Camp. Biochem. Phyriol., 1973, Vol. 46A, pp. 171 to 181. Pergamon Press. Printed in Great Britain

EFFECT OF VARIOUS GASTROINTESTINAL HORMONES AND VASOACTIVE SUBSTANCES ON VILLOUS MOTILITY*

W. L. JOYNER and E. KOKAS

Department of Physiology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27514, U.S.A.

(Received 7 November 1972)

Abstract-l. The effects of some gastrointestinal hormones and vasoactive substances on villous motility were tested in an exteriorized jejunal loop of pentobarbital-anesthetized dogs. The substances were administered intra- venously and topically.

2. The gastrointestinal hormones pentagastrin, secretin, cholecystokinin- pancreozymin, enterogastrone and gastric-inhibitory peptide inhibited villous motility.

3. Human gastrin slightly stimulated it. 4. The vasoactive peptides caerulein, ranatensin and eledoisin inhibited

villous motility. 5. The differences between the action of villikinin and the above substances

are clarified and emphasized. 6. The results support the concept that villikinin is unique and is of physio-

logical significance.

INTRODUCTION

STUDIES on villikinin have indicated that it is a low molecular weight acidic peptide which specifically stimulates the contraction of intestinal villi without acting on the tunica muscularis (Kokas & Johnston, 1965; Kokas et al., 1971). Crude villikinin- like substances have been prepared from the intestinal mucosa of various animal species and refined by electrophoresis and resin chromatography (Kokas, 1938; Kokas & Johnston, 1965; Kokas et al., 1971). Since the chemical nature of refined villikinin has not yet been established, it seemed of importance to compare the action of villikinin with other gastrointestinal hormones and vasoactive substances.

In recent years, human gastrin, secretin and cholecystokinin-pancreozymin (CCK-PZ) have been isolated and synthetized (Gregory & Tracy, 1961; Jorpes et al., 1962, 1964; Gregory, 1964; Mutt & Jorpes, 1967). Fragments (tetra- and penta-peptides) from the C-terminal portion of gastrin and the C-terminal octa- peptide in CCK-PZ have been found to exert physiological and pharmacological activities which resemble the parent molecule (Grossman, 1966). These substances influence not only secretory activities, but also motility, in different parts of the gastrointestinal tract (Grossman, 1966).

* This paper is dedicated to Professor Dr. G. Ludlny on his 65th birthday.

171

172 W. L. JOYNER AND E. KOKAS

Caerulein, a vasoactive decapeptide from the skin of the amphibian HyZu caemlea, was found to contain the same C-terminal pentapeptide sequence as gastrin and the octapeptide of CCK-PZ (Anastasi et al., 1968; Mantovani, 1970). Due to its close resemblance in chemical structure and physiological action, the effect of this substance on villous motility was investigated and compared to the action of the gastrointestinal hormones.

Ranatensin, isolated from the skin of the American frog Rana pi&ens, was found to act on smooth muscles in different portions of the gastrointestinal tract (Nakajima et al., 1970; Clineschmidt et al., 1971). It stimulates various smooth muscle preparations but relaxes the rat duodenum (Clineschmidt et al., 1971). Its chemical composition was shown to bear a distant resemblance to eledoisin- like peptides (Bertaccini, 1970) such as Substance P which is naturally occurring in the intestinal tract (Euler & Gaddum, 1931; Chang et al., 1971). Substance P and eledoisin were shown to influence villous motility (Ludiny et al., 1960, 1968).

MATERIALS AND METHODS Twenty-eight mongrel dogs (10-12 kg), fasted for 24 hr, were anesthetized with sodium

pentobarbital (30 mg/kg, i.v.). Mean arterial blood pressure was registered in the femoral artery and the test substances were injected into the saphenous vein. Villous motility was observed in a jejunal loop prepared according to the method of Kokas & Johnston (1965). Villous activity was recorded by counting the number of villous contractions in a 9 mm2 optical field with a stereomicroscope (45 x magnification). Prior to administering any substance, the basal rate of villous contractions (B) was recorded by counting the number of contracting villi for 1 min. This procedure was repeated every other minute until the difference between two consecutive counts was less than 5 per cent and the average of these counts was used as the basal level. After the administration of the test substance(s) experi- mental counts (E) were recorded by counting the number of villous contractions per minute (CPM) at predetermined time intervals throughout the test period. The Contrac- tion Index (C.I.) was computed for each period by subtracting the basal count (B) from the experimental count (E) and dividing by the basal count (B). (C.I. = E-B/B) (Kokas & Johnston, 1965). The C.I. of zero indicates an increased or a decreased villous motility, respectively. The physiological variations of C.I. are in the range of +0-l to - 0.03. All of the C.I.‘s are expressed in the illustrations as arithmetic means f S.D. of five to eight observations.

.

Experimental procedure The test substances were administered intravenously, or topically on the exposed

mucosal surface (Table 1). After the basal count was established, the test substance was injected into the saphenous vein. Counts, each lasting 1 min, were made at 1,3,5,8,12 and 15 min after the intravenous injection of the test substance. For each period the C.I. was computed as described above. When the counts returned to within 5 per cent of the basal count, another test substance was injected. For topical administration a small cylindrical “collar”, made of thin cardboard, was placed on the exteriorized mucosal surface (Gordon & Kokas, 1968). After basal counts were established within one quarter of the visual field of this “collar”, 0.2 ml of the test substance at 38°C was placed inside the “collar” and the test period immediately began. Counts, each lasting 30 set, were taken at 2, 4, 6 and 8 min after the application of the test substance. The mucosal surface was then washed with saline (0*85%, pH 7.4, 38”(Z), and another count was made at 10 min. Before another test substance was applied, the new basal level was established. Throughout a 3-4 hr experi- ment, the basal level was usually very stable.

GASTROINTESTINAL HORMONES AND VILLOUS MOTILITY 173

TABLE I-SUMMARY OF THE EFFECT OF GASTROINTESTINAL (G.I.) HORMONES AND VASOACTIVE SUBSTANCES

Substance Route of

administration V.M. M.S. Anemia Peri. B.P.

G.I. hormones Human gastrin

Pentagastrin

Secretin

Cholecystokinin-pancreozymin

Enterogastrone

G.I.P.

Villikinin*

Vasoactive substances

Eledoisin*

Caerulein

Ranatensin

Substance P *

Bradykinin *

Histamine *

Kallidin* Serotonin*

Intravenous Topical Intravenous Topical Intravenous Topical Intravenous Topical Intravenous Topical Intravenous Topical Intravenous Topical

Intra-arterial Intravenous Topical Intravenous Topical Intravenous Topical Intravenous Topical Intra-arterial Intravenous Topical Intravenous Topical Intra-arterial Intra-arterial Intravenous Topical

+ + - - - -

-- -- - - - _

+++ +++

+ + +

--- --

--- -- -- --

++ + 0

++ +-

++ ---

+ ---

0 0 + + 0 0

+ +

+ +

+ 0

0 0

0 + +

+ +

+ 0

t++ ++ 0 0 0

+ + 0

0 + +

0 0 0 0

0 0

0 0

++ +

+ 0

0 0

Jr

+ +

+

+

+

+

+

0 0 0 0

0 + - -

+ 0

0 0

0 0

f

t

T

t

r” t

+ , Stimulation; - , inhibition; 0, no effect; +, slight; + +, moderate; + + + , strong stimulation; - , slight; - - , moderate; - - - , strong inhibition.

f , Increase; .J , decrease; V.M., villous motility; M.S., mucus secretion; B.P., arterial blood pressure; Peri., peristalsis.

* See text for literature references.

174 W. L. JOYNER AND E. KOKAS

RESULTS

The results of this study were organized into two groups: (1) the effect of the gastrointestinal hormones on villous motility and (2) the effect of vasoactive pep- tides on villous motility. Each substance was tested by intravenous injection and/or topical administration. This latter method of application was used to avoid changes in the mean arterial blood pressure since it is known that changes in the arterial blood pressure may affect villous motility (Kokas & Johnston, 1965).

I. Gastrointestinal hormones

Human gastrin. The biphasic effect of an intravenous injection of human gastrin (1 pg/kg) on villous motility is shown in Fig. 1. There was an initial decrease (C.I. = -O-2), followed by a slight increase (C.I. = +0+4). Villous motility returned to the basal level in 15 min. Topically, human gastrin (l&2*0 pg) caused only a slight increase (maximal C.I. = O-17) (Fig. 2).

- 0.1

-0.2

-0.3 -0.4

-0.5

-0.6 i

-0.9 F

u HUman gastrin (I.Opg/kg) - Wntagostrin(I~0 g/kg) .--* Secretin (0.5~ M /kg) o--q CCK-PZ(I .O units/ kg)

-I.OL’ ’ ’ ’ ’ ’ ’ ’ 1 I I I 0 I , ,

0 I 2 3 4 5 6 7 8 9 IO II 12 13 14 15

Time, min

FIG. 1. This compares the effect of villous motility of different gastrointestinal hormones with that of villikinin. The substances were all administered i.v. The ordinate shows the time of successive countings of contracting villi in a defined visual field. The Contraction Index (C.I.) computed for all time intervals is

shown on the abscissa. Values are mean f S.D.

Pentagastrin. Pentagastrin (O*l-1.0 pg/kg) intravenously (Fig. 1) and topically (lG2.0 pg) (Fig. 2) ’ d m uced an immediate decrease invillousmotility (C.I. = - 0.49 respectively - O-6). Th e inhibition lasted throughout the test period.

GASTROINTESTINAL HORMONES AND VILLOUS MOTILITY 175

&cretin. Intravenously, secretin (O*l-O*S clinical units/kg), like human gastrin, displayed a biphasic action, an initial decrease (C.I. = -0.5) followed by a slight (C.I. = -tO.25) increase (Fig. 1). The C.I. returned to the basal level within 15-20 min. Topically, in different dosages, no significant change in villous moti- lity was registered (Fig. 2).

0.9

0.6

0.7

0.6

0.5

- Human gastrio @Opg) - Pentogostrln (2.0 g) c--4 Secretin ( I .o ““I& P--Q CCK-PZ (2.Ounit)

‘. - Enterogartrone lo+g) b--4 Vllllkinin (0.1 mg)

\ ‘. (Crude CME) 8.

a ‘.

-0.3

-0.4

-0.5

-0.6

-0.7

-09 Wash -1.0 1 *, ,

0 I 234567 6 9 IO

Time, min

FIG. 2. This shows the effect of various gastrointestinal hormones on villous motility after topical application in comparison to villikinin. Ordinate: time

intervals of countings. Abscissa: the Contraction Index (C.I.).

Cholecystokinin-pancreoxymin (CCK-PZ). Intravenously, CCK-PZ (O-l-1.0 Ivy units/kg) caused a biphasic change in villous motility (Fig. 1). An initial stimulation (C.I. = + 0.5) was followed by a rapid long lasting decrease in activity. CCK-PZ (0.1-2.0 Ivy units), topically, inhibited villous motility (C.I. = - 0.4) in all doses (Fig. 2).

Enterogastrone and Gastric Inhibitory Peptide (GIP). Partly purified entero- gastrone preparation (0.5 mg/kg) (L ucien et al., 1968, 1969) and “Gastric Inhibi- tory Peptide” (0.1 pg/kg) (G.I.P.) (B rown et al., 1969) intravenously (Fig. 1) and topically (Fig. 2) inhibited villous motility. The inhibition lasted throughout the test period.

176

II. Vasoactive peptides

w. L. JOYNER AND E. KOKAS

Caerulein. Caerulein (Osl-1.0 pg/kg) intravenously (Fig. 3) caused a dramatic inhibition of villous motility; all motility stopped (C.I. = - 1.00). The tonus of the villi increased; the villi contracted and remained in this state. They regained their original length and activity 20-25 min after the injection. The mean arterial blood pressure decreased 20-30 mm Hg. The peristaltic activity of the observed jejunal loop was greatly enhanced. Caerulein (O*Ol-0.3 pg) topically, also inhibited villous motility (C.I. = -0.4) (Fig. 4).

Ranatensin. Ranatensin (I$)-lO.Opg/kg), intravenously, as topically (0.2-0.5 pg), caused a slowly developing inhibition (Figs. 3 and 4). The villi became con- tracted and motility stopped (C.I. = - 1.0). The mean arterial blood pressure increased 20-25 mm Hg.

Eledokin. Eledoisin (1.0 pg/kg) intravenously initiated a triphasic response (Fig. 3). An initial decrease was followed by an increase and a subsequent decrease in villous motility (C.I. = - 0.3). The second inhibitory period was stronger and longer lasting than the first. Topically, eledoisin (0.1 pg) caused a slight stimulation (C.I. = 0.2) (Fig. 4).

Dose-response testing

It is known that the various actions of gastrointestinal hormones and vaso- active peptides depend upon the dose; therefore, a dose-response test was per- formed with some of the substances. The results are summarized in Fig. 5, depicting the log of the maximal or minimal experimental CPM (E) divided by the basal CPM (B) for each substance.

Pentagastrin. Pentagastrin, injected intravenously, inhibited villous motility at all doses tested (l-0, 0.5 and O-1 pg/kg) (Fig. 5). There was a linear relationship between dose and Contraction Index.

CCK-PZ. This inhibited villous motility at higher doses (1.0 and 0.5 Ivy units/kg) (Fig. 5). H owever, at a lower dose (0.1 Ivy units/kg) CCK-PZ stimulated it slightly.

Caerulein given intravenously at various doses (1.0,O.l and 0.01 ,ug/kg) inhibited villous motility (Fig. 5). There was a linear relation between the log dose and the Contraction Index.

Villikinin given intravenously at lower doses (O-5, 1.0, 1.5 and 2.0 mg/kg) stimulates villous motility while higher doses (2.5 and 5.0 mg/kg) initially inhibit it, but this inhibition is followed by a stimulation.

DISCUSSION

Villikinin can be differentiated from other smooth muscle active substances by the mode of its action. The characteristics of villikinin action when given intra- venously are: (1) the number of contracting villi starts to increase l-3 min after the injection (aa depicted in Fig. 1 and 2), (2) the maximal effect is reached 5-8 min after the injection, (3) the rate of contractions returns to the basal level within

GASTROINTESTINAL HORMONES AND VILLOUS MOTILITY 177

raptI! uoplJ+uo3

‘\ ??.

‘. ‘.

%

T : I

I

& .’

.’ c /

178 W.L. JOYNERAND E. KOKAS

IO.0 : - PentwJartrin(/vJ/kg) 1 e-a CCK-Pi! (units/kg) _ - Corruleln (/.&kg) _ W-Q Villikinln (mg/kg)

4 / \

3” ‘1,

‘l \

FIG. 5. Represents the dose-response curve of pentagastrin, cholecystokinin, caerulein and villikinin after intravenous administration. Ordinate: the different doses in pg, units or mg/kg wt and abscissa : the appropriate contraction ratio (E/B),

E = experimental CPM and B = basal CPM.

1.5 min after the injection, (4) the tunica muscularis of the gut is not affected, (5) there are no obvious changes in mean arterial blood pressure, (6) no tachyphylaxis is observed, (7) there is no increased mucus formation on the exposed mucosal surface (Kokas & LudAny, 1933, 1934; Kokas & Johnston, 1965), (8) the response is blocked with hexamethonium (Ludiny et al., 1960), (9) villikinin causes vaso- dilation in villi (Ludany & Obal, 1945), (10) it acts topically and intraarterially in the same way, (11) in very high doses, above 2.5 mg/kg crude material causes an inhibition with subsequent stimulation (Fig. 5) and (12) refined material is only stimulatory (Kokas & Johnston, 1966). These criteria were used in this study to differentiate between the action of villikinin and the effect of other substances.

In order to present a summary, the effect of various substances on villous motility contained in the intestinal mucosa and tested in earlier experiments are included in Table 1.

Pentagastrin and enterogastrone-like substance G.I.P. inhibited villous motility while human gastrin, CCK-PZ and secretin displayed a biphasic effect, inhibition followed by stimulation. In earlier investigations unrefined secretin, cholecysto- kinin preparations (Kokas & Ludiny, 1934) and crystallized secretin (Ludiny & Leone, 1940), caused no changes in villous activity. The effect of secretin and human gastrin is similar to the injection of large doses of crude intestinal mucosal extracts (CME), inhibition followed by stimulation (Kokas & Johnston, 1966).

GASTROINTESTINAL HORMONES AND VILLOUS MOTILITY 179

Most of the substances decreased mean arterial blood pressure, but the changes were not enough to influence significantly the villous motility. Changes in villous motility were also evident after the topical application of the substances; therefore, the effect is a direct action on the smooth muscle of the villi rather than the result of changes in systemic blood pressure. It was shown that changes in intestinal motility produced by the injection of vasoactive substances are independent of changes in intestinal blood flow (Shehadeh et al., 1969).

One of the powerful stimulators of villous motility is histamine (Verzir, 1936). Histamine stimulates villous motility within 1 min after the injection. All the villi in the visual field contract simultaneously; its action is short-lasting. Serotonin (Ludany et al., 1959) inhibits villous motility. Serotonin causes a sustained con- traction and the villi become shorter. Substance P is similar in action to villikinin, the difference being that its action is not blocked by hexamethonium (Ludany et al.,

1960). Recent observations with synthetic Substance P, however, showed an inhibition of villous motility with increased mucous secretion, while only a very small dose (5 ng/ml) topically stimulated villous motility (Kokas & Crepps, personal communication). Bradykinin, kallidin and eledoisin inhibited villous motility (Ludiny et al., 1968). The response was either a biphasic response, stimulation followed by inhibition or vice-versa, or a triphasic response, inhibition, stimulation and inhibition again, but the dominant effect was inhibition. The action of caerulein was similar to CCK-PZ and pentagastrin. All of these sub- stances inhibited villous motility. In avians (Angelucci et al., 1972), low doses of caerulein (l-5 pg/kg per min) stimulated villous motility. Ranatensin effectively inhibited villous motility by causing the villi to remain in a contracted state, similar to the action of caerulein. Ranatensin is an alytensin-like peptide and this group of peptides is quite different in chemical composition and pharmacological action from other active polypeptides (i.e. caerulein) found in amphibian skin (Bertaccini, 1970).

Analyzing the results, it can be stated that none of the substances had an effect on villous motility identical with that of villikinin.

Pentagastrin, CCK-PZ and caerulein, which have similar C-terminal peptides, inhibited villous motility and this inhibition was dose related. Human gastrin, however, stimulated villous motility. The difference in the action of pentagastrin and human gastrin indicates that a region other than the C-terminal portion of the gastrin molecule is of importance for the stimulatory effect.

This study supports the concept that villikinin is unique and is of physiological significance.

SUMMARY

The effect of the gastrointestinal hormones (cholecystokinin-pancreozymin, human gastrin, pentagastrin, secretin and enterogastrone) and vasoactive peptides (caerulein, ranatensin and eledoisin) on villous motility were studied and their action was compared to that of villikinin. The substances were injected intraven- ously or applied topically to the exteriorized intestinal mucosa of the jejunum in

180 W. L. JOYNER AND E. KOKAS

anesthetized dogs. The changes in villous motility were expressed by computing the Contraction Index. The results show that: (1) the dominant effect of the gastrointestinal hormones (secretin, CCK-PZ, pentagastrin, enterogastrone-like substances) was inhibitory on villous motility; (2) human gastrin slightly stimu- lated it; and (3) the vasoactive peptides (caerulein, ranatensin and eledoisin) inhibited villous motility.

All of the data indicate that villikinin has a physiological action which is not identical to the tested substances present in the mucosa of the small intestines.

Acknowledgements-We would like to thank the following for their generosity: Dr. H. Lucien for the enterogastrone preparations; Dr. J. Brown for the Gastric Inhibitory Peptide; Ayerst Laboratories for the pentagastrin; and Farmitalia (M&no) for the caerulein. This study was supported by U.S.P.H.S. Research Grant No. AM04675.

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GASTROINTESTINAL HORMONES AND VILLOUS MOTILITY 181

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Key Word Index-Villous motility; secretin; gastrin; cholecystokinin; pancreozymin; caerulein; ranatensin; eledoisin; villikinin.