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, corticosteroids 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 zona glomerulosa cells. To allow a better comparison of the characteristics ofthe re- ceptors in glomerulosa and fasciculata cells, binding-inhibition In addition to stimulating aldosterone 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.