Proc. Natl Acad. Sci. USA Vol. 78, No. 1, pp. 592-596, January 1981 Medical Sciences

Characterization ofangiotensin receptors on bovine adrenal fasciculata cells (corticotropin/glucocorticosteroids/cyclic AMP/calcium/potassium) M. B. VALLOTTON*, A. M. CAPPONI*, CH. GRILLET*, A. L. KNUPFER*, R. HEPP*, M. C. KHOSLAt, AND F. M. BUMPUSt *Division ofEndocrinology, Department ofMedicine, Laboratory ofClinical Investigation, University Hospital CH-1211 Geneva, Switzerland; and tResearch Division, Cleveland Clinic Foundation, Cleveland, Ohio 44106 Communicated by,Irvine H. Page, August 28, 1980

ABSTRACT We have further characterized angiotensin re- by a series of peptide analogs with antagonistic properties was ceptors on bovine adrenal fasciculata cells whose presence was examined and compared with the biologic effect of these pep- previously demonstrated by the intrinsic agonistic activity ofangi- tides on steroidogenesis. The roles of cyclic AMP, Ca2+, and otensin II (AI), des-Asp'-AII, angiotensin I (AI), and des-Asp'-AI on steroidogenesis. The specific binding of All and des-Asp -All K+ on the steroidogenesis induced by angiotensin also were labeled with`2'I to dispersed bovine fasciculata cells was studied. examined. For both peptides, a single class ofbinding sites accounted for the data with a mean (± SEM) K. value of 0.23 ± 0.123 x 10' liters/ MATERIALS AND METHODS mol for All and 0.68 ± 0.19 x 0I liters/mol for des-Asp'-AH. The [Asp',Ile5]- and [Asn',Val5]-AII and AIII were iodinated with concentration at which unlabeled All and des-Asp'-AII displaced 125I by the chloramine-T method (10) as described (11). Sources 50% of the tracers (K.) was similar to that at which they induced half-maximal stimulation ofsteroidogenesis (K.,,). For AI and des- of the peptides were: [Asn',Val5]-AII and ACTH (1-24), CIBA, Asp'-AI, I& > Kac. Analogs of All or des-Asp'-AII with antago- Basel, Switzerland; [Asp',11e5]- and [Sar',Ala8]-AII, Bachem, nistic properties upon steroidogenesis competed also with binding Torrance, CA; [Ile5]-AI, Schwarz/Mann. AIII and other pep- of the tracers. Corticotropin (ACTH) did not inhibit binding. Al- tide analogs were synthesized at the Research Division of the though ACTH stimulated the formation ofcyclic AMP, none ofthe Cleveland Clinic. Fasciculata and glomerulosa cell suspensions angiotensins with intrinsic activity did so. Calcium, but not potas- were prepared by the method ofKloppenborg et al. (12) as mod- sium, appeared to potentiate the steroidogenic activity of AII. These data suggest that there is a single class of receptors for an- ified by Sayers et al. (13), as described (8). After separation by giotensins and analogs in zona fasciculata. These receptors show paper chromatography, were measured by the characteristics that differentiate them from ACTH receptors in protein-binding method according to Leclereq et al. (14). zona fasciculata or angiotensin receptors in cells. To allow a better comparison of the characteristics ofthe re- ceptors in glomerulosa and fasciculata cells, binding-inhibition In addition to stimulating biosynthesis in the zona experiments were performed the same day with both types of glomerulosa, angiotensin II (All) has been shown to stimulate cells prepared from the same adrenal glands. The incubations steroid biosynthesis in the zona fasciculata in canine and bovine were for 10 min and were at 22°C. Approximately 20,000 cpm species (1-5) and, under certain circumstances, in man (6). (10 fmol) oflabeled peptide was added per tube. Specific bind- High doses of All amide (hypertensin) also increase steroido- ing usually amounted to 2-6% of total added radioactivity. genesis in rat fasciculata-reticularis cells but this effect has been Binding-inhibition experiments for the estimation of dissocia- attributed to a corticotropin (ACTH)-like impurity (7). There- tion constants were performed by adding increasing amounts of fore, before considering any effect on steroidogenesis to be of the various peptides over a wide range ofconcentrations (10`0 physiological significance, it is important to ascertain that pure to 10' M). Nonspecific binding was measured in the presence angiotensins, possibly ofdifferent sources, have such an effect ofa large excess ofangiotensin II (10-5 M). Cell-bound and free at low concentration. We previously reported (8) that, in the radioactivities were then separated either on HAWP Millipore zona fasciculata from bovine adrenals, not only All (9) but also filters (0.45 ,um) or by centrifugation (700 X g at 4°C for 10 min). its precursors, the decapeptide angiotensin I (Al) and AI nona- The pellet was resuspended gently in 2 ml ofKrebs-Ringer glu- peptide 2-10 (des-Asp'-AI), possessed intrinsic activity without cose/bicarbonate buffer and centrifuged again. The radioactiv- being converted to All and angiotensin heptapeptide 2-8, des- ities in the combined supernatants and in the cell pellet were Asp'-AII (AIII), respectively. AIII was as potent as All in stim- counted for 10 min. All estimations were performed in tripli- ulating steroid biosynthesis in the zona fasciculata. Both curves cate. Each experiment was repeated at least twice for the var- were similarly displaced to-the right in the presence of the pep- ious peptides and four times for All and AIII. The number of tide analog [Sarl, Ala8]AII which behaves as a true competitive cells per incubation tube ranged from 1.37 to 6.10 x 105 (mean analog. ± SD: 3.57 ± 1.08 X 105). In addition, subcellular fractions For better characterization of these specific membrane re- from both zones, prepared as described elsewhere (15, 16), ceptors for angiotensin in the cells from the zona fasciculata, we were also used for comparative binding studies. Binding data studied the hormone-receptor interaction in binding-inhibi- were analyzed by using the computer programs SCATFIT and tion experiments using '25I-labeled All or AIII. The specific dis- SIGMOID conceived by Rodbard et al. (17, 18). The values are placement of these tracers by either of these two peptides and reported as mean ± SEM. The publication costs ofthis article were defrayed in part by page charge Abbreviations: All, angiotensin II; ACTH, corticotropin; AI, angioten- payment. This article must therefore be hereby marked "advertise- sin I; des-Aspl-AI, AI nonapeptide 2-10 (des-Aspl-AI); AIII, des-Asp'- ment" in accordance with 18'U. S. C. §1734 solely to indicate this fact. All (angiotensin III). 592 Downloaded by guest on September 28, 2021 Medical Sciences: Vallotton et al. Proc. Natl. Acad. Sci. USA 78 (1981) 593

RESULTS AND DISCUSSION 1200 - 220C Binding and Displacement Studies. The binding of labeled AIII was linearly related to the number offasciculata cells incu- bated (r = 0.99, P < 0.0001, n = 33) (Fig. 1). Steady state of 1000- specific binding of All (Fig. 2) was reached after 10 min when the cells were incubated at 220C, and the binding maintained the plateau for up to 20 min. At 370C, binding occurred just as w 800- rapidly but reached a maximum after 5 min and thereafter de- 0u: creased regularly with a half-life of 18 min. At 00C, only weak binding was observed even after 20 min ofincubation (0.46%). x 600- The dissociation of the peptides from the receptors was in- - vestigated by adding an excess (10-5 M) ofunlabeled All or AIII after 10-min incubation offasciculata cells with 125I-labeled All 400 - or AIII, respectively. When cell suspensions in triplicate were 370C incubated for 0-18 min with the labeled peptide alone, the binding ofthe tracer reached a plateau after 10 min; this plateau 200 - was maintained up to 18 min (Fig. 3). Immediately after the ad- 0°C 0o dition of the unlabeled peptide the specific binding decreased 0- rapidly; this decrease followed an exponential curve. The dis- I I I sociation rate constant calculated for both All and AIII was 5.75 1 3 5 7 9 12 15 20 X 10-3sec-1. Time, min Inhibition of binding of labeled All or AIII was studied at steady state with peptide concentrations ranging from 10-10 to FIG. 2. Time-course ofthe specific binding of "25I-labeled AII to bo- 10-5 M (Fig. 4). Data obtained under these conditions were vine adrenal fasciculata cells at three different temperatures. The cells analyzed by Scatchard plot. In every instance, a single class of were incubated for 10 min. Each point represents the mean of results binding sites accounted for the data. The binding affinities were obtained with duplicate incubations. In this experiment, 18 fmol ofla- characterized by an apparent Ka of 0.23 ± 0.123 x 10' M- (n beled All had been added to each tube. The nonspecific binding, amounting to 1.4%, has been subtracted. = 4) when All competed with labeled All and of 0.68 ± 0.19 X 10' M-l (n = 4) when AIII competed with labeled AIII. The difference between these Ka values was significant by the Stu- calculated as the reciprocal of the peptide concentration giving dent t test (P < 0.05). Similar values were obtained when Ka was 50% inhibition ofbinding (ID50 or Kd). In another series ofexperiments, Kd values for All binding to both glomerulosa and fasciculata cells were determined within the same experiment by using both isolated cells and subcellu- lar fractions. The values for Kd calculated in the zona glomeru- losa (7.8 x 10- M with isolated cells; 6.4 x 10' M with mem- branes) were close to those measured in the zona fasciculata (5.5 x 10' M with isolated cells; 3.7 X 10-9 with membranes). As also indicated in Fig. 4, the binding affinities of Al, the nona- peptide, and the hexapeptide were approximately 3 orders of -e magnitude lower than the affinities ofAll and AIII. a ax 0 Studies with Angiotensin Analogs. It was first demonstrated -4 that none of the following analogs displayed an agonistic effect in terms of stimulation of steroid biosynthesis at a concentra- tion of 10-5 M: des-Asp'-[Ala¶]-AII, des-Asp'-[Thr8]-AII, des- Asp'-[Ile8]-AII, des-Asp'-[Ala8]-AII, [Sar',Thr8]-AII, and [Sar',Thr(Me)8]-AII. However, all behaved as competitive an- tagonists both for inhibition of steroid biosynthesis stimulated 0 - by All and for binding ofeither labeled All or AIII (Table 1). By contrast ACTH (1-24) displayed no binding-inhibition activity up to a dose of 10-5 M. Effect ofCa2+ and K+ Concentration in the Incubation Me- dium upon Steroidogenesis Induced by All. The production of steroids by five different preparations of fasciculata cells was studied at various Ca21 concentrations in the medium, from near zero to 2.85 mM (Table 2). In the absence of added All, basal steroid production was not affected by changes in Ca2+ concentration. In the presence of i0' M All, an effect ofCa2+ concentration was observed. However, there was considerable 1 5 10 variation among experiments. An effect ofCa2+ was already no- Cells, no. x 10-5/ml ticeable at a concentration as low as 0.13-0.3 mM. No change in basal production (19.98 ± 0.49 FIG. 1. Relationship between the number ofbovine adrenal fasci- ng/ml, n = 8 triplicates) was observed when All was absent and culata cells incubated and the amount ofbound '25I-labeled des-Asp1- the concentration ofK+ was changed from 0.9 to 8.4 mM. In the AIL. The cells were incubated for 10 min at 22°C. Each point represents of of in K+ concen- the mean ofresults obtained with five incubates. + = 0.99; P < 0.001. presence I0- M All, no clear effect change Downloaded by guest on September 28, 2021 594 Medical Sciences: Vallotton et al. Proc. Natl. Acad.-ScL USA 78-(1981)

220 - 100 -

200 - 180- 80 - 160- W

10-140- 0 e.) 0 60- -0 120-

X 100- 0 4 0-a) a) co 80- .o.4 E. 60- 40- / 20 20-

0- r 0 2 4 6 8 10 12 14 16 18 0-

10-10 10-9 10-8 10-7 10-6 1o-5

1000- I

800- 6q 1-

o 600-

x "0 a O cq 400- 0

200- "0 Uola a) .-- Or- 2 4 6 8 10 12 14 16 18 Time, min

FIG. 3. Time-course of association (o) and dissociation (0) of 125I- labeled All (Upper) and AIII (Lower) to bovine fasciculata cells. At the . time indicated by the arrow, unlabeled peptide was added in excess 0 I (10-5 M)-to displace the tracer. Each point represents the mean of re- 10-9 10-8 10-7 10-6 10-5 suits obtained with triplicate samples. Peptide, M FIG. 4. Displacement from bovine adrenal fasciculata cells, of125I- tration was observed, but the mean corticosteroid production labeled All (Upper) and AIII (Lower) by increasing quantities ofunla- beledACTH (1-24) (L), des-Asp', or increased to 227.8 ± 11.0 ng/ml (n = 14). des-Arg2-AII in Upper des-Asp1-AI in Lower (A), Al (m), All (o), or AIII (x). The incubation was for 10 min Effect of Angiotensins upon Cyclic AMP Formation. The stimulation of steroidogenesis from fasciculata cells by either at 2200. The points represent the mean oftriplicate samples. AI, All, des-Aspl-AI, or AIII was not preceded by any increase in cyclic AMP in the medium after 5-min incubation. By con- the shorter agonist without the necessity to postulate the exis- trast, ACTH induced a. significant increase in nucleotide pro- tence of a second class ofsites with higher affinity. duction within 5 min (Table 3). As shown previously (20), a sig- Previous results from this laboratory have indicated super- nificant increase in corticosteroid production can be observed imposable dose-response curves of steroid production for All after only 5-10 min ofincubation with All. and AIII and identical displacement to the right ofthese curves These data with those of our previous studies (8) clearly in- by the antagonist analog [Sar',Ala8]-AII (8, 20). In zona glomer- dicate the existence, in adrenal fasciculata cells, ofspecific and ulosa cells from rats, two classes of binding sites, with one of operational receptors for angiotensin. These cells bound la- higher affinity for AIII, have been proposed by Devynck et al. beled AIl and AIII in direct proportion to their number; this (21). Catt and his collaborators (22-24), on the other hand, ob- binding occurred rapidly with a maximum around 10 min, the tained data from rat and dog adrenal cells that favor a single class time when the first significant increase ofsteroid in the medium ofbinding sites for All and AIII and do not support the proposal is observed (20). The labeled peptides were selectively dis- that the formation ofAIII is a requisite intermediate step in the placed in proportion to the concentration ofunlabeled All, 50% action of All. Douglas et al. (25) reported that, in rat adrenal inhibition of binding occurring at the same concentration as zona glomerulosa cells, AIII was less potent than All but that half-maximal stimulation of steroidogenesis induced by All. for each of these agonists the biological potency of the com- With labeled AIII, the Kd for All or AIII itself was also similar to pound was increased when valine was the substituent in posi- the. K.,, ofthese peptides. These data suggest the presence of a tion 5 instead of isoleucine. Yet the reverse was true when rel- single population of binding sites for the octapeptide and the ative binding affinity was considered. -In these studies, in *heptapeptide. However, Scatchard analysis ofthe binding data contrast to those ofAguilera et al. (23), a-difference in the me- from four separate experiments revealed a small but signifi- tabolism of these peptides by the adrenal cells has not been cantly higher association constant for AIII than for All. This dif- taken into consideration. The latter authors reported a more ference could-be accounted for by a reduced steric hindrance for rapid degradation ofAIII than ofAll. Because in our study All Downloaded by guest on September 28, 2021 Medical Sciences: Vallotton et al. Proc. Natl. Acad. Sci. USA 78 (1981) 595

Table 1. Effects ofangiotensin analogs ciculata cells, did not displace labeled All or AIII, thus giving ID5o, M x 107 more evidence of the selectivity of these receptor sites. When 1251_[Val5]_ desoxycorticosterone (I), (11), 1 1-deoxycortisol '251-des-Aspl- and were in me- Peptide ED50, M x 108 All [Ile5]-AI (III), (IV) separately determined the dium, the same proportion ofthese steroids and the same ratio [Val5]-AI 3.83 ± 0.35* 0.47 0.485 I/I and Ell/IV were observed whether steroidogenesis was [H1e5]-AI 8.57 ± 1.06* 250 950 stimulated by ACTH or by All, a suggestion that these peptides des-Asp'-[Ile5]- act at the same or at close enzymatic steps (unpublished data). ± AII 3.93 0.9* 0.45 0.438 These results are consistent with those obtained with dis- des-Asp1-[1le5]-AI 2.0 100 50 persed glomerulosa cells from rats and dogs by Aguilera and [SarWAla8]-AII 8.5t 0.46 2.5 Catt (26). ACTH is known to exert its action on the zona glom- des-Asp1-WAla8]-AH - - 1.85 erulosa and fasciculata through stimulation of adenyl cyclase des-Asp1-[Thr8]-AII - 7.3 8.Ot 0.18 0.054 and the intermediary action ofcyclic AMP, whereas in the zona des-Asp-[41e8]-AH glomerulosa the stimulation of aldosterone All is [Sar1,Thr8]-AH 25t 0.47 - synthesis by [Sar',Thr(Me)8]_ not accompanied by the stimulation ofadenyl cyclase or the for- AII 2.5t - _ mation of cyclic AMP (27). Accordingly, we investigated the possibility of a link between the stimulation ofcortisol produc- Data are from single experiments unless indicated otherwise. ED50, tion by All in the zona fasciculata and the formation of cyclic concentration for half-maximal stimulation; ID50, concentration for AMP. Our negative results suggest that this nucleotide has no displacement of50% ofbound label. * Mean ± SEM, n = 3. role in the mediation of angiotensin-stimulated steroidogene- t For 50% inhibition ofsteroidogenesis induced by All at 10-7 M. sis, thus confirming previous reports (27-31). Only few reports have mentioned an increase ofcyclic AMP output with All stim- ulation (9, 32-34), usually only at high concentrations (33, 34). carried isoleucine, the small difference in binding affinity could When cyclic AMP was not involved, intracellular calcium has also be partly accounted for by this difference. been considered in some hormonal systems as the "second mes- This could also at least partially explain the higher Kd for All senger" after bindingofhormone to its receptor. Although basal observed in the three more recent experiments in which we steroid production was independent of added calcium, a compared the binding to fasciculata and glomerulosa cells be- marked increase in steroid output between 0 and 0.3 mM cal- cause in these studies we used [Ile5]-AII instead of [VaP]-AII. cium was observed in the presence ofAll. This supports a per- Consistent with previous studies with the zona glomerulosa missive effect of calcium. A similar observation was made by cells, a marked decrease in affinity was observed when the Fakunding et al. (31) with glomerulosa cells from rats and dogs. chain length was increased to 9 and 10 amino acids or decreased Potassium plays a major role in directly stimulating steroid to 6 amino acids (25). production in vitro from glomerulosa cells but not from fasci- Among the analogs tested that were devoid of any agonistic culata cells (35). In the present study, variations ofthe concen- activity but had antagonistic properties in terms ofsteroidogen- tration ofK+ in the medium affected neither basal nor AII-stim- esis, des-Asp'-[Ile8]-AII was the most potent inhibitor of bind- ulated corticosteroid production. This absence ofeffect of K+ is ing ofboth All and AIII. [Ile5]-AI and des-Asp'-[I1e5]-AI previ- consistent with previous findings in rat (35) and dog adrenals ously were found to possess an intrinsic partial agonist activity (36) and permits the operational differentiation of fasciculata without having to be converted to All and AIII in stimulating and glomerulosa cells. steroidogenesis in fasciculata cells (8, 20). We showed that these These specific binding sites for angiotensin in bovine adrenal peptides also display a much weaker ability to displace labeled zona fasciculata cells therefore have both remarkable similari- All or AIII. We have no explanation for the marked discrepancy ties to and also important differences from receptors for ACTH between Al binding and activation potencies. Discrepancies and receptors for angiotensin in the zona glomerulosa. There between relative affinity and steroidogenic potency have been appears to be a single class of receptors of angiotensin and its observed, although to a lesser extent, particularly when the po- congeners or analogs. These binding sites fulfill the criteria for sition 5 substituent was changed (25). Also, the time of incuba- true receptors because the binding is specific, saturable, and tion and the temperature are different for these two types of reversible and is followed, within the expected time lag, by a measurements. biological effect, in this case corticosteroid production. Unlike ACTH, the primary factor stimulating steroidogenesis in fas- that of ACTH, this biological effect is not mediated by cyclic

Table 2. Effect ofCa2+ on steroid production induced by 10-1 MAII Steroid production, ng/ml medium Ca2, mM Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5 0.08-0.2 40.03 (23.9) 226.7 (17.8) 107.1 99.3 92.5 0.28-0.38 95.43 (18.75) 386.2 (15.75) 125.8 112.5 101.9 0.53-0.83 110.23 (23.3) 427.7 (20.2) 130.7 125.6 127.4 0.98-1.25 152.25 (38.4) 428.3 (12.3) 130.2 142.2 123.3 1.38-1.4 134.38 (28.1) 435.1 (11.4) - - - 1.75-1.93 102.9 (16.1) 371.1 (26.9) 166.3 169.8 129.4 2.25-2.33 97.77 (16.1) 371.1 (26.9) 166.3 169.8 129.4 2.63-2.85 79.8 (11.8) 301.7 (15.45) 180.7 171.2 98.0 Values are the means ofthree separate determinations ofcorticosteroids released into the medium after 60 min ofincubation oftriplicate samples ofdispersed bovine zona fasciculata cells at 37°C. Values in pa- rentheses are from control experiments in the absence ofAll. Downloaded by guest on September 28, 2021 596 Medical Sciences: Vallotton et al. Proc. Natl. Acad. Sci. USA 78 (1981)

Table 3. Production ofcyclic AMP by bovine adrenal 8. Hepp, R., Grillet, C., Peytremann, A. & Vallotton, M. B. (1977) fasciculata cells 101, 717-725. 9. Peytremann, A., Brown, R. D., Nicholson, W. E., Island, D. P., Cyclic AMP, pmol/ml Liddle, G. W. & Hardman, J. G. (1973)J. Clin. Invest. 52,835-842. Control 14.8 ± 1.2 10. Hunter, W. M. & Greenwood, F. C. (1962) Nature (London) 194, Al 15.38 ± 1.8 495-496. 17.4 ± 3.8 11. Vallotton, M. B. (1971) in Immunological Methods in Endocrinol- des-Asp1-[Ile5]-AI ogy, Hormones andMetabolic Research, eds. Federlin, K., Hales, All 15.18 ± 1.13 C. N. & Kracht, J. (Thieme, Stuttgart, Germany), Suppl. 3, pp. Al 15.14 ± 0.7 94-100. ACTH (1-24) 36 ± 4.8* 12. Kloppenborg, P. W. C., Island, D. P., Liddle, G. W., Michelakis, A. M. & Nicholson, W. E. (1968) Endocrinology 82, 1053-1058. Cells were incubated for 5 min at 370C in the presence (10-6 M) or 13. Sayers, G., Swallow, R. L. & Giordano, N. D. (1971) Endocrinol- absence ofpeptide hormones. Results are the mean (±SEM) offour sep- ogy 88, 1063-1068. arate experiments in which three tubes were incubated for each pep- 14. Leclereq, R., Copinschi, G. & Franckson, J. R. M. (1969) Rev. Fr. tide concentration and cyclic AMP was determined in duplicate by the Etud. Clin. Biol 14, 815-819. method ofGilman (19). 15. Glossmann, H., Baukal, A. J. & Catt, K. J. (1974)J. Biol Chem. * For difference from control, P < 0.0025. 249, 825-834. 16. Capponi, A. M. & Catt, K. J. (1979)J. Biol Chem. 254,5120-5127. 17. Rodbard, D., Munson, P. J. & De Lean, A. (1978) Radioimmu- AMP and, contrary to the effect of angiotensin upon the zona noassay and Related Procedures in Medicine 1977 (International glomerulosa, its effect on the zona fasciculata does not appear to Atomic Energy Agency, Vienna), pp. 469-504. be regulated, at least in vitro, by K+. 18. Rodbard, D. & Hutt, D. M. (1974) Radioimmunoassay and Re- We conclude that glomerulosa and fasciculata cells possess lated Procedures in Medicine (International Atomic Energy distinct receptors for ACTH and angiotensin and distinct me- Agency, Vienna), pp. 165-192. 19. Gilman, A. G. (1970) Proc. Natl Acad. Sci. USA 67, 305-312. diators of these two agonists (27, 31). This allows us to suggest 20. Hepp, R., Grillet, C., Peytremann, A. & Vallotton, M. B. (1976) that they derive from common precursor cells, such as the in- Prog. Biochem. Pharmacol. 12, 41-48. termediary cells described recently, f which in the course ofspe- 21. Devynck, M. A., Pernollet M.-G., Matthews, P. G., Khosla, M. cialization evolve into cells for which either angiotensin (zona C., Bumpus, F. M. & Meyer, P. (1977) Proc. Natl Acad. Sci. USA glomerulosa cells) or ACTH (zona fasciculata) becomes the pri- 74,4029-4032. mary stimulus with the other peptide hormone playing a sec- 22. Saltman, S., Fredlund, P. & Catt, K. J. (1976) Endocrinology 98, or 894-903. ondary vestigial role. 23. Aguilera, G., Capponi, A., Baukal, A., Fujita, K., Hauger, R. & Catt, K. J. (1979) Endocrinology 104, 1279-1285. We are grateful to Miss V. Nicolet for her secretarial assistance. This 24. Douglas, J., Bartley, P. & Catt, K. (1978) Endocrinology 102, work was supported by the Swiss National Science Foundation (Grant 1921-1924. 25. Douglas, J. G., Michailov, M., Khosla, M. C. & Bumpus, F. M. 3.230-0.74 and 3.845-0.77) and the Fondation Suisse de Cardiologie. (1979) Endocrinology 104, 71-75. The collaboration ofDr. J. Durr in setting up the method for separation 26. Aguilera, G. & Catt, K. J. (1979) Endocrinology 104, 1046-1052. ofcell-bound and free radioactivities is acknowledged. The help of Dr. 27. Fujita, K., Aguilera, G. & Catt, K. J. (1979) J. Biol Chem. 254, P. J. Munson in analyzing data is acknowledged with thanks. 8567-8574. 28. Shima, S., Kawashimo, Y. & Harai, M. (1978) Endocrinology 103, McDougall, J. G., Butkus, A., Coghlan, J. P., Denton, D. A., Muller, 1361-1367. J., Oddie, C. J., Robinson, P. M. & Scoggins, B. A. Proceedings ofthe 29. Saruta, T., Cook, R. & Kaplan, N. H. (1972) J. Clin. Invest. 51, 6th International Congress of Endocrinology, Melbourne, 1980 2239-2245. (abstr. 839). 30. Espiner, F. A., Liversley, J. H., Ross, J. & Donald, R. A. (1974) Endocrinology 95, 838-846. 1. Carpenter, C. C. J., Davis, J. 0. & Ayers, C. R. (1961)J. Clin. In- 31. Fakunding, J. L., Chow, R. & Catt, K. J. (1979) Endocrinology vest. 40, 2026-2042. 105,327-333. 2. Slater, J. D. H., Babour, B. H., Henderson, H. H., Casper, A. G. 32. Bing, R. F. & Schulster, D. (1978) Biochem.J. 176, 39-45. T. & Bartter, F. C. (1963)J. Clin. Invest. 42, 1504-1520. 33. Albano, J. D. M., Brown, B. L., Ekins, R. P., Tait, S. A. S. & Tait, 3. Bravo, E. L., Khosla, M. C. & Bumpus, F. M. (1975) Am.J. Phys- J. F. (1974) Biochem.J. 142, 391-400. iol. 228, 110-114. 34. Tait, S. A. S., Tait, J. F., Gould, R. P., Brown, B. L. & Albano, 4. Kaplan, N. M. & Bartter, F. C. (1962)J. Clin. Invest. 41, 715-724. J. D. M. (1974) Steroid Biochem. 5, 775-787. 5. Kaplan, N. M. (1965)J. Clin. Invest. 44, 2029-2039. 35. Haning, R., Tait, S. A. S. & Tait, J. F. (1970) Endocrinology 87, 6. Ames, R. P., Borkowski, A. J., Sicinski, A. M. & Laragh, J. H. 1147-1167. (1965)J. Clin. Invest. 44, 1171-1186. 36. Lobo, M. V., Marusic, E. T. & Aguilera, G. (1978) Endocrinology 7. Bing, R. F. & Schulster, D. (1977)J. Endocrinol 74, 261-272. 102, 1061-1068. Downloaded by guest on September 28, 2021