Activation of rat lung particulate guanylate cyclase by cholesterol-sequestering agents

Vol.

97, No.

December

BIOCHEMICAL

3, 1980

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Pages

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1199-1205

ACTIVATION OF RAT LUNGPARTICULATEGUANYLATE CYCLASE BY CHOLESTEROL-SEQUESTERING AGENTS Pushkaraj J. Lad* John M. Dalton Research Center, University of Missouri-Columbia, Columbia, Missouri 65211 U.S.A. Received November 3,198CJ Naturally occurring cholesterol-sequestering agents, digitonin, cereolysin and streptolysin 0, activated rat lung particulate guanylate cyclase. Particulate enzyme treated with digitonin and cereolysin was Digitonin and cereolysin lowered further activated by sodium nitroprusside. sodium nitroprusside activation of the rat lung soluable guanylate cyclase. Activation of the particulate guanylate cyclase by digitonin and cereolysin was not due to the solubilization of the enzyme. SUMMARY:

Certain membraneactive with membranelipid

INTRODUCTION toxins and natural products act by interacting

components. The lipid

(phospholipid and cholesterol)

components of cell membrane

have been implicated in the modulation of

many membrane-associated enzyme, including phate lyase (cyclising) cyclic

EC 4.6.1.21 which catalyses synthesis of 3',5'-

GKP (cGKP) from GTP. Melittin,

venom and antibiotic

guanylate cyclase [GTP pyrophos-

a cytotoxic

a cholesterol

Here, stimulatory

guanylate cyclase by naturally

tight

0 are reported.

complex (4).

cereolysin

by interacting

sequestering polyene, also activates

late guanylate cyclase (3).

streptolysin

from bee

polypeptides alamethicin and gramicidin S activate

membrane-associated guanylate cyclase (l-2) Filipin,

polypeptide

occurring Digitonin

Streptolysin

effects

rat lung particu-

of particulate

agents, digitonin, interacts

with phospholipids.

cereolysin

with cholesterol

and

to form a

0 (from Streptococcus pyogenes) (5) and

(from BaciZhs cereus) (6) are oxygen labile

which are activated

by thiol-treatment.

action in altering

membranestructure;

hemolytic peptides

These toxins have a commonmode of cholesterol

is the suggested membrane

binding site for them (7). * Present address:

The Salk Institute,

P. 0. Box 85800, San Diego, CA 92138. 0006-291X/80/231199-07$01.00/0 1199

All

Copyrighr 0 1980 rights of reproduction

by Academic Press, Inc. in any form reserved.

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MATERIALS AND METHODS GTP was purchased from P. L. Biochemicals, and [u-~'P]GTP and [3H]cyclic GMPwere obtained from New England Nuclear. Cyclic GMP, creatine phosphate, creatine phosphokinase, digitonin and other unspecified biochemicals were from Sigma. Bio-Gel A-5m was obtained from Bio-Rad. Cereolysin and streptolysin 0 were generous gifts of Dr. Alan Bernheimer, New York University Medical Center & Dr. James Duncan of Northwestern University, reSpectiVelYa Enzyme Preparation. Male rats weighing in the range of loo-150 g were decapitated and lungs were perfused in situ with 10 ml of homogenizing buffer (50 mM Tris-HCl, 0.5 mM Nan EDTA, 10 mM 2-mercaptoethanol, pH 7.6) by injecting it in the right ventricle. The lungs were removed and washed twice, blotted, placed in 3 volumes of homogenizing buffer and homogenized for 10 seconds at 10,000 revfmin with a Willems Polytron equipped with a PT 20 ST generator. The homogenatewas filtered through a 40-mesh stainless steel wire screen and centrifuged for 10 min at 8000 X g, after which the supernatant solution was centrifuged at 160,900 X g for 40 min. The supernatant was used as the soluble enzyme and the pellet (microsomes) was chromatographed on a Bio-Gel A-5m column (1.6 X 15-cm). The column was eluted with 50 mMTris-HCl, pH 7.6 buffer containing 30 mM2-mercaptoethanol. The void volume fractions were pooled and used as the particulate enzyme. Assays and Biochemical Determinations. Guanylate cyclase assays were performed by determining the conversion of [o-32P]GTP to [a-32P]cyclic GMP. The assay mix contained 1.2 mMGTP, 6 mMMnC12, 5 mMcyclic GMP, 50 mMTrisHCl, 15 mMcreatine phosphate and 10 units of creatine phosphokinase (8). Enzyme activity was assayed for 5 to 6 min at 37°C with lo-30 pg protein. The [a-32P]cyclic GMPformed was separated from substrate by sequential chromatography on Dowex 50 and neutral aluminum oxide columns according to White and Karr (9). Proteins were determined by the method of Lowry et ai!. (10) after precipitation with silicotungstic acid (11). Bovine serum albumin was used as the standard. Specific activities are expressed as picomoles cyclic GMPformed/min/mg protein. All assays were in triplicate, and the activities are given as mean 2 standard error of the mean. Data presented here are representative of at least 2 or 3 experiments. RESULTSANDDISCUSSION Guanylate cyclase is found in particulate lung homogenate (12,13).

Most of the particulate

detergents (Lubroi PX or Triton 8-12 fold

(14).

activated

by nitric

X-100) and lysolecithin

oxide or nitric

cause only small stimulation

activation

non-ionic

increase the activity

oxide generating compoundslike nitro-

derivatives

Digitonin

activated

(15,16).

of the particulate

treatment changed the rat lung particulate 1).

enzyme is latent;

of rat

The soluble enzyme from rat lung and other sources is

prusside and nitrosoguanidine

(Fig.

and soluble fractions

enzymes (15).

the enzyme at low concentrations,

the optimal

range (0.02-0.04%), which

usually used for solubilization

1200

Digitonin

guanylate cyclase activity

being evident in a narrow concentration

is far below the concentration

However, these compounds

of membrane

Vol.

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01 DIGITONIN

CONCENTRATION

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02

P/o)

Fig. 1. The effect of digitonin on particulate guanylate cyclase activity. Enzyme was added to tubes containing different concentrations of digitonin and preincubated for 3 min at 37'C, then guanylate cyclase activity was assayed for 6 min at 37°C with (a) and without (0) 1 mM sodium nitroprusside.

proteins.

The optimal

(1 mM) activated 2.4

fold

acEivation

the basal

and 3.0 fold,

activated

the

of bacterial

guanylate

cyclase

about

fold,

5-6

respectively.

together

toxins,

Streptolysin

0 produced

cereolysin

(Table

Effects First, cyclase

6 toxin

activity

in Table

membrane-associated

Cereolysin

was further

together

and

activity.

0 on the particulate activated

stimulated

activated

some activation

Digitonin

of the basal

and streptolysin

enzyme

nitroprusside

3 fold.

activation

I.

nitroprusside

the enzyme

by nitroprusside.

the enzyme about

although

to a lesser

11.8

extent

fold.

than

I).

of these

lytic

7-8 fold

activity

and nitroprusside

Sodium

In some experiments,

cereolysin

are shown

Cereolysin

fold.

enzyme more than

caused

and this

2.4

and digitonin-treated

digitonin-treated

nitroprusside Effects

was about

agents (from

(data

solubilized

the

guanylate

particulate

preparation

not

cannot

be explained

Stap~~ococcus shown). cyclase

aurous)

Second,, activity.

was chromatographed

1201

neither Third,

by their did

not digitonin

lytic

property.

alter

guanylate nor

cerelolysin

when a digitonin-treated

on a Bio-Gel

A-5m column,

most

of

Vol.

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8lOCHEMlCAL

Table

I.

AND

8lOPHYSlCAL

Effect of cereolysin and streptolysfn particulate guanylate cyclase Concentration Hu

Agent None Cereolysin None Streptolysin

RESEARCH

picomoles

0 on

cGMP formed/min/mg

- SNP 117.0 663.3 143.4 143.5 216.9

100 0

COMMUNICATIONS

10 100

2 t 2 2 +

+ SNP

10.0 8.0 2.1 5.7 6.9

300.2 1506.0 363.3 389.8 514.2

Rat lung particulate fraction was treated with streptolysin cereolysin and assayed with and without 1 mM sodium nitroprusside 6 minutes at 37°C. The activity is expressed as mean + standard mean. HU = hemolytic unit.

the enzymatic

activity

was eluted

in the void

Furthermore,

such enzyme was further

treated

detergents

with

stimulated Lubrol

particulate

increase 2).

activity

by solubilizing form

These

studies

cholesterol tissues intestine

guanylate membrane activation

0.01%; which

PX like

other

that

membrane

it

antibiotic,

bound

is mostly filipin

of non-ionic Lubrol

detergent

PX produced

stimulated

non-ionic

detergents

guanylate

cyclase

is proposed the

agents

guanylate

a

by nitro-

increases into

in a membrane

the

non-

guanylate that

toxin-cholesterol

cyclase

thiol-activated complexes

1202

interact

bound

with

Most mammalian

cyclase;

in small Cholesterol

form.

the membrane

fluidity

Such a mechanism

which

cyclase.

guanylate

the membrane

(18).

of the particulate

the membrane,

Enzyme Nitroprusside

further

seem to activate

by increasing

disorganization

It

was not

the natural

some membrane-associated

here.

above 0.01X,

enzyme.

(14,17). demonstrate

cyclase

by nitroprusside.

in the presence

7.0 18.4 5.6 2.9 2.1

0 or (SNP) for error of

as an insoluble

to nitroprusside.

the particulate

and stomach

sequestering

below

Lubrol

can modulate contain

sensitive

in activity,

(Fig.

sedimentable

activated

enzyme activity

prusside

in

not

PX, at concentrations

progressive

studied

is

volume

t t ? 2 +

associated

or increasing

also

may explain

by the natural toxins aggregate

bind

the the

agents to cholesterol

in the plane

of

Vol.

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3, 1980

WOCHEMICAL

0L

O’ooo1P

AND

0.01 LUBROL

BIOPHYSICAL

RESEARCH

01 CONCENTRATION

PX

COMMUNICATIONS

1.0

i

(%)

Fig. 2. The effect of Lubrol PX on sodium nitroprusside activation of The particulate enzyme was treated with particulate guanylate cyclase. different concentrations of Lubrol PX and assayed for activity at 37°C with (0) and without (0) 1 mM sodium nitroprusside.

the membrane

and cause

disorganization phospholipids

core

of the membrane

outer

liposome, half

membranes,

trapped

(6),

which

the

this

then

increase inside

the in the

because

of cholesterol

from

aggregates

may explain

is a hydrophobic do not

structural

(6).

layer If

results guanylate

the membrane

it

that

cause

reported cyclase

its

being

here

for argue

activity

vesicle.

1203

of the

filipin,

of substances

trapped

by them seen only and do not

their

action

against is

interaction

activator

Unlike

the release

membrane

in the hydrophobic

better

produced

true

normal

mostly

of the membrane is

of increased

protein.

alterations

of the hydrophobic bilayer

occurs

Cereolysin

0 and cereolysin

the membrane

that

(7,19).

cyclase

streptolysin in the

Lysis

due to removal

with

guanylate

lysis.

merely

transverse

with

rat

an unlikely the

in the

soluble

lung possibility enzyme

Vol.

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3, 1980

BIOCHEMICAL

Table

II.

Effect

AND

BIOPHYSICAL

of digitonin guanylate

and cereolysin cyclase

COMMUNICATIONS

on soluble

picomolescGMP

Concentration

Agent

RESEARCH

formad/min/mg

- SNP . None Digitonin

537.9 523.3 462.9 440.1 482.2 464.5

0.003% 0.01 % 0.03 % 0.1 % 100 HU

Cereolysin

t 2 r 2 ? ?

+ SNP

19.9 23.1 4.9 14.3 11.1 11.1

8361.3 7610.4 7168.1 4886.1 2025.9 4127.9

f 2 2 ? t 2

791 165 174 626 244 261

About 48 ug of soluble enzyme was treated with water, digitonin or cereolysin. Guanylate cyclase activity was determined at 37°C with and without 1 mM sodium nitroprusside (SNP). The activity is given as mean f standard error of mean. HU = hemolytic unit.

Ability

of these

probably

other

specific

functional

to play

a role

of both

small

natural

similar

agents

enzymes role

for

in smooth and large

to modulate

may partially cyclic

muscle

relaxation

cyclase

can impair

suggested

to bind

cholesterol,

are

peptide

metridiolysin

A lytic

to bind

cholesterol

Digitonin cyclase

and cereolysin

by nitroprusside

with to

effect

did

of digitonin

fluidity;

but

it

nature.

excitation

and secretion

which

Other

A

has been suggested

modulate

bacterial

pneumolysin

from

and

toxic

the agents

functions.

not

toxins

and thuringiolysin

sea anemone

also

these

soluble

agents

it

in nitroprusside

to digitonin

concentration.

only

on the particulate

and (b)

the basal

The decrease

was activated

cholesterol

alter

with

in the case of untreated

concentrations, changing

II).

was proportional

fold

their

, neuronal

tetanolysin,

but when treated (Table

0.1% digitonin 15.5

some vital

cyclase

is

suggested

(22-23).

activity,

to digitonin

unknown,

Therefore,

guanylate

(7,19-21).

explain

GMP is

molecules.

guanylate

binding at higher

4.2

fold

enzyme.

was less

This

increase

due

Enzyme treated

by nitroprusside

concentrations,

1204

activated

activation

may explain

enzyme seen in Fig. compounds

guanylate

as compared the biphasic

1: (a)

the activity they

decrease

at

low

by nitro-

Vol.

97, No.

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BIOCHEMICAL

3, 1980

activation

hydrophobic

probably

properties

AND

BIOPHYSKAL

by interacting

with

the

RESEARCH

COMMUNICATIONS

enzyme which

has

(14).

ACKNOWLEDGMENT This

work Service Grant John M. Dalton laboratory this criticism; and

was supported in part by the United States Public Health HL 15002, from the National Institutes of Health and by the Research Center. I am grateful to Dr. Arnold White, in whose work was done, for his generosity with support, advice and to Mrs. Lorna White for preparing this manuscript.

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2. 3. 4. 5. 6.

Lad, P. Lad, P. 315-323. Lad, P. Haslam, Duncan, Cowell,

J. and White, .I. and Shier, J. R. J. 3.

Acta 507, 7. 8. 9. 10.

12.

14. 15.

White, A. A. (1979) J. Cyclic NucZeotide Res. 5, 315-325. and Klyne, W. (1953) Biochem. J. 55, 340-346. and Schlegel, R. (1975) J. Cell Biol. 67, 160-173. Kim, K.-S. and Bernheimer, A. W. (1978) Biochim. Biophys.

230-241.

Chem. 193, 265-275.

White, A. A., Northup, S. J. and Zenser, T. V. (1972) in Methods in CycZic NucZeotide Research (Chasin, M., ed.) pp. 125-167, Marcel1 Dekker, New York. Chrisman, T. D., Garbers, D. L., Parks, M. A. and Hardman, J. G. (1975)

J. Biol. 13.

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Bernheimer, A. W. (1974) Biochim. Biophys. Acta 344, 27-50. White, A. A., Crawford, K. M., Patt, C. S. and Lad, P. J. (1976) J. BioZ. Chem. 251, 7304-7312. White, A. A. and Karr, D. B. (1978) Anal. Biochem. 85, 451-460. Lowry, 0. H., Rosebrough, N. H., Farr, A. L. and Randall, R. J. (1951)

J. Biol. 11.

and M. L. L.,

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White, A. A. (1975) in Advances in Cyclic NucZeotide Research (Drummond, G. I., Greengard, P. and Robison, G. A., eds.) Vol. 5, pp. 353-373, Raven Press, New York. Lad, P. J. and White, A. A. (1979) Arch. Biochem. Biophys. 197, 244-252. Katsuki, S., Arnold, W., Mittal, C. and Murad, F. (1977) J. Cyclic

Nucleotide Res. 3, 23-35. 16. 17. 18. 19.

20. 21.

De Rubertis, F. R. and Craven, P. A. (1977) J. BioZ. Chem. 252, 5804-5813. Lad, P. J. (1976) Ph.D. Dissertation, University of Missouri-Columbia. Lad, P. J. (1980) Biochem. Biophys. Res. Commun.96, 203-210. Rottem, S., Hardegree, M. C., Grabowski, M. W., Fornwald, R. and Barile, M. F. (1976) Biochim. Biophys. Acta 455, 876-888. Smyth, C. J. and Duncan, J. L. (1978) in Bacterial Toxins and Cell Membrane(Jelijaszewicz, J. and Wadstrsm, T., eds.) pp. 129-183. Pendleton, I. R., Bernheimer, A. W. and Grushoff, P. (1973) J. Invert.

Path. 21, 131-135. 22. 23.

Bernheimer, 96-106. Bernheimer,

A. W. and Avigad, A. W., Avigad,

L. S. (1978)

L. S. and Kim,

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