Differential effect of sodium butyrate on cyclic AMP phosphodiesterase activities in butyrate sensitive and resistant mastocytoma cells

Vol. 138, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1030-1036

August 14, 1986

DIFFERENTIAL EFFECT OF SODIUM BUTYRATE ON CYCLIC AMP PHOSPHODIESTERASE ACTIVITIES IN BUTYRATE SENSITIVE AND RESISTANT MASTOCYTOMA CELLS Yoichi Mori¼ Yumiko Tanigaki~

Atsuko Yamaguchi I

and Hitoshi Akedo 2

i Department of Cell Biology, 2 Department of Biochemistry, Research Institute, The Center for Adult Diseases,Osaka, 1-3-3 Higashinari,Osaka 537,Japan Received May 17, 1986

SUMMARY:The effect of sodium butyrate on the intracellular cyclic AMP levels and the activities of cyclic AMP-regulating enzymes were examined in two types of mastocytoma p-815 cells in culture: one type (S cell) was sensitive and the other (R cell) was resistant to the induction of differentiation by sodium butyrate. In the presence of sodium butyrate, adenylate cyclase activity increased in both S and R cells to the same degree, whereas the level of cyclic AMP was elevated only in S c e l l s . Cyclic AMP phosphodiesterase activity increased in R cells but not in S cells. Cyclic AMP phosphodiesterase activities of two cell populations differed in their response to sodium butyrate and they seem to have an important role in regulating cellulr level of cyclic AMP that might be an importan t factor in controlling cell differentiation. ©1986AcademicPress, Inc.

Sodium butyrate at millimolar concentrations has been reported to change morphology,

growth rate and gene expression in

a variety of mammalian cells in culture reported effects of sodium butyrate,

(1-8). Of all the

the most remarkable action

of the agent is the induction of differentiation cells

of cultured

(5-8).Although the mechanism of action remains unclear,

sodium butyrate has been reported to increase intracellular cyclic AMP levels

(2). Changes induced by sodium butyrate have

been shown to be similar to those produced by other agents which increase the cellular level of cyclic AMP or by analogs of cyclic AMP (2). Previously we reported that sodium butyrate inhibited cell proliferation and induced differentiation of mastocytoma p-815 cells accompanied with metachromatic granulopoiesis and an 0006-291X/86 $1.50

Copyright © 1986 ~ A c a ~ m ~ Pre~, ~c. Aft r~hts ~ r~roduction in a ~ ~ r m reserved.

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increase in intracellular histamine,

serotonin and heparin

(6,7).

In this study we isolated butyrate-resistant mastocytoma cells (R cell)

and investigated the effect of sodium butyrate on cyclic

AMP and cyclic AMP-regulating enzymes in both S and R cells in relation to differentiation. Materials and Methods Cells: Mastocytoma p-815 cells were cultured as previously reported (6). S cells were cloned and maintained in suspension culture. R cells were cloned from S cells which had been cultured for 6 months in the presence of 2 mM sodium butyrate. The growth rate of cells was initially inhibited but after 4 months cells started to proliferate regardless of the presence or absence of 2 mM sodium butyrate. Determination of histamine and serotonin: The cellular histamine and serotonin contents were determined by the methods described by Shore et al (9) and by Devis (10),respectively. Determination of cellular cyclic AMP: Cyclic AMP was extracted from 5 X 106 cells with cold 6~ trichloroacetic acid. The extract was shaken vigorously 3 times with 5 ml of ether saturated with distilled water to remove trichloroacetic acid and the aqueous solution was subjected to cyclic AMP assay by using a commercially available cycilc AMP assay kit (Yamasa Shoyu,Tokyo) based on the method of Honma et al (ii). Determination of adenylate cyclase: Adenylate^~yclase activity ~ by th-e ~ of c o ~ s i o n of [~_3zP]ATP to [3 p] cyclic AMP using the method of Krishna et al (12). Determination of cyclic AMP phosphodiesterase: Cyclic AMP phosphodiesterase activity of cell~ was measured by the rate of conversion of [3H] cyclic AMP to [ZH] 5'-AMP using the method of Mark and Raab (13). The low Km activity was assayed with the substrate concentration at 1 ~ M and the high at 400 ~M.

Results and Discussion When S cells were cultured in the presence of 2 mM sodium butyrate, entiation,

the optimal dose for the induction of cell defferthe growth rate of cells was inhibited, whereas that

of R cells was almost unchanged

(Fig.l).

The doubling time of S

cells prolonged to 49 hours from 24 hours. As shown in

Table i,

S and R cells contained the same quantity of histamine and serotonin before adding sodium butyrate. When these cells were cultured for 4 days in the presence of 2 mM sodium butyrate,

the

cellular contents of histamine and serotonin in S cells increased 137 times and 7.6 times, respectively, 1031

but those

in R cells

Vol. 138, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10 5 E

\ _m J/ /

~0

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:E 1

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I I K 1 2 3 days of cell culture

1 4

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0

I 12

I 24

I 48

I

7 2 hrs

F i ~ . l E f f e c t of s o d i u m b u t y r a t e on the g r o w t h of S and R cells. S and R cells (4 X 104 cells) w e r e c u l t u r e d in the p r e s e n c e and a b s e n c e of 2 m M s o d i u m b u t y r a t e in 35 m m p l a s t i c p e t r i d i s h e s and cell n u m b e r s w e r e c o u n t e d in t r i p l i c a t e by a C o u l t e r Counter. e - - m e S cells;B---41 R cells; o o S cells w i t h s o d i u m butyrate; D ~ R c e l l s w i t h s o d i u m butyrate. Fi9.2 C h a n g e s of i n t r a c e l l u l a r c y c l i c A M P in S and R c e l l s c u l t u r e d in the p r e s e n c e and a b s e n c e of 2 m M s o d i u m b u t y r a t e . o o S cells; D----D R cells; • ~ S cells w i t h s o d i u m butyrate; R c e l l s w i t h s o d i u m butyrate.

increased only slightly.

In S cells over 2 mM concentrations of

sodium butyrate not only inhibited cell growth but also caused cell death. Whereas,

in R cells the increase of concentration of

this agent up to 8 mM did not result in killing of cells but in an increase of the cellular amine contents. Cyclic AMP levels of S and R cells at 12 hours after subculture were 2.0 and 1.2 pmoles/106cells,

Table

respectively.

They increased

i. E f f e c t of s o d i u m b u t y r a t e on the a m i n e c o n t e n t s S and R c e l l s

butyrate (mM)

histamine (~g/107cells)

ratio

serotonin (~g/107cells)

in

ratio

S cell 0 2

0.02 2.75

1 137

0.02 1.51

1.0 7.6

R cell 0 2 4 8

0.02 0.23 0.42 0.80

1 ii 21 40

0.02 0.23 0.50 0.45

1.0 i.i 2.5 2.2

S and R c e l l s w e r e c u l t u r e d for 4 days in the p r e s e n c e and a b s e n c e o f s o d i u m b u t y r a t e . H i s t a m i n e and s e r o t o n i n in the cells w e r e m e a s u r e d .

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 2. Effect of sodium butyrate on adenylate cyclase activity in S and R cells butyrate (mM)

adenylate cyclase pmoles/min/106cells

S cell 0 2 R cell 0 2

ratio

15 42

1.0 2.8

32 85

1.0 2.7

S and R cells were cultured in the presence and absence of sodium butyrate for 24 hours.

slightly during growth.

In the presence of 2 mM sodium butyrate,

the cellular level of cyclic AMP in S cells increased to 5.2 pmoles/106cells within 24 hours cells

, whereas it did not change in R

(Fig.2). Effect of sodium butyrate on

and cyclic AMP phosphodiesterase, AMP level, were examined.

adenylate cyclase

the enzymes regulating cyclic

Basal adenylate cyclase activity was 15

and 32 pmoles/min/106cells

in S and R cells, respectively.

Adenylate cyclase activity in both S and R cells increased almost 3 times during 24 hours after the addition of 2 mM sodium butyrate

(Table 2).

S and R cells contained two kinetic forms

of cyclic AMP phosphodiesterase, Vmax,

71.4 pmoles/min/106cells)

Vmax, 25.0 pmoles/min/106cells) affinity

(Km,

affinity

(Km, 250 ~M;

R cells

16.6 ~M; Vmax, Vmax,

a high affinity

(Km, ii.0 ~M;

and a low affinity

(Km, 125.0 ~M;

enzymes in S cells and a high

13.3 pmoles/min/106cells) 66.6 pmoles/min/106cells)

and a low enzymes in

(Fig.3). Both enzyme activities in S cells did not change

in the presence of 2 mM sodium butyrafie.

In R cells the activity

of low affinity enzyme increased to a level almost

1.9 times that

of the control at 24 hours after the addition of sodium butyrate (Table 3), but the activity of high affinity enzyme was not affected. R cells were derived from the culture of S ceils in the presence of sodium butyrate for 6 months. 1033

Although the molecular

Vol. 138, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

o.o'~

i

m

//

//

// / // i/I / / ~,//

| 0.I

o

I O.2 cyclic A M P

0.4

.3

Fig.3 Lineweaver-Burk plots of cyclic AMP phosphodiesterase activity in S and R cells. Michaelis constants (Km) and maximum velocities (Vmax) were determined from the linear portions of the plots, o o S cells; • = R cells. mechanism butyrate

which

Sodium AMP

to

study

butyrate

the

cyclic

cell

regulation

(2).

of c e l l s

lines may

of c e l l

reported

cyclase

activity AMP

sensitivity both

has been

and adenylate

phosphodiesterase affinity

the

is n o t u n d e r s t o o d ,

opportunity

cyclic

controls

sodium

offer

an

differentiation.

to i n c r e a s e

activity

to

but

Here

we r e p o r t

phosphodiesterase

activity

intracellular

not

cyclic

that

the

AMP

low

increased

in R

Table 3. Effect of sodium butyrate on cyclic AMP phosphodiesterase activity in S and R cells butyrate (mM)

cyclic AMP phosphodiesterase pmoles 5'AMP/min/106 cells high concentration low concentration substrate substrate

S cell 0 2

122 143

3.2 3.9

R cell 0 2

342 662

6.4 6.0

S and R cells were cultured for 24 hours in the presence and absence of sodium butyrate. The treated cells were harvested and assayed for enzyme activity at substrate concentrations of 400 ~ M and 1 ~M. 1034

Vol. 138, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

~o

"E=- m 0

a

Q,

I

0

I

I

12

I

I

24

I

48hrs

F i 9 . 4 C h a n g e s of c y c l i c A M P p h o s p h o d i e s t e r a s e a c t i v i t y in S and R c e l l s c u l t u r e d in the p r e s e n c e and a b s e n c e of 2 m M s o d i u m b u t y r a t e . D---[] S cells; o o R cells; H S cells with sodium butyrate; • • R cells with sodium butyrate.

cells but not in S cells.

Although

cyclic AMP phosphodiesterase destruction

it is not clear which kind of

is actually functioning

of cyclic AMP in the cells,

for

the invariability

of the

level of cyclic AMP in R cells appears to be at least the consequence

of elevation of cyclic AMP phosphodiesterase.

reported that 3-isobutyl-l-methylxanthine, AMP phosphodiesterase, in S and R cells cells

(14).

increased cellular

an inhibitor of cyclic cyclic AMP levels both

and induced differentiation

An important

factor limiting

We also

of mastocytoma

the magnitude

and

duration of action of cyclic AMP is the rate of destruction the nucleotide

by cyclic AMP phosphodiesterase.

of

The resulting

high level of cellular cyclic AMP appears to be linked with the differentiation

of mastocytoma

cells by sodium butyrate(Fig.

4).

References i. 2. 3. 4. 5. 6.

Wright,J.A. (1973) Exp. Cell Res. 78,456-460. Prasad,K.N.,and Sinha,P.K. (1976) In Vitro 12,125-131. Storrie,B.,Puck,T.T.,and Wenger,L. (1978) J.Cell Physiol.94,69-76. Riggs,M.G.,Whittaker,R.G.,Neumann,J.R.,and Ingram,V.M. (1977) Nature 268,462-464. Leder,A.,and Leder,P. (1975) Cell 5,319-322. Mori,Y.,Akedo,H.,Tanaka,K.,Tanigaki,Y.,and Okada,M. (1979) Exp. Cell Res.l18,15-22.

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7. 8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mori,Y.,Akedo,H.,Tanigaki,Y.,Tanaka,K.,Okada,M.,and Nakamura,N. (1980) Exp. Cell Res. 127,465-470. Tanaka,H.,Mori,Y.,and Akedo,H. (1982) Cell Biol. Int.Rep.6,8590. Shore,P.A.,Burkhalter,A.,and Cohn,V.H.jr. (1959) J.Pharmacol. Exp.Therap. 127,182-186. Davis,R.B. (1959) J.Lab. Clin.Med. 54,344-351. Honma,M.,Satoh,T.,Takezawa,J.,and Ui,M. (1977) Biochem. Med. 18,257-273. Krishna,C.,Weiss,B.,and Brodie,B.B. (1968) J.Pharmacol.Exp. Therap. 163,379-385. Mark,F.,and Raab,I. (1974) Biochim.Biophys.Acta 334,368-377. Mori,Y.Tanigaki,Y.,Yamaguchi,A.,Okada,M.,and Akedo,H. (1981) Proc. Jap. Cancer Assoc. 40,159.

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