Tetanus toxin affects the K+-stimulated release of catecholamines from nerve growth factor-treated PC12 cells

Vol.

128,

No.

1, 1985

April

16,

1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

249-256

TETANUS TOXIN AFFECTS THE K+-STIMULATED RELEASE OF CATECHOLAMINES FROM NERVE GROWTH FACTOR-TREATED PC12 CELLS + and Alfonso Grass0 Bruna Figliomeni* + Istituto di Biologia Cellular-e, C.N.R. Via G.D. Romagnosi 18/A 00196 Rome, Italy Received

February

25,

1985

Tetanus toxin specifically binds to neuronal surfaces and Summary. interferes with the release of transmitters. The effect of tetanus toxin pretreatment of PC12 cell line, taken as a model of neuronal cells in culture, was studied and found that it depresses This effect is depolarization-dependent catecholamines secretion. by the action of Nerve limited to PC12 cells fully differentiated Growth Factor (NGF) and is indicative of the expression of specific binding sites for tetanus toxin during @nsition from the undifferentiated state. Specific binding of [ I] tetanus toxin45to Ca NGF-treated PC12 was demonstrable. The toxin has no effect on the accumulation coupled with the depolarization dependent release of catecholamines. 0 1985 Academic Press, Inc.

When the

cells

pheochromocytoma

cells

neurone.

Both

membrane the

release is

neurone-like

(2,3). cell

system

derived

excitable

neurites

of

and

and immunological

from

a rat

NGF arrest

transform properties

into of a

and -untreated PC12 cells have been 2+ in a Ca dependent manner when the

by a high

concentration activated

Therefore, in which

PC12 (1) concentrations

catecholamines

is

line

nanomolar

NGF-treated

receptor

charbamilcholine

with

the

biochemical

depolarized

acetylcholine

of

electrically several

to

culture

grown

possessing

mature found

are

sprout

division,

in

by

of the

PC12 cells to

examine

external

K+ or when

cholinergic constitute

secretory

agonist an useful

events

induced

*

Present address: Fidia Research Laboratories, Abano Terme To whom all correspondence should be addressed Abbreviations: DA, Dopamine; FUDR, 5-Fluoro-2' deoxyuridine; HEPES, 4(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid; NGF, Nerve Growth Factor; a-LTx, a-Latrotoxin; NE, Norepinephrine; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis. +

0006-291X/85

249

$1.50

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 128, No. 1, 1985

BIOCHEMICAL

by physiological

means.

investigate

the

presynaptic release

ionophore

toxin

presynaptically

surfaces

(6)

initially

(5)

is

weight and

modifying

a -LTx primarily

catecholamines.

by studying

toxin

exclusively

using

displays

indeed

not

The ability

of

of[3H]

but

found

been

naturally dependent

toxin

tetanus

for

to

catecholamine

considered

release:

to

the

binds

functions

toxin to

act is a

neuronal

(7,8).

It

was

useful

agent

capable

of

in the

present

study,

to

release

of

toxin is

used occurring

depolarization-coupled

NE or[3H]DA

affinity

has

specifically

not consequence of a reduced 125 [ I] labelled tetanus toxin nanomolar

a

a potentially

if

is

studies

be

RESEARCH COMMUNICATIONS line

transmitter

some presynaptic

on PC12 ,

with

natural

which

action

release

NGF and

with

protein

to

K+ stimulated

cell of

another

depresses

considered

this

properties

by interfering

molecular

affect

addition

neurotoxin a-LTx, 45 and Ca uptake (4).

Tetanus

large

In

AND BIOPHYSICAL

to

reduce

limited

Ca

2+

to PC12 cells

calcium

uptake.

indicate

that

NGF-treated

-dependent grown Binding the

toxin

PC12.

MATERIALS AND METHODS General. Tetanus toxin was a generous gift of Dr. B. Bizzini (Institute Paris).The liophylized toxin was equilibrated in 50 mM HEPES Pasteur, buffer pH 7.4 by extefaive dialysis. Aliquots of the resulting solution in mice S.C. were kept frozen at -3OOC. In all having LD50 10 ng Kg the experiments reported the concentration of tetanus toxin is taking 150.000 dalton its molecular weight. expressed on a molar basis, Mouse NGF was kindly supplied by Dr. C. Cozzari of this Institute. Veratridine was purchased from Sigma and FUDR from Serva. BoltonHunter reagent was purchased from Amersham at452000 Ci/mmol as well as [3H]NE (30 Ci/mmol) orf3H] DA (40 Ci/mmol) and CaC12 (30 mCi/mg). All other reagents were of the highest purity available. Cell culture. PC12 cells were kindly supplied by Dr. Calissano of this These cells were subcloned from the PC12 cells (clonal rat Institute. pheochromocytoma) established by Dr. Greene and Tishler (1). PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with and 5% heat-inactivated horse serum in a 10% fetal calf serum One week before experiment humidified atmosphere of 8% CO2 and 92% air. the cells were subcultxred on a polylisine-coated Falcon dish (35 mm) at a density of 1.5x10 cells/dish. NGF in nmolar concentration (2.5 nM) was present in the medium and maintained for the all lenght of the culture. NGF-untreated cells were plated at the same density 48 hrs before experiment. Assay of transmitter release. Tetanus toxin-treated or untreated cells were washed and incubated at 37OC in 1 ml of the assay medium pH 7.3 (126 mM NaCl, 5.4 mM KCl, 2.2 mM CaC12, 1.2 mM MgS04, 1.2 mM K2HP04, 250

Vol.

128,

No.

1, 1985

BIOCHEMICAL

AND

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RESEARCH

COMMUNICATIONS

30-yM HEPES, 5 ~JM glucose and 1 mM Ascorbic acid) supplemented with of 0.2 mM Pargyline (3). Following 10 M ? H] DA or[ H] NE in presence incubation the cells were washed three times with 1 ml aliquots of incubation medium or high K+ medium. High potassium media were prepared by reducing sodium chloride molarity accordingly with the desired molarity of potassium chloride. The releasing medium was collected from the dishes directly into scintillation vials, by careful suction. At the end of the experiment cells were solubilized with 1 ml of 0.5% SDS and assayed for radioactivity in five volumes of Pica-Fluor 30 (U.T. Packard). The radioactivity released was expressed as percentage of total radioactivity: radioactivity released plus that remaining in the cells. The cells remain firmly attached to the substrate during the whole Parallel experiments performed centrifuging the experiment. sucked media indicated that the total of radioactivity was found in the fluid phase. 45 Assay of calcium accumulation. Experiments of Ca uptake were performed essentially as descr' &ed (4). 125 Labelling tetanus toxin with I. Tetanus toxin was labelled with I according to the method of (6) using the Bolton and Hunter reagent. The reaction mixture was then chromatographed on-y Sephadex G25 column, 30 mM Tris HCl, pH 8.4, 10 mg ml gelatin. The pooled ;i;;:Fo;ip5 I] tetanus toxin eluting as the first ra$oactive peak off 195 column had a specific radioactivity of 3 PCi ,g . The purity of [ I] tetanus toxin was measured by analyzing the distribution of radioactivity on SDS-PAGE and developing the gels by autoradiography.

RESULTS AND DISCUSSION PC12 cells for

studying

have the

been extensively

molecular

addition

when PC12 cells

a normal

medium

neurotoxin they

profoundly

calcium

we have

studies

indicated

in NGF-treated by

a

that

showed,

as when

and

the

reproducible

basis

tetanus

with reduction

at

(9).

In

exposed

in

presynaptic

nerve

endings,

external

medium

of

observations

these

inhibitory

the of the

in

effect

a

of

secretory

induced by a-LaTx. Preliminary 3 of[ H]DA induced by 8 nM a-LaTx

PC12 cells

of

of neurons

the

of studying but

secretion

a consequence

the

possibility

transmitters

with

pretreated

in

toxin,

a -LaTx,

secretion

On the

tetanus of

of

mediator

the

and -untreated

pretreatment

differentiated

clear

considered

secretion

with[

(4).

system

transmitter secretion 3 3 H]DA or[ H]NE were

transmitter the

like

of

as a model

concentrations

stimulate

manner

toxin

on the

preloaded

released

a presynaptic event

to

dependent

initially

mechanism

to nanomolar

known

used

is not

toxin.

affected PC12

prolonged

which

had

fully

NGF treatment

(4-7

days)

)JM concentrations in

2.51

the

substantially

amount

of

tetanus

of[3H]

toxin,

DA or[

a

3H] NE

Vol. 128. No. 1, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

0

5

10

15

20

0

TIME

5

10

15

20

tmln)

Fig. 1. Influence of tetanus toxin on L3H] DA release in response to K+ depolarization. At the end of the uptake period control culture (a) or tetanus toxin treated cultures (b) were washed and brought to 25'V, incubated for 5-min periods with l-ml aliquots of assay medium. After two 5-min intervals, a medium containing 36 mM K+ was added (filled bars) to the cultures. Incubation condition are describe5 in the text. Tetanus toxin-treated PC12 showed to take up[ H]DA as effectively as control ~$11~. A statistically significant reduction (P > 0.01) in[ H]DA release in response to K+ was produced by tetanus toxin pretreatment.

released

upon high

replaceable either

as a releaser agent

was

pretreatment

as

different

agent

depressed shown

certain Experiments release

(2

hr)

was

1,

substantially time

chosen

that

a culture

2,

the

the

majority

dish

a collection

response

agents.

of

cells

maximal on

the

.time that

252

length

transition

days),

of toxin

for

three

tetanus

toxin

and within

of

the

treatment.

inhibition

for

3 hr

was the

reported.

PC12 (4-6 slow

data

37OC,

2 hr and this

effect

tetanus

of

incubation

of experiments

in

by

at

with

K+ medium was The

The efficacy

being

after

of NGF-treated

(10).

2 reporting

showed

effect

High

degree

depending

3 hr)

1).

a comparable

pretreatment

improve for

(Fig.

by veratridine

dependent

correlating (0.5,

Fig. of the

temperature

limits

to

in

concentrations

was strongly

degree

(36 mM) K+ stimulation

of did

not

incubation

The consideration reflects

from

the

in various round

shape,

BIOCHEMICAL

Vol. 128, No. 1, 1985

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

b

3 w

20

iii a

25

50

25

75

50 VERATRIDINE

K+ hd

TiE. 2. Dose response relationship between three a) and veratridine b) concentrations in control and tetanus toxin treated cells (filled bars). cond'tion as in Fig. 1. Each bar represents the 3 of [ H]DA released during the 5 min incubation corresponding with the filled indicated agent, 1. The S.D. for each group was less than respective mean.

undifferentiated

cell

sensitive the

to tetanus

inhibitory

synchronized

by

experiment.

While

inhibitor

toxin,

non

dependent

with

that

of

tetanus

consequence

There

are

differential of

the

constituent

test

prediction

of

ones

acetylcholine

fact

to only

acts

as a receptor

which we used

tetanus 253

It

toxin

NGF-treated

toxin

not

the

for jodinated

of K+-

different conditions

by depolarizing

tetanus

that

will

35% that

both

(3).

an

induced

that is

in

receptor

response

to

cells

stimulated

release with

of[3H]NE

believe

since

then

NGF committed)

reduced

NGF-treated

the

treatment

release

to

were

before

(not

is

reasons

can be equally

of the

membrane's this

poisoning

untreated

release

(2) or by agonists

FUDR the

to augment

cells

the

ones

toxin

mechanism the

dividing

Following

controls.

catecholamines

the

the

48 hrs

survive

one,

in order

NGF-treated

FUDR, cells

neurone-like

important

toxin.

dividing

(11).

secretory

very

[@d

different K+ (open bars ) Incubation mean (D = 4) with the area of Fig. 10% of the

differentiated

tetanus

treatment

and perish

toxin-untreated

fully

resulted

of

a

by 50 mM K+ after

that

the

of DNA synthesis,

degenerate

from

to

effect

100

agents

was concluded was

cells

probably express

tetanus by

a

toxin.

To

Bolton

and

Vol. 128, No. 1, 1985

BIOCHEMICAL

11

AND WOPHYSICAL

9

10

6

7

[UNLABELED

-log

RESEARCH COMMUNICATIONS

Toxm

I

125 Fig. 3. Displacement of[ I]tetanus toxin binding to NGF&ated PC12 by unlabeled tetanus toxin. [ I] tetanus toxin (20.000 cpm) was incubated with NGFtreated PC12. Incubations were performed in triplicate in 16 mm5wells of tis_slue culture cluster originally plated with 3 x 10 cells well for 2 hr. The cells were then washed twice with incubation medium resuspended, centrifuged in a Eppendorf microfuge and counted in a LKB gamma counter after ;y$jion and wash of the supernatant. Specifically bound I] tetanus toxin is defined as the excess #{ding over control wells containing the same amount of[ I] tetanus toxin, but an increasing excess of unlabeled toxin. Each point is the mean of 2 triplicate determinations.

Hunter

procedure

in

sites

in

cells.

these

homogenous

procedure

degradation

and

to

show the presence of 125 I] labeled tetanus The [

on SDS-PAGE and autoradiographs

labeling

cells

order

did

products.

was determined -untreated

not

by displacing[ PC12

with

cells

of

toxin

was 4x10m8M for

tetanus bound indirect cells results

toxin to

of 125

unlabeled

toxin

NGF-untreated immunofluorescence

showed

to

give

allow

to

conclude

showed

to

bands

attributable

tetanus

toxin

unlabeled

This

a positive that,

i) 254

the to

for

from

NGF-treated

on the was

whereby

the

that

affinity

while

observation

reaction

was

PC12

(Fig. 3). The 125 50% of[ I] tetanus

effect

experiments

used

toxin

PC12 cells,

displacing

cells.

toxin

displaced

NGF-treated

had a negligible

toxin

the gels

I] tetanus

that

binding

of

rise

The evaluation

concentration binding

give

specific

only

(data plasma

even

radioactivity confirmed

NGF-treated

not

membrane

10e7M

shown). of

by PC12 These

PC12 cells

Vol. 128, No. 1, 1985

BIOCHEMICAL

before

and after

of

affinity

its

treatment

with

NGF can

to tetanus

toxin,

ii)

specific

recognition

promoted

by NGF (12).

nature

investigated.

neuronal

arising

susceptible

of

sites

(gangliosides),

mechanism

by

the

which

depressed.

is

To

release

untreated values out

given

cells

In PC12

cell

surface

toxin

action

which

influx

evokes of

ACKNOWLEDGMENT:

of

in the

This

after

next

toxin,

questions

and of

the

cell

transmitters

work

from

These

action,

effect

the

a direct

possibility entry

that

following

receptors and

into

the

of

calcium.

release (4)

from is not

was supported

the

toxin

at a step

toxin,

depolarization-evoked

extracellular

rule

NGF treatment,

tetanus

has however

the

cells.

prolonged for

of

findings

of an effect

The toxin

cytosol

that

~=8 respectively;

are means _+SEM).

ca'techolamines

calcium

the

and 7266_+1002

calcium

shown

depressed.

associated

level

after

express

is

of open

we have

catecholamines

several

13,14,15),

different

1'8

min.

leave

release

to

in

treatment might affect 45 we studied Ca accumulation

influx,

(6898+854

summary

vulnerable

on the

but

occur

toxin

significantly

mechanisms

fluxes

to

not

action

not

as cpm/dish/l5

in transmitter

the

is

as a plausible

The

of 56 mM K+. While K+-dependent DA 45 1 and Fig.2). the Ca accumulated by tetanus

(Fig.

control

on calcium

Latx,

calcium

cells

the

action.

has however

release

tetanus

PC12 upon the

toxin-poisoned

of

toxinresponses

to toxin

Thus,

action

basis

tetanus

delayed

alia,

internalized.

test'wether

is reduced

inter

on the of

of

known

depolarization-coupled

depolarization-dependent by NGF-treated

(see

molecular the

is

as

culture

toxin

concern

to a group

cells

in

the

expression

makes the these

RESEARCH COMMUNICATIONS

be distinguished

the

belonging

Conceivably,

preparation

interaction,

is

sites,

and composition

been

AND BIOPHYSICAL

become release

little

of

or no effect

The action

PC12 probably

of e-

by raising

affected.

in part

by OTAN grant

no 1764

REFERENCES 1) 2) 3)

Greene,L.A., 73, 2424-2428. Ritchie,A.K. Jumblatt,J.E., 152154.

and Tischler,A.S. (1979) J. Physiol. and Tischler,A.S.

(1976) 286,

255

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Natl.

541-560. (1982) Nature

Acad.

Sci.

(London)

USA

297,

Vol. 128, No. 1, 1985 4) 5)

6) 7)

8) 9) 10) 11) 12) 13) 14) 15)

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Alema,S.,Rufini,S., and Senni,M.I. (1980) Nature Grasso,A., (London) 283, 774-776. Bizzini,B. (1979) Microbial. Rev. 43, 224-240. (1981) J. Biol. Chem. 256, 2402Rogers,T.B., and Snyder,S.H. 2407. Dreyer,F.. and Bigalke,H. (1980) NaunynHaberman,E., Scmiedenberg's Arch. Pharmacol. 311, 33-40. Pearce,B.R., Gourd,A.L., and Dutton,G.R. (1983) J. Neurochem. 40, 887-890. Stallcup,W.B. (1979) J. Physiol. 286, 525-540. and Raiteri,M. (1980) Neurochem. Res. 5, 281Levi,G., Gallo,V., 295. and Walker,I.G. (1975) Biochim. Biophys. Acta 395, Chan,A.C., 422-437. Greene,L.A; (1984) TINS 7, 91-94. Mirsky,R., Wendon,L.M.B., Black,P., Stolkin,C., and Bray,D. (1978) Brain Res. 148, 251-259. Yavin,E., Yavin,Z., Habig,W.H., Hardegree,M.C., and Kohn,L.D. (1981) J. Biol. Chem. 256, 7014-7022. Nicholas,D., Neuhoff,V., and 0sborne.N.N. (1982) Brain Beale,R., Res. 248, 141-149.

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