Cervical gangliectomy does not affect in vitro trytophan hydroxylase activity in rat brain base arteryes

ELSEVlER

TllC

csistencc ilS Well ;IS Ilk

g9rigin

ot‘ ;I st’rc~loncrgic

inllcf\TiUion in nl;ijor ccrcbral artcrics has been ,~lso in\*cstigiItd bv showing the prcscncc 01’tryptc,phiul ISI.JII~x~I~w J’I‘t’OH)

in IIICII~. This

i~~I/Jr~)it~hhigh ~hc ild\-i~n~a~~01’

c~lrcumvcnfing the prohlciii 01’rhc pm~iblc take

by sylllpalhctic

SCt’Cl-ill

groups

hcrolollitl

up-

ncrvc endings.

I1rrk.c rL>portcd the prc\cnc’c01.‘lW)lH-like

iillrli~rtlc~rc;rc~i\~i~y iii Ilc’n’c cIklillg\

prc\cnl iii l11iljO1. c-crc’-

bral art.crics from cat and rat [2.X 121. Regarding ~hc origin The presence 01’serotonin

bouird to nerve endings iii

pial and major cc~cbral blood vcsscls ws

also dcscribcd

of the l’ibcrs contilining TPOH-like

it, Cohen

;\I. [I+] fluted that imlnunorcacticity in rat major ccrcbral artcrics ct

which sllggcsts il

by other reports although they dif’fcxredon the origin of the

disappeared after cervical

putative srrotoncrgic:

serotoncrgic innervation of peripheral origin. but they could

innervation.

gilllgliCCt~illy.

[Jsing ii similar cxperi-, mental approach as described before, scrotonin was found to be associated in rat pial vessels to fibers of central

not show the same kind of immunoreactivity in supcrioi cervical ganglia. On the other hilnd, Mathiau ct :.I!. [ Iz]

origin, but not to sympathetic nerve endings [S. 181. On the

discarded that this TPOH-like

other hand, superior cervical ganglia removal as well as

the prcscntx of 11 true cenlral scroloncrgic inncrvalion

dorsal and median raphc nuclei destruction

rat major ccrcblal artcrics

significant reduction of scrotonin braI arteries. suggesting that fclinc cerebral arteries might receive serolonergic innervation of dlliki origin [ IO]. Howcvcr. (hcsc results wcrc ;hallcngcd by morphologi-

fro111 dorsal raphe II~C~CUS by

brought about ;I levels in cat major ccrc-

cal reports indicating that S-HT chemistry

revcalcd by immunohisto-

in major cerebral arteries of

scvcral animal

species was only the result of its uptake from blood by

(NM)6-sr1~.1/97i$l7.cH) Copyriphl PI/

SOOOtJ-8993(9h)O

1382-O

:Q tW7

glutinin

iri~lnuiic,rcl:cti\.IIty indicztcd in

because anterogradc tracing

PIMsw~~~s r~l~l,~c~~.i.~ I~UCOLI~and scrotonin inlinuliocytochcnlistlv rcvcnled no

seroloncrgic projections from vessels of rat. However.

ruphe nuclei to large pial

they did nr)l cxplorc

whcthcr thar was related 10 sympathk

imnlunorcactivity TPOH-like fibers. TPOH activity has been measured in ceil-free extracts of rul brain base arteries [ 131. This cnzymrrlic activity was at least rclatcd to an innervation of central origin. since thC destruction of dorsal raphc nucleus, bul not that of median rqthc nucleus. decreased it. However. the possible exis-

Elwvier ScietlLT R.V. All righlh resend.

tellce of enzyIne suprior haS

cervical

&en

alSo

Cerebral

associated

with

fibers originating

ganglia was not explored.

TPOH

it1 cell-free

of

assayed

arteries

and found

[ 131

e?dkXtS

lessened by lesions of the brainstem gangliectomy.

Cervical

Cat

nlilj
in ncrvc

origin.

8s it was

nuclei and by Superior

Furthermore,

atso appraised in cell-free

activity

to be located

endings of fibers of central and peripheral

from

enzyme

activity

extracts of cat superior

was

cervical

ganglia. The aim of the present work was to explore whethe,: rat cerebral arteries receive a serotonergic

innervation

i.Irisil:g

the animals.

in Eppendorf

tubes containing

300 ~1 of 0.4

cervical ganglia. To achieve ihis, TPOH

N HOCI,

with i).OO2% of ascorbic

acid. homogcni/.cd

activity was assayed in brain base arteries ;1s well ~1s in

sonication

and spun. Noradrenaline

was assayed in wpcr-

superior

natants. The pellets were resuspended

from

superior

cervic:!I

ganglia

from

ml/kg.

rats. weighting

with 30% chloral

i.p.1 and superior

aseptic conditions.

until tion.

ganglia

The sham-operated without

removed

under

group was submitexcising

Ihc ganglia.

were frozen on dry ice and stored at - 15°C

performing

surgical

130- i 80 g. were

the enzyme

procedure,

The

brain

assay. Two

weeks after the

the rats were sacrificed

was removed

by decapita-

and the circle

arteries and its branches dissected

of

Willis

out, frozen on dry ice

wiIs iIssiIycd

a~ indicated

elsewhere

tissues were homogcnizcd

by sonicntion

M Tris

containing

(pH

7.4) buffer

ethiIn(ll and O.OSc/cTriton

141. The

[

10 ’ M

Inercapto-

centrifuge

iIt I2 000

P SCICCliI).ChX hundred @I of SllpC!IliltillltS WL‘I’Cilddtd ~1 of iI stiIndiIrd rcuction

mixture

Tris (~1-1 7.4) hut’fcr. tryptophiul drtrptcrinc

(

I6 mM),

luse (2.5 ~ngJmI).

The smplcs arId 5-HTP liquid

Proteins

first

honIogeIIrrtes

HPLC

20

after 90 min ol’ incubation ut 37°C.

(HPLC)

with

for 3 nlin

by high pre:;sure

ODS. 5 micron.

LLGILSON

model

reference

in the rel’ersc

Brownlce

141 electrochemi-

electrode.

set at -t- 0.72 V

The Inobile

w;Is mi\.de of 0. I M citric aci< .‘0.2 M disodium ( I : 1) n ith 0.3 HIM rI-octyl

sodium

eluted

sulfilte

under

Two

weeks after gitngliti

showed

iI reduced

and iI final pH

these conditions

iIt

removal.

noradrenaline

rat cerebral

content

artc’rics

whereas TPOH

iulilllalS (‘l’i~hle I 1.

TPO, -1 ilcliVit>. cXNlld !I()( hb nic~lSllrcd in illdividuill g;lllglii\

II = 3) or when three g;urglii\

1: vc gangli:I

\vh

prot/h.

wcrc pooled for Ihc

were

pooled

(8.07 rf: 3.47

~IIIO~/III~

11= 8).

Thes : results indicitte cervical

ganglia

that the contribution

to the overall

bral artlbries is either

TPOH

nil or very

of superior

activity

small,

of rat cere-

because ponglia

remova

in the prccipitatcs

of the

of ! .owry et ill. [7,9].

enzymrtic nctivity found in the ganglia was much lower fl>‘)!- ‘hat present in the blood vessels which reduces the

referred

ilTlpWtiulce of il putative serotonergic innervation of pe-

being

to them.

module (GILSON.

The

model 305)

model SOS). giving

iI Rheodyne

injectiorl

valve

ripheral neurona

activity

C , ,/Cora-

that

affect

it. Furthermore.

cervical

gtingli;;

Ci111 be

of

the TPOH

located

in

activity

measured

intragaI@ioniIr

cells [ 191. The luck of a functional

serotonerpic

innervation

trophotometer. Hitachi) at 286 nm excitation and 343 nn: emission wavelengths. The mobile phase w;1s prepared xcording to Lxkovic et al. [8] with 4% methanol.

is also supported

of the ganglia

removal

was checked

animals by measuring noradrenaline levels in cerebral blood vessels. These were placed, after sacrificing

cerebral

arteries, [

though

peripheral

on rat cerebral arteries

by the fact that superior

does not affect

noradrenaline

saotonin it signific;Intly

uptake

cervid

gan-

in rat major

decreases that ot

I].

The low TPOH ganglia

impinging

III the

serotonin-

synthesizing

gliectomy

S-HT

to this, OIIC must bear in

[IS]. in iiddition

SOIIW

have ti low

S-Hydroxytryptophan was detected with ;I spectro11uorornetric detector (F-2000 Fluorescence SpecWrrtets).

the

This latter finding agrees with data show-

origin.

rat superior

giulglii1

(p BondiIpiIk

does not significantly

ing that

C Is. Waters) \;$lith guard column

in some

phosphate

itctivity was similar to that found in cerebral hloc!~J Vessels

miIId

The effectiventiss

phase

8:1S iii n.

with a 20 TVI hop and a reverse phase column ( PBoIldiIpak Sil.

Labs.)

dercc.-

system consisted of a pump (GILSON.

LI flCW rate of I.200 Inl/min.

described

The sys-

fluorolnetric

by the method

t’inal concentrations

detection.

with 3 glitssy carbon electrode

vs. an Ag/AgCI

high prcssurc

1’11L~lllCilSSiiy (II = 3). SOlllC Cll/~IllilliC ilCliVity WiIh liNllld

Ci\ti\-

was stopped by adding

were determined

with a manonietric

iII 0.05 M

( I IIIM). d

IllcrciIptoethiIIlol

was assayed in supernatants

(ion.

S-HTP

coIltiIitlIng.

wcrc centrifuged ilt I2OOO r.pm

chromstogtaphy

and in the detection.

(0

(2 rnM), ~-tnethyltctrrthy-

The rctiction

p.1 ol‘ 11.64 M HCIO,

from the previously

1’l’~~lll41 Im-operated

(Mcdil’rigcr.

conc’cI~tr;ifioIi~

by ion-pair

with clcctrochcmical

phase column (SPHEKI-S

in 300 PI of 0.05

IOU-X and centrifuged

r.p.rn. for IO nIin in iI refrigerated 20

was mci\sIIrcd

chror.Istography

cal deti:ctor

for

to them

Nl jradrenaline tem c iffercd

in 0.5 N NuOH

l’inal iioradrcnulii~c

of 4.0( . Noradrenaline

and stored tit - 15°C. TPOH

bciny referred

hydrate in saline soh.Ition ( 1

cervical

ted to the same procedure The gq$ia

to cervical

protel n determination,

gangliectomy. Male Sprague-Dawley anesthetized

rats submitted

by

activity

might explain

assayed in rat superior cervical

the absence of TPOH-like

immuno-

-.

‘r

-_

--T 7 -

-z 3

-

-C -

-

Cl

_-

-.

-