the ultrastructural bases for coordination of intestinal motility

The Ultrastructural Bases for Coordination of Intestinal Motility

E. E. Daniel, PhD, G. Duchon, MSc and Ruth M. Henderson, Phd

The electrical control activity (slow waves) of dog small intestine is characterized by phase locking of potential changes in a frequency plateau in the upper intestine. In the distal intestine, phase locking does not occur, though frequencies of each segment are pulled up (increased) by adjacent, more proximal segments. This suggests poorer coupling in the distal compared to the proximal intestine. Electron microscopic studies of the intestine revealed no differences in appearance or number of nexuses (found only in circular muscle) or of intermediate contacts (found in both muscle layers) in duodenum and upper jejunum as compared with ileum. Thus, differences in cell to cell contacts could not explain poorer coupling in the ileum. No difference in innervation of these two regions was observed. However, evidence was obtained that circular muscle cells of the ileum, unlike those of the duodenum, are not oriented perpendicular- ly to the longitudinal muscle layer. This could provide a structural basis for poorer coupling and for the observed phase lag of potentials around the circumference of the ileum.

In the small bowel of the (tog, the electrical control activity, designated as slow waves or pacesetter potentials, originates from periodic d e p o l a r i z a t i o n s of the l o n g i t u d i n a l m u s c l e cells (1-3). In the d u o d e n u m and upper je-

i u n u m , these waves are of constant frequency and are phasedocked (4 7); this region has been called the frequency plateau. More distal segments are not phase-locked and have lower and more variable frequencies. Th e intr insic frequencies of surgically separated segments de- crease from duodenum to i leum (6, 7).

An appropr ia te model for intest inal electrical control activity, which duplicates near ly all of its observed characteristics, is a chain of re- laxation oscillators which are mutua l ly c o u -

From the Department of Pharmacology, University of Alberta, Edmonton 7. Alberta.

Address for reprint requests: E. E. Daniel, PhD, De- partment of Pharmacology, University of Alberta, Edmon- ton 7, Alberta.

Supported by Alberta Heart Fund.

pied (6, 7). Th i s model is capable of account ing for more characteristics of the intest ine than a chain with only forward coupl ing (8, 9). T h e abil i ty of such mutua l ly coupled relaxat ion oscillators to become phase-locked depends, in- versely, upon the difference in their intr insic frequencies and directly upon the extent of mu- tual coupl ing (the fraction of the ou tput of one oscillator fed into the other). Th i s led us to study further the intr insic frequencies of intes- t inal segments and also the degree of coupl ing between cells as evidenced by the number s and types of cell contacts.

MATERIALS AND METHODS

Female mongrel dogs were anesthetized with chloralose- urethane supplemented by pentobarbital, the abdomen was opened and monopolar-punctate electrodes were placed on intestinal segments. Details of these procedures have been given (6, see also 7). Various regions of the intestine were uncoupled, usually by transection of the muscle layers, as previously described (6). Recording and analysis of records were carried out as before.

Digestive Diseases, Vol. 17, No. 4 (April 1972) 289

DANIEL ET AL

Electron Microscopy Appropriate segments of the intestine were perfused

intra-arterially via the vasa recta, with a diluted Krebs- Ringer solution containing 1.2% glutaraldehyde (325 mOsm). After fixation the mucosa was dissected off and rectangular pieces of known orientation were placed in phosphate-buffered gtutaraldehyde fixative. One or more days later, l-ram strips of tissue were postfixed in OsO 4, dehydrated in graded ethanols and embedded in flat molds, oriented so that a known muscle layer was obtained in longitudinal section and the other layer in cross section. Details of the fixation and embedding have been described previously (10).

To obtain relative numbers of cell contacts in the intestinal segments, the number of contacts per 400-mesh grid square was counted. The nexuses were counted on 6-grid squares from each sample, uniformly covered with cells of the desired (circular) layer in cross section; the intermediate contacts on 3-7 fields per sample.

R E S U L T S

If the f requency p la teau ends at the midje-

j u n u m because the intrinsic f requencies of

more distal segments are too low to be pulled up

to the p la teau f requency by the avai lable coupling, reduct ion of the f requency of the lower

par t of the f requency-p la teau region should ex-

tend the p la teau distally. Such an extension of

the p la teau had been found (4, 5). In our ex-

p e r i m e n t s , w h e n p l a t e a u f r e q u e n c i e s w e r e

d iminished by cut t ing the muscle layers in the

lower por t ion of the plateau, the p la teau was

extended for several cent imeters in the je junum

(Figure 1). However , when cuts were made 15

cm apar t in the i leum, the frequencies below

each cut were lowered but the regions between

cuts did not become phase-locked (Figure 2). In

the oscilIator model, uncoupl ing in the region

below the f requency pla teau (by reducing the

coupl ing coefficient to zero) did lead to phase

locking of oscillators below the point of uncou-

pl ing (7). Th i s suggested that some factor not

incorpora ted in the model had accounted for

this phenomenon . Decreased coupl ing between cells, because of

differences in numbers or types of cell contacts,

could also provide an explana t ion for the ob- servations. W e had previously showed that, in

the canine duodenum, nexuses were found only

in the c i rcular muscle layer (10). However , both

c i rcular and longi tudinal muscle have a second

type of cell contact, which is designated the in-

te rmedia te contact. T h e appearances of both

nexuses and in termedia te contacts were s imilar

in duodenum and i leum (Figures 3 and 4). T h e

nexuses have been shown to be gap junct ions

and are character is t ical ly associated with gly-

cogen granules and mitochondria . These mito-

chondr ia and others located near the plasma

m e m b r a n e are found near single membrane -

l imited spaces which may correspond to sarco-

plasmic ret iculum. W h e n the number of nex-

19

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Fig 1. Effect on f requency of slow waves of mak ing a single cut in the f requency plateau region of the intest ine of a nor- rnal dog. Frequencies recorded pr ior to the cut (ar row) are in- dicated by (o) and those af ter cut by (I-I). The second frequency plateau fo rmed distal to the cut was at a lower frequency and extended beyond the original plateau. The average f requencies distal to the s e c o n d f r e q u e n c y p l a t e a u varied wi th t ime as before.

~' 40 80 120 160 PYLORUS DISTANCE FROM PYLORUS (cm)

200

290 Digestive Diseases, Vol. 17, No. 4 (April 1972)

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10 20 30 40 50 ELECTRODE Number

40 80 120 160 200 240

DISTANCE FROM PYLORUS (crn)

Fig 2. Effect on f requency of a series of cuts a long the ent ire small intestine. Frequencies of control electrical act ivi ty in the intact intest ine (o) recorded at 53 electrodes implanted th roughout the ent ire length of the small intestine. All f requencies were averaged over 1- minute periods. Height of each vertical line shows the range of var iat ion of 1-minute average frequencies over a m in imum t ime interval of tO minutes. Intr insic f requencies a f ter surgical uncoupl ing (x) of regions just distal to each cut (arrows) are shown as averages. There was no phase locking in distal intestine in areas between cuts.

uses and contacts per grid square of duodenum or upper jejunum was compared with that of the ileum, no great difference was observed, though the range in each was considerable (Table 1). Thus, /here was no uhrastructural evidence for a difference in coupling in the two regions.

While no quantitative studies were done on nerve fibers, the number of studies made between the duodenum and ileum showed no marked difference. In both, nerve fibers parallel to the smooth cells were seen within the muscle layers, completely surrounded by muscle cells, (Figure 5) as well as in the connective tissue between layers. The nerves were seen more commonly in circular muscle than in longitudinal muscle.

We did observe, however, a difference in the alignment of the circular muscle. Longitudinal sections of circular muscle could be easily oh-

tained from the duodenal segments by appropriate orientation of the tissue before section- ing. In the ileum, identical procedures fre- quently produced oblique sections of short segments of circular muscle, with ragged ends passing out of the plane of section (Figure 6). With the tissue sample illustrated, true longitudinal sections of muscle cells were obtained by tilting the block approximately 10 ° before sec- tioning. Low-power views of circular muscle in cross section demonstrated the presence of discrete bundles of muscle cells separated by connective tissue, each bundle having slightly different orientation. Similar bundles were not prominent in the duodenum.

Discrete bundles of circular muscle, especial- ly if not perpendicular to longitudinal muscle cells (ie, if spiraling or in random disarray), provide a structural basis for uncoupling of distal oscillators (see Discussion). If this structural


DANIEL ET AL

Fig 3. Appearance of nexus in circular muscle of duodenum (3A) and ileum (3B). Note proximity of mitochondria and glycogen granules and presence of single membrane-l imited spaces near mitochondria and the plasma membranes (arrows) (x 120,000).


INTESTINAL MOTILITY

Fig 4. Appearance of intermediate contacts in longitudinal muscle of duodenum (4A) and ileum (4B). Note increased density of cytoplasm at contacts and central dense line. Such contacts also appear in circular muscle (× 30,000).


Table 1A. Number of Nexuses in Intestinal Segments*

DANIEL ET AL

Duodenum (D) or jejunum (J) Ileum

Experiment No. Average Range Average Range

1 13 10-18(J) 11 2 11 6-16(J) 10 3 16 10-19(D) 12 4 12 7-19(D) 9 5 13 8-16(D) 9

4-19 4-15 9-15 5-12 4-17

*Values are numbers per 400-mesh grid square

Table lB. Number of Intermediate Contacts in Intestinal Segments*

Duodenum Ileum

Experiment No. Average Range Average Range

3 22 15-26 21 12-26 4 36 29-51 37 25-49 5 37 34-40 34 23-50

';Values are numbers per field at x 2,800

arrangement occurs, there should be an appre- ciable phase lag around the circumference in any region in which circular muscles are out of alignment. This prediction has been verified in studies on the region below the frequency plateau in 5 clogs (Figure 7).

D I S C U S S I O N

The results appear to rule out a simple difference in intrinsic frequencies of slow waves as the explanat ion for the failure of phase locking in the ileum. It is still possible that because of greater cycle-to-cycle variat ion in the periods of slow waves from isolated segments of ileum, a higher degree of coupling would be required to achieve phase locking.

Differences in the number or structure of cell contacts also do not appear to provide an explanat ion for the observed physiologic differences between the segments. Our results do not, of course, el iminate more subtle structural differences or purely functional differences of

cell contacts as possible mechanisms. Likewise, there is no obvious difference in the nerve sup- ply to the muscle layers in the two regions.

Although there is probably good electrical coupling through the nexuses between circular muscle cells within a bundle, the structural ar- r a n g e m e n t of c i r c u l a r - m u s c l e bund le s at somewhat variant angles could account for the phase-locking failure in the ileum. The longitudinal muscle at any given transverse plane of the ileum would be in contact with several circular muscle bundles of different orientation, each of which might have received electrical input from slightly different levels of longitudinal muscle. Such input would have different phase relations and might interfere with phase locking in the overlying longitudinal muscle. No direct evidence is as yet available to prove or disprove that slow waves in circular muscle can influence the activity of overlying longitudinal muscle. However, the structures that provide a current path for longitudinal muscle slow waves to drive circular muscle will also provide a re-


INTESTINAL MOTILITY

Fig 5. Appearance of nerves between circular muscle cells of duodenum (5A) and ileum (5B). The nerves generally run parallel to the muscle cells and may contain large dense-cored vesicles and small agranular vesicles (× 45,000).


DANIEL ET AL

Fig 6. Appearance of circular muscle of i leum cut with plane of section exactly perpendicular to longitudinal muscle layer. The actin filaments are seen in nearly longitudinal section, but the cell outlines are truncated, passing out of the plane of section (× ]2,000).


INTESTINAL MOTIL ITY

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I I I l l

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Fig 7. Control electrical activity from ileum about 45 cm above terminal ileum. Electrodes 1, 2 and 5 placed along the longitudinal axis on antimesenteric side of intestine at distances shown below figure. Electrodes 2, 3 and 4 placed circumferent ial ly in the same plane around intestine. The distances around the intestine are shown below the figure, For convenience in estimating phase lag. the points of maximum negative-going deflections are shown by l ines left in the figure and maximum positive-going deflections are similarly shown at right. Cali- brations at lower left.

turn current path unless marked rectification exists. Results of recent studies on the computer model demonstrate that circumferential phase lags do disrupt longitudinal phase locking and support this explanation hw physiologic obser- vations.

R E F E R E N C E S

1. I)aniel EE, Honour ..\J, Bogoch A: Electrical activity of the hmgitudinal muscle of dog small intestine studied in viw~ using microelectrodes. AmJ Physiol 198:113-118, 1960

2." Prosser CL, Bortoff A: Electrical activity of in-

testina] muscle under in vitro conditions, chap 99, Handbook of Physiology. Sect 6, Alimentary Canal, VoI IV, Motilhy. Edited bv C.F. Code. VVashington, DC, American Physiological So- cietv. 1968

3. Holman ME: An introduction to electro- physiology of visceral smooth muscle, chap 83, Handbook of Physiology, Sect 6, Alimentary Canal, Vol IV, Motilitv. Edited by C.F. (;ode. Washington, DC, American Physiological So- ciety, 1968

4. Diamant NE, BnrtoffA: Nature of the intestinal slow-wave frequency gradient. Am J Phvsiol 216:301 307, 1969

5. Idem: Effects of transection on the intestinal

Digestive IDiseases, Vol. 17, No. 4 (Aprj l 1972 ) 297

DANIEL ET AL

slow-wave frequency gradient. Am J Physiol 216:734-743, 1969

6. Sarna SK, Daniel EE, Kingma Y J: Simulation of slow-wave electrical activity of small intestine. AmJ Physiol 221:166-175, t971

7. Sarna SK: Computer models of gastrointestinal electrical control activity. PhD Thesis, Univer- sity of Alberta, 1971

8. Nelsen TS, Becker JC: Simulation of the elec-

trical and mechanical gradient of the small intestine. Am J Physiol 214:749-757, 1968

9. Diamant NE, Rose PK, Davison E J: Computer simulation of intestinal slow-wave frequency gradient. Am .1 Physiol 219:1684-1690, 1970

10. Henderson RM, Duchon G, Daniel EE: Cell contacts in duodenal smooth muscle lavers. Am ,] Physio1221:564 574, 1971


the ultrastructural bases for coordination of intestinal motility

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