evidence fr tw different mechanisms. Science 174: , WILLIAMS, G.H., MCDONNELL, L.M., TAIT, S.A.S., AND TAIT, J.F.: Effect f medium cmpsitin and in vitr stimuli n the cnversin f crticsterne t aldsterne in rat glmerulsa tissue. Endcrinlgy 91: , HANING, R., TAIT, S.A.S., AND TAIT, J.F.: In vitr effects f ACTH, angitensins, serntnin and ptassium n sterid utput and cnversin f crticsterne t aldsterne by islated adrenal cells. Endcrinlgy 87: , SNEDECOR, G.W., AND COCHRAN, E.C.: Statistical Methds, 6th ed. Ames, Iwa, The Iwa State University Press, 1967, p DUNNETT, C.W.: New tables fr multiple cmparisns with a cntrl. Bimetrics 20: , HOLLENBERC, N.K., BARBERO. L.R., AND HlNRlCHS, K.P.J.: Angitensin tachyphylaxis and vascular angitensinase activity. Experientia 27: , STREWLER, G.J., HINRICHS. K.J., GUIOD, L.R., AND HOLLEN- BERG. N.K.: Sdium intake and vascular smth muscle respnsiveness t nrepinephrine and angitensin in the rabbit. Circ Res 31: , BUMPUS, F.M., KHAIRALLAH, P.A., ARAKAWA, K., PACE. I.H., AND SMEBY, R.R.: Relatinship f structure t pressr and xytcic actins f isleucine 5 -angitensin ctapeptide and varius analgues. Bichim Biphys Acta 46:38-44, KHAIRALLAH, P.A., TOTH, A., AND BUMPUS. F.M.: Analgs f angitensin II: II. Mechanism f receptr interactin. J MedChem 13: , JORCEEN. E.C., RAPAKA, S.R., WlNDRIDCE, G.C., AND LEE. T.C.: Angitensin II analgs: 6. Sterechemical factrs in the 5 psitin influencing pressr activity 1. J Med Chem 14: , JORCEEN. E.C., RAPAKA, S.R., WlNDRIDCE, G.C., AND LEE, T.C.: Angitensin II analgs: 7. Sterechemical factrs in the 5 psitin influencing pressr activity 2. J Med Chem 14: , LlN S.-Y., AND GOODFRIEND, T.L.: Angitensin receptrs. AmJ Physil 218: , RECOLI, D., PARK, W.K., AND RIOUX, F.: Pharmaclgy f angitensin antagnists, Part II. Can J Physil Pharmacl 51: , GLOSSMAN. H., BAUKAL, A., AND CATT, K.J.: Angitensin II receptrs f the adrenal crtex (abstr.). Clin Res 21:492, MARSHALL, G.R., VINE, W., AND NEEDLEMAN, P.: Specific cmpetitive inhibitr f angitensin II. Prc Natl Acad Sci USA 67: , PALS, D.T., MASUCCI, F.D., SIPOS. F., AND DENNINC, G.S., JR.: Specific cmpetitive antagnist f the vascular actin f angitensin II. Circ Res 29: , TURKER,R.K., YAMAMOTO.M., AND BUMPUS. F.M.: Newshrtacting antagnist f angitensin II. Arch Int Pharmacdyn Ther 201: , Riux, F., PARK, W.K., AND REGOLI, D.: Pharmaclgy f angitensin antagnists, Part I. Can J Physil Pharmacl 51: , BRAVO. E.L., KHOSLA, M.C., AND BUMPUS, F.M.: Angitensin (All) receptrs in the adrenal crtex and vascular smth muscle: Dissciatin f respnses t a cmpetitive All antagnist (abstr.). Clin Res 21:865, PEACH, M.J., AND CHIN, A.T.: Stimulatin and inhibitin f aldsterne bisynthesis in vitr by angitensin II and analgs. Circ Res (suppl.), in press. 24. KHAIRALLAH, P.A.: Actin f angitensin n adrenergic nerve endings: Inhibitin f nrepinephrine uptake. Fed Prc 31: , HOLLENBERC, N.K., CHENITZ, W.B., ADAMS, D.F., AND WIL- LIAMS. G.H.: Salt intake: Determinant f adrenal and renal vascular respnses t angitensin II in nrmal man (abstr.). Am Sc Nephrl 6:50, NEEDLEMAN, P., FREER, R.J., AND MARSHALL, G.R.: Angitensin-receptr interactin: Influence f ph n angitensin inhibitin by Phe 4 -Tyr 8 -angitensin II in rat uterine smth muscle. Arch Int Pharmacdyn Ther 200: , NEEDLEMAN, P., JOHON, E.M., JR.. VINE, W., FLANICAN, E., AND MARSHALL, C.R.: Pharmaclgy f antagnists f angitensin I and II. Circ Res 31: , MlMRAN. A., CUIOD, L., AND HOLLENBERG. N.K.: Angitensin's rle in the cardivascular and renal respnse t salt restrictin. Kidney Int, in press. Circulatin Research, Vl. XXXIV, March 1974