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184

ON THE PHYSIOLOGY AND PHARMACOLOGYOF THE EARTHWORM GUT

BY K. S. WUFrom the Department of Zoology and Comparative Anatomy,

University College, London

(Received 19 August 1938)

(With Eleven Text-figures)

THE physiology of the gut movements in invertebrates has been little studied. Inthe case of the earthworm, although numerous investigations have been publishedon the physiology of locomotion and of the neuro-muscular structures of the bodywall, nothing has yet appeared on the gut movements. It was with the intention ofthrowing light on this problem that the present investigation was undertaken.

Two species of earthworms were studied, Lumbricus terrestris and Allolobophoralonga.

I. THE NORMAL MOVEMENTS OF THE ISOLATED GUT

For most of the experiments, the gut was arbitrarily divided into the followingfour parts:

(a) The buccal cavity and pharynx.(b) The oesophagus.(c) The crop and gizzard.(d) The front part of the intestine (occupying about twenty segments).

Each part was dissected out free of septa, major blood vessels, and so forth, tiedat both ends and suspended in a bath designed to allow of easy change of fluid(see Wells, 1937, Fig. 4). The preparations were suspended in ordinary frog Ringer,and the movements were recorded by very light isotonic levers. Aeration wasemployed in the later experiments, and was found to improve the responses todrugs. Rhythmic activity continues for many hours in the absence of aeration.

Owing to the small size of the gut it is difficult to work with smaller portionsthan those listed above. However, during the experiments certain correlations werenoticed, e.g. the crop leads the gizzard, and confirmatory experiments were done inthese cases by pinning down the middle of each preparation and simultaneouslyrecording the contractions of the two ends.

The nature of the spontaneous rhythm varies greatly from part to part of the gut(Fig. 1). In all cases the contractions become stronger during the first half-hour ofthe experiment, as the preparation stretches slightly under the weight of the lever.Activity continues for many hours, in frog Ringer, in some cases for more than tenhours.

On the Physiology and Pharmacology of the Earthworm Out 185

The "buccal cavity and pharynx" preparations give rather irregular records,sometimes exhibiting prolonged tonic contractions, at long and irregular intervals,in addition to the briefer contractions shown in the figure. The pharynx appears to

l| l| » MM-I-M-V4-160 170

s^\• /

\ n \

30\ \ \ \ 1 ) )

80

Fig. 1. Spontaneous activity of various parts of the earthworm gut suspended in frog Ringer.A. Buccal cavity and pharynx, Lumbricus terrestris. B. Oesophagus, Lumbricus terrestris. C. Cropand gizzard, Lumbricus terrestris. D. Intestine, Lumbricus terrestris. E. Intestine, Allolobophoralonga. F. Intestine, Eisema venata.

In all records: Read from left to right; upstroke of lever means shortening of preparation; timesignal marks once a minute.

act as pace-maker for the buccal cavity. The major contractions appear first in thepharynx and spread to the buccal cavity.

The oesophagus gives a somewhat similar record. The oral end of the oeso-phagus is apparently more excitable and more contractile than the aboral. Thelarger contractions in Fig. 1 involve the whole oesophagus; they appear first at the

jEB-rviii 13

186 K. S. Wu

oral end and spread backwards. The smaller contractions are due to the oral endonly, and do not spread back.

The "crop and gizzard" preparation gives a very vigorous and regular rhythm,with a tendency, especially towards the end of the experiment, to grouping of thecontractions. The thin-walled crop seems to act as pacemaker for the thicker moremuscular gizzard, much as the auricle leads the ventricle in the vertebrate heart.If the preparation is watched the crop can be seen to contract first and the gizzard tofollow it. The amplitude of the contractions of the crop is very great; when fullycontracted the crop appears very small, as if withdrawn into the cavity of thegizzard. The gizzard contracts less and is responsible for a small part only of theupstroke of the lever. Sometimes the gizzard may fail to contract after the crop, ormay go into contracture, producing irregularities in the record.

In the case of the intestine, the type of record obtained varies according to thespecies of worm employed (Fig. i). In Lumbrtcus the contractions are comparativelybrief. In Allolobophora slow tone waves appear. A few experiments were also carriedout on Eisenia venata; in this species the intestine shows relatively sharp contrac-tions followed by slow relaxations.

Each of the four parts studied seems to act as a functional unit as regardsmuscular activity. However, no correlation was noted between these four parts,which all appeared to act independently of each other, when the entire excised gut wascarefully watched lying in a Petri dish of Ringer, or suspended in the bath andobserved, or when experiments with two levers were carried out along the linesdescribed above. It is, however, possible that in the intact worm the parts arecorrelated, either through the agency of the nervous system or by mechanicalfactors such as the presence of food in the lumen.

II. THE ACTION OF CHEMICAL AGENTS ON THE GUT MOVEMENTS

In recent years there have been several studies of the action of adrenaline andacetylcholine on invertebrate organs, in order to determine how far our views on therole of these substances in vertebrates can be generalized to apply to the inverte-brate phyla. However, such studies have mostly been made on somatic muscles andnot on the gut. Experiments were therefore carried out to test the action of thesesubstances on the earthworm gut. As the gut was found to be exceedingly sensitiveto acetylcholine, experiments were also done with a number of other substanceslikely to be present in tissue extracts, in order to find out whether the earthworm gutwould prove to be a suitable preparation for the detection and assay of acetyl-choline.

Acetylcholine, eserine, prostigmine and atropine

Acetylcholine produces a sharp contracture in all parts of the gut, accompanied,except in the lowest concentrations, by abolition of the rhythmic movements.

The "crop and gizzard" preparation is exceedingly sensitive to acetylcholine,the effect being detectable in concentrations of io~* and sometimes even down toIP"11, With high concentrations there is a. sharp tone rise and abolition of the

On the Physiology and Pharmacology of the Earthworm Gut 187

rhythmic contractions. With low concentrations the tone rise is less pronounced andthe rhythm persists with diminished amplitude but increased frequency. There istherefore a graded series of responses over a wide range of concentrations (Fig. 2),and in any one preparation the response to any one concentration is quite accuratelyrepeatable. It is therefore possible, by comparison with standard solutions, toestimate the amount of acetylcholine in an "unknown" solution with a considerabledegree of accuracy, using the crop and gizzard of the earthworm.

m

Ringer's, fluid Acetylcholine MO"7 Rinqer's fluid IRinger'sfluid Acetylcholine MO""* Rinqer\ fluid

Fig. 2. Action of acetylcholine (without eserine) on the crop and gizzard of Lumbricus. Each recordbegins in Ringer; at first signal, acetylcholine is added; at second signal, return to Ringer. Acetyl-choline concentrations: A, io~"; B, io~*; C, io~'; D, io~*.

The other parts of the gut are also sensitive, but the threshold concentrationswere not accurately determined.

Eserine by itself, in 1 : 500,000 concentration, has a pronounced effect on themotility of the crop and gizzard, causing a slight rise in tone, an increase in fre-quency and a decrease in amplitude. The whole picture is very like that produced bysmall doses of acetylcholine, and is probably due to potentiating the action of cholineesters already present in the preparation (see Fig. 7 D). Lumbricus is more sensitivethan AUolobophora, and shows these effects even with 1 : 1,000,000 eserine. Ininvestigating the interaction of eserine and acetylcholine, the following concentra-tions of eserine were therefore used: 1: 10,000,000 for Lumbricus and 1 : 1,000,000for Allolobophora,

188 K. S. WuThe action of acetylcholine on tone is not augmented by eserine in these con-

centrations, even after an exposure to eserine of four hours. There is a slight increasein the effect on frequency and amplitude. The case seems to be similar to that of therabbit intestine (Matthes, 1930), where it is impossible to apply sufficient eserine toaugment the action of acetylcholine without producing a contraction.

The action of prostigmine alone is similar to, but seems a little stronger than,that of eserine. As in the case of eserine, prostigmine causes no noticeable augmenta-tion of the action on the rhythmic contractions.

Fig. 3. Crop and gizzard of AUolobophora. Record begins in Ringer. First signal, acetylcholine1: 100,000,000; second signal, the same with atropine I : 50,000 added; and third signal, Ringeralone.

The action of acetylcholine is abolished by atropine. On adding drops ofatropine 1 : 1000 from a hypodermic syringe to the bath containing the preparation(final concentration approximately 1 : 50,000 or 1 : 100,000) the acetylcholine effectdisappears (Fig. 3). By itself, atropine in these concentrations has a slight excitingaction.

Adrenaline, ergotoxine and ephedrine

Preliminary experiments were made with Parke Davis's adrenaline solution,which contains adrenaline 1 : 1000 and chloretone 1 :200. However, controlexperiments showed that the crop and gizzard of the earthworm are very readilydepressed by chloretone 1 : 20,000 in frog Ringer, so in subsequent work a drypreparation of adrenaline ("adrenalina B.P." from British Drug Houses Ltd.) wasused.

By the action of adrenaline, the gut can be differentiated into two regions—thebuccal cavity, pharynx and oesophagus on the one hand, and the crop, gizzard andintestine on the other.


The "buccal cavity and pharynx" responses by contracture to adrenaline are3 : 10,000 to 1 : 1,000,000. The oesophagus is even more sensitive, responding to1 : 10,000,000 adrenaline, contracting more vigorously and promptly than theformer part, and maintaining a longer contracture (Fig. 4).

The "crop and gizzard" preparation is inhibited by adrenaline in concentrationsabove a threshold which varies considerably in different preparations. If theapplication is repeated, the inhibition often becomes more marked. Below thatthreshold the effect of adrenaline is variable; sometimes there is no effect, but oftenthere is a definite exciting effect (Fig. 4). Usually inhibition occurs in concentrations

U Adrenaline 110 ' Ringers fluid

Fig. 4. Action of adrenaline on the gut of Lwnbricus. A. Crop and gizzard; adrenaline io~7. B. Cropand gizzard; adrenaline io~5. C. Oesophagus; adrenaline io~*.

down to 1 : 100,000 and excitation down to 1 : 1,000,000, but in very sensitiveworms these values change to 1 : 1,000,000 and 1 : 10,000,000 respectively. Theaction of adrenaline on the intestine resembles that just described, although thecontractions are not so powerful.

Addition from a hypodermic syringe of sufficient ergotoxine to bring the con-centration in the bath to 1 : 50,000 or 1 : 100,000 abolishes the inhibitory action ofhigh adrenaline concentrations on the crop and gizzard (Fig. 5). Applied in theabsence of adrenaline, ergotoxine has no action on the preparation in these concen-trations. Ergotoxine also antagonizes the exciting actions of adrenaline on theoesophagus and on the buccal cavity and pharynx.

It was found by Blaschko et al. (1937) and Gaddum (1938) that low concentra-tions of ephedrine increase the action of adrenaline on certain vertebrate tissues, inmuch the same way that eserine increases the action of acetylcholine, i.e. by in-hibiting the destruction of the drug by an oxidase. In the earthworm gut, there is aninteresting parallel between the actions of ephedrine and eserine. In most cases

K. S. Wuephedrine from i : 10,000 to 1 : 100,000 causes slight excitation of the buccalcavity and pharynx, but slight inhibition of the crop and gizzard (Fig. 6); that is to

v r~t i~i~t 1 1 rAdrenaline 10~5 + Ergotoxine 5 xio4

~r~r~r 1 1 1 1 1 1 1 t t~ri~i~t i I ~r~i I i -TTTI~I I I n

Fig 5 Crop and gizzard of Allolobophora. Record begins in Ringer. First signal, adrenaline1 : 1 oo^ooo;second signal, the same with ergotoxine i: 50,000 added; and third signal, Ringer alone.

Buccal cavity and pharynx

Fig 6. Action of ephedrine on the "buccal cavity and pharynx" (above) and "crop and gizzard(below) of Allolobophora. In both: left, adrenaline 1 : 1,000,000; right, ephednne 1 : 100,000followed by adrenaline 1 : 1,000,000 and ephedrine 1 : 100,000.

say, the action of ephedrine by itself resembles that of adrenaline, much as the actionof eserine by itself resembles that of acetylcholine. Possibly the effect is due topotentiating the action of an adrenaline-like substance already present After


ephedrine, there is a slight augmentation of the action of adrenaline. In somepreparations ephedrine has no action by itself, and in these cases it does notaugment the action of adrenaline.

If adrenaline and acetylcholine are applied simultaneously to the crop andgizzard, the inhibitory action of adrenaline diminishes the exciting action of acetyl-choline. An experiment on a single preparation illustrating the interaction ofadrenaline, acetylcholine and eserine is shown in Fig. 7. It will be seen that the

ill! II 1 I

Ad.i6"5" Ach.10-81 r r r r~r t~t 1 i~t 1

Fig. 7. Crop and gizzard of Lumbricus. Effect of adrenaline on the action of acetylcholine. A, adre-naline; B, acetylcholine; C, adrenaline with acetylcholine; D, adrenaline and acetylcholine witheserine after eserine. The concentrations are 1: 100,000 for adrenaline, 1 : 100,000,000 for acetylcholineand 1 : 1,000,000 for eserine,

separate actions of adrenaline and acetylcholine appear to be added together whenthe drugs are simultaneously applied. Eserine sways the balance in the expecteddirection. However, in the case of the oesophagus or buccal cavity and pharynx theposition is not so clear. Either adrenaline or acetylcholine alone produces contrac-ture, but when the two drugs are applied simultaneously the resulting contraction isless than those produced by separate application.

Choline, histamine, potassium and calcium

Because the crop and gizzard has certain advantages as a test preparation for theassay of acetylcholine, experiments were made to determine the action of some othersubstances likely to be present in tissue extracts.

Choline chloride tends to slow slightly the rhythm in concentrations above about

192 K. S. Wu1: 100,000 but the effect is somewhat variable. At the same time there may be aslight, rather transient tone-rise immediately after the application of choline. It willbe noted that the action of choline does not even qualitatively resemble that ofacetylcholine because the two drugs affect the frequency of the rhythm in opposite

E.1CT6 Ach.10~8+H.1(r*+E.10"6

' ' t f / 1 1 1 1 1 1 t t t f r T} t ;

Fig. 8. Crop and gizzard of Lumbricui. Effect of histamine on the action of acetylcholine. A, hista-mine; B, acetylcholine with histamine; C, acetylcholine; and D, acetylcholine and histamine witheserine after eserine. The concentration! are 1 : 100,000 for histamine, 1 : 100,000,000 for acetylcholineand 1 : 1,000,000 for eserine.

The action of histamine is very variable. In some cases there is no clear effect ontone or frequency in concentrations up to 1: 100,000. In others, lower concentra-tions may produce either a definite inhibition of rhythm and tone, or excitation,especially of tone. Certain preparations appear to exhibit a conflict between the twotendencies, showing alternating excitation and inhibition under the influence ofhistamine. In two cases, the drug (1 : 10,000 and 1: 1,000,000 respectively)produced sharp contracture with abolition of the rhythm.


The experiment of Fig. 8 shows that histamine, even when its effect on thepreparation is very slight, tends to inhibit the action of acetylcholine, and that thebalance between the two drugs is altered by eserine in the expected direction.

The action of excess of potassium and calcium was studied by adding variousamounts of isotonic KC1 or CaClj to the Ringer before applying it to the preparation(Fig. 9). In the crop and gizzard, potassium excess at first excites and then, inhigher concentrations, inhibits. Calcium causes slowing of the rhythm, loss of tone,and, in high concentrations, stoppage in the completely relaxed condition. Therecords obtained in these experiments are exceedingly like those got for the heartof the spider crab Maia squinado by Wells (1928). The "buccal cavity and pharynx"preparation resembles the crop and gizzard in its reaction to excess of K or Ca.

Fig. 9. Crop and gizzard of Lumbricu*. Effect of raising the normal potassium or calcium concentra-tions (0-0018 M KC1, o-ooi MCaCIO to the following values: A. KC1 to 0-00765 M. C. CaCl, to000159 M. B. KC1 to 00369 M. D. CaCl, to 000685 M.

The effect of potassium-free and calcium-free Ringer was also studied. Thecrop and gizzard reacts very similarly to lack of K or Ca, i.e. by tone-rise withoutnoticeable disturbance of the rhythm during several minutes (Fig. 10). Lack of Kcombined with increased Ca excess leads to a rapid, twitching, inco-ordinated beat.

Experiments were also carried out to find out whether disturbance of the normalelectrolyte ratio would affect the responses of the preparation to acetylcholine andadrenaline. It was found that moderate potassium excess slightly reinforces theaction of acetylcholine but partially antagonizes the action of adrenaline. Withcalcium excess, the reverse is the case. These results are to be expected if we supposethat the ions and drugs act independently on the tissue and that their effects aresimply added together.

194 K - S. Wu

Fig. 10. Crop and gizzard of Lumbricus. Effect of: A, K lack; B, Ca lack.

III. THE INNERVATION OF THE GUT

According to Stephenson's monograph (1930), a subepithelial nerve plexusexists in the wall of the alimentary canal throughout its length. Six small nervespass from each pharyngeal connective and unite on the wall of the pharynx in apharyngeal ganglion, whence fibres pass forward and backward to enter the plexus.Nerve fibres also reach the alimentary tube by way of the blood vessels and from thesegmental nerves via plexuses in the septa. Experiments were therefore carried outto determine whether the movements of the crop and gizzard could be modifiedby impulses passing either (a) along the gut plexus from its anterior end, or (b) fromthe nerve cord along the blood vessels and septa.

The animal was opened dorsally and the body wall was pinned out sideways.The crop and gizzard were still linked to the body wall by the ventral parts of theirsepta. In this region of the body the nerve cord was untouched, but it was dissectedaway from the body wall in front of and behind this, region, and finally cut a fewmillimetres behind the subpharyngeal ganglion, and a couple of centimetres pos-terior to the gizzard. The cut ends were tied and the two ends of the cord were liftedon to chloride-coated silver wire electrodes. By stimulating these, the influence ofimpulses reaching the crop and gizzard through the septa could be studied. Thecircumpharyngeal ring was also stimulated; as the nerve cord had been dividedbetween the ring and the crop, the results of the stimulation could only reach thecrop and gizzard by way of the plexus in the wall of the gut, or possibly along thedorsal blood vessel. Weak faradic stimulation was employed.

Stimulation of the circumpharyngeal commissures was not always successful.Owing to the small size of the parts and the necessity for keeping them moist it iseasy to set up a short circuit. However, in several cases, stimulation of the ringproduced a definite exciting action on the crop, shown by a tone rise and an increasein the rate of spontaneous contractions (Fig. 11). Stimulation of the nerve cordinhibits the rhythm and may stop it altogether. In many cases, as in the upper halfof Fig. 11, when the anterior part of the nerve cord is stimulated there is also an


apparent tone rise, but careful inspection shows that this is due to a contraction ofthe septa, which pull the crop downwards. If the preparation is so pinned that thecrop and gizzard do not stretch the septa, which therefore do not carry the weight ofthe lever, stimulation of the cord produces a slight loss of tone in the crop.

It will be noted that the nerve fibres coming from in front excite the crop andtheir action therefore resembles that of acetylcholine, while those coming through

1 1 i i y Mil I .liJvliiJiA

Stimulate

Fig. 11. Effect of electric stimulation of nerves. A, crop of AUolobophora. First, stimulation ofcircumpharyngeal commissure; second, stimulation of anterior part of the nerve cord; third, stimula-tion of circumpharyngeal commissure. B, crop of Lumbricut. Stimulation of anterior part of thenerve cord. The first effect (labelled " Pull") is produced by lifting the nerve on to the electrodes, thesecond by electrical stimulation.

the septa inhibit the crop and resemble adrenaline. The possibility therefore arisesthat these nerves are respectively cholinergic and adrenergic. This suggestion wasconfirmed by the following experiments.

The preparation was made on cork glued to the bottom of a finger bowl. Aftertypical inhibition had been obtained by stimulating the ventral nerve cord, ergo-toxine was added in amount sufficient to produce a concentration of 1 : 100,000. Theresult was to abolish the effect of stimulating the nerve cord. Addition of atropine,on the other hand, did not block the action of stimulating the nerve cord. It was,however, found that atropine 1: 100,000 abolished the exciting effect of stimulatingthe circumpharyngeal commissures.

196 K. S.As the two kinds of nerves resemble adrenaline and acetylcholine respectively,

both as regards the effect of stimulation and as regards the effect of atropine andergotoxine; and as annelid worms have been shown to contain acetylcholine(Bacq, 1935) and chromaffin cells (Gaskell, 1914), it is concluded that the hinderpart, at least, of the earthworm gut is provided with a double autonomic innervationsimilar physiologically to that of the vertebrates.

As regards the front part of the gut—i.e. the buccal cavity, pharynx and oeso-phagus—the position is not so clear. These organs resemble the crustacean heart(Bain, 1929; Maclean & Beznak, 1933) in being excited both by adrenaline and byacetylcholine, so that if inhibitory nerves exist their effect is presumably transmittedby some other substance. In the case of the crustacean heart, it is known that suchnerves exist (Polimanti, 1913; Carlson, 1909); in the case of the earthworm gut,although no experiments on the point could be done, their existence is likely.

IV. SUMMARY

1. The gut of Lumbricus terrestris and AUolobophora longa was studied.2. The normal movements of the different parts of the gut, isolated and

suspended in Ringer's fluid, are described.3. Acetylcholine excites all parts of the gut. The "crop and gizzard" prepara-

tion is very sensitive to acetylcholine and shows a graded series of responses over awide range of concentrations. It may therefore prove to be useful in the assay ofacetylcholine.

4. The action of acetylcholine on the crop and gizzard is only slightly increasedby eserine or prostigmine. The potentiation is especially noticeable in the presenceof other drugs (adrenaline, histamine) which interfere with the acetylcholine action.

5. The action of acetylcholine on the crop and gizzard is abolished by atropine.6. Adrenaline excites the buccal cavity, pharynx and oesophagus in all concen-

trations. On the crop, gizzard and intestine it has two actions, inhibition above andexcitation below a certain threshold concentration, whose magnitude varies some-what from worm to worm.

7. The action of adrenaline is slightly increased by ephedrine, and abolished byergotoxine.

8. The actions of choline, histamine, potassium and calcium on the crop andgizzard are described.

9. The crop and gizzard receive two sets of nerves:(a) Exciting nerves, coming along the gut wall from the circumpharyngeal

nerve ring; action abolished by atropine; probably cholinergic.(b) Inhibiting nerves, coming along the septa from the ventral nerve cord;

action abolished by ergotoxine; probably adrenergic.

I wish to express here my sincere thanks to Prof. Watson for allowing me towork in his Department, to Mr Wells for his suggestion of the problem and forhis interest and advice during the investigation, and to Prof. Gaddum for valuablesuggestions.


REFERENCES

BACQ, Z. M. (1935). Arch. int. Pkytiol. 42, 24.BAIN, W. A. (1929). Quart. J. exp. Pkytiol. 19, 297.BLASCHKO, H., RICHTER, D. & SCHLOSSMANN, H. (1937). Biochem. J. 31, 2187.CARLSON, A. J. (1909). Ergebn. d. Pkytiol. 8, 371.GADDUM, J. H. (1938). Brit. med.J. 1938, 1, 713.GASKEIX, J. F. (1914). Pkilot. Tram. B, 205, 153.MACUJAN, M. N. & BEZNAK, A. B. L. (1933). Arb. Biol. Fortchgintt. 6, 258.MATTHES, K. (1930). J. Pkytiol. 70, 338.POLIMANTI, O. (1913). Arch. Anat. Pkytiol. 1913, Physiol. p. 117.STEPHENBON, J. (1930). The Oligochaeta. Oxford: Clarendon Press.WELLS, G. P. (1928). J. exp. Biol. 5, 258.

(1937)- 7- exp. Biol. 14, 117.

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