0022-1554/79/2705-0913$02.00/O ThE

JOURNAL

Copyright

OF

HlsTocHeMIsmY

© 1979 by The

Studies

AND

Histochemical

on the

Inc.

DepartmeiU

of Immunohistochemical and Cyclic GMP1

M. ROSENBERG2,

ofMedicine

Received

and

1979 in U.S.A.

Printed

Specificity

EDWARD

Vol. 27, No. 5, pp. 913-923,

CYTOCHEMISTRY

Society,

GARY

BiOchemistry

for publication

LaVALLEE, and

November

Anatomy,

1, 1978 and

Techniques

PETER

WEBER

Albany

in revised

form

STEVEN

AND

Medical

for Cyclic

College,

January

AMP

M. TUCCI

Albany,

New

10, 1979 (MS

York

12208

78-232)

hnmunohistochemical

studies employing antibodies against cyclic nucleotides indicate that cyclic are localized to distinct subeellular sites. These antibodies, however, cross-react weakly with nucleotides (eg. AlP, GTP), and therefore we investigated the specificity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncycic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Conclusion: Both nitrous acid and acetylation studies support the specificity of the iminunohistochemical method for cyclic nucleotides. AMP

Recently several double antibody

examine

the

In these

studies

cyclic

GMP noncycic

and

studies fluorescence

intracellular

found

The

extremely relative tides.

first

antiserum

priate with

the

et

consequent

antibodies

our

(cAMP)

al.

utilized

appropriate cyclic and have

found

tissue

(8).

AMP

lation

passage

loss

of

immunofluorescence

(1).

the observations,

affinity

(10).

chromatography

we

techniques First,

although

for their

affinity in the

are

stifi

for the the

respective

purification

with

the high

of the

affinity

that

nucleotides

antibodies

cyclic

for other nucleotides cell in concentrations

Second,

concerned

cyclic

have

(eg., ATP) detected antibody

ligand,

by

does

Restoration

and

pattern

the

antibody.

restored cence.

they which by the

nuclei caused

The

may do

acid groups

bodies

a much

nucleotide,

Indeed,

we have found by adsorption

cross-reactivity we have used

other

affinity

for the

not insure

it was This investigation was supported by grant 1R01CA22690-O1 awarded by the National Cancer Institute, Departments of Health, Education and Welfare. 2 To whom reprint requests should be addressed: Edward M. Rosenberg, M.D., Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208. I

purification cAMP-agarose

with other methods

technique and

In the

for cAMP an acety-

has

been

(6).

In

reported the

study,

Only

cyclic found

to

modify

exposure

free

of

to nitrous acid before application of the a marked reduction in immunofluorescence. of the original immunofluorescent intensity

was then attempted of nucleotides before the

previously

present

exposure

original

nucleotides that

appropriate

and

the

cyclic

intensity

themselves

acetylating

and

by incubating the slides with treatment with the appropriate

to the pattern

nucleotide

immunohistochemistry of succinylated a much greater cyclic nucleotides (4). In the

cyclic

present

nucleotides

in rat

of acetylation of nucleotides for these nucleotides in the

913

Downloaded from jhc.sagepub.com by guest on April 8, 2015

the

effect

antibodies

denyaffinity than

we also examined the affinity of our On comparing

a

of immunofluores-

of the

the effect antibodies

fetal

anti-

slices before the application intensity of immunohistochemical

tube.

of

nucleotides. to examine

procedure

Antibodies used in cyclic nucleotide are generated in the rabbit by injection atives ofcAMP or cGMP (7), and have for the acetylated derivatives of the

may anti-

that on

procedure.

amino mouse

variety

bind to other substances of in higher concentration in the

also

of the immunohistochemical including a nitrous acid

Nitrous

of the

not

present

antibody

specificity and cGMP,

of an

cyclic nucleotide, noncycic nucleothat

specimen. anti-cAMP

does not eliminate the present study

have

be eluted from the Sepharose histochemical fluorescence pattern.

specific.

have some be present body

nucleotides

cyclic

can subsequently

immunofluorescent be

the

for the for other

Steiner

used to restore Despite these

greater

both

that the antibody does lower affinity which are

through a Sepharose column to which the approcyclic nucleotide is attached removes the antibody,

antibody

not

of cyclic

that

is that

high affinity to its affinity Secondly,

in which the been used to

exist in discrete loci within cells. the specificity of the immunofluorescyclic nucleotides has been mainly

cyclic GMP (cGMP) Evidence supporting cent techniques for the types.

been reported technique has

localization

it was

and

two

have

fluorescence.

of acetylation

study liver

increased

the

In addition,

in the

on test

immuno-

914

ROSENBERG

ET

AL.

swelled

in excess

PBS,

pH 7.5 at 4#{176}C for 12 hr, centrifuged at 2500 and the supernatant was discarded and then rewashed with excess PBS at 4#{176}C until the spectrophotometric absorpnofluorescence and in affinity of the antibody to the approtion (Ao) of the supernatant was equal to the buffer. The gel was pniate cyclic nucleotides, which further supports the specificity diluted 1:1 v/v with PBS, and 100 tl of the undiluted anti-cAMP of the immunohistochemical technique. antibody were added to a 5 ml aliquot of the gel, which was gently rotated (Multi-Purpose Rotator, Model 150, Scientific Industries, METhODS Springfield, Mass.) for 12 hr at 4#{176}C. The gel was centrifuged at 2500 The histochemical method utilized is that described by Wedner et RPM for 15 mm and the supernatant separated. Five milliliters of al. (11) and recently reviewed by Steiner et al (8). PBS was then added, and the gel was resuspended and gently rotated Preparation of tissue sections: Male Sprague-Dawley rats, for 20 mis, centrifuged, and the supernatant was pooled with the weighing approximately 200-250 g, were obtained from Taconic initial supernatant. The gel was then washed three times with an Farms, Germantown, N.Y. The animals were anesthetized with soexcess of PBS, 4#{176}C, at which time the spectrophotometric adsorption dium pentobarbital, 0.1 mg/g, given s.c. A hepatic lobe was removed, (A) of the supernatant equaled that of the buffer. and a small cube (1 x 1 x 2 mm) was cut out, placed on the tissue To elute the antibody from the gel, 10 ml of guanidine HC1, 6 M, support of an LEC “minotome” cryostat #3398 (Needham Hth., pH 3.0, was added to the gel and the mixture was rotated for 1 hr. Mass.), and rapidly frozen on the precooled freezing bar or in dry ice The gel was centrifuged at 2500 RPM for 15 mm at 4#{176}C, and the and acetone. Tissue sections were cut 6 microns in thickness, placed eluate was removed and dialyzed against PBS, 4#{176}C, 1:200 v/v for 48 on slides precoated with egg albumin, dried, and heated on a hot plate hr. Nucleotide cross-reactivity studies were performed directly with atSO#{176}Cfor5min. the dialyzed eluate. Preparation of fetal nuclei: Nuclei were prepared by a modifiImmunofluorescence: Tissue slices or nuclear preparations were cation of the method of Tucci and Skalko (9). Female and male incubated with rabbit antibody of high affinity for either cAMP or albino mice, ICR strain, weighing 25 g, were obtained from Camm cGMP, or with “control serum” from rabbits not injected with cyclic Research Institute, Wayne, New Jersey, and males and females were nucleotide. The slices or nuclei were then exposed to fluorescein housed together. Females were checked daily for vaginal semen plugs. labeled goat anti-rabbit iminunoglobulin (Miles Laboratories, ElkFemales with evidence of insemination were separated and sacrificed hart, md.) as described by Wedner et al. (11). by cervical dislocation on various days of fetal development. The Microscope: Slides were viewed with a Zeiss photomicroscope III fetuses were removed, placed in a phosphate-saline buffer (PBS), pH with a vertical illuminator III R.S., a 10 x eyepiece, 1.25 optivar 7.0, 37#{176}C, and the intact fetuses pushed through a 60 micron nylon setting, and a planapochromat 40/1.0 oil less (1-46-17-46) (Carl Zeiss mesh (Tablet, Ernst and Traber, Elinsford, N.Y.) with a glass rod to Co., New York, N.Y.). disperse the cells. The cell suspension was then centrifuged at 500 Photography: Pictures were taken with KOdak Technical Pan RPM in an IEC centrifuge Model PR-6 for 3 mm at 20#{176}Cand the Film 50115 (Eastman Kodak Co., Rochester, N.Y.), usingan exposure supernatant was discarded. The cells were resuspended in PBS, 37#{176}C, index of 100, and developed with Perfection Micrograin Film Develrecentnifuged, and the supernatant discarded. The cells were then oper (Perfection Photographic Products, Beverly Hills, Ca.) for 20 resuspended in a solution of methanol and glacial acetic acid, 6:1 v/v, mis at 82#{176}F. Prints were made on ilford bospeed multigrade paper and rapidly dropped onto glass slides precooled to 0#{176}C, and allowed MGIM, glossy (ilford, Inc., Paramus, N.J.), with a No. 5 or No. 7 filter to dry. (Ilford Multigrade filters) and developed with ilford Ilospeed MultiGas phase nitrous acid exposure: Nitrous acid was formed by grade Developer. mixing equal volumes of sodium nitrate, 1 M, and 50% glacial acetic, Determination ofantibody affinity for nucleotides: Antibody both precooled to 4#{176}C, and poured into a covered Wheaton jar affinity for various nucleotides was determined by displacement of precooled to 0#{176}C and placed on ice. Slides were placed in a precooled the ‘9 cyclic nucleotide radioligand by the method of Steiner et al. carrier and expooed only to the gas phase of the nitrous acid for (8). Acetylation of nucleotides was by the method of Harper and variable times. Short-term gas phase exposure was used to minimize Brooker (4). Whether this method results in substitution of acetyl damage to the receptor and other proteins. Maintaining the coldness group in noncycic nucleotides is unknown. However, the method of all constituents and prevention of gas loss was critical. does increase the affinity of the antibody for noncycic nucleotides as Gas phase acetylation: A gauze pad at the bottom oftwo covered noted in the above study (4). Wheaton jars, maintained at room temperature, were saturated with either triethylamine (TEA) (Eastman Kodak, Rochester, N.Y.), or RESULTS acetic anhydnide (AA) (Aldrich Chemical Co., Milwaukee, Wiac.). Nucleotide cross-reactivity of anti-cAMP antibody Slides supported in a carrier were first exposed to the gas phase of adsorbed to cAMP-agarose: We first determined whether TEA for 5 mm, followed by immediate transfer to the second jar and prior adsorption of the antibody by affinity chromatography exposure to the gas phase of AA for approximately 40 sec. The

fluorescence found

that

studies

acetylation

and the radioimmunoassay produced

a parallel

studies,

increase

it was

in inunu-

duration of exposure to AA is critical, and the optimum duration varies with the thickness of the tissue slice and the nature of the tissue specimen. Overexposure resulted in distortion of the cyclic nucleotide fluorescence pattern and tissue histology. Cyclic nucleotide antibodies: Antibodies were raised in rabbits against succinylated cyclic nucleotide conjugated to keyhole limpet hemocyanin, by the method of Steiner et al. (7). Antibodies were further purified by isolating the y-globulin fraction using Pluromc Polyols F-38 (BASF Wyandotte Chemical Corp.), by a modification Of the method of Garcia and Ordonez (2). Preparation of anti-cAMP antibody by adsorption to cAMPagarose: Adenosine 3’,5’-cycic monophosphate-agarose was obtained from Sigma Chemical Co., St. Louis Mo. The gel was

RPM

for 15 mis,

eliminate cross-reactivity 1 summarizes the percentage radioligand from the previously would

produced antibody nucleotides.

by still

trates the fluorescence

nucleotides.

various showed

to gas adenosine

effect of gas in mouse

phase fetal

the normal nuclear reported (7). Figure

Downloaded from jhc.sagepub.com by guest on April 8, 2015

As

significant

ofnuclei with

Exposure

by incubation

with other nucleotides. Table ofdisplacement ofthe ‘I cAMP adsorbed and eluted antibody,

shown,

cross-reactivity

phase nitrous nucleotides: nitrous nuclei.

pattern and lB demonstrates

the

adsorbed with

other

acid followed Figure 1 ifius-

acid on cAMP iminunoFigure 1A demonstrates

is similar to that previously the effect of exposure

to

CYCLIC TABLE Displacement Antibody

nitrous

I

and

vapor

for

5 mm

before

to fluoresceinated

pattern. and then

shown

acid,

exposure

anti-rabbit

to

IgG

and staining

in Figures

slides

monophosphate

1D

were

anti-rabbit pattern

crease

and

1E,

1 mM,

or adenosine

(5’AMP),

anti-

pattern. the imthe histo-

and

incubation

shows

the

with

effect

of nitrous

to

tniphosphate

guanosine nucleotides: acid on the cGMP immunofluores-

fluorescence

varies

with

the

2

day

of fetal

devel-

opment, Figure

and this variation is the subject of a separate study. 2B demonstrates the effect of exposure to nitrous acid

before

staining

for cGMP.

the fluorescence. trous acid, and before ceinated of the

to

the

5’guanosine tniphosphate In

neither

cGMP

liver

anti-rabbit is an ment

nitrous

instance

was

treated IgG

adjacent with the

illustrates with

acid,

diminution

nuclei cGMP, antibody Of note Figures

slides

of

exposed to ni1 mM, for 1 hr and the fluoresis the restoration 2D and 2E, after

were

incubated

with

(5’GMP), 1 mM, or guanosine for 1 hr before staining for cGMP. there

a restoration

of

the

on

with

anti-cAMP

slide same

edly enhanced the 3D show acetylated

without

of hepatic dilution

antibody

and

prior

acetylation.

fluonesceinated Figure

tissue acetylated before of antibodies. Acetylation

intensity of fluorescence. Figures and unacetylated hepatic slices,

3B

treatmark3C and which

produced

absolute

Figure

acetylation the IgG

a nonim-

IgG antibody, significantly in-

for cAMP, nucleotides

the the

anti-cAMP

threshold

of

a comparison of the for the anti-cAMP

approximately

20%

displacement

affinity

for

4 shows

cGMP

on the

remained

cGMP

effect

much

of

in this

(5

on

cGMP

im-

liver examined for 4B is hepatic tissue In contrast to the effected by prior

fact that there is more intense fluorescence Figure 3C is due to the fact that the IgG concentration

lower

immunohistochemis-

of acetylation

is modest. Figure 4C is hepatic tissue fraction of serum from nonimmunized

treated rabbits.

with The

in Figure 4C than fraction was used in

experiment.

Figure

4D

shows

hepatic from

tissue acetylated before exposure to the IgG fraction nonimmunized rabbits. Nonspecific fluorescence was minimally increased by acetylation. Effect of acetylation on affinity of the anti-cGMP antibody for nucleotides: Table III lists the threshold of sensitivity of the antibody for cGMP, a comparison of the relative affinity for other nucleotides, and the effect of acetylation on antibody, cGMP

the relative as expected,

when

effect there

affinity for has a marked

compared

to other

of acetylation on was only a ten-fold

antibody

for

cGMP produced

tochemical terestingly,

fluorescence the increase

less than antibody degree

(4). the

fact

increases

the

affinity

explains

that

both cyclic nucleotides ity for their respective

acetylation

on

(Tables cyclic

fact

4A and (20 x)

the affinity as might be

of cAMP expected, of the

a more

and

II and III), nucleotide

that

4B). Inwas also

is a characteristic

of the

cAMP

to the

in immunohis-

(Figures for cAMP

demonstrating

of the

the

increase

to acetylation

antibodies

response Despite

perhaps

of acetylation (100 x increase);

In contrast

The for

of the cAMP antibody, in affinity of the cGMP

a modest

for cGMP in affinity

of response

various nucleotides. preferential affinity

nucleotides.

affinity increase

this

only

the effect for cGMP other

the

and

acetylation

antibody,

cAMP immunofluorescence Figure 3 shows the effect of gas phase cAMP immunofluorescence. Figure 3A shows antibody

from

affinity of Table II lists

munohistochemistry. Figure 4A is rat cGMP without prior acetylation. Figure acetylated before examination for cGMP. cAMP study, the increase in fluorescence

the

normal

pattern.

of acetylation

in hepatocytes: acetylation on

is a significant

the anti-cGMP IgG antibody. pattern. In

monophosphate (GTP), 1 mM,

staining

Effect

rat

Figure 2C then incubated

staining with anti-rabbit initial staining

exposure

There

which

a higher

followed Figure

cence pattern in mouse fetal nuclei. Figure 2A illustrates the normal nuclear pattern for cGMP. It is of note that there is a significantly greater amount of intranuclear fluorescence than in the cAMP studies (Fig. 1). The amount and pattern of intranuclear

on

of the antibody affinity of various

the

try:

(ATP), 1 mM, for 1 Kr, before treatment with anti-cAMP and anti-rabbit IgG antibodies. In neither instance was there a restoration of the normal cAMP staining pattern. ADP also

by

of serum anti-rabbit did not

fluorescence.

x 1O_8) than for cAMP. Effect of acetylation

5’adenosine

acid

fraction

the 1125 labeled marker from its antibody. The antibody, as expected, has a marked preferential affinity for cAMP, when compared to other nucleotides. Acetylation significantly increased the affinity ofthe antibody only for cAMP and cGMP,

There

exposure

failed to restore the initial pattern (not shown). Exposure of nuclei to gas phase nitrous

IgG

of acetylation for nucleotides:

cleotides

to nitrous 1 Kr, before

either

the

antibody, and the effect of acetylation on the relative affinity of these nucleotides. Relative affinity of various nucleotides was determined by comparing the concentration of the flu-

cAMP

following

with

with

nonspecific

antibody sensitivity relative

IgG antibodies. is evident. In the

incubated

treated

Effect

antibody.

of the cAMP fluorescence to nitrous acid eliminated completely, but destroyed

the anti-cAMP of the initial

nitrous

bound

23. 58. 24. 75. 35. 72. 33.1

Figure 1C illustrates nuclei exposed incubated with 1 mM cAMP for

staining with A restoration studies

counts

10#{176}M 103M 102M 103M 102M 103M 102M

915

GMP

munized rabbit, plus fluorescemated and it is apparent that acetylation

100.

is a marked diminution More prolonged exposure munofluorescence more logic acid,

CYCLIC were

% of

None cAMP 5’AMP 5’AMP ADP ADP ATP ATP

acid

body

AND

of I’-cAMP by Nucleotides from Anti-cAMP Purified by Adsorbtion on cAMP.Agarose Nucleotide

1 2 3 4 5 6 7 8

AMP

cyclic

cGMP the is

dramatic nucleotides

antibodies absolute much

to

affingreater.

Similarly, the immunohistochemical fluorescence pattern remains similar to the unacetylated patterns, with no tendency to cross over from a cAMP to a cGMP pattern or the reverse. This can acetylated

be seen more clearly in Figures 5A and patterns are compared. As previously

Downloaded from jhc.sagepub.com by guest on April 8, 2015

5B where reported

the (8),

1. Effect

of gaseous nitrous acid and incubation with nucleotides on cAMP immunofluorescence in mouse fetal nuclei. Nuclei in B E were exposed to nitrous acid for 5 mm. Slides in A and B were then incubated with PBS; C) exposure to cAMP, 1 mM, in PBS; D) to 5’AMP, 1 mM, in PBS; and E) exposure to ATP, 1 mM, in PBS. All incubations were for 1 hr at 20#{176}C. All slides were then stained for cAMP as described in the Methods section. All slides were exposed to film for the same duration of time, and all development and printing FIG.

through exposure were

done

under

identical

conditions. 916

Downloaded from jhc.sagepub.com by guest on April 8, 2015

Fic.

1E.

917

Downloaded from jhc.sagepub.com by guest on April 8, 2015

918

FIG.

ROSENBERG

2.

Effect

of nitrous

acid

and

incubation

exposed to nitrous acid for 5 mm. The slides 5’GMP, 1 mM in PBS; E) exposure to GTP, same duration of time, and all development

with

nucleotides

on cGMP

ET

AL.

immunofluorescence.

Slides

of B through

E show

nuclei

in A and B were then incubated with PBS; C) exposure to cGMP, 1 mM, in PBS; 1 mM in PBS. All incubations were for 1 hr at 20#{176}C. All slides were then exposed and printing were done under identical conditions.

Downloaded from jhc.sagepub.com by guest on April 8, 2015

which

were

D) exposure to to film for the

CYCLIC

AMP

AND

CYCLIC

GMP

Downloaded from jhc.sagepub.com by guest on April 8, 2015

919

920

I

) Effect IgG, acetylated nonimmunized under identical

ROSENBERG

of acetylation on immunohistochemical tissue (triethylamine (TEA)): 4 min.; rabbit, acetylated tissue. Pictures were conditions (see Methods).

ET

AL.

staining for cAMP in rat liver A)nti cAMP IgG A A 40 sec.); C) IgG from nonimmunized rabbit, exposed for the same duration of time, and development

Downloaded from jhc.sagepub.com by guest on April 8, 2015

unacetylated tissue B) anti cAMP unacetylated tissue; D) IgG from and picture printing were done

CYCLIC

TABLE A nti-cAMP

Antibody-Relative Effect

2

3

4

AND

II ofOthe

Affinity

r Nucleotides

and

of Acetylation Affinity

1

AMP

Increase

Produced Acetylation

by

two other techniques. The first was gas phase exposure nitrous acid, to destroy cyclic nucleotides. Nitrous acid known to deaminate primary amino groups, deamination

ucts

cAMP cAMP

unacetylated#{176} acetylated

5’AMP 5’AMP

unacetylated acetylated

1 x 10 5 x iO

5x

ADP ADP

unacetylated acetylated

5 X iO 5 x 10#{176}

0

ATP

unacetylated

ATP

acetylated

5 x iO 5 X i0#{176}

0

1. 50.

yielding hypoxanthine, predominantly xanthine (10).

Exposure

50x

their modified Alternatively decreased acidity

itself

cGMP

cGMP Threshold

acetylated ofsensitivity:

(unacetylated):1

x 103M.

a

1 x iO-#{176}

unacetylated”

1 x i07

cAMP

lOOx

(unacetylated):1

x 10’2M;

cGMP

immunoreactive cAMP appears predominantly in the cytosol and the bile canaliculi, with little appearing in the nucleus or cell membrane. In contrast, immunoreactive cGMP is predominantly intranuclear, perinuclear, and in the cell membrane.

of cyclic tween these

techniques potential value

nucleotides

within

cells of varying methods, it is

specific,

that

the specific nucleotides nucleotides.

is, that

cells,

function. important the

and

within the cross-reactivity

of tissues before

eliminated nucleotides

cell

method chemistry

to hydrolysis for

the that react

validity they

its receptor.

the

staining

only

because and the

the specificity demonstration that

the bound

(5). The

ing

patterns

(8).

Purification

of our

anti-cAMP

is consistent

cyclic

possible that the cyclic nucleotide

our the

away. have a

ability

to restore

appropriate

cyclic

of other

nucleotides

to restore

with

hypothesis

that

due not

to the presence know whether

the

nal nuclear fluorescent pattern cyclic nucleotides. Currently

was do

we

the

the

origiof the

specificity lies in the antibody, the nuclear binding sites, or both, i.e., whether the binding site will bind other nucleotides which are not recognized by the antibody, or binds only the appropriate cyclic nucleotide. The second method used to explore the question of specificity was acetylation. Acetylation markedly increased the

did not occur (Fig. 5). increased the affinity

of not

cyclic form main

antibody

cGMP, and cent staining. in antibody acetylation

by

cGMP,

but

if this

increase

immunofluorescence, pattern to change

were

respon-

one would have toward that of cGMP,

In contrast, acetylation of the cGMP antibody

only for

only modestly increased cGMP immunofluoresThe parallelism between the degree of change affinity for the cyclic nucleotides produced by and the degree of change in the intensity of

effect of acetylation nucleotides to the fixative, vention

is due to increased fixing of the slide, since acetic acid is a known

and that of washing

an

increase

fact

that

the out

in their acetylation

AMP

than

increased of the

affinity

fluorescence was cyclic nucleotides for

increased with

cyclic

the

antibody.

GMP

makes

due to prerather than

more it more

with likely

increases in fluorescence were due to enhanced cyclic nucleotides for their antibodies. In summary we have examined the specificity fluorescent techniques for the cyclic nucleotides, method employing treatment with gas phase

first nitrous

followed

nucleotides,

by incubation

with

and second by a gas phase of our nitrous acid studies fluorescent techniques for results

of our

acetylation

conclusion.

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cyclic

and

noncycic

the

cyclic tissue

However,

fluorescence

the the

the by

for increased staining

immunohistochemical fluorescence produced by acetylation, is consonant with our conclusion that the immunofluorescence is specific for the cyclic nucleotides. It is possible that

cyclic

cross-reactiv-

of specificity

failure

and this modestly

with

of immunohistoprior adsorption

antibody

the

the the

the eluted antibody cross-reacts with in addition, some antibodies which are chromatography give identical stain-

adsorption on cAMP-agarose, did not eliminate ity with other nucleotides (Table I). We have therefore examined the question

was with

sible for expected

ofanti-cycic nucleotide antiserum by cyclic nucleotide affinity chromatography eliminates the capacity of the antiserum to generate immunofluorescence, while the antibody eluted from the affinity column restores immunofluorescence (1). However, it is possible that other nucleotides, and, not adsorbed by affinity

This

interest

cAMP

by demonstrating that is destructible by phoshave found that prein-

perhaps protein

and

It is also of the

by incubating

of are

against cyclic with other of some of

under a variety nucleotides, has

Of greater

pattern

decreased

them to be washed nucleotides may

affinity for the antibody. caused the dissociation

be-

in comparison with the cyclic poses a problem. In the im-

by phosphodiesterase

demonstrating has been the

cyclic nucleolocalization

distribution

employed

with phosphodiesterase examination for cyclic

immunofluorescence, are bound to receptor

is resistant

their

Antibodies raised low cross-reactivity high concentration

munoassay, specificity can be insured the immunoreactive material in tissue phodiesterase (3). Unfortunately, we cubation conditions,

the

In considering to establish

antibodies

cyclic nucleotides. often demonstrate Because of the

these substances nucleotides, this

for identifying for studying

markedly

It is possible that the diminis due to a decreased affinity of the binding sites, as a consequence of

affinity of the cAMP antibody for cAMP, and markedly enhanced immunofluorescent staining for cAMP. While haying minimal or no effect on the affinity for other noncycic nucleotides, acetylation also increased the affinity of the

DISCUSSION Immunofluorescent tides are of great

acid

to is of

deamination of guanine other unidentified prod-

structure, permitting the modified cyclic

and

pattern.

and and

to nitrous

nucleotide immunofluorescence. ished immunofluoreseence cyclic nucleotides for their

nucleotide, 5

921

GMP

adenine yielding

in Affinity

Relative to cAMP

CYCLIC

the the that

affinity

of

of immunoby a acid

acetylation procedure. The results strongly suggest that the immunocAMP and cGMP are specific, and studies

are

consistent

with

this

922

ROSENBERG

ETAL.

Th\*

FIG.

4.

acetylation; acetylation.

Effect

of acetylation

on

cGMP

immunohistochemistry

C) IgG fraction of serum from nonimmunized rabbits, Time of film exposure and conditions of development

of rat

liver.

unacetylated; and printing

A)

cGMP

antibody,

D) IgG fraction were identical.

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unacetylated; of serum from

B)

cGMP

nonimmunized

antibody,

rabbits,

prior

prior

CYCLIC

AMP

AND

CYCLIC

923

GMP

____

FIG.

5. Effect

of acetylation

TABLE AnticGMP

Antibody-Relative the

on cyclic

2

3

4

a

Nucleotides

ration

of Acetylation Increase Produced

in Affinity by Ace-

tylation

1.

unacetylated#{176}

cGMP

acetylated

5’GMP 5’GMP

unacetylated acetylated

0.5 x 10_b 0.2 x iO

4x

GTP GTP

unacetylated acetylated

2.5 x iO 7.5 x l0

3x

unacetylated#{176}

acetylated

Threshold

cAMP

of

(unacetylated):

lOx

10.

cAMP

1.25

x

cGMP

3. Goodman

7.

3

x

8.

10’4M;

2.5 x 10#{176}M. 9.

ACKNOWLEDGMENT The authors gratefully wish to express thanks to Dr. A. David Goodman for his thoughtful suggestions and critical review of the text, and to Kathleen Maney and Laura Barton for secretarial assistance. LITERATURE

by

immunofluorescence.

Science

GA:

of plasma

of plasma AD,

148:1089,

1974

The

Steiner

AL,

acetylated.

of pluronic polyols its application in the Transfusion 16:32, 1977

proteins

derivatives.

use

in the

and

prepa-

Pagliara

AS:

Effect

of acidosis

10.

of free and J Biol

kinase-complexed cyclic Chem 246:6183, 1971 G: Bestimmung der biologisch

and Am

J

acetic 1:207,

phos-

adenosine

3’,

wirksamen Tabakmosaikvirus auf chemischem Wege. Naturforsc 13B, 697, 1958 Steiner AL, Parker CW, Kipnis DM: Radioimmunoassay for cyclic nucleotides. J Biol Chem 247:1106, 1972 Steiner AL, Ong SH, Wedner HJ: Cyclic nucleotide immunocytochemistry, Advances in Cyclic Nucleotide Research vol. 7. Edited by Greengard, P, Robison, GA. Raven Press, New York, 1976, p 115-155 Tucci SM, Skalko RG: Demonstration of lateral asymmetry in mouse embryo chromosomes. Experienta 33:1437, 1977 Weber G, Jackson RC, Williams JC, Goulding FJ, Eberts TJ: Enzymatic of purse ulation

VH, Schramm in der Ribosenucleinsaure

des

markers of neoplastic transformation and pyrimidine metabolism, Advances vol. 15. Edited by Weber, G. Permagon

New York, 1977, p 53-77 11. Wedner JH, Haffer BJ,

CITED

1. Fallon EF, Agrawal R, Furth E, Steiner AL, Cowden R: Cyclic guanosine and adenosine 3’, 5’-monophosphates in canine thyroid: localization

Ordonez

B) cGMP,

alkalosis on 3’, 5’-GMP and 3’, 5’-AMP in renal cortex. Physiol 223:620, 1972 4. Harper JF, Brooker G: Femtomole-sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2’-acetylation by anhydride in aqueous solution. J Cyclic Nucleotide Res 1975 5. O’Dea RF, Haddox MK, Goldberg ND: Interaction with

6. Schuster Einheit

20x

(unacetylated):

LA,

acetylated;

phodiesterase 5’-monophosphate.

l0

2.5 x l0

sensitivity:

A) cAMP,

precipitation

and

cGMP

cAMP

pattern.

2. Garcia of Other

Affinity Relative to CGMP

1

staining

III Affinity

Effect

nucleotide

Battenberg

Bloom FE: A method for detecting monophosphate by immunofluorescence. 20:293, 1972

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and

regulation

in Enzyme RegPress, Elmsford, AL,

intracellular cyclic J Histochem

Parker adenosine Cytochem

CW,