the possible role of close contacts (nexuses) in the propagation of control electrical activity in...

The Possible Role of Close Contacts (Nexuses) in the Propagation of Control Electrical Activity in the Stomach and Small Intestine

E. E. Daniel, PhD, Kathleen Robinson, HA, G. Duchon, MSc and Ruth M. Henderson, PhD

The hypotheses that intraarterial perfusion of hypertonic solutions would uncouple slow waves of the dog intestine and that this uncoupling would be associated with disruption of nexal connections were studied. The first hypothesis was affirmed, but reserpinization decreased the uncoupling effect of hypertonic solutions. This suggested that release of endogenous norepinephrine might play a role. However, after sufficient hypertonic solution to uncouple slow waves, the nexuses were found to be morphologically intact, though the cells were markedly shrunken as shown by histologic examination and their electrolyte contents. Nexuses may have been damaged or other changes may account for the uncoupling caused by hypertonic solutions.

The purpose of this study was to deter- mine (a) if slow wave transmission in intestinal muscle was affected by hypertonic solutions, and (b) if such changes as oc- curred could be a consequence of disruption of the close contacts (nexuses) between smooth muscle cells.

Intestinal slow waves are omnipresent in the small intestine of a variety of mammal - ian species (1-11) and arise from periodic depolarizations of longitudinal muscle cells, t ransmit ted electrotonically into the un- derlying circular muscle (12-16). When distal portions of the intestine are separat- ed in various ways from proximal portions, the frequencies of their slow waves dimin- ish (3, 17-19). The re appears to be a gradi-

From the Department of Pharmacology, Univer- sity of Alberta, Edmonton 7, Alberta, Canada.

Supported by a grant from the Life Insurance Medical Research Fund.

Address for reprint requests: E. E. Daniel, PhD, Department of Pharmacology, University of Al- berta, Edmonton 7, Alberta, Canada.

ent of inherent rhythmici ty decreasing down the intestine (19, 20). T h e frequency of slow waves in distal portions of the intestine can be increased by electrical coupling to proximal intestine (18-20), and the model of coupled relaxat ion oscillators has been suggested (18, 20, 21) as an appro- priate model for this interaction.

Transmission of action potentials in taenia coli can be disrupted reversibly by hypertonic solutions, associated with rever- sible disrupt ion of nexuses and increase in resistance across a sucrose gap (22). These are areas of fusion of the outer surfaces of cell membranes of adjacent cells believed to provide low resistance pathways for current flow (22-30). Others have been unable to disrupt ei ther nexal conections (31) or electrotonic connections between smooth muscle cells with hypertonic solutions (31, 32). However, in nonintest inal smooth muscle, hypertonic solutions disrupted electrical and mechanical activity (33) and in- hibited submaximal responses to drugs

Digestive Diseases, Vol. 16, No. 7 (July 1971) 611

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CONTROL ELECTRICAL ACTIVITY

GONTROL

A

kcrER I 8 i i . . , , , , , t ~ l , , , I , •

Fig 2. Effects of hypertonic and isotonic solutions on slow waves of duodenum. Top panels show slow waves before and just after perfusion at 3.88 ml/min of hypertonic KR. Bottom panels show slow waves before and just after similar perfusion of isotonic KR. Note uncoupling at top and lack of uncoupling at bottom (compare frequencies at electrodes one and two to those at five and six. Slow waves were depressed at electrodes three and four in perfused area. Electrode distances were: ® to ®, 1.4 cm; ® to ®, 0.6 cm; ® to ®, 0.4 cm; ® to @, 1.2 cm; and ® to ®, 1.2 cm. Distance from electrode @ to pylorus was 8 crn. Calibrations were as in Fig 1.

Digestive Diseases, Vol. 16, No, 7 (July 1971) 613

(34); b u t t h e s e ef fec ts c o u l d b e o v e r c o m e by

e l e v a t i o n of e x t e r n a l K + (31, 33 - 35 ) . T h e

s u g g e s t i o n was m a d e t h a t h y p e r t o n i c so lu -

t i o n s h y p e r p o l a r i z e d t h e ce l l b y c o n c e n t r a t -

i n g i n t e r n a l K + to a c c o u n t f o r t h e s e effects.

T h e s e f i n d i n g s a n d h y p o t h e s e s l e d t o t h e

p r e s e n t s t u d y .

MATERIALS AND M E T H O D S

Jejunal or duodenal electrical activity of healthy female dogs under pentobarbi tal anesthesia was studied, using six silver wire electrodes and mono- polar recording as previously described (3, 5, 36, 37). Solutions were perfused intraarterially into one of the vasa recta by a technic previously described (36, 37). Two electrodes each were proximal to, in or distal to the perfused segment. From 10 to 60 milliliters of heparinized Krebs Ringer solution (KR) (36), made hypertonic by adding 350 mmoles/ l i ter sucrose (total osmolarity about 750 mOsmolar), were perfused at a rate of 3.9-7.8

DANIEL ETAL

ml/min, depending on the size of the intestinal segment. This disrupted coupling of slow waves in and through the perfused segment (Fig 1 and 2, Table 1). Recovery required 1-35 minutes (segments which failed to recover because of ischemia or other technical problems were not included in this analysis). Then a similar volume of ordinary heparinized KR was perfused at the same rate. The frequency of intestinal slow waves was sometimes disrupted by this procedure, but the decline in frequency below the perfused segment was less, and coupling was restored much faster--within 60 seconds in all but I case (Fig 1 and 2, Table 1). In many experiments, this procedure could be re- peated one or more times.

Finally, ligatures were tied around the intestine above and below the perfused area, its vein was cut and the segment perfused with 20 ml of either KR or hypertonic KR containing 2% glutaraldehyde, each diluted to 1 volume of Ringer with 0.7 volumes of H~O. The tissues became yellow and leathery within a few seconds. The glutaraldehyde solution emerging from the vein was collected

Table 1. Effect of Hypertonic Perfusion on Intestinal Slow Waves

No. of Dogs, intestinal

No. segments Perfusate

Wave freq uency

Decrease Above below

Rate of Volume of perfused perfusion Time to perfusion perfusion area area recover (ml/min) (ml) (min -1) (min -1) (min)

Normal 6

Reser- pinized

3

Normal 1

12 KR 12 KR + 350 mmolar

sucrose

6 KR 10 KR + 350 mmolar

sucrose 1 KR 3 KR -I- 350 mmolar

sucrose

2 KR (Ca-free) 2 KR (Ca-free) + 350

mmolar sucrose 2 KR + 2--4 X K 2 KR + 2-4 X K + 350

mmolar sucrose

3.9-7.8 10-20 16.4 + 0.7 0.8 ± 0,3 0.6 ± 0.3 3.9-7.8 10-20 16.5 ± 0.7 2.6 ± 0.3 16.8 ± 4.3

7.8 20 18.0 ± 0.2 2.4 ± 0.9 1.1 ± 0.4 7.8 20 18,0 ± 0.3 2.6 ± 0,6 1.5 ± 0.5

7.8 55-65 18.0 2.0 0.5 7.8 55-65 18.2 3.0 16.5

3.9-7.8 15-20 17.7 0.6 1.0 3.9-7.8 15-20 17.7 2.5 1.8

3.9 10 13.5 2.5 3.9 3.9 10 13.6 2.9 2.0

614 Digestive Diseases, Vol. 16, No. 7 (July 1971)


on gauze sponges to avoid damage to adjacent segments. After fixation, the segme~]t was cut out, opened, the mucosa removexl and the muscle sub- divided. Two strips (circular and longitudinal) were placed in buffered glutaraldehyde for electron microscopy, and two larger strips were taken for analysis of H~O (by drying overnight), Na and K (using the EEL flame photometer), Ca and Mg (using the Unicam SP90A atomic absorption spec- trophotometer) and C1 (Cotlove titrator) (7, 38) .

Tissues were prepared for electron microscopy by postfixation for 1 hour in OsO,, dehydration, and oriented embedding in El)on. Blocks of longitudinal and circular muscle were cut, both longitudinally and in cross section, on a Porter-Blum MT 2 ultramicrotome. Sections were mounted on 400 mesh grids and double stained with uranyl acetate and lead citrate. They were examined on a JEM-7A microscope, and the number of nexuses per grid square was counted at moderately high magnification (approximately × 40,000 on the screen). Pho- tographs were taken at low and high magnification to show cell outlines and the structure of the nexuses.

R E S U L T S

In tissues perfused with d i lu ted K R and glutaraldehyde, the s tructures were very well preserved and nexuses were readi ly observed (Fig 3 and 4, T a b l e 2). All the nexuses observed were p laquel ike (32). T h e electrolyte contents of such tissues (Tab le 3) were near normal values. Tissues fixed with d i lu ted hyper ton ic K R and glutaraldehyde had obviously larger ext racel lu lar spaces and sh runken cells (Fig 5), and these changes were reflected in the values for electrolyte contents (Table 3). N a and C1 contents per k i logram wet weight were increased, while H 2 0 contents were decreased (the H 2 0 con ten t / l i t e r was less in hyper tonic compared to no rma l KR). How- ever, nexuses were regular ly observed (Fig 5 and 6, T a b l e 2); indeed, they ha d the appearance (Fig 6) of being very stable points of contact despite the forces pu l l ing ceils apar t on shrinkage. T h e n u m b e r of nexuses per gr id square was no t detectably deci, eased after hyper ton ic perfusion, even

Table 2, Number of Nexuses in Cross-Sectional Areas of Intestinal Smooth Muscles*

After Nexuses per Segment perfusion (ml) grid square

1 Hypertonic (55) 12, 19, 14 (mean 15) 2 Isotonic (55) 19, 26, 18 (mean 21) 3 Hypertonic (65) 21, 31, 18 (mean 23) 4 Isotonic (20) 7, 8, 9, 11, 13, 21

(mean 11.5)

* All segments are from 1 reserpinized dog; except for the last segment, each was perfused with the solution indicated, the last 15 ml of which contained glutaraldehyde. The last segment was perfused only with isotonic KR aand glutaraldehyde. The total number of nexuses per grid square (400 mesh grids) was determined.

when hyper ton ic K R was given, fol lowed immedia te ly by fixation as above (Tab le 2). Similar results were ob ta ined when the perfus ing solutions were Ca-free and con- ta ined 1 m m o l e E G T A ; bu t recovery of electrical activity after hype r ton ic solutions seemed faster in such cases. E leva t ion of K in the perfusates (fi 'om 4.6 to 9.2 mmotes or higher) did not prevent the effects of hyper tonic solutions in electrical activity, and indeed elevat ion of K itself d i s rup ted slow waves as previously described (Tab le 1) (37).

These results suggested tha t some factor o ther t han d i s rup t ion of nexal connect ions was responsible for the effects of hype r ton ic solutions. In t raa r te r i a l n o r e p i n e p h r i n e un- couples slow waves in vivo. Af te r reser- p in iza t ion (0.1 m g / k g in t ravenous ly for each of 3 days), larger volumes of hyper tonic solut ions were requ i red to uncoup le slow waves (Table 1), bu t nexal connect ions were still intact in such cases (Table 2). Figures 3 to 6 were taken f rom reserpinized animals, bu t similar figures could have been presented f rom norma l animals.


Fig 3. Low-power view (X 15,000) of cross section of circular intestinal muscle after perfusion with isotonic KR. Muscle was from segment 2 of reserpinized dog from which data for Table 2 were taken. Slow waves of this segment were not uncoupled when fixed, and proportions of cellular and extracellular spaces should be compared to Fig 5. Numerous pinocytotic vesicles, mitochondria and one or two myelin figures are present. At least four nexuses are also present in this control segment.

Fig 4. Higher-power views from same preparation as in Fig 3 (top, X 210,000; bottom, X 60,000). Bottom view shows nexus, pinocytotic vesicles, mitochondria, dense bodies and myelin figure. At top, typical five-lined structure of nexus is shown at higher power. Note that at left, membranes of two cells separate to show two unit membranes. In nexus, central dark line is about as thick as outer lines.

Fig 5. Low-power view ( X 15,000) of cross section of circular intestinal muscle after perfusion with hypertonic solutions. Muscle was from segment 3 of reserpinized dog from which data for Table 2 were taken. Slow waves of this segment were uncoupled when this segment was fixed. Note large proportion of extracellular space and shrunken cells with numerous processes. At least three nexuses were identified in this view, as well as other structures characteristic of smooth muscle.

%

~7 #

b 6

Fig 6. Higher-power views from same preparation as in Fig 5 (top, X 210,000; bottom, X 60,000). Bottom view shows nexus connecting extended processes from two cells. Pinocytotic vesicles, mitochondria, etc, are present. At top, higher-power view shows details of nexal structure, which are identical to those in Fig 4. At left, two unit membranes separate.

Table 3. Electrolyte Contents of Fixed and Unfixed Intestinal Muscle*

DANIEL ET AL

Perfusate No. K Mg Na CI H=O Ca

None 4 75.04-4.3 5.74-0.5 63.94- 6.7 58.1-+-5.8 7894-17 2.24-0.6 KR + gluta- ra ldehyde 4 61.3 4- 1.0 6.8 4- 0.5 77.0 4- 11,9 60.0 ± 5.7 740 ± 13 3.7 ± 0.8 KR Jr gtutaralde- hyde (after hypertonic perfusion) 8 55.2 -4- 2.6 6.4 ± 0.2 69.0 4- 3.3 52.2 ± 1.4 795 4- 4 3.4 -4- 0.5 Hypertonic KR Jr g lu tara ldehyde 4 75.0 4- 1.8 8.3 4- 0.2 87.1 4- 3.1 67.1 4- 4.8 710 4- 22 3.0 4- 0.4 Hypertonic KR Jr g lutara ldehyde (after hypertonic perfusion) 8 44.6 4- 2.5 6.2 4- 0.5 90.5 4- 2.5 62.8 4- 1.7 771 =E 11 2.9 4- 0.2 Hypertonic KR Jr g lutara ldehyde (after KR and hypertonic perfusion) 14 58.64-3.3 6 .84-0 .2 94.74- 3.7 72.34-3.6 740=L L 9 3 .54-0 .5

* Values are means i standard errors expressed in mmoles/kg wet weight except for H20 which was expressed in g/kg wet weight.

D I S C U S S I O N

Hyper tonic solutions, perfused intraar- teriaIly, uncouple slow waves in the dog intestine in vivo, but this is not related to the breaking of nexal contacts. Failure of nexal contacts to be broken by hypertonic solutions has been repor ted by others (31, 32), but in the one instance where it was studied, action potent ial propagat ion and electrotonic spread of electrical activity was also preserved (31). However, our experiments with reserpinized dogs suggest that release of norep inephr ine by such solutions may be involved in uncoupl ing of slow waves in some of our experiments. With or wi thout reserpinization, uncoupl ing of slow waves could be obtained, but nexal contacts were preserved. This does not suggest that coupling of slow waves is not via pathways of low resistance through nexuses. (The critical exper iments would be demonst ra t ion of preservation of coupl ing after disrupt ion of nexuses.) Instead, our

data suggest that changes more subtle than physical separation of nexal contacts may be involved. Other technics (eg, measure- ments of resistance between cells) will be required for demonstra t ion of these changes.

R E F E R E N C E S

1. Bozler E: Electrophysiological studies on the motility of the gastrointestinal tract. Amer J Pbysiol 122:614, 1938

2. Armstrong HIO, Milton GW, Smith AWM: EIectropotential changes in the small intestine. J Physiol (London) 131: 147, 1956

3. Daniel EE, Carlow DR, Wachter BT, et al: Electrical activity of the small intestine. Gastroenterology 37:268, 1959

4. Haladay DA, Votk H, Mandel J: Electrical activity of the small intestine with special reference to the origin of rhythmicity. Amer J Physiol 195:505, 1958

5. Daniel EE, Wachter BT, Honour A J, et al: The relationship between electrical and mechanical activity of tile small intestine



of dog and man. Canad J. Biochem 38:777, 1960

6. Bortoff A: Slow potential variations of small intestines. Amer J Physiol 201:203, 1961

7. Bass P, Code CF, Lambert EH: Motor and electric activity of the duodenum. Amer J Physiol 201:287, 1961

8. Christensen J, Schedl HP, Clifton JA: The small intestinal basic electrical rhythm (BER) frequency gradient in normal men and in patients with a variety of diseases. Gastroenterology 50:301, 1966

9. Daniel EE, Chapman KM: Electrical activity of the gastrointestinal tract as an indication of mechanical activity. Amer J Dig Dis 8:54, 1963

10. Daniel EE: Pharmacology of the gastrointestinal tract, chap 108, Handbook of Physiology, Sect 6, Alimentary Canal. Vol IV, Motility. Edited by CF Code. Wash- ington, DC, American Physiological Society, 1968

11. Daniel EE: The electrical activity of the alimentary canal. Amer J Dig Dis 13:297, 1968

12. Daniel EE, Honour AJ, Bogoch A: Elec- trical activity of the longitudinal muscle of dog small intestine studied in vivo using microelectrodes. Amer J Physiol 198:113, 1960

13. Bortoff A: Electrical activity of intestine recorded with pressure electrodes. Amer J Physiol 201:209, 1961

14. Bortoff A: Electrical transmission of stow waves from longitudinal to circular intestinal muscle. Amer J Physiol 209:1254, 1965

15. Kobayashi M, Prosser CL, Nagai T: Elec- trical properties of intestinal muscle as measured intracellularly. Amer J Physiol 213:275, t967

16. Kobayashi M, Nagai T, Prosser CL: Elec- trical interaction between muscle layers of cat intestine. Amer J Physiol 211:t281, 1966

17. Bass P: In vivo electrical activity of the small bowel, chap 100, Handbook of Physiology, Sect 6, Alimentary Canal. Vol

IV, Motility. Edited by CF Code. Wash- ington DC, American Physiological Society, 1968

18. Nelson TS, Becker JC: Simulation of the electrical and mechanical gradient of the small intestine. Amer J Physiol 214:749, 1968

19. Diamant NE, Bortoff A: Nature of the intestinal slow-wave frequency gradient. Amer J Physiol 216:301, 1969

20. Diamant NE, Bortoff A: Effect of transec- tion on the intestinal slow wave frequency gradient. Amer J Physiol 216:734-743, 1969

21. Daniel EE: Digestion: motor function. Ann Rev Physiol 31:203, 1969

22. Barr L, Berger W, Dewey MM: Electrical transmission at the nexus between smooth muscle cells. J Gen Physiol 51:347, 1968

23. Barr L: Transmembrane resistance of smooth muscle cells. Amer J Physiol 200: 1251, 1961

24. Barr I,: Propagation in vertebrate visceral smooth muscle. J Theor Biol 4:73, 1963

25. Barr L, Berger W: The role of current flow in the propagation of cardiac muscle action potentials. Pflugers Arch 279:192, 1964

26. Barr L, Dewey MM: Electrotonus and electrical transmission in smooth muscle, chap 85, Handbook of Physiology, Sect 6, Ali- mentary Canal. Vol IV, Motility. Edited by CF Code. Washington, DC, American Physiological Society, 1968

27. Dewey MM: The anatomical basis of propagation in smooth muscle. Gastroenterology 49:395, 1965

28. Dewey MM, Barr L: Intercel lular connec- tion between smooth muscle cells: the nexus. Science 137:670, 1962

29. Dewey MM, Barr L: A study of the structure and distribution of the nexus. J Cell BioI 23:553, 1964

30. Dewey MM, Barr L: Structure of vertebrate intestinal smooth muscle, chap 81, Handbook of Physiology, Sect 6, Alimen- tary Canal. Vol IV, Motility. Edited by CF Code. Washington, DC, American Physio- logical Society, 1968

31. Tomita T: Electrical responses of smooth


DANIEL ET AL

muscle to external stimulation in hypertonic solution. J Physiol (London) 183:450, 1966

32. Cobb JLS, Bennett T: A study of nexuses in visceral smooth muscles. J Cell Biol 41: 287, 1969

33. Johansen B, Ljung B: Spread of excitation in the smooth muscle of the rat portal vein. Acta Scand Physiol 70:312, 1967

34. Carroll PM: The effect of hypertonic solution on the wet weight and contractions of rat uterus and vas deferens. J Gen Physiol 53:590, 1969

35. Johansson B, Jonsson o : Cell volume as a

factor influencing electrical and mechanical activity of vascular smooth muscle. Acta Physiol Scand 72:456, 1968

36. Daniel EE: The electrical and contractile activity of the pyloric region in dogs and the effects o[ drugs. Gastroenterology 49: 403, 1965

37. Daniel EE: Effects of intra-arterial per- fusions on electrical activity and electrolyte contents of dog small intestine. Canad J Physiol Pharmacol 43:551, 1965

38. Batra SA: Cation exchange and binding in smooth muscle. PhD thesis, University of Alberta, 1969


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