J. Physiol. (1981), 314, pp. 501-511 501With 7 text-figuresPrinted in Great Britain

TRANSPORT OF CHOLECYSTOKININ-OCTAPEPTIDE-LIKEIMMUNOREACTIVITY TOWARD THE GUT IN AFFERENT VAGAL

FIBRES IN CAT AND DOG

BY G. J. DOCKRAY, R. A. GREGORY, HILDA J. TRACYAND WEN-YU ZHU*

From the Physiological Laboratory, University of Liverpool, Liverpool

(Received 24 October 1980)

SUMMARY

1. The distributions of gastrin- and cholecystokinin-like immunoreactivities in thedog and cat vagus nerves have been studied after nerve section and ligation.

2. In dogs, there was an increase in cholecystokinin-octapeptide-like immuno-reactive material on the cranial side of ligatures on the thoracic or cervical vagi.When pairs of ligatures were tied on the cervical vagi there was accumulationproximal, and a slight decrease distal to, the upper ligature. There was also a modestincrease distal to the lower ligature.

3. In cats, section of the vagus above the nodose ganglion, and hence degenerationof the efferent fibres, did not prevent increases in cholecystokinin-octapeptide-likeimmunoreactivity on the cranial side of ligatures which were later tied below theganglion. Removal ofthe superior cervical ganglion had no effect on the accumulationof immunoreactive material above the ligatures. Section of the vagus below thenodose ganglion, and hence degeneration of both afferent and efferent fibres,abolished the accumulation on the cranial side of ligatures which were later tied belowthe section. Cholecystokinin-octapeptide-like material is therefore localized to afferentfibres with cell bodies in the nodose ganglion.

4. Immunoreactive forms were characterized by gel filtration and ion exchangechromatography, and the use of region-specific antisera. In all cats, and all but onedog, a molecule with the properties of sulphated cholecystokinin octapeptide wasfound to predominate. In some cats (30 %) and dogs (26 %) a molecule with theproperties of heptadecapeptide gastrin (G17) was identified; concentrations of G17were generally low compared with cholecystokinin octapeptide. In three dogs (20 %)there was an accumulation of heptadecapeptide gastrin above the ligatures.

5. Axonal transport of cholecystokinin octapeptide in the vagus is consistent witha neuro-regulatory role for this peptide. However, the functional significance of itslocalization in afferent fibres, and transport towards the periphery, remains to bedetermined.

* Visiting Scholar on leave from Department of Physiology, Peking Medical College, Xue YuanRoad, Hai Dian District, Peking, People's Republic of China.

0022-3751/81/5060-1289 $07.50 D 1981 The Physiological Society

G. J. DOCKRA Y AND OTHERS

INTRODUCTION

The widespread distribution of biologically active peptides in the central andperipheral nervous systems is now well established (H6kfelt, Johansson, Ljungdahl,Lundberg & Schultzberg, 1980; Snyder & Innis, 1979). Many of these peptides alsooccur in other cell types, notably in endocrine cells of the gut (Dockray & Gregory,1980). Recently, we isolated from sheep brain one such peptide corresponding to theCOOH-terminal octapeptide (CCK8) of the intestinal hormone cholecystokinin(Dockray, Gregory, Hutchison, Harris & Runswick, 1978), together with a closelyrelated slightly less acidic octapeptide. CCK8 in brain accounts for the 'gastrin-likeimmunoreactivity' first described by Vanderhaeghen, Signeau & Gepts (1975) andlater shown to be due to cholecystokinin-like peptides which share a commonCOOH-terminal pentapeptide with gastrin and react with many gastrin antisera(Dockray, 1976; Rehfeld, 1978; Muller, Straus & Yalow, 1977). There is a growingbody of evidence to suggest that CCK8 in brain has a neuro-regulatory function. Thus(1) immunohistochemical studies have localized CCK8-like immunoreactivity tonerve cell bodies, fibres and nerve endings (Straus, Muller, Choi, Paronetto & Yalow,1977; Larsson & Rhefeld, 1979; Loren, Alumets, Hakanson & Sundler, 1979; Innis,Correa, Uhl, Schneider & Snyder, 1979), (2) CCK8 is released by depolarizing stimulifrom brain slices and synaptosomes by a calcium-dependent mechanism (Dodd,Edwardson & Dockray, 1980; Emson, Lee & Rehfeld, 1980; Pinget, Straus & Yalow,1979), (3) CCK8 has neuronal actions (Ishibashi, Oomura, Okajima & Shibata, 1979)including depolarization of CA1 pyramidal cells in the hippocampus (Dodd & Kelly,1979), induction ofsatiety after intracerebroventricular injection in sheep (Della-Fera& Baile, 1979) and release of acetylcholine, and possibly Substance P from themyenteric plexus of guinea-pig ileum (Vizi, Bertaccini, Impicciatore & Knoll, 1973;Hutchison & Dockray, 1981). If CCK8 is indeed a neurotransmitter synthesized innerve cell bodies and released at nerve endings it should be possible to demonstrateaxonal transport. The present studies were undertaken to examine this idea byinvestigating the distribution of immunoreactive material in the vagus nerve of catand dog, before and after ligation of the nerve. An earlier report (Uvniis-Wallensten,Rehfeld, Larsson & Uvniis, 1977) has suggested that heptadecapeptide gastrin (G17),rather than CCK8 occurs in peripheral neurones, notably in the vagus of cat and dog.Therefore in the present experiments particular attention was given to thecharacterization of CCK-gastrin immunoreactive factors. The results presented hereindicate that, while a small amount of G17 may occur in the vagus of some cats anddogs, CCK8-like immunoreactivity predominates. The results further indicate axonaltransport toward the gut in afferent fibres with cell bodies in the nodose ganglion.Preliminary reports of this work have been published (Dockray, Gregory & Tracy,1980; Dockray, Gregory & Zhu, 1980).

METHODS

All the operative procedures were performed with aseptic technique under Nembutal anaesthesia.Crystamycin (Glaxo) was injected intramuscularly at the end ofeach operation and daily thereafterfor up to 4 days.Dog experiments. Two types ofexperiments were performed to study transort ofCCK-like peptides

in dog vagus. First in five dogs one branch of the vagus was ligated in the thorax, 3-5 cm above

502

CCK8 IN THE VAGUSthe diaphragm. A loop of thread was passed, but only loosely tied around a second, control, branchofthe vagus; the dogs were allowed to recover and 48 hr later they were killed and segments (3-4 cm)of the vagi removed above and below the point of ligation for extraction of CCK-like peptides.Secondly, in ten dogs pairs of ligatures were tied 4-5 cm apart on the cervical vagus one one side.The vagus of the other side served as a control with loose ligatures in corresponding positions. Theanimals were allowed to recover and 48 hr later were killed and segments of nerve about 2 cm longwere carefully removed from above, between and below the ligatures, and from correspondingpositions on the control side.

Cat experiments. Four groups of cats were studied. In the first group (n = 7) the cervical vaguswas ligated on one side about 5 cm below the nodose ganglion. Forty-eight hours later the cats werekilled and segments of nerve were removed and extracted, as described in the dog experiments. Inthe second group of animals (n = 6) the vagus on one side was cut above the nodose ganglion. Theanimals were allowed to recover and 2 weeks later (when efferent vagal fibres had degenerated) thenerve which had been sectioned was ligated about 5 cm below the nodose ganglion at a secondoperation. The animals were again allowed to recover and 48 hr later were killed and samples ofnerve removed for extraction. In a third group (n = 6), the vagus was sectioned about 4 cm belowthe nodose ganglion. The cats were allowed to recover for two weeks during which time both afferentand efferent fibres distal to the cut degenerated. At a second operation the vagus was then ligatedabout 4 cm below the original section, and after 48 hr samples were taken for extraction. In thefourth group (n = 6) the superior cervical ganglion was removed on one side, and after 2 weeksthe effects of vagal ligation on the same side were studied as in the previous experiments.

Extracts. Samples of nerve were frozen on dry ice immediately after dissection, and were storedat -40 0C prior to extraction. The frozen tissues were weighed quickly and while still deeply frozenwere added to 1-0 ml. boiling water. The samples were boiled for about 30 sec, then cut into smallpieces and homogenized (Ultra-Turrex) for 15 sec. The extracts were centrifuged (2000 g, 10 min),the volume made up to 2-0 ml., and supernatants stored at -200C before assay.

Assays. Radioimmunoassay methods have previously been described in detail (Dockray & Taylor,1976; Dockray, 1980). Samples were assayed routinely with two antisera; one of these, L48, isCOOH-terminal-specific and reacts equally with G17 and CCK8 and so gives an estimate of totalCCK-gastrin immunoreactivity (Dockray, 1980). Sensitivity of assays using L48 was approximately0 3 f-mole/ml. in the assay tube, and tissue extracts were assayed at a dilution of 1: 20, or moreif appropriate. Dog vagal samples generally weighed at least 20 mg so that the detection limit ofCCK-gastrin immunoreactivity in the tissue was approximately 0-3 p-mole/g. Cat samples wereoften only 10-20 mg and so detection limit was up to 0-6 p-mole/g. The second antiserum, L6, isspecific for intact G1 7 and does not show significant reactivity with CCK or other forms of gastrin(Dockray & Taylor, 1976). The sensitivity of assays with L6 was similar to those with L48.Column fractionation. After initial assays of total CCK-gastrin immunoreactivity with L48, and

G17 with L6, the remaining extracts were pooled, lyophilized and reconstituted in 1-0 ml. distilledwater for fractionation by ion-exchange or gel-filtration chromatography. Satisfactory results weregenerally obtained with the pooled samples from a single animal, but in a few instances samplesfrom several animals were pooled. Gel filtration was performed on Sephadex G50 (1 x 100 cm) ineither 0-02 M-sodium barbitone, or 0-05 M-ammonium bicarbonate, at 4 'C. Samples were fortifiedwith bovine serum albumin to provide a marker for the void volume, detected by absorption at280 nm, and with Na'251 to indicate the salt region. The columns had been previously calibratedwith natural human unsulphated heptadecapeptide gastrin (G17) and synthetic sulphated chole-cystokinin octapeptide (CCK8). Ion-exchange chromatography was carried out on amino ethyl (AE)cellulose (1 x 10 cm) equilibrated with 0 05 M-ammonium bicarbonate and eluted with a gradientto 0 5 M using a 150 ml. conical flask as a mixing vessel. Routinely, 1251-labelled human G17 and'251-labelled desulphated CCK8 were added to samples to provide markers. The columns were alsocalibrated in separate runs with synthetic sulphated CCK8 and natural human unsulphated G17.

RESULTS

Dog experiments

Effects ofvagalligation. After ligation ofthe thoracic vagus in dogs, the concentrationsof total immunoreactive CCK in the segment of nerve above the ligature (9-27 + 2-3p-mole/g, mean + S.E. of mean, n = 5) were significantly higher (t-test, P < 0 01) than

503

G. J. DOCKRA Y AND OTHERSin either the segment distal to the ligature (2-26 ± 0'42 p-mole/g), or the correspondingsegment of nerve on the control side (4-36 ± 1-3 p-mole/g). In contrast, theconcentrations below the ligature were markedly less than in the correspondingcontrol segment (5-4+ 2-20 p-mole/g), although the difference weas not statisticallysignificant. In these animals assays with antibody L6 failed to show significantamounts of immunoreactive material in all but a single animal (and in this animalligation did not cause a clear-cut accumulation of G17-like activity).

2 Tof05n G! ar LeA E50 EN

E~

t ,> - ControlE

00co

. 5.4-

0 3.E 2i

Ligatured48 hr

0 2 4cm

- Cranial Caudal

Fig. 1. Distribution of total CCK-gastrin immunoreactivity measured by radioimmuno-assay using antiserum L48 in segments of dog vagus nerve 48 hr after ligation. Pairs ofligatures were tied on one cervical vagus (lower panel); on the other side thread was passedbut not tightly tied (upper panel). Mean values are shown, and vertical bars representS.E.M.

When pairs of ligatures were tied on the cervical vagus there was a significant(P < 0 01) accumulation of total CCK-like immunoreactivity proximal to the upperligature compared with both the control side and the segments between ligatures ordistal to the lower ligature (Fig. 1). There was no significant accumulation proximalto the lower ligature, although it is of interest that in these experiments there wasa modest accumulation below the second ligature, possibly due to some retrogradetransport (HMkfelt, Elde, Johansson, Luft, Nilsson & Arimura, 1976). There was aslight (20 %) but not statistically significant depletion below the first ligaturecompared with the control side (Fig. 1). The results shown in Fig. 1 were obtainedfrom seven dogs in which there were no significant amounts of immunoreactiveis aterial measured with the G17-specific antiserum, L6 (< 0 3 p-mole/g). However,in three other dogs, in which pairs of ligatures were tied on the cervical vagus, therewere significant quantities of immunoreactive G17 in all vagal samples. Theconcentrations were 0-5-2 p-mole/g throughout the control side, and between anddistal to the ligatures, but on the cranial side of the first ligature there was a marked,and statistically significant, accumulation (P < 0 05) of immunoreactive G17(4-1 + 2-2 p-mole/g) compared with controls (1-2+ 0-46 p-mole/g).

Characterization of immunoreactive material. Fractionation of pooled dog vagalextracts by gel filtration typically showed a major well resolved peak of activity

504

CCK8 IN THE VAGUS

eluting in a similar position to synthetic sulphated CCK8 and reacting with L48 butnot L6 (Fig. 2). In seven of fifteen samples studied this was the only peak ofimmunoreactivity. In five of the remaining eight samples there was a minor peak ofactivity eluting in a similar region of G17 (< 150 total activity). In the other threesamples the G17 peak was over 250 total activity measured with L48,--and in onecase it was the only peak of immunoreactivity (Fig. 2). In four extracts fractionatedby ion-exchange chromatography on AE cellulose a single major peak was found thateluted in the characteristic position of sulphated CCK8 (Fig. 3). There was also aminor and poorly resolved peak eluting earlier than the major peak, but no significantamounts of immunoreactive material were found in the G17 region of the eluates.

j G17s jCCK 8S

30ACCK 8

20-

10,

0E

,50 MixtureO l l CCK 8-G17co

00.

50

0

E60E

40 G17

20-

0 20 40 60 80 100 120Elution volume (%)

Fig. 2. Fractionation of three different extracts of dog vagus nerve on Sephadex G50(1 x 100 cm). In the upper panel the elution profile is shown of a typical extract containingonly CCK8-like material. In a middle panel is shown the elution profile of an extract withboth CCK8- and G17-like activity, and in the lower panel is shown the single extract whichcontained only G17-like activity. All assays with L48. Elution volume is expressed as apercentage from the void volume (0%) estimated from bovine serum albumin, to 1251(100)

Cat experimentsEffects of ligation. Ligation of the cervical vagus nerve in the cat caused a marked

accumulation of immunoreactive CCK from about 5 to 20 p-mole/g in the segmenton the cranial side of the ligature (Fig. 4, top). When the vagus has been sectionedpreviously above the nodose ganglion and efferent fibres had degenerated, theaccumulation still persisted above the ligature, although it was reduced by about 25%(Fig. 4, bottom). In a further group of cats, in which the vagus was sectioned below

505

G. J. DOCKRA Y AND OTHERS

[1251]CCK8 | CCK8S

200

E& 150co

100

.1'01.4.

500 50.

E

E

20 30 40 50

[1251] G171

60 100

Elution volume (ml.)

Fig. 3. Fractionation ofan extract ofdog vagus nerve on AE cellulose (1 x 10 cm). Columnswere equilibrated with 0 05 M-ammonium bicarbonate and eluted with a gradient to 0 5M-ammonium bicarbonate. '251-labelled G17 and CCK8 were added to the sample as

markers. Column assayed with antiserum L48.

20

15.

10

015 _

co I,,~i 0 IIntact

020 n=7

15.

0

E 10E

5. 12 , T2

Vagotomized=4 14 days

0 2 4 n=6

- Cranial Caudal-

48 hr post-ligation

Fig. 4. Distribution of total CCK-gastrin immunoreactivity measured by radioimmuno-assay using antiserum L48 in segments of cat vagus 48 hr after ligation. In the upper panel(n = 7) the vagus was intact and the cats had not previously been operated upon. In thelower panel (n = 6) the vagus nerve had been sectioned above the nodose ganglion 2 weeksprior to ligation. For details see Fig. 1 and text.

506

(iI

cl

CCK8 IN THE VAGUS 507the nodose ganglion, subsequent ligation of the nerve at a lower level did not resultin an accumulation ofimmunoreactivity (Fig. 5). Concentrations ofimmunoreactivitybetween the section and the ligature were virtually immeasurable, presumably dueto degradation of CCK in the degenerating fibres. In a fourth group of cats in whichthe superior cervical ganglion had been removed 2 weeks before vagal ligation there

10

E 0.tl t IL _ ControlW 20

15 -

0C

=t==ti > ~~~~~Vagotornized

EE hr ~~~~~~~~~4daysCranial Caudal-48 hr Post-l igation

n = 6Fig. 5. Distribution of total CCK-gastrin immunoreactivity in vagus 48 hr after ligation.The ligated nerve had been sectioned below the nodose ganglion 2 weeks prior to ligation.In the upper panel is shown the distribution of immunoreactivity in the unoperated,unligated, vagal nerve; in the lower panel is shown the distribution in the sectioned andligated branch of the vagus. For details see Figs. 1, 4 and text.

TABLE 1. Effect of vagal ligations on the distribution of immunoreactive CCK in cats with superiorcervical ganglion removed 2 weeks previously*

Ligated side Control(p-mole/g) (p-mole/g)

Nodose ganglion 2-88 + 055 3-11+ 0 50Proximal segment 21-92+4-00 7-27 +1-59Distal segment 4-28 + 0-98 7-56 + 1-44

* Samples taken 48 hr after ligation. Values are means +S.E. of means, n = 6. Assay withantiserum L48.

was still a marked and significant accumulation of immunoreactivity on the cranialside of the ligature (Table 1). The immunoreactive CCK in the vagal trunk is nottherefore localized in sympathetic fibres which run in the vagus. Of twenty-nine catsstudied, antiserum L6 showed measurable amounts of Gl7-like immunoreactivity innine (31 %), although concentrations were close to the limit of detection (about1 p-mole/g); in no case did vagal ligation produce a clear increase in G17-likeimmunoreactivity.

Characterization of immunoreactive material. Of eleven samples fractionated onSephadex G50, eight showed a single peak ofimmunoreactivity eluting in the positionof CCK8 and reacting with L48 (Fig. 6). In the remaining four samples there were

G. J. DOCKRA Y AND OTHERS

80

-

100

E 60a

cooy4

0

E

E20

09

G174sI CCK 8sI

0 20 40 60 80 100Elution volume (%)

Fig. 6. Fractionation on Sephadex G50 of an extract of cat vague nerve. See Fig. 2 andtext for details.

[1251] CCK 8 1

- 50-500

,E 40a

coi 30.C.)0

*t 2000C 10*EE

0-

[1251] G171

0 20 40. 60 80Elution volume

(ml)

16o

Fig. 7. Fractionation on AE cellulose of an extract of cat vagus nerve. See Fig. 3 and textfor details.

ON

508

CCK8 IN THE VAGUS 509small amounts of G17-like activity (10% total in three samples and 25% total in thefourth). Fractionation of four samples on AE cellulose gave a single major peakeluting as sulphated CCK8 in every case (Fig. 7).

DISCUSSION

The present results establish the transport of CCK8-like activity toward the gutin the vagus nerve of cat and dog. The accumulation of CCK8-like activity on thecranial side of vagal ligatures persisted after previous section of the nerve proximalto the nodose ganglion and consequent degeneration ofefferent fibres. The CCK8-likeactivity can therefore be localized to afferent fibres whose cell bodies lie in the nodoseganglion. The present results provide quantitative support for previous immuno-histochemical studies that revealed nerve cell bodies in the rat and guinea-pig nodoseganglion which reacted with COOH-terminal-specific gastrin antisera (Lundberg,H6kfelt, Nilsson, Terenius, Rehfeld, Elde & Said, 1978).

Characterization of immunoreactive material by gel-filtration and ion-exchangechromatography clearly demonstrated that CCK8 was the principal immunoreactiveform of the gastrin and cholecystokinin group of peptides in the cephalic and thoracicvagi of cat and dog. These observations are in keeping with earlier reports that CCK8occurs widely in the central and peripheral nervous systems (Dockray, 1976; Dockrayet al. 1978; Rehfeld, 1978; Larsson & Rehfeld, 1979; Dockray & Hutchison, 1980).However, in the past Uvniis-Wallensten et al. (1977) have reported that G17-likeactivity occurred in concentrations of 16-272 p-mole/g in the abdominal vagal fibresofcat and dog, and 0-2 p-mole/g in the cervical and thoracic vagi; these authors failedto find CCK8 in the vagus. In the present work small quantitites of G17 (up to2 p-mole/g) were found in the cervical and thoracic vagi of about 26% of dogs and30% of cats. In contrast, CCK8-like activity predominated in all cats and all but onedog. In a few dogs there was an accumulation of G17 on the cranial side of ligaturescomparable to that of CCK8, but no accumulation of G17 could be demonstrated inthe cat. The present findings do not suggest that G17 is transported down the vagusin quantities sufficient to account for the concentrations found by Uvniis-Wallenstenet al. (1977) in abdominal fibres. It is also unlikely that G17 is generated by conversionfrom an inactive precursor during vagal transport, since there was no significantaccumulation in the isolated segment of nerve in the double ligature experiments.Clearly, further work is needed to establish the factors governing the relativedistribution of gastrin and CCK in the vagus nerve.

Recently, axonal transport of several other neuropeptides in the vagus nerve hasbeen demonstrated, e.g. somatostatin, Substance P and vasoactive intestinalpolypeptide (Gilbert, Emson, Fahrenkrug, Lee, Penman & Wass, 1980; Brimijoin,Lungberg, Brodin, Hokfelt & Nilsson, 1980; Gamse, Lembeck & Cuello, 1979). Theevidence suggests that these peptides, like CCK8, are localized to afferent fibres.However, the functional significance ofthe transport ofCCK8 and other neuropeptidestowards the gut in afferent fibres has yet to be demonstrated. It should be emphasizedthat the vagus is likely to make only a very minor contribution to total intestinalCCK8, most ofwhich is localized in intestinal mucosal endocrine cells, although somealso occurs in intestinal nerve fibres arising from intrinsic gut neurones (Schultzberg,

510 G. J. DOCKRA Y AND OTHERSHdkfelt, Nilsson, Terenius, Rehfeld, Brown, Elde, Goldstein & Said, 1980). It seemsprobable that at least some of the CCK8 synthesized in cell bodies of the nodoseganglion is transported toward the central nervous system, but the amount anddestination of this pool of vagal CCK8 is unknown.

It is significant that after double ligation of the dog cervical vagus there was onlya modest (20 %) decrease of activity distal to the first ligature. It seems possible thata relatively high proportion of CCK8 in the vagus is transported very slowly, andthat the material which accumulated above ligatures represents a sub-fractionationoftotal CCK which is rapidly transported. However, the possibility that ligation itselfinfluences the rate of transport, particularly distal to the constriction, cannot yet beexcluded. Further studies on the time course of accumulation should help clarify thekinetics of axonal transport of CCK8.The present findings are of general interest in that they clearly reveal the capacity

of nerve fibres to transport CCK8. These observations therefore provide a functionalbasis for interpretation of the immunohistochemical demonstration of CCK8-likepeptides in nerve cell bodies, fibres and endings (Straus et at. 1977; Loren et al. 1979;Larsson & Rehfeld, 1979; Innis et at. 1979). In particular the results directly supportthe notion that CCK8 is synthesized in cell bodies and can be transported to its siteof release at nerve endings, and so contribute to the idea of a neuroregulatory rolefor this peptide.

We are grateful to the Medical Research Council for financial support and to Carol Higgins andGavin Laing for skilled technical assistance. Mrs Eva McElroy very kindly typed the manuscript.

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