Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8251-8255, November 1988 Medical Sciences Evidence for extracellular, but not intracellular, generation of II in the rat adrenal zona glomerulosa ( release/subceilular fractionation/renin-angiotensin system) HIDENORI URATA, MAHESH C. KHOSLA, F. MERLIN BUMPUS, AND AHSAN HUSAIN* Department of Heart and Hypertension Research, Research Institute of The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195-5071 Communicated by Irvine H. Page, July 18, 1988

ABSTRACT Based on the observation that high levels of system in the adrenal ZG. The major goal of this study was renin and angiotensin II (Ang II) are found in the adrenal zona to directly examine the previously proposed models of Ang glomerulosa (ZG), it has been postulated that Ang H is formed II formation (intracellular versus extracellular) in the rat intracellularly by the renin-converting enzyme cascade in this adrenal ZG. We also describe a biochemical mechanism for tissue. To test this hypothesis, we examined renin-angiotensin inactivation of Ang II in the ZG. system components in subcellular fractions of the rat adrenal ZG. Renin activity and immunoreactive-Ang II (LR-Ang II) were observed in vesicular fractions but were not colocalized. MATERIALS AND METHODS In addition, angiotensinogen, angiotensin I, and converting enzyme were not observed in the renin or IR-Ang 11-containing Subcellular Fractionation Studies. Female Sprague-Dawley vesicular fractions. These data do not support the hypothesis rats (-300 g) were used in this study. Adrenal glands were that Ang II is formed intracellularly within the renin- dissected on ice to yield the capsular ZG and the decapsular containing vesicles of the ZG. Rather, since modulatable renin zona fasciculata medulla tissue. The ZG tissue was sus- release from adrenal ZG slices was observed and renin activity pended in 0.25 M sucrose and homogenized in a glass/Teflon was found in dense vesicular fractions (33-39% sucrose), it is homogenizer. The homogenate was centrifuged at 800 x g likely that Ang II formation in the ZG is extracellular and for 10 min to remove unbroken cells and nuclei, and the initiated by the release of vesicular renin. Receptor-mediated resultant supernatant was layered on either a discontinuous endocytosis and subsequent degradation of Ang II in ZG sucrose density gradient system 1 (3 ml each of 65%, 37%, lysosomes have been shown by others. The presence of IR-Ang and 15% sucrose) or system 2 (2 ml each of 65%, 39%, 33%, II in light vesicular fractions (15% sucrose) and the finding of and 15% sucrose) and centrifuged at 85,000 x g for 1 hr at a high correlation between ZG IR-Ang H and Ang II receptor 4°C. After centrifugation, the gradients were fractionated levels suggest that the primary occurrence ofthis peptide in the from the bottom. Renin, angiotensinogen, and converting ZG is by receptor-mediated endocytosis. In ZG lysosomal enzyme were measured in subcellular fractions after vesic- fractions '2MI-labeled Ang II was degraded to '2,I-labeled ular disruption. To disrupt vesicles in subcellular fractions, des-[Phe8]Ang II. Since Ang II antibodies do not recognize distilled water was added to a portion of each fraction (1:1, des-[Phe8]Ang II, these rmdings explain why IR-Ang II in the vol/vol) and the samples were freeze-thawed three times. ZG is due predominantly to Ang II and not to its C-terminal Measurement of Renin-Angiotensin System Components. immunoreactive fragments. Renin activity was measured by RIA (New England Nuclear) ofthe generated Ang I after incubation with excess rat plasma angiotensinogen at pH 7.4 as described (4). The incubations The secretion of aldosterone from the adrenal zona glome- were for 16 hr at 37°C in 100 mM NaH2PO4 buffer containing rulosa (ZG) appears to be predominantly under endocrine 5 mM N-ethylmaleimide, 0.5 mM phenylmethylsulfonyl flu- control imposed by the renal renin-angiotensin system and oride, and 10 mM EDTA to prevent Ang I breakdown. the pituitary corticotropin (ACTH) system (1). During the last Angiotensinogen concentration was estimated by Ang I two decades the possibility of autocrine and/or paracrine produced after incubating the sample with excess rat kidney regulation of aldosterone secretion by angiotensin II (Ang II) renin for 1 hr at 37°C in the renin incubation buffer. The has gained support in view of the demonstrations of the indirect method for estimation of angiotensinogen was used components of the renin-angiotensin system and Ang II since it, unlike the direct RIA, quantifies only the prohor- receptors in the adrenal gland (2-5). Recent studies have mone and not the des-Ang I angiotensinogen. Using a 100-,ul provided considerable information on the regulation of ad- sample, the minimum level of angiotensinogen that could be renal Ang II and renin, the primary processing enzyme ofthe detected by this assay was 20 pM. Converting enzyme renin-angiotensin system. Interpretation of the regulation activity was measured with a radioassay kit (Ventrex Labo- data, however, has been difficult. For example, in potassium ratories, Portland, ME). Acid protease activity was measured chloride-loaded rats adrenal renin activity is increased and by the method ofAnson (7) with [14C]hemoglobin as substrate adrenal Ang II is reduced (3); thus, it is not clear whether in in 500 mM sodium acetate buffer (pH 3.2). Protein concen- potassium chloride-loaded rats the adrenal production ofAng tration was measured by the method of Lowry et al. (8). For II is increased or decreased. A similar divergence between the measurement ofAng I and Ang II in subcellular fractions, renin and Ang II has been reported in the rat adrenal ZG after 200 mM HCl/ (1:3) was added to a portion of each bilateral nephrectomy (5, 6). The elucidation of the mecha- fraction (10:1, vol/vol) and the mixture was mixed vigorously nism of formation of Ang II in the adrenal gland is therefore for 5 min, clarified by centrifugation, and lyophilized. The necessary and is likely to be an important first step in residues were resuspended in 100 mM NaH2PO4 buffer (pH understanding the physiological role ofthe renin-angiotensin Abbreviations: Ang II and I, II and I; IR-Ang II, The publication costs of this article were defrayed in part by page charge immunoreactive Ang II; Ang-(1-7), des-[Phe8]Ang II; ZG, zona payment. This article must therefore be hereby marked "advertisement" glomerulosa; 'l25-Ang II, "N-I-labeled Ang II; ACTH, corticotropin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

8251 Downloaded by guest on September 29, 2021 8252 Medical Sciences: Urata et al. Proc. Natl. Acad. Sci. USA 85 (1988) 7.4), and Ang I and Ang II in the solution were partially RESULTS purified on a C18 Sep-Pak cartridge and then quantified by RIAs as described (4). Subcellular Localization of Renin-Angiotensin System Com- Adrenal Slice Studies. Fresh adrenal capsules containing ponents. Subcellular fractionation of the rat adrenal ZG by the ZG were obtained from rats nephrectomized 48 hr sucrose gradient system 1 yielded a discrete renin activity were and peak (fractions 5-8) at the 37%/65% sucrose interface (Fig. previously. Individual capsules cut into quarters 1). In two different separations, the average recovery of the preincubated in 2 ml of Krebs-Ringer buffer without bicar- measured renin activity and immunoreactive-Ang II (IR-Ang bonate containing 10 mM Hepes and 1 mg of glucose per ml II) applied to the gradient was 84% and 75%, respectively. (pH 7.4) at 370C for 30 min. Four adrenal capsules were used About 60% of the recovered renin activity and <5% of the per incubation tube. The supernatant was discarded and recovered IR-Ang II was present in the renin peak. The replaced with 2 ml of Krebs-Ringer buffer without bicarbon- majority (>80%) of the IR-Ang II was recovered in a broad ate containing 10 mM Hepes (pH 7.4), 0.1 mg ofbovine serum peak (fractions 11-19) within the 15% and 37% sucrose albumin per ml, with or without 55 nM epinephrine, 50 nM layers. To examine the presence of the other components of ACTH, or 20 mM K+, and incubated at 370C for 3 hr. The the renin-angiotensin system in the renin-containing subcel- tubes were then centrifuged at 1900 x g for 5 min at 4TC. lular fractions, the rat adrenal ZG tissue was fractionated by Supernatants were stored at - 900C until assayed for renin sucrose gradient system 2, which was more shallow than activity. sucrose gradient system 1. When gradient system 2 was used, 12,I-Labeled Ang II ('25I-Ang II) Metabolism Studies. Ang renin activity was enriched in the 33% and 39% sucrose layers II, des-[Phe8]Ang II [Ang-(1-7)], des-[Pro7,Phe8]Ang II [Ang- (Fig. 2, fractions 4-6). In some renin-containing fractions, (1-6)], des-[His6,Pro7,Phe8]Ang II [Ang-(1-5)], des-[Ile5, such as fraction 3 in Fig. 2, a small amount ofangiotensinogen His6,Pro7,Phe8]Ang II [Ang-(1-4)], des-[Asp']Ang II [Ang- was observed. The level of angiotensinogen obtained in this (2-8)], des-[Asp1,Arg2]Ang II [Ang-(3-8)], and des-[Asp', fraction was <1/10,000th of the Km of the renin-angioten- Arg2,Val3]Ang II [Ang-(4-8)] were synthesized and purified sinogen reaction, which is -1 puM. However, IR-Ang I, as described (9). These peptides were all labeled with 1251 by converting enzyme activity, and angiotensinogen were all the lactoperoxidase method and the monoiodinated products undetectable in the peak renin-containing fractions (Fig. 2). were purified by reverse-phase high-pressure liquid chroma- The recovery of applied renin activity was 54% + 5% (n = tography (HPLC) (10). The specific activity ofeach iodinated 3). Angiotensinogen, converting enzyme, and IR-Ang I could angiotensin was 2200 Ci/mmol (1 Ci = 37 GBq). 125I-Ang II not be detected in the supernatant applied to the sucrose purified adrenal ZG lysosome-containing fractions prepared gradient. Whereas angiotensinogen and converting enzyme by sucrose gradient system 2. Lysosome-containing fractions activity could not be detected in the adrenal ZG tissue, the (50 ,ul), identified by the enrichment ofacid protease activity, recovery of IR-Ang I after the initial glass/Teflon homoge- were incubated with 300 fmol of 1251I-Ang II in 250 ,ul of 50 nization of the tissue was <1%. mM sodium acetate buffer (pH 5.0) for 0-30 min. To Renin Release from Adrenal ZG Slices. Fig. 3 shows renin determine the nature of enzyme activity involved in the release into culture medium from adrenal ZG slices. Basal degradation of 1251I-Ang II, the incubation medium also renin release over a 3-hr period was not modified in the contained either 1 mM phenylmethylsulfonyl fluoride, 1 mM presence of 50 nM ACTH or 20 mM K+. Epinephrine (55 pepstatin, 1 mM EDTA, or 1 mM N-ethylmaleimide. The nM)-induced renin release (1.96 + 0.3 ng ofAng I-hr- '-ml - `; reactions were terminated by the addition of 2 ml of ice-cold n = 4) was 2-fold higher than basal (0.88 + 0.14 ng of Ang ethanol, and the precipitated lysosomal proteins were re- moved by centrifugation. The supernatant was lyophilized and the resulting residue, containing synthetic iodinated Ang II and the generated 1251I-Ang II fragment(s), was resus- I= ;;Is0. pended in 100 ,ul of 25 mM triethylammonium phosphate .EC*ac (TEAP) buffer (pH 3) and applied to a Vydac (Hesperia, CA) C cj- C4 HPLC column (4.6 x 25 cm) preequilibrated with 25 mM u6 TEAP (pH 3) containing 10% acetonitrile. The column was developed with a linear acetonitrile gradient (10-32% aceto- nitrile in 20 min) at a flow rate of 1 ml/min. The column was calibrated with 125I-Ang II and 125I-labeled fragments of Ang II representing all potential initial radioactive cleavage prod- ucts of 125I-Ang II. 12'I-Ang II and 12SI-Labeled Ang-(1-7) [`2I-Ang-(1-7)J C Binding Studies. Adrenal ZG (10 adrenal capsules) and zona fasciculata medulla (10 decapsular adrenal glands) mem- branes were prepared and Scatchard analysis of specific 4. 125I-Ang II and 125I-Ang-(1-7) binding to adrenal membranes O6 was carried out as described (11). The assay buffer was 50 u mM NaH2PO4 (pH 7.2) containing 100 mM NaCl, 1 mM EGTA, 10mM MgCl2, 0.2% bovine serum albumin, and 10 jig ofbacitracin per ml. Either 125I-Ang II or 125I-Ang-(1-7) (0.2- 2 nM) was incubated at 22°C for 60 min in assay buffer, with 1 2 3 4 5 6 7 8 9 101112131415161718192021 100 ,ug of membrane protein in a final vol of 250 ,ul. Fraction number Nonspecific binding of 125I-Ang II and 125I-Ang-(1-7) was determined in the presence of 1 ,uM Ang II and Ang-(1-7), FIG. 1. Distribution ofrenin activity and IR-Ang II in subcellular fractions of the rat adrenal ZG. (A) Renin activity; (B) IR-Ang II in respectively. subcellular fractions. Fraction volume was 0.5 ml. (Inset) Sucrose Statistical Analysis. Results are expressed as mean ± SEM. concentration of the gradient before centrifugation. Essentially Comparisons ofthe mean were by Student's t test and values identical results were obtained in two independent experiments. of P < 0.05 were considered significant. Adrenal ZG tissue from 40 glands was applied to each gradient. Downloaded by guest on September 29, 2021 Medical Sciences: Urata et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8253

|Sucrose concentration I 65% 1 39% 33% 15% 1 9% ou A 1 u'=2 'w =20 -IL

4_ - 10 CF. loE

300

w E 200 Basal Basal ACTH EPI K+ 0 0 Ohr 3hr 100I FIG. 3. Effects of ACTH, epinephrine (EPI), and K+ on basal renin secretion from rat adrenal ZG slices. Adrenal capsules con- taining the ZG were obtained from rats nephrectomized 48 hr earlier. Adrenal capsule quarters (four capsules per incubation) were prein- CS cubated in Krebs-Ringer buffer (pH 7.4) containing 10 mM Hepes U and 1 mg of glucose per ml at 370C for 30 min. The supernatant was replaced with 2 ml ofKrebs-Ringer buffer (pH 7.4) containing 10 mM A I Hepes, 0.1 mg of bovine serum albumin per ml with either buffer (basal, 3 hr), 50 nM ACTH, 55 nM epinephrine, or 20 mM K+ for 3 0.4 hr at 37°C. A basal incubation for 1 min (basal, 0 hr) at 370C was also D carried out. Renin released into the medium was estimated as 0.2 - described. Bars represent means and the vertical bars represent SEM. Values from four independent incubations are reported in each 0.0U group. 2 -E aspartyl (pepstatin), sulfhydryl (N-ethylmaleimide), or metal " ion-requiring (EDTA) proteases. WU 1 l Adrenal 1"I-Ang II and '25I-Ang-(1-7) Binding. Fig. 5

il . . . shows the specific binding of '25I-Ang II and 1251I-Ang-(1-7) 1 2 3 4 5 6 7 8 9 10 11 12 to pooled rat adrenal ZG membranes and to zona fasciculata Fraction number medulla membranes. High-affinity 1251I-Ang II binding sites were present in the ZG (Kd = 198 pM; Bmax = 80 fmol/mg) FIG. 2. Distribution of renin-angiotensin system components in and zona fasciculata medulla (K = 805 pM; Bma = 168 renin-containing vesicular fractions of the rat adrenal ZG. (A) Renin fmol/mg) membranes, but binding of 125I-Ang-(1-7) was not activity; (B) protein concentration; (C) angiotensinogen (Aogen); (D) saturable. converting enzyme (ACE) activity; (E) IR-Ang I in subcellular fractions. Fraction volume was 1 ml. (Inset) Sucrose concentration ofthe gradient before centrifugation. Bars are means and the vertical DISCUSSION bars are SEM ofthree separate experiments. Adrenal ZG tissue from 14 glands was applied to each gradient. In peak renin-containing It is generally believed that in the adrenal ZG Ang II fractions, levels ofangiotensinogen, converting enzyme, and IR-Ang formation occurs intracellularly by the renin-converting en- I were undetectable. zyme cascade since renin and Ang II occur in one cell type. However, recent reports showing a divergence between the Ihr- '-ml -'; n = 4) (P < 0.05). Simultaneous angiotensino- regulation of renin and Ang II in the adrenal gland have gen release into the media from ZG slices could not be suggested that if intracellular renin-dependent Ang II forma- detected-thus, the level of angiotensinogen in the medium tion occurs in the adrenal ZG, renin is not rate-limiting (3, 5, was <20 pM (i.e., detection limit of the assay), which at 6). Alternatively, it has been proposed that Ang II production maximum represents a level ofprohormone corresponding to by the adrenal cells occurs extracellularly, initiated by renin 1/50,000th of the Km the renin-angiotensinogen reaction. release, and that Ang II in adrenal ZG cells represents 1"I-Ang II Metabolism in Adrenal Lysosomes. Rat adrenal endocytosed hormone (4). We provide direct evidence sup- ZG lysosomes were purified by sucrose gradient system 2. A porting the concept that Ang II formation in the rat adrenal band occurring immediately below the interface of 33%/39% ZG does not occur through an intracellular pathway, but sucrose was highly enriched in acid protease activity (a likely occurs through an extracellular pathway, by demon- marker for lysosomes) and was used as a source oflysosomal strating that (i) the renin-containing vesicular fractions ofthe enzymes after vesicles were disrupted by three freeze/thaw rat adrenal ZG do not contain the other components of the cycles. Fig. 4 shows the metabolism of 125I-Ang II by ZG renin-angiotensin system, and (ii) renin release occurs in lysosomal enzymes. 1251-Ang II was degraded by lysosomal vitro from adrenal ZG slices. enzymes to a single radioactive product, which was identified Rat adrenal ZG renin activity was enriched in a single as 125I-Ang-(1-7) by HPLC using pure radioactive iodinated dense vesicular fraction. The ZG renin-containing vesicular Ang II fragments as standards. Further degradation of 1251I fractions were of a high density, similar to the renin- Ang-(1-7) was not observed. The rate of 1251I-Ang-(1-7) containing vesicular fractions obtained from the rat kidney formation was 56.2 + 7.6 fmol per 20 min per ,ug oflysosomal cortex (12). In subcellular fractionation studies, >95% of the protein (n = 3). In one experiment, lysosomal degradation of recovered IR-Ang II was observed in vesicular fractions '251-Ang II and the formation of '25I-Ang-(1-7) was 100% lighter than renin-containing fractions. Angiotensinogen, suppressed by the serine protease inhibitor phenylmethyl- converting enzyme, and IR-Ang I were also not detected in sulfonyl fluoride, but <5% suppressed by inhibitors of the ZG renin-containing vesicles. The lack of prohormones Downloaded by guest on September 29, 2021 8254 Medical Sciences: Urata et al. Proc. Natl. Acad. Sci. USA 85 (1988)

3 co, 1. Ang-(1-6 p- 2. Ang- (1-4 x Ang-(1-7 4. Ang-(l-5 5. Ang-(2-8 5 6: Ang-f 4.8 7. Ang 8. Ang-(3-8) 0 r.

B. 0 min incubation 0 50 100 150 20_ 1251-Ang II bound, fmol/mg protein 8

04 It I- 6 U x tfasciculata-medufla 4

U C. 5 min incubation 0 * 0 - .0 2 200 4 zona glomerulosa

. . 0 .- 0 1 2 3 10- 1251-Ang-(1-7) bound, fmol/mg protein

FIG. 5. Scatchard analysis of '25I-Ang II binding (A) and 125i- Ang-(1-7) binding (B) to rat adrenal ZG and zona fasciculata medulla (fasciculata-medulla) membranes. 125I-Ang II binding to adrenal ZG (Kd = 198 pM; Bmax = 80 fmol/mg) and zona fasciculata medulla (Kd D. 20 mmnincubation = 805 pM; Bmax = 168 fmol/mg) membranes were of a high affinity and low capacity, but 125I-Ang-(1-7) binding was not saturable. 20- inadvertently degraded. If Ang I was present in the extra- cellular or cytosolic cell compartments, degradation is likely due to the presence of high levels of aminopeptidases and 10 carboxypeptidases in these compartments. Therefore, since neither the prohormones, the active hormone, nor the final processing enzyme of the renin-angiotensin cascade are present in the renin-containing vesicular fractions, it is unlikely that Ang II formation occurs in the renin-containing 8 12 0 i6 vesicles of the rat adrenal ZG. Retention time, mmn. Extracellular Ang II formation initiated by renin release is

FIG. 4. Metabolism of 1251-Ang in adrenal ZG lysosomal a well-established pathway for Ang II formation by juxtaglo- fractions. (A) Separation of standard 1251-Ang and its known merular cells of the kidney cortex (14). However, release radioactive metabolites by reverse-phase HPLC. (B-D) HPLC studies with adrenal tumor cells (15) or with collagenase- radioactivity profiles of 0-, 5-, and 20-mmn incubations of 1251I-Ang II dispersed adrenal ZG cells (3) have suggested that renin may (300 fmol) with purified adrenal lysosomal fractions, respectively. not be released by the adrenal gland. Because transformed or chemically treated cells sometimes display altered patterns of in vesicles is un- and the active hormone renin-containing protein secretion, we reinvestigated the possibility of renin likely to have resulted from premature rupture of the renin- release by the adrenal gland by using adrenal ZG slices. To containing vesicles, since only a low level of renin activity maximize the chance of demonstrating adrenal renin release was observed in the application zone (cytosolic cellular and minimize contamination of the adrenal gland by circu- compartment) of the sucrose density gradient. The lack of lating kidney renin, adrenal glands were obtained from rats converting enzyme in ZG renin-containing vesicular fractions nephrectomized 48 hr earlier; nephrectomy has extensively is consistent with autoradiographic studies showing that been shown to elevate adrenal ZG renin activity levels (5). converting enzyme is absent in the cells of the rat adrenal ZG Basal renin release from ZG slices was =1% of the stored (13). Also, the absence of angiotensinogen in ZG homoge- content during a 3-hr in vitro incubation at 370C. Basal renin nates is consistent with the observed lack of angiotensinogen release was not affected by ACTH and K +, factors known to production by adrenal ZG slices. Unlike the high recovery of increase adrenal renin content in vivo (3, 16). Epinephrine IR-Ang 11 (75%) in subcellular fractions, the recovery of produced a 2-fold increase in basal renin release. These IR-Ang I was low (<1%). The differential degradation of Ang studies suggest that renin in the adrenal ZG cell layer is II and Ang I during fractionation suggests that both peptides releasable and that its release can be modulated. A limitation may occur in different subcellular compartments. Since in the interpretation of these release data is that some renin renin-containing vesicles were not ruptured during fraction- release during the incubation may have occurred from cells ation, it is unlikely that Ang I initially present in vesicles was injured during slice preparation. Although cell injury was not Downloaded by guest on September 29, 2021 Medical Sciences: Urata et al. Proc. Natl. Acad. Sci. USA 85 (1988) 8255

quantified, to minimize this problem a 30-min preincubation endocytosed Ang II may be autocrine and endocrine. The period was included to stabilize the tissue and remove renin consequence of renin release rather than the release of the potentially released during initial tissue handling. Because active hormone as an autocrine/paracrine mechanism of renin release from the slices could be modulated, it is likely hormone formation has obvious significance. Since elevation that renin release from slices is not simply due to cell death. of extracellular Ang II levels is likely to take a longer time if Thus, Ang I formation in the adrenal ZG can potentially be Ang II is made extracellularly than ifactive Ang II is released, initiated by renin release. and since renin probably has a longer half-life than Ang II The major form of IR-Ang II in the rat adrenal gland is Ang once released, effects oflocally produced Ang II are likely to II; angiotensin III and other IR-Ang II fragments represent have a slow onset and to be prolonged in their duration. The '20% of the total immunoreactivity (4). We have recently cortical sinuses provide a mechanism for adrenal cortical demonstrated that in normal and sodium chloride-loaded rats hormones to directly influence the adrenal medulla (22). IR-Ang II levels in the adrenal ZG were highly correlated Thus, the mode of formation of Ang II in the ZG and the with ZG Ang II receptor binding activity (r = 0.94) (4). The presence of a circulatory connection between the cortex and finding that subcellular fractionation of the ZG cell layer the medulla may facilitate the occurrence of high levels of yielded low density IR-Ang 11-containing vesicular fractions, Ang II in the blood that drains into the adrenal medulla. High together with our previous correlation studies, suggested that levels of Ang II receptors are present in the rat adrenal the IR-Ang 11-containing vesicular fractions may be endo- medulla and Ang II can stimulate the secretion of medullary somes or plasma membrane fractions containing receptor- and bound Ang II. Support for the conclusion that Ang II in cells epinephrine norepinephrine (23). The adrenal ZG renin- of the adrenal ZG represents endocytosed peptide is also angiotensin system may therefore play a role in regulation of provided by the finding that 1251-Ang II is rapidly internalized medullary catecholamine secretion as well as cortical aldos- by Ang II receptors in rat adrenal ZG cells (17). However, terone secretion. because the endocytosed peptide terminates in lysosomes (17) and we observed no IR-Ang II in lysosomal fractions, we This research was supported in part by the Reinberger Foundation examined the possibility that the primary mode of inactiva- and by National Institutes of Health Grant HL 6835. tion of Ang II in the lysosome may render the peptide 1. Reid, I. A. & Ganong, W. F. (1977) in Hypertension, eds. immunologically unreactive. Incubations of 1251-Ang II with Genest, J., Koiw, E. & Kuchel, 0. (McGraw-Hill, New York), the soluble extracts of ZG lysosomes, at a pH of 5, produced pp. 265-293. a time-dependent formation of a single radioactive product, 2. Ryan, J. W. (1967) Science 158, 1589-1590. 125I-Ang-(1-7). Since 125I-Ang-(1-7) was not further degraded 3. Nakamaru, M., Misono, K. S., Naruse, M., Workman, R. J. & by lysosomal peptidases, it is possible that Ang-(1-7) may Inagami, T. (1985) Endocrinology 117, 1772-1778. accumulate in lysosomes and be released from ZG cells by 4. Husain, A., DeSilva, P., Speth, R. C. & Bumpus, F. M. (1987) lysosomal exocytosis. Further degradation of Ang-(1-7) may Circ. Res. 60, 640-648. thus occur extracellularly. The N-terminal heptapeptide 5. Doi, Y., Atarashi, K., Franco-Saenz, R. & Mulrow, P. (1983) Ang-(1-7) is unlikely to be biologically active in the rat Clin. Exp. Hypertens. Part A 5, 1119-1126. adrenal gland since Ang-(1-7) is not an 6. Aguilera, G., Schirar, A., Baukal, A. & Catt, K. J. (1981) aldosterone secreta- Nature (London) 289, 507-509. gogue (18) and saturable binding sites of 1251I-Ang-(1-7) were 7. Anson, M. L. (1938) J. Gen. Physiol. 22, 79-89. not observed in the rat adrenal cortex or medulla. The 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. formation of 125I-Ang-(1-7) by lysosomal fractions was in- (1951) J. Biol. Chem. 193, 265-275. hibited by phenylmethylsulfonyl fluoride, suggesting that the 9. Khosla, M. C., Nishimura, H., Hasegawa, Y. & Bumpus, enzyme involved was a serine peptidase. Since angiotensi- F. M. (1985) Gen. Comp. Endocrinol. 57, 223-233. nase C, a lysosomal serine carboxypeptidase (EC 3.4.16.2), 10. Husain, A., Pajka, S. F., Taylor, S. M. & Speth, R. C. (1986) inactivates Ang II by releasing the C-terminal phenylalanine J. Pharmacol. Exp. Ther. 239, 71-77. (19), it is likely that the primary mode of inactivation of the 11. Pucell, A. G., Bumpus, F. M. & Husain, A. (1987) J. Biol. octapeptide Ang II in the adrenal lysosomes occurs by Chem. 262, 7076-7080. 12. Sagnella, G., Price, R. & Peart, W. (1980) Hypertension 2, 595- angiotensinase C or a similar enzyme. All reported Ang II 603. antisera (20), including the one used in this study (21), appear 13. Strittmatter, S. M., DeSouza, E. B., Lynch, D. R. & Snyder, to be'directed to the C-terminal portion of the Ang II S. H. (1986) Endocrinology 118, 1690-1699. molecule, since sequential deletions of the first three N- 14. Keeton, T. K. & Campbell, W. B. (1981) Pharmacol. Rev. 31, terminal amino acids of Ang II do not immunologically 81-227; inactivate the peptide, but the deletion of the C-terminal 15. Naruse, M., Shizume, K. & Inagami, T. (1985) Acta Endocri- phenylalanine renders the hormone immunologically inac- nol. (Copenhagen) 108, 545-552. tive. The primary fragmentation of Ang II to Ang-(1-7) by 16. Baba, K., Doi, Y., Franco-Saenz, R. & Mulrow, P. J. (1986) adrenal lysosomal enzymes provides an explanation for the Hypertension 8, 997-1002. 17. Bianchi, C., Gutkowska, A., DeLean, A., Ballak, M. B., lack of IR-Ang II in adrenal lysosomes. Anand-Srivastava, J., Genest, J. & Cantin, M. (1986) Endocri- Collectively, these studies provide evidence that Ang I nology 118, 2605-2607. formation by the rat adrenal ZG may be initiated by renin 18. Kono, T., Taniguchi, A., Imura, H., Oseko, F. & Khosla, release and thus occurs extracellularly. It is not clear whether M. C. (1986) Life Sci. 38, 1515-1519. the source of angiotensinogen in the extracellular compart- 19. Yang, H. Y. T., Erdos, E. G. & Chiang, T. S. (1968) Nature ment is. from the adrenal gland or from blood, but both are (London) 218, 1224-1226. possible. Conversion of extracellularly generated Ang I, 20. Vallotton, M. B. (1974) in Handbook of Experimental Phar- however, may occur by circulating converting enzyme. macology, eds. Page, I. H. & Bumpus, F. M. (Springer, New Intracellular Ang II formation is to since the York), Vol. 37, pp. 185-200. unlikely occur, 21. Husain, A., Bumpus, F. M., DeSilva, P. & Speth, R. C. (1987) renin-containing vesicles in the ZG do not contain the Proc. Natl. Acad. Sci. USA 84, 2489-2493. prohormones or the active hormone of the renin-angiotensin 22. Coupland, R. E. (1975) in Handbook of Physiology, eds. cascade. Furthermore, this and our previous studies (4) Blaschko, H., Sayers, G. & Smith, A. D. (Am. Physiol. Soc., suggest that'intracellular Ang II in the adrenal ZG probably Washington, DC), Section 6, pp. 282-294. represents receptor endocytosed hormone; the origin of the 23. Peach, M. J. (1977) Physiol. Rev. 57, 313-370. Downloaded by guest on September 29, 2021