0022-1554/79/2707-1103102.00/0 THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1979 by The

AND

Histochemical

Vol. 27, No. 7, pp. 1103-1107,

CYTOCHEMISTRY

Society,

Inc.

Histochemical Detection of Carbonic Dimethylaminonaphthalene-5-Sulfonamide1 CHRISTIAN Institute

ofMolecular

POCHHAMMER,

Biology

Received

A new reaction fluorescent

and

for

PETER

Biochemistry,

publication

DIETSCH

Free

May

22,

and

Several

methods

tissues

have

by

Kurata

cobalt

method

controversial even

show at

Progress carbonic

of

carbonic

et

(e.g.

needing

and We

al.

(5).

of

in

a

the

to the and

was

Recently

is not

specific

for

These

(14)

suitably

the

application was

or

are

use

was stated.

in

for

the

tissues.

anhydrase

has

been

reaction

for

the

disc

described

detection

was

by

carbonic

and

isoelec-

(7).

METhODS

AND

140

equipped

and

an

from

Serva

(Heidelberg,

sulfonylchloride ethanol, I Some

anhydrase

Germany).

according

m.p. results

209#{176}C.The of this

in osteoclasts

to

Weber

absence work have

DNSA (15) of

been

filter

47

aperture observe

the light

compounds

occurrence

1976,

at

the

and

7th

1,2-1,4

470

nm

540

taken lens

Inter-

Optovar

(2).

an

the

films

with

were

a Zeiss immersion

field

immersion

the

system,

magnification

dark use

of

fluorescence background For

Camera

measuring

the

at

470

used

The

measure-

200

G 40-

watt

(Osram)

to examine

a KP

500

exciter

field

condensor

the

filter,

the

with

the

filters allowed us to of the CA-DNSA-complex

these

fluorescence

of unbound

documentation

Rollei

nm.

Universal

HBO

con-

of excitation

fluorimetric

was

was

450 Watt)

XBO

microscope

microscopy

quenched.

The

Germany)

wavelength for

microscopy

greenish

nm was

in solutions: (Osram

Hg-lamp

The

10were

observed

A Zeiss

used.

the

35 B, and

Ilford

microscope HP

4 (ASA

used.

microscopy:

and

was

light

the and

substances

W. For

the

pressure

for

Twelve

of DNSA

The

lamp

quartz-cells

blue specific

equipped

with

a wide

at the 6th

and were

while

at

Light

of carbonic

tube

barrier

was also 400-800)

from

fluorescence

a diet Berlin,

ethoxzolamide,

Oberkochen,

Xenon

special

a high

For

orally.

fed

Zitrig,

application.

Zeiss,

microscopy:

additional

DNSA the

recrystallized

in short

Hamburg, 1977

from

fluorescent the

shown

prepared

and

other

concerning

national Congress of Endocrinology, Parathyroid Conference, Vancouver,

was

purchased

for

fluorescence

with

as

Berlin,

experiment.

35 mg/kg

(M4QIII).

into

used

were

from

DNSA-fluorescence

pressure

the

put

slices.

at

was

Dimethylamino-naphthalene-5-sulfonylchloride

and

stained

the

and

CA-DNSA-complex,

ments. Fluorescence

proved

of

nm

III

starting

given

quartz-monochromators

were

animals

of DNSA were

(Carl

of the

the

before

capsules

a high

Germany

F. E. v. Bernuth,

(Universal

sulfonamides,

of the

with

gift car-

1500 g. fluorescence

The

gelatine

a

sex-sal-link)

from

of antibiotics

starved.

into

320

=

Bovine

Mannheim,

(Warren,

purchased

of ethoxzolamide

two

F 254 65:

Michigan.

Boehringer,

chickens

background

were

solutions

forming

already

electrophoresis

gels

MATERIALS

spe-

a 1:1 ratio,

Siegmund

in acrylamide

loca-

sulfonamide

with that

during

histochemical

from

The were

content

centration

DC,

was

Kalamazoo,

approximately

diminish

equipped

dimethylaminona-

This

obtained

animals weight

Co.

spectrophotofluorimeter

of antibodies)

of

Pharmaceutical

Measurement

consuming

Siicagel

6-ethoxybenzothiazole-2-sulfonamide,

otherwise

weighed

by

on

chloroform:methanol:water

system.

anhydrase

and

the

and

isoenzymes

time

preparation

(1). Holke this

published

with

bonic

mg/kg

it pos-

by filters has been

anhydrase osteoclasts.

chromatography

if not

20

Germany

makes

layer

Upjohn

animals

of an mdi-

also

however,

exposure

the

33,

78-160)

wavelength

Germany) for at least 3 weeks hours before the administration

and

of

compound

of

Gay

(MS

Germany)

from

a low

Berlin

in tissues is described. The (DNSA) forms a highly ofthis fluorescence with

the

thin

as solvent

with

autoradiography

(DNSA)

Kernohan

by

by

Germany;

localization

achieved

in

Darmstadt,

experimental

carbonic

inhibitor

methods, time

the

D-1000

2, 1979

Ethoxzolamide,

black

a specific

technique,

fluorescent

focussing

proved

25:4

subject

Muther

for

was

antibody

to carbonic

usefulness

tric

conversion modified

13).

method,

binds

anhydrase

catalyzed

acetazolamide,

skillful work. like to introduce

and

its

Another

anhydrase

highly

Chen

by

tissues

and

carbonic

cifically

be

of methods

phthalene-5-sulfonamide tion

of

reaction

tritiated

long

require now

precipitation

(ii,

development

tissues.

Gay

the

been

anhydrase

fluorescent

used

by

anhydrase

rect

who

to

introduced

To

the

22,

February

difference

(Merck,

was

reaction

often

was

anhydrase

one

followed

this

carbonic

first

has

that

(3, 4) using

prepared

of

SIEGMUND

Arnimallee

form

The

all.

in

Mueller

in revised

R.

USA.

with

dimethylaminonaphthalene-5-sulfonamide have been examined. Carbonic cells, proximal tubule cells, and

The

this

discussions

anhydrase

of

(6)

(believing

This

could

localization

H#{224}usler

anhydrase)

sulfide.

the described.

and

carbonate

carbonic to

for been

(9)

Berlin,

(6-ethoxybenzothiazole-5-sulfonamide).

sible to absorb the fluorescence of the unbound Kidney, proventriculus, and bone from chicken detected in the cytoplasm of the columnar lining

in

PETER

AND

University

1978

Anhydrase

specific method for the detection ofcarbomc anhydrase, EC 4.2.1.1, of carbonic anhydrase with dimethylaminonaphthalene-5-sulfonamide complex. The specificity ofthe method is proved by the quenching

ethoxzolamide

1979

Printedin

Photographs Photomikroskop oil were factor

used. 0.63,

changer

1103

Downloaded from jhc.sagepub.com at East Tennessee State University on June 10, 2015

ofthe

stained

III

Ilford

The and

the

was set

on

tissue Pan

section

F films.

microscope

was

equipped

projective

factor

was

at

1 .6 or 2.0,

respectively.

were A xlOO with 3.2.

The

POCHHAMMER,

1104 Determination cent

of

complex

anhydrase serum. was

as anti

mmol/1 solution

levels the

coagulant.

solution pH

=

of

of

7.4,

were

DNSA.

as described

above.

with After

Three

The

1. a, Fluorescence

of the

20-gil

portions

fluorescence

microscopic

highly

fluores-

with

carbonic

of DNSA

of a brachial of an in

into

addition,

The DNSA

concentration

milliliters

anhydrase

pipetted each

DNSA:

combination

carbonic

was titrated

10 imol/1

FIG.

by

was also used to determine Blood was obtained by puncture

used

buffer,

serum

forming

20

vein.

mmol/1

ofa

chicken fluorescence

of a solution

in

Heparin

approximately cuvette. serum was

containing

AND

The

containing measured

approx-

SIEGMUND

imately

0.1

served

as

mmol/1 a

determination having

0.1

phosphate

a fluorescence the

DIETSCH

received

mixtures

was

by anaesthesia

DNSA

with

proventriculus

as described

The the

exactly

was

300 il of the

in

serum

at

the

were

measured values

1 imol/1

set

decapitation

under

7 to 24 hr after

put

obtained

from

The 20-40

administration

of chicken proventriculus after the oral administration of 35 mg/kg body 10 hr after being fed a DNSA filled gelatine capsule. Excitation wave length (m), lumen (1), columnar lining cells (c) (x 800). b, Azan stained section from in a. Muscularis mucosae (m), lumen (I), columnar lining cells (c) (x800).


470 nm. Muscularis mucosae chicken

and

concentration

solution.

compared

concentrations

Preparation butal,

fluorescence

DNSA,

anhydrase

sacrificed

anhydrase

Its

of the

carbonic known

carbonic

standard.

of solutions

animals

of the these with

were

mg/kg

Nem-

of the

sulfon-

weight of DNSA. 500 nm, emission the same piece of

DETECTION -

.

:

.

.

OF

CARBONIC

ANHYDRASE

1105

.

;;‘_‘

r.

.

‘

:

--

FIG. 2. a, Fluorescence fluorescent cells (xl000).

microscopic picture of chicken kidney. b, Azan stained section from the same

amide.

Approximately

10- x 10- x 5-mm

excised

from

organs

and

had

been

the

b.p.

-76#{176}C, which

The

maximal

time

prepared

required

pieces

immediately for

of the

immersed by cooling

the

were

At

the

time

when

to a cryostat un

were

embedded without

kidney

observed

liquid

nitrogen.

microscope.

until

this

step

was

cut

sections

were

required,

microtome

that

was

and

thawed

immediately (8) any

and

with

covered

staining

with for

on glycerol cover

fluorescence

the

kept

clean

glass

gelatine slides.

pieces

around

were

trans-

slides. prepared

They

of

were

according

The

sections

microscopy.

To

were interpret

The

microscopic

2.5%

glutaraldehyde

and

dehydrated

stained to

of Figure

with

blue

orange

the

and

xylene.

Proximal

sections

frozen

sections and

in 0.05

G and

washed

Cover

slides

sodium 70%

sections with were

Downloaded from jhc.sagepub.com at East Tennessee State University on June 10, 2015

ethanol, mounted

tissue

with

were

the

light

glycerol

fixed

for

5 hr with

buffer,

pH

ethanol. After

counterstained isopropanol, with

of

on albumin were

of

by

(x1500).

piece

cacodylate

ethanol. were

surrounded

(pt)

examined

They

amounts in

the

same and

mounted

dried.

moles/l

increasing

(pt)

tubuli

the (12)

were air

borax-carmine acid,

from

staining

slides

tubuli

in a. Proximal

azan

with

phosphotungstic

used

la.

as described

by a modified

Staining:

20

-30#{176}C;slices

legend

fluorescence,

stained

coated

ferred

Kaiser

see

of chicken

propane,

mm.

5-12

conditions

in liquid with

preparation

tissues

For

piece

Canada

Nuclei treatment with methylbenzoate Balsam.

7.3 were with

aniline

1106

POCHHAMMER,

RESULTS

DIETSCH

DISCUSSION

AND

AND

of 1.2 tmol/l results

Absorption

and

ministration DNSA in water ticable; after nous injections, after

the

plasma

concentration

after

of DNSA: Because of the its application by injections some we

application

unsatisfactory preferred the of 35 mg/kg

weight

tion had reached a value of 3.1 imol/l. showed also a marked diuresis indicating

microscopic (o) (x2000).

picture b, Azan

ad-

with intraveTwo hours the

At this time a significant

of the DNSA to the renal carbonic anhydrase. tion in the plasma then slowly decreased and

FIG. 3. a, Fluorescence polynucleated osteoclast

oral

poor solubiity of appeared imprac-

experiments oral application. body

SIEGMUND

concentrathe bird binding

The concentrareached a value

after

pearance

plasma

inhibitor

after

prepare

the

other

chose

of chicken tibia. For conditions stained section of chicken tibia.

body

Gay

When inhibitor,

and

fluids,

and

from

dogs

that

loosely while

is

the

release

of the process.

10 (maximal

to

Mueller

(3) sacrificed

using only

disap-

inhibitor

slower

DNSA

assuming anhydrase.

similar

this

bound

a much

of approximately of

see legend of Figure 1. Bone Compact bone (c), polynucleated

Downloaded from jhc.sagepub.com at East Tennessee State University on June 10, 2015

concluded in

of free

a time

24 hr after the injections, is then bound to carbonic hr to be sufficient. DNSA as the

(10)

anhydrase

application tissues.

Maren

loss

carbonic

we the

hr.

acetazolamide

the

and

from

Therefore

1/2

with

reflected

from

hr

8

obtained

kill

the

birds

the

24) and

to

chicken

that most of the inhibitor We found the time of 10

the fluorescence the CA-DNSA

marrow (m), osteoclast

technique complex

compact (o).

and was

bone

(c),

DETECTION

detected

and

disturbed.

the

free

Therefore

of the below

inhibitor show that

cal

simultaneous

the

body

The were

supernatants

of

of the tissues

of 10 mg/kg of

DNSA.

the

some

a

dose

of

20

mg/kg

that

body

ences

extreme

the

the

for (Fig.

occurred

nized

some

the

in

and

the

results

Gay

2): The

markedly

less

ulus. The in variance :IHlabeled

nuclei again did to the results acetazolamide.

the

and

than

and the

several of the

the

the

fluoresrecog-

of fluores-

the

of

was The

found

Gay

in the

intensity

of the

was

proventric-

unclear

who

kidney

between

(1 1). the

the

We

method

inhibitor

in

These et

al.

and cartilage. those cells results

The which

bone are

correspond

well

(3).

This is used also in

determined described

CV,

Mueller

tissues

chem Cytochem 4. Gay CV, Mueller

IC:

Combination sulfonamide. PR: Mechanism

of bovine carbonic anhyJ Biol Chem 242:5813, 1967 of activation of carbonic

by

of Calcium Excerpta

WJ: Cellular localization of carbonic anhydrase by labeled inhibitor autoradiography. J Histo21:693, 1973 WJ: Carbonic anhydrase and osteoclasts: local-

labeled

inhibitor

autoradiography.

Science

183:432,

1974 5. Gay CV, Faleski EJ, Schraer H, Schraer R: Localization of carbonic anhydrase in avian gastric mucosa, shell gland and bone by immunohistochemistry. J Histochem Cytochem 22:819, 1974 6. H#{228}usler G: Zur Technik und Spezifit#{227}t des histochemischem Carboanhydrase-nachweises im Modellversuch und in Gewebes-

von Rattennieren. Histochemie P: Detektion von

7. Holke M, Siegmund men in Acrylamidgelen

FEBS-Let

16:304,

mit

einem

1:29, 1958 Carboanhydrase

fluoreszierenden

IsoenzySulfonamid.

1971

8. Kaiser latine. 9. Kurata activity. 10. Maren

E: Verfahren zur Herstellung einer tadellosen GlyceringeBiol Tbl 1:125, 1948 Y: Histochemical demonstration of carbonic anhydrase Stain Technol 28:231, 1953 TH: The binding of inhibitors to carbonic anhydrase in vivo; drugs as markers for enzymes, Proc of the 1st International Pharmacology Meeting. Edited by B Uv#{228}s.Macmillan Co, New York. 1963, p 39-48

11. 12. 13.

Maren

TH:

Carbonic

anhydrase:

bition. Physiol Rev 47:595, 1967 Romeis B: Mikroskopische Technik. 1968, p368 Muther TF: A critical evaluation

chemistry,

physiology

Oldenburg, of the

and

inhi-

M#{252}nchen, Wien

histochemical

methods

for carbonic anhydrase. J Histochem Cytochem 20:319, 1972 14. Muther TF: On the lack of specificity of the cobalt-bicarbonate method for carbonic anhydrase. J Histochem Cytochem 25:1043, 15.

and

CITED

by parathyroid hormone. Endocrinology Edited by DH Copp and RV Talmage. Amsterdam-Oxford, 1978, p 412

in avian

whether

complex

Kernohan a fluorescent P, Siegmund

chnitten of

muscularis

fmdings

It is still

whereas

Medica,

ization

influ-

we

RF, with

2. Dietach

3. Gay

by different chemical properties of and acetazolamide in penetrating the specificity of our method. The may also be eliminated faster by in

of Gay

Metabolism.

1-3).

not show any fluorescence. of Gay and Mueller (3) They found radioactivity

complex,

in (3) does not distinguish the free inhibitor.

and

cells

cells.

hemopoiesis.

anhydrase

(5).

appropriate

in the fmdings

of a

osteoclasts

mineral. As in the other examined occurs in the cytoplasm. There

in bone mineral fluorescence

also

picture

The generous help and advice of Professor W#{252}stenfeld and Dr. Grunz is gratefully acknowledged. The authors are indebted to Miss Petra Ott and Miss Sabine Pohle for skillful technical assistance.

the

show any fluorescence; in the zymogemc cells

cells.

the

drase

differ-

blue

intensity

with

polynucleated

large,

Acknowledgements

after

before

There

fltorescence

tubule

secretion

CA-DNSA

accord

involved

It is possible

cells.

propna,

tubule

in the

collecting

not

fluorescence showed

1. Chen

The

either

(Figs.

in the

al.

proximal

the differences are caused the two inhibitors DNSA the different cells or by more soluble acetazolamide only

in et

of

filtration

hr

to

light

lining

specific

cytoplasm

distal

the

applibefore

coefficients

specific

tumca

are

(3) and (Fig.

After

only

membranes.

membranes The

differences

glands,

Mueller Kidney

15

due

partition

of the cells did any fluorescence

submucosal These

cell 1):

columnar

structural

The nuclei was there

mucosa.

the

cell

microscopic

shows

LITERATURE

10 hr osteoclasts.

given

in the

permeability

cence

the

be

fluorescence

tibia

of ethoxzolamide was not sufficient

is complete

may

various

difference

Proventriculus

cence. neither

This

and inhibi-

of the carbonic anhydrase from or, more probably, differences

in passing

an

in

weight

DNSA.

in the affinity to ethoxzolamide

left

was no marrow with

thousandDNSA (1).

osteoclasts.

fluorescence

The

tissue

is an

administered is

specific of

inhibitors

in the

fluorescence

of the

administration ences tissues

fluorescence of ethoxzolamide

3): from

method by the

of ethoxzolamide

an approximately anhydrase than

(Fig. section

at the border to the bone cells the specific fluorescence

chemi-

the

Ethoxzolamide

with carbonic

Bone

tissues

with

1107

ANHYDRASE

cross

described

of the specificity fluorescence in

for quenching DNSA,

results right.

estimated

dose of 10 mg/kg body weight at the same time as DNSA

quenching

excess

of the CA-DNSA tissues which were

However, the and application

of 15 mg

longer

an

proof of this

tor of carbonic anhydrase fold greater affinity for

cation

no

remove

appropriate

weight

CARBONIC

DNSA

in several

anhydrase,

application

mg/kg

tissue.

anhydrase

carbonic

in

to

light blue fluorescence found in sections of

to contain

methods

bound

necessary

of carbonic The only

homogenates. Another was the quenching 35

not

by rinsing the these assumptions

Localization

of chicken: complex was known

or unspecific it was

OF

1977 Weber G. Fluorescent

Biochem

Polarization of the fluorescence conjugates of ovalbumin and J 51:155, 1952

Downloaded from jhc.sagepub.com at East Tennessee State University on June 10, 2015

of macromolecules. bovine serum albumin.

2.