Cation-dependent substance P activation of the enzyme guanylate cyclase

Vol.

128,

No.

April

16,

1985

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

Cation-Dependent Substance of the Enzyme Guanylate

Activation

David

February

25,

47

P Cyclase

L. Vesely

Department of Medicine of Arkansas for Medical Sciences Little Rock, Arkansas 72205

University

Received

141-l

1985

Substance P enhanced guanylate cyclase (E.C.4.6.1.2) two - to %!Z%Zd in pancreas small intestine, cerebellum, liver,,kidney, and lung. Dose response relat;onship revealed that substance P caused a maximal augmentation of guanylate cyclase activity at concentration of 1 micromolar. Increasing substance P's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for substance P’s enhancement of guanylate cyclase activity. Substance P could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of substance P. 0 1985 Academic Press, Inc.

Substance (l),

but

P was

its

completely

mechanism

of

elucidated.

including smooth

discovered

of

and

the

(2).

With

respect

has

been

reported

to

adenylate

cyclase

activity

does

increase

cyclic

not

messenger increase

to

cyclic in

45Ca and release

to

determine

that

catalyses

part

of

substance

of

ago

by Von Euler

cellular

P has

level

numerous

contraction

stimulation

of

and

increase the

(5,6).

In

brain

(4).

In the but

dispersed

The

guanylate

cyclase

(E.C.

of

guanosine

P's mechanism

of

action.

triphosphate

stimulate substance

another

associated

investigation

peptide

to

acinar

present

conversion

and

increases

P is

secretion

P, this

pancreas,

pancreatic

substance

intestinal

pancreatic

(3)

been

effects

of

substance

levels

(5,6)

to (6).

of

AMP

AMP levels

of

action

cyclic

in

amylase

salivary

of

not

pharmacological

motorneurons,

mechanism

and Gaddum

has still

spinal

activation the

50 years

at the

GMP secondary

of

if

the

GMP

cyclic

action

Substance

excitation muscles

over

second

cells with

the efflux

was designed

4.6.1.21,

the

to cyclic

enzyme GMP, is

0006-291X/85 141

All

P

Copyright 0 1985 rights oJ reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol. 128, No. 1, 1985

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS Methods

Tissues utilized in this investigation were obtained from male SpragueDawley rats weighing 150-200 g that had been maintained ad libitum on Purina laboratory chow. Substance P was obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN. Substance P was dissolved immediately before use in triple distilled water to prepare the concentrations specified in the text. Substance P was added at zero time lie, without any preincubation) to the various particulate and soluble enzyme preparations in the present in vitro investigation. Alumina, neutral activity I for co&mn chromatography, was obtained from E. Merck, Darmstadt, Germany. The (a PI-GTP was from International Chemical and Nuclear Corporation, Irvine, California. Guanylate cyclase activity was measured as previously described (7,8). The respective tissues were homogenized with a Polytron homogenizer using 10-s bursts in cold 0.03 M Tris HCl (pH 7.6) and centrifuged at 37,000 g in a refrigerated centrifuge at 4°C for 15 minutes. The supernatant and particulate fractions were then assayed at 37°C for 10 minutes for guanylate cyclase activity. The reaction mixture consisted of 20 mM Tris HCl (pH 7.61, 4 mM MnCl (unless feecified otherwise), 2.67 mM cyclic GMP (used to minimize destrugtion of ( PI-GMP, a GTP regenerating system (5mM creatine phosphate and 11.2 U creatine phosphokinasy2(E.C.2.7.3.2), 100 pg bovine gerum albumin, 20 mM caffeine, and 1.2 mM (CX P)-GTP, approximately 5x10 cpm. The final pH of the reaction mixture was 7.6. The volume of the supernatant or particulate fractions was 25 ~1 and the final volume of the cyclase assay which included the above supernatant or particulate fractions, the reaction mix and the radioactive isotopes was 75 I.I~. The enzyme preparations contained 0.1 to 0.2 mg/ml protein. The reaction was terminated b% adding 10 ~1 of 0.1 M EDTA (pH 7.6) containing about 30,000 cpm of ( HI-cyclic GMP (to estimate recovery in the subsequent ss$ps) and boiling for 3 minutes. After cooling in an ice bath, the ( PI-cycjic GMP formed was isolated by sequential chromatography on Dowex 50 Wx4 H (200-400 mesh) and alumina using the modification described in detail previously (7). In this assay system, cyclic GMP production was linear with t@e for 20 minutes and with added protein from 50 to 400 vg. All of the P-containing material was identifiable as cyclic GMP as determined by thin-layer chromatography on PEI-cellulose (Brinkman, Westbury, NY) using 1 M LiCl as solvent and on Chromar sheets (Mallinckrodt Chemical Works, St. Louis, MO) developed with absolute alcohol and concentrated NH40H (5:2 v/v). All reagents were of analytical grade and from the same sources as described previously (7,8). Results Substance this to

P activated

enzyme the

(5,6),

isolated

pancreas

four

fold

in

I).

Substance

in

particulate

Dose soluble

from

where

substance

soluble

it

a variety

of tissues

has

reported

P enhanced small

as

well

response-relationships guanylate

been soluble

intestine,

P caused

cyclase

guanylate

as

to

soluble

that

by substance

142

I).

cyclase

of

fractions

revealed

vitro

increase

liver,

enhancement

in

(Table

guanylate

cerebellum,

a similar

cyclase

the

these

half-maximal

added

to

in addition GMP levels

activity

guanylate

P was at the

Thus, cyclic

kidney,

from

when

two

and

- to

lung

(Table

cyclase

activity

various

tissues.

activation

10 nanomolar

-

concentra-

of

Vol.

128,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

Table Substance

P enhances

soluble

guanylate

Tissue

Without

cyclase

activity

1762 Intestine

in

a variety

of

tissues

cyclase*

GMP / mg protein

substance

Pancreas Small

cyclic

COMMUNICATIONS

I

Guanylate (pm01

RESEARCH

/ 10 min)

With

P added

substance

9

P

P§

390 -+ 14


596 + 25

1193

+ 20


Cerebellum

488 + 22

982

+ 29


Liver

280 + 13

601 + 18


Kidney

291 + 15

655 + 32


1296

Lung

* 5

Each three

value is separate

Significance unpaired

tion

the mean experimen& of

further


+ 43

S.E.M. of triplicate samples on three animals (N = 9). Assay conditions are as described in with

comparisons

controls

was

determined

by

Student's

confirmed the text. t

1). with

Maximal

micromolar

augmentation

enhancement

of

concentrations

of this

guanylate of

enzyme with

cyclase

substance

increasing

test

P.

substance

activity

for

There

was was

no

P's concentra-

P--P-P-P

E

2”

2 loo-

2 2:: 00

0 SUBSTANCE l CONTROL z ,P

FIGURE

in

values.

(Figure

observed

t

5191

+ 36

1.

O

.OOl

.Ol

P

0.1 1 Micromolar

10

100

4 1000

Dose response relationships of substance P on soluble guanylate cyclase activity. Each value represents S.E.M. of triplicate samples from 3 animals in each separate experiments.

143

pancreatic the mean group in

t 3

Vol. 128, No. 1, 1985

FIGURE

Cation requirement for substance P enhancement (at the 1 micromolar concentration) of soluble pancreatic guanylate cyclase activity. Each bar represents the mean + S.E.M. of triplicate samples from three animals in each group inthree separate experiments. The groups without any cations also had 2.5 mM EGTA added to them. With calcium (1 mM) or manganese (1 mM), substance P's augmentation of guanylate cyclase activity was significant Ip
2.

tion

to

ships

were

the

The

millimolar

observed

of

determine

calcium

or

as the

enhanced

by

guanylate

cyclase

utilized

Mn2'

pancreatic from

to the

the

cyclase

tissues

usual of

of

2.5

and

calcium

of

guanylate

concentration

(data

cyclase

cyclase utilized

but

The

data

similar and

both

in this 144

(Figure

substance than

when

increased

shown results forms

are were

of

investigation.

on found

guanylate

P was With

1

could

be

1 mM manganese

by

shown).

co-

stimulated

as the

cyclase not

P

to

Without

2).

cyclase

and 4 mM calcium

were

cyclase

substance

guanylate

basal

the done

cyclase.

activity

stimulation

guanylate

requires were

mM EDTA,

pancreatic

with

enzyme.

quanylate

guanylate

cyclase

less

relation-

of experiments

2,3

activity

guanylate

hormones

some

Utilizing

1 mM Mn2+ .

to

by

the

both

were

2).

P stimulated

other

but

1 mM calcium

compared

guanylate

activity

of this

presence

co-factor, P,

increased

the

dose-response

preparations

activation

guanylate

cationic

(Figure no

in

Similar

A series

manganese,

P's

soluble

substance

(9).

or

and

1).

cyclase

co-factor

manganese

mM calcium

substance

guanylate

substance

stimulate

(Figure particulate

calcium

to

compared

the

of

whether affected

revealed

with

a cationic

factor,

was

range

activation

presence

unable

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

co-factor

substance

Both

P

basal

and

somewhat

with

soluble

pancreatic

with cyclase

4 mM

particulate isolated

Vol. 128, No. 1, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Discussion Although

substance

(pancreas)

previously

present

The

nature

of

effects action tissue.

present

findings

role

induces

of

as

to

the

potency

of

substance

with

parallel

amino

in

spike

itself

able

cervical

than is

thought

Purkinje

cells.

sympathetic

to

be associated

of

guanylate

bullfrog with

GMP levels

a doubling

cyclase,

thus,

P

(10). when

depolarizing (10)

the

which

excitatory

Both

glutamate

in the cerebellum

ganglion

of cyclic is

its

spinal

neurons

glutamate mediate

in

transmission

of

to

substance

The

to

cyclic

one

depolorization

(11).

that

in only

mammalian

to cause

ganglion

higher

of

of

of the

as

mechanism

related

isolated

discharges

is

glycine

the

to increase

been

has also

GMP (131.

consistent

been

Substance

with

substance

P's

guanylate

cyclase,

the

as a neurotransmitter.

respect

to

how

investigation for

substance

guanylate

cyclase.

cyclase

when

cations

all

requirement it guanylate

substance

would

soluble

enhance

motorneurons

be

enzyme

and having

its

application

shown

role

distinguishes

Local

to

the

ubiquitous

ubiquitous)

appear

have

requirement

cation

effects

the

present for

also

of

P activation

present

is

modulations

demonstrated

With

acid

unlikely

(that

other

P being

fibers

Synaptic

possible

P's

much

tissues

given

be very

frequency

P is

the

expect

substance

superior

activate of

with

tissue

P can number

ie,

one (5,6),

a

one might

would

in

GMP metabolism

and climbing

and glycine (12).

rabbit

the

in

what

GMP, likewise,

applied

but

neurotransmitter.

by high

cyclic

cyclic

substance

on a enzyme

of

investigated

are

it

a depolarization

Dibutyryl

that

substance

a

to

pancreas

P (21,

accompanied

along

the

effects

A number

been

respect

so many tissues involve

possible

cord,

in

substance

to

only

indicates

cyclase

in

has

with

investigation

guanylate well.

P

for from

P interacts

indicate P to

were

was

some hormones activity

to no

removed

activation

cyclase

that

be able

There

with

of

is

activate

from

the

guanylate

without

as the any

an

absolute

either

enhancement

such

145

there

of

particulate either

incubation cyclase

or

guanylate media.

by

This

substance

glucocorticoids divalent

cation

which cation

P can

present

Vol. 128, No. 1, 1985 (14).

Although

does

not

divalent

the

obtain

enhancement

the

cofactor

significant guanylate of

BIOCHEMICAL

maximal is

cyclase

a cationic

as has been

increase

in

the

was

the

cyclase

cyclic

depends

line)

requires

the

levels

(15);

vitamin

augment

guanylate

calcium

to enhance

nes

can

activate

respective that

respective the

guanylate

ionic of

D and

cyclase

enzyme

without

mileau of

cyclase-cyclic

different

will

each

to

hormones

and

when

specific

other

for

guanylate (carbamylchocyclic to

GMP

able

to

manganese

or

glucocorticoid ionic

to

manganese

activate

either

whereas

tissue

presence

investiga-

manganese

hormo-

requirement.

determine

do so at a specific

target

present

increase

P requires

helps

therefore,

cyclase at

any

to

need

activity,

of

specific

acetylcholine

metabolites

cyclase

the

not

greater

be able

and substance

(9),

requires

In the

agents

to

a

carbamylcholine-stimulated

since

calcium

environment,

guanylate

mileau

its

guanylate this

ionic

specificity

on the

when

The activation

was

actually

various

seen

a statistically

(14).

(15).

P was

is

cause

hand,

the

kidney

of

presence

ionic

activate

ability

for

glucocorticoids

that

requirement

in the

by substance The

do

other

previously

GMP levels

cofactor. thus

cofactor

with

activity

cations

P, on the

reported

augmentation

of

any divalent

This

RESEARCH COMMUNICATIONS

cyclase

glucocorticoids

by substance cofactor.

guanylate

augmentation

without

calcium,

tion

of

present,

activation

AND BIOPHYSICAL

point

which

The agents

in time.

The

may therefore

contribute

to

agents

activate

the

that

GMP system.

Acknowledgements Dr Vesely is a Senior Fogarty International Fellow supported by the International Center, National Institutes of Health, John E. Fogarty Bethesda, Md., (IF06TW00905=01) who is in residence at the Institut National de la Sante et de la Recherche Medicale, Nice, France. The author thanks Steve Meiners for excellent technical assistance and L. Capolongo for her valuable secretarial assistance. References 1. 2. 3. 4. 5.

Von Euler, U.S., and Gaddum, J.H. (1931) J. Physiol. 7.2, 74-87. Physiology 1, 235-251. Pernow, B. (1981) Clin. Narumi, S., and Maki, Y. (1978) J. Neurochem 30, 1321-1326. and Powell, D. (1975) Biochim. Biophys. Acta 385, 275-280. Duffy, M.J., Albano, J., Bhoola, K.D., and Harvey, R.F. (1978) J. Physiol. 275, 6OP-61P. 146

Vol. 128,No.

1, 1985

8lOCHEMlCALANDBlOPHYSlCALRESEARCH

COMMUNICATIONS

6. Sjodin, L., Conlon, T.P., Gustavson, C., and Uddholm, K. (1980) Acta Physiol. Stand. 109, 107-110. 7. Vesely, D.L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3491-3494. 8. Vesely, D.L. (1981) Am. 3. Physiol. 240, E79-E82. 9. Vesely, D.L., and Juan, D. (1984) Am. J. Physiol. 246, E115-E120. 10. Otsuka, M., and Konishi, S. (1976) Cold Spring Harbor Symp Biol 40, 135-143. 11. McAfee, D.A., and Greengard, P. (1972) Science 178, 310-312. 12. Mao, C.C., Guidotti, A., and Costa, E. (1974) Brain Res. 79, 510-514. 13. Weight, F.F., Petzold, G., and Greengard, P. (1974) Science 186, 942-944. 14. Vesely, D.L. (1980) J. Pharmacol. Exp. Ther. 214, 561-566. 15. De Rubertis, F.R. and Craven, P.A. (1976) Metabolism 25, 1113-1127.

147