Influence of prostaglandins and of cyclic nucleotides on the metabolism of 25-hydroxyvitamin D3 in primary chick kidney cell culture

Vol.

93, No.

April

29,

4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1980

INFLUENCE

OF PROSTAGLANDINS

METABOLISM

AND OF CYCLIC

OF 25-HYDROXYVITAMIN

D3 IN PRIMARY

Trechsel,

CHICK

KIDNEY

Carol M. Taylor, Jean-Philippe and Herbert Fleisch

CELL

Bonjour

Department of Pathophysiology, University of Murtenstrasse 35, 3010 Berne, Switzerland Received February

ON THE

1

CULTURE Ulrich

NUCLEOTIDES

1210-1216

Berne,

20, 1980

SUMMARY Prostaglandins (PG) are likely to be involved in a number of regulatory mechanisms in the kidney, which may be mediated by cyclic nucleotides. The present study describes the effects of prostaglandins and cyclic nucleotides on the hydroxylases of 25-hydroxycholecalciferol (25(OH)D3) in a primary chick kidney cell culture. 3-30 nM PG E produced significant increases in the 25(OH)D -lhydroxylase issociated with decreases in the 25(OH)D3-24-hyJroxylase at 6 hours but not at 1 hour. in concentrations between 0.3 nM and 3 PM affected the in a similar manner. A significant increase of l-hydroxylase activity was observed with 0.1 mM cyclic AMP (CAMP) or dibutyryl cyclic AMP (dbcAMP) in 6 hours, but again no effect on either hydroxylase was observed when the incubation time was reduced to 1 hour. These results suggest that PG E2 and PG F might be involved in the regulation of renal 25(OH)D metabolisiN and that the effects on the 25(OH)D3hydroxylases mig ;i t be mediatkd by CAMP. INTRODUCTION The kidney

is

D3 (25(OH)D3) active (7,8).

form

the

site

to

dihydroxyvitamin

of can

vitamin also

conversion

suggestions

as to

in the 3 be metabolized

(11,12)

but

defined

no well

the

of

25-hydroxyvitamin

D3 (l,25(OH)2D3),

D

D3 (24,25(OH)2D3)

several

1

of

1,25-dihydroxyvitamin

known

25(OH)D3

(1,2,3,4)

intestine in

(9,lO).

function

This work has been supported by the Foundation (3.725.76), F. Hoffmann-La Switzerland, and by the AusbildungsArbeitsgemeinschaft fiir Osteosynthese

has

(5,6)

the

kidney

There

physiological

role yet

the

been

have of

most

and

in

bone

to

24,25-

been

this

metabolite

discovered.

Swiss National Science Roche & Co. AG, Basel, und Forderungsfonds der (AO), Chur, Switzerland.

Vol. 93, No. 4, 1980

BIOCHEMICAL AND BLOPHYSICAL RESEARCH COMMUNICATIONS

The development tories of

of

kidney

(13,14,15,16)

long-term

has

mechanisms In a previous

chick

cell

to

hormone the

(PTH)

and

are

in

suggesting

that

alternative

to

ases In

under the

in

increased

the

study vitro study and of

in primary

chick

in

and many of suggested

to

a number the

with kidney

the

for

we have

In

investigation

of

this

a primary 3H-25(OH)D3

system

reduced

1,25W-U2D3

obtained culture of

in

4 hours.

in

vivo

represents the

parathyroid

and reduced

24-hydroxylase

regulation

25(OH)D3

described

-l-hydroxylase

results

labora-

of metabolizing

6 hours.

cell

the

regulation

capable

25(OH)D3

activity

(PG)

involved

the

chick

the

(15)

by several

tool

3H-24,25(OH)2D3.

agreement

present

glandins ases

in

in

system

stimulated

1-hydroxylase

systems

a new

report

and to

24-hydroxylase

effects

provided

culture

3H-l,25(OH)2D3

culture

involved

metabolism. kidney

cell

renal

the These

(17,18,19), a useful

25(0H)D3-hydroxyl-

conditions. we have cyclic

physiological

be mediated

the

nucleotides

kidney of

investigated

cell

on the

culture.

regulatory

by cyclic

of

these

nucleotides

of

prosta-

25(0H)D3-hydroxyl-

PG are

mechanisms

effects

effects

in

thought the

to be

kidney

compounds

(20) have

been

(21).

METHODS AND MATERIALS __The cell cultures were produced and the 25(0H)D3 hydroxylases assayed as described previously (15). Briefly, kidneys of 10 day old chicks were digested with collagenase and hyaluronidase; the 6 cells were cultured in Petri dishes, 3.5 cm in diameter, 1.2 x 10 cells, in 1.5 ml of minimum essential medium (MEM) with Earle's The medium salt solution, antibiotics and 10 % fetal calf serum. contained 1.2 mM Ca. 2 x 250 pmoles of 25(OH)D3 were added 24 Cultures were kept in a humidified and 48 hours after p&ating. C02-incubator, at 37 C. Confluence was obtained 3 days after PG and cyclic nucleotides were addded to confluent culplating. Control dishes received tures in 15 ~1 EtOH or distilled water. 15 1~1 solvent. Incubation with the agents was terminated by refetal calf serum and placing the medium by 1 ml of MEM without The cultures were incubated with without PG or syclic nucleotide. 100 pmolles of The reaction was stopped H-25!OH)D3 for 30 min. with methanol, tritiated vitamin D3 metabolites were extracted on small-particle silica into chloroformjmethanol and separated

1211

Vol.

93, No.

BIOCHEMICAL

4, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

The eluent chromatography (HPLC). high pre ssure liquid Results are expressed in n-hexane. consisted of 1 2.5 % isopropanol recovered from the HPLC. as the percent age of the radioactivity using

25(OH)[26,27-3B]D3 (specific activity 7.3 Ci/mmol) was obtained Synthetic 25(OH)D from the Radiochemical Centre, Amersham, U.K. a. was kindly donated by Hoffmann-La Roche fi Co., Basel, Switzerlan AMP (CAMP), dibutyryl cyclic AMP (dbcAMP) and PG E PG F2u, cyclic dibu r yryl cyclic GMB (dbcGMB) were from Sigma Chemical Co., St. medium, antibiotics and Louis, MO, USA. Eagle's minimum essential fetal calf serum were from Gibco, Paisley PA3 4EP, Renfrewshire, Scotland, U.K. Collagenase (type I) and hyaluronidase (type I) were Organic solvents and all other chemicals from Sigma Chemical Co. were analytical grade. RESULTS Effect

25(0H)D3-hydroxylases. associated tained that

Figure

of prostaqlandins:

with

with

a decrease

incubation

of

the

3 nM PG E2 in 6 hours.

that

shows the effect

A significant

30 nM PG E2 produced

demonstrates

1

were not

observed

Separate

when the

0.001

Fig.

I-hydroxylase was ob-

experiments (not

showed Figure

shown).

incubation

6 hours

of

time was reduced

2 24.25 1 hour

Values

the activity

of PG E2 seen after

:

Fig.

of

24-hydroxylase

effects

maximum

the effects

increase

of PG E2 on the

1.25

24.25 6 hours

1.25

Effect of PG E (6 hours) on the metabolism of 3H-25(OH)D3 cell culture in primary chi .e k kidney l p < 0.05; l * p < 0.01; *** p < are means + SEM, n = 3. by Student's t-test for unpaired values.

1:

2: Evidence for a delayed hydroxylases to PG E Values are means + SEM, n = 3. unpaired values. -

response **

1212

of

p < 0.01

the

renal

by Student's

25(OH)D3t-test

for

2

Vol.

93, No.

4, 1980

BIOCHEMICAL

AND

PGF>,

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

(nM)

Fig.

3: Effect of PG F (6 hours) on the metabolism of 3H-25(OH)D3 in primary chi$? kidney cell culture Values are means + SEM, n = 4. ** p < 0.01 by Student's t-test for unpaired values. to

one hour.

PG F2u,

Similarly

and 3 b!M,. produced under

idential

Effect

of

produced

of

the

hydroxyl3se. hour

did

(Fig.

up to 1

not

of

24-hydroxylase

concentratior,s

in

? mM failed

mM CAMP added

influence

(Fig.

As shown

increase

the

to

between

in hydroxylase

conditions

nucleotides:

a significant

concentrations

changes

experimental

cyclic

a decrease in

significant

in

nM

activities

3).

in Table

the

0.3

I,

CAMP and dbcAMP

25(0H)D3-1-hydroxylase

6 hours. to

In

produce

confluent

contrast

any

effect

cultures

25iOH)D3-hydroxylase

for

and dbcGMP on either only

1

activities

4J.

DISCUSSION Our

rescits

of

the

of

agent.s,

been

renal

shown

sugqest

that

PG E 2 and PG F2,L

25(0H)D3-hydroxylases suc9

as bradykinin,

to modulate

renal

at

the

noradrenalin PG synthesis

1213

act

may

tissue

as modulators level.

and vasopressin (22).

It

is

A number have therefore

Vol.

93, No.

4, 1980

Table

BIOCHEMICAL

Effect of cyclic in kidney cell

I:

AND

BIOPHYSICAL

nucleotides

on

RESEARCH

COMMUNICATIONS

of 3H-25(OH)03

metabolism

culture % conversion

Cyclic

Concentration

nucleotide

3H-24,25(0H)2D3 __ __.._______

(mM)

CAMP (Exp. 1)

0

2.6 + 0.2 2.5 + 0.2 1.8 T- 0.2

0.1 1

dbcAMP (Exp. II)

3.9 3.4 2.5 2.3

0

0.01

0.1 1

dbcGMP (Exp. II)

n = 3.

0

Values

Student's conceivable

metabolism.

As

to

the

might

of

action

of

of

10

evidence

of

m+lmM

: : ”

act

s

2 0

lllrir

I 24.25 lhour

Fig.

1.25

*

p

c

factors

‘T 0.1

**

T __ 0.4 **

by

which

at

the

regulate in renal

cellular

.:-: .: ::: :.: :_ :j: ::: _‘. a:.: -_

been

accumulated

are

Especiaily

suggesting

involved

those

.::. . 1:: :: !!.I-

24.25 Ghours

*

+ 0.4 T 0.2

Q.ifl

alterations

interactions

CAMP

:: ::: ::: .I. ::: ::: :.:

l

prostaglandins

prostaglandins.

6

::_: :::: :::: :::: :.:.

0.05;

through

has

*

l

+ 0.2 5.1 -T 0.3 5.6 T 0.2 5. 7 7-. 0.1

T 0.2 T - 0.2 p ”

l

s . ‘7

+ 0.1

8

4

**

+ 0.3

: .- VI c

0.1

4.7 5.7 6.7 7.6

+- 0.2 **

nucleotide

effects

z ::

T

OH) 2D3

6.7 t 0 1 8.0 T 0 2 8.5 -T 0 3

*

physiological

vivo

amount

cellular

the

l

values.

in

prostaglandin-cyclic

several

+ SEM.

unpaTred

of

mechanism

a large

means

some

metabolism

PG

that

for

that

25(OH)D3

level,

are

t-test

3H-1,25 - _----.- ~--_-- -

+ 0.2 T 0.2

3.8 4.0 4.2 4.0

0.01 0.1 1

to

1.25

4: Evidence for a delayed response of the renal 25(OH)D3hydroxylases to CAMP Values are means + SEM, n = 4. l * p < 0.01; **‘* p .. 0.001 by Student's t-test Tar unpaired values.

in

of

Vol.

93, No.

the

.4, 1980

BIOCHEMICAL

E-series

number

have

of

been

systems

of

kidney

cell

culture

tained

in

of

results

lend

senger

the

hydroxylase

already

Further

work in

25(OH)D3

is

the

in

a

play

effect Thus

this

that

is

enzyme

elevating

modulation

study of

whether

the

second

that

CAMP.

PTH and PG E2 in

mes-

25(OH)D3-lPTH,

PG E2

Furthermore tubules

suggests

CAMP levels

prostaglandins

stimulatory

of

an increase

renal

conceivable through

the

PG E2 and PG F2a

to be the

kidney

to

in

stimulator

through

isolated

needed

a

ob-

in vitro

a role

of PTH on the

it

with

findings

as

hypothesis

suggested

in

tubules

metabolism

been

obtained

between

kidney

25(0H)D3-l-hydroxylase

(23,24,25).

interaction

earlier

CAMP might

to the

stimulatory

evidence

a role

that

support

and PG F2e stimulate recent

cyclase

25(0H)D3-hydroxylases with

isolated

25(OH)D3

CAMP has

for

COMMUNICATIONS

adenylate

on the

renal

the

stimulate

CAMP.

RESEARCH

stimulate

agreement

and with

the

Our

in

in

suggested

1 -hydroxylase.

might

are

(23)

which

the

to

CAMP and dbcAMP

vivo

regulation

shown

BIOPHYSICAL

(21).

The effects

(24,251,

AND

effect

(26).

might of

an

play

PTH on the

-1-hydroxylase.

ACKNOWLEDGEMENTS We thank Miss A. Monod, J. Portenier, C. Steiner and M. Tschannen for their skilled technical assistance, Mrs. C. Stieger for assisof tance in illustration, and Mrs. B. Gyger for the preparation the manuscript. REFERENCES 1. Omdahl, 2. Norman,

J.L., A.W.,

and DeLuca, H.F. (1974) and H. Henry

(1973)

Physiol.Rev. 53, Recent Prog. Horm.Res.

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431-480.

3. Fraser, 4. De:Luca,

D.R. H.F.,

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(1975)

45,

631-666.

5. Bo,yle, H.F.

I.T., (1972)

Miravet, L., Endocrinology

Gray, R.W., 90, 605-608.

121.5

Holick,

M.F.,

and DeLuca,

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4, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

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COMMUNICATIONS

Wong,

R.G., Norman, A.W., Ready, C.R., and Coburn, J.W. J.Clin.Invest. 51, 1287-1291. Raisz, L.G., Trummel, C.L., Holick, M.F., and DeLuca H.F. (1972) Science 175, 768-769. Reynolds, J.J., Holick, M.F., and DeLuca, H.F. (1973) Calcif. Tiss.Res.12, 295-301. Holick, M.F., Schnoes, H.K., DeLuca, H.F., Gray, R.W., Boyle, and Suda, T. (1972) Biochemistry 11, 4251-4255. I.T., Taylor, C.M., Mawer, E.B., Wallace, J.E., St. John, J., Cochran, M., Russell, R.G.G., and Kanis, J.A. (1978) Clin.Sci. Molec.Med. 55, 541-547. Norman, A.W., and Henry, H.L. (1979) Vitamin D, Basic Research and Its Clinical Application, pp. 571-578, de Gruyter, Berlin. Kanis, J.A., Cundy, J., Bartlett, M., Smith, R., Heynen, G., Warner, G.J., and Russell, R.G.G. (1978) Brit.Med.J. 1, 1382(1972)

7. 8. 9. 10.

11. 12.

1386. 13. 14. 15. 16. 17. 18.

Juan, D., and DeLuca, H.F. (1977) Endocrinology 101, 1184-1193. (1979) J.Biol.Chem. 254, 2722-2729. Henry, H.L. Trechsel, U., Bonjour, J.-P., and Fleisch, H. (1979) J.Clin. Invest. 64, 206-217. Spanos, E., Barrett, D.I., Chonq, K.T., and MacIntyre, I. (1978) Bi0chem.J. 174, 231-236. Garabedian, M., Holick, M.F., DeLuca, H.F., and Boyle, I.T. (1972) Proc.Natl.Acad.Sci.USA 69, 1673-1676. Fraser, D.R., and Kodicek, E. (1973) Nature (Lond.)New Biol. 241,

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163-166.

Tanaka, Y., Lorenc, R.C., and DeLuca, H.F. (1975) Arch.Biochem. Biophys. 171, 521-526. Dunn, M.J., and Hood, V.L. (1977) Am.J.Physiol. 233, F169-F184. Samuelsson, B., Goldyne, M., Grandstrom, E., Hamberg, M., Hammarstrom, S., and Malmsten, C. (1978) Ann.Rev.Biochem. 47, 997-1029.

22. 23. 24. 25.

26.

Danon, A., Chang, L.C., Sweetman, S.J., Nies, A.S., and Oates, (1975) Biochim.Biophys.Acta 388, 71-83. J.A., Horiuchi, N., Suda, T., Takahashi, H., Shimazawa, E., and Ogata, E. (1977) Endocrinology 101, 969-974. Rasmussen, H., Wong, M., Bikle, D., and Goodman, D.B.P. (1972) J.Clin.Invest. 51, 2502-2504. Larkins, R.G., MacAuley, S.J., Rapoport, A. Rapoport, Martin, T.J., Tulloch, B.R., Byfield, P.G.H., Matthews, E.W., and Clin.Sci.Molec.Med. 46, 569-582. MacIntyre, I. (1974) Biddulph, D.M., Currie, M.G., and Wrenn, R.W. (1979) Endocrinology 104, 1164-1171.

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