Pharmacology of the forgotten nervous system

iIppfISKllC!3 to human

carwr

ma?

IW

cvolvcd. Inhibition of the wnthesi~cw function of the anrigcn may rcbrc\cnt discriminatory approach

and

a highl!

rather

fcl cirsm&on

specific

of human cancer

cells. It is possible that these antigens ma! bc tht- type of chcmclthcrapcutic that has been searched

‘targel’

for over a long

hcrics of 5,tudies. An

intriguing

opportunity

approaches to thcrapl would

appear

to he associated

antigen!, found in non-tumor noteworthy

for

of human neop!asia with the

tissues. Ir is

that several ‘benign’

tumor\.

which exhibited the presence of the human tumor the

nucleolar

nucleolar

human

antipcn.

antigen

liver.

The

repression

tlctor

in normal

suggestion

made that (hi> antigen ‘modulating’

alho contamcd

found

has hccn

may represent

which

of nucleolar

is involved

If this

function.

were the case. it would

a in

be of particular

interest to attempt to isolate the livtr factors .md cxaminc their possible functions in eithct pcrmcabilized Although

repressive

been rcporred

or intact tumor cells. mechanisms

for nucleolar

have

function

in

several normal ccIlsB. fractions uith specific inhibitory

effects in physiological

have not been demonstrated.

systems

If inhibition

of nucleolar function could be achieved bl administration

of a factor(s)

inhibitory

to

One of the potent:all!

Important

aqx~s

of these stud& hd\ hrcn the demon\trdtion of an antigen in human cancer a-II\ that is not presenr in the rodrnt rumcjr\ wed previous& in our studs and m \lmlldr tumors The

used in tc>tmg anricanier

absence

tumors

of this an&en

supges;s that our currcnr

system\.

control of neoplasia might then be evolved which would be like hormonal control of

rodent tumor>at cme pomf IX amjrhrr.

There

have been interesting

commen-

of \rhlch

arc

~~tmg

normal cells. a mechanism for maintenance

deficiency diseases.

man!

drug\.

in rt\Jcnr bard

on ma)

be inadequate fo te$;t fx drug M’czt\ on Important t!fcments of the hundn ianc‘r‘r

taries on the human tumor nucleolar anti-

process. AccordingI!. ~Juahlc Irug\ m&t> not be detected or drugs !h,it NC &:cLw;~

Fen. One

suggestion uas

may be less useful for humAn c’anccr.

Important.

it would have been discovered

th&

if it Has

earher. Another was that if ir is found in humans but not in animal species. it is probably not important tu the cancer prob-

R. Alan North

This expand

area

IS one Hhich

although

mined I: hethcr immunotherapeuric

it remdin\

certaInI>

~111

ICYbe deter-

immunc~diagnrwtc. or w_wt’nin$ uwr ~111

ever. be moJufated by affcrent and cffi:rent connections which entcric neurons make with ~~ut~?n~~nli~ ;ganglia and spitlaf crrrd. An ~natomicaf substrale for such autonomy can be found in the structure of the gut nerves, which reiembfe much more ~loscly those of the central than ~riphe~1 parts of the ncrvou; system. The indepettdence of function al.%>implied the presence within the enterit nervous sysremof &er= cnt cells (responding to distension by intes= tinaf .:ontentsf. in:egrative cdls (which. amoryg #their things. progam the!~1tphisticated unidirectional faeristafticreflex) and eifferent neurons which are either excicat0~ f almost exclusively cflol~nergic~ or inhibitory Iperhaps utilizing adenosine triphosphate (ATP) or vasoactive intestinal pofypeptide (VW)1 to one or both of

the muscle layers. Mare itten!fy it has beconre cfear that an impressive diversity of neuronal types’. whether cfassilied by elect!rophysiof~gy or ultrastructure. immtutohistochemistry. are av~afable to co-orclinateand effect control c&he mixing and p;,op~sjve fun~tio~sof the gastrointestinal t,act.

Acetyfcholine certainly serscs as the major excitatory transmitter to suth muscle layers. and also mediates thi: synaptic tmnsnI~sion - the ‘fast’ excita::ory postsynaptic potential (ep.s.p.) - between neurons H ithin the myeiatericand submucous pliexuses.which is essential for normal peristaltic activity. In this respct. synaptic transmissionwithin thi: plexusc: is similar to that studied in other autonomic ganglia: just ES nicotinic blockers prevent:transmksion in autonolmic ganglia and cause the expected physiologicaf sequelae, so these agents block tlte peristaltic reflex of the intesttie. The postganglioniesympathetic neurons that reach the intestine terminate mostly’ among the neuronsof the myenteric plexus ratfrer than directly on the smooth muscle layer?,.Stimuiat~)n of these nerves directly relaxes the muscle in mon regions of the gut. but this effect is weak compared with the influences of the inhibitory neurons whiclt are intrinsic to the gut &all. Rather. sympathetic nerve stimulation causespfesynaptic inhibition of acet\-lchofinerelease at the cfrolinergic synapses within the myenteric gangfia’; application of exogenow catecholamiu~s has the same effect *%frin the ganglia and at the sites from which acetylchoIine is released to ad on the musefe layers. it seems likely tkat the potent effe4z.sof The sympathetic nervous system on gastrointestinal motility first

described by Bayliss and Starling are chofocystokinin tctrapeptide; there ir fess largely due to presynaptic inhibition of the good evidence for y=aminobutytic acid and release of the predominant excitatory ~=hydr~?xytryptamit~e.Two of thew pep transmitter from nerve to nerve and nerve tides. VIP and SST. are c~~ntainedwithin to muscle. Vagal influcnccs on gut motility cells which project almost exclusively in an ate presumed to be mediated hy ac~?tyl= anal dire&on -so providing the first struccholine acting on the nicotinic receptors on tural evidence for the polarity of ~~stafsis the myenteric and submucous neurons, first described by Bayliss and Starling and though it is generally considered that the elaborated elegantly by Hit% and Hofman number of such vagal inp& on to myen- at Monash’. From physi~~logica1experiteric neurons must be small compared with ments, Costa and Furness propsed that SST may occur within intemcurons which that of intrinsic cholinergic neurons. on the non-ad~nergjc inhibitor ?%tt:main value of the enteric nervous te~i~te system to pharmacologistslies in the ready neurons. They rven suggested that VIP accessibilityof nervoustissueextraordinar- may act as the inhibitory transmitter conily similar in many respcts to that of the tained in the nerves to the circufar muscle central nervous system. It affords an but there is considerable evidence. though opportunity to study the mwhanism of not universally accepted. that ATP adedrug action in the expectation. thus far fuf- quately fuilils this role. mere is accumulatfilled, that this would prove to be the same ing support for the h~~tl~esis that SP can. at the more therapeutically relevant sites in under some circumstances. act as a tram= the centrat nervous system. Tftis value is mitter between the enteric nerves and the well recognized. and the isolated intestine IongitudinaI muscle. The possibility that any of these subhas long heen staple fare in the educational aknd research diet of the classicaf phar- stances may function as transmitters betmacologist. Until a few years ago the action ween neurons within the enteric nervous of drugs ‘n the enteric nervous system had systemhas been studied by comparing their been studied only by indirect met.hods - effects with those of substancesreleased by stimulation of presynaptic either by observing their effects on the electriCa peristaltic reflex (see the scholarly review nerves. A vari&y of non-cholinergic synap by Kosterhtz and Leesa) or by measuring tic potentials can be recorded intracellutheir ability to cause or inhibit the release larly from myenteric neurons in response of acetyfcholine from nerves of the myen- to such stimufati~~~.and these are about teric plexus which were excited bv efectri- 1000 times sfower in time course than the cal field or transmuraf stimulation. The fast chohnergic e.p.s.p. The slow e.p.s.p. is acet~fchoiine released may be me-asured a de~la~z~g synaptic potential resulting directly. (r. more commonly. by using the from inactivation of the resting potassium contractile response of the longitudinal conductance of the membrane: depofarizamuscle layer as a convenient bioassay. tions of identical ionic nlechanism can be Drug ar=tionshave also been studied more produced in most neurons by sp6. Chymodirectly bv tixtracelfufar and intracellular trypin. which destroys SP, revemibiy prerecordings from the single neurons of the vents the slow e.p.s.p. Other substances enteric nervous system: such techniques present in the plexus. including VIP and offer the advantagesof a direct approach to SST. also depolarize some neurons by inacmembrane actions- which can then be cor- tivating their potassium conductance - as related with the ~pulat~n responseof the does S-hydroxyt~ptam~e (ts-HT). But neurons, and the functional responseof the these agents can either depofarize or intact tissue. Moreover, the simple. two- hypetpolarize different myenteric neurons. dimensional layout of the plexuses affows and there is poor correlation between their such recordings to be carried out with vis- effects on the membrane potential and the ual placement of electrodes wvthin and potential change during the slow e,p.s,p. around ~div~d~al neurons and t& applica- VIP and S§T are present in amounts mush tion of drugs to specific regions of the Iess than those of SP, whilst the evidence neuronal surface. for the presence of S-HT within neurons of

A large number of substances fuffil at least one criterion for transmitter identiffcation in the en&c nervous system namely, their presence within neurons. These include substance P (SP), VIP, ~mat~tat~ (SST). enkephalic (ENK), neurotensin, bombesin and gas&in/

the myenteric plexus is still controversial (although cells certainly exist which can take up and decarboxylate aromatic amines). According to available physiolog icaf knowiedge, the peristaltic reflex is completely blocked by nicotinic antzgonis& therefore, the function of any or all of these non~hofi~e~~ transmitter substances is not proven. Indeed, an over=

whelming need renains

to establish ‘func-

tional’ roles for these substances - as dis-

use massive electrical evoke

transmitter

field stimulation

release

from

lo

a large

tinct from mere presence, retcase and post-

population

synaptic actions.

difficult to interpret as expcrlment\ in \\hich high potassium uMion\ arc cm-

This

will

require

elcc-

tr~)physi~~i{~g~al work in V&O.

of neurons are. alas. almost as

ploycd for the same purpox.

Model system for opiate ectbns

effect on transmitter

‘The findings of Paton and Schaumann that

narcotic

analgesics

choline (ACh)

The findings

can in neither case be extrapolated

inhibit

acetyl-

output from the electrically

to the

release by a single

action potential in a single nerve cell. Hhich is presumably more relevant to our undcrs~nd~g of the effects of morphine in rivn.

stimulated guinea-pig iirum has been fully exploited by later workers. Kosterlitz’s6’

ates and opioid peptides hyperpoiarize

group carefully

the opiate

membrane

antagonist

centrations similar to those that inhibit the

receptor

on

characterized the

basis

of

fntracellular

recordings show that opl-

of myenteric

affinities and rank orders of agonist poten-

acetylcholine

release

cies: those studies indicate that the recep

stimulation*.

Experiments

tar (now

tophoretic

known as the F-receptor)

was

evoked

by

animals including man. but was clearly dif-

the soma membrane.

ferent

ization.

through

which

renaline release is inhibited

norad-

in the mouse

especially

vent excitation field stimulation.

neuroblastoma

in cultures

cells (&receptor).

of

Binding

evoked

of myenteric

varicose

guinea-pig

ileum.

Recently

rhe

it has been

found that the rabbit and rat ileum. tissues in which aceIylchoiine

release is not de-

pressed by morphine, p-receptors - emphasizing

contain

feu

the fortuitous

nerve

transmitter

A mem-

occurring

processes

b)

reduce the

release of acetylchol~ne.

preponderance

in

accom-

neurons

and therebv

brane hyper@arization

e-receptors

Such a hlperpolar-

a5 it is often

studies with radioactive ligands confirm the of

iu gencr-

panied by a fall in input resistance. can pre-

late

is inhibited

mdr-

ated on the cellular processc\ rather than

vas deferens or that through which adenycyclase

irtn-

applicat~~~n of enkephalin

cate that the hyperpolarizaatmn

that

field

uith

pharmacologically indistinguishable from that subserving opiate anti-nociception in from

the

neurons in con-

on the

from

Hhich

is released could also account

for the inhibItion of ACh release. and such a mechanism of action mighr con~~bute IO the presynaptic

effects ot opiates

central nervous

system. In the hyperpc,larization

in the

m!enteric results in

nature of the original choice of the guinea-pig for studies of opiate action. The

neurons.

ilea of the rat and rabbit do contain substantial amounts of et~kephalin. and also

and this action of opiates and opioid pep-

appear to have &receprors.

The pkysiolog-

ical sequelae of activating

b-receptors

the rat and rabbit

intestine

in

have hardly

begun to be investigated. Intriguing morphine

early observations

is more effective

the a~~lc~lol~e

were that

in depressing

release from the guinea-

pig myentexic plexus when submaximal

electrical field stimulation is employed. and that, even when supramaximal stimu. lation is used, the depression of acetylcholine output by morphine can be overcome

by a fitrther

increase

in stimulus

an inhibition of action potential discharge. tides has been the subject of several studies. The inhibition of firing (and underlying hypetpolarization)

is not dependent

on extracellular c&ium ions. and is apparently not mediated by uav crf a reduction in intracelluiar ~noph~~s~hate

adenosine-3

+clic IeAs.

.5

-

The clari;): and ease t~fdern~~nst~ti(~n of these acute effects of opiate3 on m!entrnc neurons let several group4 to investigate the sequelac of long-term rxpohure

tll rtp~-

ate agonists. When experiments arc conducted with ileum which is rctmoued fn\m

guinea-pig which has been made ph?sical& dependent

on morphine during the pry I-

morphine may act by preventing excitation

ous

days,

of a part

be--response curve to morphme is shifted to the right. i.e. tolrrdncc has drwlnped. &I long as the in virrn medium contains an

strength,

These of the

findings

indicated

population

thti.

of enteric

neurons which are releasing acetylcholine during field stimulation. mechanism

of action

Support

for this

is the finding

that

few

it

is

found

that

opiate in order to simulate rhe in viva envi-

morphine does not affect the rate of efflux

ronment,

of tritiated ACh during field stimulation,

marked excitation of myenteric

but does reduce the size of the pool from

which release

which ACh is released. The size’of this pool may simply represent the number of cells brought to threshold for action potential ~~~~. ResuIts from experiments that

the

addition

contracture

of naloxone

sufficient

ACh

of the longitudinal

caust‘s a neurons

to C~)UIC a muscle*.

caused by naloxone is not the result of acetylcholine The neuronal

excitation

released from other Iu~ente~~ neurons -it

TIPS -November 1980

442

Other stud&s Space does not permit even a perftmctory acco\mt of the many studies in which acetylcholine release from emetic neurons has ‘been measured. This release is inhiconcentrations of low bited by S-hydroxytqptamine. even though larger concentraions themselves can bring some enteric neurons to threshold and thereby produce I Iew asynchronous aetion-potential-dependent ACh release (the action mediated by Gaddum’s M-receptor). Adenosinl’ and a wide range of analogs are of acetylcholine effective inhibitors release. Presynaptic a-agonists such as clonidine ate potent. as are muscarinic agonists like oxotremorine. But. whereas the titie of a paper may state that such agents re,luce transmitter release from enteric neurons. consideration mus:l be afforded to the possibility that these agents simplly reduce the number of neurons excited by the given experimental. highly artificial circumstances of electrical field stimulatioo. Such experiments can give valuable information regarding pharmaccrogic II specificity - for example, the effect of cl&dine is blocked by yohimbine but not naloxone - but may be misleading as regards underiying mechanism. Careful electrophysiological studies of membrane actions offer hope of determining mechanism - stsb as the hyperpolarization

observed with opiates - but these studies suffer the substantial limitation of analysing phenomena occurring chiefly on the neuron somata. COIlCIUSIOtlS There acan be few pharmacologists who have never experimented with a segment of isolated intestine. In some controlled experimental circumstances. the preparation approaches a simple nerve-muscle system, and this relative facility of experimentation may have contributed to the general neglect of the other neuronal types and functions over the years. The enteric nervoussystem wasforgotten for fifty years; the most extensive collection of neurons in the periphery (approximately the same number as n the spinal cord) found its way out of the textbooks, relegated to facile explanations of borborygmi. Earlier igttorante of structure and function has been considerably assuaged in the last ten years. and several close structural and functional similarities between the enteric and central nervous r+tems have become apparent. The tissue provides an easy mEthod of studying drug effects on transmitter relczsc from intact neurons, allows drug actions to be studied 9: the membrane level, permits biochemical studies such as radioligand binding, and yet will survive in explants for substantial periods. These advantages a.ffer

hope that the enteric netvous system may be a rich south of further understanding of the mechanism of action of neuropeptides, narcotic analgesics, biogenic amines - and perhaps other drugs undiscovered or already forgotten. Rcadhglrst 1 Fumes\ J. B. and Costa, M. (IYBO) Ncumseirnrc 5. t-20 2 Hirst. 0. D. S. and McKirdy, H. C. (I 974) Nclh.rn (Lnndonj 250.430-431 3 Kostcrlilt. H. W. and Lees. G. M. (lY64) P&maccd. Rev. It]. 301-339 4 Hiixt.6.D.S. (IY79)Br. Med. Bull. 35.263-266 5 Katayama. Y.. North, R. A. and Williams, J. T. (197Y)Pmr.R.~.Lo~nS*r.2206.~91-208 6 Kosteditz, H. W. and Waterfield. A. A. (1975) AMK Rev. Phomrruol. 15.29-45 7 bxd. J. A. H.. Waterfkld, A. A.. Hughes, J. and Kosteditz H. W. (1977) Nanvc (London) 2h7. 495-499 8 North, R. A. and Tonini. M. (I 977) Br. 1. P/wmacol. 61154 l-549 9 Schulz R. and Hen. A. (1976) I.& Sci. 19. 1117-1128 i0 North, R. A. and Kanai. P. J. (1978)in &AICr&tics and Fuxhnu of Opioids (Van Rec. J. M. and Terenius. L. eds), pp. 25-36. Ekvier/No&HoUand Biomedical Pmss, Amsterdam, New York and Oxfurd

is,4sm&te Ro&sor of Phamocdqy 01 h3pl.a Vnivers~ of Chicago. P&r 10 moving to lk Alon North

VnkdStares. hespentscvcmlywsin the hbomrory Hans Koswi~ (u the Vniversiry ofAbe&wz.

of