PAPERS

Nitrinergic and peptidergic innervation of thehuman oesophagus

C Singaram, A Sengupta, M A Sweet, D J Sugarbaker, R K Goyal

Division ofGastroenterology,William S MiddletonVA Hospital,University ofWisconsin-Madison,Wisconsin, USAC SingaramM A Sweet

Division of PediatricGastroenterology andNutrition, BostonUniversity School ofMedicine, Boston,USAA Sengupta

Department ofSurgery, Brigham andWomen's Hospital,Boston, USAD J Sugarbaker

Division ofGastroenterology,Beth Israel Hospital,Harvard MedicalSchool, Boston, USAR K Goyal

Correspondence to:Dr C Singaram, Division ofGastroenterology, H6/5 10,CSC University of WisconsinHospitals, 600 HighlandAvenue, Madison, WI53792, USA.Accepted for publication11 March 1994

AbstractThe distribution, colocalisation, and inter-connections of nitrinergic and peptidergicneurons and nerves in the human oesoph-agus were examined. Cryosections ofsurgically resected tissues from eight sub-jects were studied with indirect immuno-fluorescence for the presence of 11neuropeptides and neuron specificenolase. After immunohistochemistry,nitric oxide synthase was shown on thesame sections with the ,B nicotinamideadenine dinucleotide phosphate (NADPH)diaphorase histochemical reaction. Thehistochemical findings were verifiedimmunohistochemicaily on other sectionswith an antiserum against nitric oxidesynthase. Most myenteric neurons (55%)were nitrinergic. Most (96%) receivedterminations positive for vasoactiveintestinal polypeptide (VIP), calcitoningene related peptide (CGRP) (80%), andgalanin (59%). The neuronal somata of140/o also contained VIP, while 10% hadgalanin. Of the NADPH-diaphorasecontaining fibres seen in the musclelayers, many had closely associated VIPand galanin, but only rarely CGRP andsubstance P. Thus, despite abundantrepresentation of both peptidergic andnitrinergic systems in oesophageal smoothmuscle, only VIP and galanin colocalisedto any significant extent with the nitri-nergic elements. These findings providemorphological support for the role ofnitricoxide as the non-adrenergic non-choliner-gic inhibitory mediator in the humanoesophagus and for its possible interactiverole with the peptidergic system.(Gut 1994; 35: 1690-1696)

The chemical nature of the nerves responsiblefor relaxation of the oesophageal smoothmuscle, lower oesophageal sphincter, latenciesof oesophageal peristaltic contractions, andoesophageal 'off contractions is not known. Asthe mediator of these nerves is neither anadrenergic nor a cholinergic substance, theyare categorised as non-adrenergic non-cholinergic (NANC) nerves.1 Functionalstudies have suggested that the peptides,vasoactive intestinal polypeptide (VIP), andcalcitonin gene related peptide (CGRP) may

be candidate NANC inhibitory neurotransmit-ters in the oesophagus.2-4 Morphologicalstudies in the human oesophagus and inexperimental animals show abundant VIP andCGRP in the myenteric plexus, and VIP andCGRP positive fibres in the muscle layers.5-12

It has now been shown that nitric oxide or arelated product of the L-arginine-nitric oxide-synthase pathway may participate in theoesophageal smooth muscle relaxation,latencies of oesophageal contraction, andoesophageal off contraction in the opossum aswell as in humans. 13-16 Furthermore, theinvolvement of the nitric oxide synthase path-way in swallow induced peristaltic contractionsin the opossum have been shown.17 Also, elec-trophysiological studies support the role ofnitric oxide as the inhibitory mediator in theoesophageal smooth muscle. 18-23 In sharp con-trast, there is no morphological information onthe nitrinergic innervation of the humanoesophagus.

It is possible that the inhibitory neurotrans-mitters VIP and CGRP may act in concert withnitric oxide in the oesophageal smooth muscle.In some tissues neurally released VIP isthought to exert its effect by releasing nitricoxide from the smooth muscle.24 Inhibition ofnitric oxide synthase has been shown toenhance CGRP induced relaxation in vascularsmooth muscle.25 26 It is known, however, thatat least in the oesophageal smooth muscle, theinhibitory action of VIP or CGRP does notinvolve nitric oxide.14 Recently nitric oxidesynthase activity has been reported to becolocalised in the VIP reactive neurons in theguinea pig ileum.27The aims of this study are to characterise

the nitrinergic innervation of the humanoesophagus by (a) identifying nitric oxidesynthase containing neurons and nerve fibresusing the NADPH diaphorase reaction as wellas a specific antiserum against nitric oxidesynthase; (b) to investigate the colocalisation ofNADPH diaphorase and various peptides inmyenteric neurons; and (c) to evaluate thepresence and colocalisation of nitrinergic fibreswith various peptides in the external musclelayer of the human oesophagus.

MethodsTissues were obtained from the loweroesophagus of eight subjects (five male and

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Nitrinergic and peptidergic innervation of the human oesophagus

three female) aged 38-66, who had surgicalresections for various malignancies. The tissuesamples were obtained from between five and15 cm proximal to the squamocolumnar junc-tion. These patients had no motility disordersand were also free of diabetes, scleroderma,and other degenerative neurological disorders.Tissues were taken from sites at least 4 cmaway from areas showing the presence oftumour cells as shown by cryosectioning andrapid haematoxylin and eosin staining. Themucosa delaminated or was removed bypeeling in most cases. Fixation was by immer-sion in cold buffered picro-formaldehydefollowed by thorough rinsing in phosphatebuffered saline and overnight immersion in15% sucrose enriched phosphate bufferedsaline. Tissues from each patient werefreeze blocked in OCT Tissue-Tek (MilesDiagnostics, Elkhart, IN) in two orientationsso as to permit cross sections of both longi-tudinal and circular muscle layers. Tissuesfrom five of these eight patients have also beenused in a previous study on oesophagealpeptidergic innervation.12

Several sets of slides were prepared fromeach patient. Each set contained about 15slides and each slide bore at least five 16 ,umthick cryosections. One slide from each set wasinitially incubated at 40C for four hours withpolyclonal rabbit antiserum samples raisedagainst cholecystokinin, CGRP, galanin,gastrin releasing peptide, leucine enkephalin(L-ENK), methionine enkephalin (M-ENK),neuropeptide Y, peptide YY, somatostatin,substance P, and VIP (all from PeninsulaLabs, Belmont, CA). Details of antiserumsamples and their dilutions are as reportedpreviously.28 Rabbit anti-neuron specificenolase (Polysciences, Warrington, PA) wasused similarly at 1:1000. After thoroughrinsing, the second incubation was done ingoat antirabbit fluorescein isothiocyanate(Calbiochem, La Jolla, CA) (1:60, one hour,room temperature). All immunostaining wascarried out with a Shandon 'Cadenza' auto-mated immunostainer (courtesy Shandon,Pittsburgh, PA). Sites of antigenic bindingwere visualised under epifluorescence withan Olympus BHT-2 research microscope(Olympus, Tokyo, Japan).

After immunocytochemistry, the sectionswere rinsed and subjected to a histochemicalexamination of NADPH diaphorase. Afterthorough rinsing in phosphate buffered saline,they were incubated with 0-25 mg/ml nitroblue tetrazolium+ 1 mg/ml P nicotinamideadenine dinucleotide phosphate, reduced,tetrasodium salt (NADPH) (both from SigmaChemical, St Louis, MO)+05% TritonX-100 in 0-1 M TRIS buffer, pH 7-6, for15-20 minutes at 370C.27 The reaction wasstopped by washing in tap water. The methodstains sites ofNADPH diaphorase, indicativeof nitric oxide synthase, a dark blue.29-31Sections were then coverslipped in carbonatebuffered glycerol, and enzyme and antigenpositive structures were counted (respectivelyunder brightfield and fluorescence) andphotographed.

Several sections from three oesophaguseswere incubated with an antiserum againstnitric oxide synthase (Gift of Snyder andDawson of John Hopkins University). Thispolyclonal antiserum was raised against the Cterminal 14 amino acid sequence to neuronalnitric oxide synthase and has been shown pre-viously to be specific for all neuronal nitricoxide synthase including that in the gastro-intestinal tract. The lyophilised antiserumwas diluted 1:50 and incubated with 4%paraformaldehyde fixed sections for 24 hoursat 370C. This was followed by an antirabbitfluorescein isothiocyanate conjugate. Most ofthese sections were then stained for NADPHdiaphorase as already described to find out ifboth methods stained the same structures.As the sharing of immunodeterminant

sequences by more than one moiety cannottheoretically be excluded, a statement in thispaper such as 'CGRP immunoreactive' or'CGRP positive' means that tissue showedCGRP like immunoreactivity. Except for thecolocalisation experiments with NADPHdiaphorase and the nitric oxide synthase anti-serum, all other findings were made using theNADPH diaphorase histochemical reaction.NADPH diaphorase histochemistry waschosen over nitric oxide synthase antibody incolocalisation studies because the bright bluereaction particle could easily be studied alongwith peptide immunofluorescence in quantita-tive studies with both small amount of brightlight as well as the ultra violet light and onlyoccasionally was there a need to shut one orthe other light completely off.

QUANTITATIONAfter the myenteric plane was identified, thetotal number of NADPH diaphorase positiveneurons was counted per 20X field usingbrightfield transillumination. Fluorescenceepi-illumination was then used on the samefield to enumerate the peptide (or enolase)positive neurons. Because the blue stain forNADPH diaphorase was visible against thefaint fluorescence background, it was possibleto count neurons negative for NADPHdiaphorase but positive for various peptides.Another field was then selected until a count ofone 'unit' (100 or more NADPH diaphorasepositive neurons) was reached. The peptideneuronal counts were then normalised to 100NADPH diaphorase neurons. At least five'units' (500 nitrinergic neurons) were countedfrom each slide, and at least four slides werecounted from each patient for each peptideexamined. A simple mean was calculated fromeach patient and the standard deviation andmeans from eight patients were calculated bythe single Student's t test on a Minitabstatistical program (State College, PA).

CONTROLSA series of controls were carried out. Thesequence was reversed for a few experiments(enzyme reaction first, immunohistochemistrysecond), and counts taken as before to rule out

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I1

A

Figure 1: The same field showing the human oesophageal intermyenteric plane underbrightfield illumination after staining histochemically forNADPH diaphorase (A) andimmunohistochemically for neuron specific enolase (B). All neurons that stainedforNADPH diaphorase also stainedfor neuron specific enolase (some arrowed). Enolasestained showed additional neurons that did not stain forNADPH diaphorase(arrowheads).

the theoretical objection that immunohisto-chemistry for peptides may compromisethe subsequent enzymatic reaction. In theseexperiments identical results were obtainedregardless of the sequence.

Several sections 'kissing sections' from twooesophaguses were studied for the theoreticalobjection that the NADPH diaphorase stainingmay be interfering with the number of peptidecontaining neurons. On one of these mirrorimage sections only NADPH diaphorase reac-

tion was performed and the other sectionunderwent both NADPH diaphorase as wellas immunohistochemistry reaction to VIP.Several areas from each section was carefullyevaluated for the number of VIP positive neu-

rons. These studies showed that there is no

decrease in the number of peptide neuronswhen the sections are simultaneously stainedfor NADPH diaphorase.One step controls for the enzyme reaction

were performed (elimination of NADPH or

nitro blue tetrazolium salt), which yielded neg-ative results, confirming the validity of theenzymatic localisation.The substitution of NADH for NADPH

totally abolished the staining.5

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Figure 2: The same field showing NADPH diaphorase staining (A) andimmunofluorescent localisation of VIP (B). Arrows point to neurons that show both kindsofpositivity, while arrowheads point to those that are only nitrinergic. T-peptideterminations on NADPH diaphorase positive, VIP negative neurons. (C) and (D) Thesame field from oesophageal intermyenteric plane stained histochemically forNADPHdiaphorase (C) and immunohistochemically for galanin (D). Arrows point to neuronscontaining both substances, arrowheads to those that have only NADPH diaphorase orgalanin. Note also the galanin containing terminations (T) on some nitrinergic neurons(D). Bar=50 Am and applies to all parts ofFigures 1 and 2, exceptfor 1(A), 2(A), and2(B), where it equals 100 ,um.

Testing for specific time and temperaturefor the NADPH histochemical reactionshowed that the specific staining of theneurons that colocalises with nitric oxidesynthase antibody staining was seen onlybetween 15-30 minutes at 37°C. Incubationlonger than 30 minutes or other temperaturestested resulted in non-specific staining ofseveral neurons and other non-neuronalstructures.

All immunohistochemistry results were sub-jected to omission of the first antibody thatyielded negative reactions except for non-specific background.

All immunohistochemistry reactions forpeptides were confirmed by preabsorption oftheir antiserum samples with their respectiveantigens (10 and 100 ,ug/ml working strengthprimary antiserum), with negative results.The research protocol has been approved by

the committee on clinical investigations, BethIsrael Hospital, and the human subject com-mittee of the University of WisconsinHospitals.

Results

MYENTERIC NEURONSNADPH diaphorase positive myentericneurons were clearly identifiable by theirsharply defined dark blue cytoplasmic stainwith almost no background; the nucleiappeared as colourless 'holes'. They wereabundantly present in the oesophageal myen-teric plexus (Fig 1). For every 100 neuronscounted with neuron specific enolase (totalneuronal population), mean (SEM) 55-8(14.8) were NADPH diaphorase positive.Experiments for colocalisation with peptidesyielded clearly differentiable populations ofNADPH diaphorase positive, NADPHdiaphorase and peptide positive (Fig 2) andonly peptide positive neurons.

For every 100 NADPH diaphorase neurons,there were 27-4 (11-6) VIP, 34.9 (9 3) galanin,21-6 (7 5) CGRP, and 13-9 (6-4) substance Pneurons. Of every 100 NADPH diaphoraseneurons counted, 14 1 (3-5) also containedVIP, while 10-5 (1 6) contained galanin (Fig3). Only VIP and galanin colocalised to anysignificant extent with NADPH diaphorasepositive neurons, while other prominently pre-sent peptides such as CGRP, substance P, andneuropeptide Y did not. Other peptides suchas M-ENK and somatostatin colocalised withless than 1% of the nitrinergic neurons.

TERMINATIONS ON MYENTERIC NEURONSMany NADPH diaphorase positive neuronsreceived terminations containing peptides.For every 100 NADPH diaphorase positiveneurons counted, 92-4 (7 3) received termi-nations containing VIP (Fig 2), 79*3 (4-8)received terminations with CGRP, and 64-4(1 6-5) received galanin immunoreactiveterminations (Fig 4). These were seen asbright fluorescent points attached to thesomata of the neurons in question, and often

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Nitrinergic and peptidergic innervation of the human oesophagus

501Peptide neurons

| |_ Colocalised neurons

VIP GAL CGRP SP NPY Pyy M-ENK L-ENK SOM GRP

Figure 3: Numbers of neurons containing various peptides in human oesophageal myentericplexus and numbers ofpeptide containing neurons colocalised with NADPH diaphorasepositive neurons per area containing 100 NADPH diaphorase positive neurons.GAL=galanin, SP=substance P, NPY=neuropeptide Y, PYY=peptide YY,

SOM=somatostatin, GRP=gastrin releasing peptide.

their fibres were visible for a short distance(Figs 2B and 2D). Substance P (09 (08))and M-ENK (0.7 (0 3)) also sent termina-tions to somata of nitrinergic neurons, butneuropeptide Y, peptide YY, L-ENK,somatostatin, gastrin releasing peptide, andcholecystokinin provided very few, if any,terminations on NADPH diaphorase positiveneurons.The peptidergic neurons in turn received

NADPH diaphorase positive terminations, butthese were difficult to study with as muchclarity because of the presence of a bright'halo' effect of immunofluorescence seen

around the brightly stained neurons. It seemedsubjectively that most peptidergic neurons

received NADPH diaphorase positive fibresand terminations upon their somata.

FIBRES IN CIRCULAR MUSCLEThe human oesophageal circular muscleshowed abundant NADPH diaphorase inner-vation in the form of varicose fibres. Several ofthese fibres were associated with VIP (Fig 5)and galanin. CGRP and substance P, even

though significantly present in the circular

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Figure 4: Numbers ofNADPH diaphorase positiveneurons, per 100 counted, which received terminations fromvarious peptides. Note that many nitrinergic neuronsreceived terminations containing several peptides.Abbreviations the same as in Figure 3.

muscle, were only rarely seen in close associa-tion with the nitrinergic fibres.

FIBRES IN LONGITUDINAL MUSCLEThe density of fibres containing NADPHdiaphorase positive fibres seemed to be lessthan in circular muscle. Consistent with our

earlier findings very few fibres containingL-ENK, somatostatin, and gastrin releasingpeptide were found in the longitudinal muscle.CGRP, galanin, substance P, and neuropep-tide Y were found but none of them were seen

to be in any close association with theNADPH diaphorase positive fibres in alloesophaguses studied. Several of the VIP andgalanin positive fibres similar to that seen inthe circular muscle were once again found tobe closely associated with the nitrinergicfibres.

SUBMUCOSAL NEURONS AND FIBRES

The submucosal region could not be includedin this report as it tends to delaminate from themuscularis externa before fixation, and inmany cases was separated to permit good sec-

tioning of the thick oesophageal musculature.From fragments attached to the luminal face ofthe circular muscle, however, it can be saidthat submucous neurons in general tended tobe less frequently NADPH diaphorase positive

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Figure 5: Oesophageal circular smooth muscle. The same field stainedforNADPH diaphorase (A) and VIP (B). Arrowspoint to some fibres showing colocalisation of these substances. Bar=50 ,um.

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Singaram, Sengupta, Sweet, Sugarbaker, Goyal

than myenteric neurons, and nitrinergic fibres,though unquestionably present, seemed to beinfrequent.

NITRIC OXIDE SYNTHASE AND NADPHDIAPHORASE STAININGThe antibody to nitric oxide synthase stainedseveral of the myenteric neurons in the humanoesophagus. All these neurons were alsostained by the NADPH diaphorase reaction.Also all the neurons that stained positively forNADPH diaphorase were positively immuno-stained by the anti-nitric oxide synthase(result not shown). This close association wasverified in at least 100 neurons from threesubjects.

STAINING IN THE MUSCLENo specific staining was seen in the musclefibres usingNADPH diaphorase histochemicalreaction or using the antiserum against nitricoxide synthase under our experimental condi-tions. All the cells, however, including themuscles showed non-specific blue staining ofvarying intensity on prolonged incubation withNADPH and nitro blue tetrazolium salt.

DiscussionThis study used neuron specific enolase stain-ing as a marker for all myenteric neurons,NADPH diaphorase staining as a marker fornitric oxide synthase in colocalisation experi-ments, and the standard fluorescent antibodytechnique to localise VIP, CGRP, galanin9andother peptides. Though the precise function ofneuron specific enolase remains unclear, it isknown to stain neurons and their glial cells,and is accepted as a functional marker forneuronal differentiation.32The NADPH diaphorase staining technique

has long been used to stain certain populationsof neurons.33 NADPH is essential for theactivity of nitric oxide synthase, which partici-pates in neurotransmission and neuromodula-tion. It was shown recently that neuronsaccessible to immunostaining with antibodiesagainst nitric oxide synthase also stain with theNADPH diaphorase reaction.29 30 Althoughenzymes other than nitric oxide synthasepossess diaphorase activity, studies haveshown3l 33 that all neurons in the central andperipheral nervous system that stain fordiaphorase activity also show nitric oxide syn-thase activity. It is thus evident that NADPHdiaphorase staining under these conditionsprovides a reliable marker for nitrinergic (nitricoxide synthase containing) neurons and theirfibres. Also our experiments on colocalisationof NADPH diaphorase and anti-nitric oxidesynthase staining in the human oesophaguscorroborate the close association that we haveseen between these two kinds of staining inseveral other tissue systems.

Certain limitations upon technique areimposed because this is a human study. Forinstance, pretreatment with colchicine was notpossible. It was also not practical to make

whole mounts (peels), and thus distributionwas studied from sections alone. Despite theselimitations, animal studies have shown that theimmunohistochemical reactions of peptidescorrelate very closely with results of in situhybridisation for mRNAs of certain peptidessuch as VIP and substance P. Thus in humanmaterial, even though a statement regardingthe positivity of a neuron for a given peptidecan be made with certainty, the absence of apeptide in a particular neuron should be takenwith caution.Most interestingly, VIP (96%) and CGRP

(80%) terminations were seen on the nitriner-gic neurons. On the other hand only 14% ofthe NADPH diaphorase neurons also had VIPand only 3% of the neurons had CGRP inthem. The abundance of these terminationssuggests a regulatory system, where VIP andCGRP may act by controlling the release ofnitric oxide from nitrinergic neurons. VIP andCGRP have been presumed to serve such afunction. VIP has been shown to release nitricoxide from within the smooth muscle cells,however, but not from the nerve fibres.24 Thesite at which CGRP releases nitric oxide isnot known. In any case, in the opossumoesophageal body, inhibitors of nitric oxidesynthase do not modify the inhibitory action ofeither VIP or CGRP.20 Further physiologicalstudies are needed to explain the functionalsignificance of the morphological interconnec-tion that are described in this study.

This study shows that NADPH diaphorasepositive neurons are widely distributed in themyenteric ganglia of the human oesophagus.Moreover, abundant NADPH diaphorasepositive nerve fibres were seen to supply theoesophageal circular muscle. These findingsprovide a morphological support for functionalstudies'3 14 suggesting that a product of the L-arginine-nitric oxide synthase pathway may bean NANC inhibitory neurotransmitter in theoesophagus. These experiments showed thatinhibition of nitric oxide synthase by analoguesof L-arginine (for example, L-NNA and L-NAME) caused (a) a decrease in the latencyand amplitude of the oesophageal 'off' contrac-tion; (b) a decrease in the latencies of contrac-tion during physiological swallowing inopossums, and (c) antagonism of hyperpolari-sation of the oesophageal smooth muscle,which follows electrical stimulation of theintramural nerves.

Nitrinergic neurons accounted for some-what more than half of the total myentericneurons identifiable by neuron specific enolasestaining. The other clearly non-nitrinergicneurons may include cholinergic and pepti-dergic neurons. The comparative abundanceof the total peptidergic neuron population incontrast with the nitrinergic one is difficult toestimate because (a) peptides colocalise withnitrinergic neurons, as shown by this study,and (b) when a neuron is purely peptidergic, itmay contain a multiplicity of peptides. Thisstudy did not include triple localisation for twopeptides and the NADPH diaphorase reactiveneurons. This study shows, however, thatnitrinergic neurons were five times as

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abundant as VIP neurons and six times asabundant as CGRP neurons. On the otherhand, the number of nitrinergic nerve fibresinnervating the oesophageal circular musclewas similar to that of some ofthe peptide fibresexamined such as VIP and CGRP fibres. It isnot clear whether this difference in thecomparative proportions of nitrinergic, VIPcontaining, and CGRP containing neuronsand fibres is significant from a physiologicalpoint of view.

Colocalisation studies on the myentericneurons yielded information on the coexis-tence and interconnection of the peptidergicand nitrinergic systems of the oesophagus. Ourfindings showed that most of the nitrinergicneurons received fibre terminations fromCGRP and VIP neurons. About 50% of theseneurons received terminations from galaninneurons, but had no input from substanceP, the enkephalins, or the other peptidesexamined. The nitrinergic terminations on anNADPH diaphorase negative, peptide positiveneuron could readily be identified. In neuronswith both peptide and NADPH diaphorasepositivity, it was difficult to identify the peptidetermination, especially if the terminationhappened to lie directly upon the neuron. Inmost of the neurons, however, the termina-tions were extensive and usually could be seenall around the soma. Higher resolution usingelectron microscopy is needed to confirmthese findings. The reverse condition alsoexists: nitrinergic fibres terminated upon thesomata of peptidergic neurons, but they werenot as easily studied, and could not be quanti-tated.

In contrast, about half of the total popula-tion of VIP positive neurons containedNADPH diaphorase activity. Similarly, galaninand NADPH diaphorase were colocalised inneuronal somata. There was no colocalisationof NADPH diaphorase activity with CGRP.Interestingly, VIP and nitric oxide were alsocolocalised in about 50% of the VIP positivefibres. Colocalisation of NADPH diaphoraseand VIP has been reported in guinea pigileum.27 These findings suggest the possibilityof a simultaneous (non-cascade) action bypeptides and nitric oxide as cotransmitters.Clearly, further work is needed to fully definethe functional significance behind nitrinergicand peptidergic NANC neurons in theoesophagus.

Physiological studies have suggested asignificant role for nitric oxide in the longitudi-nal muscle of the gastrointestinal tract. 15 18 20Our findings of very few nitrinergic fibres inthe longitudinal muscle compared with that ofthe circular muscle may implicate a limitedrole of nitrinergic innervation in the longitudi-nal muscle layer in the human oesophagus.

Neither the nitric oxide synthase antiserumnor the diaphorase reaction stained the musclecells under the conditions specified here. Thiscannot be sufficient, however, to exclude thepossibility that muscle cells may produce nitricoxide. This could happen by other forms ofnitric oxide synthase that are not detectable bythe present two methods, or the amount of

nitric oxide synthase present here may bebelow the threshold values needed for eithermethod.

In summary, these findings show thatNADPH diaphorase positive neurons arewidely distributed in the myenteric plexus andthat nitrinergic fibres innervate both circularand longitudinal oesophageal muscle. Thenitrinergic neurons constitute more than halfof the myenteric neuronal population. VIPoccurs in about a seventh of these neurons,galanin in about a tenth, and CGRP in none.These nitrinergic neurons receive input fromVIP and CGRP neurons, and peptidergicneurons receive input from nitrinergicneurons. The detailed functional significanceof these findings remains to be elucidated.

The kind gift of the nitric oxide synthase antiserum providedby Drs Solomon Snyder and Ted Dawson of Johns HopkinsUniversity is gratefully acknowledged. Part of this study hasbeen presented at the annual meeting of the AmericanGastroenterological Association in Boston in May, 1993 andappears as an abstract in Gastroenterology 1993; 104: A583.This work was supported by the VA Merit and the

Department of Medicine, University of Wisconsin-Madison,the Madison VA GRECC and NIH grant # DK 31092.

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