Vol. 24. No. 9, pp.

THE JOURNAL OF HI5T0cHEMI5TRY AND CYTOCHEMISTRY Copyright © 1976 by The Histochemical Society. Inc.

A

COMPARISON

OF

ATP-DEGRADING

RAT

INCISOR

GOSTA

Laboratory

of Oral

In

active

for

odontoblasts

calcification,

two

phosphate

(ATP)

exist.

phenylimidazo

(2.1-b)

dihydroimidazo alkaline

(2.1-b)

phosphatase

dependent Ca2-ATPase

diphosphate,

phosphate

were

concerned

with

In a recent

study

in

tissue

these phosphatases activated ATPase

of Ca2

or Mg2

specific

the

the forming

activities

in

formed. have

of these rat

Preliminary been

presented MATERIALS

One

is APase, the other that is unaffected

inhibitors R 8231 and Levamisole. In ent investigation a further characterization comparison

of

(11).

two

ATP

incisor

degrading

odontoblasts

results earlier AND

of this

of

a Ca2 by the the

presand

by

per-

investigation

(9). METHODS

Odontoblast-predentin tissue was dissected out from the maxillary incisors of male Sprague Dawley rats (body weight 200 g) as described earlier (10). The This investigation was supported by the Swedish Medical Research Council and the Faculty of Odontology, University of Gothenburg. 2 Author to whom reprint requests should be sent. The abbreviations used are: APase, nonspecific alkaline phosphatase (EC 3.1.3.1.);AMP, adenosine monophosphate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; GTP, guanosine triphosphate; ITP, inosine triphosphate; PP,ase, inorganic pyrophosphatase; EDTA, ethylenediamine tetraacetic acid; EGTA, ethyleneglycol-bis-($-amino ethyl ether) N,N’-tetra acetic acid; Levamisole (1-tetramisole), (±)-2,3,5,6,-tetrahydro-6-phenylimidazo (2.1-b) thiazole hydrochloride; R 8231, bromophenyl)-5.6-dihydroimidazo (2.1-b)

was

37#{176}Cfor

enzyme is

ions

mono-

with

named ions.

The

pH

R against

is

optimum

8231,

whereas

adenosine

the

Ca2

mono-

-ATPase

mineralization

transferred

to

of

ouabain,

ATP,

and that

to the

nonspecific

Ca2-ATPase,

is

front.

a teflon-glass

homogenizer

and homogenized in 500 izl 0.1 M glycine-NaOH buffer. The reaction was performed in microtubes, containing 105 Ml 0.1 M glycine-NaOH buffer with a total Na2-ATP concentration of 3 mM. The pH of the mixture was 9.8 except when optimal pH was studied. In this case pH was varied between 7.4 and 11.8. The reaction mixture was preincubated in a water bath at

two

degradation cells

identical

triphosphate

of Ca2

biologic

(± )-6(m-bromophenyl)-5.6-

found

speculated

for

adenosine

Levamisole,

be

tissue

for

of at least

by

inosine may

system

enzyme,

was

3, 1976

(± )-2,3,5,6,-tetrahydro-6-

by these

unaffected

transport

inhibitors

presence

other

is activated

It

model degrading

and

activity

rate.

transmembranous

using

and

a of

is probably

of the

Maximal

lower

as

May

form

1-tetramisole,

and

triphosphate,

at

of Goteborg,

revised

capable

8231)

is

ions.

in

used

by

activity

Ca2-ATPase K

University

(Levamisol) (R

The

guanosine

mediating hard

3.1.3.1.).

hydrolysed

we suggested

phosphatases

hydrochloride

and

the

incisor,

be inhibited

can

and

1975,

activities

oxalate

The

Na

adenosine

APase3 ATP

9.8.

red,

rat

thiazole

presence

is

ruthenium

the

thiazole (EC

on the

18,

enzyme

One

LINDE2

of Histology,

December

from

IN

33, G#{243}teborg33, Sweden

S-400

distinguishable

ANDERS

AND

Department

publication

ACTIVITIES

ODONTOBLASTS’

GRANSTROM

Biology,

Fack, Received

ENZYME

1026-1032, 1976 Printed in U.S.A.

20

and

the

Ml of the

enzyme

was

homogenate

started per

tube.

measurements.

To enzyme with

partially activities and

the the of

degrading

APase 8231) activity.

Dr.

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

ATP

activity was

ATP were

incubation

mediated

The

pH

(obtained from optimum

Incubations linearity

medium. degradation

subtracted

mined as described above. up to 5 hr to study the 1026

degrading performed, of 0.1 mM R 8231 M. Borgers, Janssen

in the

Ca2-ATPase R

the tests

addition

by

Belgium)

obtain

discussed,

the

donated

Pharmaceutica, To

characterize the following

without

(generously

ATP

oxalate.

reaction

tissue

The incubation was performed at 37#{176}Cfor 2 hr. In the blanks the homogenate was exchanged for 20 Ml buffer. The reaction was stopped by cooling in ice and adding 200 Ml 10% trichloroacetic acid containing 5 mM CuSO, followed by the addition of 300 Ml 1% ammonium molybdate in 0.575 M H2SO4 containing 80 mg FeSO4 per ml. The liberated orthophosphate was assayed after 30 mm at 700 nm in a standard spectrophotometer equipped with a house for micro cells. The optical absorption of 20 MI homogenate diluted to 125 zl with buffer and treated with trichloroacetic acid and molybdate was subtracted from all

presence (±)-6(mthiazole

5 mm

adding

in the the was

total deter-

were followed of the enzyme

ATPS

reactions.

ACTIVITIES

IN

effects of increasing amounts of Mg2, K ions in the incubation medium were studied, and increasing amounts of EDTA and EGTA were also tested. The ability of the odontoblast homogenate to degrade other substrates such as ADP, AMP, ITP and GTP was investigated. Ca2,

The

Na

and

The tions

results

clearly

of two

different

responsible

for

inhibitable probably activity

in the

of these

Ca2-ATPase

will

be evident

from

experiments the results

from

One

is

or R 8231 and is most APase. The remaining

presence

named

All

of ATP.

inhibitors

due the

has

to reasons

that

following.

material

were performed in triplicate these were virtually identi-

out

was

Ca2-ATPase: ated phosphate

1.90 per

mediated

degradation:

Mmol DNA.

liberated The DNA

dentine ml

ATP

(n

±

(mean

0.45 (n mm and

phosphate content

homogenates

values

±

9) Mmole mg DNA;

per of the 24.7

2.62 (n

±

= 9)

mm and odontoblast-pre±

6.5

g

mg

Effect

of

1). The

did

pH:

Both

APase

mediated showed

DNA! ATP

maximal

1. Influence

of

pH

on

ATP-splitting

enzyme

of Ca2 found

degradation

of

of Ca2

i.e.,

3 mM.

inhibited When

The at

Mg2

centrations lar

ions.

obtained all ions

concentration

and

were

shown

the

Na

presence of Mg2’ what higher than

activity.

NaOH buffer. (a) Ca2 -ATPase, 3 mM Ca2 added to the medium mediated ATP hydrolysis, no Ca2 added, (d) APase mediated medium. P,, inorganic phosphate.

i’i

4).

Ca2

on added

activa-

ratio

other at

4). con-

and

APase

at equimo-

substrate

(Fig.

of Ca2-ATPase

ions was found in the presence

was

different

a maximum the

of 1:1,

hand (Fig.

Ca2-ATPase with

to be

pH 9.8 by the

Maximal

concentrations

were

Ca2-

medium. In buffer was

ATP the

the

or

Tris-HC1 buffer was activated

APase

activation

for

APase of

had 2).

to 6 mM

Both

a Ca2:

with

maximal

(Fig.

of ATP

(Fig.

at

both activation

times

linearity

concentration in the incubation these tests the 0.1 M glycine-NaOH

was

a of

incubation

the Ca2-ATPase at least 5 hr

independent

tion

with

degradation

with

time

for the

also

concentration

the

APase activity. Effect of cations:

ib FIG.

substrate

increase

ATPase

was

The

linear

in the

addition

pH

time:

was

the

not

both

concentrations

any

optimal

with

Changing

9.8,

enzyme activity ATP concentration.

by APase

The and Ca2-ATPase

same

total ATP-splitting 2 mM or 5 mM

revealed

= 9).

degradation

without

ATP

(Fig.

addition SD):

liberAPase

=

5.98

was

and

with

pH

equimolar

exchanged for 0.1 M (Fig. 3). The Ca2-ATPase

total activities of the two ATP degrading activities in the odontoblast-predentine dissected

in

completely

cal. The enzyme

at

of Ca2

up to 45 mm. In contrast, a linear relationship for

sugges-

in odontoblasts

degradation

been

and

previous

enzymes

the

activities

presence

ATP

support

by Levamisole identical with

ATP-splitting

Linearity

RESULTS

1027

ODONTOBLASTS

to be of Ca2

5).

in the someions.

12

The enzymes were assayed in 0.1 M glycine(b) Ca2-ATPase, no Ca#{176} added (c) APase ATP hydrolysis, 3 mM Ca2 added to the

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

1028

GRANSTR#{246}M

AND

LINDE

E a.

incubation

Fic.

2. Changes

in reaction hydrolysis (b) ATP hydrolysis Ca2 -ATPase activity is linear

velocity mediated for at least

time

of ATP hydrolysis by Ca2-ATPase 5 hr whereas APase

(ii)

dependent (c) ATP mediated

on incubation time. (1) total ATP hydrolysis mediated by APase. The ATP hydrolysis is linear for at most 45

mm.

0

E a. iO(

-.----.------------u...----.

-/F’

c___

d

-IF

Ca2

in a activity

3:1,

and

concentration concentration ties

was with

were

Mg2 Effect

Mg2

1:1 and was obtained

virtually

ions

were

added

together

1:3 proportion, maximal when the combined ion 3 mM, i.e., the substrate. equal

to that

at equimolar These activiobtained

with

alone. of ATP

concentration

and

(mMJ

oncentraton

FIG. 3. Effect of different concentrations of monovalent effect on Ca2-ATPase (b) K effect on APase mediated Na effect on APase mediated ATP hydrolysis. Monovalent AP-ase. Note that the abscissa is logarithmic.

When

i#{246}o’

io on

substrate

cations on ATP-splitting ATP-hydrolysis (c) Na cations have virtually

specificity: degrade ADP, (Fig. mM

The other AMP,

enzyme activities. (a) K effect on Ca2-ATPase (d) no effect on Ca2 -ATPase or

ability

of the

phosphates GTP

and

Ca2-ATPase

than ITP,

6). The highest activity Ca2 was found with

ATP, was

in the ATP.

although

the

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

activity

was

as

investigated presence of 3 An optimal

activity was reached with a Ca2: ratio of 1:1. The same result was AMP,

to such

70%

substrate found with less.

ADP,

ATPS

ACTIVITIES

IN

1029

ODONTOBLASTS

0

a a. V

a4 a a

FIG. 4. Effect of different concentrations of Ca2 on ATP-hydrolysis. (a) APase mediated ATP degradation (b) Ca2-ATPase. The Ca2-ATPase is activated by Ca2 ions whereas APase is inhibited. Note that the abscissa is logarithmic.

0

E c

a.

2-

1

Mg

Ftc. 5. Activation of ATP hydrolysis mediated ATP hydrolysis. Both enzymes abscissa is logarithmic. GTP

and

ATPase any

ITP

were

although

optimal substrate Effect of EDTA

ATPase and was completely mM

EDTA

also at

degraded

a lower

or 1.25

activities could be either Ca2 or Mg2

mM

EGTA,

and

concentrations of Mg2 ions. (a) Ca2-ATPase by Mg2 ions with a maximum at 3 mM.

Ca2-

Both

DISCUSSION

Ca2-

ATP degradation the addition of 0.3 but

the

(b) APase Note that the

without In an

concentration. and EGTA:

APase mediated inhibited by

by increasing are activated

by the

rate

10 (mM)

concentration

original

restored by the addition ions (Figs. 7 and 8).

engaged of

earlier

study

(7)

it was

shown

that

the

APase isoenzymes present in bone, calcifying cartilage, odontoblasts and the enamel organ have several properties in common. The APases tissues Similarly

in thus

the seem

PP1ase

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

formation to

be

activities

of the

different

same

hard

ioenzymes.

in odontoblasts

and

1030

GRANSTR#{246}M

AND

substrate

FIG.

ATP,

LINDE

concentration

mM

Ca2-ATPase activity with different nucleotides as substrates and in the presence (b) ADP, (c) GTP, (d) ITP, (e) AMP. Maximal activity was reached with ATP. Note

of 3 mM Ca2. (a) that the abscissa is

6.

logarithmic.

C.

15,

100 ion

Ftc.

in the presence of 0.3 mM Mg2 ions added to APase mediated ATP hydrolysis, (d) Ca2 ions added to APase mediated ATP hydrolysis. At 3 mM concentrations of divalent cations added, the original enzyme activities were restored. Note that the abscissa is logarithmic. The level of the original activities of Ca2-ATPase and APase are given to the right in the figure. EDTA.

7. Reactivation (a)

Mg2

of ATP

ions

added

hydrolysing

concentration

to Ca#{176}-ATPase,

the two major zones of the rat organ were shown to have very ties (6, 8). Levamisole were recently shown noncompetetive

and its to be very

inhibitors

These compounds and PP1ase in active

of

completely odontoblasts

incisor similar analogue potent, APase inhibit (11)

enzyme

(b)

Ca2

activity

ions

added

enamel proper-

cells

R 8231 specific,

tity

(1,

19).

APase and the

with

divalent

to Ca2

of the

support

cations

-ATPase,

enamel

previous between

(c)

organ suggestions

APase

and

(6).

Our (5,

PP1ase

calcification loci. In histochemical and biochemical phosphatase activities (11, 12) the ing activity

in osteoblasts,

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

results

21)

odontoblasts

thus

of an at

ATP

iden-

different studies of degradand

the

ATPS

ACTIVITIES

IN

1031

ODONTOBLASTS

ATPsc

too on

concentration

mM)

FIG. 8. Reactivation of ATP-hydrolysis with divalent cations in the presence of 0.25 mM EGTA. (a) Mg2 ions added to Ca2-ATPase, (b) Ca2 ions added to Ca2-ATPase, (c) Mg2 ions added to APase mediated ATP-hydrolysis, (d) Ca2 ions added to APase mediated ATP-hydrolysis. The original enzyme activities shown to the right in the figure could be restored by the addition of divalent cations. Note that the abscissa is

logarithmic. of the

cells

stratum

intermedium

of the

enamel

Mg2

in the presence the activity in

organ was somewhat decreased of levamisole or R 8231, while

striated muscle fibers and cells of blood vessel walls was unaffected. It was suggested that the ATP

degradation

mediated

hard

in

by two

tissue-forming

enzymes,

activated ATPase (11). The data presented here previous rate,

Ca2

activated

recorded

when

ity with

time:

relation ATP

ATPase

Ca2-ATPase

hydrolysis

decreased exactly

the

APase phosphate

same

showed

by

short

We

time

of

already found

linearity

could

be

stimulated

ions, while

activated

only

EDTA

is

by

in contrast,

for

by

Mg2.

known

to

either

Ca2

the APase

chelate

several

ATPase Ca2

and totally. chelator

the

APase

EGTA with

Granstr#{246}m G Linde

mediated

has

been

relatively

ATP

or was

little

A: unpublished

affinity data.

be

enzyme

the

incubation

inhibition Ca2

pattern

or

(7) or inorganic

substrate

may

EDTA

and

phate

is used

be

by

the com-

(8)

Our

to Ca2

as

inhibited

if p-nitrophenyl

EGTA

as substrate.4

the sensitivity

divalent erythrocytes

results

or Mg2

by phos-

suggest for the

is similar in magnitude, but that gives a slightly higher activation. is observed with other (15). However, Ca2, vary considerably in their

cation requirements (2, 13, 16). In it has been shown that for the localized Mg2

Ca2,

is required

Mg2 for

activated

Ca2

transport,

but Mg2 itself is not transported (16). The Ca2-ATPase activity in odontoblasts was unaffected by ruthenium red and by oua-

from for

be

pyrophosphate

completely

(12). This

shows

that the Ca2-ATPase

different from Ca2 activated ATPase rocytes (20) and Na, K activated a

as

to

Mg2 could

activities

phosphate

bain

hydrolto

adding

the

to

same

restored in both cases. We have earlier that APase assayed with p-nitrophenyl

ATPase,

cations

reported

medium, pletely shown

membrane

including Ca2 and Mg2 (3). The addition of EDTA to the medium inhibited both the Ca2ysis

After

EGTA the

Such Mg2 preference Ca2, Mg2-ATPases Mg2-ATPases may

the

have

EDTA.

Ca2-ATPase Mg2 always

a linear

whereas

Adding

showed

that

was linear-

APase

45 mm.

hard

incubated at 37#{176}C with p-nitrophenyl as substrate.4 The Ca2-ATPase ac-

tivity Mg2

after

in

difference systems

still

mediated

strongly

the

of a sepa-

activity

after 5 hr,

of hydrolysis

is

a Ca2

support

existence

cells. A marked investigating the

tissue-forming

and

further

of the

suggestion

cells

APase

(17).

medium

other

substrate Although be

tissues for

the

(4). ATP odontoblast

other nucleotide

utilized, the

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

degree

is the

is

in erythATPases preferred

Ca2-ATPase.

phosphates of hydrolysis

may

also

is much

GRANSTR#{246}M

1032 lower

than

ATPase

of

which any

that the

of

red

ATP. blood

does not hydrolyse

strate

specificity

Ca2,

Mg2-ATPases

is

cases,

ATP

The optimal suggest that the preferred either Mg2

common

substrate

to 18).

to

of sub-

many

in

at pH

all

8.

ATP

found strong indicais different from

degradation.

The

it is thus

speculated

9. Granstrbm

the transmembranous

Ca211.

that

12.

transport

of Ca2

with ions to

the mineralization front. The subcellular localization of Ca2-ATPase in odontoblasts is at the present being investigated.

13.

14.

ACKNOWLEDGMENTS

The authors are grateful for the given by Mrs. Carina Bostr#{246}m.

assistance

G,

odontoblastic

10.

the

G,

Linde

pyrophosphatase

A:

Determination

in the

of rat by a radiochemical Res 83:365, 1976

to Ca2in Ca2’

is concerned

of odontoblasts

Res in press Granstr#{246}m

ganic

9.8.

1:1 may complex is may thus be

LINDE

odontoblasts from the rat incisor. Acta Odontol Scand 33:143, 1975 7. Granstrbm G, Linde A: A comparative study of alkaline phosphatase in calcifying cartilage, odontoblasts and the enamel organ. Calcif Tissue

other

However,

preferred

we have Ca2-ATPase

and

Ca2-ATPase

(2),

degree

in odontoblasts is very similar in other tissues involved

transport

Ca2

or Ca2.

mediated

ATPase ATPases

low

ratio of cation:substrate a divalent cation-ATP substrate, the cation

In conclusions tions that the APase

this (14,

is the

the

membrane

other triphosphates

extent,

appreciable

Unlike cell

AND

15.

Linde layer

method.

A: ATPase of rat

incisor.

of

odontoblast Scand

inor-

layer J Dent

activity

in the

Determination

with a radiochemical and a colorimetric method. Acta Odontol Scand in press Linde A: A method for the biochemical study of enzymes in the odontoblastic layer during dentinogenesis. Arch Oral Biol 17:1209, 1972 Linde A, Magnusson BC: Inhibition studies of alkaline phosphatases in hard tissue-forming cells.J Histochem Cytochem 23:342, 1975 Magnusson BC, Linde A: Alkaline phosphatase, 5-nucleotidase and ATPase activity in the molar region of the mouse. Histochemie 42:221, 1974 Ma SWY, Shami Y, Messer HH, Copp DH: Properties of Ca#{176}-ATPasefrom the gill of rainbow trout. Biochem Biophys Acta 345:243, 1974 Nakamaru Y, Kosakai M, Konishi K: Some properties of brain microsome adenosine triphosphatases activated by magnesium and calcium. Arch Biochem Biophys 120:15, 1967 Parkinson DK, Radde IC: Properties of a Ca2 and Mg2 -activated ATP-hydrolyzing enzyme in rat kidney cortex. Biochim Biophys Acta 242:238, 1971

LITERATURE

16. Schatzmann ments across

CITED

1. Borgers M: The cytochemical application of new potent inhibitors of alkaline phosphatases. J Histochem Cytochem 21:812, 1973

17.

2.

18.

Cha

YN,

Shin

BC,

Lee

KS:

Active

uptake

of

Ca2-activated Mg2 ATPase in red cell membrane fragments. J Gen Physiol 57:202, 1971 3. Drews GA, Engel WK: Reversal of the ATPase reaction in muscle fibers by EDTA. Nature (Lond) 212:1551, 1966 4. Duggan PF: Some properties of the monovalentcation-stimulated adenosine triphcisphatase of frog sartorius microsomes. Biochem Biophys Acta 99:144, 1965 5. Eaton, RH, Moss DW: Partial purification and some properties of human bone alkaline phosphatase.

Enzymologia

35:31,

1968

6. Fred#{233}n H, Linde A, Magnusson BC: Inorganic pyrophosphatase in isolated enamel organ and

Physiol Schmid titration sium. Shami

HJ, Vincenzi the membrane 201:369, 1969

FF: Calcium moveof human red cells. J

RW, Reilley CN: New complexon for of calcium in the presence of magneAnal Chem 29:264, 1957 Y, Radde IC: The effect of the Ca2/Mg2

concentration ratio on placental (Ca2 -Mg2)ATPase activity. Biochim Biophys Acta 255:675, 1972

19. van Belle H: Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochim Biophys Acta 289:158, 1972 20. Watson EL, Vincenzi FF, David PW: Ca2activated membrane ATPase: selective inhibition by ruthenium red. Biochim Biophys Acta 249:606, 1971 21.

Wbltgens JHM, Bonting SL, Bijvoet OLM: Relationship of inorganic pyrophosphatase and alkaline phosphatase activities in hamster molars. Calcif Tissue Res 5:333, 1970

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