Effects of Angiotensin II and Angiotensin II Antagonist Saralasin on Cell Growth and Renin in 3T3 and SV3T3 Cells PIERRE SCHELLING,' DETLEV GANTEN,' GEORG SPECK ' AND HANS FISCHER * ' Department of Pharmacology, Uniuersity of Heidelberg and Institute for Virus Research, German Cancer Research Center, Heidelberg, 6900 Heidelberg, West-Germany

ABSTRACT Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian viruk 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin I1 stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin I1 may be involved in cell growth regulation, (2) that a negative feedback exists between angiotensin I1 added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.

.

I

'

Enzymes similar to kidney renin (EC 3.4.4.15) can be synthetized in extrarenal tissue; they are part of a complex reninangiotensin system (RAS) consisting of the enzyme renin, its substrate angiotensiogen from which the decapeptide angiotensin I (ANG I) is cleaved, and converting enzyme (CE), which converts ANG I to the biological effector peptide angiotensin I1 (ANG 11). Specific ANG I1 receptors have been described in many tissues and the ANG I1 degrading enzymes, angiotensinases, are almost ubiquitous (Oparil, '76; Ganten et al., '77; Peach, '77). The enzyme renin is an acid protease which specifically cleaves the leu-leu bond of the angiotensinogen leading to ANG I production. Like other acid proteases, e.g., cathepsin D and pepsin, it is inhibited by pepstatin (Umezawa et al., '70; Barrett and Dingle, '72). The ANG I and ANG I1 inactivating angioJ. CELL. PHYSIOL. (1979)98: 503-514.

tensinases comprise a group of enzymes including angiotensinase A (aminopeptidases), angiotensinase B (endopeptidases), and angiotensinase C (carboxypeptidases) (Ledingham and Leary, '74). In the classical RAS, renin is released from the kidney into the blood stream, where generation of ANG I and conversion to ANG I1 occurs. It has been suggested that tissue renins may exert their effects a t the cellular level by local production of ANG I1 (Ganten et al., '76). We have investigated the RAS in 3T3 cells (Todaroand Green, '63) and in Simian virus 40 (SV40) transformed oncogenic 3T3 cells (SV3T3) (Todaro et al., '65) which are established cell culture lines of mouse fibroblasts. The purpose of this study was to see whether Received Aug 29, '78 Accepted Nov 17, '78 Present address Institut de Recherche Cardio-Angexolooque, Chemin du Musee,CH 1700 Fnbourg, Swtzerland

503

504

P. SCHELLING, D. GANTEN, G. SPECK AND H. FISCHER

these cells do synthetize renin and whether ANG 11, t h e ANG I1 receptor antagonist [sar', va15, ala"]-ANG I1 (saralasin), and whether ANG I1 fragments influence the intracellular RAS and cell function. Since tumor-transformed cells a r e known to undergo changes in their proteolytic enzyme activities (Bosmann, '69; Ossowski e t al., '73), not only renin but also the cathespin D-like acid protease activity and angiotensinase activity were measured. Parts of t h e results have been reported previously (Schelling et al., '75; Fischer et al., '78).

per petri dish. Twenty-four hours later, t h e petri dishes were infected with SV40 (Fischer and Sauer, '721, while the controls were mockinfected with conditioned medium. After 24, 48, 72, 96 and 168 hours, the samples of infected and mock-infected cells were harvested and stored at -30°C for enzyme estimations. Estimation of renin and ANG Z

Renin concentration was estimated by incubation of the enzyme with angiotensinogen according to the micromethod of Boucher et al. ('67) as described previously (Ganten e t al., MATERIALS AND METHODS '78). Different angiotensinogens were preCell culture pared from plasma of nephrectomized animals Cloned 3T3 and SV3T3 cells were grown in as described by Haas e t al. ('66).The affinities Eagle's basal medium (BME) with doubled were tested and found to be the highest with concentrations of amino acids and vitamins, sheep angiotensinogen. All estimations were and containing 10%fetal calf serum (FCS) for therefore performed with sheep substrate, 3T3 and 5%FCS for SV3T3 cells. For some ex- which was added in excess in order to obtain a periments, 3T3 and SV3T3 cells were main- first-order reaction with respect to the entained in culture medium without FCS for 72 zyme. The frozen cell pellet was resuspended hours. Furthermore, a calf was bilaterally in incubation buffer and sonified. Cells were nephrectomized and bled 16 hours later to ob- incubated for four hours a t 37°C with 1 cm3 tain a renin-poor serum for some tissue cul- DOWEX 50 W x 2NH,+ and with 2 ml of 0.2 M ture studies; 2 X lo5 cells were seeded per tris-hydroxymethylaminomethan (TR1S)petri dish. Cells were counted under the micro- maleate buffer, pH 5.5, containing 100 mg scope or by a coulter counter. Cells were har- sheep angiotensinogen and 8.6 mmol ethylvested and washed in a phosphate-buffered ene-diamine-tetraacetic acid (EDTA) for insaline (PBS), pH 7.2 (0.12 M NaCl; 0.018 M hibition of angiotensinase activity. The ANG I Na,HPO,; 0.0025 M KH,PO,); they were then formed during the incubation was eluted from frozen and thawed three times and kept a t DOWEX, lyophilized and stored at - 30°C until assayed. The lyophilized incubation - 30°C until assayed. In some cases, the supernatants of t h e cell cultures consisting of BME product was dissolved in 2 ml of cold 0.1 M with and without FCS were collected and TRIS-acetate buffer (radioimmunoassay frozen for peptide and enzyme measurements. (RIA) buffer), pH 7.4, and was measured by a specific RIA for ANG I. The ANG I antibody Addition of ANG 11, saralasin and ANG I1 was used in a final dilution of 1.08 x infragments to thegrowth medium terference with ANG 11,ANG-(2-8)and ANGTwo x lo5 cells were seeded per petri dish. (4-8) fragments and the ANG I1 analogue Cells were examined macroscopically and saralasin was less than 0.001%. Values are excounted 24, 48, 72, 96 and 120 hours after pressed as pmol ANG I per number of cells, or seeding. They were harvested and stored a t mg protein and hour. Proteins were measured by the method of Lowry et al. ('51). - 30°C. Drugs were dissolved in BME containIdentification of the incubation product as ing 0.1% human serum albumin and were sterile filtered; 50 pl of drug solutions were ANG I was performed (1)by injection into a added to the cell cultures 48 and 72 hours nephrectomized bioassay rat with and without after seeding at 10 A.M. and 4 P.M. The final addition of a specific ANG I antibody; (2) by drug concentrations in the growth medium for heating the incubation product for 20 minutes ANG 11 were 50.0, 5.0, 0.5 and 0.005 nmol/ml, to 100°C; (3) by serial dilution in the RIA and for saralasin 1.0 nmol/ml, for ANG-(2-8) 5.5 (4) by digestion with trypsin. Recovery of ANG I during t h e incubation of nmol/ml and for ANG-(4-8) 7.5 nmol/ml. 3T3 and SV3T3 cells was verified as follows: Infection of 3T3 cells with SV40 Homogenates of 3T3 and SV3T3 cells (0.2 ml) Cloned 3T3 cells were seeded a t 3 x lo5cells or 0.2 ml of 0.02% bovine serum albumin

RENIN, ANGIOTENSIN AND CELL GROWTH IN FIBROBLASTS

(BSA) (control) were incubated with 3 pmol ANG I under standard conditions but without substrate added. ANG I was eluted from the columns, lyophilized, resuspended in buffer and assayed as described. ANG I recovery was calculated as percent of the amount added to t h e incubation. pmol ANG

I Img prot./h

505

Possible interferences of intracellular inhibitors or activators on the reaction between renin and renin substrate were tested by incubating 3T3 and SV3T3 cell homogenates as indicated but with and without addition of 0.2 units of purified hog kidney renin. Measurement of angiotensinase activity

3T3 and SV3T3 cells grown under standard conditions as well as in BME without FCS were tested. The protein content was adjusted with bidistilled water to be equal for both cell types; 0.2 ml of the homogenates (3T3 or SV3T3) in 3.0 ml 0.2 M TRIS-maleate buffer, pH 5.5, containing 4 pmol ANG 1 were incubated in a shaking bath a t 37°C; 0.5 ml aliquots were taken at 0,5,10,15 and 30 minutes (cells kept under standard conditions) and 0, 30, 60 and 120 minutes (cells kept in BME without FCS), boiled immediately for five minutes and centrifuged at 7,500 rpm. ANG I was measured in the supernatant by RIA. From the disappearance of ANG I, the angiotensinase activity could be calculated and expressed as ANG I degraded per hour and mg protein.

T

Estimation of acid protease activity sv 3 3

3T3

Fig. 1 Renin concentrations in cell pols (n:4) of 3T3 and SV3T3 cells incubated with sheep substrate during four hours at 37"C, **p < 0.01.

Cathepsin D-like acid protease activity was measured by a modified method of Anson ('37). Cells were incubated with acid denatured bovine hemoglobin substrate for 0 to 300 minutes. The enzyme reaction was

pmol ANG I I mg pr0t.I h 3T3

infection

---.

3T3

elk-------" *svm I

t

2

I

L8 72 96 120 HOURS AFTER SEEDING

I

1U

I

I1

168"after multiple passages

Fig. 2 Renin concentrations in cloned 3T3 cells with time after seeding and after multiple passages which were infected with SV40 and mock-infected with conditioned medium, respectively, 24 hours after seeding.

506

P. SCHELLING, D. GANTEN, G. SPECK AND H. FISCHER

TD

3T3

Std

+A8

3T3

3T 3

STD

3T3

3T3

+A6

STD

+A8

Fig. 3 Characterization of the product formed during the incubation of 3T3 cell homogenate with sheep substrate as ANG I in the rat pressor assay; the type of pressor response of the incubation product was identical to synthetic ANG I and could be inhibited by a specific ANG I antibody. STD,ANG I1 standard; 3T3, incubation product from 3T3 cells with renin substrate; AB, specific ANG I antibody.

TABLE 1

Renin concentration in 3T3, SV3T3 cells and in the supernatant of virus transformed 3T3 cells. FCS, fetal calf serum; CS, calf serum; NX-CS, serum from a nephrectomued calf; BME, Eagles basal medium; numbers of measurements are given in parenthesis Culture medium

3T3

SV3T3

Supernatant from SV3T3 cells

BME BME BME BME BME BME BME BME BME BME

+ +

FCS (10%)

+ +

CS NX-CS FCS (5%)

+ + +

CS NX-CS FCS (5%)

stopped by adding aliquots of the incubation mixture to cold trichloroacetic acid. Non-acidprecipitable products were measured after neutralization by the method of Lowry et al. ('51). Results are expressed as pmol BSA equivalents generated per time and mg protein. Statistics Results are expressed as means 2 standard error of the mean (SEM). Statistical significance of differences was calculated by Student's unpaired t-test. RESULTS

Components of the RAS in 3T3 and SV3T3 cells Renin concentrations in 3T3 cells were 22.64 -t 4.44 pmol ANG I/mg proteinh, and in SV3T3 cells 5.17 -t 0.38 pmol ANG I/mg prot e i n h (p < 0.01) (fig. 1). From the kinetic studies i t is evident that renin decreased with time after virus infection, while enzyme levels continuously increased in mock-infected cells

Renin concentration pmol ANG Ilmg protein&

7.40 20.47 (8) 3.99 k0.15 (4) 1.62 (4 pools) 0.72 (4 pools) 0.40 20.12 (9) 0.72 20.10 (9) 0.20 (4 pools) (4 pools) 0.16 0.016 20.0013 (3) 0.012820.0019 (3)

TABLE 2

Angiotensinase activity in 3T3 and SV3T3 cells grown in BME with and without FCS; ***p < 0.001; n 2 6 pmol ANG I degraded/mg proteinh

BME

3T3 cells SV3T3 cells

Ratio SV3T313T3

iFCS

BME without FCS

23.522 1.36 82.6021.54***

7.69k2.30 23.18k 1.32***

3.51

3.02

(fig. 2). The incubation of homogenates from 3T3 and SV3T3 cells together with hog kidney renin led to the same generation rate of ANG I (236.6 -t 12.39 ng ANG I h , 3T3 cells versus 225.6 -t 3.69 ng ANG I/h, SV3T3 cells). These amounts were not different from the ANG I generation rate of hog kidney renin and angiotensinogen without addition of cell homogenates (226.3 2 9.03 ng ANG I k ) . The incubation product of the renin substrate reaction was identified as ANG I, since the blood pressure increases observed after in-

507

RENIN, ANGIOTENSIN AND CELL GROWTH IN FIBROBLASTS

tt tt

0 24

48

72

96

120 hrs

1 =

O

L

- 24 J

w 4 8L

- 72

96

120 hrs

tt

3.7sJ

"i 25

I

SV 3T3

**

125

0

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.

,

Fig. 4 Effects of ANG I1 and ANG I1 antagonist saralasin (SAR) on cell growth and on intracellular renin concentration in 3T3 and SV3T3 cells; (a) shows t h e influence on growth in 3T3 (*-*) and (b) in SV3T3 ( 0- O f cells, respectively. The corresponding renin levels are presented in (c) for 3T3 and in (d) for SV3T3 cells. ANG I1 and saralasin were added a t 5 nmol and 1 nmol, respectively. Points represent 3 or more determinations of one typical experiment. *p < 0.05; **p < 0.01.

travenous injection into an anaesthetized and nephrectomized bioassay rat were blocked by a specific ANG I antibody. Total blockade could be achieved if the antibody was given in large excess (fig. 3). The incubation product was heat-stable but was destroyed after digestion with trypsin. Its serial dilution curve was superimposable to that of ANG I in the RIA.

Since the standard growth medium itself (BME with 10% FCS) contained renin in low concentrations of 0.09 f 0.01 pmol ANG I/ml BME/h, cells were kept in BME without FCS or in BME with low renin serum from a nephrectomized calf. SV3T3 cells grew under all circumstances, but the cell number of 3T3 cells scarcely increased in BME without FCS.

508

P. SCHELLING, D. GANTEN, G. SPECK AND H. FISCHER

I

, , I

II

ANG

0.

t

*:I '+I 75

ANG

t t

. ,,

8

t

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**

0

co 0005 05 5 50 nmol ANG fi / m i Fig. 5 Dose-response curves of ANG I1 on cell growth (a) and on renin levels (b) in 3T3 cells; 50.0, 5.0, 0.5 and 0.005 nmol ANG I1 were added. *p < 0.05; **p < 0.01.

However, renin could still be found in cells kept under these modified conditions (table 1). The difference between renin content in 3T3 and SV3T3 cells holds true even though the absolute values varied under the different culture conditions. This indicated that the renin measured in 3T3 and SV3T3 cells was not taken up by the fibroblasts from the medium, but was synthetized intracellularly. No ANG I

was generated upon incubation of 3T3 and SV3T3 cells without exogenous angiotensinogen added. SV3T3 cells secreted small quantities of renin into the supernatant (0.016 f 0.001 pmol ANG I/mg proteinh) but angiotensinogen and ANG I in the supernatant were below detection limits of the assay (10fmol). Angiotensinase activity was high in fibro-

RENIN. ANGIOTENSIN AND CELL GROWTH IN FIBROBLASTS

blasts and differed markedly between cells grown in standard medium from cells kept in BME without FCS (table 2), but under both culture conditions, angiotensinase activity ceU numbers lo5

PANG)) 12-81

0

+

0

?

L8

2L

+

+

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hrs

96

71

Fig. 6 Effects of ANG 11, ANG-(2-8) and ANG-(4-8) fragments on cell growth in 3T3 (*-*) and SV3T3 ( 0- 0 ) cells; 5 nmol ANG 11, 5.5 nmol ANG-(2-8)and 7.5 nmol ANG-(4-8) were added.

509

was about three times higher in SV3T3 cells than 3T3 cells. The angiotensinase activity did not affect ANG I recovery during incubation and had no influence on the RIA of ANG I. After incubation of ANG I with 3T3 and SV3T3 cell homogenates, 80-85% of t h e amount added was recovered. These values did not differ significantly from the amount of ANG I recovered after incubation with angiotensinase-free BSA. Cathepsin D-like acid protease activity and renin concentrations were measured in parallel at different times after seeding in 3T3 and SV3T3 cells. The following acid protease activities were found in 3T3 cells: 37.97 2 8.55 pmol BSA/mg protein/h (n:7), and in SV3T3 cells: 33.77 2 8.55 pmol BSA/mg protein/h (n:6). The corresponding renin levels in 3T3 and SV3T3 cells were: 14.29 2 3.20 nmol ANG I/mg protein/3 h (n:7) and 4.32 % 0.79 nmol ANG I/mg protein/3 h (n = 51, respectively (p < 0.02). Influence of ANG ZI, saralasin and ANG fragments on proliferation and renin in 3T3 and SV3T3 cells Cell growth in 3T3 cells was increased when 5 nmol/ml ANG I1 were added to the cell culture medium (fig. 4a). This effect was dose-related (fig. 5a). The specific ANG I1 antagonist saralasin administered at a dose of 1nmol/ml inhibited cell proliferation in 3T3 cells (fig. 4a). Cell growth in the fast growing SV3T3

log of cell number

i

5.5

**

I *-\:

6.0

*

\

5.0-

4.5J f

I

15

30

mol A T / 4 h /1OOOcells

45

Fig. 7 Relationship between renin concentrations and log of cell numbers at different points of the growth curves; 3T3 ( 0 )and SV3T3 (*)cells treated with ANG I1 and saralasin, respectively, as well as untreated controls were taken into account; n:22; r = -0.85;p < 0.001.

510

P. SCHELLING, D. GANTEN, G. SPECK AND H. FISCHER

cells was completely inhibited by the ANG I1 antagonist saralasin and partially inhibited by ANG I1 (fig. 4b). Neither the addition of ANG-(2-8)heptapeptide nor of ANG-(4-8) pentapeptide fragments at doses of 5.5 nmol/ml and 7.5 nmol/ml, respectively, altered cell growth of 3T3 and SV3T3 cells (fig. 6). Microscopically the cells showed no signs of toxic changes under any drug and a t the concentrations used in this study. ANG I1 treatment suppressed intracellular renin content in 3T3 cells (p < 0.01).When the ANG I1 antagonist saralasin was added, renin levels were markedly increased (p < 0.01) (fig. 4c). The effect of ANG I1 on renin was dose-related and the lowest concentration of 0.005 nmol ANG II/ml did no longer suppress renin nor stimulate growth (fig. 5). There was a negative feedback between exogenous ANG I1 and endogenous renin levels in 3T3 cells. The renin content in SV3T3 cells was equally increased after saralasin (p < 0.01). ANG I1 administration slightly increased renin in SV3T3 cells (p < 0.05) when compared with untreated controls (fig. 4d). In both cell types cell growth and cellular renin concentrations were related to each other as shown by the negative linear correlation between renin content per 1,000 cells and the log of cell number (r = -0.85, p < 0.001) (fig. 7). DISCUSSION

Fibroblasts which have been kept in tissue culture for many generations synthetized renin under all experimental conditions tested. Renin was measurable after withdrawal of FCS from BME as well as after its replacement by renin-poor serum obtained from a 16 hours previously nephrectomized calf. The lack of serum suppressed growth in 3T3 cells (Gospodarowicz and Moran, '75) and their renin content was diminished when compared with the appropriate controls. Plasma factors are known to be of minor importance for growth in tumor-transformed cells (Temin, '67; Holley and Kiernan, '68). Accordingly, cell proliferation of SV3T3 cells and their renin levels were hardly affected by serum withdrawal from the nutritional medium. Low but significant renin levels were secreted from SV3T3 cells as measured in the supernatant independently of whether FCS was added to the BME or not. Juxtaglomerular cells from diseased kidneys which were repeatedly subcultured have been shown to release renin in declining

amounts into the culture medium. Cells derived from healthy kidneys quickly lost their ability to secrete renin though they contained typical renin granules (Robertson e t al., '66). Cells subcultured from a juxtaglomerular cell tumor were reported to secrete renin in very high amounts over the observation period of 25 days (Conn et al., '72). In vitro cultures from human chorion and uterine muscle (Symonds e t al., '68) were also shown to synthetize renin. Recently, Viol et al. ('78) could neither find renin content in, nor renin secretion from subcultured monkey and hamster kidney cells. They also were not able to show renin in human fibroblasts and in cells derived from a human uterine tumor (HeLa cells). This is in contrast to results indicating renin in uterine cell cultures (Symonds et al., '68) i n HeLa cells and in established cell culture lines originating from monkey kidneys (CV-1) (Fischer et al., '75). Several factors such as t h e type of cells and growth medium or details in renin measurements may in part explain the different results. The increased angiotensinase activity i n SV3T3 cells is in agreement with reports of enhanced proteolytic activity in virus transformed cells (Bosmann, '69; Ossowski et al., '73). The withdrawal of serum from the growth medium led to a decrease of angiotensinase activity in 3T3 and SV3T3 cells but did not alter the relative difference of enzymatic activity in both cell types. In contrast t o angiotensinases, renin was lower in SV3T3 than in 3T3 cells. Since no activators and no inhibitors interfered with hog kidney renin added to the homogenates, the lower enzyme concentration in SV3T3 cells probably reflects a reduced synthesis rate. This is supported by the kinetic studies with cloned 3T3 cells which showed decreasing intracellular renin levels with time after infection with SV40, while renin content of mock-infected controls increased with time. It had previously been reported that renin and a cathepsin D-like enzyme reacting with denatured hemoglobin substrate are acid proteases with similar physico-chemical properties. In fact, even highly purified kidney and extrarenal tissue renin preparations showed acid protease activity (Day and Reid, '76; Hackenthal et al., '78). Recently, however, t h e cathepsin D-like acid protease activity could be separated from renin by further purification of kidney renin (Murakami e t al., '77) and of brain renin (Osman et al., '78; Ganten and

i

RENIN, ANGIOTENSIN AND CELL GROWTH IN FIBROBLASTS

Speck, '78). In this study, we cannot decide whether interference of both enzymes occurs in the renin and acid protease assay, since no enzyme purification from 3T3 cells has been performed. It may be pointed out, however, that i n t h e renin assay a natural enzyme substrate, angiotensinogen, is used and a specific product (ANG I) was identified and measured. This is in contrast to acid protease measurements, where a denatured hemoglobin substrate is incubated and unidentified products are measured colorimetrically as non-specific copper-phenol amino acid complexes. Addition of ANG I1 accelerated growth and decreased intracellular renin content in normal fibroblasts in a dose-related manner. The addition of the ANG I1 antagonist saralasin to the culture medium caused a decrease in cell proliferation and an increase in intracellular renin levels. These results indicate a negative feedback of ANG I1 on the renin levels in 3T3 cells and support the specificity of enzyme measurements. Neither the ANG-(2-8) heptapeptide nor t h e ANG-(4-8)pentapeptide fragments had any influence on growth in 3T3 and SV3T3 cells. The ANG-(2-8) fragment is reported to be biologically active, especially on t h e zona glomerulosa of the adrenal gland (Peach, '77). Its lack of effect on endogenous renin and on cell growth in 3T3 and SV3T3 cells indicates differences in ANG receptors which need to be studied more closely in these cells. I t has been reported that ANG I1 stimulates protein synthesis and increases the rate of incorporation of labelled nucleotides into DNA and RNA of isolated rat atria (Khairallah e t al., '72). Recently, Gill and coworkers ('77) have demonstrated that ANG I1 stimulates DNA synthesis and cell growth in monolayer cultures of adrenocortical cells. The ANG(2-8) heptapeptide was shown to be also effective. The [sar',ile81-ANG I1 analogue slightly stimulated growth, though it inhibited ANG I1 effects. In t h e same study, growth was not stimulated in other cell cultures such as balb/ c 3T3 cells after ANG I1 application. Different factors such as serum, fibroblast growth factor, glucocorticoids and insulin are known to control cell growth i n fibroblasts (Todaro e t al., '67; Holley and Kiernan, '68; Thrash and Cunningham, '74; Gospodarowicz, '75; Armelin and Armelin, '75). The relative potency of these factors varies with cell cycle, with confluency as well as from cell clone to cell clone of 3T3 cells (Holley and Kiernan, '74; Noonan,

511

'76). It may therefore be t h a t the ANG I1 effects on growth of 3T3 cells is clone-specificor is related to other circumstances not yet understood. The virus-transformed cells differed to some extent from normal fibroblasts since ANG I1 partially decreased cell proliferation and increased intrinsic renin content. Saralasin on the other hand, inhibited cell growth almost completely in SV3T3 cells as in normal fibroblasts. There is no explanation for these different responses in 3T3 and SV3T3 cells, but several virus-transformation induced changes of e.g., cell surface and receptors may be involved (Buck e t al., '70; Burger, '73; Barnett et al., '74). The significant correlation between the log of cell number and endogenous renin content may indicate that intracellular renin participates in the basic regulation of cell growth. Indirect evidence for such an interpretation is presented here since the effector peptide of the RAS, ANG 11, and its antagonist saralasin inversely influence growth and renin content in 3T3 cells. Evidently, further studies, e.g., enzyme measurements during the various phases of the cell cycle, will be necessary to determine the influence of cell growth rate on renin concentration. ANG I1 is known to have many biochemical effects (for review see Goodfriend e t al., '73). It is of interest in this respect t h a t ANG I1 may influence cyclic nucleotides, causing an increase of cyclic AMP levels in rat hypophysis (Gagnon et al., '75) and, contradictory, a decrease of cyclic AMP in uteri and arteries of rats (Volicer and Hynie, '71; Angles d'Auriac et al., '72). Low cyclic AMP levels or, perhaps more relevant, a low ratio between cellular cyclic AMP and cyclic GMP seems to be compatible with cell growth, while the opposite is true for contact-inhibited resting cells (Rudland et al., '74; Anderson et al., '74). Controverse results are reported, however, indicating that cyclic AMP is increased during cell growth (Wray and Glinos, '78). More recently, ANG I1 was shown to stimulate prostaglandin (PG) production in cell cultures (Gimbrone and Alexander, '75; Zusman and Keiser, '77). A PG-related effect of ANG I1 may be possible since PGF,, administered together with insulin initiates DNA synthesis and growth in fibroblasts (Jimenez de Asua e t al., '77). On the other hand, PGE, and PGE, diminish uridine uptake and augment cellular cyclic AMP content in 3T3 cells (Jimenez de Asua and Rozengurt, '74; Claesson et al., '77).

512

P. SCHELLING, D. GANTEN, G. SPECK AND H. FISCHER

Fischer, H., R. M. Flugel, P. Schelling and D. Ganten 1975 Difference in endogenous isorenin in normal and SV40 transformed 3T3 mouse cells: Correlation with cell growth. Int. Res. Commun., 3: 328. Fischer, H., and G. Sauer 1972 Identification of virusinduced proteins in cells productively infected with SV40. J. Virol., 9: 1-9. Fischer, H., P. Schelling and D. Ganten 1978 Suppression of endogenous isorenin by the oncogenic virus SV40 after infection of 3T3 cells. Int. Res. Commun., 6: 11. Gagnon, D. J., P. Sirois and P. J. Boucher 1975 Stimulation by angiotensin I1 of t h e release of vasopressin from incubated rat neurohypophyses - possible involvement of cyACKNOWLEDGMENTS clic AMP. Clin. Exp. Pharmacol. Physiol., 2: 305-313. D., P. Schelling and U. Ganten 1977 Tissue These studies were supported by t h e Ganten, isorenins. In: Hypertension. J. Genest, E. Koiw and 0. Deutsche Forschungsgemeinschaft (DFG) Kuchel, eds. 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In spite of these possible interrelationships between ANG 11, cyclic nucleotides and PGs, the mechanisms by which ANG I1 influences cell proliferation remain unknown. Our results show, however, that ANG I1 can act as a specific growth factor. Renin is synthetized intracellularly and may be responsible for local generation of ANG I1 provided that angiotensinogen is available.

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Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Effects of Angiotensin II and Angiotensin II Antagonist Saralasin on Cell Growth and Renin in 3T3 and SV3T3 Cells PIERRE SCHELLING,' DETLEV GANTEN,' G...
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