0022- 1544/78/2608-0628$02.00/0 THE

JOURNAL

OF

© 1978

Copyright

HISTOCHEMISTRY by

The

AND

Histochemical

Vol. 26, No. 8, pp. 628-634, 1978 Printed in U.S.A.

CYTOCHEMISTRY

Society,

IMMUNOPEROXIDASE

Inc.

L. MORRISSEY”

ROBERT

Letterman Received

for

Calcium

OF VITAMIN PROTEIN

LOCALIZATION

CALCIUM DAVID

2,

Army

T.

Institute

publication

binding

BINDING

THOMAS

J.

Presidio

(CaBP)

was

BUCCI

of San

7, 1977, and in revised

November

protein

ZOLOCK,

of Research,

localized

the

DANIEL

AND

Francisco,

CA

May

23, 1978 (MS

form

by

D DEPENDENT

indirect

D. BIKLE

94129

77-225)

peroxidase-labeled

after administering 32.5 nmol of cholecalciferol to vitamin D-deficient chicks. CaBP was observed in cytoplasm and nuclei of absorptive cells but was absent from goblet cells. Our results are consistent with the suggested functional role for CaBP in the prevention of intracellular accumulation of calcium by preventing mitochondrial accumulation of calcium, enhancing removal of calcium from absorptive cells, and/or preventing the “leaking” of calcium into cells through the lateral borders. They are not consistent with an extracellular functional role for CaBP. antibody

method

in

chick

duodenum

72

hr

The cellular and subcellular localization of the vitamin D-dependent calcium binding protein (CaBP) is a key factor for establishing the biological role of this protein. Taylor and Wasserman (15) localized CaBP in goblet cells and in the surface coat microvillar region of the intestinal epithelial cells by an immunofluorescent

may

with duodenal tissue from vitamin D-deficient diet and

This observation prothe basis for the suggestion that CaBP function by sequestering calcium from the

localization vided

denum 72 hr after treatment of rachitic chicks with 32.5 nmol of cholecalciferol (D3) (15), whereas we localized CaBP at various times after treatment of rachitic chicks with 2.5 nmol of 1,25-dihydroxycholecalciferol (1,25(OH)2D3) (8). The current investigation was performed

technique.

death with 32.5 nmol Taylor and Wasserman that this study would

gut lumen so that CaBP presents a higher concentration of the ion to the absorbing surface of the intestinal cell. However, since this initial observation,

other

groups

localization

of CaBP

in

chick

(8,

Table

1. Some

14,

cellular

as

of CaBP.

While

15)

and

of these well

as some

have pig

hen

examined

(1), (6)

reports

human as

may be of species,

suits

(8)

(15), chick Taylor

each using antiserum prepared against intestinal CaBP is difficult to interpret. and Wasserman localized CaBP in duo-

those

of Taylor

basis between and

Antibody

and

AND

retech-

treatment

sched-

METHODS

preparation:

combination

of ion

filtration

of differour re-

tiserum

Wasserman

was

mately and

0.1-0.2

Titers

ml

usually

specificity

trophoresis

of soluble

electrophoresis

ton,

Mass.) acid,

0.00025

0.01

to remove been

present

any

sodium

nonspecific at

concentrations

628

Downloaded from jhc.sagepub.com by guest on June 5, 2016

from M sodium

adsorption:

each and

by

0.07 dibasic

saline intervals.

sodium

and booster

specificity

method

(Millipore

M thimerosal

Antiserum

Council.

M

ad-

8 weeks,

after

titers

buffer

contained

An-

approxi-

day

6 to

evaluated

proteins

in

30

Ouchterlony

was also

The boric

within

the

gel

(8).

of normal at

Antiserum by

by a and

subcutaneous

antigen

7 to 10 days

thereafter. evaluated

by of

adjuvant

collected

purified earlier

of 1:1 homogenate

developed

was

tibody

doses

complete

antiserum injection

in rabbits

100 g

of

was

chromatography as reported

prepared

Freund’s

were

CaBP

exchange

chromatography

ministration

‘The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 2 In conducting the research described in this report, the investigators adhered to the Guide for Laboratory Animal Facilities and Care, as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences, National Research

D form

MATERIALS

localization

results ences

and

the

CaBP localization in localization

intra-

localization

different

explained on the difference

in an

of D3 as described by (15). It was anticipated determine whether the

duodenal to differences

or vitamin

raised on a 72 hr before

ule.

7, 13),

summarized

extracellular of the

nique

the (5,

indicate

different chick sults were due

chicks treated

An-

(12). immunoelec-

chick

duodenum.

Biomedica, barbital,

Ac0.05

phosphate,

M and

salt at pH 8.6. Antiserum was adsorbed antibodies

that below

might the

have

detection

VITAMIN

D DEPENDENT

CALCIUM TABLE

Calcium

Binding

Protein

BINDING

PROTEIN

I

(CaBP)

Localization

in Intestine Localization

Investigator

Antibody

Species

Used Goblet

Taylor and Wasserman (70) Helmke et al. (74)

Lippiello Wasserman Morrissey

Chick Human

and (75) et

Anti-Chick gut CaBP Anti-Human kidney CaBP Anti-Chick gut CaBP Anti-Human kidney CaBP

Hen Human

al.

(75)

Piazolo

et al.

(75)

Human

Results

Brubor-

cells

Nucleus

+

+

-

-

-

?

+

+

-

-

+

+

-

-

-

?

+

-

-

+

+

+

-

-

-

?

+

+

+

-

?

+

+

+

+

+

-

-

-

-

Anti-Human

629

kidney CaBP Arnold

et al.

Morrissey (78) Taylor Intosh

(76)

Pig

Anti-Pig gut CaBP

al.

Chick

Anti-Chick gut CaBP

Mc-

Chick

Anti-Chick gut CaBP

et and

(77)

limits of our methods. One-day-old White Leghorn cockerels were raised on a vitamin D-deflcient diet (Teklad Test Diet TD 75007, Madison, Wise.) containing 0.43% calcium and 0.3% phosphorus for 3 weeks. The chicks were anesthetized, the duodenum removed, and the mucosa scraped from the underlying tissue, homogenized in 0.04 M tris, pH 7.4, containing 0.08 M sodium chloride, and centrifuged at 100,000 x g for 30 mm. Ten volumes of 0#{176}C acetone were added for each volume of supernatant. The precipitate was filtered and washed three times with six volumes of 0#{176}C acetone a

per

wash,

filtered,

powder

fine

marble.

One

washed

three

in

dried

a

gram

overnight,

and

ground

mortar

and

pestle

made

of powder

per

10 ml

of serum

times

with

a volume

to

M

dibasic

sodium

phosphate,

night

in phosphate

dium

phosphate,

0.85

M

were

dehydrated

ethanol,

were

sodium then

embedded

phosphate, pH

7.4

for

buffered 0.2

pH

stepwise in graded

Nakane

and

duodenum.

normal

Step except

at room

goat

hydrogen Step

a total

of xylene,

they

sections

were

dry

the

goat

(anti-RGG).

A control

anti-

slide,

in

gauze,

area

wipe

the

PBS

from

the

slide

of tissue.

10 d of 1:1 diluted

rabbit

antiserum

or NRS) in PBS to the tissue area, mix residual PBS, and allow to react for 20 mm temperature

anti-RGG

so-

Five-micron

peroxidase-conjugated

in a moist

chamber.

diluted

1:7 in PBS

as in Step

2.

Step 5. Wash the slide in three changes of cold PBS for a total of 15 min. Step 6. Incubate the slide for 20 mm in a solution of 20 mg/100 ml of 3,3’-diaminobenzidine tetrahydro chloride in 0.05 M tris buffer, pH 7.6, and 0.005%

M

concentrations

of

Step 3. Wash the slide in three changes of cold PBS for a total of 15 mm. Step 4. React the section with peroxidase-labeled

(0.2

of

method

rabbit

2. Apply

(anti-CaBP with the

saline

they

procedure

1. With from

so-

After

antibody

used to localize CaBP in of Nakane and Kawaoi (10)

serum (NRS) was substituted for anti-CaBP, was prepared for each specimen evaluated. Tissue from rachitic chicks was used as an additional control. The protocol for each slide is as follows: which

over-

concentrations

a and

(11) was

was used to prepare rabbit gamma globulin

washed

(PBS).

Pierce The

through ethanol

immunolocalization.

peroxidase-labeled

monobasic

7.1)

of

before

M

phosphate,

hydrated

indirect

and sodium

xylene,

concentrations

in PBS

Step

dibasic

with

decreasing

The

0.028

in graded

in paraffin.

placed

24 hr

M monobasic

chloride,

of

of homogenizing

buffer equal to the amount of antiserum being treated. The antiserum was then added to the washed powder and the mixture was incubated at 4#{176}C for 30 mm with slow mixing on a shaker, centrifuged at 25,000 x g, and the supernatant was removed and stored at -20#{176}C. Localization technique. Three-week-old vitamin D-deflcient chicks were given 32.5 nmol of D3 in propylene glycol by intramuscular injection. After 72 hr, chicks were killed by decapitation and tissue specimens were collected within 1 mm. Tissues were fixed in 10% formalin (3.7% formaldehyde) containing 0.088 dium

series

from was

deparaffinized

cut,

Step tetroxide

Step a total

peroxide

(added

7. Wash in three of 10 mm. 8.

Incubate

in

in Zetterquist’s

9. Wash

in three

of 10 min.

Downloaded from jhc.sagepub.com by guest on June 5, 2016

just

changes a

1.0% buffer

changes

before

use).

of distilled solution (4)

for

water of

for

osmium

5 min.

of distilled

water

for

630

MORRISSEY

Step 10. Dehydrate clear in xylene, and Scientific

Co.,

Fair

in

graded

mount

Lawn,

ethanol

with

solutions,

Permount

N.J.).

The

antiserum

1,a) or reacted from neither against tamin ficity intestinal

specificity

illustrated occurred

is demonstrated

in Figure when either

pre(Fig.

non-adsorbed (Fig. 1,b) antiserum was against duodenal mucosal supernatant

D3-treated

chicks

antiserum

(Figs. formed

duodenal D-deficient

of the antibody CaBP was

D of Figs.

C),

while

precipitin

supernatant (Fig. 1, B).

line

from viThe speci-

to vitamin D-dependent further supported by

the

(12) double immua single precipitin

band was formed against both supernatant from D3-treated purified chick intestinal CaBP. from D3-treated chicks was (part

1, A and a

mucosal chicks

results of the Ouchterlony nodiffusion method, wherein

CaBP

by

1. A single adsorbed

duodenal chicks When stained

2, 3, and

4),

mucosal and highly duodenum with antiCaBP

cells and was not obThe CaBP is distinctly confined to the glycoca-

lyx. Within absorptive increased concentrations portions of the cell, and

cells, CaBP occurred in in the apical and basal was particularly concen-

along

the

lateral

cell

borders

(Fig.

in the

3D).

apical

portion

of the

cells

was

distinctly intracellular beneath the brush der. Brush border staining was occasionally served, but since such staining was also sionally observed in sections treated with we

are

not

certain

for CaBP. the same readily

that

Nuclei degree

such

staining

were stained as cytoplasm

distinguishable

from

boroboccaNRS,

is specific

to approximately and were cytoplasm

not

in cells

on the villus. However, in the crypt region, nuclei were occasionally stained very densely (Fig. 4D). Duodenal tissue from rachitic chicks does not stain

when

3A, and

treated

4A)

Duodenal not stain CaBP

tissue when in

with

either

or anti-CaBP

the

from NRS

(Fig.

NRS 2B,

3B,

D3-treated chicks was substituted

staining

and 4C), with the specific stain along did not stain under tions studied.

procedure

(Fig.

2A,

and

4B).

also did for anti-

(Fig.

exception of occasional tissue borders. Goblet any of the treatment

2C,

3C,

noncells condi-

DISCUSSION

was

localized in absorptive served in goblet cells. intracellular and is not

trated

AL.

CaBP

The

(Fisher

RESULTS

the results cipitin band

ET

Localization

of CaBP

in the

cytoplasm

of ab-

sorptive cells and its absence from goblet cells 72 hr after administration of 32.5 nmoi of D3 is similar to earlier reports of CaBP localization in chick CaBP results

after

1,25(OH)2D3

localization do not

administration

in the normal agree with those

(8)

and

pig (1). These of Taylor and

-,

.T FIG. 1. Immunoelectrophoresis of soluble proteins from duodenum of chick: A) 72 hr after treatment with 32.5 nmol D3; B) vitamin D-deficient; C) 72 hr after treatment with 32.5 nmol D3. After electrophoresis, the antiserum troughs were filled with: a) anti-chick gut calcium binding protein (CaBP); b) anti-chick gut CaBP after adsorption with an acetone powder from vitamin D-deflcient chick duodenum. The precipitin arcs (arrow)

indicate

specific

antibody

against

a vitamin

D-dependent

protein

to be present

Downloaded from jhc.sagepub.com by guest on June 5, 2016

in both

a and

b.

FIG. 2. CaBP localization in chick duodenal mucosa. Antiserum applied, specimen source and results were as follows: A) NRS, vitamin D-deflcient chick, no specific stain; B) anti-CaBP, vitamin D-deficient chick, no specific stain; C) NRS: D3-treated chick, stain on edge of viii is non-specific; D) anti-CaBP, D3-treated chick, dark stain indicates presence of CaBP. Magnification is x102.

631

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MORRISSEY

632

ET

AL.

-I’

S FIG. 3. Localization of CaBP and results were as follows: A) deficient chick, no specific stain; dark stain indicates presence of stained lateral cell borders (LCB)

Wasserman

(15)

who

in cells along the intestinal villus of chick duodenum. Antiserum, tissue source NRS, vitamin D-deflcient chick, no specific stain; B) anti-CaBP, vitamin DC) NRS, D3-treated chick, no specific stain; D) anti-CaBP, D3-treated chick, CaBP. Vacuoles indicated by arrows are unstained goblet cells. Some densely are also identified. Magnification is x998.

reported

localization

of

CaBP in goblet cells and brush borders of chick intestine 72 hr after administration of 32.5 nmol of D, to vitamin D-deficient chicks. Thus, the differences observed between our results (8) and those of Taylor and Wasserman (15) do not appear D

to be related

administered.

to dose Further

embedment specificity

methodology will be required

and

form

studies

of

of vitamin fixation

and

as well as antibody to resolve these dif-

ferences.

The noted

major

differences

between

specimens

with D:, (32.5 nmol) and were as follows: a) CaBP from nuclei of absorptive viius after 1,25(OH)2D3 present in the nuclei to as

cytoplasm

ment. membranes

b)

in

CaBP

absorptive

in

CaBP

from

basal

chicks

treated

1,25(OH)2D3 (2.5 nmol) was notably absent cells located along the treatment (8), but was about the same degree cells

accumulation

in the

localization

portion

after D:3 treatalong the lateral of absorptive

cells (Fig. The than

was more 3, D) than

prominent after after 1,25(OH)2D3

D3 dose was approximately the 1,25(OH)2D3 dose.

D3

treatment treatment.

12.5-fold Therefore,

greater with the

D3 dose, we speculate that a considerably longer duration of effect may occur. Thus, our results may suggest that CaBP localization in nuclei and along the to the amount

lateral and

cell borders may duration of vitamin

able to the cells. The implications localization

of

of cytoplasmic CaBP

been discussed earlier localization of CaBP

in

be related D availand

absorptive

nuclear

cells

(8). Briefly, is more likely

have

intracellular than extra-

cellular localization because the apparent intrinsic association constant of CaBP for calcium (2.5 x 106M’) (2) approximates the normal range of intracellular calcium concentration. The occurrence of CaBP in the nuclei of intestinal absorptive tein

cells

may

reflect

synthesizing

Downloaded from jhc.sagepub.com by guest on June 5, 2016

either

ribosomes

presence

the

in

the

of pronuclei

of

VITAMIN

D

DEPENDENT

CALCIUM

BINDING

633

PROTEIN

FIG. 4. Localization of CaBP in chick duodenal crypts. Antiserum, tissue source and results were as follows: A) NRS, vitamin D-deficient chick, no specific stain; B) anti-CaBP, vitamin D-deficient chick, no specific stain; C) NRS, D3-treated chick, no specific stain; D) anti-CaBP, D3-treated chick, dark stain indicates presence of CaBP. Vacuoles are unstained goblet cells. Arrows indicate CaBP-containing nuclei. Magnification is x777.

young

cells

or suggest

that

the regulation of nuclear and RNA synthesis. Implications CaBP lateral

of

in apical borders

1,25(OH)2D3

is involved

increased

such concentration

treatment

observations: initial increase absorptive (9).

b) CaBP

in

as DNA

and basal portions and along of the cell must be considered

light of several other not required for the permeability of be essential intracellular treatment

CaBP

processes

of the in

a) CaBP is in calcium cells after appears

to

to the cell’s ability to maintain a low calcium content after 1,25(OH)2D3 (9). c) In vitro mitochondrial studies

demonstrated

that

accumulation logic studies

in mitochondria suggested that

occurrence dna3. e) concentrated

CaBP

may

of mineralized Mitochondna in the

cell. The mechanism bility of intestinal

prevent (3). CaBP

granules of

basal

calcium d)

Morphoprevented

in mitochon-

absorptive and apical

cells portions

are of

the

by lation

through which cells to calcium

1,25(OH)2D3 of excess Federation

is not intracellular Proceedings,

Downloaded from jhc.sagepub.com by guest on June 5, 2016

yet

the permeais increased

understood. calcium 36:1097,

Accumuunder 1977.

these

634

MORRISSEY

conditions

may

be prevented

by several

nisms, including enhan#{235}ed removal from the cell, inhibition of mitochondnal of

calcium,

changes. into cells while

and

differential

hanced.

inflow

The

and

distribution

of CaBP

a possible role to minimize

of intracellular calcium. Micapable of serving as a relatively “storage” site for calcium.

CaBP was found in the pied by the mitochondria.

regions of the cell However, optical

olution

to determine

was

inadequate

cytoplasmic determination dna, lular

en-

location of its

intracellular organelles.

of CaBP relationship

membranes,

the and to

occuresprecise

assistance

of Joseph

Herman

LITERATURE

for

4. Hayat

MA: microscopy.

(vitamin

D:).

Aust

J Exp

encour-

Cellular

lo-

protein

in

Biol

Piazolo P, Hotz M: Calcium-binding cosa of uremic

14.

Taylor

ney Int 8:110, croscopic intestinal tein. In

15.

and techniques applications.

TJ,

Fluorescent

and antibody

of the 1975

Empson,

calciumlaying

Jr. RN,

hen.

J.

Lufkin

II. protein.

Jr. RN, Zolock DT, Bikie response to la,25-dihyCellular Biochim

I. RNA

13.

Med

1975

Principles Biological

J,

protein: Its cellular localizakidney and pancreas (38742). Proc Med 149:56, 1975

droxycholecalciferol: calcium binding

2. Bredderman PJ, Wasserman RH: Chemical composition, affinity for calcium, and some related properties of vitamin D dependent calcium-binding protein. Biochemistry 13:1687, 1974 3. Hamilton JW, Holdsworth ES: The role of calcium binding protein in the mechanism of action of cholecalciferol Sci 53:469,

Stroder of calciumby immunoflu-

P,

D-dependent

oviduct 23:111,

Soc Exp Biol 8. Morrissey RL, Empson, DD, Bucci TJ: Intestinal

CITED

1. Arnold BM, Kovacs K, Murray TM: calization of intestinal calcium-binding pig duodenum. Digestion 14:77, 1976

p

localization Biophys

of Acta

polymerase,

alkaline

phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation. Biochim Biophys Acta 538:23, 1978 10. Nakane PK, Kawaoi A: Peroxidase-labeled antibody: A new method of conjugation. J Histochem Cytochem 22:1084, 1974 11. Nakane PK, Pierce, Jr. GB: Enzyme-labeled antibody for the light and electron microscopic localization of tissue antigens. J Cell Biol 33:307, 1967 12. Ouchterlony 0: Diffusion-in-gel methods for immunological analysis. Prog Allergy 6:30, 1962

Bruzynski,

their

RL, Bucci

1970,

Calcium-binding in jejunum,

droxycholecalciferol:

Elwyn Chadwick and Gary Herr, and the clerical assistance of Mrs. Anne Twitchell is gratefully acknowledged. We also thank Howerde E. Sauberlich and Robert H. agement and counsel.

RH:

vitamin

protein in the Cytochem

7. Morrissey

intracel-

ACKNOWLEDGMENTS

The technical

binding Histochem

of the

York,

538:34, 1978 9. Morrissey RL, Zolock DT, Bikle DD, Empson, Jr. RN, Bucci TJ: Intestinal response to la,25-dihy-

precluded mitochon-

or other

New

vitamin-D-deficient 15:875, 1974

L, Wasserman

localization

EG: tion

Co.,

in normal, subjects. Gut

6. Lippiello

demonstrated

in this study was consistent with in each of these three mechanisms the accumulation tochondria are large intracellular

are

Reinhold

K, Federlin K, Piazolo R, Franz HE: Localization protein in intestinal tissue

orescence uraemic

of calcium inhibited

outflow

Nostrand

336 5. Helmke Jeschke binding

permeability

basalar

AL. Van

mechaof calcium uptake

In the latter case, “leakage” via lateral cell borders maybe

apical

ET

of electron Volume 1.

J, Helmke K, Franz protein in the patients and normal

HE, Schleyer duodenal musubjects. Kid-

1975

JE: Light and electron miimmunoperoxidase localization of chick vitamin D-induced calcium-binding proVitamin D, Biochemical, Chemical and

AN,

McIntosh

Clinical

Aspects

Related

Edited

by AW

Norman,

to

Calcium

K Schaefer,

Metabolism,

JW

Coburn,

HF DeLuca, D Fraser, HG Grigoleit, Dv Herrath, Walter de Gruyter, Berlin and New York. 1977, p 303-312 Taylor AN, Wasserman RH: Immunofluorescent localization of vitamin D-dependent calcium-binding protein. J Histochem Cytochem 18:107, 1970

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