Relationships between spermidine, glucocorticoid and milk proteins in different mammalian species

Vol. 90, No. 4, 1979 October

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

29, 1979

Pages 1131-1135

RELATIONSHIPS

BETWEEN

PROTEINS

IN DIFFERENT

Franklyn National

Institute

National Received

August

30,

SPERMIDINE,

MAMMALIAN

F. Bolander, of Arthritis,

Institutes

GLUCOCORTICOID

AND MILK

SPECIES

Jr. and Yale J. Topper Metabolism

of Health,

and Digestive

Bethesda,

Diseases,

Maryland

20205

1979

SUMMARY: It was shown previously that induction of milk proteins in mouse mammary explants has an absolute requirement for glucocorticoid and spermidine, and that the latter can largely substitute for the hormone. Induction in the rabbit system was reported to be enhanced by, but not absolutely dependent on, glucacorticoids and to be independent of spermidine. It is demonstrated here that this enhancement is dependent on spermidine, although the polyamine cannot substitute for glucocorticoid. Moreover, it is also shown that the rat system represents an intermediate state: it has an absolute requirement for both glucocorticoid and spermidine, but the latter cannot mimic the steroid. INTRODUCTION The role differ

from

insulin, could

of spermidine

species

to species.

glucocorticoid partially

a potent synthesis

by

spermidine. require

mouse Houdebine

cortisol

explants,

that

for milk protein

(5). The role of polyamines tissue is not known, rise during

hypothesis

mammals.

Specifically, I,

this that

synthesis,

nor spermidine

serum

concerning

the

F,

inhibition rabbit

cortisol;

was mammary

these

that MGBG, milk

protein

by exogenous do not another

synthesis

in order

compounds

among

MGBG,

by rat

gland content

species

the role of spermidine

by such

of milk proteins

of this investigation

gland of several

prolactin;

spermidine

diaminohexane,

synthesis

to

requires

gland explants

milk protein

The purpose

P,

that

reversed

and that neither

of

explants

(3) reported

levels (6) and mammary

function

this study evaluates insulin;

mammary

appears

They also observed

affected

and lactation.

in the mammary

explants

the hormone-induced

in hormone-induced

although

pregnancy

study the role of polyamines a general

abolished

--et al. (4) reported

explants

by mouse

in the mouse system.

and

synthesis,

by mammary

Oka and Perry

synthesis,

of spermidine

I Abbreviations:

(I ,2).

(F)t

inhibitor

spermidine

synthesis

Such synthesis

of spermidine

glucocorticoid

mammary

protein

and prolactin

replace

inhibitor

in milk

(7) of was to

to develop different

and glucocorticoid methylglyoxal

in bis-

(guanylhydrazone). 0006-291 1131

Copyright All rights

X/79/201

131-05$01.00/O

@ 1979 by Academic Press, Inc. of reproduction in onyform reserved.

Vol. 90, No. 4, 1979

the synthesis

BIOCHEMICAL

of secretory

the role of spermidine MATERIALS

proteins

AND BIOPHYSICAL

by isolated

in the corresponding

RESEARCH COMMUNICATIONS

rat mammary

rabbit

tissue,

and further

examines

system.

AND METHODS

Ovine prolactin (NIH-P-S-13) was kindly provided by the Hormone Distribution Program, NIAMDD, and crystalline porcine insulin (lot 615-08E-220) was a gift from the Eli Lilly Company. Cortisol and spermidine were purchased from Calbiochem. Medium I99 was obtained from Grand Island Biological &ompany and MGBG (lot 020817&as from Inc. UDP-[ ClgqQctose 6200 mCi/mmol), [ Plorthothe Aldrich Chemical Compan F’tetramethyleneand Triton X-100 were phosphoric acid (carrier-free), I ,4- Clspermidine purchased from New England Nuclear. Mammary gland explants were prepared from either IO-I 2 day pregnant rats (Sprague-Dawley) or 12-14 day pregnant rabbits (Dutchland) and incubated in Medium 199 as described previously (I); the media were changed daily. All experiments involved a double incubation, as described in the tables. Lactose synthetase activity was determined as described previously (8), except that the homogenization buffer contained 2% Triton X-100 $jv). Casein was measured by calcium-rennin precipitation (I); the concentration of [ Plorthophosphoric acid in the medium was IO rJCi/ml. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (91, 88% and 92% of the radioactivity in this precipitate was identified as rat and rabbit casein, respectively. Spermidine was measured by the method of Pegg et al. (I 0). All data are expressed as the mean + one S.E. and comparisons were made by analysis of the variance. RESULTS

AND DISCUSSION

Mammary

gland explants

lactose

synthetase

cannot

replace

concentrations

activity

equally

induction

of milk protein

inhibiton.

This suggests effect

In agreement rabbits

do not require

study,

the

minimized

and casein synthesis

F in the culture were

to its inhibitory

from mid-pregnant

ineffective

synthesis;

on spermidine with

earlier

exogenous

possibility

that

by incubating

higher (data

I). Exogenous

not shown).

a non-specific

of both

spermidine

were MGBG

spermidine

toxic,

(0.4 mJ) while

lower

(IO JJM) abolished -

the

partially

this

could toxic

reverse

influence

in addition

synthesis. results

(4,5),

mammary

F for the induction endogenous

the tissue in the absence investigations

Although

F for the induction

concentrations

may exert

residual,

40% above the IP system (Table medium.

(Table

and exogenous

that MGBG

was the case in previous

incubation

medium;

rats require

(4,1 I).

MGBG

of milk

proteins

mid-pregnant

(Table

2).

In this

in such

induction

of F for a period

2-3 times

longer

than

induction

30-

F does stimulate

spermidine

does not reduce

1132

from

F participates

However,

2); again, exogenous

explants

could not replace

synthesis

below

that

is

F in the in the IP

Vol.

90,

No.

BIOCHEMICAL

4, 1979

AND

Table The Effects and Casein

Culture

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

of Cartisol,

Spermidine

and MCBG

Synthesis

in Mammary

Gland

on Lactase

Explants

from

Synthetase

Activity

Mid-pregnant

Rats

Conditions Lactose Synthetase (pmol lactase formed/ 30 min/mg wet wt)

Second Incubation

First Incubation

Casein kpm/mg

Synthesis wet wt)

I

I

0

1492

8

I

IP

0

205 +

9

I

IFP

I I + MCBG I+MGBG+ Spermidine

IFP + MGBG Spermidine

9429

ll38;63

IP + Spermidine

0

2022

II

IFP + MGBG

0

351:

38

+

5125

612243

Mammary gland explants from midpregnant rats were cultured for ane day during the first incubation, followed by three days during the second incubation. The medium contained I (5 uglml), F (5 ug/ml), P (5ug/ml), spermidine (0.4 mM) and/or MGBG (I O~r1, as indicated. The data from four experiments, each assay performed in triplicate, were pooled and expressed as the mean + one S.E.

Table The Effects and Casein

Culture First Incubation

of Cortisol,

Spermidine

Synthesis

in Mammary

2

and MGBG Gland

an Lactose

Explants

from

Synthetase Mid-parent

Activity Rabbits

Conditions -

Lactose Synthetase (pmal lactose formed/ 30 min/mg wet wt)

Second Incubation

Casein kpm/mg

Synthesis wet wt)

I

I

I .3 + 0.4

I

IP

9.8 + 0.9

766 + 23

I

IFP

13.1 + 0.4’]

998 = 40’

I

IP + Spermidine

IO.0 + 0.6b

7642

I + MGBG

IFP + MGBG

I + MGBG + Spermidine

IFP + MGBG Spermidine

772

9.8 + O.Eb +

3

lZb

778 + 20b

I I.7 + o.zc

8772

10d

Mammary gland explants from mid-pregnant rabbits were cultured for three days during the first incubation, followed by three days during the second incubation. The medium contained I (5 rJg/ml), F (5 ug/ml), P (5ug/ml), spermidine (0.4 mM) and/or MGBG (lore&) as indicated. The data from four experiments, each assay performed in triplicate, were pooled and expressed as the mean + one S.E. The p values were calculated using the analysis of the variance. (Ip < 0.001 bNot

vs. IP.

significant

‘p < 0.05 vs. IP and IFP. vs. IP.

dp< 0.002

1133

vs. IP and p
vs. IFP.

Vol.

90,

No.

4, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

Table The Effects

of MGBC Explants

Culture First

Content

Mid-pregnant

of Mammary

Rats

Gland

and Rabbits

Conditions

Incubation

COMMUNICATIONS

3

on Spermidine

from

RESEARCH

Spermidine

Second

Incubation

(nmol/g

wet

Rat

wt)

Rabbit

I

I

469 + 26

663%

14

I

IFP

5102

14a

8192

3Sb

I + MGBG

IFP + MGBG

1292

2c

2522

18’

Mammary gland explants from mid-pregnant rats or rabbits were incubated for one and three days, respectively, during the first incubation, followed by three days during the second incubation. The medium contained I (5~~g/ml), F (5 pg/ml), P (Spg/ml) and MGBG (IO mM), as indicated. The experiment was performed in triplicate and the data expressed as theean + one S.E. The p values were calculated using the analysis of the variance. ‘Not significant b p< 0.005 vs. I. $<

0.001

system,

vs. I.

vs. I.

it does

block

the

this

inhibition.

The

cited

above.

It should

Table for

2 are rabbit The

of

tissues

spermidine mouse

conditons. respectively;

for

rabbits the

glucocorticoid

are

on rat

for

Table

be noted

in Tables milk

spermidine

used

although

the

agreement

spermidine

was

lactose

with

MGBG

of

0.4

partially

mM

for

synthetase

values

induction enhancement

F (3).

by

rat

and

However,

substitute

a

effect

that

the

reverses

same

activities

reported

spermidine

rabbit

resemble

for

spermidine

produced

reported

in

tissues

the

a similar was

rabbit

it cannot

3 presents

2 demonstrate

proteins

actions

while

hormonal

that

in good

I and

and

the

system,

of

exogenous

reasons shown

by other

in

investigators

(5,121.

data

the

by F, while

concentration

they

glucocorticoids

respect,

nant

low,

stimulation

the

the

and

of

in 69%

of milk

gland, can

reduction

in

(4).

synthesis

1134

The in

residual

rat

which

tissue

in rabbit

and

explants

rabbit

spermidine (Table

requires

tissues.

various

from

this

F in the

or rabbit

rat

effects In

also

under

tissue

the

replace

in rat tissue

for explants.

partially

mammary

proteins

protein

mouse

on mammary

--et al.

milk

gland

glucocorticoid

of diaminohexane Houdebine

mammary

spermidine

levels

75%

is necessary

culture explants,

pseudopregis insufficient

I) (Table

and 2).

for Although

the

Vol.

90,

No.

4, 1979

IP-induction

of

conclusion

about

residual It

level

was

these

not

concentrations

work

for

reduce

reduced

cell

insulin

system

can

demonstrates

cannot

substitute

intermediate

the

that

enhancement

this

between for

not

glucocorticoid

that

in

be

(Table drawn,

for

prolactin

rabbit

tissue,

(3).

required

in

the

rabbit

occurs require

system.

mouse

spermidine,

that for

21, no since

the

stimulation. since

higher

that had

in the

presence

the

but

the

rabbit: latter

it cannot

that

steroid,

that

that

does

the

glucocorticoid

but

shown

both

largely

shown

of I and

spermidine, It is also

and

this

of can

been for

of

and

induction

spermidine

also

absence

glucocorticoid

the

requirement

the

F does

required

It

an absolute

which

showed

and

have

in the

and

are

synthesis,

by

of

levels

MGBG

can

required

prolactin,

induction

steroid

induction

explants

system

not

enhance

for

this

does is

gland

casein

in

by

COMMUNICATIONS

viability.

and

and

such

spermidine

mammary

apparently

study

requirement

to

RESEARCH

is unaffected

concentration

further

mouse

BIOPHYSICAL

for

threshold

glucocorticoid

spermidine

rabbit

requirement

activity

rabbit

glucocorticoid

is

on

synthetase

corresponding that

to

of MGBG

substitute

explants

the

in addition

lactose

in

proteins

exceed

possible

spermidine,

AND

a spermidine

may

Earlier

BIOCHEMICAL

have

replace

(4), and

that

P (5).

This

spermidine

the

rat an

the

system absolute

hormone.

REFERENCES I. 2.

2: 5. 6. ;: 9. IO.

1::

Juergens, W.G., Stockdale, F.E., Topper, Y.J., and Elias, J.J. (1965) Proc. Natl. Acad. Sci. USA 54,629-634. Turkington, R.W., Brew, K., Vanaman, T.C., and Hill, R.L. (1968) J. Biol. Chem. 243, 3382-3387. Oka, T., and Perry, J.W. (1974) J. Biol. Chem. 249, 7647-7652. Houdebine, L.-M., Devinoy, E., and Delouis, C. (1978) Biochimie 60, 735-741. Devinoy, E., and Houdebine, L.-M. (I 977) Eur. J. Biochem. 75, 41 l-41 6. Lundgren, D.W., and Oka, T. (1978) Am. J. Physiol. 234, E451-E456. Russell, D.H., and McVicker, T.A. (1972) Biochem. J. 130, 7 l-76. Freeman, C.S., and Topper, Y.J. (I 978) Endocrinology 103, I86- 192. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. Pegg, A.E., Lockwood, D.H., and Williams-Ashman, H.G. (1970) Biochem. J. I 17, 17-31. Delouis, C., and Denamur, R. (I 972) J. Endocrinol. 52, 3 I I-3 19. Delouis, C. (1975) Mod. Probl. Paediat. 15, 16-30.

1135