Angiotensin-converting enzyme and other enzymes in livers of mice with experimental schistosomiasis

EXPERIMENTAL AND MOLECULAR PATHOLOGY 35, 199-210 (1981)

Angiotensin-Converting Enzyme and Other Enzymes in Livers of Mice with Experimental Schistosomiasis

AKIRAHARA, KIMIE FUKUYAMA, AND WILLIAM L. EPSTEIN’

Department of Dermatology, University of California, San Francisco, California 94143

Received January 8, 1981, and in revised form March 4, 1981

Angiotensin-converting enzyme was measured in mice infected with Schistosoma mansoni by a fluorometric technique. Enzyme levels increased in the liver as granulomas devel- oped, attaining an increase of about ll-fold over the normal level by 14 weeks after infection, whereas activity in the serum showed no change up to 20 weeks. The enzyme was found to localize in granulomas and was mostly bound to membranes. Chemical and catalytic properties of the enzyme from granulomatous tissue and in normal liver or serum were identical. Among other enzymes of hepatic cells measured, glucose&phosphate dehydrogenase was increased and succinate-cytochrome c reductase and glucose&phosphatase were decreased. Elevation of glucose-&phosphate dehydrogenase appeared in both the liver and serum as early as 4-5 weeks atI& infection. The enzyme in granulomatous liver increased as much as 7-fold over the normal level and showed an interconversion of three isozymes by electrophoresis. The findings indicate that tissue angiotensin-converting enzyme may be used as a marker of granulomatous inflammation. They also suggest the importance of combining analysis of this and other enzymes known to be altered by chronic or subacute injury for chronic inflammation of tissues.

INTRODUCTION

Angiotensin-converting enzyme (EC 3.4.15.1, peptidyl dipeptidase) is a membrane-bound glycoprotein located primarily in the vascular endothelium of almost all organs, and in the epithelial cells of the renal tubules (Ryan et al., 1975; Caldwell et al., 1976). Accumulation of the enzyme in granulomatous lymph nodes of patients with sarcoidosis (Silverstein et al., 1976), and in spleen of patients with Gaucher’s disease (Liberman and Beutler, 1976; Silverstein and Friedland, 1977), has been reported. The serum levels of angiotensin-converting enzyme also were found elevated in these patients (Silverstein et al., 1976, 1977; Silverstein and Friedland, 1977; Lieberman and Beutler, 1976; Lieberman et al., 1979; Gronhagen-Riska, 1979), and synthesis of the enzyme by epithelioid cells in the sarcoid granulomas was suggested as the source for the increase in serum (Sil- verstein, 1976; Silverstein et al., 1976, 1979). Specific types of macrophages were considered to be involved in angiotensin-converting enzyme production because other types of granulomas such as tuberculosis (Silverstein et al., 1976) and subcutaneous Freund’s adjuvant granulomas (Silverstein et al., 1978) did not cause an increase.

Subcutaneous injection of cercariae of Schistosoma mansoni (S. mansoni) in mice induces hepatic egg granulomas (Warren, 1961; Li Hsti et al., 1972; Epstein et al., 1979). Granulomatous lesions consist of activated macrophages and epithelioid cells and attain a maximum size between 10 and 14 weeks (Epstein et al., 1979). The animal model was used by Perrotto et al. (1976) to correlate elevation of lysozyme with granuloma development, and by Cha et al. (1980) to assay hepatic drug metabolizing enzymes in terms of heavy schistosome egg de-

’ To whom all correspondence is to be addressed.

199

0014-4800/81/050199-12$02.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in my form reserved.

200 HARA, FUKUYAMA, AND EPSTEIN

position. In this study, we report modulation of several enzymes in liver during the course of schistosomiasis in mice. Some chemical properties of angiotensin- converting enzymes and glucose-6-phosphate dehydrogenase were compared in serum, and in granulomatous and nongranulomatous regions of the liver.

METHODS

General experimental procedure. Groups of 30 to 50 male BALB/c mice were infected by subcutaneous injection of 50 cercariae of the Puerto Rican strain of S. mansoni freshly hatched from snails (Epstein et al., 1979). At different times between 2 and 20 weeks after infection, the mice were bled from the femoral artery and sacrificed, and the serum was separated by centrifugation within 1 hr. Liver and lung were removed from the mice and rinsed in cold 0.25 M sucrose in 50 mM Tris-HCl buffer (pH 7.5). Unless otherwise noted, each tissue was weighed and stored at -70°C for up to 1 month prior to use. All the following procedures for tissue extraction were performed at 0-4°C. The tissue (0.5 g) was minced, homogenized in 6 ml of buffered sucrose with a Potter-Elvejhem homogenizer, and then centrifuged at 600g for 10 min. The supernatant was removed and the pellet rehomogenized by the same technique. The homogenate and the first 600g supernatant were combined and centrifuged at 105,OOOg for 60 min. Two fractions, (1) the supernatant and (2) the pellet, which was further homogenized in 5 ml of buffered sucrose by a Polytron homogenizer, were obtained.

Schistosome egg granulomas were isolated from the livers of mice infected for 8- 12 weeks by the method of Pellegrino and Brener (1956) with minor modifica- tions. The fresh livers obtained were subjected to portal perfusion with cold saline to remove the blood. Each liver was homogenized with 50 ml of saline in a Waring blender at low speed for 30 set and placed in a 50-ml cylinder. The granulomas sedimented while homogenized hepatic cells remained in the supernatant. Repro- ducibility of cell components in the two fractions was occasionally confirmed by histological techniques. The granulomas were homogenized with saline in a tissue grinder. Protein concentration in the separated granuloma-rich fraction was 14.6 ? 3.1% of that contained in the total homogenate. (The data is based on the mean ? SE from three experiments.)

In the study of intracellular distribution of the enzyme activity, the livers were homogenized with 9 vol of 0.25 M sucrose and then fractionated into nuclear, mitochondrial, lysosomal, microsomal, and soluble fractions by differential centrifugation by a modification of the method of Hogeboom (1955). The lysosomes and microsomes were sedimented by centrifuging at 15,000g for 20 min and 105,OOOg for 1 hr, respectively. Finally the sediments were suspended in 5 ml of 0.25 M sucrose.

Analytical procedure. Angiotensin-converting enzyme was assayed using hippuryl-L-histidyl-L-leucine (Hip- His - Leu) as a substrate with a modified method of Friedland and Silverstein (1976). In order to prevent degradation of histidyl-leucine (His-Leu) by peptidase (Lee et al., 1971), we added 2.5% n- butanol to the reaction mixture (Hara et al., 1981). The amount of the enzyme required to liberate 1 pmole of His-Leu was defined as 1 U. Glucose-6-phosphate dehydrogenase was assayed by determining the rate of reduction of NADP at 340 nm (Taketa and Watanabe, 1971). Succinate-cytochrome c reductase was measured by determining the rate of reduction of cytochrome c at 550 nm (De Duve et al., 1955). One unit of these enzymes was expressed as 1 kmole of NADPH and

LIVER ENZYME LEVELS AND SCHISTOSOMIASIS 201

reduced cytochrome c formed per minute at 37°C respectively. Glucose 6- phosphatase was measured under the conditions described by Takahashi and Hori (1978) and the liberated inorganic phosphate was determined by the method of Lowry and Lopez (1946). Acid phosphatase was assayed by the method of Bergmeyer (1974), after treatment of the 105,000g pellet fraction with 0.5% Triton X-100 at 4°C for 1 hr. One unit of these hydrolases is defined as the amount of enzymes required to liberate 1 pmole of organic phosphate and p-nitrophenol at 37°C respectively. Suitable enzyme and substrate blanks were carried out for all assays. Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. The enzyme activities in livers were calculated as unit of an enzyme in 1 g of the liver (content in whole homogenate) and milliunits of the enzyme in 1 mg protein (sp act).

The kinetic constants were determined by the method of Lineweaver and Burk (1934) for K, value and by that of Dixon (1953) for Ki value.

Analytical gel filtration was performed on a Sepharose 6B column (1.5 x 90 cm) equilibrated with 0.25 M sucrose in 50 mM Tris-HCl buffer, pH 7.5. Samples for gel filtration were prepared by homogenization of 1.5 g liver with 6 ml of 0.25 M sucrose in 50 mM Tris-HCl buffer (pH 7.5). The supernatant was separated after 105,OOOg centrifugation and the pellet was subjected to five cycles of freezing and thawing and centrifuged at 105,OOOg for 60 min. The supernatants from the two centrifugations were combined and fractionated by addition of ammonium sulfate; the protein that precipitated between 35 and 75% saturation was collected by centrifugation at 12,OOOg for 15 min and suspended in 2 ml of the sucrose solution. (Recoveries for angiotensin-converting enzyme and glucose-6-phosphate dehydrogenase were about 70%.) The suspension was dialyzed against the sucrose solution, diluted to 5 ml, and 1.5 ml was applied to a Sepharose 6B column, which had been calibrated with Blue Dextran 2000, Escherichia coli p-galactosidase (mol wt 500,000), bovine catalase (mol wt 244,000), yeast glucose-bphosphate dehydrogenase (mol wt 104,000), bovine hemoglobin (mol wt 66,000), and cytochrome c (mol wt 12,400).

Disc gel electrophoresis in 7% polyacrylamide was performed at 4°C with bromphenol blue as a tracking dye according to the method of Davis (1964), but 10 PM NADP+ was added into the cathodic electrophoresis solution. The glucosed- phosphate dehydrogenase activity on the gels was stained by the method of Taketa and Watanabe (1971).

Reagents. All chemicals for the enzyme assay and standard proteins were obtained from Sigma Chemical Company (St. Louis, MO.). Pyr-Trp-Pro-Arg- Pro-Gln-Be-Pro-Pro (SQ 20881) was purchased from Peninsula Laboratories, Inc. (San Carlos, Calif.) and Sepharose 6B from Pharmacia Fine Chemicals (Piscataway, N.J.). All other chemicals were reagent grade.

Statistical methods. Data are expressed as the mean ‘-c SE. Student’s t test and analysis of variance were used to compare the significance of the differences.

RESULTS

Enzyme activity in subcellular fractions of normal and granulomatous liver. The total angiotensin-converting enzyme activity detected in normal livers was low (0.093 U/g liver). After 10 weeks of infection with S. mansoni a marked increase (P < 0.02) in activity per gram of liver was found in granulomatous livers (Table I). The livers of infected mice were enlarged and weighed about 1.3 times

TABL

E I

Angi

oten

sin-C

onve

rting

En

zym

e an

d Se

vera

l M

arke

r En

zym

e Ac

tiviti

es

in S

ubce

llula

r Fr

actio

ns

of L

iver

Ex

tract

s of

Nor

mal

an

d In

fect

ed

Mice

Succ

inat

e-

Angi

oten

sin-c

onve

rting

G

luco

se&p

hosp

hate

cy

toch

rom

e c

Acid

G

luco

se

6-

enzy

me

dehy

drog

enas

e re

duct

ase

phos

phat

ase

phos

phat

ase

Frac

tion

Sp a

ct

Rec

over

y Sp

act

R

ecov

ery

Sp a

ct

Sp a

ct

Sp a

ct

mU

/mg

%

mU

/mg

%

mU

/mg

mU

/mg

mU

/mg

F

Nor

mal

N

ucle

ar

0.35

k 0

.07”

16

.4 ‘

- 5.

3 2.

2 2

0.6

17.9

2 7

.2

143

k 59

21

.6

f 2.

6 61

.0 2

5.5

,a

mic

e M

itoch

ondr

ial

0.27

k 0

.01

13.7

k 0

.8

1.1

* 0.

5 5.

1 ?

3.9

240

2 24

39

.7 2

3.0

88

.1 +

4.9

Ly

soso

mal

0.

86 +

0.1

4 12

.0 2

1.

9 2.

9 +

1.5

6.2

k 3.

1 18

2 2

66

.0 t

9.

2 17

4.7

? 53

.2

2 M

icros

omal

0.

67 e

0.0

7 11

.8 k

1.

3 1.

1 *

1.2

6.0

? 4.

2 8k

-2

36.4

c 5

.7

185.

9 k

39.2

z

Solu

ble

0.54

k 0

.01

46.1

? 2

.7

5.2

+ 0.

2 69

.8 2

4.2

0

21.2

t

2.1

0 2

Con

tent

in

5 ”

whole

;P

ho

mog

enat

e*

0.09

3 2

9.01

0.

47

t 0.

04

18.3

2 3

.7

5.95

t

0.39

12

.1 2

0.7

In

fect

ed

Nuc

lear

16

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t 5.

8 22

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4.7

3.

6 k

0.2

3.8

2 0.

3 52

2 9

26

.0 t

3.

8 12

.2 +

2.8

s

mic

e M

itoch

ondr

ial

3.1

+ 0.

3 5.

4 iz

1.6

3.1

L 0.

2 3.

2 k

0.3

186

2 7

43.1

2

2.5

36.0

2

1.9

g

Lyso

som

al

20.9

-+

4.8

11.7

2

1.0

6.5

2 0.

2 2.

4 2

0.2

48 2

5

80.0

+- 6

.6

86.5

k 6

.7

Micr

osom

al

18.4

r

2.7

26.7

k

1.5

5.7

k 0.

8 5.

4 ?

0.8

19 t

3

62.0

rt

14.1

89

.0 f

2.

0 3

Solu

ble

6.6

+ 1.

4 31

.3 k

1.

1 26

.7 k

1.

0 85

.4 k

1.

2 0

12.4

r

0.9

0 2

Con

tent

in

wh

ole

hom

ogen

ate

2.14

2

0.94

2.

65 f

0.

12

7.51

2 0

.69

5.29

2

1.15

5.

3 2

0.8

a Al

l nu

mbe

rs

repr

esen

t th

e m

ean

2 SE

fro

m t

hree

ex

perim

ents

of

nor

mal

liv

ers

and

four

exp

erim

ents

of

gra

nulo

mat

ous

liver

s ob

tain

ed

at 1

0 we

eks

afte

r in

fect

ion

of th

e m

ice

with

5.

man

soni

cerc

aria

e.

* C

onte

nt

of e

nzym

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exp

ress

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as U

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f liv

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each

enz

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more than those of noninfected mice. The enzyme activity was greater in all cell fractions of granulomatous liver, but the ratio of increase in the nuclear and granular fractions was proportionally much higher than that in the soluble fraction. Changes in other enzymes known to be associated with the subcellular organelles, including glucose-6-phosphate dehydrogenase, succinate-cytochrome c reductase, acid phosphatase, and glucose 6phosphatase also are summarized in Table I. A significant increase of glucose-6-phosphate dehydrogenase (E’ < 0.0001) and decreases of succinate-cytochrome c reductase (P < 0.005) and glucose 6- phosphatase (P < 0.0001) were detected.

Angiotensin-converting enzyme and glucose-t%phosphate dehydrogenase levels in liver and serum at various times after infection with S. mansoni. Both enzyme activities of control mouse livers showed constant levels ranging from 0.31 to 0.81 mU/mg for angiotensin-converting enzyme and from 2.2 to 6.1 mU/mg for glucosed-phosphate dehydrogenase during the 20 weeks’ observation. In infected mice, angiotensin-converting enzyme activity did not change for 6 weeks, while glucosed-phosphate dehydrogenase activity showed a slight increase (P < 0.08) by 5 weeks of infection (Fig. 1). About the time (7-8 weeks of infection) that granuloma sizes increased sharply, angiotensin-converting enzyme activity also rose significantly (7 weeks, P < 0.01; 8 weeks, P < 0.0008). The ratio of angiotensin-converting enzyme assayed in the supernatant fraction over the re- suspended pellet (the nuclear through microsomal fractions) of mouse livers up to 7 weeks of infection was similar to that found in noninfected mice, but the enzyme

2 4 6 8 10 12 14 16 18 20

Weeks After Infection

FIG. 1. Modulation of angiotensin-converting enzyme (ACE) (A) and glucose&phosphate dehydrogenase (G6PDH) (B) activities during development of hepatic granulomas. Values are mean 2 SE from three noninfected and four to six infected mice. Tissue contents were estimated from the sum of enzyme activity in 105,flWg supematant and pellet of control (0) and infected (0) mice. Sp act of the enzymes (A---A) of infected animals showed a similar pattern of increase.


7sot A

: 250 1, , , , , , ~

4 6 8 10 12 14 16 18

40

2 30

z- E 20 E a u” 10

0

B

;T #& I L

4 6 8 10 12 14 16 18 Weeks After Infection

FIG. 2. Serum angiotensin-converting enzyme (ACE) (A) and glucose-6phosphate dehydrogenase (G6PDH) (B) levels at different times after infection. The enzyme values are mean 2 SE from noninfected (0) and infected (0) mice.

in the granular fractions increased thereafter. Glucose-6-phosphate dehydrogenase continued to increase (6 weeks, P < 0.0002) and peaked at the 11th week. Both enzyme activities remained high for more than 16 weeks.

In serum, angiotensin-converting enzyme activity did not change after infection, while glucose-dphosphate dehydrogenase increased by 6 weeks of infection and its peak occurred about 9 to 11 weeks after infection (Fig. 2).

Localization of angiotensin-converting enzyme and glucose-6phosphate dehydrogenase in granulomatous livers. Enzyme activities measured in the total homogenate, granuloma-rich fractions and granuloma-poor supernatant are dis-

TABLE II Localization of Angiotensin-Converting Enzyme and Glucose-6-Phosphate

Dehydrogenase in Infected Mouse Liver

Samples

Angiotensin-converting enzyme

Total activity” Sp act

Glucose-&phosphate dehydrogenase

Total activity Sp act

% mU/mg % mU/mg Homogenate 100 15.5 + 2.0* 100 23.2 + 3.9 Supematant 35.7 2 2.8 8.4 k 0.9 79.4 k 3.7 21.5 + 3.5 Granuloma-

rich fraction 57.5 + 3.4 82.7 f 4.8 20.6 ? 3.7 32.9 k 4.6

” Total activity measured in the homogenate was set at 100%. b Values are the mean ? SE from three experiments of granulomatous livers obtained at 9 weeks

after infection.


20 30 40 SO 60 70

50

z 2

25" 3 .d > .rl e,

02 20 30 40 50 60 70

Fraction Numbers

FIG. 3. Gel filtration of angiotensin-converting enzyme (ACE) (0) and glucose-bphosphate dehydrogenase (G6PDH) (0) from normal (A) and granulomatous (B) liver extracts on a Sepharose 6B column. The column was eluted at the rate of 20 ml/hr. Fractions (2.1 ml) were analyzed for protein, OD,, (- - -). The arrows show void vol (VJ and elution positions of the following standard proteins: (1) g-galactosidase, (2) catalase, (3) yeast glucose-6phosphate dehydrogenase, (4) hemoglobin, and (5) cytochrome c.

played in Table II. Specific activity of angiotensin-converting enzyme was much higher in the granuloma-rich fraction, compared to the hepatic cell fraction. Al- most 80% of the glucose&-phosphate dehydrogenase in granulomatous liver appeared in the supernatant, indicating hepatic cells as well as cells in granulomas contributed to production of the enzyme. The specific activity was, however, slightly higher in the granuloma-rich fraction, suggesting that the granuloma pro- duces significant amounts of the enzyme.

GelJltration of angiotensin-converting enzyme and glucose-&phosphate dehydrogenase. A mixture of two cell fractions (the supematant fraction and the frozen and thawed pellet fraction) on the column gave only one peak each with enzyme activity (Fig. 3). The elution patterns for both enzymes also were identical for the livers of mice with and without schistosomiasis. Mol wts were estimated as 330,000 and 128,000 for angiotensin-converting enzyme and glucose-&phosphate dehydrogenase, respectively. In a separate experiment we used the same column for serum and enzyme samples obtained from lung of noninfected mice. The elution patterns were similar to that indicated in Fig. 3.

Comparison of catalytic property of angiotensin-converting enzyme of mouse serum with that of livers of mice with and without schistosomiasis. Rates of hydrolysis of Hip-His-Leu by angiotensin-converting enzyme in serum were maximal at pH 8.0-8.3. The pH optima were the same for the enzyme not only in extracts of the granulomas isolated from livers at 10 weeks after infection, but also partially purified enzyme of normal liver with gel frltration. The K,,, values for this substrate were 2.6 and 2.7 mM for the enzymes from granulomatous and normal livers, respectively. The activities of the enzymes from three different sources were inactivated to about 50% at 55°C for 20 min, and to nearly 100% at 60°C for 5


TABLE III Effects of Various Inhibitors on Angiotensin-Converting Enzyme Activities of Serum,

Normal Liver, and Granuloma Extracts

Inhibition (%)

Inhibitor Concentration

(PM Serum Normal liver Granuloma

extract extract

Albumin 200 29 39 22 EDTA 100 96 80 81

10 5 23 15 Dithiothreitol 200 68 72 69

SQ 20881 1 93 94 91 0.1 56 55 45

Without NaCl - 99 99 99

min. The patterns of inhibition by several known inhibitors such as albumin (Klauser et al., 1979), EDTA, dithiothreitol and SQ 20881 (Bakhle, 1974; Soffer, 1976) were similar for these different samples (Table III). The most effective competitive inhibitor was SQ 20881, for which Ki values of angiotensin-converting enzymes from granulomas and normal liver were 29 and 32 nM, respectively. Angiotensin-converting enzymes from these samples were also inactivated by deletion of NaCl from the reaction mixture.

Electrophoresis of glucosed-phosphate dehydrogenase. Relative‘mobility (Ry) of the enzymes isolated from two sources (homogenates of normal and granulomatous livers) was essentially the same and showed three bands (R, of 0.45,0.47, and 0.50) (Fig. 4). However, by scanning the gels we observed some quantitative changes among the isozymes after infection. In normal liver, the fast-moving band (RM 0.50) contained the least activity and the slow-moving band (R, 0.45) the most activity, whereas in granulomatous liver the two fast-moving bands showed greater activity compared to the slow-moving band.

FIG. 4. Electrophoretic patterns of soluble glucose-6-phosphate dehydrogeases from livers of control (A) and infected (B) mice. 50 and 200 ~1 of the soluble fractions of livers with and without schistosomiasis at 10 weeks after infection, respectively, were loaded on the acrylamide gels, stained for activity, and scanned at 645 nm.


DISCUSSION

An increase in angiotensin-converting enzyme activity was found in hepatic granulomas of mice with schistosomiasis. The increase occurred in association with development of granulomas but not dilatation of vessels and proliferation of endothelial cells observed during the first 7 weeks of infection (Epstein et al., 1979). The magnitude of the increase was about l&fold that of enzyme activity found in normal liver cells, but the increase in the tissue did not result in an elevation of enzyme in sera. This situation contrasts with that in sarcoidosis in which the enzyme levels were elevated in both tissue and serum (Silverstein et al., 1976). It is possible that the enzyme is not secreted from the granuloma cells into the bloodstream. On the other hand, since mice have relatively high serum angiotensin-converting enzyme levels compared to man (about 600 nmole/min/ml vs 20-32 nmole/min/ml) (Silverstein et al., 1976, 1977; Lieberman et al., 1979; Gronhagen-Riska, 1979), additional enzyme from the granulomas may not influ- ence their serum level while a small increase in man would be detected. Angiotensin-converting enzyme derived from granulomas did not differ from the enzymes in serum or normal liver in mol wt, K, values, pH optima, and heat stability. This is again different from the findings in lymph nodes of sarcoidosis; the enzyme varied in heat stability from that found in normal lung and lymph nodes (Silverstein et al., 1976). The mol wt determined by Sepharose 6B gel filtration was approximately 330,000 and is similar to the values reported for the enzymes prepared from calf lung (Stevens et al., 1972) and hog lung (Dorer et al., 1972), but somewhat less than that from human lung (Fitz and Overture, 1972).

The source of angiotensin-converting enzyme in the egg granulomas is uncer- tain, but it most likely derives from activated macrophages which populate the lesion. Sivlerstein et a/. (1980) recently demonstrated that angiotensin-converting enzyme may be inducible in the mononuclear phagocytes with appropriate in vitro stimuli, such as glucocorticosteroids (Friedland et al., 1978), lymphocytes, their secretory products, and some serum factors (Friedland et al., 1979).

Angiotensin-converting enzyme (1) hydrolyzes angiotensin I to angiotensin II and (2) inactivates bradykinin (Bakhle, 1974; Soffer, 1976). From the findings of Izaki et a/. (1979a, b) we propose the latter function of angiotensin-converting enzyme is most likely involved in granulomatous inflammation. The hepatic lesions begin with deposition of eggs and leakage of serum factors into the tissue, which may stimulate macrophages to synthesize angiotensin-converting enzyme. Macrophages may decompose bradykinin, and possibly other tissue kinins. Angiotensin-converting enzyme thus may play a modulating role in molding the structure and function of specific granulomas.

Glucose-6-phosphate dehydrogenase, which is elevated in a variety of hepatic disorders including CCI, hepatitis in rat (Taketa et al., 1976a) and mouse (Issel- bather and Jones, 1964), cirrhosis and hepatitis in man (Taketa et al., 1976b), and viral hepatitis in mouse (Taketa et al., 1976a; Jones and Cohen, 1962), was found to increase in livers of mice with schistosomiasis by Cha et al. (1980). In this study we detected the enzyme to increase in hepatic tissue rather than in the granulomas; the enzyme level also increased in serum as the tissue levels increased. The enzyme of normal and granulomatous liver was found to have the same mol wt and mobility by gel filtration and electrophoresis, respectively (Hizi and Yagil, 1974). However, interconversion of three isozymes by electrophoresis, reported by Taketa and Watanabe (1971) in other hepatic disorders was observed in granulomatous liver.


Other enzymes measured showed a decrease in granuIomatous liver, indicating that other molecular and biological changes occur in hepatic and possibly granulomatous cells. We believe that chemical and enzymatic characterization of granulomatous lesions, particularly angiotensin-converting enzyme, will provide im- portant information as to pathogenesis of granulomatous inflammation.

ACKNOWLEDGMENT This study was supported by Department of Health, Education and Welfare, Public Health Service

Grant AM 07939, and Biomedical Research Support Grant RR 05355 (Dr. Fukuyama).

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Angiotensin-converting enzyme and other enzymes in livers of mice with experimental schistosomiasis

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