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Preparation and characterization of adrenocortical plasma membrane angiotensin 11 recept~rsl.~ GILLESFORGET AND SEYMOUR HEISLERS De'partemc.nt dc Physiolngie et Plzurnlacologie, Centre Ilospirc~licrklnivcr.\itaire, Universitc; de Sllerbrr~oke,Sller-brooke (Qzle'.) Cancrdn JIH 5hT4
Received Feb~xiary3, 1976 FORGET,G., and HEISLFR,S. 1976. Preparation and characterization of adrenocortical plasma membrane angiotensin I1 receptors. Can. 9. Physiol. Pharmacol. 54. 698-707. A bovine adrenocortical particulate fraction prepared by zonal ultracentrifugation and banding between p" 1.013 and 1.101 in a linear sucrose gradient bound 7.3 times more ["Hlangiotensin I1 (AT,,) per milligram protein than the original homsgenate. Enzyme marker and electron microscope studies indicated that this fraction was largely devoid of mitochondria while being enriched in smooth membranes s f predominantly plasmalemmal origin. The binding of labeled AT,, was maximal after 10 min incubation (22 "C) and r'emained at equilibrium for at least 20 min thereafter. ['H]ATI, binding was completely inhibited by saturating concentrations of nonradioactive ATII. The high-affinity binding site in the preparation had a specific binding capacity of 2.38 pmol.rng-', with an equilibrium constant of 2.36 x 10hM-I. Inhibition-displacement studies with unlabeled AT,, AT,,, AT,, fragments. analogs, and antagonists show that the receptor fraction has the highest affinity for the intact native octapeptide. ACTH and bradykinin had no specific effects on [HMT,, binding. The current study suggests that the receptor fraction may be sf use in a highly sensitive AT,, radioligand assay. FORGET,G . et HEISLER,S. 1976. Preparation and characterization of adrenocortical plasma membrane angiotensin I1 receptors. Can. J . Physiol. Pharmacol. 54, 698-707. Une fraction particu1C.e du cortex surrdnalien bovin isolde par ultracentrif~agationzonale, et se retrouvant entre p" l e 0 8 et 1.101 dans un gradient de sucrose. est capable de lier 7.3 fois plus de ["Hlangiotensine 11 (AT,,) par milligramme de prothine quc l'homoghnat original. L'htude de marqueurs enzymatiqtles et la rnicroscopie Clectronique ont indiquC qtme cette fraction contient trks peu de mitochondries, alors q~a'elleest riche en membranes lisses, B prkdominence plasmalemmique. Idaliaison dlAT,, isotopique atteint un maximum aprks 10 min d'incubation (22 " C ) ,et reste ensuite h l'kquilibre pour au moins 20 min. La liaison de [W]ATIIapeut itre compli!tement inhibke par l'addition d'une quan1itC saturante d'AT,, nonradioactive. 1-e site de liaison 2 haute afinit6 de la prkparation, dont la constante d'equilibre est de 2.36 x 10" M ' a une activitC specifique de liaison cgale 2 2.38 p m o l - m g Les Ctudes d'inhibition-dkplacement avec 17ATI, l'AT,,, des fragments, analogues et antagonistes non isotopiques de ]'ATrI dCmontrent que la fraction-liante a sa plus grande afinit6 pour l'octapeptide naturel. L'ACTH et la bradykinine n'ont pas d'effet spicifique sur la liaison de ["HJAT,,. Les Ctudes dCcrites ici suggkrent que la fractionrkcepteur pourrait Ctre utilisCe dans une mkthode de dosage trks sensible de l7ATI1.
Introduction ATII is a physiologically important stimulator of adrenocortical steroid secretion (Genest et al. AUUKI VIATIONS: ATII, angiotensin TI; S'AMP, adenosine monophosphoric acid; cyclic AMP, adenosine 3',5'-cyclic monosphosphoric acid; AC'PH, adrenocorticotropin; AT,, angiotensin I. 'Supported by a grant to Dr. S. Heisler from the Quebec Heart Foundation and the Medical Research Council of Canada. "Some of the results from this study were presented at the 19th annual meeting of the Canadian Federation of Biological Societies, Halifax 1976. "Present address: Departement de Pharmacslogie, UniversitC Laval, CitC Universitaire, Quibec (QuC.) Canada C 1K 7P4.
1960; Ganong et al. 1962; Kaplan and Bartter 1962; Dufau and Kliman 1968; Peytremann et al. 1973; Forget et al. 1975). According to current understanding, measurable hormoneinduced effects are initiated by the interaction of the hormone with specific receptor sites. Studies by Arakawa et al. (1962) and Slneby (1963), suggest that ATII receptor sites are localized in cell membranes of target tissues, since responses to polymerized ATII or to ATII covalently linked through its N-terminus to a gamma globulin, are identical with those produced by ATr, itself. With respect to adrenocol-tical ATII receptors, early studies (Bumpus et al. 1964) indicated that the rat gland as a
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whole was capable of taking up [:iH]ATIIirz vivo. In more recent studies, Glossman et al. (19741, Catt c2r ctl. (19741, and Saltman et al. ( 1 975 1 have localized these specific ArFll binding sites to subcelHerlar fractions c ~ frat or bovine adrenal cortex, sedimentinag between 200 and 20 000 X g. The present study was directed toward the bulk preparation of adrenocortical plasma membranes capable of binding AT,,, with a view towards subsequent purification and solubilization of the hormone-binding entity. Methods and Materials prepurcrkion of ATII Billding Fructions Ten to 12 bovine adrenal glands were obtained 1530 niin post-mortem. and were stored on ice after removal of the surrounding adipose tissue. Each gland was hissected and demedullated; thc cortex was separated from the capsule with a scalpel, and kept at 4 " C in 0.25 M sucrose buffered at pH 7.4 with 1 rn-l4 NaHC03. The cortex was washed over two layers of cheesecloth, weighed, and homogenized in buffered sucrose ( 1:9, w/v) in a Bouncc homogenizer with 15 up and down strokes of the loose pestle. The suspension was filtered over two layers of cheesecloth and the residue washed with 5 rnl of buffered sucrose. The filtrate was homogenized with a single stroke of the tight pestle. The homogenate was centrifuged at 400 x g for 10 niin, and the supernatant was kept at 4 " C until injected directly into the core of a JCF-7; zonal rotor (Beckman Instruments, Palo Alto, CA) turning at 3000 rpm and containing 800 ml of a linear sucrose gradient (96096, w i w ) , over a 1. l-litre i~nderlayof 60% sucrose. The rotor was spun at 15 000 rprn for a period of 2 h, decelerated to 3000 rprn, and 12.5-ml fractions collected by displacement with 60% sucrose. The fractions banding between p2n 1.08 and 1.101 were diluted to 8.59% sucrose with water and centrifuged at 105 000 X g for 60 min. The pellets were resuspended gently with 0.25 1%4buffered sucrose using a Teflon pestle and glass homogenizer. Protein content was determined (Lowry et u1. 1951) and fractions were used either immediately (enzyme marker studies) o r stored in liquid nitrogen for subsequent binding studies. No loss of AT,, binding activity was observed in samples frozen for 1 month. Enzylne Marker SStztdies Marker-enzyme activities were assayed on the fractions collected fron-a the isopycnic gradient. 5'Nucleotidase (EC 3.1.3.5) was measured according to Emmelot et ul. (1964) with the following modifications. The reaction was initiated by addition of membrane protein to a buffer containing 50 mM Tris-HC1 ( p H 7.4), 5 mM MgCI,, 5 mM 5'-AMP, and 10 m M sodium-potassium tartrate (final volunle 600 p l ) . The reaction was allowed to proceed for 20 min at 37 "C and was stopped by addition of 1.4 ml 10% trichloro-
acetic acid. After centrifugation of the suspension, the supernatants were assayed for inorganic phosphate by thc method of 'faussky and Shorr (1953). Results are expressed as micromoles Pi produced per milligram per minute. Sraccinic dehydrogenase (EC 1.3.99-1) was assayed according to Maeno et ul. (1971 ) and expressed as AODWK, per milligram per minute. Cytochrome c oxidase (EC 1.9.3.1 ) was assayed by the method of Cooperstein and Lazarow ( 1951) and the results expressed as AODj50per milligram per minute. NADH cytochrome c reductase (EC 1.6.99.3) was assayed by the method of Ernster et al. (1962) and the results per , ~milligram per minute. expressed as L I O D ~ Adenylate cyclase (EC 4.6.1.1 ) activity was measured over 10 min at 37 "C in a buffer consisting of 50 rnM Tris-HCl (pH 7.4)- 5 mM MgCl,, 50 mM KC], 18 inM theophylIine, 1 mM ATP, 4 0 mM phosphoenol pyruvate, 8 U of pyruvate kinase (EC 2.7.1.40), and 0 - 1 0 0 p g of fractional protein in a final volume of 200 p1. Reactions were started by addition of fractional protein and terminated with 200 pl of 10% trichloroacetic acid. The resulting siaspensions were centrifuged and the supernatants extracted three times with five volumes of water-saturated ether. The samples were warmed at 60 "G to remove residual ether and 40- 100p1 aliquots were used to quantitate cyclic AMP formed. Controls to which no protein was added or to which protein was added after acidification were run simultaneously. The nucleotide was measured using a cyclic-AMP binding protein isolated hy the method of Tsang ef al. (1972). The reaction mixture was made by adding the following to small tulses in the order: 1 ml of 75 m M Na-acetate buffer (pH 4.0), 100 p1 of cyclic AMP standard (0.5-3.0 gmol) or sample, cyclic ["HIAMP ( 100 p1 containing 2.44 x lo4 dprn ) , bovine serum albumin (4.5 mg/200 pl ) , and finally 100 p1 of binding protein. After incubation for 30 min in ice, the reaction was ended by filtration over Millipore (0.45 p m ) ; the filters were dried, dissolved, and counted in a liquid scintillation spectrometer. Results are expressed as picomoles cyclic AMP formed per milligram per minute.
-
ATII B i d i n g Studics Fractions (50-150 pg) were incubated for I 0 min at 22 " C in a final volume of 0.17 ml, containing 100 mLM Tris-HGl (pH 7.4), 120 ITIM NaC1, and 5 x 10 M [.'HIAT,, (specific activity 24 and 40 Ci/mmol) csntaining bacitracin (0.0 1 9% )I. Test agents were added in 30 p1 aliquots. The incubation was terminated by quickly adding 4 ml of ice-cold NaC1-Tris buffer and filtering over Millipore (0.45 p m ) . The incubation tube was washed with 4 n ~buffer, l and the filter washed with this and an additional 4 ml of buffer. The filters were dried and dissolved in 10 rnl Bray's scintillation medium and counted. Controls were run simultaneously to eliminate nonspecific adsorption of [%]AT,, to the filter, and nonspecific binding of hormone to fractional protein ( by including 1.7 x 10 ' M unlabelled ATlI in the control media). Nonspecific adsorption to fractional protein or filters was about 1% of total isotope used; these values were subtracted from total binding @
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CAW. J. PHYSIOL. PKAWM'ACOL. VOL. 54, 1976
Fractlon n u m b e r
F r a c ( i ( > ~ in u m b e r
Fre;. 1. Distribution of protein, cytochrome c oxidase, and sraccinic dehydrogenase activities in a linear (9--6O%, w/w ) sucrose gradient.
values. Results are expressed as ferntomoles AT,, bolmnd per milligram protein.
E~PCPTOII M i c r ~ ~ c o pStudies ic Pellets obtained after centrifugation at 3105 808 x g were fixed in 3.0% glutaraldehyde solution, postfixed in osn~icacid (Palade 31952), and embedded in Epon (Luft 1961 ). Fine sections were stained with lead monoxide (Karnovsky 3196 I ;method A ) , and visualized with a Phillips electron microscope. Stat istics
Marker enzyme and binding studies were performed in duplicate and triplicate, respectively, on a minimum of two difTerent preparations. Student's t test was used as a measure of statistical significance. material.^ Asp1-Ile"AT,, and ACTH (porcine pituitary 250 IU/mg) were obtained from Schwarz Mann (Orangeburgh, NY). AT,, [Sarl]AT,,, [LeuR]AT,,, des-Asp1AT,,, des-Pheh-AT,,. Asp-Arg-Val-Tyr, Ile-His-ProPhe, and bradykinin were generous gifts from Dr. W. K. Park, of orar department. [Tyro.~yl-.?.5-~H (N)]AT,, and cyclic [G-'HIAMP (30 Ci/mmol) were obtained from New England Nuclear, Boston, MA. The following compasrsnds were purchased from Signla Chemicals (St. Louis, M 6 ) : cyclic AMP, 5'-
Fro. 2. Distribution of protein and 5'-nucleotidase activity in a linear (9-60%, w/w) sucrose gradient. AMP, ATP (Tris salt), bovine scrum albumin (fraction V ) , cytochron~ec (type IIT), 2,6-dichloroindophenol (Wa-salt), NADH, phosphoenolpyruvate, pyruvate kinase, theophylline, Tris-HCl. and Trizma base. Flurarna3, Roche (6phenyl-spiro[furan-2 (3H), I 'phtalanl-3,3'dione) was obtained from Fisher Scientific (Montreal, P.Q.), as was sucrose and all other reagent grade chemicals,
Results E n z y l - ~A4arkc.r a~ Studic.s The proteins in the gradient, as illustrated in Figs. 1-3, were principally segregated in a single Barge peak at a concentration of sucrose equal to about 35 5% ( p m I. 3 5 3 ) . This peak contained high specific activities of succinic dehydrogeaaase and cytoshrome c oxidase, the two mitochondrial rnarkcrs tested (Fig. I ), while the zone banding at p2" 1,08-1.181 sucrose had specific activities 9- and I I-fold lower (succinic dehydrogenase and cytochromc c oxidasc? respectively) than those found in homogenatss (Table 1 ) . I n boala cases, the total enzyme activity (yield) present in this latter zone was less than 0.2% of that in the original komogmate. 5'-Nucleotidase activity was more uniformly
FORGET AND HEISLEK 9
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'TABLE I . Characteriration of a bovine adrenc~corticalmembrane fraction banding between a sucrow cten4ity of p" 1 .08 and 1 .I01 Specific activity E n ~ y m emarker
Hornogenate
Adeny late cyclase, pmoI cyclic A M P formed . mg ( ( 1 ) basal (6) NaF (10 -2 [If)stirnrilated
-1
Yield,
Membranes
09 /i 1
. 1 0 m~n-1
Cytochrome c c~xidase, A O D ~- ~mg-1 , -r-nin-1 Succinic dchydrogenase, AOH),,, .rng-l- min-1
1 .60$_:0.10
0.1850.04
0.19 i 0 . 0 1
NADH cytochrome c reductase, ~OP),~,.rng-l.min
1.40-t0.01
2.70k0.05
2.80k0.30
Nors: Values are means
+
S E M of at least Four observations (yield i s calculated rclntivc t o the homogenate
ztx,
-( -
.=
LOOTA).
times relative to the hornogenate in the membrane fraction studicd (Table 1 ) . NADH cytochrome c reductase activity was found throughout the gradient (Fig. 3 ) . The pa) 1.Q8-1.101 sucrose band exhibited an approximate twofold incrcasc in this enzymic activity ccmpared with the specific activity in the horncjgenate. but contained less than 4% of the original honsogenate activity (Table 1 ) . The protein yield of the fractions banding at p2" 1 -08-1.10 1 sucrose was 1.8 % of the starting material in homogenates. This is equivalent to 4.1 1-: 0.6 mg protein per gland (n 7).
C
-
U
loo 2
O
10
20
30
40
00
60
70
f rdcrlall i l ~ ~ n i h c r
PIC;.3. Distribution of protein and NABH cytochrome c reductase activity in a linear (9-GO%, w/w) sucrose gradient.
Electron Microscopic Studies Electron microscopic observation of the fixed pelleted fraction shows it to be mainly composed of small to large smooth-membrane vesicles (Fig. 4 ) . No nuclei, mitochondria, or free or attached ribosomes were observed in this fraction.
Binding Studies AT,, binding studies demonstrated that the protein fraction in pa) 1.08-1.101 sucrose specidistributed in the gradient (Fig. 2 ) ; however, a fically bound 7.3 times more [3H]ATIIthan the maxiinum was observed in the pal 1.08-1 -101 homogenate, and 7.6 times more than the fracsucrose band; here, the specific activity was 3.4 tion corresponding to the mitochondria1 enzyme times higher than in the homogcnatc; the yield marker peak. For the homogenate, the p3' 1.08was 5.9 96 (Table 1 ) . The area corrcspondirlg 1.10 1 fraction, and the mitochondria1 fraction to the mitochondria1 peak did not contain the respective amounts of octapeptide bound significant 5'-nucleotidase activity. Basal activ- (ferntomoles bound per milligram 2 SEM) 117 (13% ity of aderlylate cyclase, the second plasma were 213.4 -+ 16.5, 1547.1 membrane marker tested, was increased 2.4 yield), and 204.6 2 25.7. All subsequent
*
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702
CAN. J.
PHYSIOE. PHAWMACOL.
VCPI,. 54, 1976
PIG.4. Electron micrograph of a peileted, fixed mernbrane fraction banding at in a linear (9-6895, w/w )l sucrose gradient ( x 19 600).
binding studies were performed only with the pa) 1.08-1 .I01 fraction, which we have terxned 'the ATIIparticulate-binding fraction.' Binding fraction preparations, boiled in a water bath at 100 "C for 3 mima, lost their capa-
p"
L08-1.101
city to specifically bind [:5H]A%II.Binding sf the labeled hormone was linear to at least 200 pg of fractional protein. The time required for completion 0%binding of ATIIto the particulate fraction is illustrated
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FOlICrET AND MIXISL,EK
L
0
I
10
I
eo
i
30
1 BME: ('imin) FIG.5. Time course of ['HIAT,, ( 5 x 10 " M ) binding to a ~ n e n ~ b r a nfraction e derived from bovine adrenal cortex. Values are means -t- SEM of six observations.
in Fig. 5. Specific binding was, statistically, at equilibrium after 7.5 ~ n i nof incubation; maximal binding was observed at 10 min. Specific binding of [:{H]ATIIto the receptor fraction was rapidly reversed by the addition of an excess amount of unlabelcd AT,, when binding equilibrium had been reached (Fig. 6).
703
The aflinity of [:3HqATIIfor binding in the particulate fraction is presented graphically in Fig. 7 in a Scatchard ( 1949) plot. Thc curve was analyzed according to the method of Chamilcss and McGuire (1975). The highaf'fimity binding sitc had a binding capacity ( N I) of 2.38 pmol . mg-I, and an ecluilibriurn constant K l of 2.36 X 1O8 M - l . The affinity for binding of ATI. unlabeled A?',,, and several KTI,fragrneilts viz. des-AspdAT,,, des-Phe"ATII, ( Asp-Arg-Val-Tyr) , and ( Tle-His-Pro-Phe ) was tested in inhibitiondisglace~nent studies. The above agents werc added simultaneously with ["]AT,,. RcsuIts are illustrated in Fig. 63. Bound [SH]ATIpwas, with exception of the two tetrapcptides, displaced cor~lpletelyupon addition of all thc substances tested: the displacement curves were parallel. The coilcentrations of ligands required to displace 50% of the bound [:3W1ATI, are presented in Table 2. Des-Asp1-AT,, was about 8 % as effective as unlabeled ATII (considered 100%) effective) in displacing the bound isotopic ligand. AT, was 1% effective and desPhe"ArI',, only 0.25 9% effective. Thc specificity and affinity of binding of [:W1ATI1 \.\;as further tested by using bradykinin, ACTH, [Sarl]ATIr, [Leuk]ATI1, and a fluorescanline dcrivativc of ATIP (Forget c9t uk. 1975) in another series of inhibition-displace-
FIG.6. Displacement of bound 13HIATII (5 x 10 ' 1rM) by excess unlabeled AT,, (1.7 x 10 M i added at binding equilibrium (arrowhead). Data fronl a representative experiment.
C A N . J . P N Y S I O L . P H A R M A C O L . VOL. 5 4 . 1976
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TABLE 2. Effects of ATI, ATI1,and various ALPI1 analogs, antagonists, and fragments on the binding of [%]ATII to adrenocortical particulate binding fraction
Compound
Half-maximal displacement of [3H]ATI, binding, n M
plasmic reticular contaminants than other preparations described to date (Glossman et a/. 1974; Saltman et a/. 1975). The current data suggest that the binding sites for AT,, are concentrated on the plasma membrane of cells from the bovine adrenal cortex, as previously suggested by other workers (Arakawa et al. 1962; FIG.7. Scatchard plot of AT,, binding to a particu- Smeby 1963; Catt et a/. 1974). late membrane fraction derived from bovine adrenal The rapidity with which binding occurs and cortex. Observed values, ; corrected values, 0.K1 = the displacement of isotopic ATII by the nonhigh-affinity equilibrium constant (2.36 x 10"M - ' ) ; radioactive octapeptidc indicates a possible N1 = high-affinity binding capacity (2.38 x lo-'' mol mg-') . relationship between the binding sites in the particulate fraction banding between sucrose 1.OR and 1.101 and the'physiological ATII ment studies. The results obtained are presented receptor. The equilibriuin constant of the highin Fig. 9. Neither bradykinin nor ACTH had affinity binding site is similar to the constant significant effects on ["]ATII binding in condetermined by Glossman and coIlaborators centrations lower than 1 0 - W . In higher con(1974) using a 20 000 g sediment from bovine centrations, however, both could inhibit ["HIATII binding, though not completely, and not adrenal cortex homogenate. When these authors in a parallel fashion. Binding of [:SH]AT,I was further fractionated their 20 000 (: pellet on a coizlpletely inhibited by [Sarl]AT,,, [Leu8]ATII, discontinuous sucrose gradient, they obtained and fluorescarnine-ATII. [Sarl]ATII was four an ATII binding fraction with a similar K I but with a two- to three-fold increased binding times less effective than ATII in displacing capacity. The binding capacity of this preparalabeled ligand (Table 2 ) ; [Leu8]ATI, was nine tion was similar to that observed with the prepatimes less effective, and fluorescamine-ATII, 9.5 ration currently studied, i.e., zonal ultracentiines less effective. trifugation reduced the time necessary to produce a preparation that was purer, and had Discussion similar ATII binding affinity and capacity with The specific activities of 5'-nucleotidase and respect to other preparations. adenylate cyclase are higher in the ATII particuATII displacement studies undertaken with late binding fraction than in the homogenate, analogs and fragments of the octapeptide and indicating a relative purification of plasma mem- the decapeptide ATI indicate that none of the branes. These findings are also supported by peptides tested displaces ["]ATII as effectively electron inicroscopic observation of the fraction, as unlabeled ATII; however, with the exception which was found to be mainly con~posed of of the two tetrapeptides, the other peptides tested smooth-membrane vesicles of various sizes. The displaced ATIIin a parallel manner, thereby indifraction contains fewer mitochondria1 and endo- cating a competitive process at the receptor level.
p2~
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FORGET AND HEISLER
FIG.8. Inhibition-dispIacenlent of ["MATlI binding to a particulate, membrane fraction derived from bovine adrenal cortex. 0, Unlabeled AT,,; @, des-Aspf-AT,,; n, AT,; A , desLeu8-ATII;A, Asp-Arg-Val-Tyr; W , Ile-His-Pro-Phe. Values are means -F- SEM of six observations.
The displacement of ["HIAT,, by the ( 1-4) and (5-8) tetrapeptides of ATII is not appreciable. These fragments can be derived from the action of trypsin on the ATII molecule but their minimal effect on 13H]ATlI binding would tend to indicate that if cIeavage of ATII by trypsin-like enzymes in the preparation were to occur, the products of the reaction would not interfere with binding of the labeled octapeptide. The marked inability of des-PheR-ATII to displace L3H]ATII from its binding sites similarly indicates that the possible production of this ATII metabolite, as a result of the presence in the preparation of endogenous carboxypeptidases, would not interfere with [:
Preparation and characterization of adrenocortical plasma membrane angiotensin 11 recept~rsl.~ GILLESFORGET AND SEYMOUR HEISLERS De'partemc.nt dc Physiolngie et Plzurnlacologie, Centre Ilospirc~licrklnivcr.\itaire, Universitc; de Sllerbrr~oke,Sller-brooke (Qzle'.) Cancrdn JIH 5hT4
Received Feb~xiary3, 1976 FORGET,G., and HEISLFR,S. 1976. Preparation and characterization of adrenocortical plasma membrane angiotensin I1 receptors. Can. 9. Physiol. Pharmacol. 54. 698-707. A bovine adrenocortical particulate fraction prepared by zonal ultracentrifugation and banding between p" 1.013 and 1.101 in a linear sucrose gradient bound 7.3 times more ["Hlangiotensin I1 (AT,,) per milligram protein than the original homsgenate. Enzyme marker and electron microscope studies indicated that this fraction was largely devoid of mitochondria while being enriched in smooth membranes s f predominantly plasmalemmal origin. The binding of labeled AT,, was maximal after 10 min incubation (22 "C) and r'emained at equilibrium for at least 20 min thereafter. ['H]ATI, binding was completely inhibited by saturating concentrations of nonradioactive ATII. The high-affinity binding site in the preparation had a specific binding capacity of 2.38 pmol.rng-', with an equilibrium constant of 2.36 x 10hM-I. Inhibition-displacement studies with unlabeled AT,, AT,,, AT,, fragments. analogs, and antagonists show that the receptor fraction has the highest affinity for the intact native octapeptide. ACTH and bradykinin had no specific effects on [HMT,, binding. The current study suggests that the receptor fraction may be sf use in a highly sensitive AT,, radioligand assay. FORGET,G . et HEISLER,S. 1976. Preparation and characterization of adrenocortical plasma membrane angiotensin I1 receptors. Can. J . Physiol. Pharmacol. 54, 698-707. Une fraction particu1C.e du cortex surrdnalien bovin isolde par ultracentrif~agationzonale, et se retrouvant entre p" l e 0 8 et 1.101 dans un gradient de sucrose. est capable de lier 7.3 fois plus de ["Hlangiotensine 11 (AT,,) par milligramme de prothine quc l'homoghnat original. L'htude de marqueurs enzymatiqtles et la rnicroscopie Clectronique ont indiquC qtme cette fraction contient trks peu de mitochondries, alors q~a'elleest riche en membranes lisses, B prkdominence plasmalemmique. Idaliaison dlAT,, isotopique atteint un maximum aprks 10 min d'incubation (22 " C ) ,et reste ensuite h l'kquilibre pour au moins 20 min. La liaison de [W]ATIIapeut itre compli!tement inhibke par l'addition d'une quan1itC saturante d'AT,, nonradioactive. 1-e site de liaison 2 haute afinit6 de la prkparation, dont la constante d'equilibre est de 2.36 x 10" M ' a une activitC specifique de liaison cgale 2 2.38 p m o l - m g Les Ctudes d'inhibition-dkplacement avec 17ATI, l'AT,,, des fragments, analogues et antagonistes non isotopiques de ]'ATrI dCmontrent que la fraction-liante a sa plus grande afinit6 pour l'octapeptide naturel. L'ACTH et la bradykinine n'ont pas d'effet spicifique sur la liaison de ["HJAT,,. Les Ctudes dCcrites ici suggkrent que la fractionrkcepteur pourrait Ctre utilisCe dans une mkthode de dosage trks sensible de l7ATI1.
Introduction ATII is a physiologically important stimulator of adrenocortical steroid secretion (Genest et al. AUUKI VIATIONS: ATII, angiotensin TI; S'AMP, adenosine monophosphoric acid; cyclic AMP, adenosine 3',5'-cyclic monosphosphoric acid; AC'PH, adrenocorticotropin; AT,, angiotensin I. 'Supported by a grant to Dr. S. Heisler from the Quebec Heart Foundation and the Medical Research Council of Canada. "Some of the results from this study were presented at the 19th annual meeting of the Canadian Federation of Biological Societies, Halifax 1976. "Present address: Departement de Pharmacslogie, UniversitC Laval, CitC Universitaire, Quibec (QuC.) Canada C 1K 7P4.
1960; Ganong et al. 1962; Kaplan and Bartter 1962; Dufau and Kliman 1968; Peytremann et al. 1973; Forget et al. 1975). According to current understanding, measurable hormoneinduced effects are initiated by the interaction of the hormone with specific receptor sites. Studies by Arakawa et al. (1962) and Slneby (1963), suggest that ATII receptor sites are localized in cell membranes of target tissues, since responses to polymerized ATII or to ATII covalently linked through its N-terminus to a gamma globulin, are identical with those produced by ATr, itself. With respect to adrenocol-tical ATII receptors, early studies (Bumpus et al. 1964) indicated that the rat gland as a
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whole was capable of taking up [:iH]ATIIirz vivo. In more recent studies, Glossman et al. (19741, Catt c2r ctl. (19741, and Saltman et al. ( 1 975 1 have localized these specific ArFll binding sites to subcelHerlar fractions c ~ frat or bovine adrenal cortex, sedimentinag between 200 and 20 000 X g. The present study was directed toward the bulk preparation of adrenocortical plasma membranes capable of binding AT,,, with a view towards subsequent purification and solubilization of the hormone-binding entity. Methods and Materials prepurcrkion of ATII Billding Fructions Ten to 12 bovine adrenal glands were obtained 1530 niin post-mortem. and were stored on ice after removal of the surrounding adipose tissue. Each gland was hissected and demedullated; thc cortex was separated from the capsule with a scalpel, and kept at 4 " C in 0.25 M sucrose buffered at pH 7.4 with 1 rn-l4 NaHC03. The cortex was washed over two layers of cheesecloth, weighed, and homogenized in buffered sucrose ( 1:9, w/v) in a Bouncc homogenizer with 15 up and down strokes of the loose pestle. The suspension was filtered over two layers of cheesecloth and the residue washed with 5 rnl of buffered sucrose. The filtrate was homogenized with a single stroke of the tight pestle. The homogenate was centrifuged at 400 x g for 10 niin, and the supernatant was kept at 4 " C until injected directly into the core of a JCF-7; zonal rotor (Beckman Instruments, Palo Alto, CA) turning at 3000 rpm and containing 800 ml of a linear sucrose gradient (96096, w i w ) , over a 1. l-litre i~nderlayof 60% sucrose. The rotor was spun at 15 000 rprn for a period of 2 h, decelerated to 3000 rprn, and 12.5-ml fractions collected by displacement with 60% sucrose. The fractions banding between p2n 1.08 and 1.101 were diluted to 8.59% sucrose with water and centrifuged at 105 000 X g for 60 min. The pellets were resuspended gently with 0.25 1%4buffered sucrose using a Teflon pestle and glass homogenizer. Protein content was determined (Lowry et u1. 1951) and fractions were used either immediately (enzyme marker studies) o r stored in liquid nitrogen for subsequent binding studies. No loss of AT,, binding activity was observed in samples frozen for 1 month. Enzylne Marker SStztdies Marker-enzyme activities were assayed on the fractions collected fron-a the isopycnic gradient. 5'Nucleotidase (EC 3.1.3.5) was measured according to Emmelot et ul. (1964) with the following modifications. The reaction was initiated by addition of membrane protein to a buffer containing 50 mM Tris-HC1 ( p H 7.4), 5 mM MgCI,, 5 mM 5'-AMP, and 10 m M sodium-potassium tartrate (final volunle 600 p l ) . The reaction was allowed to proceed for 20 min at 37 "C and was stopped by addition of 1.4 ml 10% trichloro-
acetic acid. After centrifugation of the suspension, the supernatants were assayed for inorganic phosphate by thc method of 'faussky and Shorr (1953). Results are expressed as micromoles Pi produced per milligram per minute. Sraccinic dehydrogenase (EC 1.3.99-1) was assayed according to Maeno et ul. (1971 ) and expressed as AODWK, per milligram per minute. Cytochrome c oxidase (EC 1.9.3.1 ) was assayed by the method of Cooperstein and Lazarow ( 1951) and the results expressed as AODj50per milligram per minute. NADH cytochrome c reductase (EC 1.6.99.3) was assayed by the method of Ernster et al. (1962) and the results per , ~milligram per minute. expressed as L I O D ~ Adenylate cyclase (EC 4.6.1.1 ) activity was measured over 10 min at 37 "C in a buffer consisting of 50 rnM Tris-HCl (pH 7.4)- 5 mM MgCl,, 50 mM KC], 18 inM theophylIine, 1 mM ATP, 4 0 mM phosphoenol pyruvate, 8 U of pyruvate kinase (EC 2.7.1.40), and 0 - 1 0 0 p g of fractional protein in a final volume of 200 p1. Reactions were started by addition of fractional protein and terminated with 200 pl of 10% trichloroacetic acid. The resulting siaspensions were centrifuged and the supernatants extracted three times with five volumes of water-saturated ether. The samples were warmed at 60 "G to remove residual ether and 40- 100p1 aliquots were used to quantitate cyclic AMP formed. Controls to which no protein was added or to which protein was added after acidification were run simultaneously. The nucleotide was measured using a cyclic-AMP binding protein isolated hy the method of Tsang ef al. (1972). The reaction mixture was made by adding the following to small tulses in the order: 1 ml of 75 m M Na-acetate buffer (pH 4.0), 100 p1 of cyclic AMP standard (0.5-3.0 gmol) or sample, cyclic ["HIAMP ( 100 p1 containing 2.44 x lo4 dprn ) , bovine serum albumin (4.5 mg/200 pl ) , and finally 100 p1 of binding protein. After incubation for 30 min in ice, the reaction was ended by filtration over Millipore (0.45 p m ) ; the filters were dried, dissolved, and counted in a liquid scintillation spectrometer. Results are expressed as picomoles cyclic AMP formed per milligram per minute.
-
ATII B i d i n g Studics Fractions (50-150 pg) were incubated for I 0 min at 22 " C in a final volume of 0.17 ml, containing 100 mLM Tris-HGl (pH 7.4), 120 ITIM NaC1, and 5 x 10 M [.'HIAT,, (specific activity 24 and 40 Ci/mmol) csntaining bacitracin (0.0 1 9% )I. Test agents were added in 30 p1 aliquots. The incubation was terminated by quickly adding 4 ml of ice-cold NaC1-Tris buffer and filtering over Millipore (0.45 p m ) . The incubation tube was washed with 4 n ~buffer, l and the filter washed with this and an additional 4 ml of buffer. The filters were dried and dissolved in 10 rnl Bray's scintillation medium and counted. Controls were run simultaneously to eliminate nonspecific adsorption of [%]AT,, to the filter, and nonspecific binding of hormone to fractional protein ( by including 1.7 x 10 ' M unlabelled ATlI in the control media). Nonspecific adsorption to fractional protein or filters was about 1% of total isotope used; these values were subtracted from total binding @
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CAW. J. PHYSIOL. PKAWM'ACOL. VOL. 54, 1976
Fractlon n u m b e r
F r a c ( i ( > ~ in u m b e r
Fre;. 1. Distribution of protein, cytochrome c oxidase, and sraccinic dehydrogenase activities in a linear (9--6O%, w/w ) sucrose gradient.
values. Results are expressed as ferntomoles AT,, bolmnd per milligram protein.
E~PCPTOII M i c r ~ ~ c o pStudies ic Pellets obtained after centrifugation at 3105 808 x g were fixed in 3.0% glutaraldehyde solution, postfixed in osn~icacid (Palade 31952), and embedded in Epon (Luft 1961 ). Fine sections were stained with lead monoxide (Karnovsky 3196 I ;method A ) , and visualized with a Phillips electron microscope. Stat istics
Marker enzyme and binding studies were performed in duplicate and triplicate, respectively, on a minimum of two difTerent preparations. Student's t test was used as a measure of statistical significance. material.^ Asp1-Ile"AT,, and ACTH (porcine pituitary 250 IU/mg) were obtained from Schwarz Mann (Orangeburgh, NY). AT,, [Sarl]AT,,, [LeuR]AT,,, des-Asp1AT,,, des-Pheh-AT,,. Asp-Arg-Val-Tyr, Ile-His-ProPhe, and bradykinin were generous gifts from Dr. W. K. Park, of orar department. [Tyro.~yl-.?.5-~H (N)]AT,, and cyclic [G-'HIAMP (30 Ci/mmol) were obtained from New England Nuclear, Boston, MA. The following compasrsnds were purchased from Signla Chemicals (St. Louis, M 6 ) : cyclic AMP, 5'-
Fro. 2. Distribution of protein and 5'-nucleotidase activity in a linear (9-60%, w/w) sucrose gradient. AMP, ATP (Tris salt), bovine scrum albumin (fraction V ) , cytochron~ec (type IIT), 2,6-dichloroindophenol (Wa-salt), NADH, phosphoenolpyruvate, pyruvate kinase, theophylline, Tris-HCl. and Trizma base. Flurarna3, Roche (6phenyl-spiro[furan-2 (3H), I 'phtalanl-3,3'dione) was obtained from Fisher Scientific (Montreal, P.Q.), as was sucrose and all other reagent grade chemicals,
Results E n z y l - ~A4arkc.r a~ Studic.s The proteins in the gradient, as illustrated in Figs. 1-3, were principally segregated in a single Barge peak at a concentration of sucrose equal to about 35 5% ( p m I. 3 5 3 ) . This peak contained high specific activities of succinic dehydrogeaaase and cytoshrome c oxidase, the two mitochondrial rnarkcrs tested (Fig. I ), while the zone banding at p2" 1,08-1.181 sucrose had specific activities 9- and I I-fold lower (succinic dehydrogenase and cytochromc c oxidasc? respectively) than those found in homogenatss (Table 1 ) . I n boala cases, the total enzyme activity (yield) present in this latter zone was less than 0.2% of that in the original komogmate. 5'-Nucleotidase activity was more uniformly
FORGET AND HEISLEK 9
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'TABLE I . Characteriration of a bovine adrenc~corticalmembrane fraction banding between a sucrow cten4ity of p" 1 .08 and 1 .I01 Specific activity E n ~ y m emarker
Hornogenate
Adeny late cyclase, pmoI cyclic A M P formed . mg ( ( 1 ) basal (6) NaF (10 -2 [If)stirnrilated
-1
Yield,
Membranes
09 /i 1
. 1 0 m~n-1
Cytochrome c c~xidase, A O D ~- ~mg-1 , -r-nin-1 Succinic dchydrogenase, AOH),,, .rng-l- min-1
1 .60$_:0.10
0.1850.04
0.19 i 0 . 0 1
NADH cytochrome c reductase, ~OP),~,.rng-l.min
1.40-t0.01
2.70k0.05
2.80k0.30
Nors: Values are means
+
S E M of at least Four observations (yield i s calculated rclntivc t o the homogenate
ztx,
-( -
.=
LOOTA).
times relative to the hornogenate in the membrane fraction studicd (Table 1 ) . NADH cytochrome c reductase activity was found throughout the gradient (Fig. 3 ) . The pa) 1.Q8-1.101 sucrose band exhibited an approximate twofold incrcasc in this enzymic activity ccmpared with the specific activity in the horncjgenate. but contained less than 4% of the original honsogenate activity (Table 1 ) . The protein yield of the fractions banding at p2" 1 -08-1.10 1 sucrose was 1.8 % of the starting material in homogenates. This is equivalent to 4.1 1-: 0.6 mg protein per gland (n 7).
C
-
U
loo 2
O
10
20
30
40
00
60
70
f rdcrlall i l ~ ~ n i h c r
PIC;.3. Distribution of protein and NABH cytochrome c reductase activity in a linear (9-GO%, w/w) sucrose gradient.
Electron Microscopic Studies Electron microscopic observation of the fixed pelleted fraction shows it to be mainly composed of small to large smooth-membrane vesicles (Fig. 4 ) . No nuclei, mitochondria, or free or attached ribosomes were observed in this fraction.
Binding Studies AT,, binding studies demonstrated that the protein fraction in pa) 1.08-1.101 sucrose specidistributed in the gradient (Fig. 2 ) ; however, a fically bound 7.3 times more [3H]ATIIthan the maxiinum was observed in the pal 1.08-1 -101 homogenate, and 7.6 times more than the fracsucrose band; here, the specific activity was 3.4 tion corresponding to the mitochondria1 enzyme times higher than in the homogcnatc; the yield marker peak. For the homogenate, the p3' 1.08was 5.9 96 (Table 1 ) . The area corrcspondirlg 1.10 1 fraction, and the mitochondria1 fraction to the mitochondria1 peak did not contain the respective amounts of octapeptide bound significant 5'-nucleotidase activity. Basal activ- (ferntomoles bound per milligram 2 SEM) 117 (13% ity of aderlylate cyclase, the second plasma were 213.4 -+ 16.5, 1547.1 membrane marker tested, was increased 2.4 yield), and 204.6 2 25.7. All subsequent
*
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702
CAN. J.
PHYSIOE. PHAWMACOL.
VCPI,. 54, 1976
PIG.4. Electron micrograph of a peileted, fixed mernbrane fraction banding at in a linear (9-6895, w/w )l sucrose gradient ( x 19 600).
binding studies were performed only with the pa) 1.08-1 .I01 fraction, which we have terxned 'the ATIIparticulate-binding fraction.' Binding fraction preparations, boiled in a water bath at 100 "C for 3 mima, lost their capa-
p"
L08-1.101
city to specifically bind [:5H]A%II.Binding sf the labeled hormone was linear to at least 200 pg of fractional protein. The time required for completion 0%binding of ATIIto the particulate fraction is illustrated
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FOlICrET AND MIXISL,EK
L
0
I
10
I
eo
i
30
1 BME: ('imin) FIG.5. Time course of ['HIAT,, ( 5 x 10 " M ) binding to a ~ n e n ~ b r a nfraction e derived from bovine adrenal cortex. Values are means -t- SEM of six observations.
in Fig. 5. Specific binding was, statistically, at equilibrium after 7.5 ~ n i nof incubation; maximal binding was observed at 10 min. Specific binding of [:{H]ATIIto the receptor fraction was rapidly reversed by the addition of an excess amount of unlabelcd AT,, when binding equilibrium had been reached (Fig. 6).
703
The aflinity of [:3HqATIIfor binding in the particulate fraction is presented graphically in Fig. 7 in a Scatchard ( 1949) plot. Thc curve was analyzed according to the method of Chamilcss and McGuire (1975). The highaf'fimity binding sitc had a binding capacity ( N I) of 2.38 pmol . mg-I, and an ecluilibriurn constant K l of 2.36 X 1O8 M - l . The affinity for binding of ATI. unlabeled A?',,, and several KTI,fragrneilts viz. des-AspdAT,,, des-Phe"ATII, ( Asp-Arg-Val-Tyr) , and ( Tle-His-Pro-Phe ) was tested in inhibitiondisglace~nent studies. The above agents werc added simultaneously with ["]AT,,. RcsuIts are illustrated in Fig. 63. Bound [SH]ATIpwas, with exception of the two tetrapcptides, displaced cor~lpletelyupon addition of all thc substances tested: the displacement curves were parallel. The coilcentrations of ligands required to displace 50% of the bound [:3W1ATI, are presented in Table 2. Des-Asp1-AT,, was about 8 % as effective as unlabeled ATII (considered 100%) effective) in displacing the bound isotopic ligand. AT, was 1% effective and desPhe"ArI',, only 0.25 9% effective. Thc specificity and affinity of binding of [:W1ATI1 \.\;as further tested by using bradykinin, ACTH, [Sarl]ATIr, [Leuk]ATI1, and a fluorescanline dcrivativc of ATIP (Forget c9t uk. 1975) in another series of inhibition-displace-
FIG.6. Displacement of bound 13HIATII (5 x 10 ' 1rM) by excess unlabeled AT,, (1.7 x 10 M i added at binding equilibrium (arrowhead). Data fronl a representative experiment.
C A N . J . P N Y S I O L . P H A R M A C O L . VOL. 5 4 . 1976
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TABLE 2. Effects of ATI, ATI1,and various ALPI1 analogs, antagonists, and fragments on the binding of [%]ATII to adrenocortical particulate binding fraction
Compound
Half-maximal displacement of [3H]ATI, binding, n M
plasmic reticular contaminants than other preparations described to date (Glossman et a/. 1974; Saltman et a/. 1975). The current data suggest that the binding sites for AT,, are concentrated on the plasma membrane of cells from the bovine adrenal cortex, as previously suggested by other workers (Arakawa et al. 1962; FIG.7. Scatchard plot of AT,, binding to a particu- Smeby 1963; Catt et a/. 1974). late membrane fraction derived from bovine adrenal The rapidity with which binding occurs and cortex. Observed values, ; corrected values, 0.K1 = the displacement of isotopic ATII by the nonhigh-affinity equilibrium constant (2.36 x 10"M - ' ) ; radioactive octapeptidc indicates a possible N1 = high-affinity binding capacity (2.38 x lo-'' mol mg-') . relationship between the binding sites in the particulate fraction banding between sucrose 1.OR and 1.101 and the'physiological ATII ment studies. The results obtained are presented receptor. The equilibriuin constant of the highin Fig. 9. Neither bradykinin nor ACTH had affinity binding site is similar to the constant significant effects on ["]ATII binding in condetermined by Glossman and coIlaborators centrations lower than 1 0 - W . In higher con(1974) using a 20 000 g sediment from bovine centrations, however, both could inhibit ["HIATII binding, though not completely, and not adrenal cortex homogenate. When these authors in a parallel fashion. Binding of [:SH]AT,I was further fractionated their 20 000 (: pellet on a coizlpletely inhibited by [Sarl]AT,,, [Leu8]ATII, discontinuous sucrose gradient, they obtained and fluorescarnine-ATII. [Sarl]ATII was four an ATII binding fraction with a similar K I but with a two- to three-fold increased binding times less effective than ATII in displacing capacity. The binding capacity of this preparalabeled ligand (Table 2 ) ; [Leu8]ATI, was nine tion was similar to that observed with the prepatimes less effective, and fluorescamine-ATII, 9.5 ration currently studied, i.e., zonal ultracentiines less effective. trifugation reduced the time necessary to produce a preparation that was purer, and had Discussion similar ATII binding affinity and capacity with The specific activities of 5'-nucleotidase and respect to other preparations. adenylate cyclase are higher in the ATII particuATII displacement studies undertaken with late binding fraction than in the homogenate, analogs and fragments of the octapeptide and indicating a relative purification of plasma mem- the decapeptide ATI indicate that none of the branes. These findings are also supported by peptides tested displaces ["]ATII as effectively electron inicroscopic observation of the fraction, as unlabeled ATII; however, with the exception which was found to be mainly con~posed of of the two tetrapeptides, the other peptides tested smooth-membrane vesicles of various sizes. The displaced ATIIin a parallel manner, thereby indifraction contains fewer mitochondria1 and endo- cating a competitive process at the receptor level.
p2~
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FORGET AND HEISLER
FIG.8. Inhibition-dispIacenlent of ["MATlI binding to a particulate, membrane fraction derived from bovine adrenal cortex. 0, Unlabeled AT,,; @, des-Aspf-AT,,; n, AT,; A , desLeu8-ATII;A, Asp-Arg-Val-Tyr; W , Ile-His-Pro-Phe. Values are means -F- SEM of six observations.
The displacement of ["HIAT,, by the ( 1-4) and (5-8) tetrapeptides of ATII is not appreciable. These fragments can be derived from the action of trypsin on the ATII molecule but their minimal effect on 13H]ATlI binding would tend to indicate that if cIeavage of ATII by trypsin-like enzymes in the preparation were to occur, the products of the reaction would not interfere with binding of the labeled octapeptide. The marked inability of des-PheR-ATII to displace L3H]ATII from its binding sites similarly indicates that the possible production of this ATII metabolite, as a result of the presence in the preparation of endogenous carboxypeptidases, would not interfere with [:
