Degradation of substance P by neurones and glial cells

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1984

Pages 728-733

DEGRADATION OF SUBSTANCE P BY NEURONES AND GLIAL Bernhard Institut

Received

Horsthemke,

fUr Biochemie FranklinstraBe

October

22,

Michael

Schulz

and Karl

CELLS

Bauer

und Molekulare Biologie, Technische Universitat, 29, D-1000 Berlin (West) IO, W. Germany

1984

and astroblast-rich cultures from rat brain degrade exoSUMMARY: Neuronal genously added substance P. The rate of degradation is decreased by diisopropylfluorophosphate, phosphoramidon and bacitracin, but not by N-ethylmaleimide or bestatin. When diisopropylfluorophosphate, phosphoramidon and bacitracin are simultaneously present in the culture medium, the degradation of substance P is completely inhibited. These results indicate that the hydrolysis of substance P by intact cells is catalyzed by the post-proline dipeptidylaminopeptidase (EC 3.4.14.51, the thermolysin-like metallopeptidase ("enkephalinase", While EC 3.4.24.11) and a yet uncharacterized bacitracin%nsitive activ ity. the thermolysin-like metallopeptidase is mainly associated with gl i al cells, the specific activity of the other enzymes is five times higher in the neuro0 1984 Academic Press, Inc. nal culture.

Substance

P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2)

tributed

in the

central

cells

of the

endocrine mission the

and other

dation

intestine.

forms

inactivation

extracellular

and peripheral

of substance

capable brain,

but their

proline

dipeptidylaminopeptidase substance

EC 3.4.24.11)

P (21,

between

(3),

Gln6-Phe7,

have indicated glial

cells

0006-291x/84 Copyright All rights

P.

function

the

peptide

and that

thermolysin-like the

$1.50 728

in

known about

in

;he degra-

particle-bound

has remained

have been

isolat-

obscure.

A post-

Arg-Pro

and Lys-Pro

(enkephalinase", the

amino-side cleaves

Previous

metallopeptidase

"substance-p-degrading

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

three

enzyme" (4).

found

of acetylcholine,

in vitro

involving

and Phe8-Gly'

is

hydrolysis

releases

and a "substance-P-degrading

the

Little

To date,

dis-

in neurotrans-

metallopeptidase bonds

is also

may be involved

the undecapeptide

(EC 3.4.14.5)

Phe7-Phe'

that (5)

substance

(1).

to the

enzymes

a thermolysin-like

hydrolyzes

Phe8 and Leu"

bound

physiological

and it

seems to be involved

By analogy

of hydrolyzing

ed from

from

P.

released

system,

communication

membrane

of synaptically

peptidases

It

of cellular

or plasma

nervous

is w dely

the

studies, is mainly

enzyme

of Phe7, peptide

however, present

is a mitochondrial

on

1

Vol. 125, No. 2, 1984

enzyme which

(6).

Using

BIOCHEMICAL

intact

cells

substance-p-degrading

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in culture

enzymes

are

MATERIALS

we have now tried present

on neuronal

to determine, and glial

cells.

AND METHODS

Materials: [Phe8- 3 HIsubstance P (20 Ci/mmol) was a generous gift from Dr. R. Wade, Ciba-Geigy, Horseham, England. Phosphoramidon and bestatin were kindly provided by Dr. Umezawa, Microbial Chemistry Research Foundation, Tokyo, Japan. Bacitracin and N-ethylmaleimide were purchased from Sigma Chemical Company. Unlabelled substance P and phenylalanine were products from BACHEM AG, Bubendorf, Switzerland. Oiisopropylfluorophosphate, organic solvents and TLC plates silica gel 60 (layer thickness 0.25 mm) were purchased from Merck AG, Darmstadt, W. Germany. Cell culture: Neurones (7) and astroblast-rich cultures (8) from rat brains were grown on plastic Petri dishes (diameter 50 mm) as described. The protein content per dish was 0.25 mg for the neurones and 0.99 mg for the astroblasts. At the start of the experiments, the cells were washed with 5 ml of HEPESbuffered medium and then maintained at 37" C in 2 ml of this medium, which consisted of 145 mM NaCl, 5.4 mM KCl, 1.8 mM CaC12, 1.0 mM MgC12, 20 mM glucose and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES) adjusted of the medium was 330 mOsmol/l. As estito pH 7.4 with NaOH. The osmolarity mated from the percentage of lactate dehydrogenase in the 100,000 g supernatant of media which had been conditioned for 4 hours, the viability during the experiments was 96 % for the neurones and 98 % for the astroblasts. Degradation of substance P: All incubations were performed at 37" C. When inhibitors were used, the cultures were preincubated without the substrate for 15 min. [Phe8-3 HI substance P (IO ucij was added to the cultures (final concentration 250 uM) and after 0, 30, 90 and 240 min., aliquots of 10 ul were withdrawn from the media and spotted on silica gel plates. After development in ethyl acetate-pyridine-water-acetic acid (100:100:60:20 v/v/v/v), the plates were scanned for radioactivity using a Berthold Linear TLC Analyzer LB 2832. The amount of intact [3H] substance P was then determined by integration with the help of a computer program. Unlabelled substance P and phenylalanine were used as marker substances and localized by spraying the TLC plates with ninhydrin. For determining the soluble substance-p-degrading activity resulting from cell leakage, the degradation of [3H] substance P by conditioned media was also determined. Preparation of cell homogenates: The cells of 10 culture dishes were off in 10 ml of HEPES-buffered medium and homogenized by sonication.

scraped

Lactate dehydrogenase: The activity of the lactate dehydrogenase in the homogenates and conditioned media was determined as described (9). Protein determination: procedure (IO) using

Protein was determined by a modification bovine serum albumin as standard.

cell

of the Lowry

RESULTS AND DISCUSSION Although brain

the is not

represent 11). drolyzed

structural conserved

a useful

When

in

model

L3Hlsubstance

by the

the breakdown

and functional

cells.

of substance

primary for

cultures

studying

P is added The neuronal P than

integrity

glial

of ce 1 interactions

of neuronal

the

inactivation

to the

culture

cultures

are

cultures 729

and glial

in the cells,

of neuropeptides med ium, three

times

it

is

(5,

rapidly

more active

(56 % hydrolysis/h

they

hyin

x mg of

Vol.

125, No. 2, 1984

BIOCHEMICAL

Effect

of

Table on the

inhibitors

inhibitor

AND

BIOPHYSICAL

1 degradation

of

concentration (mM)

RESEARCH

COMMUNICATIONS

substance

P

relative

activity

neurones

no additive

astroblasts

100

100

N-ethylmaleimide

1.0

107

130

diisopropylfluorophosphate

0.1

71

83

phosphoramidon

O.Ul

97

78

bestatin

0.1

bacitracin

0.1

phosphoramidon bacitracin diisopropylfluorophosphate

0.01 0.1 0.1 >

100

105

28

27

0

6

The degradation of substance P was determined as described under values are the mean of duplicate determinations, which differed IO %, and are corrected for soluble enzyme activities resulting leakage.

protein

versa

18 % hydrolysis/h

alanine

rapidly

accumulates

In order

on these

culture

medium

enhances clear.

the

between

cells, (see

x mg of protein). as the

to differentiate

present

various

Table

degradation

individual

inhibitors

The SH-reactive

of substance

phosphoramidon,

which

dase

decreases

(13),

has hardly

also

any effect

inhibitors

the

degradation

tent

by the

did

completely the

inhibits rate

not

lead

leads the

culture.

to stronger P by intact

dipeptidylaminopeptidase

metallopeptidase. 730

this the

in the

and the

is not enzyme 10 uM

metallopepti-

higher

is catalyzed

effect

inhibition.

by the glial

it

slightly

serine

thermolysin-like

inhibition, cells

for

to partial

Since

enzymes

included

inhibits

of degradation

on the neuronal

of substance post-proline

(121,

13Hlphenyl-

N-ethylmaleimide

the reason

which

dipeptidylaminopeptidase

cultures

P-degrading were

agent

P, but

Methods. The by less than from cell

end product.

substance

peptidase

1).

In both

main radioactive

0.1 mM diisopropylfluorophosphate,

post-proline

these

(%)

culture,

but

concentrations

can be concluded only

to a small

thermolysin-like

it of

that ex-

Vol. 125, No. 2, 1984

BIOCHEMICAL

The arninopeptidase of substance This

inhibitor

P, but

result

susceptible

involved

The degradation than

the

of substance 0.1

P is

but

P, these

hibits

with

results

these

results

(15,

clearance

16).

So far,

been characterized

When bacitracin,

pletely

Since

in accordance a mitochondrial

and glial of substance

of the

(which

these

en-

by bacitracin inhibits

the

can be completely

considerably

thermo-

the

the

presence

observations P in vivo bacitracin

blocked

to the degradation of a highly

enzyme on intact

earlier

that (14)

cells.

In

bacitracin

and its

sensitive

for

in Table

mainly

previous

thermolysin-like be involved

compounds

isolated

in-

degradation

activities

this

have

associated reports

P degrading

The phosphoramidon with

on the

glial

cells.

heterogeneous

metallopeptidase in the

studies

simultaneP is com-

any effect

(4),

this

cells.

enzyme cannot This

(6),

on the

result

which

is

indicated

enzyme.

substance 2.

are without

and glial

fractionation

are

of substance

by Lee et al.

of neurones

subcellular

shown

the degradation

three

is

with

zyme may only

these

of the

P is

not

post-proline-dipeptidylaminopeptidase.

indicate

medium,

distribution cells

accordance

the

localization

The cellular

P are

that

and diisopropylfluorophosphate

plasmamembrane

with

compound

however,

enzyme"

on the

This

of substance

culture

"substance-p-degrading be present

effectively

but

shown).

further.

in the

inhibited.

most

the

phosphoramidon

present

(5),

inhibited

not contribute

are

(not

cells

P fragments.

therefore

of degradation

of substance

substance

the

rate

bonds

substance-p-degrading

the metabolic

in vitro

on these

not

does

on the

[3H]phenylalanine

peptide

metallopeptidase

bacitracin-sensitive

agreement

of

mM concentration).

mM phosphoramidon)

active

X-Pro

present

thermolysin-like

of substance

ously

the

metallopeptidase,

by 0.01

not

that

has no effect

formation

in hydrolyzing

70 % at

lysin-like Since

the

to aminopeptidases

zymes are

(more

bestatin

inhibits

demonstrates

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(5,

degradation 731

enzymes

between

sensitive This cellular

neuronal

degradation

observation

is

in

distribution

11) and suggests

that

of neuropeptides

outside

this

enthe

syn-

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 2 of substance-p-degrading

Distribution enzyme activity

enzymes

specific

activity

(m oun'~~tein)

neurones total

56

18

< 1

4

16

3

39

9

activity

phosphoramidon-sensitive activity (thermolysin-like metallopeptidase) diisopropylfluorophosphate sensitive (post-proline dipeptidylaminopeptidase) "uncharacterized

activity

bacitracin-sensitive

astroblasts

activity"

The degradation of substance P was determined as described under Methods. 1 unit is defined as 1 % hydrolysis of substance P per minute under the conditions used.

aptic

cleft,

function also

thus

might

of degrading

synapse,

by 2 enzymes, likely

the

the

the

lead

to the

Therefore,

C-terminal

synaptic

best

candidate

inactivation

functionally

intact

to evaluate

this

which

sensitive

activity

(which

is supported five-fold

higher

aminopeptidase,

inactivation

of substance retains

full

and aminopeptidases

only

biological

concerted

action would

neuronal

and Lys-

however, P but

that

in the

of the Arg-Pro

dipeptidyl

P the

and the

by the observation

removal

which

most

does not to the activity. of the

be required.

bacitracin-sensitive-substance-P-degrading as a peptidase P.

Since

likely

and,therefore,we 732

enzyme

to be important

the cell

immunohistochemical

hypothesis

are

be catalyzed

aminopeptidase)

of substance

of substance synapses,

potentially

The sequential

aminopeptidase active

cells,

P might

is nearly

heptapeptide

highly

the

notion

enzymes

inactivation

dipeptidyl the

is probably

This

biological

The same

on endothelial

dipeptidyl

cultures.

biological

post-proline

of substance

by the post-proline

of the

For the

of both

cell

directly

formation

needed

enzyme.

glial

present

function.

P (17).

post-proline

activity

Pro residues

substance

clearance

diisopropylfluorophosphate

represents

the specific

to enzymes

the degradation

bacitracin-sensitive

than

a more general

be attributed

capable

At the

fulfilling

cultures

and cytochemical are

presently

in the

do not contain studies attempting

are

Vol. 125, No. 2, 1984 to isolate

and to characterize

BIOCHEMICAL the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

bacitracin-sensitive

substance-p-degrading

enzyme.

ACKNOWLEDGEMENT We are grateful to Dr. R. Wade for providing supported by the Deutsche Forschungsgemeinschaft.

['HIsubstance

P.

This

work

was

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

Nicoll, R.A., Schenker, C. & Leeman, S.E. (1980) Annu. Rev. Neurosci. 3, 227-268. Heymann, E. & Mentlein, R. (1978) FEBS-Letters 91, 360-364. Matsas, R., Fulcher, I.S., Kenny, A.J. & Turner, A.J. (1983) Proc. Natl. Acad. Sci USA 80, 3111-3115. Lee, C.M., Sandberg, B.E.B., Hanley, M.R. & Iversen, L.L. (1981) Eur. J. Biochem. 114, 315-327. Horsthemke, B., Hamprecht, B. & Bauer, K. (1983) Biochem. Biophys. Res. Commun. 115, 423-429. Horsthemke, B., Leblanc, P., Kordon, C., Wattiaux-De Coninck, S., Wattiaux, R. & Bauer, K. (1984) Eur. J. Biochem. 139, 315-320. Reiser, G., Lijffler, F. & Hamprecht, B. (1983) Brain Res. 261, 335-340. Van Calker, D., MUller, M. & Hamprecht, B. (1978) J. Neurochem. 30, 713-718. Bergemeyer, H.U. (1970) Methoden der enzymatischen Analyse, Vol. 1, p. 533538, Verlag Chemie, Weinheim, W. Germany. Peterson, G.L. (1977) Anal. Biochem. 83, 346-356. Lentzen, H., & Palenker, J. (1983) FEBS Lett. 153, 93-97. Kenny, A.J., Booth, A.G., George, S.G., Ingram, J., Kershaw, D., Wood, E.J. & Young, A.R. (1976) Biochem. J. 157, 169-182. Malfroy, B. & Schwartz, J.C. (1982) Biochem. Biophys. Res. Commun. 106, 276-285. Lembeck, F., Holzer, P., Schweditsch, M. & Gamse, R. (1978) NaunynSchmiedeberg's Arch. Pharmacol. 305, 9-16. Berger, H., Fechner, K., Albrecht, E., & Niedrich, H. (1979) Biochem. Pharmacol. 28, 3173-3180. Lee, C.M., Arregui, A., & Iversen, L.L. (1979) Biochem. Pharmacol. 28, 553-556. Johnson, A.R. & Erdijs, E.G. (1977) in Substance P (v. Euler, U.S. & Pernow, B., eds.) pp. 253-260, Raven Press, New York.

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