Bayliss & Starling (1899, 1900) were the first to report the

existence of polarized reflexes in the gastrointestinal tract.

They showed that stimulation of the canine small intestine

evoked a relaxation in the smooth muscle lying anal to the

stimulus site, but it was noted that a contraction was evoked

orally. Such observations led them to formulate their now

widely accepted ‘law of the intestine’ which has formed the

basis of our understanding of the peristaltic reflex. Since

their study, other investigators, particularly those using

intracellular microelectrodes from the smooth muscle layers,

have reported the existence of polarized reflex pathways

that appear to generate ascending excitation (depolarization)

and descending inhibition (hyperpolarization) along the

small intestine (Hirst & McKirdy, 1974; Hirst et al. 1975;

Smith & Furness, 1988; Smith et al. 1990, 1991; Yuan et al.

1991, 1995; Johnson et al. 1998).

Despite Bayliss and Starlings’ seminal observations, a

number of investigators in the first half of this century have

found difficulty in demonstrating the ‘law of the intestine’,

at least in its most simplistic form, during mechanical

recordings from the small bowel of many species (see

chapter 3 in the review by Alvarez, 1948). For example,

Alvarez & Starkweather (1919) reported that local

stimulation produced contractions both above and below the

stimulus site of rabbit small intestine. Similarly, Hukuhara

et al. (1936) found that local stimulation of the dog small

intestine produced a contraction that propagated in both

directions, with no evidence of the descending relaxation

reported earlier by Bayliss & Starling (1899). One difference

between these studies was that Bayliss & Starling (1899)

used castor oil to purge the intestinal lumen. Even in the

small bowel of man, White et al. (1934) could not see any

wave of inhibition that preceded spontaneous propagating

contractions. Later, R�oden (1937) suggested that descending

inhibition in the human small bowel ‘does not seem to be

valid under physiological conditions’. In fact, Alvarez (1948)

was of the opinion that the widening of the small bowel that

is sometimes observed to precede a wave of contraction was

Journal of Physiology (1999), 517.3, pp.889—898 889

Does the guinea-pig ileum obey the ‘law of the intestine’?

Nick Spencer, Michelle Walsh and Terence K. Smith

Department of Physiology and Cell Biology, University of Nevada School of Medicine,

Reno, NV 89557, USA

(Received 27 January 1999; accepted after revision 9 March 1999)

1. We report the first simultaneous mechanical reflex responses of the longitudinal muscle (LM)

and circular muscle (CM) layers of the guinea-pig ileum following mucosal stimulation and

distension in vitro.

2. Dissection techniques were used to prevent mechanical interaction between the LM and CM

layers both oral and anal to a stimulus site.

3. All graded stimuli produced graded contractions of both the LM and CM orally and anally to

the stimulus. Contractions occurred synchronously in the LM and CM and under no

circumstances were inhibitory responses recorded in either muscle layer, despite the presence

of ongoing cholinergic tone in both the LM and CM. Contractions were abolished by

tetrodotoxin (1·6 ìÒ).

4. Local brush stroking of the mucosa evoked a peristaltic wave which readily conducted

distally over 13 cm, without the presence of fluid in the lumen. No descending relaxation

was observed.

5. Apamin (300 nÒ) disrupted evoked peristaltic waves and significantly increased the rate-of-

rise of the LM and CM contractions anal to a stimulus, and the LM oral to a stimulus.

6. Nù

-nitro-¬_arginine (100 ìÒ), a nitric oxide synthesis inhibitor, had no overall significant

effect on the characteristics of the LM and CM contractions, although on occasion an

enhancement in their peak amplitude was noted.

7. It is suggested that the guinea-pig ileum does not conform to the ‘law of the intestine’ as

postulated by Bayliss & Starling (1899). Rather, local physiological stimulation of the ileum

elicits a contraction both orally and anally to a stimulus, which occurs synchronously in both

the CM and LM layers. Apamin-sensitive inhibitory neurotransmission modulates the rate-

of-rise of the anal contraction of the CM, possibly to generate distal propulsion.

9196

due ‘not to inhibition but to distension by the long column

of intestinal contents which is forced ahead by the wave of

contraction’. The presence of a contraction and absence of

relaxation anal to a local stimulus has not been restricted to

mammals, but has also been reported to occur following

distension of the avian small bowel (Hodgkiss, 1986).

However, despite difficulty in reproducing the ‘law of the

intestine’ in the small intestine, it seems that following a

local stimulus, an oral contraction and anal relaxation is

more readily recorded in the large bowel (Bayliss & Starling,

1900; Crema et al. 1970; Mackenna & McKirdy, 1972;

Smith & McCarron, 1998). This is quite likely to be due to

regional differences in the composition of the intestinal

contents, since propulsion of a solid pellet in the distal colon

necessarily requires different mechanisms for propulsion

than the rapid movement of fluid in the small bowel (see

Crema, 1970).

Since the pioneering observations of Bayliss & Starling at

the turn of the century (Bayliss & Starling, 1899, 1900), we

have gained significantly more knowledge about the enteric

nervous system of mammals. In particular, the guinea-pig

ileum has emerged as the model preparation for

understanding how enteric neurons are integrated to

regulate peristalsis; use of this model has implied that the

stereotypical ascending excitatory and descending inhibitory

reflexes underlie peristalsis in the small intestine (Furness et

al. 1994). Immunohistochemical studies in this tissue have

shown the existence of polarized projections of enteric

motoneurons to the circular muscle (CM) layer, since

inhibitory motoneurons project anally along the ileum

(Costa et al. 1992), while the excitatory motoneurons project

orally, or locally, to the CM layer (Furness et al. 1994).

Further support for the existence of these polarized

motoneural projections to the CM comes from intracellular

recording from the CM layer, where it has been shown that

distension (Hirst et al. 1975; Smith et al. 1990, 1991) or

mucosal stimulation of the ileum (Smith & Furness, 1988;

Smith et al. 1991; Yuan et al. 1991) elicits inhibitory junction

potentials (IJPs) anal to a stimulus, while excitatory

junction potentials (EJPs) are elicited orally. It has therefore

been assumed that distension, or muscosal stimulation, of

the guinea-pig ileum may generate similar mechanical

responses in the smooth muscle (i.e. contraction orally and

relaxation anally), consistent with the ‘law of the intestine’,

originally postulated by Bayliss & Starling (1899).

Recently, we showed that, in the guinea-pig distal colon, a

mucosal stimulus was sufficient to elicit an anal relaxation

response and oral contraction of the smooth muscle (Smith &

McCarron, 1998), consistent with the ‘law of the intestine’

suggested by Bayliss & Starling (1899). Remarkably,

however, no studies have investigated whether the guinea-

pig ileum conforms to the ‘law of the intestine’ under control

conditions. These experiments most probably have been

overlooked, due to an expectation that IJPs would cause

relaxation anally and EJPs would cause contraction orally.

Therefore, we have developed a preparation that allows

simultaneous recording of the mechanical activity of the

longitudinal muscle (LM) and CM layers of the guinea-pig

ileum, both oral and anal to a stimulus site, and that avoids

the possibility of any mechanical interactions between the

two muscle layers. This technique has been utilized to

investigate (1) whether the mechanical reflex responses of

the guinea-pig ileum conform to the ‘law of the intestine’

proposed by Bayliss & Starling (1899), and (2) whether the

LM and CM layers are reciprocally innervated, as suggested

by Kottegoda (1969).

METHODS

Guinea-pigs weighing 250—350 g were killed by inhalation of a

rising concentration of COµ, approved by the animal ethics

committee of the University of Nevada School of Medicine. The

abdominal cavity was opened and the terminal 15 cm of distal

small bowel was removed, the mesenteric attachment trimmed

away, flushed clean with Krebs solution, and then placed

immediately into a modified Krebs solution (composition below). To

record the simultaneous responses of the two muscle layers, the LM

and myenteric plexus (MP) were dissected free of CM and mucosa at

each end to create an LMMP preparation (see Smith & Robertson,

1998). To do this, a longitudinal incision (approximately 15 mm in

length) was made along the oral and anal extremities. These regions

were pinned mucosal side uppermost so that the mucosa and

submucosa could be delicately removed from either extremity to

expose the underlying CM layer. Strips of CM were then delicately

removed from the exposed oral and anal regions, to reduce the

possibility of any mechanical interactions between the movements

of the LM and CM muscles. Therefore, preparations consisted of a

flap of LM with attached myenteric plexus that remained in

continuity with the oral and anal regions of ileum (Fig. 1). The free

ends of the dissected region (see LMMP, in Fig. 1) were connected

via thread to independent tension transducers (see below) mounted

orally and anally to the stimulus. To examine the propagation of

neural reflexes along the ileum, a long segment (approximately

15 cm in length) was removed from the animal and a region

(20 mm) was cut open along the mesenteric border, the mucosal

surface opened and pinned (facing uppermost) approximately

30 mm from the oral end of the segment (Fig. 4). The mechanical

activity of the CM was monitored using small clips (Micro-

serrefines No. 18055-04; Fine Science Tools Inc., Foster City, CA,

USA) mounted oral (2 cm from the opened region) and anal (3 clips

placed 2 cm apart) from the opened stimulating region. These were

attached via the serosal surface to the underlying CM of the intact

ileum (Figs 1, 4 and 5). Tension in the LM and CM was recorded

using Grass (Quincy, MA, USA) FT03 tension transducers and

recorded on a 4-channel Gilson Medical Electronics 5Ï6H Recorder

(Middleton, WI, USA). Initial tensions of the LM and CM were

routinely set to 1 g, so that we could directly compare the reflex

responses of the guinea-pig ileum with those we observed in the

guinea-pig distal colon, where we used identical stimulating and

recording methods and reflex-evoked relaxations of the LM and CM

were shown to occur (Smith & McCarron, 1998). Preparations were

bathed in oxygenated Krebs solution at 37°C.

Protocol for stimulation of the ileum

Oral and anal reflexes were elicited by distension (radial stretch) or

by mechanical stimulation of the mucosa; the mucosa was

stimulated with an artist’s brush and the gut distended using a

plastic plate (12 mm ² 5 mm) under the serosa of the opened

pinned segment (Fig. 1). This enabled mechanoreceptors to be

N. Spencer, M. Walsh and T. K. Smith J. Physiol. 517.3890

stimulated, without distortion of the mucosa, and therefore prevent

mucosal stimulation. The plate was connected via strings that

pierced the lumen to a pulley system to which weights (5—30 g)

were added.

Drugs and solutions

The following drugs were used in the current study: atropine

sulphate, apamin, hexamethonium bromide, histamine, Nù

-nitro-

¬_arginine (L-NA), sodium nitroprusside (SNP) and tetrodotoxin

(TTX), all obtained from Sigma. Stock solutions were prepared in

distilled water.

The composition of the modified Krebs solution was (mÒ): NaCl,

120·35; KCl, 5·9; NaHCO3, 15·5; NaHµPOÚ, 1·2; MgSOÚ, 1·2;

CaClµ, 2·5; and glucose, 11·5. The solution was gassed continuously

with a mixture containing 3% COµ—97% Oµ (vÏv), pH 7·3—7·4.

Measurements and statistics

Mann—Whitney U test and Student’s (paired or unpaired) t tests

were used where appropriate. A minimum significance level of

P < 0·05 was used throughout. The use of n in the results section

refers to the number of animals on which observations were made

and data are presented as means ± s.e.m. (standard error of the

mean). Measurements of the rate-of-rise of contractions were

performed by dividing the peak contractile amplitude by the time-

to-peak. The latter was derived by the time taken for contractions

to reach peak amplitude, taken from 10% of peak amplitude (on

the rising phase).

Descending excitationJ. Physiol. 517.3 891

Figure 1. Anal and oral responses of longitudinal and circular muscle to mucosal and distension

stimuli

A, diagrammatic representation of the preparation used for simultaneous recording of the tension in the

longitudinal (LM) and circular muscle (CM) oral and anal to a stimulus site. The CM at the oral and anal

extremity of the tissue was dissected away, so that a flap of LM remained in continuity with the myenteric

plexus (LMMP). TLM and TCM refer to isometric tension transducers for the LM and CM, respectively; g is

applied radial stretch in grams. B and C, control responses of the LM and CM oral and anal to a mucosal

stimulus (B; 5 brush strokes, arrow) and radial distension (C ; 30 g, arrow). Note that the LM and CM

contracted synchronously above and below the stimulus.

RESULTS

Mechanical responses of the longitudinal and circular

muscle to mucosal stroking and radial distension

We investigated whether the mechanical reflex responses in

the guinea-pig ileum conform to the ‘law of the intestine’

postulated by Bayliss & Starling (1899). We found that, in

73 out of 80 trials (n = 8), brush stroking the mucosa (see

Methods) induced a synchronous contraction in the LM and

CM, both orally and anally to the stimulus (Fig. 1B). In the

remaining seven trials, the anal contractions of the LM or

CM were faint, or within the level of the recording noise.

The amplitude and rate-of-rise of the CM contraction oral to

the stimulus were significantly enhanced compared with the

CM contraction anal to the stimulus (the oral CM amplitude

was 13·2 ± 3·6 mN and rate-of-rise, 6·6 mN s¢ compared

with an anal CM amplitude of 4·5 ± 0·5 mN and rate-of-rise

of 2·3 ± 0·4 mN s¢). There was, however, no significant

difference between the amplitude and rate-of-rise of the LM

contraction oral (amplitude, 7·0 ± 0·2 mN; rate-of-rise,

4·0 ± 0·6 mN s¢) or anal (amplitude, 6·9 ± 2·2 mN; rate-

of-rise, 3·7 ± 1·1 mN s¢; P > 0·05; n = 8) to the stimulus,

when recorded under control conditions. Under no

circumstances were any relaxations recorded in either

muscle layer, orally or anally to the mucosal stimulus. A

maximal contractile amplitude was typically obtained with

two to three brush strokes, delivered at a frequency of

approximately 1—2 Hz. To examine whether distension of

the ileum also elicited similar mechanical responses to those

elicited by mucosal stroking, we radially distended the

ileum underneath the serosal surface to avoid stimulation of

the mucosa (see Methods). We found that distension also

consistently evoked synchronous contractions in the LM and

CM, orally and anally to the stimulus (Fig. 1C). A consistent

observation was that grading the number of brush strokes

(1—5 strokes) and radial distensions (5—30 g) consistently

produced graded changes in the amplitude of the contractions

in both the LM and CM orally and anally to the stimulus.

We examined the possibility that inhibitory responses

(relaxations) may only occur within short distances (< 2 mm)

anal to a stimulus. We found that, in 24 out of 24 trials of

N. Spencer, M. Walsh and T. K. Smith J. Physiol. 517.3892

Figure 2. Effects of mechanical and

pharmacological increases in resting tone on

anal reflex responses of the longitudinal and

circular muscle

A, effects of increasing the resting tone on the LM

and CM anal to a mucosal stimulus. Progressive

increases in the resting tone applied to the LM and

CM (1, 2 and 4 g) consistently elicited powerful

contractions in the LM and CM following mucosal

stimulation (3 strokes; arrow). B, effects of pre-

contracting the ileum with histamine on anal reflex

responses of the LM and CM. Brush stroking the

mucosa (3 strokes; arrow) elicited a synchronous

contraction in the LM and CM. Subsequent addition

of histamine (3 ìÒ) caused a rapid increase in the

resting tone of the LM and CM. In the presence of

histamine, mucosal stimuli delivered on 3 occasions

blocked contractions in the LM. In the CM, faint

contractions were still elicited in the presence of

histamine. No relaxations were recorded, despite

enhancement of the resting tone. C , application of 3

brush strokes to the mucosa (arrow) elicited a

synchronous contraction in the LM and CM anal to

the stimulus. Addition of atropine (1 ìÒ) reduced the

resting tone of both the LM and CM, suggesting the

presence of ongoing cholinergic tone. Contractions to

mucosal stroking were abolished in the LM, and in

the CM, faint non-cholinergic contractions persisted.

mucosal brush stroking (n = 4), each stimulus always

generated a powerful contraction of the CM, 2 mm anal to

the stroking site, with no sign of muscular relaxation.

It might have been argued that the absence of muscular

relaxations in the LM and CM anal to the stimulus was due

to insufficient resting tone applied to the muscles. Therefore,

we delivered a mucosal stimulus, after progressively

applying increased levels of resting tension (1, 2 and 4 g) to

the LM and CM (Fig. 2A). Regardless of the level of applied

resting tone, we consistently recorded a powerful contraction

in the LM and CM anal to each mucosal stimulus (n = 3;

Fig. 2A).

We then examined whether pharmacological pre-contraction

of the ileum with histamine (1—6 ìÒ) would induce

relaxations in the LM and CM anal to a mucosal stimulus

(see Costa et al. 1986). From 11 animals tested, we found

that addition of histamine caused an immediate increase in

the resting tone (> 10 mN) of both the LM and CM

(Fig. 2B). In eight of these animals, contractions of the LM

and CM were essentially blocked with no sign of relaxation

(Fig. 2B). In one preparation, we did record a faint

relaxation in the CM, and surprisingly also in the LM (as the

LM does not receive an intrinsic inhibitory innervation (see

Hirst et al. 1975; Bywater & Taylor, 1986), relaxations in

both the LM and CM of this particular animal were resistant

to atropine (2 ìÒ), but blocked by apamin (300 nÒ)). In the

remaining two animals, the anal contractions of the LM and

CM were not blocked, but were reduced in amplitude by 83

and 73%, respectively, by histamine. To test that the

muscles were capable of relaxation, we examined the effects

of the nitric oxide donor SNP on the tone of the LM and CM.

SNP (10 ìÒ) was found to induce an immediate and powerful

relaxation of both the LM (by 5·4 ± 1·1 mN; 4 preparations,

n = 2) and CM (by 2·3 ± 0·6 mN; 4 preparations, n = 2) of

the ileum, as it does in the guinea-pig distal colon (Smith &

McCarron, 1998).

Effects of blockade of inhibitory neurotransmission

on reflex responses of the longitudinal and circular

muscle

The distension- and mucosal stimulus-evoked IJP in the CM

layer of the guinea-pig ileum is abolished by the bee venom

apamin (Smith & Furness, 1988; Smith et al. 1990). We

examined the possibility that apamin-sensitive inhibitory

neurotransmission may modulate the characteristics of the

contractions of the LM and CM, oral and anal to a mucosal

stimulus. Addition of apamin (300 nÒ) enhanced small

spontaneous background contractions of the LM and CM

and in half of the animals tested (4 out of 8) induced

spontaneous motor complexes (see below). Apamin (300 nÒ)

significantly increased the rate-of-rise of the contractions in

the LM (3·7 ± 1·1 to 5·7 ± 1·0 mN s¢; P < 0·05; n = 8)

and CM (3·2 ± 0·4 to 5·5 ± 1·1 mN s¢; P < 0·05; n = 8)

anal to the mucosal stimulus, and in the LM oral to the

Descending excitationJ. Physiol. 517.3 893

Figure 3. Effects of apamin on oral and anal responses to mucosal stimulation

A, control responses of the LM and CM layers, oral and anal to a mucosal stimulus. Brush stroking the

mucosa (3 brush strokes; arrow) elicited a synchronous contraction in both the LM and CM, oral and anal to

the stimulus. B, addition of apamin (300 nÒ) increased the amplitude of the LM contraction oral and anal

to the stimulus, and the CM contraction anal, but not oral, to the stimulus.

stimulus (Fig. 3B). While in the presence of apamin

(300 nÒ), no significant difference was found in the rate-of-

rise of the oral CM contraction (Figs 3B and 4B). Further

addition of L_NA (100 ìÒ) had no significant (P > 0·05;

n = 7) effect on the amplitude or rate-of-rise of the

contractions oral and anal to the stimulus, although on

occasion an increase in the amplitude of the LM and CM

contractions was noted. To examine the involvement of

nicotinic neurotransmission in these ascending and

descending reflexes, hexamethonium (300 ìÒ) was applied to

the ileum. Addition of hexamethonium (300 ìÒ) significantly

reduced the amplitude of the oral contractions of the LM (by

80·0 ± 11·7%; P < 0·05; n = 4) and CM (by 86·2 ± 9·6%;

P < 0·05; n = 4). However, overall the amplitudes of the

anal contractions of the LM and CM were not reduced

significantly (by 45·3 ± 22·5 and 53·0 ± 21·2%, respectively)

although in one preparation contractions were abolished.

Atropine (1—2 ìÒ) reduced the resting tone of the LM (by

2·3 ± 0·3 mN; n = 5) and CM (by 3·6 ± 1·4 mN; n = 5)

(Fig. 2C) and consistently blocked contractions in the LM

(n = 5) and in four out of five animals abolished the

contraction in the CM oral to, and anal of, the mucosal

stimulus. In one animal, contractions of the CM were

reduced oral (by 60%) and anal (by 92%) by atropine.

To examine whether mucosal stimuli elicited only local

contractions, or responses that propagated along the ileum,

multiple CM recording sites were located (2 cm apart) anal

to the mucosal stimulus (see Fig. 4). In these preparations,

local brush stroking of the mucosa was sufficient to elicit a

wave of contraction that propagated aborally, despite the

absence of fluid in the lumen. These waves exhibited a

propagation velocity of 28·7 ± 9·6 mm s¢ (n = 4), consistent

with the measurements for peristaltic waves reported by

Tsuji et al. (1992) in this tissue. These descending waves

readily conducted over 13 cm anal to the local stimulus,

often with no decrement in amplitude (Fig. 4A). These

evoked peristaltic waves did not occur in the presence of

TTX (1·6 ìÒ; n = 3), suggesting they were dependent upon

activity within the enteric nervous system. In six out of

seven animals tested, apamin (300 nÒ) significantly

increased the amplitude (site ii: 6·0 ± 1·1 to 15·1 ± 5·1 mN;

site iii: 8·6 ± 2·2 to 13·5 ± 4·5 mN; site iv: 5·8 ± 1·1 to

16·3 ± 4·4 mN) and rate-of-rise (site ii: 1·8 ± 0·3 to

5·8 ± 1·5 mN s¢; site iii: 2·2 ± 0·4 to 4·7 ± 1·0 mN s¢; site

iv: 1·0 ± 0·2 to 4·2 ± 1·0 mN s¢) of the CM contractions

recorded at three sites anal to the stimulus (Fig. 4B). In

contrast, apamin (300 nÒ) had no effect on the activity of

the CM oral (site i) to the stimulus (P > 0·05; n = 7). In one

preparation, apamin (300 nÒ) did not appear to alter the

N. Spencer, M. Walsh and T. K. Smith J. Physiol. 517.3894

Figure 4. Effects of apamin on a peristaltic wave evoked by local stimulation

Mechanical recording of CM activity oral (i) and anal (ii, iii and iv) to a local mucosal stimulus. A, mucosal

stimulation (3 strokes; arrow) elicited a contraction that propagated at least 60 mm anally despite the

absence of fluid in the lumen. The rate-of-rise of the descending contraction was shown to decrease, and the

time-to-peak to increase, the further the recording site was from the site of stimulation. The time-to-peak

of the response increased with distance giving rise to an effective propagation of the response. B, reflex

responses of the CM after addition of apamin (300 nÒ). Apamin increased the amplitude of the responses

of CM anal, but not oral, to the stimulus and disrupted the apparent propagation of the peristaltic wave

(dashed line).

characteristics of the anal or oral contractions of the CM. We

also noted that apamin unco-ordinated evoked peristaltic

waves, and contractions often occurred almost

synchronously at all sites (Fig. 4B), leading to infinite

apparent conduction velocities. Also, four out of eight

preparations showed spontaneous motor complexes in

apamin (300 nÒ), which did not appear to have any

consistent preferential site of origin or direction, often

originating from oral or anal regions of ileum. On other

occasions, it appeared that spontaneous contractions almost

occurred simultaneously at all sites in the ileum (Fig. 5). In

two animals, these motor complexes were recorded in

control solution (i.e. in the absence of apamin). These events

appear to be similar to the intracellularly recorded

myoelectric complexes that also propagate spontaneously

along the isolated mouse colon (see Bywater et al. 1989;

Spencer et al. 1998).

DISCUSSION

A primary finding of this study is the demonstration that

local stimulation of the guinea-pig ileum (via radial

distension or mucosal distortion) elicits a contraction in both

the LM and CM layers, both orally and anally to a stimulus.

No inhibitory responses were recorded in either muscle layer

anal to a stimulus site (despite the presence of ongoing

cholinergic tone in both muscle layers), suggesting that the

guinea-pig ileum does not conform to the ‘law of the

intestine’ as originally postulated by Bayliss & Starling

(1899). Moreover, the LM and CM layers are not reciprocally

innervated, as suggested by Kottegoda (1969), but rather

contract synchronously during reflex stimulation.

We have shown that two different stimuli applied to the

ileum elicit the same mechanical response in both muscle

layers oral and anal to the stimulus. In the current study, we

radially distended the muscle layers underneath the serosal

surface, rather than using a balloon, to avoid direct

distortion of the mucosa. This enabled us to selectively

stimulate stretch-sensitive mechanoreceptors in the muscle,

and avoid inadvertent stimulation of the mucosa (which

occurs with balloon distension). Conversely, brush stroking

the mucosa selectively stimulated ‘mucosal’ sensory neurons

and avoided activation of stretch receptors. The observation

that both stimuli generated the same mechanical response

implies that both types of sensory neuron may converge

onto the same population of motoneurons to the LM and CM

(see Smith et al. 1992).

In the guinea-pig ileum, the characteristics of the oral

mechanical reflex responses (ascending excitatory reflex) of

the CM have been examined (see Tonini & Costa, 1990).

However, the only study of mechanical reflex responses anal

to a stimulus in the guinea-pig ileum that we are aware of

was by Costa et al. (1986). These investigators reported that

Descending excitationJ. Physiol. 517.3 895

Figure 5. Spontaneous motor complexes recorded from circular muscle in the presence of apamin

Mechanical recording of CM activity at 4 sites (i—iv) along the ileum in the presence of apamin (300 nÒ).

Spontaneous contractions appeared to have reached peak tension at the same time along the length of the

bowel.

the CM relaxed anal to a distension stimulus. However,

relaxations were only reported to occur when the muscle

was pre-contracted with histamine. In the current study, we

found that identical concentrations of histamine also pre-

contracted the ileum, but abolished, or markedly attenuated,

the amplitude of the descending contractions recorded in

control solution, revealing faint relaxation in only one out of

11 animals. This suggests that the pharmacological

intervention required to induce such relaxations is not

consistent with any view that such inhibitory responses are

physiologically significant. There is evidence that histamine

can exert indirect actions on the enteric nervous system, by

either presynaptically inhibiting the release of excitatory

neurotransmitters, or postsynaptically stimulating the

muscle (Daniel, 1982; Tamura et al. 1987).

In the guinea-pig small intestine, the presence of IJPs anal

and EJPs oral to a local stimulus is well documented (Smith

& Furness, 1988; Smith et al. 1990, 1991; Yuan et al. 1991,

1995; Johnson et al. 1998). This is due, in large part, to the

fact that inhibitory motoneurons project anally to the CM

layer along the guinea-pig ileum (Costa et al. 1992) and

excitatory motoneurons project orally, or locally, to the CM

(Furness et al. 1994). It has been assumed that stimulation

of the inhibitory motoneurons would generate relaxation of

the CM, especially since IJPs have been shown to occur at

least up to 40 mm anal to a local distension or mucosal

stimulus site (Smith & Furness, 1988). In direct contrast to

the implications of immunohistochemical and electro-

physiological studies on the guinea-pig ileum, the findings

of the current study have shown an absence of polarized

mechanical reflex responses in the LM and CM. Rather, we

report the presence of ascending and descending excitation

in the ileum. Descending excitation is consistent with the

studies of Hirst et al. (1975) on the guinea-pig ileum, where

it was reported that anal to a distension site, intracellular

recording from the CM revealed an IJP which was

truncated by a cholinergic EJP and if this ‘reached

threshold a muscle action potential was initiated.’

A major observation of this study was the ability to elicit a

wave of contraction from any site along the intestine that

readily conducted, often without decrement, over 13 cm anal

to a mucosal stimulus. These waves were not accompanied

by descending relaxation of the CM (Fig. 4A), despite the

presence of ongoing cholinergic tone in this muscle layer. It

is particularly interesting that the apparent conduction

velocity of these waves was 25—30 mm s¢, which is similar

to the values reported for peristaltic waves evoked in this

tissue by fluid distension using the classical Trendelenburg

technique (Tsuji et al. 1992). Our results suggest that

distension caused by the presence of fluid in the lumen is

not essential for the initiation and maintenance of

propagation of a peristaltic wave. It is quite likely, however,

that the local stimulation (stretch or mucosal) produced by a

moving fluid may modify propulsive activity. Our findings

appear to differ somewhat from the mechanism proposed by

Waterman et al. (1994), where it was suggested that

‘ascending excitatory pathways, activated by distension at

each point along the intestine, are necessary for the

peristaltic contraction to propagate. Descending excitatory

pathways, if involved, are not sufficient to mediate the

peristaltic contraction.’ Our results suggest that descending

excitatory pathways are important in peristalsis and that

apamin-sensitive descending inhibitory pathways modulate

descending excitation, most probably to co-ordinate distal

transit in the small bowel. This was shown by the effects of

apamin, which preferentially enhanced the amplitude of

anal contractions and disrupted the phase lag between their

onset at distal sites. This is consistent with the work of

Waterman & Costa (1994), where it was noted that, in the

presence of apamin, ‘the circular muscle appeared to contract

almost simultaneously along the length of intestine’.

Are the longitudinal and circular muscle layers

reciprocally innervated?

There has been some controversy in the literature as to

whether the LM and CM layers of the gastrointestinal tract

are reciprocally innervated. This is due in part to the

hypothesis put forward by Kottegoda (1969), where it was

argued that as the CM contracted, the LM layer relaxed, in

the guinea-pig ileum. Gregory & Bentley (1968) first drew

attention to the possibility of passive mechanical

interactions between the two muscle layers that can lead to

false interpretations of the relative movements of the two

muscles. In this study of the ileum, we paid particular

attention to the mechanical isolation of the muscles by using

similar dissection techniques to those we had developed for

the guinea-pig colon (Smith & Robertson, 1998; Smith &

McCarron, 1998). We found that, in the small intestine, the

longitudinal and circular muscles contract synchronously

following local stimulation. This strongly suggests that the

local excitatory LM and CM motoneurons must receive a

near-synchronous burst of fast excitatory postsynaptic

potentials (from ascending and descending interneurons)

(Smith et al. 1999), to generate synchronous contractions in

both muscle layers oral and anal to a local stimulus. Under in

vivo conditions, propulsion of the chyme in the ileum is

most probably mediated by an imbalance in the rate-of-rise

of the oral and anal CM contractions, since it was noted that

the mean rate-of-rise of the CM contraction oral to the

stimulus was significantly more rapid than that of

contractions of the CM or LM recorded anally. The results of

the current study suggest that this is likely to be mediated

by the apamin-sensitive inhibitory neurotransmitter(s) anal

to a local stimulus, to delay the rate-of-rise of the anal

muscle contractions, facilitating aboral transit of liquid

chyme. This is supported by the observations that apamin

significantly enhanced the rate-of-rise of the LM and CM

contractions anal, but not the CM contraction oral, to a

stimulus (Figs 3 and 4). In contrast to the CM, the LM layer

of the guinea-pig ileum does not receive an inhibitory

intrinsic innervation following distension (Hirst et al. 1975),

or transmural nerve stimulation (Bywater & Taylor, 1986).

Therefore, the enhancement of the oral and anal contractions

N. Spencer, M. Walsh and T. K. Smith J. Physiol. 517.3896

of the LM in the current study by apamin remains unclear.

One possibility is that apamin may facilitate the release of

acetylcholine, by increasing the excitability of the LM

motoneurons (Smith et al. 1999).

We found insignificant evidence that nitric oxide modulated

the characteristics of the reflex-evoked contractions of the

LM and CM, although, on some occasions, we did observe an

enhancement in their amplitude. This overall insignificant

effect may be due to the fact that the ‘slow’ IJP, which has

been suggested to be due to nitric oxide, is not revealed until

the membrane potential actions of the known excitatory

neurotransmitters (acetylcholine and substance P) have been

prevented (Lyster et al. 1992; Crist et al. 1992).

Physiological significance of ascending and

descending contractions

Recently, we showed that, in the guinea-pig distal colon,

addition of atropine or nitric oxide donors reduced the

resting tone of both the LM and CM (Smith & Robertson,

1998; Smith & McCarron, 1998). This is consistent with the

findings of the current study, in that both muscle layers of

the ileum also appear to be under tone, due to cholinergic

drive, since they relaxed in the presence of atropine

(Fig. 2C) or SNP. The presence of ongoing cholinergic tone

in the ileum is supported by direct intracellular recordings

from LM and short CM motoneurons from this tissue, which

have shown that these neurons appear to be highly excitable,

often discharging spontaneous action potentials (Smith et al.

1999). Despite the presence of tone in both muscles of the

ileum and distal colon, the identical stimulus and recording

conditions applied to both tissues elicited opposite responses.

In the distal colon, we showed the presence of a relaxation

in both the LM and CM anal to a mucosal stimulus and a

contraction orally (Smith & McCarron, 1998). We believe

these responses are consistent with the slow aboral transit of

solid faecal pellets into the accommodating region (Smith &

McCarron, 1998). In the ileum, however, we found that

mucosal stroking elicited contractions both orally and anally

to the stimulus. The absence of similar anal reflex-evoked

relaxations in the ileum (as those recorded from the distal

colon) is unlikely to be due to insufficient tone in the muscle.

This is because the smooth muscle in both regions of the

intestine exhibited a similar level of ongoing cholinergic

tone and, furthermore, pharmacological enhancement of the

tone (with histamine) usually abolished contractions, and

rarely revealed relaxation. Moreover, we have shown that

when an identical stimulus and recording conditions were

applied to the distal colon, relaxations anal to the stimulus

were clearly observed (Smith & McCarron, 1998). We

suggest that the lack of a mechanical relaxation in the ileum

is likely to be due to the fact that the contents of the small

bowel are in liquid state and muscular relaxation is

unnecessary for propulsion of a fluid.

In conclusion, the guinea-pig small intestine does not

conform to the ‘law of the intestine’ as postulated by Bayliss

& Starling at the turn of the century (Bayliss & Starling,

1899, 1900). Rather, physiological stimulation of the

guinea-pig ileum elicits a contraction both orally and anally

to a stimulus, and contractions occur synchronously in the

LM and CM layers. That is, the two muscle layers are not

reciprocally innervated as suggested by Kottegoda (1969).

Furthermore, apamin-sensitive inhibitory neurotransmission

facilitates co-ordinated distal transit, by modulating the

amplitude and rate-of-rise of the CM contraction anal to a

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Acknowledgements

Michelle Walsh is a visiting research scholar from the University of

Ulster (Coleraine, UK). We wish to acknowledge Professor Kenton

Sanders and Dr Fivos Vogalis for helpful suggestions and Dr Kirk

Hillsley for suggestions with data analysis. The National Institutes

of Health (USA) provided financial support of the project (grant no.

RO1 DK45713).

Corresponding author

T. K. Smith: Department of Physiology and Cell Biology,

University of Nevada School of Medicine, Reno, NV 89557, USA.

Email: tks@physio.unr.edu

N. Spencer, M. Walsh and T. K. Smith J. Physiol. 517.3898