Inhibition of guanylate cyclase and cyclic GMP phosphodiesterase by cholera toxin

Vol. 66, No. 3, 1975

BIOCHEMICAL

INHIBITION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

OF GUANYLATE CYCLASE AND CYCLIC GMP

PHOSPHODIESTERASE BY CHOLERA TOXIN * Helen C, Kiefer, Robin Northwestern University

Atlas, Daniel Moldan, Department of Biochemistry Medical and Dental Schools, Chicago, 111. 60011 and Division of Infectious Disease, Department of Medical School, V. A. Hospital, Downey, Ill. 60064

Harvey S, Kantor, Medicine, Chicago

Received

August

8,1975

SUMMARY: Results of cholera toxin exposure in rabbit small intestinal epithelial cells, following 4 to 6 hours of incubation, indicate that there is simultaneous dose-dependent activation of adenylate cyclase and deactivation cyclic GMP phosphodiesterase activity is of guanylate cyclase. In addition, These data indicate that cholera toxin interacts with a binding repressed. site of dissociation constant Kd=3.8+1.3 X 10mgM to produce multiple coordinated events in the cells.

INTRODUCTION: of adenylate

Cholera cyclase

and subsequently, Elevation

cyclase

the

GMP, has not been

small

recognized

intestinal

cell

type

of cyclic

rate

as a potent epithelial

of mammalian

in the

of cyclic

rate

activator

cells origin

AMF are achieved

of synthesis

change

other

naturally

considered

(1)

(21,

tested

by cholera

(3). toxin

AMF by adenylate

of hydrolysis

(5)

between

hypothesis supported Dickerson

occurring

to be influenced

of cyclic

a considerable

* This work is to Ms. Rosetta manuscript.

Copyright All rights

only

concentrations

"Yin-Yang"

been

by phosphodies-

(2).

levels

thymocytes

Yet,

levels

any apparent

(EC 3.1.4.-)

tissue

in every

of an enhanced

To date,

toxin,

(EC 4.6.1.1)

in virtually

without

terase

rat

has recently

of intracellular

as a function

state

toxin

GMP were 5 minutes

of cyclic body

(6)

(71,

nucleotide,

toxin.

after

mucosa

exposure

1017

(4)

and

to cholera

unchanged. has accumulated that

a reciprocal

in support

of the

biological

in part by RR-05370 USPHS. Appreciation and Ms. Doris Roberts for aid in preparing

o I9 75 by Acudemic Press. IK. of reproduction in anv form reserved.

cyclic

When steady-

in dog jejunal

and 3 hours

of evidence

cyclic

by cholera

measured

GMP were

of Goldberg

3'5'

is

expressed this

Vol. 66, No. 3,1975

dualism

exists

between

nucleotides

within

this

it

reason

toxin

epithelial

sure,

a time

mum (8). paralleling Cyclic

cyclase

indicate

cyclase teracting coordinated

is

dose-response

and inhibition with

within

which

the

effects

activation

also

activity

inhibited

behavior

between

cyclase

reveals

has a pervasive

and rates

in rabbit

reached

cyclase

role

toxin

cholera

in regulating

of in-

expo-

its

activity

activation that

small

to toxin

at similar

the

occuring

activity

subsequent

by cholera

state

on enzyme

has normally

in guanylate

cholera

In the present

examined

of 4-6 hours

For

steady

of processes

cyclase

were

cyclase

of guanylate

a receptor

events

on guanylate

reductions

in adenylate

maintaining

nature

of the two

whether

of enzyme products.

at periods

GMl? phosphodiesterase The similar

of the

GMP phosphodiesterase

when adenylate

increases

for

ratio

functions.

to determine

demonstrated

levels toxin

cells,

The data

responsible

the

many cellular

interest

Directly

cell

GMP, so that

regulate

more revealing

of cholera

by cyclic

testinal

GMP.

of whole

effect

hydrolysis

thus

enzymes

frequently

are studies the

AMP and cyclic

might

of cyclic

are

study,

cells

those

concentrations

than

cyclic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

seemed of considerable

influenced

function

BIOCHEMICAL

maxi-

directly

toxin

doses.

exposure. of adenylate toxin

is

in-

multiple

a cell.

MATERIALS AND METHODS: Toxin Exposure: Purified cholera toxin was obtained from the NIH Cholera This material had been prepared by Advisory Committee, Bethesda, Maryland. Stock solutions of cholera toxin the method of Fink&stein and LoSpalluto (9). were serially diluted in phosphate-buffered saline (0.85% NaCl, 0.15 g Phosphate, Laparotomy was performed on 2-3 Kg male New Zealand rabbits (ScientipH 7.5). Ill.) under intravenous pentobarbital fic Small Animals, Arlington Heights, anaesthesia. Segments of the distal ileum were flush:d with saline and ligated to construct 4 cm closed loops interfaced with 2 cm intermediate spacing loops at both ends. Injections of 2 ml of cholera toxin solutions of varying concentrations were made into the lumen of the 4 cm loops with 25 gauge needles. Alternate injections of high and low toxin concentrations were made to randoAnimals were sacrificed after 4-6 mize toxin concentration along the ileum. hour incubation periods by overdose of pentobarbital. Tissue Preparation: Ileal loops were immediately excised from sacrificed animals, slit open, blotted and quickly rinsed three times in 75mM Tris buffer containing &Medithiothreitol, pH 7.5: at 4’. Loops were placed epithelial cell side up on filter paper in a bed of ice; epithelial cells were scraped and homogenized in 2 volumes of the previous buffer by 10 strokes in a chilled Homogenates were immediately aliquotted glass Tenbroeck tissue homogenizer. to prepared assay mixtures held at 4'C. Time between sacrifice of animals and completion of assays was held to one hour. Protein Determinations: Protein content of each homogenate was determined

1018

Vol. 66, No. 3,1975

BIOCHEMICAL

AND BIOPHYSICAI. RESEARCH COMMUNICATIONS

in quadruplicate by the method of Geiger and Bessman (10). This method is a modification of the procedure by Lowry et al (11) in which hydrogen peroxide is used to destroy all dithiothreitol present in the homogenates. Dithiothreitol has been shown to artifactually elevate protein values by standard Lowry determinations (10). Bovine Serum Albumin (Sigma) was used as a protein standard. Adenylate Cyclase Assay: Adenylate cyclase was assayed as previously reported by this laboratory (12). The phosphodiesterase inhibitor, aminophylline, previously used, was replaced in this assay by 1mM MIX (l-methyl, 3-isobutyl xanthine). Guanylate Cyclase Activity: Guanylate cyclase was assayed in 30 mM Tris GMP and 1mM MIX at pH 7.5. containing 10 mM MnCl , 5 rnM KCl, 0.2 n&I cyclic Reactions were starte i? by the addition of 1 mM GTP, (Sigma) containing [32PIGTP (New England Nuclear) for a final specific activity at 90-110 cpm/pmol. Final assay volumes were 50 ul; assay tubes were incubated at 37' for 5 min and stopped by boiling for 3 min. On one occasion reactions were stopped by the addition of 50 mM sodium acetate buffer, pH 4, and 1 mM cyclic GMP (13), to prevent non-enzymatic conversion of L3*Pl-~~p to cyclic [32P]-GMP during boiling. Following centrifugation, aliquots of 25 ~1 from each assay mixture were removed and quantitatively spotted on cellulose-polyethylenimine sheets (Brinkman) for chromatography by the method of Keirns et al (14). [32P]-cyclic GMP spots were then cut out and counted by liquid scintillation spectrometry. The identity of the [32P]-cyclic GMP spot was confirmed by hydrolysis with cyclic 3'5'-iiucleotide phosphodiesterase (Sigma). The method of Thompson and Appleman -Assax of Cyclic GMP Phbsphodiesterase: (15) was used. ['HI-cyclic-3'5'-GMP was obtained from New England Nuclear, cobra Results are expressed as venom and 5'- nucleotidase were obtained from Sigma. pmole cyclic GMP hydrolyzed per minute per mg homogenate protein. For each experimental point triplicate determinations of each Statistics: velocity assay were preformed, while protein was determined in quadruplicate. Standard deviations of the mean (am) for each final velocity and protein value were combined in the equation, in which R is the relative R;pgqq error of the final result expressed in pmol/min/mg homogenate protein, and p and v denote protein (mg) and velocity (pmol/min), respectively. Error bars depicted were calculated in this way. In addition, each group of experiments were performed at 4, 5 and 6 hr intervals subsequent to toxin exposure, with identical results. RESULTS AND DISCUSSION: at varying Also

incubation

depicted

standard

binding

concentration, pg/nil a linear

is

analysis, and values

of l/v

of guanylate

concentrations

a theoretical

and the maximal plot

Results

curve

[T]l.

pmol/min/mg,

For comparison

are shown

constructed

V max

Vmax= 10.5

determinations

toxin

where I + [T] / Kd of the apparent dissociation

velocity vs.

of cholera

binding v =

cyclase

Fig.

[T]

according represents constant have been

1 also

obtained in Fig.

1.

to a toxin Kd = 0.37 obtained

shows assays

from of

The data given in Fig, 1 are for whole-cell homogenates. When cells were fractionated in a preliminary experiment, reduction in guanplate cyclase activity appeared slightly more marked in the soluble cell fraction than in the particulate fraction (16).

1019

Vol. 66, No. 3,1975

BIOCHEMICAL

-G-a’

-2 LOG

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-I

0

[CHOLERA

+I

TOXIN,

+2

” + 00

pg/ml]

Figure 1. Changes in guanylate (a) and adenylate (0) cyclase activities in rabbit intestinal epithelial cells subsequent to 5 hr incubation with varying concentrations of cholera toxin. Experimental points are contrasted with theoretical binding curves: guanylate cyclase (- - - ), Kd"G.37 ug/ml toxin, v max= 10.5 pmol/min/mg protein; adenylate cyclase Kd=o.42 ug/ml toxin, ( -), V = 21 pmol/min/mg protein. Both assays were performed using whole cell hzggenates with substrate at lo-fold excess over Km, in 3GmM Tris, pH 7.5, see Methods.) Identical results were lmM MIX, 37'. (For metal ion additions, obtained for incubation times of 4 and 6 hrs.

adenylate toxin

cyclase

which

guanylate

were

velocities performed

cyclase binding

The results

delineate cyclase

The activity GMP was also (Fig.

obtained

from

construct

curve

the

profile

by cholera

incubation same time

with

intestinal

loops

replot

die&erase

activity

assayed upon

(4 - 6 hrs)

as the

are compared

to a

and Vmsx= 21 pmol/min/mg.

established

catalyzing

activation

of

the hydrolysis

fashion

subsequent

of Vmax= 3300 pmol/min/mg

Values

the theoretical

data

of cholera

toxin.

in a dose-dependent

a linear

intervals

Kd=0.k2ug/ml

of the well

of phosphodiestera,se(s)

2).

concentrations

The experimental

constructed

the

reduced

exposure

after

determinations.

theoretical

adenylate

at varying

curve failed cholera

(inset, shown.

Fig.2),

Approximately

to show a depression toxin

exposure,

1020

to cholera

and Kd=0.17

and these

of cyclic

values

toxin

pg/ml were

used to

one out of every in

regardless

cyclic

were

15

GMP phosphoof toxin

concen-

Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

\

\

\ ’f

[Tls

/a/ml

\

0 -m

-2.0

-1.0 log

‘Z-+t I.0

0 [T,~g/mll

Figure 2. Changes in cyclic GMP phosphodiesterase activity levels (e) in rabbit intestinal epithelial cells subsequent to 5 hr incubation with varying concentrations of cholera toxin. The inset shows a linear double-reciprocal binding determination yielding values of Kdz0.17 ug/ml toxin and Vmsx=33CC pmol/min/mg protein. tiperimental points are contrasted with a theoretical binding curve ( - - -) constructed from these Vmax and K values. Assay of whole-cell homogenates were performed in 40 mM Tris, p H 8.9 with 2 mM mercaptoethsnol, 5mM Mg++ and C.lmM [3H]-cyclic GM?, at 30'. Identical results were obtained for incubation times of 4 and 6 hrs.

tration. high

As a result, experimental

point

is unclear,

but

of the

in the

loop

Results related

each of three

it

standard

of experiments

deviation.

was shown experimentally

The meaning

to be unrelated

above

clearly

GMP metabolism

indicate

that

in rabbit

modified

subsequent

to cholera

apparent

dissociation

constant

representing

the

molecule

(M.W. 84,000)

receptors

mediating

are very

guanylate

cyclase

repression,

with

similar: inhibition,

the

adenylate

toxin

cyclase

Kd= 4.4 X lO-'E,

Kd= 2.1 X 10-qM.-

1021

the

intestinal

strikingly

processes

contains

a single of this

finding

to the position

ileum.

presented

to cyclic

of high

groups

exposure.

cellular

activities

epithelial

cells

Values

interaction all

activation, cyclic

are

of the

of the toxin three

observed

Kd= 5.0 X lo-%-; GIQ phosphodiesterase

Vol. 66, No. 3, 1975

BIOCHEMICAL

The above findings et al

(4) and Boyle

respond

to cholera

change

in --whole

to 3 hrs

over

et al

cell

levels

simultaneous

which

steady

state

cyclase,

clear

that

class

of sites

value

corresponds

and 1.1

all

three

and liver

result

membranes

The biological

meaning

of cyclic

this

inhibition,

the

face

ratio

of rising

between

meaningful

cyclic

to be an event

AMP levels,

between

cyclic

ulatory

machinery.

nucleotides

of profound which

at which (b)

cyclase

the and cycl.ic for

GMP at any given behavior

time. of guanyappears

toxin

by Kd = 3.8kl.3

at a

X 10bqg.

constants

of cholera

apparent

it

by cholera

dissociation

inhibition

GMP and cyclic

appears

obtained

and account

binding

of both

to a single

GMP metabolism

In summary,

5 min

toxin

(4.6 with

This X IO-l"M -

fat

cell

(17).

of the

cyclic

were

a time

of

current

GMP phosphodiesterase,

interaction

GMP in response

cyclic

signal.

the

by Cuatrecasas

destruction double

for

results

dose-dependent

represented reported

from of the

guanylate

to

on the lack

completed;

of cyclic

a saturable

to the

obtained

our

compensate

and cyclic

from

based

contact,

of both

of the

affinity

closely

(a)

could

does not appear

Two features

toxin

levels

cyclase,

of average

X 10-gM)-

cell

analysis

adenylate

toxin.

of Schaefer

at intervals

known to have been

we found

whole

From the mathematical late

GMP, assayed

reductions

findings

were

reports

initial

is

previous GMP system

difference:

following

dose-dependent

unchanging

cyclic

these

of

this

activation

GMP phosphodiesterase

the

to cholera

for

a 4 to 6 hr period

to the

of cyclic

exposure

cyclase

that

Both

may account

adenylate

in contrast (5)

toxin.

following

experiments

are

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

these AMP levels binding

is

tend

constitutes

to the

such a vital

and the Since

with

paralyzed

in

subtle.

be virtually

would

of cholera

importance may play

agent

would data

the production

to imply

that

the

the biologically toxin

to

intracellular role

receptor

its

balance

in cellular

reg-

REFERENCES: 1. 2.

Schafer, D.E., Lust, W.D., Sircar, B., and Goldberg, N.D. Acad. Sci. U.S.A. 67, 851-856. Sharp, G.W.G., and Hynie, S. (1971) Nature 229, 226-269.

1022

(1970)

Proc.

Nat.

Vol. 66, No. 3, 1975

7.

G. 9.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Finkelstein, R.A., C.R.C. Critical Rev. Microbial. 3, 553-623. Schafer, D.E., Lust, W.D., Poison, J.B., Hedtke, J., Sircar, B., Thakur, A.K., and Goldberg, N.D. (1971) Ann. N.Y. Acad. Sci. 135, 376-385. Eoyle, J.M., and Gardner, J.D. (1974) J. Clin. Invest. 53, 1149-1158. Goldberg, N.D., Haddox, M.K., Hartle, D.K., and Hadden, J.W. (i973) "Cellular Mechanisms," in Proc. of the Fifth International Congress on Pharmacolo,T, San Francisco, 1972 (Karger, Base_?), 5, lhb-149. Goldberg, M.D., Haddox, M.K., Dunham, E., Lopez, C. and Hadden, J.W., in R. (eds), (1971~) The Cold Spring Harbor Symposium Clarkson, B., Beserga, on the Regulation of Proliferation in Animal Cells, pp. 609-625, Academic Press, New York. Kantor, H.S. (1974) Clin. Res. 22, 614~. Finkelstein, R.A., and Lo Spalluto, J.J. (1970) J. Infect. Dis. 121 (Suppl.

s63-~72.

12.

Geiger, P.J. and Bessrnan, S.P. (1972) Anal. Biochem. 49, 467-473. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol Chem. 193, 265-275. Kantor, H.S., Tao, P., and Kiefer, H.C. (1974) Proc. Nat. Acad. Sci. 1J.S.A.

13. 14.

Kimura, Keirns,

10. 11.

71, 1317-1321. H., and Murad, J.J., Wheeler,

F. (1974) J. Biol. M.A., and Bitensky,

Chem. 21~9, 329-331. M.W. (1974) Anal.

Biochem.

61, 336-348. 15. 16. 17.

Thompson, W.J., and Appleman, M.M. (1971) Biochem. Kantor, H.S., Tao, P., and Gorbach, S.L. (1974) J. Cuatrecasas, P. (1373) Biochem. 12, 3547-3558.

1023

10, 311-316. Infect. Dis.

129, l-9.