0022- 1554/78/2604-0298/$02.00/0 THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1978 by The

THE

AND

Histochemical

Vol. 26, No. 4, pp. 298-312,

CYTOCHEMISTRY

Society,

CYTOCHEMICAL

Printed

Inc.

LOCALIZATION FACT

H. J. M. KEMPEN, Departments

J. J. H. H. M. ofBiochemistry

for publication

September

S. L. BONTING

Submicroscopic

29,

ADENYLATE

CYCLASE:

ARTIFACT?

PONT,

DE

and

Nmegen, Received

OF

OR

1978

in U.S.A.

AND

Morphology,

A.

M.

STADHOUDERS

University

of Nijmegen,

the Netherlands and

1977,

in revised

form

December

1, 1977

(Ms

77-205)

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et aL (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin, NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [a’2P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complete Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is too low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.

Adenylate conversion into cyclic By

cyclase (EC 4.6.1.1) catalyses the of adenosine triphosphate (ATP) AMP and inorganic pyrophosphate.

allowing

insoluble

the

pyrophosphate

complex

cytochemical

with

detection

should in principle (21) first reported adenylate cyclase modified

ion

a heavy of this

metal enzyme

be possible a cytochemical in rat liver

Wachstein-Meisel

to form

as

medium

To

overcome

this

activities comparable cyclase. problem,

when to Howell

a

$ and

ion,

ATPase

et al. of of a

cle

(37)

substrate.

This

instead

‘y phosphorus atoms activity in broken and

liver

with

tissues The

(18, 2). following

of the

cytochemical

(24),

Precipitate of glucagon

a

between

the

it is converted

rat

into

(PNP,)

liver

observations

addition

Downloaded from jhc.sagepub.com by guest on June 20, 2015

containing

atom

(36), is resistant to cell fractions of mus-

while

from

cyclase.

298

(33) intro(AMP-PNP)

imidodiphosphate

liver (21) and islets dense deposits are brane, the presumed

Whit-

Bitensky

substance,

of an oxygen

cyclic AMP and adenylate cyclase

these are or higher and

the

nitrogen

ATP as substrate and lead ions as precipitating agent. This method is not specific, since ATP can also be cleaved by phosphohydrolase and pyrophosphohydrolase present in activities than that of adenylate

(11) and Wagner and adenylyl-imidodiphosphate

an

activity

(23). Reik localization by means

(31)

field duced

(24)

favor

detection

and the

method.

by

other validity

Both

in

of Langerhans (11) electronlocated at the plasma memlocation of the adenylate formation or

fluoride,

is enhanced which

by stimu-

ADENYLATE

lates

the

adenylate

CYCLASE

cyclase

biochemical assays. ance of precipitate

in

these

Furthermore, in isolated fat

subsequently

been

localization

applied

pad

kidney

(12,

(6). with

lead

ions

(1), colonic mucosa (7), salivary frog pancreas (29), liver subcellular (4) and two fungal species (30, 9).

The

validity

of the

method

of

segments

has,

(33), gland frac-

pad

capillaries,

and

however,

nonenzymatic (and

hydrolysis

even

however,

moreover

cyclic

that

been

it causes

of AMP-PNP

AMP)

maintain

and

that

PNP1.

the

Other

Reik-Howell

localization

of

pancreozymin-sensitive ity

in rat

pancreas.

Reik-Homell indicate that

(R-H) cyclic

the

adenylate We

find

no

(Ba2 part

instead of Pb2; pH of the enzyme activity

experiments, into insoluble effective incubation

as

8.9),

in the R-H of pancreatic

medium. cells

from

ml

10

until

sodium

cacodylate

Osmium

tetroxide

a 1%

in 50 mM tion

England.

on

dehyde

and

equal

rat

pancreatic

half

(pH

7.4),

the

same

the

cacodylate

medium

6.5%

(w/v) are

buffer

homogenized

by no

from

these 5 min

homogenates

hereafter

late

the

pieces

(ca.

blades)

of

formation prepared

from

fresh

after

heating

for

5 mm

of the

coagulated

1 mm3; fresh

(pH

overnight 7.4),

briefly

6.5% in

rat

rat cells

or

x

for the

at

particu-

pancreas out

are

homog-

with

the

100#{176}Cand

carried by

pancreas,

liver

pancreatic

in different

a similar

same

rehom-

acinar

out

with

cutting

and cells.

small

razor

with kidney,

and

Tissue

frag-

are prefixed in 1% formaldehyde or for 5-45 min, and are then washed and at 4#{176}C in 50 mM sodium cacodylate (w/v) glucose. They are rinsed 2 times

medium

cyclase

8000 fraction,

is used

rat

obtained

without

incubated in the cytochemical Biochemical adenylate ate

This

particles.

incubations

and

phase-

at

(25).

with

carried

glutaraldehyde

are loose

is prepared

AMP-PNP

is studied

Cytochemical

stored

from

media

are

isolated

of a

2.5 mM

by

fraction

fraction,

assays

ogenizing

in

3 ml

7.4),

centrifugation

Control

material

in

assay.

AMP

fraction

stored

suspensions

observed

washing

particulate

cyclase

cytochemical

by

an

buffer

homogenizer, are

by

followed

called

adenylate

(pH The

cells

at 4#{176}C.

2 times in

Tris-HCI

A particulate

sus-

with

and

washed

(Dounce

broken

microscopy.

g for

glucose

Na2EDTA.

hand

10 mm

cacodylate

then

de-

cell

is mixed

resuspended

10 mM

mM

2.5

until

and

on as

of 1% formal-

for

sodium

cells

The

containing

Cyclic

left

way.

fixa-

resulting

volume

containing

and

contrast

of the

equal

mM

7.4.

is studied prepared

suspension

of a 50

pH solution

of aldehyde

cells

and

of the

is 5 mM

(w/v)

activity

Half an

is

7.4.

effect

acinar (15).

2

pH

also

a 2%

pH

cyclase

with

in adding

glucose,

to

The

adenylate

solution

Glutaraldehyde

(w/v)

cacodylate,

volume

ments

4.5%

All

sources

The

containing

is dissolved

the

other

with

6 M HC1.

or glutaraldehyde,

The

is obtained.

solution

preparation:

ing

Materials: Unlabeled AMP-PNP is used as the tetrasodium salt (ICN Pharmaceutical Div., Eschwege, W. Germany) or as the tetralithium salt (Boehringer, Mannheim, W. Germany) without any apparent difference in our experiments. PNP1 (the lithium salt) is obtained from Boehringer, Mannheim, W. Germany, [a-32P}-ATP (5-10 Ci/mmole) from the Radiochemical Centre, Amersham, England; [a-32P1-AMPPNP (5 Ci/mole) from ICN Nuclear Division, Pitts-

(w/v)

sodium

Tissue

pestle)

METHODS

syn-

paraformalde-

to 65#{176}Cand

solution

to 7.4 with

to

enate.

AND

Pa.;

water

a clear

adjusted

material in this medium does not lead to localized precipitation at the electron microscopic level. These findings throw further doubt on the validity of the results obtained in lead-contain-

MATERIALS

of Squibb

N.J.;

Reading,

distilled

diluted

process. medium

media.

Princeton,

Warrington,

of double

then

MgCl2

Cytochemical and particulate

The

other chemicals are obtained from commercial in the highest available grade of purity. Solutions: A fresh 1% (w/v) formaldehyde is prepared by heating 100 mg of paraformaldehyde

studies medium

of PNP1 a third as

octapeptide

Ondetti,

TAAB-laboratories,

is mixed

in which a large in test tube

M.

Research,

Polysciences,

hyde

previously

survives

and in which the capture complexes is still about

from

pension

in the

C-terminal of Dr.

Medical

scribed

activ-

synthetic

thetic secretin is a gift of Dr. M. Wunsch, Max Planck Institute for Biochemistry, Munich, W. Germany. Glutaraldehyde (8% (w/v) solution in water) is purchased

method

cyclase

is due to a nonenzymatic, lead-catalyzed We have, therefore, developed a revised

for

299

ARTIFACT?

is a gift

authors,

and

activity

The

isolated

secretin-

medium. Chemical AMP formed in this

OR

in AMP

can be safely used for cytochemical adenylate cyclase detection (8, 4). We have tried to apply the method to the cytochemical

Pa.

M NaOH

questioned by Lemay and Jarett (17). They have shown that lead nitrate in mfflimolar concentrations completely inhibits adenylate cyclase activity in fat cell ghosts, islets of Langerhans and fat

burgh, Institute

has

in a number

outer

FACT

pancreozymin

ultrastructural

cyclase

16), rod

in

appearcapillaries

by addition of inhibitor of the

to the

of adenylate

tissues: uterus (16), tions

in rat liver method

tissues the

can be prevented almost entirely alloxan (32), which is a potent adenylate cyclase The cytochemical

LOCALIZATION:

activity

aldehyde-fixed

Downloaded from jhc.sagepub.com by guest on June 20, 2015

in the isolated

AMP-PNP

and

are

then

incubation medium. cyclase assay: Adenylparticulate acinar

fraction cells

of fresh

is determined

KEMPEN

300 by the

method

et al. (25)

of Rutten

substrate. Hormone-stimulated mined in the presence of 1 mg/mi

with

a-32P-ATP

ET

as

activities are phosphatidylserine

deter-

AL.

final

concentrations

the

medium

in the

ered

from

a constriction

plished

(13).

Cyclic ious

AMP

formation

cytochemical

following is

the

addition PNP the

final

tubes

at

tube).

The

final

10 mm,

then

the

tubes

for

2 mm

tubes

are

heated

reaction at

are

is stopped

Effect

the

case

for the

of blanks,

the

The

fo1med

2P-cyclic

AMP

according

to Woods

and

4 mM

Pb(NO:;)2

is present

in the

high

blank

values

are

found,

after

measured dehyde,

addition

fragments

(10-20

or isolated

pancreatic

milliliter)

are

one

of two

incubated

types

and

deter-

the

When

tapeptide, secretin When glutaraldehyde

incubation

medium, to 0.2%

is the

modified

by Howell

second

one

Control

strate-free

media

cells

in

of the

tissue

or

glucose

osmium

prepared assay

revised

medium

stopped

by 10 mm

lected

by

The

for

120

material

for

has

30 of

fixation,

min.

centrifugation

tissue

then

in a

for the bio-

incubated

The

reaction

in the is

matter

postflxed

are

(22),

electron

and

cut with

are

uranyl viewed

fluoride is is used instead,

recovered. less than

is left.

often

periphery of the the precipitation at the basement

of acini, ductuli and between adjacent

cells

and

between

blood acinar

plasma membrane typically occur

adjacent parenchymal cells the kidney they are located membranes of the epithelial

Effect

vessels, cells at (Fig. ib). between

(Fig. 2b), while at the basement and endothelial

microvilli

at the

apical

pole

by

of Fixation

via

Cyclase

a

(34) Philips

PMQ

is listed

Contro

pmoles

and

propylene

acetate in

II

formation:

oxide.

and

are lead

EM

300

incubation

The

stabil-

media

Activity#{176}

10

spectrophotometer.

1 to 3, and

Medium

represents

protein

20

M pancreozymin-

Glutaral-

dehyde dehyde cyclic AMP/lO

mm/mg

Basal

11

n.d.b

443

268

10

465 750

304

34

C-octapeptide

M secretin M NaF

102 a

and

added PNP1 is checked by at 400 nm, which is recorded

in Figures

of

I

on Adenylate

passing

concentrations

812

in

te-

10

their reactivity towards following the absorbance Zeiss

formaland of

by pancreozymin-C-ocor

TABLE

microscope.

a

in the cells is

fixation with basal activity

predominantly occur at the fragments. In the pancreas, granules are frequently found

is col-

on a LKB-ultramicrotome,

Dynamics of precipitate ity of the different cytochemical

composition

cells:

are treated or glutaral-

then

in osmium

dehydrated

ethanol

in Epon

stained

citrate

are

increasing

sections

cyclase acinar

Formal-

materials

be

incubations

glutaraldehyde.

particulate

and

embedded

sometimes

from

used

8%

fixed

experiment

been

10 pJ of the

cacodylate,

one

not

adenylate

acinar cells formaldehyde

activities

the outside of the In liver, precipitates

wash-

is then

In

fraction

above)

are

by briefly

sodium

mm.

particulate (see

through

Thin

7.4).

pipet

changes,

in the R-H medium: Tissue fragments from rat pancreas, liver and kidney are prefixed and incubated according to the procedure of Howell and Whitfield (11) with AMP-PNP as the substrate. The fragments are seen to contain precipitation granules that

membranes and less

fragments

incubations

and

mM

50

in

in sub-

tissue The

as

medium). described

on

for 120 min.

The are

preheated

in

(R-H

first

(21),

performed

medium

cells

The

medium are

medium.

addition

After

troxide

with the

(pH

chemical

them

is a revised

tetroxide

freshly

(11)

the

therefore

pancreatic

stimulated

Cytochemical

cells per at 30#{176}C in

et al.

Reik

activities

10% of these

milliliter)

medium. by

Whitfield

complete

ing

per million

for 30 or 60 mm

and

or

by removing

6.5%

(3-6

incubations

stopped the

weight

described

medium

“Results”. or

wet cells

can

deliv-

is accom-

from

Absorbance

3 sec,

(Table I). After some 70% of the

(35).

of cytochemical

medium The

mg acinar

mixing

air

the adenylate cyclase activity fraction prepared from these

Waitzman

initial radioactivity of AMP-PNP. The data in Table H have been corrected for these blank values. Cytochemical tests and electron microscopy: Tissue

and

is isolated

amounting

3 sec.

first

in isolated

dehyde particulate

of substraU’. mined

the

Isolated rat pancreatic for 10 mm with 0.5%

are

heating

100#{176}Cimmediately

for

of aldehydes

activity

is 50 zl;

incubated by

and of

to

are

RESULTS

by

constituents

tubes

100#{176}C.In

to

volume

various

The

medium

within

of AMP-PNP additions

pipette a stream

0#{176}C.After

is started

incubation

III.

followed.

0.2 j.Ci of a-32P-AMP-

of the

II and

occurring

pre-

These

blowing

the

in the

addition

matter

at

reaction

through

var-

or

particulate

(about

concentrations

fresh

medium

30#{176}C,the

substrate

in Tables

of

pancreatic

incubation

of the per

given

the

in the

is measured

microliters

of rat

with

placing

media

Ten

suspension

mixed

AMP-PNP

incubation

manner.

heated

from

by

after cuvette.

Isolated mm

10

particulate

subjected b

Not

in

rat pancreatic 0.5% fraction

Downloaded from jhc.sagepub.com by guest on June 20, 2015

acinar

formaldehyde is prepared

to the enzyme detectable.

602

assay

cells or

82

are treated

glutaraldehyde.

for A

from these cells and is with ATP as substrate.

ADENYLATE

CYCLASE

LOCALIZATION: ..1.

FACT

OR

..

a.

#{149},.

#{149}.

FIG. 1. Rat in Reik-Howell secretin

lead

(c),

or

pancreas. Tissue (R-H) medium with AMP-PNP

.



.)

,:i’

301

ARTIFACT?

-#..#{176},.

.4,

...,

fragments are fixed for 45 mm in 1% glutaraldehyde and incubated without substrate (a), with 0.5 mM AMP-PNP (b), with AMP-PNP and 10_2 M NaF (d). Postflxation in 2% OsO4 and staining in uranyl

citrate.

Downloaded from jhc.sagepub.com by guest on June 20, 2015

for and acetate

30 mm 10 M and

302

KEMPEN

ET

AL.

,.,---

.*;...

4

4.

.

..

‘I (.

-

,.‘.

.

-

#{149},,“:

.

-

,

-.

#{149}t

,P!I:t.

1

-

*

#{149}

.

d)

S

4



‘‘;:



4

‘1k’ .4

‘,

.

.

#{149}i-’

..

-

...

.

.

.. -

..

..‘

I..

.,

.

. . ..44.1il -

,

-

.

..

I

..,..

-,,.,

#{149}.

..

(4

-

,t..

#{149}

-

4i)

.

:4’

S

.

‘.t

._#{149}_.,

.

w::

.-.

-

1’

.

4 -.

-

..

..

-

.,

.

#{149}4,

.

#{149}.

-

#{149}

b

-

t

#{149}

...

f

:,.

.,,

,

4.

.,#.1I.#{248}

#{149}

.

P.

---w

- #{149}-

1

C FIG.

H

medium

Postfixation

‘4:, 2.

Rat

liver.

without in

Tissue

fragments

substrate

2% Os04

and

(a), staining

are with

fixed 0.5

in uranyl

in 1% glutaraldehyde AMP-PNP (b), or with acetate and lead citrate. for 45 min

mM

Downloaded from jhc.sagepub.com by guest on June 20, 2015

‘----

and

incubated

AMP-PNP

and

for 30 miii in RM NaF (c).

10_2

ADENYLATE

CYCLASE

LOCALIZATION:

FACT

OR

ARTIFACT? ...

-

..

303 .f

.

(

.,

% -

......

-

.

.

#{149}

-#{149}

g-

.4

.

I.r



‘i,

-: ‘‘



/.

.-

-

-

-

4-

,

...-. -

...

-

.7



a...

,.

-

.

... .4

:

\

-

-

-



.

4,#{149}

‘?

.

:,

a..:.

P1”

4

‘a,

‘.-

#{149},t:-

4,,

.

.,.#{149}*..

;

1

., i. -.

..4 .4

.-,.

.

-..

.

a.’-.

‘‘I I’

: L FIG. 3. Rat kidney. Tissue fragments are fixed for 45 miii in 1% glutaraldehyde and R-H medium without substrate (a), with 0.5 mM AMP-PNP (b), or with AMP-PNP Postflxation in 2% OsO4 and staining in uranyl acetate and lead citrate.

Downloaded from jhc.sagepub.com by guest on June 20, 2015

incubated for 30 mm in and 102 M NaF (c).

304

KEMPEN

the proximal precipitation

tubular is rare

particular

cells and

ET

process

(Fig. 3b). Intracellular is not confmed to

compartment

in

any

the

of

three

a tis-

heated

is absent

for

3

difference la, or

3a)

100#{176}C,but

(Figs.

ib,

2b

and

incubation Isolated fixed and AMP-PNP ules

are

but

within

altered M NaF

3b).

The

in these

by addition (Figs. id,

Instability AMP-PNP:

pattern

tion

tissues

is not

of secretin 2c and 3c)

(Fig. to the

medium. acinar cells of rat pancreas are preincubated in the same manner with as substrate. No precipitation grannow

observed the

occurs

in cisternae

ulum,

at

the

on the

cell,

fine

plasma

of rough

boundaries

precipitation

endoplasmic of

retic-

zymogen

does not result in a notable or amount of precipitation, obtained instead Cyclic

with cells prefixed of glutaraldehyde. AMP formation

the Reik-Howell described in the determine the from AMP-PNP

with from

in

formation particulate

addition

for

non-enzymatic

might

explain

these

5, the

formed

in the

Pb(N03)2

amount

presence

to the

of cyclic

of fresh

particulate

a

10-fold

increase. of cyclic

However, AMP

are

even

formed

under

can

of the AMP-PNP prepared solution as has also (4). Figure

is

been noticed 5 also shows

me-

AMP

is mat-

greater these

conditions when the particulate matter is preheated. These findings indicate that: a) lead nitrate gives rise to nonenzymatic formation of cyclic AMP from AMP-PNP, b) this non-enzymatic

lowered,

shown

become

solution. does

in

cloudy

rate

of apdepends

Addition not cause

by Cheng that the

and rate

addition of aged when the glucose or

when

glucose

is

replaced by sucrose. Cloudiness is completely prevented, however, when 6% dextran is used, as is done by Wagner et al. (32). Figure 6 shows that turbidity formation in the R-H medium is accelerated not observed

by

addition of CaCl2. This effect is in the absence of AMP-PNP, but

is aggravated mM AMP-PNP CaCl2

ter. There is no significant stimulation by hormones in this case, but addition of NaF causes amounts

medium

on the age of a freshly

to the

when and

These

the medium 10 mM NaF.

cytochemical

Results with cyclase activities

of 4 mM

which As

The turbidity

for 5 mm at 100#{176}C,indicating that the of fresh particulate matter is enzymatic. addition

or with

mechanisms

upon addition of the substrate. pearance of this spontaneous

fects, reported using the R-H nonenzymatic

a considerable

alone

observations.

R-H

slightly stimulated by pancreozymin-C-octapeptide, secretin or NaF. No activity is observed when the particulate preparation is preheated

After

re-

of Langerhans

of AMP-PNP,

looked

al. (32) does (not shown).

dium,

of

been

or fluoride, observed in cytochemical of others (see Introduction), we have

matter in the R-H medium (Table II). In the absence of lead nitrate, cyclic AMP formation is very slight (compare with Table I) and is only

activity

have

of islets

concentration

AMP-PNP

of cyclic AMP rat pancreatic

preparations capillaries.

nitrate

formation

(17) for cyclic AMP of fat cell ghosts, or

4d)

medium: The observations previous section have led us to extent by

and Jarett presence

of turbidity formation after substrate solution is increased

formaldehyde

c) lead

observations

4a). of

change in the pattern Similar results are

and

enzymatic

of the R-H medium with In view of the increased precipita-

turbidity, Farquhar

granules

and within membrane-filled vacuoles (Fig. Omission of substrate (Fig. 4b), or addition 10.6 M secretin (Fig. 4c) or lO_2 M NaF (Fig.

the

Similar

upon

hormones studies

Figure

membrane,

granular

AMP.

in com-

(Figs.

incubated

cyclic

by NaF,

inhibits

particulate and fat pad

significant

controls

fragments

of precipitation

consistently lc) or 10_2

no

pre-

of precipitation

nonsubstrate

and

medium amount

fragments

or amount

between

2a and

plete

at

mm

in location

is observed

in tissue

is stimulated

completely

ported by Lemay formation in the

sues.

Precipitation

AL.

not

lead

findings

beef adrenal timum with

medium to development

suggest

that

contains Addition

0.5 of

of Wagner

et

of turbidity the

positive

ef-

in cytochemical studies of others medium, may have been due to precipitation reactions. a revised medium: Adenylate of rat cerebral cortex (18) and

medulla AMP-PNP

strate.

Figure

for the

fluoride-stimulated

(2) have a higher than with ATP

7 shows

that

the

same

adenylate

pH opas sub-

holds

true

cyclase

ac-

tivity in rat pancreas 0.5 mM AMP-PNP

particulate matter. With optimal activity is attained

at pH

7.4 is optimal

8.9,

while

pH

ATP as substrate (25). In trying to develop

a medium

with of pH

0.4 mM 8.9

for

adenylate cyclase cytochemistry, we have looked for a metal ion which (a) at 8.9 would not precipitate

as its hydroxide

Downloaded from jhc.sagepub.com by guest on June 20, 2015

or with

AMP-PNP,

(b)

ADENYLATE

CYCLASE

LOCALIZATION:

FACT

‘.4

I,,,-..

-.

OR

305

ARTIFACT?

1 [,.

-

.4.-...

-.

,

I

/

.,

..,

C‘:T”T

a

.,

.

...

0 ‘,a*.,

‘q

,

.4

..‘

4,

a

A

..r2’

FIG. 4. Rat pancreas. Isolated acinar cells are fixed for 10 mm in 0.5% glutaraldehyde min in R-H medium without substrate (a), with 0.5 mM AMP-PNP (b), with AMP-PNP (c), or with AMP-PNP and 10_2 M NaF (d). Postfixation in 2% Os04. No staining.

Downloaded from jhc.sagepub.com by guest on June 20, 2015

I

and incubated for 30 and 10-6 M secretin

306 would

not

dation,

cause

(c)

nonenzymatic

would

leave

substrate the

adenylate

KEMPEN

ET

degra-

these

cyclase

activity maximally intact, and (d) would readily give precipitation of PNPI. We have established that barium ion meets

Formation

TABLE

II

AMP

from

of Cyclic ReikHou’ell

in the

Medium

M pancreozymin-C-octapeptide 10” M secretin 10 M NaF

10 ter

is incubated

for

rat 10 mm

8.9),

no precipitation

mM from

AMP-PNP. AMP-PNP

amount

is produced

13.6

60

0

77

0

947

particulate

at 30#{176}Cin the

mat-

Reik-Howell

presence of 4 mM 80 mM Tris-maMgSO4, 400 mM 4 mM Pb(NO3)2,

upon

cyclic

formation

AMP

NaF. It

AMP In the is stim-

NaF, the

and is partic-

enzymatic nature The addition of

but

in the that

in the

absence a

presence

enzymatic

can occur both in

presence of hormones. AMP in the presence non-enzymatic, and

and NaF intact. material, virtually

in the

secretin

is concluded

production with BaC12,

of 0.5

the same or a slightly inAMP formation, and leaves

is formed

with

(pH

addition

of cyclic Table III.

by hormones particulate

AMP

5 mM

BaCl!

The formation is given in

BaCl2 gives rate of cyclic

cyclic

contain-

4 mM

indicating the AMP production.

or

pancreatic

(R-H) medium, in the absence or Pb(NO.)2. R-H medium contains: leate, 2 mM theophylline, 2 mM glucose, 0.5 mM AMP-PNP, 0 or pH 7.4.

of BaCl2,

mones

609

and

ulated by addition of hormones and completely abolished by preheating

no

26.6

medium, theophvlline,

occurs

63

matter

a clear

10 mM

sucrose

58

M NaF or preboiled

mM

5.3

no addition

“Fresh

200

11.2

no addition 10-6

In

Tris-HC1,

the stimulation With pre-heated

matter

particulate

MgCl,

4 mM creased

pmoles cyclic AMP/lO mm/mg protein

Pre-boiled

80 mM

ulate material, of the cyclic

mMPb (NOS

particulate

requirements.

ing

absence AMP.PNP

NoPb

Fresh

AL.

of hor-

considerable of

10 mM

cyclic

AMP

the revised medium absence and in the

The production of cyclic of BaCl2 and NaF is largely is probably caused by the

catalytic action of a barium fluoride complex. Kinetics of the precipitation of imidodiphosphate in cytochemical media: The reactivity of the various cytochemical media toward

Tris-maleate

0.3

80mM

Theophylline

2mM

MgSO4

2mM

Pb(N03)2 Sugar

E

see

4mM below

C

0 0

pH

=

74

Q2 addition of

U

C 0

0.5

AMP

mM

a)

PNP

L ©

0 “I

a

0.1

Sucrose

200mM

Sucrose

400mM

Glucose

200mM

Glucose

400mM

Dextran (MW

0.0-

6#{176}/s 250.000)

10

time

(mm)

5. Spontaneous precipitation in the R-H medium, in relation to the sugar component. The composition of the media (after addition of AMP-PNP) is listed on the right. Curve 4 shows the behavior of the original R-H medium. FIG.

Downloaded from jhc.sagepub.com by guest on June 20, 2015

ADENYLATE

CYCLASE

LOCALIZATION:

FACT

OR ARTIFACT?

307

a)

0.3

additions

of

I

I

Tris-ma Theophyl

Ca Cl 2

1mM

I

80mM 2 mM

leate

line

SO4 Glucose

2mM 400mM

Mg

a)

Pb(N0&2 AMP-PNP

E

4

mM

0.5 mM

0.2

pH

#{149} 7.4

a, U

C

©

0

As

a)

AMP-PNP

without

L

0 UI

0.1

0

//

Asa),withNoF

5

10mM

10

time(min) FIG.

substrate,

6. Precipitation induced by addition of CaCl2 and (3) the same medium with 10_2 M NaF.

C

to (1) the

o

350

R-H

medium,

(2) the

same

medium

without

-

a) L

0

Tris-CI

250

0

4

80mM

MQCI2

/

200

150

U

UI

100

,0

0

/

5mM

Sucrose Theophylline

200mM 10mM

NaF AMP-PNP

10mM 05 mM pH

varies

E 50

74

7.7

80

83

8.9

8.6

9.2

9.5

pH FIG.

PNP

7. pH-dependence as the substrate.

imidodiphosphate changes turbidity

of adenylate

cyclase

is investigated

in absorbance due to precipitate

at

by

400 nm, formation.

following

phase. Both the and beginning

distance of the

in a rat

as

caused by As dem-

onstrated in Figure 8, within the first few onds after each addition of imidodiphosphate, rapid increase in absorbance occurs, followed a slower absorbance

activity

seca by

between zero trace, as well

pancreas

the

particulate

initial

slope

fraction

of the

with

trace,

increase

with

amounts to obtain

absorbance, diphosphate tochemical contrast,

about triple the amount of imidomust be added to our revised cymedium as to the R-H medium. In the cytochemical medium of Wagner

al.

(32)

is about

Downloaded from jhc.sagepub.com by guest on June 20, 2015

50

times

imidodiphosphate. initial increase

AMP-

increasing In order

et

of added the same

0.5 mM

less

sensitive

in

to

308

KEMPEN

added

PNP1

than

media, addition AMP together same increase

the

R-H

medium.

ET

In all three

clium: medium

of imidodiphosphate and cyclic in equimolar amounts causes the in absorbance as an equal addition

of imidodiphosphate Cytochemical

alone. testing

AL.

although cipitation ble

of

the

revised

The results obtained in test tube experiments

me-

it is less of PNP1,

to

the

R-H

sensitive it would

since

hormone-sensitive TABLE Formation

III

of Cyclic AMP from Revised Medium

cyclic AMP.PNP

in the

No

4mM

BaCl2

BaCl2

particulate

10-6

M

52

48

M secretin

1O2MNaF Pre-boiled

particulate

108

130

153

146

178

239

106

M secretin

1O2MNaF or preboiled

is incubated

medium

8

0

6

0

Fresh

#{176}

ter

0

for

10

pancreatic mm

at

the

in the

micrographs

the

mentioned at any (Fig.

granules

In applying

revised

in Table location in

9).

The

same

to

the

is

partichas

incubation visible

attached

the

cytochemical

et al. (21), as modified (11), for the localization

absence or presence of 4 mM medium contains: 80 mM Tris-Cl, 10 mM 5 mM MgCl2, 200 mM sucrose, 0.5 mM 0 or 4 mM BaCl2, pH 8.9.

been

of the as elecoutside

of

activity ground

method

of Reik

by Howell and of adenylate

Whitfield cyclase

in rat pancreas, we found precipitation after incubation

high

backof small

©

additions

of

80mM

Tris-maleate Theophylline

imidodiphosphate

2 mM

M9SO4 Glucose

400

Pb(N03)2

25

AMP-

0.3

2 mM mM

4 mM 0.5mM

PNP

pH=74

E 1000

C

©

0 0

(4-4-50 0.2

80mM

Iris - Cl Theophylline MgCI2

20

I) C

me-

DISCUSSION

mat-

0.4

U

revised

cells.

205

particulate 30#{176}Cin

incubation

to the medium during This leads to precipitation,

tron-dense

8.9 in the

at pH

BaCl2. This theophyllune, AMP-PNP,

rat

electron

added cells.

matter

no addition

into

PNP1.

true for incubation of unfixed pancreatic ulate matter. In control experiments, 0.1 mM PNP1

peptide 106

and

of AMP-PNP

mones in the concentrations III. No precipitate is visible the

pancreozymin-C-octa-

and

cyclase

enzymatic

dium has been carried out with isolated acinar cells, prefixed for 10 mm in 0.5% formaldehyde. The medium contains BaCl2, substrate and hor-

matter

no addition

adenylate

allows

conversion

Cytochemical

pmoles cyclic AMP/lO mm/mg protein Fresh

AMP

for it

our revised indicate that

with respect to preseem to be prefera-

medium

cytochemistry,

with

10mM 5mM

Sucrose

15

200

BaCI2

0

AMP-

0.5mM

PNP

L

pH

0 ill

mM 4 mM

=

8.9

10

a

20

01

Irismaleate A minophylline

5

10

©

FIG.

8.

2 mM

Pb(NO&2 AMP-PNP

2mM

6

/.

0.5 mM pH-72

time

medium, addition

SO4 Dextran Mg

100

80mM 5mM

(mm)

Precipitation induced by addition of PNP1 to (1) the R-H medium, (2) our revised cytochemical and (3) the cytochemical medium of Wagner et a!. (see Ref. 31). The composition of the medium (after of PNP1) is listed on the right. The final concentration of PNP1 is given in LM at the end of each curve.

Downloaded from jhc.sagepub.com by guest on June 20, 2015

ADENYLATE

:;.

:r

:.

CYCLASE

LOCALIZATION:

FACT

OR

309

ARTIFACT?

.:,

..m.

I .4

-.4.’-

y 4’.

,i,_

.

l

,‘

FIG. 9. Rat pancreas. Isolated acinar cells are fixed mm in the revised medium (see text) without substrate Postfixation in 2% OsO4. No staining.

fragments in the absence of substrate. we detected no significant increase

tissue

addition,

staining upon with hormones apparent

addition or NaF.

success

in applying this merated in the fore, made an

In in

of AMP-PNP alone or This is in contrast to the

obtained

by other

for 10 min (a) or with

increase cells

sults

(liver

Jarett

been

cell

and

kidney)

reported and 26). tissues,

for

obtained

by

which

positive

by others (liver: After incubation the

pancreas, precipitation

same

using

results

had

re-

ref. 21; kidney: of fragments

refs. 12 of these

are

as with

observations

namely the without

tissues

occurrence substrate,

made

of background and no noticea-

ble

a much

can

for 60

M secretin

(b).

stain-

no substrate-specific

be detected.

with greater

3

When

formaldehyde, part of the

these

which preenzyme activ-

does (Table I), the is noted. these findings that

same pan-

creas, liver and kidney contain material that reacts with lead ions in the R-H medium to form insoluble electron-dense complexes. Lemay and

it on

be

pre-fixed

incubated

106

intracellular

Again,

of staining are

and and

pronounced

instead.

ity than glutaraldehyde lack of specific staining It is concluded from

technique, and have attempted to modify the basis of the information obtained. First, we have checked whether different could

a more

serves

investigators

method to various tissues (enuIntroduction). We have, thereextensive investigation of this

in 0.5% formaldehyde AMP-PNP

ing is found

.

0.5 mM

whereas

:

p,..

(17)

have

ghosts,

also

noticed

incubated

in

Howell medium. not occur after is still observed indicates

that

destroyed

by

Our boiling after

some

finding of the treatment

nucleation boiling

of fat Reik-

that staining does tissue, although it with aldehyde,

sites but

staining

substrate-free

are

on

the

tissue

apparently

are pre-

effect upon addition of AMP-PNP. Secondly, we have tested the method on isolated pancreatic acinar cells, known to contain

served by fixation. Next, we have zymatic adenylate

hormoneand fluoride-sensitive clase activity (Table I), in order access of lead ions and substrate

tected biochemically under the conditions of the cytochemical incubation. We find that alpha32PAMPPNP can be converted into 32P-cyclic AMP in the presence of fresh particulate matter

matic strate,

sites. no

In this case extracellular

adenylate to obtain to the

in the absence precipitation

cyeasier enzyof suboccurs,

from

rat

pancreas

Downloaded from jhc.sagepub.com by guest on June 20, 2015

investigated cyclase

in the

whether any activity can be

complete

R-H

medium.

ende-

KEMPEN

310 However,

this

also

is performed

occurs

with

when

preboiled

the

incubation

particulate

matter

or in the absence of any tissue. cyclic AMP formation in the ions is caused by non-enzymatic

Apparently, the presence of lead hydrolysis of

the

fully

substrate,

enzymatic clusions Lemay the

and

lead

and

Jarett

adenylate

production. with

(17),

cyclase

inhibited by as little that non-enzymatic curs in the presence is the

nitrate

cyclic AMP are in agreement

(8)

claimed cyclic

that AMP

dium.

However,

have

in rat

cell

used

and

the

who

the

These fmdings

observed ghosts

Cheng

there from

in the

and

R-H

is an enzymatic AMP-PNP in the

former

ity

the

background incubations.

(7.2)

and

its

very

precipitate with PNP1 For these reasons, another

(Fig. 8). we have

cytochemical

contain lead for adenylate as substrate. lent cations,

of

activity, to AMP-PNP,

yield of precipitation AMP formation by

ration

in

the

remain and

fresh

have of me-

not

de-

a

develop not

in solution to give the

with PNP1. The enzyme prepamedium

is

enzymatic

hormones

and

the

pancreozymin-C-octapeptide

the cyclic AMP dium. However, formaldehyde-fixed

acinar

particulate matter of rat in this medium, even with

secretin

can

production cytochemical

or

pancreas hormone

mains below concentration

test for

concenactiv-

level

tube

8, it follows

0.3 (rat

to

cause

experiments

that

the

thresh-

precipitation

LM, whereas pancreas

in the

even under slices, pH 7.4,

xanthine

cellular

cAMP

and

0.3 M

concentration

re-

10 .tM (14). However, the maximal reached at or near the site may,

of course,

leaves

tigators applying strated hydrolysed PNP1).

Rat very

enough

the

effects

be that the from enzymatic

the

exceed

question

this

of why

level other

of

traninves-

have apparently had positive results in the R-H medium. We have demonin our study that AMP-PNP can be to cyclic AMP (thereby generating Furthermore, turbidity can develop in rate

this of this

cytochemical medium process is increased by

of CaCl2, especially ions (Fig. 6). Liver contain calcium (27, by

treatment

in the presence of cell plasma mem20), which can be

with

epinephrine

pancreatic plasma membranes high amounts of calcium (5).

(28).

also contain In addition,

hormone-stimulated calcium efflux has ported for liver (10), parotid gland pancreas (3). Therefore, the precipitation served upon incubation of these tissues R-H medium may be related to the and/or release of calcium ions at or plasma

membrane,

clase

activity.

cium dium

ions does of Wagner

rather The

than

finding

been re(19) and obin the presence near the

to adenylate

that

addition

not cause turbidity et al. (32) might

in the meargue against

this explanation. However, precipitation still be induced in this medium by calcium in the micro-environment of incubated fragments, sites for

offering initiation

cyof cal-

the presence of precipitation.

of

may ions tissue

nucleation

stimulate

in the revised incubation cells

the

mobilized a

may

1-methyl-3-isobutyl

addition fluoride branes

when the preparathe enzymatic nasupport for the

is that

does not exceed optimal conditions

spontaneously (Fig. 5). The

form

vis-

medium is about 10 riM. The maximal concentration in the test tube assays

siently. This

nearly completely inhibited tion is preboiled, indicating ture of the reaction. Further nature

revised product

precipitation

a high

From

formation

been cyclic tissue

does

barium-containing

reach

in Figure

ions, and has the optimal pH (8.9) cyclase activity with AMP-PNP After screening a number of divabarium ion is found to permit the

highest enzyme after addition highest cyclic

to

which

not

PNP1

10 mM

to

tried

medium,

does

concentration

to lead to precipita-

ability

revised medium of PNP, resulting

described

precipitation in the Although we have not

poor

to electron-dense

the electronmicroscope. for the negative cytochemical

old

used the medium of Wagner et al. (32) in our cytochemical experiments, this medium does not seem to offer more promise, in view of its low pH

rise

that

material has not been investigated. Irrespective of the question of the nature of the cyclic AMP formation in the R-H medium,

tion above cytochemical

in the tration

secretin)

does

of this process is too low increase in electron-dense

under A reason

(4)

scribe how the control incubations have performed, while in the latter study the AMP formation in the presence of boiled

the rate detectable

give

ible

medium.

formation the R-H

report

not

precipitation.

is fully

Farquhar

AL.

conof

as 0.1 mM lead nitrate and cyclic AMP formation ocof 4 mM lead nitrate, which

concentration

Cutler

inhibits

ET

of

ACKNOWLEDGMENT

meof unfixed

homogenate added, does

We nical

gratefully

acknowledge

assistance

of Miss

biochemical J.

A.

H.

the

experiments, A.

Dormans

Downloaded from jhc.sagepub.com by guest on June 20, 2015

excellent

tech-

Jakobs

in the

A. H. M. and and

Mrs.

the

aid C.

de

of

Dr.

Groot-

ADENYLATE Versteegden support for Netherlands

CYCLASE

in the histological studies. this study was obtained Organization for Basic

(ZWO) through Biophysics.

the

Netherlands

LOCALIZATION: Financial from the Research

Foundation

ylate cyclase the salivary

17. Lemay

for 18.

LITERATURE

CITED

1 . Abro A, Kvinnsland 5: Adenylate cyclase in an estradiol-sensitive tissue: a cytochemical study. Histochemistry 42:333, 1974 2. Birnbaumer L, Yang PC: Studies on receptor-mediated activation of adenylyl cyclases. I. Preparation and description of general properties of an adenylyl cyclase system in beef renal medullary

membranes

sensitive

to

neurohypophyseal

hor-

mones. J Biol Chem 249:7848, 1974 3. Case RM, Clausen T: The relationship between calcium exchange and enzyme secretion in the isolated rat pancreas. J Physiol 235:75, 1973 4. Cheng H, Farquhar MG: Presence of adenylate cyclase activity in Golgi and other fractions of rat liver. II. Cytochemical localization within Golgi

and ER membranes. J Cell Biol 5. Clemente F, Meldolesi J: Calcium

70:671,

and

19.

20.

11.

12.

13.

14.

pancreas

15.

adenylate

cyclase

IV.

Effect

23.

pancreas adenylate lated rat pancreatic Acta 496:52 1, 1977 iim. Kim

SK:

The

cyclase acinar

cytochemical

V. Its presence cells. Biochim

in isoBiophys

localization

of aden-

L: Pitfalls

in the

tion by competitive protein binding. phys Acta 358:154, 1974 Nielsen SP, Petersen OH: Transport the perfused submandibular gland Physiol 223:685, 1972 Nijjar MS, Pritchard ET: Calcium plasma membrane fraction isolated mandibular gland. Biochim Biophys

Reik

L, Petzold

GL,

Higgins

Hormone localization

sensitive in

JA,

use

of lead

Biochim

Bio-

of calcium of the cat.

in J

binding by a from rat subActa 323:392,

Greengard

adenyl rat liver.

P,

cyclase: Science

1970

Reynolds ES: The use of lead citrate as an electron opaque stain in electron J Cell Biol 17:208, 1963 Rogers LB, Reynolds CA: Interaction phosphate ion with certain multivalent aqueous solutions. J Am Chem Soc

24. Rodbell

M, Birnbaumer

L, Pohl

at high microscopy.

pH

of pyrocations in 71:2081, 1949

SL, Krans

HMJ:

The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. V. An obligatory role of guanylnucleotides in glucagon action. J Biol Chem 246: 1877, 1971 25.

Rutten WJ, de Pont JJHHM, Bonting SL: Adenylate cyclase in the rat pancreas: properties and stimulation by hormones. Biochim Biophys Acta 274:201, 1972

26.

Sato tural clase 27. Shlatz liver

T, Garcia-Bunnel R. Brandes cytochemical localization of in the rat nephron. Lab Invest L, Marmnetti GV: Calcium plasma membranes. Biochim

290:70, 28.

29.

30.

31.

D: Ultrastrucadenylate cy30:222, 1974 binding to rat Biophys Acta

1972

Shlatz L, Mannetti GV: Hormone-calcium interactions with the plasma membrane of rat liver cells. Science 176:175, 1972 Trandabaru T: Ultrastructural localization of adenyl cyclase activity in the pancreas of two am-

phibian species Rana esculenta

(Salamandra salamandra L.). Histochemistry 48:1,

L. and

1976 Tu JC, Malhotra SK: Histochemical localization of adenylate cyclase in the fungus Phycomyces blakesleeanus. J Histochem Cytochem 21:1041, 1973 Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiological pH with special reference to the demonstration of bile canaliculi. Am J Patho! 27:13, 1957

32.

Wagner

33.

Biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase in isolated capillary endothelium. Proc NatI Acad Sci USA 69:3175, 1972 Wagner RC, Bitensky MW: Adenylate cyclase in electron microscopy of enzymes. Principles and Methods, Vol. 2. Edited by MA Hayat. Van Nostrand, New York, 1974, p 110-131 Watson ML: Staining of tissue sections for electron

of hormones

and other agents on cyclic AMP level and enzyme release. Biochim Biophys Acta 496:65, 1977 Kempen HJM, de Pont JJHHM, Bonting SL: Rat

A, Jarett

and serous cells of Struc 4:185, 1976

nitrate for the histochemical demonstration of adenylate cyclase activity. J Cell Biol 65:39, 1975 Maguire ME, Gilman AG: Adenylate cyclase assay with adenyl imidodiphosphate and product detec-

168:382,

22.

pancreatic

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Yount

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Babcock

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actomyosin.

The cytochemical localization of adenylate cyclase: fact or artifact?

0022- 1554/78/2604-0298/$02.00/0 THE JOURNAL Copyright OF HISTOCHEMISTRY © 1978 by The THE AND Histochemical Vol. 26, No. 4, pp. 298-312, CYT...
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