Modification of neuroblastoma X glioma hybrid NG108-15 adenylate cyclase by vanadium ions

BIOCHEMICAL

Vol. 105, No. 3, 1982 April

AND BIOPHYSICAL

14, 1982

MODIFICATION

RESEARCH COMMUNICATIONS Pages 1157-1165

OF NEUROBLASTOMA X GLIOMA HYBRID NG108-15

ADENYLATE CYCLASE BY VANADIUM IONS David

Lichtstein',

*Dept. **Dept.

Received

Debra

Physiology, Physiological Molecular

March

Mullikin-Kilpatrick**

and Arthur

Hebrew University, Hadassah Jerusalem, Israel

J. Blume**

Medical

School

Chemistry and Pharmacology, Roche Institute Biology, Nutley, New Jersey 07110, U.S.A.

of

8, 1982

Vanadium ions activate as well as inhibit the activity of the NG108-15 adenylate cyclase --in vitro in the absence of any hormone. Below 5mM ion, orthoand metavanadate activate; the maximal increase in activity is 2-fold. Vanadyl sulfate, at O.l-O.lmM, activates to a similar magnitude as does vanadate over these concentrations; above O.lmM, it inhibits. Activation of the enzyme by vanadate is not additive to that induced by PGEl or PF. 5 Vanadium ions do not alter the Ka for PGE,-activation, nor the Ki for Dala met amide-inhibition, nor diminish the efficacy of opiate, muscarinic and alpha adrenergic regulation of the enzyme. However, the mechanisms by which NaF and vanadium ions activate must differ. Vanadium, unlike NaF, does not attenuate the ability of hormone receptors to direct inhibition of adenylate cyclase.

Vanadium

is an essential

at 10 -7 to 10-6M

cells

physiological attention

and (see

the +5 oxidation various like

action

stimulate action adenylate

metabolic

of

in the absence vanadate

vanadate cyclase

have

been

is complex'.

has

been

in vivo the

of hormones

(8,

guanine Although

9).

It

focus

the rate

ions of

of CAMP synthesis

in

has been proposed

many hormones

mediate

considerable in

be due

regulatory

on various

vanadate

Additionally,

to

nucleotide

in eucaryotic

Most recently,

(4-g).

postulated

present

vanadium the

[3? ).

has been shown to stimulate

CAMP synthesis of

phenomena

metal

of different

by Ramasarma and Crane

state

membranes

The effects

(1,2).

review

Group Vb transition

to

an insulinits that unit

ability

to

the site

of

(N)

of

an inhibition

the of

'For simplicity, the term adenylate cyclase will refer to a complex containing a catalytic moiety (C) and a regulatory moiety (N). It is N that confers on C sensitivity to guanine nucleotides, NaF, cholera toxin, Mg+2 and hormones (see recent review by Limbird 1101). 0006-291X/82/071157-09$01.00/0 1157

C’op.vrght 0 1982 b-v Academic Pres, Inc. ,411 rrghts of reproduction rn an-v form reserved.

Vol.

105, No. 3, 1982

adenylate

cyclase

information

various well

activity

is

particular

BIOCHEMICAL

available

which on

receptor-mediated vanadium

ions

characterized

AND BIOPHYSICAL

on the

requires

the

the

effects

of

processes.

This

adenylate

cyclase

receptor-mediated

presence

vanadium paper

of

CTP (11

ions

on any

reports

of the

activations

MATERIALS

RESEARCH COMMUNICATIONS 14),

of

on the

and inhibitions

these

effects

which

NG108-15

IJO

of

has both

(11,

15-19).

AND METHODS

The mouse neuroblastoma x rat glioma hybrid clone NG108 15 and membranes: was grown, maintained, harvested and membranes prepare9 as previously detailed (21). The final pellet was re-suspended in 75mM Tris /HCl buffer, pH 7.4 and 25mM MgC12 at a protein concentration of 3-5mg/ml. Protein measured by the method of Lowry &. al. (22). Cells (20)

Adenylate cyclase assays: Assays were performed at 3Z°C for 20 min (unless specified otherwise) in final volumes of 50~1 All reaction mixtures contain (in final concentrations) 30mM Tris/HCl buffer, pH 7.4; 1OmM MgCl ; 135mM NaCl; 2.76mM phosphoenolpyruvate; 0.26ug(O.l2IU)pyruvate kinase; 0.5ug(?.OIU) myokin(phosphodiesterase ase, O.lm 5 CAMP' and 91,;r ;;;3+;A\p;. O&M GTP i O.lmM R020-1724 The reactions were initiated by addition of inhibitor) membrane protein (O.l-0.2mg/reaction) and stopped by immersion in ice water and t e addition of lml stop solution (1.26mM CAMP; 0 35mM ATP and 10 000 cpm Subsequently, the reactions were centrifuged and the [ 9 HIcAMP, at 4OC). resulting supernatants assayed for CAMP according to Salomon -et. -al. (23). Phosphoenolpyruvate (tricyclohexylammonium salt), myokinase (rabbit Materials: CAMP, GTP (Na or Tris salts), ATP (diNa salt, made by phosphorylation muscle), of adenosine), carbachol, epinephrine and PGE1 were purchased from Sigma Chem from CalBiochem., San Diego, co. ) St. Louis, MO. Pyruvate kinase was purchased sodium orthovanadate (Na VO 1, sodium metavanadate (NaVO 1 and vanadyl ,"t;;ate (VOSC&,) porn BDH Labaratories, England; and ? he 35nkephalin Poole, Labs., San Carlos, analogue Dala me amide from Penninsula Ca., [a- P\ATP (10 -3OCi/mmol) and [4 HIcAMP (30-50 Ci/mmol) were obtained from New England Nuclear, R020-1724 was a gift from Hoffmann-La Roche, Inc., Nutley, N. J. Boston, Mass. RESULTS Orthovanadate vitro this

activity

of the

ion stimulates

constant

for

activity

occurs

visible

(Na3V04,

between

enzyme activated

at 0.2

without

30 min (Fig.

1).

with

At concentrations

which increases

PGE,)

is

twice

the

by another

the &I 1mM

2

remains

maximum stimulation

of activity,

lo-%

changes

lag and the activation

The half

Orthovanadate

and 5mM ion.

significantly

cyclase.

any apparent

10uM and a plateau

by PGE, (i.e.,

state)

adenylate

NG108-15

activity

at least

+5 oxidation

of 'basal' 'basal',

is

activity

of the

30-50%.

Inter-

2

Abbreviations: R020-1724: b-(3 butoxy-4-methoxybenzyl)-2-imidazolidinone; PGE : prostaglandin El: Hepes: (N-2 hydroxyethylpepeTazi$e-N'-2-ethanesulfonic aci .a 1; Tris: tris (hydroxymethyl)aminomethane; Dala met amide; the carbamylcholine. pentapeptide TryDalaGlyPheMetNH2; carbachol: 1158

enkephalin

BIOCHEMICAL

Vol. 105, No. 3, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30 TIME (mtn)

Fig. 1

Time course of the effects of orthovanadate on NG108-15 adenylate Activity cyclase. assayed as described (see Methods) without any further additions ( i.e., taken as 'basal' activity) (0) with 1mM orthovanadate (t~l), 1uM PGE (0) and with PGE plus 1mM orthovanaThe date CM). The 'basal' activity = 30.0 pmol/lmin/mg protein. values shown are mean + S.D. of two independent experiments.

estingly,

in

stimulate

activity.

unchanged

(i.e.,

concentrations

2).

the

presence

of

However, 0.25+0.05uM)

of ion

In the presence

PGE,,

5:

only

the in

apparent

the

presence

lOmM, inhibition

of PGE,,

this

IOpM ion Ka for of

inhibition

required

is visible

to

maximally

PGE,-activation

ImM Na3V04

of 'basal'

tog [VANADIUM

Fig. 2

is

activity

(Fig. is observed

remains 3).

At (Fig,

at 5mM ion and by lOmM,

ION] (MI

NG108-15 adenylate cyclase Response of 'basal' and PGE -stimulated to varying concentrations bf orthovanadate and vanadyl sulfate. 'Basal' (open symbols) and 1uM PGE,-stimulated (closed symbols) adenylate cyclase activity incubated for 20 min in the presence of increasing concentrations of orthovanadate (0, 0) or vanadyl sulData are from a representative experiment which has fate (A, A). been replicated three times.

1159

Vol. 105, No. 3, 1982

BIOCHEMICAL

a

AND BIOPHYSICAL

OoL

I -8

-9

I -7

1

the activity

is

reduced

inhibitions

change

significantly

in

adenylate

inhibited of the reports

cyclase

in

are

without

induced

activation by this

NGl08-15

lag

Na3V04,

of 30% in

vanadyl

ions

241,

were

of the ion compared

of

is

at

10mM NaF stimulates

membranes

--in

vitro

and

of vanadate

action

1160

than

(Table that

total

constant sulfate

(Fig.

with

doses

inhibition

those the

produced

and NaF are

at 1mM)

the enzyme 3). of NaF.

activity

1).

for (at

of the enzyme by PGE, although

or

3;

and are

to PCE,.

At higher

2).

The Ki for

larger

stimulatory

or without

l-2mM and inhibition

concentration

vanadium (17,

with

similar

increases

(Fig.

any time

As found

with

effects:

by NaF is not statistically the

acts

of vanadyl

to be qualitatively

stimulation NaVO

state)

the actions

enzyme activity.

occur

the Ka for

previous

+5 oxidation

VOS04 activates

enzyme by VOS04 is

not shown).

The actions shown

activity

VOS04 inhibits

20 min (data

does not

seen

lo-50uM

Clearly,

and PGE,-stimulated These

are

VOSO,, and Na3V04 have ident'.cal

O.lmM),

5-10mM.

(NaV03,

of concentrations,

state)

and PGE,-stimulated

'basal'

Metavanadate

range

+4 oxidation

at lOpM,

(i.e.,

is

a wide

of the vanadates.

'basal'

(M)

not shown).

over

(VOSO,,

In fact,

least

by 60%.

to Na3V04 (data When judged

those

I -4

Activation of NG108-15 cyclase by PGEl in the presence and absence of vanadium ions. Adenylate cyclase activity assayed under standard conditions (O), or with 1mM vanadyl sulfate (A), or 1mM orthovanadate (0) present; PGEl added as noted along the abscissa. This is a representative experiment that was replicated twice.

Fig. 3

sulfate

I -5

-6

iog [PGE,]

similar

RESEARCH COMMUNICATIONS

As

of

the

The degree

of

by ImM Na VO 3

not

additive.

4

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 EFFECTS OF NaF AND VANADIUM IONS ON NGlOE-15 ADENYLATE CYCLASE Adenylate

--plus DAMA

---

Additions

cyclase

(pmoI/min/mg

+ S.D.)

protein

+ '$I+

---

plus PGE, ; plus DAMA %I

70519

3826

45"

205266

l47+32

29**

NaF

161253

13254

18

209549

170260

19

NaVO 3 Na3V04

130238

102+39

22'

265584

144233

84+17

42**

245+65

174+53

29**

voso4

66+l4

79

125+110

69216

45**

14

42**

155243

Adenylate cyclase activity assayed as described in Methods with the following additions (sina3 concentrations): NaF (lOmM), NaV03+(lmM), Na VO (ImM), VOS04 (ImM), Dala met amide (DAMA, 10uM) and PGEl (1pM). + = percenz izhibition significant based on paired t test at p = 0.0005 produced by DAMA. Inhibition (a*); at p = 0.05 (*) and, not significant at p = 0.5 ( 1.

the

However,

actions

conditions,

we never

vanadium

ions

observed

in

reported

(17)

NaCl,

of

to inhibit

no such effects find

that

vanadium

inhibition

or absence

enzyme activity

is

is

to

inhibit

blockable

the

adrenergic maximal

activity

levels

antagonist

of

processes adenylate

in the presence

shown).

Although

acts

absence

of

if any effect

on

cyclase

(Table

peptide

Dala2met5amide

similarly.

is fully cases,

by 40-502:

naloxone

(15).

in NG108-15

all

[16,

251) 2).

to the action

of Dala3met5amide such

actions

In addition, (i.e.,

At

1).

Even when the

responsive

the action

enzyme

1161

been

135mM NaCl.

Metavanadate

of Na 3 VO,, (Table

NaF has

enzyme in the

adenylate

cyclase

assay

of 1mM VOSO4 is

NaF, have little

it

our

by NaF; clearly,

action

of the opioid

of the above of the

inhibitory

inhibition

the action

Under

inhibitory

NG108-15

unlike

by 1mM VOS04,

In all

identical. directed

containing

ions,

of PGE,.

inhibited

by the opiate

receptor-mediated

not

of the NG108-15

in the presence

peptide.

NaF (data

in assays

Na3V04 does not diminish

of the opioid

the full

the PGE,-stimulated occur

not

inhibition

Actually,

presence

receptor-mediated

and NaF are

see any significant

can inhibit. the

We also

lmM,

of vanadium

the

to

other

two

and alpha

muscarinic occur

are

their

normal

Vol. 105, No. 3, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 2 RECEPTOR-MEDIATED INHIBITIONS OF VARIOUS NGlOB-15 ADENYLATE CYCLASE ACTIVITIES Percent

Inhibition

---

Ligand None

0(70+19)*

of Adenylate

Cyclase

plus Na3V0,,

plus PGE,

oc144+331*

0(205+60)*

Dala*met*amide

48

42

45

Carbachol

48

50

35

Epinephrine

46

50

37

Ligands

added as follows: Dala2met5amide ClOpM), carbachol (lOOuM), epinephrine l = the activity in pmol/min/mg protein Na3V04 (1mM) and PGE (1uM). (i.e., 'basal' activity) or +-_ S. D. for the enzyme assaye d without additions at least twice. with Na3V04 or PGE,. This experiment has been replicated (lOOuM),

1mM Na3V04

Finally, Dala?net5amide

action

of PGE, (data the

(Table

Despite in the

statist

the

presence

the analysis

is noted; there

not

is a great ically

deal significant

alter

significantly

Ki=0.'75+0.2yM) In contrast,

of enzyme activity

1).

performed

(i.e.,

not shown).

inhibition

does

fact

that

of variability opiate

data

the

averaged, by

a

data

from

t test

from one experiment effect

in

the

(Fig.

of the peptide that,

to another, of

receptor experiments

indicates

presence

for

to attenuate

individual action

Ki

4) or absence

by the opiate

a partial

paired

apparent

of NaF appears

can be directed

when

of NaF are of this

in the presence

the presence

that

the

NaF.

although there

is no

We have

log [DALA2MET5AMIDE](M)

Fig. 4

Inhibition of adenylate cyclase activity by Dala 'met5amide in the Activity assayed as despresence and absence of orthovanadate. cribed in the Methods with the exception that all reactions contain (0): 1mM orthovanadate (0) present. 1uM PGE . No orthovanadate This is's representative experiment that was replicated twice. 1162

Vol. 105, No. 3, 1982

previously

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

reported (24) that less than 30%of the normal opiate inhibition

of

NGl08-15 adenylate cyclase occurs in the presence of NaF. DISCUSSION This study shows that vanadium ions modify the adenylate cyclase from Ortho-

NG108-15.

concentrations

and metavanadate enhance enzyme activity

*

5mMthey inhibit.

increases enzyme activity; that

VOSO4 is

yet,

at

Vanadyl sulfate, it

O.lmM

too,

at

from

inhibits.

It

would appear

than Na VO ; however, 3 4 of the various forms of the vanadium ions may occur under our

assay conditions

(3,

to a particular

26).

This makes it difficult

to assign absolute potencies

ionic specie.

Activations

of the enzyme of up to 6-fold by vanadate (with Ka's of

0.2mM) have been demonstrated in many other systems (4, 5, 8).

is

of the NG108-15 enzyme directed These differences

10vM.

by

degree of stimulation

Na3V04

activity

Ka

implied by the observation on 'basal'

actions of these

that

PGE,-

of the opiate, muscarinic or

processes which cause inhibition

processes are independent of the level

with equal efficacy

its

enzyme is

NG108-15

However, the stimulatory

Our data would indicate

previously

of the

either the potency or efficacy

alpha adrenergic receptor-mediated activity.

yet

The ions do not change the Ka for

are independent.

nor alter

%2-fold

by vanadate (7).

to that produced by PGE,.

activation

only

The maximal

been reported hat GTPdecreases the

The magnitude of the vanadate stimulation

two effecters

is

O.l-

may be due to the presence of NaCl (at O.lM) and/or

GTP (at 60uM) in our assays. It has recently

similar

also

O.Ol-0.1~24

times more potent as an inhibitor

5-10

interconversion

stimulation

at

O.Ol-1mM;

the receptor-mediated

of catalytic

activity

of enzyme inhibitory

of C as was

that hormone-directed inhibitions

and PGE1-stimulated

NGlO&15

occur

adenylate cyclase

(15).

Work in other systems shows that Gpp(NH)p and vanadate actions are not additive

(5, 7).

The N subunit has been shownto be the site of action of both

Gpp(NH)p and NaF (27, 28). vanadate and NaF are alike

With the (i.e.,

NG108-15

enzyme, although the actions of

both stimulate 1163

to a similar

extent,

neither

Vol. 105, No. 3, 1982 requires Sodium

BIOCHEMICAL

any hormone fluoride,

bitions

and their

but not

actions

vanadium

of the enzyme activity.

systems

The

(5-7).

cyclase

actual

that

the action

is

responsible

membranes adenylate

cyclase

catalytic

subunits

inclusion

of this

mechanisms

for

without

of receptor-mediated

of

they

which

processes

activation

of

Since

disturbing

vanadate

the

studies

inhibitory

directed

inhibition

adenylate it

capable

required

of adenylate

of activating

process(es),

at further

has been

in NGl08-15

NGTp units is

in other

NG108-15

CTP hydrolysis

active

inhi-

differ

Recently,

increases in

are not identical.

receptor-mediated

to be illucidated.

a decline

(29).

in future

to attenuate

two stimulatory

mechanisms

of opiates

activity

ion

appears

remain

RESEARCH COMMUNICATIONS

are not additive)

These

by NaF and vanadate

proposed

AND BIOPHYSICAL

the

illucidation

cyclase

would

for the

routine of

the

seem to be

most appropriate. REFERENCES 1. 2. 3.

4.

Underwood, E. J. (1977) in Trace Elements in Human and Animal Nutrition. Academic Press, N. Y. Pp. 338-397. Josephson, L. and Cantley, C. L., Jr (1977) Biochemistry l6, 4572-4578. in Current Topics in Cellular Ramasarma, T. and Crane, F. L. (1982) (B. L. Horecker and E. R. Stadtman, eds.) Academic Press Regulation. N. Y. Vol. 20 (in press. Schwabe, U., Puchstein, C., Hannemann, H. and Scohtig, E. (1979) Nature 277,

5.

143-145.

Krawietz,

W., Werdan,

K. and Erdmann,

E. (1979)

Biochem.

Pharmacol.

28,

2517-2520s 6. 7.

a. 9. 10. 11. 12. 13. 14. 15. 16. 17.

la. 19. 20.

Grupp, G., Grupp, I., Johnson, C. L., Wallick, E. T. and Schwartz, A. (1979) Biochem. Biophy. Res. Comm. 8&, 440-447. R. A. (1981) Fed. Proceedings 40, 1598. Combest, W. L. and Johnson Hackbarth, I., Schmitz, W., Scholz, H. and Wetzel, E. (1980) Biochem. Pharmacol. 3, 1429-1432. Cuthbert, A. W., Herrera, F. C., Schuz, A. D. and Wilson, S. A. (1980) Br. J. Pharmacol. 69, 8-10. Limbird, L. E. (1980) Biochem. J. 195, l-13. Blume, A, J., Lichtshtein, D. and Boone, G. (1979) Proc. Natl. Acad. Sci. USA 76, 5626-5630. Saur, W. and Schultz, G. (1980) FEBS Letts. 65, 167-170. Jacobs, K. H. Londons, C., Cooper, D. M. F., Schegel, W. and Rodbell, M. (1978) Proc. Natl. Acad. Sci. USA 75, 5362-5366. M. M., Strawbridge, R. A., Fleming, J. W., Watanabe. A. M., McConnaughey, Jones, L. R. and Besch, H. R. (1978) J. Biol. Chem. 253, 4833-4836. Sharma, S. K., Nirenberg, 1~. and Klee, W. A. (1975) Proc. Natl. Acad. Sci. USA 72, 590-594. Sabol, S. L. and Nirenberg, M. (1979) J. Biol. Chem., m, 1913-1920. Sharma, S. K., Klee. W. A. and Nirenberg, M. (1977) Proc. Natl. Acad. USA 77, 3365-3369. Life Sci. 25, 985-992. Lichtshtein, D., Boone, G. and Blume, A. J. (1979) J. Cyclic Nucleotide Lichtshtein, D , Boone, G. and Blume, A. J. (1979) Res. 5, 367-375. Klee, W. A. and Nirenberg, M. W. (1971) Proc. Natl. Acad. Sci. USA 11, 3474-3477. 1164

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Larsen, N. E., Mullikin-Kilpatrick, D. and Blume, A. J. (1981) Mol. Pharmacol. 20, 255-262. Lowry, 0. H., Rosebrogh, N. J., Farr, A. L. and Randall, R. J. 91951) J. Biol. Chem. 193, 265-275. Salomon, Y., Londos, C. and Rodbell, M. 91974) Analyt. Biochm. 5&, 541548.

24. 25. 26.

Blume, A. J. (1980) in Developments in Neuroscience 11, 369-383. Traver. J., Fischer, K., Buchen, C. and Hamprecht, B. (1975) Nature 255, 550-559. Adam-Vizi, V., Varadi, G. and Simon, P. (1981) J. Neurochem. 36, 16161620.

27. 28.

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Ross, E. M., and Gilman, A. G. (1980) Annl. Rev. Biochm. 49, 533-564. Sternweis, P. C., Northrup, J. K., Smigel, M. D. and Gilman, A. G. (1981) J. Biol. Chem.256, 11517-11526. Koski, G. and Klee, W. A. (1981) Proc Natl. Acad. Sci. USA 78, 41854189.

1165