Renin biosynthesis by human tumoral
juxtaglomerular cells. Evidences for a renin
precursor.
F X Galen, … , F Mounier, J P Camilleri
J Clin Invest.
1984;
73(4)
:1144-1155.
https://doi.org/10.1172/JCI111300
.
Renin biosynthesis was studied in a juxtaglomerular cell tumor. The tumoral tissue had a
high renin content (180 Goldblatt Units/g of tissue), was heavily stained by
immunofluorescence using human renin antiserum, and exhibited numerous characteristic
secretory granules by electron microscopy. In one series of experiments, renin biosynthesis
was studied in tissue slices, by following the incorporation of radiolabeled amino acids into
specific immunoprecipitable renin. Time course studies showed that renin was first
synthesized in a high molecular weight form, 55,000 mol wt, i.e., 10,000 mol wt higher than
that of active renin, and was then converted into a 44,000-mol wt form. In a second series of
experiments renin tumoral cells were cultured. Small, round, birefringent cells obtained after
collagenase digestion produced renin in both primary culture and subculture media. After 5
d most of the renin found in the culture medium was inactive, but could be activated by
trypsin treatment. The tumoral tissue exhibited a strong renin immunofluorescence and
numerous secretory granules were observed by electron microscopy. In contrast, the
renin-producing cells isolated from this tumor and grown in culture showed little renin
immunofluorescence and no secretory granule could be observed. The renin-producing
cells in primary culture and subculture were pulsed with radiolabeled amino acids, and
immunoprecipitable radiolabeled renin was found in the culture media, thus demonstrating
the actual biosynthesis […]
Research Article
Renin
Biosynthesis by Human
Tumoral Juxtaglomerular Cells
Evidences for a Renin Precursor
F. X. Galen, C. Devaux, A. M. Houot, J. Menard, and P. Corvol
Inserm U36, Paris, France 75005
M.T. Corvol
Inserm U30, Paris, France 750015
M.C. Gubler and F.Mounier Inserm U192, Paris, France 75015
J. P. Camiller.
HopitalBroussais, Paris, France 75014
Abstract. Renin
biosynthesis
was
studied in
ajuxtaglomerular
cell
tumor.
The
tumoral tissue had
ahigh renin content (180 Goldblatt
Units/g
of
tissue),
was
heavily
stained by
immunofluorescence using
human
renin antiserum, and
exhibited
numerous
characteristic
secretory granules by
electron
microscopy.
In
oneseries
of
experiments,
renin
biosynthesis
was
studied in tissue
slices,
by
following
the
incorporation
of radiolabeled
amino
acids
into
specific immunoprecipitable
renin. Time
course
studies showed that renin
was
first
synthesized
in
a
high
molecular
weight
form, 55,000
mol wt,
i.e., 10,000
mol wt
higher than that
of
active
renin,
and
was
then
converted into
a
44,000-mol
wt
form. In
a
second
series
of experiments
renin tumoral cells
were
cultured.
Small,
round,
birefringent
cells
obtained after
collagenase
diges-tion produced
renin in both
primary
culture and
sub-culture
media. After 5 d
most
of
the
renin found in the
culture
medium
was
inactive,
but
could be activated
by
trypsin
treatment. The tumoral tissue exhibited
a
strong
renin
immunofluorescence
and
numerous
secretory
granules were observed by electron
microscopy.
In
con-trast, the
renin-producing
cells
isolated from this
tumor
and grown in culture showed
little renin
immunofluo-rescence and
no
secretory
granule could be observed. The
renin-producing cells in primary culture and subculture
Address correspondence to Dr. Galen at Inserm U36, Pathologie
Vas-culaireetEndocrinologie.
Receivedfor publication26January 1983 and in revised form 12 December 1983.
were
pulsed with radiolabeled amino acids, and
immu-noprecipitable
radiolabeled
renin
was
found in the culture
media,
thus
demonstrating
the
actual
biosynthesis of
the
enzyme. This
renin
was not
stored
inside
cultured cells
but
was
rapidly
released
into
the
medium and had
a
molecular weight of
55,000.
No
conversion of
this inactive
high
molecular weight
renin into
the
active, 44,000
mol
wt
form
of
renin was
observed. We postulate the
existence
of two pathways for the
processing, packaging,
and
se-cretion of renin in the tumoral cells: in
juxtaglomerular
cells
oftumoral tissue renin
is
synthesized
as a
preprorenin
and
rapidly
converted into
prorenin (55,000
mol
wt),
which is in turn packaged in
secretory
granules where it
is processed into
active renin
(44,000 mol wt) and
finally
secreted; in the cultured tumoral cells
renin is
still
bio-synthesized
as a
preprorenin
molecule and
then
converted
into
prorenin,
but
is
neither stored
as
granules
nor
pro-cessed
into active
renin.
In
this
case
the
renin is
released
in
an
inactive
form.
Introduction
Although
the syndromeof primary reninism is
anexceptional
cause
of hypertension it is interesting
tostudyfor
many reasons.It provides the best example of a purely renin-dependent
hy-pertension,
asremoval of therenin-producing
tumorcuresthehypertension (for
review, seereference
1). It alsopermits
ex-tensivebiochemical
andmorphological
studies that wouldoth-erwise
beextremely difficult
toperform
duetothescarcity
ofrenin-producing
cells in the normal kidney. For this reason,juxtaglomerular
cell(JGC)'
cultures have only been recently 1. Abbreviations usedin thispaper: AI, angiotensin I; EM, electron microscopy; FCS, fetal calfserum;GU, GoldblattUnit, JGC, juxtaglo-merularcell;MEM,minimumessentialmedium; PRA, plasmareain
activity; RPMI,Roswell ParkMemorial InstituteMedium;SDS,sodium dodecyl sulfate.
J.Clin. Invest.
© The American Society for Clinical Investigation,Inc.
described byRightseletal.(2)intherat.We observedapatient with hypertension dueto aJGCtumorandwehave
previously
described the complete purification of human renin (3), which was extracted from this tumor. The biochemical, enzymatic,and immunological characteristics of tumoral renin were in-distinguishable from those of cadaver kidney renin. Polyclonal (3) and monoclonal (4) antibodies wereraisedtothisrenin.
Endocrine tumors also permit the study of hormone
bio-synthesis, andin apioneer experiment, Steineretal.(5) dem-onstrated theexistence ofaninsulin precursor in apancreatic insulinoma. Inthecaseof human kidney reninnobiosynthetic studies have yet been performed, due to the lack ofboth a
sufficient number of renin-producing cells and specific renin antibodies. However,from previous renin biosynthesis studies
performedinmousesubmaxillary gland tissue (6)and in canine
aortic smoothmusclecells,(7)reninappearedtobesynthesized as a large precursor that was then converted into an active enzyme of-40,000 mol wt. The finding ofanother patient with arenin-producing JGC tumor offered us a unique
oppor-tunitytostudy renin biosynthesisintumorslices as well asin
cell culture. Inthe presentexperiments, humanrenin was bio-synthesizedas alarger molecular weight inactive precursor and then processed into a smaller active molecule.
Methods
Case
reportA 25-yr-old malepatient suffered from severehypertension (240/154
mmHg)discoveredduring asystemic examinationafewmonthsbefore. Therewas nohistory of cardiovascularevents.Analysis ofplasma
elec-trolytes showed anhypokalemia of 2.9 mmol/l associated with hypo-natremia of131 mmol/l and ametabolicalkalosis withabicarbonate
levelof32 mmol/l. Both plasmarenin activity(PRA) inuprightposition (18ngangiotensinI(AI)/mlperh,normalvalues<3 ng/mlper h on adaily 60-meq sodium intake), and plasma aldosterone (800 pg/ml, normal values < 300 pg/ml) were significantly elevated. Initially no tumor was foundonrenal arteriography, and treatmentwitha
com-bination of captopril (300 mg/d) and hydrochlorothiazide (50 mg/d) effectively normalizedblood pressurefor21 mo.Subsequentlythe
pa-tient'shypertensionbecamerefractorytothis therapy andasecond renal
arteriogramwasperformed, whichdemonstratedapoorly vascularized
tumor inthe lowerpole ofthe right kidney.Duringthelasthospital admissionbeforesurgery, hisrecumbentblood pressurewas 178/126
mmHg on treatmentwithcaptopril(300 mg/d), furosemide(80 mg/d),
andguanfacine (2 mg/d). Onthefifth hospitalday, on alowsodium diet without diuretics, and on the same antihypertensive drugs, the
patient's bloodpressurewas130/80 mmHg. RecumbentPRAwas30.8
ng AI/ml per h and plasma aldosterone was 153 pg/ml. After acid activation at pH 3.3 (8), total renin was 435 ng AI/ml per h. The percentageof inactive reninwastherefore 93%.Thelevel of renin
mea-suredby directradioimmunoassay (RIA)(8)was4ng/ml.Tumorectomy
wasthenperformed, whichresulted in animmediate normalization of
thepatient'sblood pressure(130/85 mmHg)andwasassociatedwitha
return to normalofPRA(1.5ng/mlperh)andplasma aldosterone(30
pg/ml). The percentage of inactive renin was90%,and norenin was
detectable by directRIA. At the presenttime,6moaftersurgery, blood pressureisnormalwithouttreatment.
Method of culture
PRIMARY CULTURES.A 3 X 2X 2-cm fragment of tumor was used forcell culture. Two methods were used to dissociate the cells: mechanical dispersion and enzymic digestion. The mechanical dispersion was per-formed by gently pressing the tissue while in a Petri dish. From the cell suspensionobtained, samples of 3 X 105-cells were layered in 35 X 10-mmPetri dishescontaining 2 ml of the incubation mediumdescribed
below. Thetumorthat remainedafter the mechanical dispersionwas cutinto smallpieces and enzymic digestionwascarriedoutwith0.05% clostridium collagenase (Boehringer Mannheim Biochemicals, India-napolis, IN) in Gey's balanced salt solution, containing 0.1 % pure bovine serumalbumin. Five successive pools of cells, referred to asC1-C5,were
released from the tumor by five successive 1 5-min periods of incubation withcollagenase,at 370C. Eachpoolof cells wascollected separately anddistributedtotwo different types of culture dishes: 25-cm2 flasks
with 8X 101cells/flask and 75-cm2 flasks containing 1.2-3X106cells,
correspondingtoa meanof 1.6-4X 106 cells/cm2. Cellwerecultured
inRoswell ParkMemorialInstitute medium (RPMI), containingafinal concentration of 0.6 mM aspartic acid, 1 mMglycine,and 3 mM glu-tamine. The medium was supplemented with 10% fetal calfserum(FCS)
andantibiotics(100IU/ml penicillin;100
Ag/ml
streptomycin).Cultures flasksweregassedwith 10%CO2in airandplacedat370C.Petri dishes wereincubatedat370Cunder 5%CO2 in air.Inallcasesthemedium waschanged three timesper week and the usedmediumwasstored at-20'Cbeforebiochemical studies.
SUBCULTURES.FlasksC2andC5weremaintainedinprimaryculture for 16 and 27 d,respectively.They were thentrypsinizedusing 0.05% trypsin (Gibco Laboratories, St. Lawrence,MA) and0.02% EDTA in
Mg++-andCa'+-freeGey's balancedsaltsolution. The cells obtained
from flasks C2 andC5 were then incubated in the culture medium
described aboveand grownforaperiod of 15-20 d. Some flasksfrom
this firstpassagewerethensimilarlytrypsinizedandmaintainedin culture
forafurtherthreesuccessivepassages.
CELL COUNTING.Sinceatdayzeroofthe culture the same number
of cells per squarecentimeter were inoculated in each flask, itwas possibletocalculatethe numberofcellsremainingafterthefirst change
ofmedium(day 3) bysubstractingthenumberofcellsin thediscarded medium in the initial cell count. At different stagesofthe primary cultureorsubculture,oneflask fromeach group was usedforcellcounting toexpress reninproductionper 106cells.
Morphological
studies
TUMOR STUDY. Forlight microscopy each specimen of tissue was
fixed in Bouin's fluidand embeddedin paraffin. Serial sections (3
gm
thick)wereprepared forstaining with either hematoxylin-eosin-safran orMasson'strichromeand silverimpregnation. Renin immunofluo-rescence wasperformedonparaffin-embedded material. Afterremoval ofparaffinwith xylene, sectionswerepassedthrough successivealcohol solutions and washed inphosphate-buffered saline (PBS). Indirect
im-munofluorescencewascarriedoutasdescribedpreviously (9) using R15
rabbitrenin antiserum. ThespecificityofR15rabbitantiserum was first
described byGalenetal.(3)andfurther documented in studies dealing withthedirectRIAof renin (8, 10)andwith theimmunohistochemistry
ofrenin in human kidney (9). Thesedata show that our antiserum
crossreactsexclusivelywith renin both inplasmaandkidney.Sections
were incubated for 1 h with a 1:50 dilution oftheantiserum. After repeated washingthetissue sections were exposed to fluoresceinated
goat anti-rabbitIgG (Hyland Laboratories,CostaMesa, CA)for 30 min. Theslidesweremounted inbufferedgelatin andexamined under ul-travioletlight microscopy (Leitz Orthoplan-Ortomat, E. Leitz, Inc.,
Forelectron microscopy (EM) the tissue was cut into 1-mm3 frag-ments andfixed in2.5%glutaraldehydewithphosphatebuffer(pH 7.4).
After postfixing in 1% osmium tetroxyde and embedding in Epon,
i-Mmtissue sectionswereprepared and stained with toluidine Blue.
Ultrathin sections of selected areas were cut and stained with uranyl acetateand leadcitrate.
CELL CULTURE STUDY.Periodically, throughoutthe culture
pe-riod,morphology ofthe cells was observed in reverse microscopy, and
photomicrographsweremade. Immunofluorescentand EM studieswere performed between days 15and27 of culture. Cultured cells grownon coversliporplasticflaskswerebrieflyrinsed with PBS and then fixed
in methanol -20'C for 15 min. After hydration in PBSthey were
incubated with R15 antiserum,diluted1:50,inamoist chamberatroom
temperaturefor1h.Theywerethen washed twice for 15 min with PBS andincubated for 30min with fluorescinated goat antirabbitIgG (Beh-ringwerke AG, Marburg, FederalRepublic ofGermany) diluted 1:4.
After beingrinsedagainin the PBS thepreparations were mounted in
buffered gelatinand viewed inaLeitz-photomicroscope using epiflu-orescence illumination. For EM studies the cultured cells were fixed in
situ with2%glutaraldehydein 0.1 Mcacodylatebufferat40C for 2h, rinsed in bufferandpostfixed 1 h in 1% osmic acid. After three rinses withdistilledwater,cells werestained insituwith 2%uranyl acetate for 30 min, then rinsedindistilledwater.Theyweredehydratedwith
ethanolandthen withhydroxypropylmethacrylate, and embedded in
Epikote 812 resin. Sectionswerecut, paralleltotheplaneof the cell
layers, withadiamond knife inaultrocut ultramicrotome(Reichert,
Austria). Theywere stained with lead citrate and examined with an Elmiskop 101 electronmicroscope (Siemens,FederalRepublicof
Ger-many).
Assay of components of the
renin-angiotensin
system in
the
tumor and in cell cultures
250 mgoftumor was homogenized with a Teflon glass homogenizer in 1 ml of0.1 M sodium phosphate buffer, pH 7.5, containing the
mixtureof protease inhibitors described by Murakami et al. (11)(buffer
A)andcentrifugedat20,000 g for 30 min. Aliquots of the supernatant werestored at-200Cbefore assay.
RENIN ASSAYS. Enzymatic assay. Theconcentration of activerenin wasassayed by its enzymatic activity on 0.5 MM renin-free human
angiotensinogen prepared as already described (8). AI produced during
the 30-min incubationwas measured by RIA (12). Inactive renin was
activated bytrypsintreatmentaccordingtoSoubrieret al. ( 13) as follows: 25 Mlculture medium (up to 1/5,000 dilution) was incubated at 4°C
for10 minwith I Mgtrypsin (Sigma ChemicalCo., St. Louis, MO) in 25 Ml 0.1 Mphosphate buffer, pH 7.5. The reaction was stopped by
adding 100 Mg soybean trypsin inhibitor in 0.1 M citrate-phosphate
buffer,pH 5.7.This concentration oftrypsin resulted in maximal renin
activation.Thus measurementof reninconcentration aftertrypsinization
provides an estimate of total renin that includes both active and inactive
renin.
Direct renin RIA. Arapid directrenin RIA was established in order to followthedailyrenin production in cultures. The modification of the previously described method (10) consisted of a decrease in the
incubationtime of renin with antiserum (2 h instead of 48 h) and an
increasein the amount of both renin and antiserum. The modification made itpossible to obtain renin measurements within 6 h.
RENIN CHARACTERIZATION. Renin produced in cell cultures was
characterized bytheeffect ofpH onits reaction with human
angioten-sinogen,and by the effect of
RI5
antiserum. These effects were comparedwiththoseofstandard human renal renin (Medical Research Council,
London, England). Theoptimum pH of trypsin-activated reninwas
determined by incubation with 0.5MMhumanangiotensinogen in citrate-phosphate bufferatvariouspH,from 4.5 to7.0. Theability ofrenin antiserum to inhibit the enzymatic activity of25 M Goldblatt Units
(GU) of trypsin-activated renin wastested byincubatingthereninand antiserum together for 24 h at4VCandthenmeasuringtheremaining
activity. The high immunospecificityof thishumanrenin antibody has
already been demonstrated, by adsorption ofR15antiserum with pure human renin, in both immunofluorescence staining (9) and in renin biosynthetic experiments (14).
ANGIOTENSINOGEN ASSAY. Angiotensinogen was measured by
direct RIA (15). The sensitivity of this method is 200pg/assaytube. CONVERTING ENZYME ASSAYS. Converting enzyme was mea-sured by both the spectrophotometric assay of Cushman and Cheung (16) and by a direct RIA (17). The sensitivity of the RIA was 1.5 ng/ assay tube.
Renin biosynthesis
LABELED AMINO ACIDS INCORPORATION. Biosynthesis experi-ments were performed both on the tumortissuedirectlyaftersurgery and on cells inprimaryandsecondary culture.
Tumor tissue. Tissue was finely chopped using scissors, and 200 mg
of minced tissuewasincubatedwith 2.8ml methionine- and leucine-free Eagle minimal essential medium (MEM) (Gibco Laboratories) sup-plemented with 110 MCi [35Sjmethionine (Commisariata l'Energie Ato-mique, Saclax, France [CFA]) and 200 MCi [3H]leucine (CEA) with
specificactivity of 1,095 and 50 Ci/mmol, respectively. Incubation was
performedat37°Cwithhorizontal agitation under5%CO2in02.The
pulse experiments lastedfor 20 min or 4h,followedby a chase of 4 or
6 h, respectively. Thechase was realized by addition of 2 Umol of
nonlabeled methionine and leucine. In a separate flask thepulse ex-perimentwasextended up to 10 h.Thereactionwasstopped by removing the medium. Tissuewashomogenized withaTeflon glass homogenizer using 1 mlbuffer A/0.1 g tissue. The extracts were centrifuged 30 min at 20,000 g. The supernatant wasdialyzed overnight againstbuffer A and stored at -20°C.
Cellcultures. On day 12of the primary culture of flask C5, andday 17ofthe secondary cultureobtainedfrom C5 cells, themedium was replaced by serum-free RPMI culture medium. 20 h later the medium was changed and cells were incubated in methionine- and leucine-free Eagle MEM (Gibco Laboratories), supplemented with 75 MCi of
[35S]methionine/ml (CEA)and 75,Ciof[3H]leucine/ml (CEA) witha specific activity of1,070 and 105 Ci/mmol, respectively. Incubations were continued for 48 h and an aliquot ofeach supernatant was removed atvarioustimes.Inparallelexperiments a 1,000-fold excess of
nonra-dioactive methionineandleucine was addedafter 1, 3, 6, 10, 24, and 48 h. Themediumwas thendialyzed overnight against buffer A,
cen-trifuged,and stored at -20°C. The cells were scrapped off the flask with arubberpolicemanand washed three times with 2 ml buffer A. Cells were then suspended in 0.5 ml of the same buffer, sonicated for 15 s
and stored at -20°C. Simultaneous measurements of radioactive-labeled proteins and renin were performed both in the medium and in the cell
sonicate. In a separate flask C5, the incorporation of labeled amino acids was performed in the presence of10-5M cycloheximide. After a 24-h incubation the reaction was stopped as described above.
DETERMINATION OF THE RADIOACTIVE-LABELED PROTEINS.
The incorporation of
["S]methionine
and[3H]leucineintoproteins inincubation media or cell extracts was measured in
5-,Ml
samples that were precipitated with 100Ml 10% trichloracetic acid in the presence of 100 Ml albumin(1 mg/ml) as a carrier protein. The precipitate was washed four times with 1 ml 5% trichloracetic acid, dissolved in 500Mlspectrometer using Picofluor 15 (Packard Instrument Co., Inc., Downers Grove, IL) as scintillation cocktail. Correction was made for the overlap of 35S in the 3H-channel.
PURIFICATION OF RADIOACTIVELY LABELED RENIN. Renin
present in the incubation medium and in cell and tissue extracts was
purified by two different methods.
Method1.Immunoprecipitationwith thereninantiserum
R15*5
,pl
oftissue extract or 25 Ml ofculture mediumor cell extract wereincubated for 24hat 40C, with 5 MI R1, antiserum in 0.1 M phosphate buffer,
pH 7.5, containing 0.2% EDTA,1% TritonX-100,and 1mg/ml albumin,
ina final volume of 200 Ml. The immune complex was then adsorbed
on 30 ul Pansorbin (Calbiochem-Behring Corp., San Diego, CA) and
washed four times with the same buffer. Incorporation oflabeledamino acids into renin was calculated as the differencebetween theradioactivity incorporated into the immune complex formedwithR15antiserumand the radioactivityincorporated into the complex formedwithpreimmune
serum. Using iodinated pure human renin (10) as aprobe, it wasverified that5ul
RI5
bound 80% of the renin containedin 5Ml of tissueextract and 90% of that contained in 25 Mldialyzed culture medium orcell extract. Radioactivity was quantitated asdescribedabove.Method 2. Affinity chromatography on antihumanreninmonoclonal antibody-Sepharose was performed using a new monoclonal antibody directed against human renin, 1A12 F37, obtained using themethod already described (4). IA12 F37 differs from the first monoclonal an-tibody, F15, by its higher affinity for human renin (association constant 109 M-'). Half-maximal binding to iodinated renin is obtained at a
concentration of 5X10-10M. The antibody is specific of primate renin anddoesnotbind renin of other species nor other acid proteases: the binding of iodinated renin to the antibody is displaced by5ng human renin whereas up to 5 ug mouse or hog renin, hog pepsin, or human cathepsin D do not modify this binding. The specificity ofIA12F37 forhuman renin was further documented by its ability to exclusively stain JGC ofhuman kidney, both in indirect fluorescence and peroxydase-antiperoxydase technique. The purified IgG fraction of the antibody was coupled to Sepharose 4-B according to March et al. (18) using 10
mgIgG/ml gel. The immunoadsorbant obtained wascharacterizedby itsability to bind human active and inactive renin at pH7.5. Active reninwasobtained from a kidney extract. The source of inactive renin wasthe supernatant of chorionic cell culture that contains renin under
a 95% inactive form (14). Both forms of renin were eluted in 6 M guanidine. Three different types of extracts were purified on 0.5 ml immunoadsorbant: (a) extracts from tumor slices (450 Ml); (b) culture medium corresponding to 48hof biosynthesis (10 ml); and (c) 500Ml
of cold medium from C5 flask, supplemented with -50,000 cpm io-dinated pure human renin. This preparation was run on the gel asa
control for the elution pattern of both active (iodinated) and inactive (culture medium) renin. Renin containing fractions were dialyzed and
lyophylized, and aliquots of the fractions were analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis according to Laemmli (19) with 10% acrylamide. Protein standards for molecular weight deter-mination were obtained from Bio-rad Laboratories (Richmond, CA). Labeled proteins were detected by fluorography (EN3 HANCE, New England Nuclear, Boston, MA) using Kodak X-ray film NS2T (Eastman KodakCo., Rochester, NY). Autoradiograms were either revealed or scanned usingcellomatic equipment (Sebia, Paris, France).
Results
Characterization of the
tumor
MORPHOLOGY. The tumor weighed 14g and was delimited byafibrous capsule.
Light
microscopy. The tumor consisted of polygonal cells arranged in cords from one to several cells thick. Tumorcells showed largevesicular nuclei
withoccasional
nuclear poly-morphism and finely granular cytoplasm. On impregnation with silver stain, cellsappeared
to besurrounded
by a basement membrane, which was particularly evident in closeproximity
to small muscular arteries. In all areas the tumor was richly vascularized, with ectasic vascular profiles in some areas. Small arteries showed intimalthickening
andhyalinosis.
Renin
immunofluorescence.
Thecytoplasm of
most tumorcells was fluorescent
(Fig.
1 B). Some groupsof
cells showed intense homogenous fluorescence, while other cells werenegative
orfaintlypositive.
Some cells hadpositive
cytoplasms in the media of intratumoral muscular arteries. Controls performed with preimmune serum were negative.Electron
microscopy. Tumor cells showed largeGolgi
com-plexes, and well developed rough endoplasmic reticulum. Mi-crofilaments and attachment bodies were present in the cyto-plasm of many tumor cells. Two types of electron opaque se-cretory granules were evident (Fig. 1A): Rhomboidcristalloids
within membrane-bound vesicles adjacent to Golgi (protogran-ules) andspherical
or irregular shaped uniformly dense granules (mature granules). In some cells the smooth endoplasmic re-ticulum was markedly dilated.BIOCHEMISTRY. Active and total renin were measured in thetumoral tissue extract by enzymatic assay: 130 and 180 GU were, respectively, obtained per gram tissue. The same concen-tration
of
total renin wasobtained
by direct RIA, i.e., 180 GU (=220gg)
per gram. This signifies that 72% of renin in thetumoris presentasanactive form. Active and total renin were reassayed after 1 mo storage of the tissue extract at -20'C. Identical results were obtained.
Characterization of cells
in
culture
MORPHOLOGY. About
30-40%
of cells werelost at the first changeof
medium on day 3. Mostof
thediscarded cells consisted of blood cells. Some of theremaining
cells started to divide and differentmorphological
aspects were observed, depending on theoriginal
technique of celldissociation.
The
mechanically
dispersed cells started to divide after 6 d in cultureforming
large monolayercolonies
of regular polygonal cells.These"epithelial-like"
cellswereconfluent
by day 10-15. Aftertrypsinization,
thesame morphologywasobserved in sec-ondary culture. Nosignificant immunofluorescence staining
wasdetected with renin antiserum.
Thecollagenase
digestion
producedamixedpopulation of
cells: (a) A very low amountof
"epithelial-like"
cells(about
onecolony per flask)wasobserved in flasks
containing
thecells extractedduring
the firsttwocollagenase
incubations(C,
andC2);
(b)
A fewelongated
cellsresembling
smooth muscle cells were growing as multilayered colonies(Fig.
2A) andwereevenly
distributed in the cultures flasks(2-3
colonies per75-cm2
flask),
pre-b
a
Ct4o
*.o4\
.,
eWC
Figure 1. Immunofluorescenceand electronmicroscope photographs oftumoraltissue and cultured cellsobtained from the tumor. (A) Electronmicroscope ofthe tumor. Portion of two cellsincluding pro-togranules (singlearrow), homogenousmaturegranules(double
ar-row), and the Golgi (G). Also shownareintracytoplasmic
myofila-ments(A). X 14,000. (B) Immunofluorescence ofthe tumoral tissue
(X 300). Numerouscellsexhibitedacytoplasmic
immunofluores-cence.(C) EMofacellin culture. Detail ofalarge elongated cell
showing peripheralbundle ofmyofilamentswithdensebodies,
pro-eminent rough endoplasmic reticulum,andvacuolescontaining amorphorm electron-dense material (X 24,000). (D) Immunofluores-cenceofthecells in culture. With renin antiserumagranular cyto-plasmicfluorescence is observed withinonebinuclear cell. Adjacent
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4
.. '# j, .
Figure 2. Phase-contrastmicroscopyof tumoral cells in culture (X 140). (A) Elongated cellsformingmono- ormultilayers. (B) Round and
birefringent
cellsaggregated in small clusters.dominant
cellsobtained in allfive series of flasks (Fig.
2 B) and were the onlycell types observed inflasks
C4 and C5. These cells showedauniform morphology.Inflasks
C4 andC5 mitosis
was observed only between days 8 and 12 of primary culture. During the following days, the cells appeared to be in a resting phase.By
immunofluorescence, carried
outafter
20 dofprimary
culture,
someof
these small round cellswerediffusely
stained with renin antiserum; in some cells, thespecific
immunofluo-rescence was moremarked(Fig. 1 D). By electron
microscopy,
alsocarriedout atthe end of theprimary culture,
nogranules
-similar
to those observed in the tumoraltissue
were found inthe cultured small round cells. Most cells had an
extremely
abundant
rough endoplasmic
reticulumcontaining
frozenma-terial. A few bundles of microfilaments
presenting
fusiform,
electron-dense bodies were present in their
periphery (Fig.
1 C). Subcultures wereperformed
on thecollagenase-dispersed
cells. After the first trypsinization of flasksC5,
thesame mor-phology of the small round cell clusterswasobserved with thepresence ofmitosis between days 5-8. Isolated cells seemedto
degenerate
veryrapidly.
Afterthe secondorthirdpassage,cellswereelongated andproliferatedvery
quickly.
BIOCHEMISTRY.No renin could be detected in
mechanically
dispersed cells grown as monolayer of "epithelial-like" cells. However, reninwasrepeatedlydetected in the culture medium of all
primary
cultures obtainedby collagenase
treatmentof the tumoral tissue (C, to C5). During the first days of culture, averylarge amount of immunoreactive renin wasfoundinthe culture medium. The renin level decreased during the first
days
andremained fairly stable from day 6 today 24 (Fig. 3).En-zymatic and immunoreactive renin wasfound in the medium ofprimary culturesup today 27 and in secondary and
tertiary
culture media. After three passages, no renin wasdetected,
whichever methodwasused (Table I).On thefirst day of culture, 45% of reninwaspresentinan active form but thispercentagerapidly decreased with increased duration of culture (Fig. 4). By day 10, <5% of reninwasactive. Asfor thetissue, measurementof total renin by directRIAor byassayof theamountof AI produced after trypsin activation gave similar results when expressed in GU (r = 0.96 for
n = 24).
Renin present in the culture medium showed
enzymatic
andimmunological properties similartothose of MRC human renin: trypsin-activated renin from the culture medium had thesameoptimum pHcurvewith human angiotensinogen (pH
5.7)
as MRC renin (results notshown); the enzymatic activity of bothMRChumanrenalrenin and renin from cell culturewere
fully inhibited by renin antiserum at a 1/10,000 dilution and
50% inhibition was obtained using a 120,000-fold dilution of the antiserum. Itwasverified thatthe culture medium
containing
FCS itself did not generate Al during the 30-min incubationand did not displace renin in the direct RIA. Finally, pure
human renin and renin from the cultures exhibited parallel
curveswhen assayedinserialdilutionsinthe direct renin RIA
(Fig.
5).
Angiotensinogenandconvertingenzyme were measuredin
the media offlask C5 at days 2, 6, 19, 22, and 23 of primary culture andincontrolincubation medium containing10%FCS. Assay ofconverting enzyme by the enzymatic method revealed the presence ofalowconcentration of converting enzyme (1.00
to1.44mU/ml)inthe culture medium, with similar levels being obtainedin controlmedium (1.69 mU/ml). Convertingenzyme
measured bythedirect RIA specific for the human enzymewas
undetectable throughout the culture as well as in the control
medium.Itistherefore assumed that converting enzyme activity detected bytheenzymatic assay was duetothe presenceofthe enzyme in FCS, and that no human converting enzyme was
secretedby the cells.
ll.
..*
.4
lk.jl
Pk.
A ..:.
V
Collagenase Extracts
5
CS
Collagenase Extracts2.5_
Days ofcu
l l l l
Figure
3. Meandaily
reninproduction
in theme-dium of
primary
cell cultures.Immunoreactivereninwasdetected
by
rapiddirect RIA fromday
2to
day
24. Resultsareexpressedinmicrogram
ND renin in the medium per day of culture. Determi-nationswere
performed
onthree separate flasks(Cl, C2, C3)for the first 10 d of culture and on 8 10 12 14 16 18 20 22 24 three separateCs flasks from day 6 to day 24. The
arrowsindicate the
change
of medium. Renin de-terminationwasnotperformed
betweenday
8 Iture and 10 inCsflasks. ND, not determined.By direct RIA
specific
for humansubstrate, angiotensinogen
wasundetectable in the medium
throughout
theculture as wellasin the control medium.
Therefore,
it is assumed that these reninproducing
cells do notrelease
angiotensinogen
into
themedium.
Biosynthesis of radiolabeled
renin
BIOSYNTHESIS EXPERIMENTS ON CELL CULTURES.
Ra-dioactive amino-acid incorporation
into totalprotein
andreninwas studied in
flask C5
at day 13 ofprimary
culture andatday 18 ofsubculture.
Fig. 6 shows the time course ofthe distribution ofradiolabeled proteins
andrenin between the cell extracts and the culturemedium. Renin,
like mostnewly biosynthesized
proteins,
was not stored in the cells but wasrapidly
secreted into themedium. Cycloheximide abolished
reninbiosynthesis
during
the 24 h of the study. Since renin was not stored inside the cell, furtherstudies
werecarried out on renin secreted into the mediumfor
up to 48 h. Reninbiosynthesis
waslinear fromTableI. ImmunoreactiveReninProduction by TumorCells inCulture (ng/10 cells/h)
C2 flasks Csflasks
Primary culture 10 38
First passage 11 ND 10 22 ND
Second passage 0 ND 5 ND 3
Thirdpassage 0 0
Fourth passage - 0
ND, not determined.
50
40
Z 30
z
.2O
2 20
10
2 4 6 8 10 12 14 16 18 2022 24 26
DAYS OF
CULTURE
Figure
4. Variation of percentage of active renin in the mediumdur-ing
primary
culture. Thepercentageof active renin isthe percentageratio of active renin
(measured
beforetrypsinization)tototalrenin(measured
aftertrypsinization).No determinationswereperformed
between
day
10and 13. The total reninproductionwas6, and2X4ng/flask
per 106cellsperday
ondays2 and4, respectively,andwasstableat .5 ng betweenday4 and 24.
5
-z 0
U
>-z ".
Z
2.5-Lu
z
LU
f 12
6 8 10cl,c2'C3
-40 20 100 5S
Figure
5. Direct RIA of renin in the cultureme-dium.(Standardcurveobtainedwithpure human
renin
(25-800
pg/assay) (.).Serialdilutionofcul-turemedium(5-40-fold dilution) (o).Abscissaare
plotted
inalogarithmicscale.6to48 h. From these experiments itwascalculated that renin represented 3-10% of the total biosynthesized proteins secreted into the culture medium. Whenbiosynthesis studieswere
per-formed again, on day 18 ofsecondary culture, renin wasstill radiolabeled but represented only 1-2% of thebiosynthesized
proteins in the medium.
Theradioactivelabeled reninwascharacterizedinSDS-gel
electrophoresis after purification by immunoaffinity chroma-tographyon anantirenin IA12 F37antibody-Sepharosecolumn (Fig. 7 A). Renin exhibited a single band with an apparent molecular weight of 55,000. In thesameconditions,pureactive
human renin hadanapparentmolecularweight of44,000.No 44,000-molwtrenin could be detectedinanyof the cell culture media assayed. Using the same antibody-Sepharose column, both iodinatedpure human renin and immunoreactiverenin from the culture mediumwere eluted in the sameguanidine
fraction (Fig. 7 B).
BIOSYNTHESIS EXPERIMENTS ON TUMOR SLICES.
Labeled reninwasalso obtained in the experiment performed
onthe tumor slices andwaspurified by thesame affinitygel, specific for human renin. Purified fractionswere analyzed on
SDS-gel electrophoresis. The patterns of thegelsafter
autora-diography and scanning are given in Fig. 8. No radioactive
material could bedetectedontheextractcorrespondingto
20-min incorporation with
[35SJmethionine
and [3H]leucine. In contrast, theextract correspondingto a4-hpulsewas charac-terizedby
thepresenceofamajor bandof55,000andaminorband of44,000molwt,while theextractcorrespondingtoa 4-hpulse + 6-h chase exhibited a minor band of55,000 and a
majorband of44,000 molwt.
Discussion
This new case ofJGC tumorshows a number ofsimilarities
withmostof the previously describedcases. The renincontent
ofthe tumor was - 1,000times higher than that found in a normal human kidney (20). By light microscopyand immu-nofluorescent staining,these tumoral cells possessed the struc-turalcharacteristicsand the immunochemical properties of ep-itheloid cells from theJGCapparatus. Almostno immunoflu-orescence wasfound in the cytoplasm of the myocytelike cells
surrounding the vessels, whereas granular and diffuse
immu-nofluorescencewasobserved in therenin-producing cells. Ul-trastructural studies of these cells showed thepresence of two
types of granule, specific rhomboidal protogranules with a paracrystalline structureandspherical dense reningranules. 100
60
0
,0
0o
Im
4020
35S
[cpm X103]
TOTAL
PROTEINS
250-CULTUREMEDIUM
RENIN
151
5-CULTURE MEDIUM
+ CYCLOHEXIMIDE
CELL EXTRACT_
1 3 6 10 24
Hours of
incorporation
Figure6.
[35S]methionine
incorporationinto totalproteinandrenin. Counts per minute of IS-labeledtotalproteins (top)and35S-labeled renin(bottom)weremeasured in culture medium (-- - -*)andcellextracts
(.-
*)after 1 to24hofincorporation. Arepresents the cpm ofI'S-labeled
renin in the culture mediumcontaining 10-0 Mcycloheximide.
This
renin
tumoroffered
aunique
opportunity
toculture and subculture the human JGC. Suchstudies
areextremely
difficult
toperform
in normalkidney
due to thescarcity
of these cells. Theavailability of
arapid direct
reninRIAgreatly
facilitated
thedetectionof renin
production
in thecultureme-dium;
results could beobtained
within 6 h. Thehigh
specificity
of the
polyclonal antibody
raised to human reninprevented
anyinterference
with renin in FCS. Since theincrease
in theenzymatic activity
of reninafter
trypsin
treatmentinvitro,
in plasma,amniotic fluid
(8), and chorionic cells(14)
isnotac-companied byachangein immunodetectable
renin,
it iscon-cluded that our antibody, as others (21)
recognizes
both theactive and inactive forms of the enzyme. The results of the enzymatic assay of
trypsin-activated renin
areidentical to those with theRIA(r=0.96). This identity has also been confirmed by Cravenetal.(22) with chorionic renin, using our antibody.Conn etal. (23) have
previously
cultured renin cellsfromaJGCtumorand were able to maintain a renin production up
to 500 h. Inthe present study, two types of cell preparations were
tried
and only collagenase treatment produced cells that synthesized renin. The small round cells observed after longexposure to collagenase are most
likely
tobe the renin-producing cells since biosynthesis of radiolabeled renin was observed in theflasks
that contained onlythis
cell type. Nofirm
conclusion can bedrawn on the ability of the smooth musclelike cells to producerenin, since no culture flask contained exclusively this typeof cells. Although it is difficult to compare the morphology of cells in culture with that observed in tissue, the morphology of the cultured "smooth musclelike" cells was close to that of themyocytelike
cellsof
the tumor that failed to exhibit im-munofluorescence with renin antiserum. Finally, the round renin-producing cells became elongated after 2-3 passages and at the sametime
losttheir ability to produce renin. Thisde-differentiation
ofrenin-producing
cellsis expected as JGC tumorsare
nonmalignant
tumors.The
Al-producing
enzyme released by the cells in culture wascharacterized as renin by its ability to hydrolyze humanangiotensinogen,
its pHoptimum,
the inhibitionof
itsenzymatic
activity byrenin
antiserum at the samedilution
as standard human renin, andits immunological identity with
standard renal and pure tumoralrenin (10)
demonstratedby the paral-lelism of the dilution curves inourdirectRIA.During
the first days of culture, 45% of renin released wasinactiveform,
but the percentageof
activerenin
in themedium
decreasedwith
theduration of
culture andby day
10essentially
allrenin
in the medium was inactive. Therelatively high
levels ofactive
renin in the medium initially maycorrespond,
atleast in part, totherelease of the enzymepreviously
storedwithin
thesecretory
granulesof
the tumor, as the tumoritselfcontains
predominantly
activerenin.
Similarly,
an initial large releaseof active
renin into the culture medium was found by Conn et al. (23). After this sharp decrease in renin release,astablerenin concentration
wasfound in the culture medium, both in this study and in the reportof Conn. However, Conn may haveunderestimated renin
concentration since
only activerenin
was measured. Thisfairly
stablerenin
concentration in the culturemedium
suggests that the cultured cellsareactuallysynthesizing renin. Definitive
proof
of
this
synthesis
wasprovided
by theincorporation of
labeled amino acids into the renin molecule in bothprimary
andsec-ondary culture. The
fact
thatnewlysynthesized renin
israpidly
released in the culturemedium
and not stored in the cellis
consistent with the low level ofimmunofluorescence
staining
in the cultured cells and with the absenceof
granules by electron microscope.Theinactive renin found in the
primary
andsecondarycul-turemediawasactivated by
trypsin,
aswith plasmaoramniotic inactive renin. After activation, renin had thesamepropertiesas
fully
active renin. Inactiverenin
(after
24 and 48 h ofin-corporation)
wasbiosynthesized
as asingle
molecule of55,000
mol wt, larger than the fully active pure human renin molecule (44,000 mol wt)(14).
By contrast, in thebiosynthesis experiments
performed
onthe tumorslices two forms of labeled reninwereA
8250
-0
x
CA)
c19
E
0.
u
40
-30
-20
10
0
.5
O Z -0
S*
2s
_ s2 10 14 18 22
B
8 12 16
Elution volume
(ml)
Figure7. Purification of the labeled culture mediu finity and determination of the molecular weight(
onSDS-gelelectrophoresis. (A)(right)SDS-gelelei
vealedbyfluorography:
(CD)
lodinatedpure humantoaffinitychromatography (B) and elutedin 6M
consists of 44,000-mol wtnormalrenin (arrow) an
nins,23,000 and 17,000 molwt, found only in dis tions(3).© Labeled total culture medium. 0 Labe
diumpurifiedonaffinity chromatography(6Mgu (A)(left)Represents the elution pattern of IS-label
dium (-). 10mlof this medium (O)wasapplied
tF
noadsorbantobtained by coupling the antireninm toSepharose. C) Renin-containingfraction elutedv
dine. (B) Control elution pattern on 0.5 mlofthe
sorbant.(0)Peakof iodinated pure human renin(
that renin was
biosynthesized
asa 54,000-molwt protein thatwasconvertedintoa44,000-mol wtmolecule after activation
by trypsin (14).
Takentogether,
theseexperiments
suggestthat reninis biosynthesizedas aninactiveprecursor(prorenin) withanapparentmolecular weight 10,000 higher than active renin. Such resultsaresupportedby therecentmodel for the processing (1tJ) ®) @) of submaxillary glandmouse renin, deduced from the cloned -- cDNA ofrenin. Mouse renin is
synthesized
as aprecursor(44,000
mol wt) and processed into active renin (38,000 mol wt) after
releaseofaprosequenceof46-amino acid length (24). Murakami
(25)
has recently shown that inman renal preprorenin synthe-tized ina cell-free reticulocytesystem hasa molecularweight
of 45,000. The apparent increase in molecular weight of the presently described 55,000-mol wt prorenin canbe explained by the fact that human renin, butcontrastwith mouserenin,
isglycosylated
(3, 26).
Glycosylation
is well known to bere-sponsible foranapparentincrease of protein molecular weight onSDS-gel electrophoresis. 55,000-molwtglycosylated
prorenin
is further processed into 44,000-mol wtglycosylated renin. Acomparative schema for thematuration ofmouseand human renin isproposedin Fig.9. Molecular cloningandstudyofthe
structureof human renin cDNA will mostlikelyconfirm this model of processing.
In this study, the cultured cells did not release
angioten-sinogen or converting enzyme into the medium: thesecom-ponentsof the renin-angiotensin systemwere notdetected by enzymatic ordirect RIA. This is incontrastwith thereportof _
1.5 -75 Rightsel et al. (2) who found angiotensin converting enzyme
activity
and(Al
andangiotensin II/III)
inaculture ofratJGC. o In thisstudy,
nodetermination ofangiotensinogen
orconverting
- 0 5 enzyme were performed on the cell extracts andtherefore
itcannotbe assessed whether tumoral cells either failtoproduce or
only
failto release theseproteins.
0 5 25 Basedonthe dataobtained during the culture
experiments
of renin-producing cells (from JGC tumor and chorion) and renin production by tumoraltissue
slices, a model for reninK.
-biosynthesis,
packaging, processing,
and secretion can be pro-20 posed. We hypothesize that renin is synthesized in a preproform, andthat theprefragment israpidlyremoved in theroughen-doplasmic reticulum. Renin could then be released via oneof
im byimmunoaf- twodifferent
pathways (Fig.
10).
Pathway
1 wouldbeasfollows:)fpurifiedfractions (a) accumulation of renin in the Golgi apparatus; (b)packaging ctrophoresis is re- of renin in the rhomboidal protogranules and then in the
spher-i renin submitted ical renin granules; (c) processing of prorenin into activerenin
guanidine. This (associated with thepackaging);and(d)secretion of activerenin
idtwo "small" re- byexocytosis.Analternative pathway
(pathway
2) involvesse-ssociatingcondi- cretion of inactive prorenin without formation of renin granules.
-ledculture me- Several observations are consistent with this two-pathway hy-anidinefraction). pothesis: the presence of both active and inactive renin inthe ledculture me- tumoral tissue has been demonstrated by biochemical
mea-o
(.5 ml
immu-Monoclonalantibodywith 6 M
guani-same
immunoad-(o). Renin
purifica-tionfrom 0.5 ml of unlabeled Cs cell culture medium is represented by absorbance peak(---) andimmunoreactivepeak(o---o).
Ar-rowsindicate changes of eluting conditions similar to A.
-mob.
S
w U z
m
co
0 U1) 0
MOUSE
(44K)
(42K)
(38K)
55
Figure 8. Scanning of
SDS-gel
pattern of radiolabeled C reninobtainedfrom tumorslices.Tissuewaspulsed with
[35S]methionine
and [3H]leucine. (A) 4-h pulse; (B) 4-h pulse + 6-hrchase;and(C) 10-h pulse. Arrow indicates
position
of iodin-2 4 6 8 ated renin run on the sameCM MIGRATION gel.
surements
performed
on the tumor homogenate. Thebiosyn-thesis experiments
have also shown the presence oftwoforms of renin(55,000
and 44,000 molwt)
in the tumor and the conversion ofthe 55,000-molwtform(prorenin) into
the 44,000-mol wt form(active
renin). In thistissue,
reninis found
in secretory granules andmorphological
data suggest that thema-ture granules were
derived
from the protogranules.Inthe kidney, also, the same two typesof
renin granules have beendescribed:
sharply angulated granules(identical
to our protogranules)in-dicative
ofahigh
rateof renin synthesis and lobulated granules(identical
to our mature granules) (27). By contrast, no reningranules could be detected by EM in cultured cells between days 15 and 27. It was not
possible,
because of the limited numberof cultureflasks,
to make serialEMstudies throughout theculture,
and no correlation could be made between renin granularity and the decreased active per inactive renin ratio in the supernatant. However, renin was not stored orprocessed in the cells at a time when the cells were still able to synthesize renin and to release it at a constant rate. We speculate that cultured cells from the JGC tumor progressively lose the capacityto package and process renin (pathway 1) and that these cells
then secrete
inactive
renin via pathway 2. Pathway 2 might also exist in vivo in chorionic cells that are characterized by the presence ofpredominantly
inactive renin both in tissue ho-mogenates (28) and incell
culture(14).
This two-pathway model, whichimplies
that mature granules contain mainly active renin,involves
several assumptions that will have to be proven. The(45K) PREPRORENIN
(55K) Ppnarh~illh
RENIN
ACELLULAR TRANSLATION
n INACTIVE
(44K) IN VITRO CELL
I I
BIOSYNTHESIS ~"ACTIVE
Figure
9.Comparative
schema for the maturation ofmouseandhu-manrenin. Maturation ofmousereninwasdeduced from the renin-cloned cDNA
(24)
and from thebiosynthetic
pathwaydescribedby
Catanzaroetal.(6).
HumanpreproreninwasobtainedbyMurakami inanacellular translation system(25). 55,000-
and44,000-molwt reninwereobtained in the JGCtumor.m,Prefragment;m,profrag-ment; cD, active
renin;
andX,
site ofglycosylation.
exact natureof renin in the renin
granules
isnotknown,
sincesomeauthors found inactive renin in
purified
ratreningranules
(29)
whereas others foundmainly
active renin ingranules
fromdog
(30)
and human(31)
JGC. It will also beimportant
to further document the fact thatprorenin
is not stored in anyother
type
ofgranule
in culturedcells,
and is indeedimmediately
secreted.Finally,
reninsecreting
cells can be compared with other endocrine cells.According
to ourhypothesis,
reninpackaging
associated with renin
processing
in JGC is similartothe anteriorpituitary
cell line that containsonly
matureforms ofcortico-tropin
and betalipotropin
in their secretory granules(32).
In these cells the presence oftwo distinct pathways have been described for the transport ofglycoproteins leading
tothese-55KInactive renin
44 K Activerenin
Golgi
Figure
10. Proposedmodel oftwopathways for the release of renin.Pathway(X wouldbepredominantin JGC of the tumoral tissue
(55,000-molwtrenin). Pathway 0) would be predominant in the
renin-producingcellsin culture (44,000-mol wtrenin).
MAN
rKUKLMIN W9
cretion in the medium ofeither ACTH precursor or mature
ACTH (33).
Acknowledgments
The skillful assistance ofMrs. F. M.Gonzalesand I.Laboulandine is
greatlyappreciated.The authors wish also tothankDr.C. Genain for the measurement of angiotensinogen andDrs. F.Alhenc-Gelasand T.
Yasui for the assays ofconverting enzyme. Finally, we express our
gratitude to Drs. J. Campbell and T. Kotchen for theirstimulating discussions duringtheredactionof themanuscript.
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