Gangliosides do not raise cyclic AMP levels during inhibition of lymphocyte mitogenesis

Vol. 105, No. 4, 1982 April

BIOCHEMICAL

AND BIOPHYSICAL

29, 1982

RESEARCH COMMUNICATIONS Pages 1453-1460

DO NOT RAISE CYCLIC AMP LEVELS DURING INHIBITION LYMPHOCYTE MITOGENESIS.'

GANGLIOSIDES

Rajabather

and Robert

Krishnaraj'

G. Kemp

Department of Biochemistry, University of Health Sciences/The Medical School, 3333 Green Bay Road, North Chicago, IL Received

March

12,

OF

Chicago

60064

1982

Summary: The effect of bovine brain gangliosides on the intrathymocyte levels of cyclic AMP as a potential mediator of ganglioside action has been studied. Commercial tri-, and disialogangliosides, at 2.5 to 5 UM were found to produce a rapid and profound increase (es., 10 fold within 2 min by trisialoganglioside). When the preparations were purified on Florisil, this effect on cyclic AMP content was lost, but not the immunoinhibitory potency of the ganglioside (as tested on Concanavalin A induced DNA synthesis) . The water soluble "ganglioside associated protein" fractions separated from commercial di- and trisialo gangliosides by Florisil chromatography did not alter the cyclic AMP levels of thymocytes. Previous reports of an effect of commercial gangliosides on the enzymes of cyclic AMP metabolism in nervous tissue should be re-evaluated. Introduction: hypothesis

This that

cells

and inhibit

whose

level

acidic

induced

duce an inhibition Others

of gangliosides action 1

2

of the

has proposed

and provided

glycosphingolipids

normal

increase

chemically

(2,3,6).

laboratory

cellular

in the

immune responses

sera

lymphomas

and tumors

(3,4),

bind

of several

--in vitro

have confirmed

the

(7-11). gangliosides

(gangliosides)

assays general

The biochemical is

however

(l-3).

thymocytes

known.

the

the

shed by tumor

spontaneous (5)

or and pro-

immune responses

immunosuppressive for

for

Gangliosides,

of cellular

basis not

are

of mice with

to murine

evidence

ability

immunosuppressive

An attractive

pos-

Supported by USPHS Grant CA 21631 from the National Cancer Institute and BRSG Grant RR-5366 from the BRS grant program of the Division of Research Resources, NIH. Special

Fellow

of Leukemia

Society

of America.

Abbreviations used are: GMl, monosialoganglioside; GDla, disialoganglioside; GTl, trisialoganglioside; Con A, Concanavalin A; CAMP, cyclic 3',5', adenosine monophosphate; MEM-TBH, Hanks' minimum essential medium with buffers TES, BES and HEPES, pH 7.4.

0006-291X/82/081453-08$01.00/0 1453

Cop.vright 0 1982 bv Academic Press, Inc. All rights of reproduction in any form resenjed.

Vol. 105, No. 4, 1982 sibility

for

Exogenous (14)

BIOCHEMICAL

a mechanism

gangliosides

brain

of exogenous

decrease

cyclic

activation

of action

cyclase

studies

from

it

(15).

is generally exert

our

an overall

laboratory

CAMP content

of thymocytes

following

preparations

of gangliosides

(3).

more extensively

the effect

of thymic

lymphocytes.

MATERIALS

AND METHODS

nucleotide

With

for

(12,

13) or inhibit

cells

regard

believed

cyclic

AMP.

negative

the

addition

In the

present

has been shown to

to lympocyte

that

have shown

of exogenous

role

phosphodiesterase.

to neuroblastoma

AMP (CAMP) content

CAMP levels

Preliminary

was a potential

or cyclic

gangliosides

and function,

intracellular

RESEARCH COMMUNICATIONS

have been shown to activate

adenylate

Addition

AND BIOPHYSICAL

elevated

influence

(16).

an increase

in

of commercial study

gangliosides

we examine on CAMP content

Chemicals: Bovine brain gangliosides were purchased from Supelco, Bellfonte, PA. Thin layer chromatographic analysis of these gangliosides revealed the presence of 8% GM1 in GTl. GDla was impure to a similar extent. Human "0" erythrocyte globoside was a kind gift from Dr. S. Basu, University of Notre Dame. N-acetylneuraminic acid was from Sigma Chemical co., St. Louis, MO. Florisil (100-200 mesh) was from Fisher Scientific Co., Fairlawn, NJ. [Methyl-3H] thymidine (5 Ci/mmol) was supplied by New England Nuclear, Boston, MA. Stock solutions of gangliosides in chloroform:methanol (2:l) were evaporated under N, gas, dissolved in MEM-TBH (see below) on the day of experiment, and briefly sonicated. For blast transformation experiments, solutions including Concanavalin A (Con A, 3x crystallized, from Miles Laboratory, Kankakee, IL) were passed through a 0.45 u pore size Millipore filter. Florisil chromatography of gangliosides: In general, the procedure of Watanabe et al-. (17) was followed. Gangliosides in dichloroethane: methanol v:n were applied to a Florisil column (0.85 g packed in a Pasteur pipet to a height of 7.5 cm) and the column was washed with 15 ml of a mixture of 1,2-dichoroethane: mehtanol: water (10:90:1, v/v/v). The column was then washed with 5 ml of the above solvent and then successively with 10 ml each of the following; CHC13 :MeOH:bO (10:90:5), CHC13: MeOH:H2C (60:40:5), and CHC13: MeOH:H*O (40:60:5). After a final wash with methanol (10 ml), the gangliaside-associated protein fraction was eluted with 5 ml of water. Ganglioside content was determined on the basis of N-acetylneuraminic acid content (18) and using 31.8% (by weight) as the sialic acid content GDla (38% for GTl). The protein fraction was monitored by fluorescamine reaction (19), and determined by the micro-Lowry procedure (20). Thymocyte Incubation: Thymocytes were obtained from young AKR/J mice of 2-5 months age as described previously (21) and were suspended in Eagle's Minimal Essential Medium with Hanks' salt containing TBH buffer, pi 7.4 (MEM-TBH). The cells were preincubated for 30 to 45 min at 37°C before additions. Gangliosides, Con A, adenosine, or ganglioside-associated 1454

of

BIOCHEMICAL

Vol. 105, No. 4, 1982

0

AND BIOPHYSICAL

J

2

I

I

30

60

Time

Time

bl.

kinetics

of

ganglioside

RESEARCH COMMUNICATIONS

I

I

120

90

(men)

induced

elevation

of

CAMP levels.

Thymocytes were incubated with gangliosides for indicated time and cyclic AMP was determined by radioimmunoassay as described under the Materials and Methods section. Results are average of 2 separate experiments each from 1.1 x log pooled thymocytes. GMl, (100~ g/ml):A& GDla, (25 ,,g/ml): o-o; and GTl, (25 Ug/ml): o-o.

proteins thymocytes

dissolved7in (2 x 10

MEM-TBH

were

added

to

microfuge

tubes

containing

/ml).

Extraction, Separation, and Radioimnunoassay of Cellular CAMP: After incubation, cells were sedimented (12000 xg for 30 set) and the supernatant removed. After one wash with 1 ml of MEM-TBH, the cell pellet was deproteinThe acid-soluble supernatant ized with 1 ml of ice cold 1% perchloric acid. was neutralized with cold 6N KOH. After at least 30 minutes at 4", the precipitate was removed by centrifugation and the clear supernatant passed through a BioRad AG 1 x 8 (ZOO-400 mesh) formate column (0.5 x 4 cm) equilibrated Cyclic AMP was eluted with 8 ml of 2N formic acid as described with 0.1 N HCOOH. by Frandsen and Krishna (22). CAMP recovery from each column ranged from 70 to 100% and suitable corrections were made using marker C3H]cAMP. The cell extracted CAMP (and standard CAMP) was succinylated (22) by adding 10 U1 of freshly made succinic anhydride (200 mg per 1.36 ml of la.36 mixture of acetone: triethylamine). C2sI] CAMP and anti-CAMP antibody [both from Collaborative Research, Inc.) were added successively with intermittent mixing. After overnight reaction at 4", antibody bound p251] CAMP was precipated by 60% saturated (NH )2 S&and radioactivity in the 12,000 x g precipitate was determined by liqui 4 beta-scintillation counting. More than 90% of the putative cellular CAMP was susceptible to cyclic nucleotide phosphodiesterase prior to assay. Blast (+ gagioside) previously

RESULTS: -__ of

GDla

transformation: Procedures and determination of (2).

Elevation and

levels

in

bovine

brain

was

noticeable

of

GTl.

Fig.

thymocytes GMl, as

thymocyte

CAMP

1 shows exposed

GDla

and

early

as

for thymocyte [3H]-thymidine

the to

levels

kinetics

fixed

GTl. 2 minutes

cultures uptake

by

commercial

of

the

A rapid

lo-fold

of

exposure

1455

of

of

A

CAMP

commercial

increase and

with Con described

preparations

elevation

concentrations

are

stayed

(Supelco)

induced

by

near

that

GTl level

Vol. 105, No. 4, 1982 for

about

to the true

30 minutes.

basal

level.

all

three

for

those

BIOCHEMICAL

to increase

By 60 minutes, This

gangliosides

CAMP levels

Loss of effect A potential studies presence

in ganglioside

proteins

by chromatography

(in

thymocyte response)

the

of 0.1 mM adenosine, compounds 3 fold (not

column

out

to the cells

along

with

Florisil

ganglioside

itself

gangliosides (Fig.

ZB).

pendent fied

remain Thus,

fully the

upon the early

commercial

results

(Fig.

by the

loss

proteins,

found

to be very of a virus

gangliophilic

of GDla's A possible

course

is the

shift

to the Under

sensitive

in presence

of inhibiting of DNA synthesis in CAMP content

a

of unpurified

GDla

(50 ,,M) not

although

observation

the

to the

In the same experiment,

reveal the

CAMP levels

1456

ability

in the presence

remained

GDla did

gangliosides.

was

experiment.

increased

(24).

of increasing

increases

potential

(2:l)

2A).

time

purified

inhibition

previous

the nature

of isobutylmethylxanthine

capable

found

dichloroethane:methanol:

in the

was observed

importance

all

of these

with

on CAMP content,

was capable

Of considerable

and eluted

CAMP levels

addition

of

(17).

the thymocytes

the

,M were

is the

were

freed

showing

that

potent

gangliophilic

regarding

of the cells

Also,

inhibitor

particularly

CAMP content

in CAMP content

purified

as with

Such contaminants

CAMP metabolism

shown).

of the

least

was

of gangliosides.

in dichloroethane:mehtanol

known to influence

increase

increase

not shown).

on CAMP metabolism

on Florisil

was ruled

same conditions,

the

as well

can be readily

Such a treatment

to influence

GM1 being

purification

of data

GDla dissolved

H20 (10:90:1).

left

(17).

a Florisil

of the

(results

results

components,

Gangliosides

through

nature

following

above

interpretation

(23).

Commercial

thymocytes

effects

preparations

receptor

passed

the

ganglioside

in the

with

returned

of GTl and GDla as low as 2.5-5

of non-ganglioside

significant

tested,

of murine

with

involving

transient

on CAMP content

artifact

RESEARCH COMMUNICATIONS

the CAMP concentration

relatively

Concentrations

tested.

AND BIOPHYSICAL

phosphodiesterase

(data

that

any influence

not

shown).

Florisil-purified

Con A-induced

mitogenesis

in this

is

that

are

system

not de-

produced by unpuri-

vol.

105,

No.

BIOCHEMICAL

4, 1982

III I2

I 15

I 5

'2.

Effect induced

of

RESEARCH COMMUNICATIONS

f 30

(mln)

TIME

Fig.

AND BIOPHYSICAL

Florisil purified blast transformation

GDla on early at 48 hrs.

CAMP levels

and Con

A

A. Assays performed as described in Materials and Methods. Each point represents mean value (6.E.) from 2-11 estimations (3 experiments). Control, 6: plus 25 ,, g/ml Florisil purified GDla, 0; plus O.lmM adenosine,A. Thymocytes (1.25 x lo6 viable cells/O.25 ml) were cultured in B. absence or presegce of Con A (0.25 ,g/culture well) for 48 hr. One IJ Ci of [methylHlthymidine was present during the last 24 hr. At 48 hr, cell viability was measured and cultures harvested in MASH as described before (2). The histograms 1 to 4 are control, plus Florisil purified GDla, plus Con A, and plus Florisil purified GDla and Con A, respectively. Values given are the mean of 3-4 cultures of pooled thymocytes from 4 mice, and represent cpm/ 104 thymocytes.

GDla

Effect

of

ganglioside-associated

failed

to

elevate

present

in

observed --et

-al.

the

commercial in

(17)

Fig.

intrathymocyte

GDla 1.

could

While

reported

the

proteins.

proteins(s)"

preparations.

Following

their

Methods")

we

"Materials

and

column

after

iodine

and

Lowry's out

stepwise

either Similarly,

45 alone

or

did along

a "protein

not with fraction"

CHC13-MeOH

general obtained with

a water

"GDla

alter

Florisil-purified (24

1457

of

the

solvents.

to

13.6

effect Watanabe

Hz0

soluble

Florisil

polar

the

the

ganglioside

fraction

GDla \)g out

as

elutable

associated

significantly

well

a

positive

compound(s)

progress,

erythrocyte

increasingly

this

in

as

approach

but

for

was

purified

other

responsible

bovine

(25)

However,

mg GDla)

of crude

resorcinol-negative

reactions. of

in

washes

been

the

levels,

investigation

presence

"gangliophilic

CAMP

have

this

Since

step from

(yield:

cellular

CAMP

to

30 min

mg GTl)

was and

protein"

up

the

It

fluorescamine

(see

from

30~

g

levels (Table commercial

1).

Vol. 105, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL TABLE

Effect

of

1

"ganglioside associated CAMP levels --in

proteins" vitro

p mol Additions

Expt.

( &ml)

RESEARCH COMMUNICATIONS

on thymocyte

cAMP/107

thymocytes

1

Expt.

2 min

30 min

None

2

15 min

30 min

1.06

2.01+0.41 (5)

2.69

Protein (O-1)

Fraction

1.71

NT

2.44fp.42

2.29

Protein (1.0)

Fraction

NT

1.62

NT

NT

GDla

(25)

NT

0.93

2.8

NT

GDla

(25)

NT

1.43

1.64

NT

t Protein (0.1)

Fraction

Ganglioside associated proteins were isolated from commercial GDla as Incubation conditions were as described except described in methods section. that indicated concentrations of the ganglioside associated proteins were present for specified time periods. Each value represents mean +S. E. of 2-5 determinations on pooled thymocytes from 3 mice in each experiment. NT = not tested.

bovine

brain

positive vate

GTl

for CAMP

levels

In have

Although

Florisil

and

unpurified respectively, even

as

to

completely

higher

0.18%

of

concentrations

led

event

in

the

bovine

brain

content

lost

when

The

protein an

would associated the

protein 1458

to

also

e le-

the

and

CAMP

action

with

were

dose about

and

25

either

GTl

pig/ml,

and

45

However,

proteins.

dependence

purified

GDla

18

about

mitogenesis

further

of

gangliosides.

a dose of

in

effective

fractions

of

brought

inhibition

GTl

have

CAMP

gangliosides

contamination

At

ganglioside

of

for GDla

was

shown).

a potential

dependence

of

fai

it

thymocyte

preparations the

ion

in

respectively.

ganglioside

but

elut

changes

of

dose

GTl

reactions not

intracellular

the

after

early

preparations the

just

Lowry's

study,

chromatography.

0.07

water

(data

examined

in

was

and

present

been

identical effect

with

thymocytes

the

increases

nearly

to

in

commercial

rapid

the

eluted

fluorescamine

DISCUSSION: content

that

alone

(2), by

corresponds the

m/ml these or

and in

com-

BIOCHEMICAL

vol. 105, No. 4, 1982 bination

with

cytes. of the

the

should

duced

is

clear

intracellular

liosides.

from by

the

use

Previous

CAMP

was

that

varied

content in

noted

although

with

the the

that

conceivable

CAMP

content

Florisil

column

a labile

of

thymo-

was

cap-

contaminant

retained the

different

RESEARCH COMMUNICATIONS

or

action

inactivated on

batches,

present

the

on

CAMP

content

of

qualitative

aspects

similar.

mitogenesis

used

is

alter from

preparation

be

gangliosides

It

of

It

not

isolated

ganglioside It

always

did

fraction

cAMP.

commercial

column.

were

no

elevating

unpurified

be

gangliosides

Furthermore,

able in

purified

AND BIOPHYSICAL

metabolism

the

data

gangliosides of

of

present

data

It

CAMP. obtained

reports should

is

of be

an

that not

is

effect

by

clear

that

commerical of

with

inhibition

mediated

also

with

examined

the

of early

changes

great

care

preparations

gangliosides these

lectin-in-

limitations

on

in must of

the in

the

gang-

enzymes mind

(12-15).

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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RESEARCH COMMUNICATIONS

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