0022-1554/90/$3.30 Thejournal of Histochemistry Copyright © 1990 by The

and Cytochemistry Histochcmical Society,

Vol.

Original

BENKOL,’

CHRISTIANE and ALBERT Laboratoires

dAnatomie

Pathologique

(PB) et Service

for

LUC

publication

(LB,LXJB,AC)

de Chirurgie

January

18,

1990

et de

digestive and

Chimie

(BS),

in revised

Hopital

form

June

Glucose-6-phosphatase (G6Pase) is a multicomponent systern that catalyzes G6P hydrolysis. To determine the specificity ofthe histochernical reaction ofG6Pase we investigated the inhibitory effect ofdiethylpyrocarbonate (DEPC), a specific and very effective inhibitor of the phosphohydrolase cornponent of the G6Pase systern, in normal human liver. The inactivation of the histochernical enzymatic activity by DEPC was monitored by determining the mean brightness of the microscopic image and the histogram oflight inten-

Introduction Glucose-6-phosphate ticomponent system This

(1975).

brane

(G6P) hydrolysis in liver is catalyzed by a mul(EC 3.1.3.9) first described by Anion et al.

system

requires

components

translocase cated inside

of the

the

coupling

endoplasmic

(Ti), an inorganic the endoplasmic

of three

integral

reticulum:

brane (Anion et al., i980b, 1983). Because histochemical phosphatase to

the reaction selective employed

have

(Hoefsmit

et al.,

Kanamura,

1986;

1971).

phosphohydrolase

Burchell, Pi,

PPi,

and

specificity

Shugyo

Sodium

1982).

rat liver

Many

microsomes:

carbamyl-P

(Anion

1986;

the

system have

phlonizin

(Anion

et al.,

1980b),

usually activity

et al.,

selectively

G6Pase

of

by using

on G6Pase enzymatic

inhibitors

it

specificity

Nichols

interacts

ofthe other

for

studies of the

et al.,

fluoride

active mem-

lack specificity,

can be confirmed

cytochemical as inhibitor

component

1980,

on isolated

This

Earlier fluoride

evidence

1984;

with

the

(Burchell

been

pynidoxal-P

1

Correspondence

to: Liliane

Pr A. Chamlian,

Bcnkoel,

Laboratoire

27 BdJean

Moulin,

d’Anatomie 13385

Marseille

(JMG,CD),

Facult#{233}de MIdecine,

de Sainte

Marguerite,

12,

accepted

1990;

Marseille, June

18,

and

Laboratoire

France.

1990

(0A1882).

distributions. The results obtained indicate that the histogram is more sensitive than the mean brightness to vaniations ofenzymatic activities, and that the percent of pixels brighter than a convenient level is directly proportional to DEPC concentration. This study indicates that DEPC can be used as an efficient inhibitor of the histochemical reacnon ofG6Pase. (JHistochcm Cytochem 38:1565-1569, 1990) KEY WORDS: Human liver; Glucose-6-phosphatase; Diethyl pyrocarsity

bonate;

Inhibitor;

Quantitative

analysis.

and Widnell, 1976), i-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (Murataliev and Vulfson, 1986), sulfhydryl reagents and taurocholate (Wallin and Anon, 1972), 4-acetamido-4’-isothiocyanostilbene-2-2’-disulfonic

acid

acid

(Zoccoli

thiocyano-1,2-diphenyl 1982).

Anion

(DEPC) hydrolase

Lygre,

and

and

4,4’-diisothiocyanostilbene-

Kamovsky,

1979),

ethane-2,2’-disulfonic

et at. (1984)

demonstrated

and

acid that

diethyl

24-4’-diiso-

(Zoccoli

et at.,

pynocarbonate

was one of the most effective inhibitors of the phosphocomponent: it inactivates through formation of a stable

covalent bond with the active site (Miles, 1977; 1966)

and

histidyl Feldman

is effective

residue of the phosphohydrolase and Butler, 1969; Nondlie

at reasonably

and

low concentrations

un-

der relatively mild conditions (30#{176}C at neutral rarely used as an inhibitor of the cytochemical

pH). DEPC has been reaction of G6Pase

(Benkoel

The

was

et at., 1989;

a quantitative

Chamlian

study

histochemical

1980a),

reaction

(Gold

Materials

PatholoCedex

year-old

et at., 1989).

aim

ofthis

work

the inhibitory effect of DEPC on the of G6Pase in normal human liver. of

After

stored

temperature

and

surgery,

2800N)

Methods

were obtained

female

nitrogen, Jung

and

specimens

Liver sue.

gique, Service 5, France.

SASTRE,

and

investigated

et al.,

Biologique

2’-disulfonic

a G6P-specific

whose reticulum

activities

confirmatory

of G6Pase.

inhibitors. sodium

mem-

phosphore (Pt) translocase (T2) loreticulum membrane, and the glucose-

6-phosphatase (G6Pase) enzyme (phosphohydrolase) site is on the luminal surface of the endoplasmic

is important

1565-1569, 1990 Printed in USA.

CHAMLIAN

d’Immunologie Received

11, pp.

Study of by Diethyl Liver

GULIAN, PIERRE BONGRAND, XERRI, JOELLE BRISSE, BERNARD

JEAN-MARC

DALMASSO,

No.

Artide

Image Analysis for Histochemical Glucose-6-Phosphatase Inactivation Pyrocarbonate in Normal Human LILIANE

38,

Inc.

by open

a 49-year-old

male)

the specimens

were

biopsy with

from

two patients

apparently

immediately

normal

flash-frozen

(a 60liver

tis-

in liquid

for 24 hr at - 80’C, sections

before

at

- 20C.

incubation.

and cut into 12-sm cryotome (Reichert Sections were dried for 5 mm at room In a first series ofexperiments, the sections 1565

Downloaded from jhc.sagepub.com by guest on August 15, 2015

BENKOL,

GULIAN,

DALMASSO,

BONGRAND,

XERRI,

BRISSE,

SASTRE,

CHAMLIAN

Figure 1. Histochemical demonstration of glucose-6-phosphatase activity. Sections unincubated in DEPC or pro-incubated for 10 mm in DEPC and then incubated in medium containing equal moles of DEPC. (A) High activity in hepatocytes unexposed to DEPC. (B,C)Loweractivitywith 4 mM and 10 mM DEPC. (D)Total inhibition ofthe reaction with 13 mM DEPC. Onlylipofuscin pigments were visible. Original magnification x 400. Bars 50 tm.

%t.

/

.: -

.

.

‘..

#

:

,

44

#{149}

:

V

M #{149} ‘

4t’.

.

,..

were incubated in a modified Wachstein and Meisel (1956) medium (Wegmann and Mello de Oliveira, 1969): 2.4 mM G6P, 10 mM acetate buffer, pH 6, 3.9 mM lead acetate for 30 mm at 37C. In a second series of experiments, sections were pre-incubated for 10 mm in acetate buffer containing only

0.5-27

medium

mM described

series ofexperiments, containing

0.5-27

DEPC

(Anon

above

equal were incubated

containing

sections mM

et al., 1984)

DEPC

without

and

then

amounts directly

pre-incubation.

incubated

in the whole

of DEPC. In a third in the whole medium After

incubation,

the sections were treated with 2 % ammonium sulfide and mounted in glycergel. Samples were examined with an Olympus IMT2 inverted microscope bearing a thesa 1036 video-intensified camera (Lhesa; Cergy Pontoise,

.

France).

,.;

The standard

CCIR signal (512 lines per frame)was digitized frame grabber(Imaging Technology; Woburn, yielding 512 x 512 pixel images with 256 gray levels. Under standard ditions, samples were examined with a x 40 objective (0.85 numerical ture). A pixel then represented a rectangular area of c. 0.9 x 1.35 The linearity ofphotometnic determinations was checked by assaying a 10 mHz

rescent ously

PCVision

solutions described

was studied

+

ofvarious

concentrations

(Andr#{233}et al., by examining

the

1990). density

under The

spatial

profile

uv

illumination

resolution yielded

Downloaded from jhc.sagepub.com by guest on August 15, 2015

apersm2.

fluo-

as previof the

by dark

strips

edges(Andr#{233}et al., 1990). The coefficient ofvariation ofpixel sity was 5% on a blank window, as obtained by repeatedly digitizing sharp

with MA) con-

system with

intenempty

INHIBON

GPase

IN HUMAN

LWER

1567

100

0. 3

80 >,

0.2 60

U) C U)

U) U

40 0.1

,-

0 20

0

0 0

2

4

6

8

10

DEPC concentration

12

14

0

2

(mM)

4

6

8

10

DEPC concentration

12

14

(mM)

Figure 2. Monitoring of DEPC inhibitory activity. A series of liver slices were stained for phosphohydrolase in the presence of different concentrations of DEPC. Three to five microscopic fields were digitized for determination of the following parameters. (A) Optical density; the mean oD was estimated (a) by calculating the logarithm of the ratio between the mean brightness of a blank control and the tested field (filled lozenges), and (b) by determining the average value of the logarithm of the ratio between the brightness of the blank control and that of each pixel of the tested field (open squares). (B) Histogram analysis; the image histogram (16 values) was used to calculate the fraction of pixels with brightness higher than or equal to 5 (filled lozenges) or 4 (open squares). The vertical bar length is twice the SEM.

fields

with

varying

dows of about of the The

x

amplification.

65 sm2

(6144

When pixels)

slides were

were

used

for

examined,

The mean

win-

brightness

b was b

=

calculated

with

the

formula:

n(i)

is the

number

where

io i5 the

intensity

The

Sixn(i)/Sn(i)

of pixels

with

brightness

i.

density OD

4 pixels where

optical

determination

parameters.

following mean

camera

115

grams

and

was calculated =

Sn(i)

with

the formula:

lg(i/i)/Sn(i)

mean brightness of a blank sample. histogram was calculated by considering

defining

16 brightness

were used to achieve

classes

a sensitive

for mean

detection

intensity

of minimal

1536

clusters

levels.

of

Histo-

enzymatic

ac-

120 L0

-i

100

Ii E 20

0 0

5

10

15

DEPC concentration Figure a Relationship between the mean variation of enzymatic Case 2), without pre-incubation (filled squares, Case 1; open -8.018, rb 0.951, pC (Case 2). Without preincubation, rb, correlation coefficient; pC, significance level.

20

30

(mM)

reaction squares, 5a

25

and DEPC concentration after 10-mm pre-incubation (filled lozenges, Case 1; open lozenges, Case 2). After b-mm pre-incubation, s -7.973, rb 0.971, pC io4 (Case 1); 5a -a756, rb 0.968, pC iO4 (Case 1); 5 - -4.205, rb 0.982, pC i0-4 (Case 2). 8, slope;

Downloaded from jhc.sagepub.com by guest on August 15, 2015

BENKO1L,

1568

tivities

as shown

below.

is characterized (St.

Five

by high

to ten

enzyme

fields

of the

activity,

were

periportal

GUHAN,

region,

bution

which

studied.

Reagents G6P and DEPC were purchased from Louis, MO). Other chemicals were of analytical

BONGRAND,

ned

Sigma Chemical grade.

Co.

the

observed

intracytoplasmic

reaction

was

in the

hepatocytes (Figure 1A). After 10-mm prein DEPC, the reaction decreased significantly (Figures

incubation lB and

1C) until

total

inhibition;

were apparent

(Figure in an inhibitory

resulted bition

a strong

ofthe

reaction

only

some

lipofuscin

was observed

tion.

In a 16 gray-scale-calculated

tions,

the intensity

with

a higher

histogram

level was 6-7 when

DEPC

and

the mean

light

percentage

intensity

of pixels

the

(expressed brighter

reaction, as a percentage

than

level

and

in Figure

2,

used

brightness value

(i.e.,

lg(i/b)

was on average

was 8%

higher

However, it was felt that sensitivity to detect minimal

Figure

0.996

2B, the appearance

(n

than

=

the

32

the

fields).

The

than

a suitable

threshold

level.

patches

The

of enzymatic

pre-incubation

enzyme

activity

distribution of enzyme

showed activity

a good linear relationship and DEPC concentration.

calculated by the least-squares method zyme activity decrease in pre-incubated out

pre-incubation

ing

a total

(Figure

inhibition

was

concentration

range,

actual

quantification

of these

ac-

These

as-

former

G6Pase

system.

are performed.

the

enzyme

inactivation

than

in those by the

was

without

of the

concentration

calculated

from

cx-

reaction Figure 3 represents reaction points

between the decrease The regression lines

demonstrated sections than

3). The

a higher enin those withof DEPC

the

equation

We thank

Ms C. Granger

relatively

yieldof the

histochemical

ensures

G6Pase

Discussion distributed within 1982), with higher

the liver paactivities in

the peniportal and lower activities in the penivenous region as demonstrated by quantitative histochemical studies in rat liver (Hildebrand 1978).

This

reaction

our conditions). that

(13

Moreover,

the histochemical

phosphohydrolase

and

C. Casino

for

G6Pase

activity

expert

secretarial

stimuli. Anion

Cell WJ,

Biophys,

Burchell

of the

heterogeneous

assistance.

in press B, Burchell

A (1984):

phohydrolase

component ofthe by diethyl pyrocarbonate.

system

Specific

inactivation

distni-

ofthe

Anion WJ, Lange A), Walls HE (1980a): Microsomal membrane and the interactions of phlorizin with the glucose-6-phosphatase

J

Biol

Anion pation

Chem

phos-

hepatic microsomal glucose-6-phosphatase Biochem J 220:835 integrity

system.

255:10387

WJ, Lange A), Walls HE, Ballas L (1980b): of independent translocases for phosphatase

phatase in the microsomal 255:10396 teristics of liver and microsomes

Evidence and

glucose-6-phosphatase

ID, Lange A), TelfordJN, glucose-6-phosphatase are identical.

J

Biol

Lange A), transport

J Biol Chem

Walls HE (1983):

The of isolated

in the envelope Chem 258:12661

Ballas LM (1975): On system in the function

Mol Cell Biochem

for the particiglucose-6-phos-

system.

the

characnuclei

involvement of microsomal

6:75

Benko#{235}lL, GulianJM, Payan MJ, Choux R, BrisseJ, Cytochemical demonstration ofglucose-6-phosphatase

Teutsch,

amount

Andr#{233} P, Capo C, Benoliel AM, Buferne M, Bongrand P: Analysis of the topological changes induced on cells exposed to adhesive or mechanical

Anon WJ, Wallin BK, of a glucose-6-phosphate glucose-6-phosphatase.

2).

1986;

higher

with the enzyme DEPC, which was

Cited

Literature

Case

G6Pase activity is heterogeneously renchyma (Jungermann and Katz,

effective

pre-incubation.

inhibitor of phosphohydrolase (Anion et at. , 1984), can be used under

ofDEPC

more

be

Acknowledgments

Anion WJ, Schulz

Schleicher,

assays

and

only if homogeneous samples could analysis and stain elution for quantitative

in fact the

regression lines: 12.6 mM (Case 1) and 13.4 mM (Case 2) in expeniments with pre-incubation and approximately twice as great in expeniments without pre-incubation (26.7 mM, Case 1; 23.4 mM,

and

biochemical

be explained

effect

represents

was thus

and without pre-incubation) the mean variation ofenzymatic The study of the experimental

the relationship between and DEPC concentration.

a wide

pre-incubation

activity

using higher

ofpixels brighter than level 5; a 100% without DEPC. In the two cases studied,

(after b-mm

over

inhibitor

10-mm

5), we can way of corn-

activity

in percent

was obtained

than

a useful

latter.

was more efficiently monitored with brightness histograms, as an objective parameter the percent ofpixels with brightness

pressed

mM with

is

allow

and irreversible microsomes

isolated

mean

the mean OD did not provide optimal enzymatic activity. As suggested by

of sparse

higher

provide

there

and a histo-

might

can

as a specific

in rat liver

that

concentration

of inhibitor diffusing in the section and reacting in pre-incubated sections. This study shows that

the inhibitory

the correlation

brightness

(1989)

parameters

3 suggests

with

experiments

findings

Figure

parameters

Noonden

ofhistograni

DEPC

den-

activities

Finally,

my(Van

from

Because

ofoptical

by Van

between pixels

that

in pre-incubated

as an efficient

Noorden, 1989) remained small. Indeed, “true” optical density and that derived

2B.

parameters provided

These

as defined the significance

(a) histogram

enzymatic

the mean optical density (oD) varied between 0.24 and 0 when the DEPC concentration was increased from 0 to 13 mM. As shown in Figure 2A, the distributional error caused by field heterogenebetween

densities 2A and

(%

that

car-

region was studied. To make of staining distribution, the

Figure

parameter

conclude

study

only the peniportal of heterogeneity

relationship

determination.

both

ofcontrols)

5. As shown

gram

in our

the variability

says would be warranted subjected both to image

inhibited.

we calculated

in

CHAMLIAN

SASTRE,

liver. To minimize

“true”

linear

tivities,

with an apwas no obvi-

.

pearance of pixels ous a priori way of quantifying

was observed

in Figure

(b) these

distnibu-

was totally

activity seemed to be correlated darker than level 5 Because there

a fairly

paring

concentra-

of intensity

G6Pase

G6Pase

Furthermore,

pigments

1D). Experiments without pre-incubation effect; nevertheless, an almost total inhi-

activity

problem

is depicted

to DEPC,

of enzyme

sity determinations,

are shown exposure

BRISSE,

pattern

“mean”

Without

XERRI,

out on human

clear

Results

DALMASSO,

Chamlian in human

A (1989): liver. Cell

Mol Biol 35:563 Burchell microsomal

A, Burchell

B (1982):

glucose-6-phosphatase.

Identification Biochem

and

purification

Burchell A, Burchell B (1980): Stabilization of partially-purified 6-phosphatase by fluoride. FEBS Lett 118:180

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