Selective effects of gold(III) on parathyroid hormone-, prostaglandin E2- and guanine nucleotide-sensitive adenylate cyclase

Vol.

130,

July

31,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

SELECTIVE

580-587

EFFECTS OF GOLD(II1) ON PARATHYROID HORMONE-, PROSTAGLANDIN E2AND GUANINE NUCLEOTIDE-SENSITIVE ADENYLATE CYCLASE F. van Valen,

Medizinische

Received

June

H. Franck,

H.L.

Klinik und Poliklinik 4000 Dusseldorf

Kriiskemper

and E. Keck

C der Universitst 1, West Germany

DUsseldorf,

14, 1985

SUMMARY: Gold(II1) (Au(III)) up to 0.25 nM increased parathyroid hormoneand prostaglandin E2 -sensitive chick osteoblast adenylate cyclase activity without affecting 5'-guanylylimidodiphosphate-stimulated enzyme activity. Au(II1) at 5-50 uM inhibited hormoneand nucleotidemediated activation of adenylate cyclase. Basal adenylate cyclase activity was not influenced by Au(II1) in the given concentrations. Treatment of membranes with 5'-guanylylimidodiphosphate prior to incubation with Au(III) prevented the inhibitory effect of Au(II1) on adenylate cyclase. Our data suggest that Au(II1) alters the response of adenylate cyclase to agonists most likely through interaction with specific sulfhydryl groups associated with the enzyme system. 0 1985 Academic Press,

Inc.

It

is

cyclase

believed

that

(ATP pyrophosphate-lyase

composed the

generally

of at least

guanine

component

three

the

distinct

proteins:

of ATP to cyclic

of the enzyme system

depends

on the complex

components.

studies

have been

proteins.

The sites

gold

compounds

proteins

are

have been zymes (3-5). pounds

with thought

shown to inhibit

responsive

reports

The activation

of Au(III)

between

these

interaction

of

of gold

(2,3).

Gold

is

still

on adenylate

with

complexes

of a variety

on the action cyclase

is

and the catalytic

on the

activity

information

the effects

receptor,

interactions

moieties

adenylate

the hormone

of interaction

the biologic

To our knowledge,

on hormone

communication

to be sulfhydryl

system

AMP (1).

reported

adenylate

EC 4.6.1.1)

component,

regulatory

conversion

Numerous

responsive

(cyclizing),

nucleotide-binding for

the hormone

of en-

of gold lacking. cyclase

comThis in

Au(III), chloroauric acid; hPTH l-34, human ABBREVIATIONS: gold(III), parathyroid hormone N-terminal peptide; PGE , prostaglandin E2; GppNHp, 5'-guanylylimidodiphosphate; NEM, N-ethylmafeimide. 0006-291X/85 $1.50 Copyright 0 I985 by Academic Press, Inc. All rights of reproduction in any form reserved.

580

Vol.

130,

BIOCHEMICAL

No. 2, 1985

response pares

to hPTH its

agent.

with

The system

chick

osteoblast

and which cyclase. lationship cyclase

for

this

membranes,

has typical

that

(GppNHp),

Au(III)

is

and comalkylating

was the adenylate

cyclase

by hPTH

of hormone

functional

COMMUNICATIONS

sulfhydryl

is activated

characteristics

of the various

nucleotide

study

which

indicate

RESEARCH

of NEM, a well-known

those

chosen

Our data

BIOPHYSICAL

PGE2, and guanine

l-34,

effects

AND

and PGE2

l-34

responsive

useful

components

from

adenylate

in probing

the re-

of the adenylate

system.

METHODS Osteoblast-like bone cells were isolated from 18-day-old chick embryo calvaria with collagenase (1 mg/ml isolation medium (6)) by the method of Nijweide et al. (7). Cell cult ures were maintained in Costar T-150 flasks in DMEM supplemented with 10% fetal calf serum, 200 ug/ml glutamine and 50 ug/ml gentamycin sulfate. Cultures were kept at 37°C with 5% CO2 for 6-7 days before being used. Osteoblast membranes were prepared as described recently (8). Adenylate cyclase was assayed in a reaction mixture containing 10 mM Na-acetate buffer pH 7.4, 0.5 mM ATP, 5 mM M&12, 5 mM creatine phosphate, 6.2 U/ml creatine phosphokinase, and 250 ug/ml membrane protein. Test substances were added at concentrations as warranted by individual experiments. When hormone was present in the assay mixture, GppNHp (200 JJM) was also included. hPTH l-34 (2600 U/mg; Bachem) and PGE (Sigma) were dissolved in 0.001 N HCl and ethanol, respectively, and 2.iluted in buffer just prior to assay. Solutions of Au(II1) (HAuC14.3 H 0; Au content min. 49%; Merck) were always prepared fresh in 0.001 N HC? and used within 15 min of an experiment. Membranes were preincubated with Au(III) or NEM for 30 min at 37°C in the assay buffer. Pretreatment of membranes with GppNHp (200 uM) was performed in 10 mM Tris-HCl pH 7.4 with 5 mM MgC12 for 20 min. Enzyme reactions were started by addition of ATP and were conducted for 20 min at 37°C. Preliminary studies showed that, under the assay conditions employed here, cyclic AMP formation was linear over this time period. Incubations were terminated with n-propanol (9) and cyclic AMP was measured with the Amersham International assay kit. Protein was determined by the method of Lowry et al. (10)

--RESULTS The dose response stimulated in Fig.

1. Au(II1)

cyclase, found

adenylate

with

curve cyclase

stimulated

a maximal

centrations adenylate

of Au(II1) cyclase

on basal,

activities

in osteoblast

at 0.25

enzyme activity inhibited

activity.

hPTH

hPTH 1-34-mediated

effect

when PGE2-stimulated

of Au(II1)

uM

membranes

activation

was measured.

581

total

is

shown

of adenylate

The same maximum was

Au(II1).

hPTH I-34-sensitive

Essentially,

and PGE2-

l-34-,

inhibition

Increasing

and PGE -sensitive 2

of hormone-

con-

Vol.

130,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

01a,-6 . -7 -6

RESEARCH

-5

-4 Au(III)

COMMUNICATIONS

* (M)

1. Concentration-effect curve for Au(II1) on adenylate cyclase Fig. activity in the absence (+) and presence of 5 rig/ml hPTH 1-34 (A) or 10 pM PGE2 (e). Other conditions were as described under 'Methods'. Values are the means of duplicate determinations in 3 separate experiments.

sensitive

enzyme

Basal

adenylate

Au(II1)

tested The

0

up of of

1

50

10

was

in not

the

presence

of

significantly

50

IJM Au(III).

influenced

by

MM.

Au(II1)

hPTH

5

observed

activity

to

effects

0.5

was

cyclase

concentrations

a

activity

l-34

50

loo

hPTH

l-34

on

adenylate

and

PGE2

cyclase are

500 bq/ml)

in

response

demonstrated

0

-6

in

-7

to Fig.

-6

2.

varying Au(II1)

-5

b

Fig. 2. Effect of A~(1111 on hPTH 1-34 and PGE2 stimulation of adenylate Values are expressed as per cent of maximal adenylate cyclase cyclase. activity. Maximal activities were (pm01 cAMP/min/mg): In the presence of 50 rig/ml hPTH I-34 and 200 flM GppNHp, control, 170; 0.25 pM Au(III), 212; 10 UM Au(III), 69; and in the presence of 100 pM PGE and 200 pM 68; 0.25 pM Au(III), 93; 10 )JM Au(III), $9. GppNHpGPPNHP, control, stimulated activity was substracted from each total activity.

-4 PGE2(M)

Vol. 130, No. 2, 1985

BIOCHEMICAL

1. Effect

Table

of

Au(III)

MIT*+ -dependent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and

NEM on hPTH

osteoblast

adenylate

cyclase

Adenylate (pm01

GppNHp-

activity

cyclase

CAMP/IO

and

activity

min/mg

of

protein)

Treatment

Control

Basal

None

120

-+ 9

483

-+

18

1432

-+

130

UM

126

-+ 6

471

-+ 20

1843

-+

197

NM

115

-+ 15

473

-+ 31

560

+ 63

UM

119

-+

17

492

-+ 23

1350

+ 101

UM

124

-+

11

488

-+ 35

612

Au(III)

0.25

Au(II1)

50

NEM

0.25

NEM

50

Osteoblast NEM and (basal) activity are the

tration

of hPTH

stimulate Au(II1)

concentration

nM) did

(10

constant

PTH + GppNHp

-+ 76

membranes were incubated for 30 min at 37°C with Au(III) or in the absence assayed for adenylate cyclase with 2 mM MnCl Control and presence of 50 rig/ml hPTH l-34 and 200 u i4 GppNHp. represents adenylate cyclase assayed with 2 mM MgC12. Values means -+ SEM of triplicate determinations.

at a stimulating

not

PM) and at an inhibiting

significantly

change

Similarly,

l-34.

adenylate

(0.25

cyclase

altered

concen-

the apparent

activation

constant

of PGE

the activation

was not

to

2

in the presence

of the

in the presence

of Mg

same

concentrations. The above experiments

co-factor strate

for the

were

carried

enzyme activity.

effects

of Au(II1)

recorded

on adenylate

cyclase

of Mn 2+ . Basal

the presence

of Mn 2+ as compared

high

(50 uM) concentrations

late

cyclase

inhibited presence tested

activity.

However,

was found to Mg

not

at 0.25

in osteoblast

cyclase,

however,

1,

the

as

1 demon-

higher (0.25

in in

pM) and

basal

activity

adeny-

and 50 uM in the

sulfhydryl

agent

Mn 2+ -dependent hPTH

2+

assayed

uM increased

membranes.

was inhibited 583

at low

cyclase

influence

activity

activity

Mn2+ -dependent

affect

in Table

in Table

to be 4-fold

. Au(II1)

adenylate

uM and 50 nM did not

cyclase

of adenylate

shown

2+

Au(III)

the hPTH I-34-sensitive

at 0.25

adenylate

activity

did

of Mn 2+ . As also

out

The results

the presence

tion

plus

l-34

l-34

NEM

basal stimula-

by NEM at 50 uM.

Vol.

130,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

10.

0-w

* 0

-7

-6

-6

-4

-5 concentration

(Ml

Fig. 3. Effects of Au(III) (O,O) and NEM (A,A) on GppNHp-activated (@,A) and on GppNHp-preactivated (O,A) adenylate cyclase. Assay conditions were as described under 'Methods'. Values represent the means of triplicate determinations in 2 separate experiments. The effect cyclase wing

of Au(II1)

by GppNHp and their

preactivation

Au(II1)

with

adenylate

at 0.25

preactivated

enzyme.

of Au(II1)

ted adenylate

of Au(II1) cyclase.

hPTH l-34

was completely

failed

in Fig.

3.

inhibited

In contrast,

GppNHp-

by Au(II1)

NEM inhibited

at the

GppNHp activation

to inhibit

on the hormone The data

responsiveness

reveal

that

and PGE2 stimulation

In the presence

of hPTH l-34

depicted

follo-

enzyme.

effects

both

activity

uM progressively

l-50

was not affected

adenylate

uM increased

are

activity.

Similarly,

2 shows the

of GppNHp-preactivated

tion

cyclase

the GppNHp-preactivated

Table

effect

from

of adenylate

cyclase

nucleotide

of cyclase

concentrations.

not

on adenylate

ranging

activation

preactivated indicated

effect

the guanine

at concentrations

GppNHp-mediated

but

and NEM on the activation

of 50 JJM Au(III), abolished.

of nucleotidethe stimulatory

The same high

PGE2 stimulation

Au(III)

concentra-

of GppNHp-preactiva-

cyclase. DISCUSSION

The results selectively

alters

described

in this

communication

agonist-sensitive

adenylate 584

show that cyclase

Au(II1)

activity

without

Vol.

130.

BIOCHEMICAL

No. 2, 1985

Table

2.

of Au(III)

Effect

in osteoblast

AND

BIOPHYSICAL

RESEARCH

on hormone sensitive

membranes pretreated

COMMUNICATIONS

adenylate

with

Adenylate

cyclase

GppNHp

cyclase

activity

(pm01 CAMP/IO min/mg of protein) Addition

No treatment

GPPNHP hPTH

+ GppNHp

l-34

0.25

423

-+ 23

419

1829

-+ 90

2323

653

-+ 73

867

PGE2 + GppNHp

)JM

50 UM Au(III)

+ 17

432

-+ 20

+ 108

490

-+ 52

-+ 58

747

-+

Au(III)

101

GppNHp-pretreated osteoblast membranes were treated for 30 min at 37°C of hPTH l-34, PGE2, and with Au(II1) prior to assay. The concentrations GppNHp were 50 rig/ml, 100 MM, and 200 uM, respectively. Values are the means -+ SEM of triplicate determinations.

affecting

basal

stimulated whereas Since its

enzyme activity.

hPTH I-34-induced it

changed

Au(II1) effects

at low

cyclase

consistent

with

regulatory

protein.

but

with

groups. basal

by our

adenylate

Furthermore,

experiments

cyclase effect preactivation

known that

is

and those

It

of GppNHp with

Identical

with

of other

of

This

is

nucleotide

that

for

Au(II1)

this

with

however,

the

view

has

added

(ll-

sulfhydryl

strongly

not

inhibit suppress

hormone.

GppNHp obstructed were

in

investigators

up to 50 uM, did

observations 585

guanine

Support

and without

of cyclase

forms

may be essential

irreversibly

did,

similarly,

as non-specific.

suggestion

that

mechanism.

NEM, tested

activity.

the

groups

to react

very

activated

on the

activity,

enzyme activity.

of the enzyme.

of Au(II1)

of our data

coupling

showed

of enzyme activity.

activity

uM, Au(III)

cyclase

be considered

the variosly

sulfhydryl

NEM, an agent

the activatory

tion

reactive

Our data

action

0.25

adenylate

GppNHp-dependent

must

the basal

aspect

receptor-cyclase

been provided 13)

not

below

and PGE2 stimulation

l-34

uM inhibited

a primary

with

nor

concentrations

An interesting interacts

basal hPTH

above 0.5

adenylate

and PGE2-induced

neither

influenced

Au(II1)

hormone

At concentrations

NEM inhibi-

made when GppNHp-

Vol.

130,

No. 2, 1985

preactivated

BIOCHEMICAL

enzyme was challenged

NEM confirm

those

by Au(II1)

is

groups

located

these

sulfhydryl

late act

cyclase at

cyclase.

of considerable the binding

adenylate

complex

less

inhibited

to its

to hPTH

receptor

to adenylate

and/or

cyclase

sulfhydryl

Presumably,

additive

not

perhaps

shown).

the response

of

to PGE2 is

selectively the

on

when

(data

but not

l-34

Au(II1)

cyclase,

with

then

at 50 uM fully

that

to NEM

of the two agents

inhibition

suggests

analogy

modifies

coupling

of the

by gold-sulfhydryl

at the receptor.

should

be stressed

gold-sulfhydryl

that

interactions

activated

adenylate

possible

mechanisms

from

general

molecular

It

dopamine-

and NEM probably

submaximal

cyclase

nucleus

and NEM when adeny-

effects were

with

adenylate

Au(II1)

inhibitory

COMMUNICATIONS

obtained

protein.

(14).

cyclase

Au(II1)

of hPTH l-34

PTH-receptor

our

the

giving

interest.

the

to Au(II1)

state

of adenylate

GppNHp-preactivated

exchange

since

that

if

of Au(III)

inaccessible

activated

at concentrations

Hence,

nucleotide-binding

are

in the

Our data

f or the caudate

an interaction

guanine

groups

RESEARCH

of the GppNHp-sensitive

with

on the

GppNHp stimulation

It

inhibition

consistent

The finding

Au(I.11).

adenylate

the same site,

combined

with (14)

the

is

BIOPHYSICAL

of Suen et al.

and GppNHp-sensitive may be extended,

AND

these

to account

for

they

cyclase, of Au(II1)

conclusion

entities

although

do not

action. that

However, is

have

the effects

exclude

Au(II1)

and interactions

studies

on

of Au(II1)

on

in any way other this

useful

responsible

focused

does not for

for

distract

elucidating

adenylate

the cyclase

activation.

ACKNOWLEDGEMENTS We thank Mrs. Ulrike This study was supported schaft (no. Ke 291/2-l).

Sch;ifer for her skilled technical assistance. by a grant from the Deutsche Forschungsgemein-

REFERENCES 1.

ROSS) E.M.

and Gilman,

A.G.

(1980)

Annu.

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C.J.

(1976)

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BIOPHYSICAL

RESEARCH

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Shaw, F.C. III (1979) Inorg. Perspect. Biol. Med. 2, 287-355. Buneaux, J.J., Buneaux, F. et Galmiche, P. (1975) Colloquia de 1'Institut National de la Recherche Medicale 40, 141-155. Kean, W.F., Kassam, Y.B., Lock, C.J.L., Buchanan, W.W., Rischke, and Nablo, L. (1984) Clin. Pharmacol. Ther. 35, 627-632. Hefley, T., Cushing, 3. and Brand, J.S. (1981) Am. J. Physiol. 24C&

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NiIweide, P.J., van der Plas, A. and Scherft, J.P. (1981) Calcif. Tissue Int. 33, 529-540. Van Valen, F. and Schiitte, P.P. (1983) Proc. Kon. Ned. Akad. Wetensch. -B 86 401-415. -’ Heersche, J.N.M., Marcus, R. and Aurbach, G-D. (1974) Endocrinology 2,

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10. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 2, 265-275. 11. Suen, E.T., Stefanini, E. and Clement-Cormier, Y.C. (1980) Biochem. Biophys. Res. Commun. 96, 953-960. 12. Stadel, J.M. and Lefkoztz, R.J. (1979) Mol. Pharmacol. 16, 709-718. 13. Schramm, M. and Naim, E. (1979) J. Biol. Chem. 245, 3225-3231. 14. Suen, E.T., Kwan, P.C.K. and Clement-Cormier, Y.C. (1982) Mol. Pharmacol. 2, 595-601.

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