ADP-ribosylation of isolated rat islets of Langerhans

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 663-668

BIOCHEMICAL

Vol. 118, No. 2, 1984 January 30, 1984

ADP-RIBOSYLATION Carl

OF ISOLATED RAT ISLETS

Bernofsky*

and David

G. Amamoo**

Laboratory of Molecular Mayo Foundation and Mayo Graduate Rochester, Minnesota Received

December

OF LANGERHANS

Biology School of Medicine 55901

19, 1983

Incubation of isolated rat islets of Langerhans with [adenine-2,8-3HlNAD+ products. The major results in rapid incorporation of 3H into acid-insoluble The reaction is site of incorporation appears to be the cell membrane. an ADP-ribosylation inhibitor, and stimulated by inhibited by nicotinamide, The results demonstrate that islet membrane arginine, an ADP-ribose acceptor. proteins can be ADP-ribosylated in the absence of exogenous ADP-ribosylating agents and suggest that ADP-ribosylation plays a role in pancreatic islet cell function. A variety eukaryotic

of microbial cells

(1-5).

Cholera

through toxin,

by ADP-ribosylating subunit,

not

cyclsse the

sites

that

clear

that

own membrane regulation

are

to

exert

ADP-ribosylation

example,

activates

MW protein

the

of

their

the

guanine

influence

toxic

specific

membrane

of

inhibitory

action

of

these

toxins

can be modified such

islet

sites

of

actually

undergo

physiological

cells

have suggesting

cell

Preparation of Islets collagenase method of Lacy

the

demonstrates

by external

under

proteins, of islet

known

effect

acceptor

bound

sites

adenylate

nucleotide

on

cyclase regulatory

the

regulatory

subunit

the

existence

of regu-

(6-8).

ribosyltransferases we show that

for

altering

Although latory

the

a 42,000

thereby

on adenylate

toxins

ADP-ribosylating modification

conditions intrinsic

capacity

that

ADP-ribosylation

MATERIALS

AND METHODS

(9).

agents, by

endogenous

In the

present

to ADP-ribosylate may be involved

it

is ADP-

work, their in the

function.

- Islets from adult Lewis rats were prepared by the and Kostianovsky (IO), as modified by Amamoo --* et al

*TO whom requests for reprints should be directed. Present address: Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112. **Present address: Kingston Hospital, Kingston, New York 12401.

663

0006-291X/84 $1.50 Copyright 0 1984 by Academic Press, inc. All rights of reproduction in any form reserved.

Vol. 118, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(11) f and were suspended in Hanks’ salts (12) at a concentration of about 1 islet/nl. Intactness of the islets was verified by microscopic examination and by the ability of similar preparations to reverse the diabetic state of streptozotocin-diabetic rats upon intrahepatic implantation (11). Reaction Conditions - Incubations were conducted in a plastic microtiter plate (Falcon) mounted on a brass box that was clamped to a shaker platform and kept at 37’ by the circulation of water. Reaction mixtures had a final volume of 150 nl and contained 40 pl of Hanks’ salts, 10 nl of 0.15 M Na-HEPES’, pH 7.4, and 100 ul of suspended islets, added to start the reaction. Additions to the reaction mixture were made at the expense of Hanks’ salts. Reaction mixtures were covered with tape and shaken at 200 rpm. Chemicals were of reagent grade quality, and solutions were prepared with Pyrex-distilled water that was previously deionized. ADP-ribosylation - Reaction mixtures contained 20 ul (2 WI, 0.6 nmol) of [adenine-2,8-JH]NAD in 50% ethanol (New England Nuclear), evaporated in the reaction well before use. Other conditions are described above and in the For sampling, duplicate IO-n1 aliquots were transferred legends to figures. to 24-mm Millipore, Type HA filter disks kept on a plastic screen that was heated by a warming tray maintained at 60’. Dried discs were washed 3 times in 500-ml changes of cold, 10% (w/v) trichloroacetic acid, blotted, placed face-up on the bottom of a scintillation vial , overlayed with 10 ml of Aquasol and assayed for radioactivity with a Packard, Model (New England Nuclear), 3310 scintillation counter. Activity is expressed as+ cpm per IO-p1 aliquot and represents the conversion of [adenine-2,8-3H]NAD to an acid-insoluble form. A correction was made for the small amount of radioactivity bound to the Millipore filter in the absence of islets. RESULTS Figure

1 (lower

acid-insoluble

curve)

products

increase

the

intracellular

proteins

disruption

resulted

in

an overall

most

of

the

cell

membrane.

the

the uptake

tion

addition

islets

X-100

1 The abbreviation sulfonic acid.

Triton

be expected the

at

of

with

to greatly

ADP-ribosylation

of How-

(4,5,13-16).

X-100

3H incorporation

3H into

(Fig.

1, upper

of only

islet

cells

a concentration

of

is

40%,

curve)

suggesting

associated

1% did

with

not

further

3H.

is a well-known

the

would

of

incubated

poly(ADP-ribose)

activity

of 20 mM nicotinamide

of 36% under

islets

0.2%

in

uptake

were

through of

with

increase

Triton of

the

formation

ADP-ribosylation

Nicotinamide the

the

of

was a rapid islets

3H incorporation and

of

that

increase

of

there

intact

Disruption

extent

that

when

[adenine-2,8-3H]NAD+.

ever,

shows

conditions

used

is

inhibitor

of ADP-ribosylation

to intact of Fig.

HEPES:

islets

1 (not

caused

shown).

reactions, an overall

In addition,

N-2-hydroxyethylpiperazine-N’-2-ethane-

664

and inhibi-

nicotin-

BIOCHEMICAL

Vol. 118, No. 2, 1984

\na

AND BIOPHYSICAL RESEARCH COMMlJNlCATlONS

addition

nicotinomida

7

0

01

00

20

40

60

Time

(mid

80

100

20

0

02

60

40

80

I

0

Time (min)

Figure 1 - Incorporation of [adenine-2,8-%l]NAD+ into acid-insoluble Islets were incubated at 37O and the acid-insoluble radioactivity products. determined in lo-111 aliquots as described in text. For the upper curve, 10 ul of 3% Triton X-100 replaced 10 ~1 of Hanks' salts. Results are means of duplicate experiments. 2 - Release of radioactivity from ADP-ribosylated Islets. incubated at 37" in two parallel wells and the acid-insoluble of 10-1~1 aliquots determined in one of them (upper curve) as text. At 60 min. 20 ul of 375 mM nicotinamide was added to the (final volume, 150 vu, and lo-u1 aliquots were assayed for radioactivity at the times indicated (lower curve).

Figure Islets were radioactivity described in second well acid-insoluble

amide

had

the

Fig.

3H.

2 shows

mixture

that

60 min,

there

shows

noteworthy

that

theophylline

from

islets.

The

ADP-ribosylation,

in a manner

was dependent

basis

presence

[adenine-2-8-3H]NAD+

is

release

for

these

of labile

was

(Table

1))

as has been known

to

that

is glucose

33% of

upon

the

effects

is

stimulate dependent

to

an incubation

[adenine-2,fG3H]NAD* radioactivity.

still

stimulate that

other

glucagon (18,19). 665

incorporated

for Table

concentration

in releasing unclear,

1 and

ADP-ribose although

they

linkages.

suggesting for

the to

nicotinamide

nicotinamide

found

found

the

than

ADP-ribose

arginine

also

of

of

was added 3H from

to incorporate

was more effective

Interestingly,

Arginine

allowed

effect

a portion

50 mM nicotinamide

was an immediate

this

the

to discharge

when

that,

was first

that

suggest

ability

arginine

%I incorporation is

a substrate

ADP-ribosyltransferases and insulin

release

from for (6,17).

from

islets

Vol. 118, No. 2, 1984

Table

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMJNICATIONS

1 - Release

of

Radioactivity

from

ADP-Ribosylated

Compound tested (final concn.)

Isletsa

Radioactivity released

Nicotinamide,

10 t&i

Nicotinamide.

100 mM

40.3

Theophylline,

10 mM

30.3

Arginine,

(f)

4.5

10 mM

-9.4b

-

“i slets 3H]NAD for

-

were

incubated at 37°C with [adenine-2,8,60 min as described in text and aliquots (50 ul) transferred to either 10 ul of Hanks’ salts (control) or 10 pl of the compound tested. Duplicate IO-u1 samples were taken 10 and 20 mfn later and assayed for acid-insoluble radioactivity. Results are expressed as the percent difference between experimental and control values and are means of duplicate experiments.

b Stimulation

of

3H incorporation.

DISCUSSION The

present

paper

ADP-ribosylate

their

transferase(s).

recognized

transferases the

a number

and

various

action

involved

in

sites

of pertussis

of

are

particular

to homogeneity

from

the

of

ability

ADP-ribosylation

microbial

ADP-ribosylated

to

ADP-ribosyl-

ADP-ribosyl-

regulatory

mechanisms,

by microbial

and

to be established. cell

functions

events

are

relevance

to

a recently

toxin,

have

sites

exogenous

eukaryotic

AMP-mediated

of

the

physiological

remains

cyclic

islets

by means of endogenous

that

important

intact

that

the

the

of

(l-5),

has been purified

described

such

known

the

to

as protein

be

present

affected

by

work

is

ADP-ribosyltransferase

the culture

medium of Bordetella

the that

pertus-

(20).

When pertussis response observable (22,23).

of

proteins

ADP-ribosyltransferases

ADP-ribosylation

sis

be

that

sugggests

through

identity

Although

activity

result

may also

endogenous

synthesis

own membrane

This

previously

although

demonstrates

to

toxin

glucose with

Because

and the of

is other

isolated this

injected

into

insulin

on

it

causes

secretagogues

and

pancreas effect

rats,

insulin

666

islets release,

a prolonged

(21,22)

of

which

toxin-treated

pertussis

toxin

insulin is

also

animals was

known

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMJNICAT~ONS

vol. 118, No. 2, 1984

as "islet-activating

only activity

was discovered.

the ADP-ribosylation cells

and isolated

However, pertussis

toxin

has a similar

ADP-ribosylating

is now known to catalyze in a variety

regulatory

of intact

appears to be a

subunit involved in the regula-

and in the presence of GTP, adenylate cyclase is

protein

is ADP-ribosylated

Although

(24-29).

cholera

Katada and Ui (24) have shown that

mode of action,

peptides ADP-ribosylated

its

The 41,000 MW protein

membranes (24).

tion of adenylate cyclase, when this

toxin

of a 41,000 MW membraneprotein

component of the guanine nucleotide

activated

or "TAP" before

protein,"

by pertussis

toxin

and cholera

toxin

the

are not the

same. The above differences multiple

regulatory

pertussis toxin insulin

indicates

specificity

point

to the existence

sites for modulating adenylate cyclase activity.

can ADP-ribosylate

release, the latter

tion in islets

in toxin

Because

one of these sites as well as promote islet

two events may be related.

The present demonstra-

of endogenous enzymes that can ADP-ribosylate

that such a relationship

of

membraneproteins

could exist under physiological

conditions.

ACKNOWLEDGMENT This work was supported, in part, by grants to C. B. from the National Science Foundation (GB-42057) and National Institutes of Health (CA-13137). REFERENCES 1.

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