do gap junctions couple interstitial cells of cajal pacing and neurotransmission to gastrointestinal...

Do gap junctions couple interstitial cells of Cajal

pacing and neurotransmission to gastrointestinal

smooth muscle?

E. E. DANIEL, J. THOMAS, M. RAMNARAIN, T. J. BOWES & J. JURY

Health Sciences Centre, McMaster University, Hamilton, Ontario, Canada

Abstract Interstitial cells of Cajal (ICC) pace gastro-

intestinal phasic activity and transmit nerve activity.

Gap junctions may couple these cells to smooth

muscle, but no functional evidence exists. The objec-

tive of this study was to use uncouplers of gap junc-

tions, 18a-glycyrrhetenic acid and its water-soluble

analogue carbenoxolone, to evaluate if gap junctions

function in pacing and neurotransmission. After

inhibition of nerve function with tetrodotoxin (TTX)

and NG-nitro-L-arginine (L-NOARG), ionomycin- or

carbachol-initiated regular phasic activities of circular

muscle strips from canine colon and ileum. In some

cases, the primary ICC network responsible for pacing

was removed. The effects of inhibitors of gap junction

conductance (10±5±10±4 mol L±1) on frequencies and

amplitudes of contraction were compared to appro-

priate time controls. Lower oesophageal sphincter

(LOS) relaxations to nerve stimulation were studied

before and after inhibition of gap junction functions.

No major changes in LOS relaxations or frequencies of

colonic or ileal contractions occurred, but amplitudes

of contractions decreased from these agents. Similar

results were obtained when the myenteric plexus±ICC

network of ileum was removed. Regular phasic activ-

ity was not obtained after removal of the colon sub-

muscular plexus ICC. These ®ndings suggest that

mechanisms other than gap junctions couple gut

pacemaking activity and nerve transmission.

Keywords electrical coupling, interstitial cells of

Cajal networks, myogenic activity, neuro-transmission,

slow waves.

INTRODUCTION

Gap junctions have been observed using electron

microscopy (structural gap junctions) between circular

muscle cells, apart from cells of the inner circu-

lar muscle of intestine and most cells of the circular

muscle of colon in the canine gastrointestinal tract.

They are also present between interstitial cells of Cajal

(ICC) in the myenteric plexus in all regions, deep

muscular plexus (DMP) of intestine and submuscular

plexus of colon.1±12 Good electrical coupling has been

observed or inferred between circular muscle2 cells,13±18

and assumed but not established experimentally

between ICC and between these ICC and circular

muscle. Although longitudinal muscle cells have no

gap junctions visible by electron microscopy, except

near the myenteric plexus of colon,2±4,8 some electrical

coupling has been observed or deduced between them

because they have regular slow waves coupled to those

in circular muscle in intestine9,15,18 and in several

species, space constants longer than the cell length

have been observed (see).9

Slow waves throughout the gastrointestinal tract

are known to be paced by the networks of ICC in the

myenteric plexus of stomach and intestine and in the

submuscular plexus of colon,19±21 as originally pro-

posed by Thuneberg.22 In the intestine, a network of

ICC in the deep muscular plexus plays a subsidiary

role15,16

as does the ICC network in the myenteric

plexus in the colon.20 However, gap junctions visible

in electron microscopy between ICC in the myenteric

plexus and circular muscle are rare and small11,12 and,

except in the colon, nonexistent between ICC and

longitudinal muscle.8 However, there are numerous

gap junctions between the ICC of the submuscular

Address for correspondenceE. E. Daniel, Room 4N51, Health Sciences Centre,McMaster University, 1200 Main St. W.,Hamilton, ON, L8N 3Z5 Canada.Tel.: 905 5259140 (22250); fax: 905 5243795;e-mail: [email protected]: 30 October 20001

Accepted for publication: 1 May 2001

Neurogastroenterol. Mot. (2001) 13, 297±307

Ó 2001 Blackwell Science Ltd 297

plexus of colon and the adjacent circular muscle6,11

and between the ICC in the DMP and the adjacent

outer circular muscle.2,3,11,12 Slow waves are lost

when ICC networks are removed.14,15 Slow wave

amplitudes decay with distance from these net-

works.14 Electrical pulses trigger slow waves only

when ICC networks are present.17 Such ®ndings

suggest that gastrointestinal smooth muscles cannot

generate their own slow waves and are driven by

current ¯ow from ICC networks.19

Furthermore, evidence has accumulated that the

intramuscular ICC play an essential role in inhibitory

neurotransmission.23±26 This was originally suggested

because of the regular occurrence of nerve endings very

close to intramuscular ICC, which were in gap junc-

tion contact with circular muscle.27 Additional obser-

vations in canine gastrointestinal circular muscle have

shown similar relationships.6±13,28,29 How the intra-

muscular ICC amplify and transmit neural inhibitory

information to the muscle is unclear, but the gap

junctions connecting them are considered likely to be

essential.11

These observations raise several structural para-

doxes:

(1) how can the rare (to circular muscle) or nonex-

istent (to longitudinal muscle) gap junctions between

myenteric plexus ICC of myenteric plexus of stomach

and small intestine pass suf®cient current to ®ll the

large capacity of the circular muscle syncytium and

drive slow waves of these muscle layers, which appear

to be syncytia?

(2) how can the numerous gap junctions between

ICC networks of the submuscular plexus of colon and

deep muscular plexus of intestine provide current to

drive slow waves in the adjacent syncytial circular

muscles and still maintain independent pacemaking

activities?

One possibility is that these different gap junctions

have different current-passing properties, including

possible recti®cation of current ¯ow, because: they

may contain different connexins; they may be hetero-

meric with more than one connexin in each connexon;

or they may be heterotypic with a different connexin

comprising each connexon to form a channel.29

Evaluation of the function of gap junctions in a whole

tissue precludes direct measurement of gap-junction

conductance by recording across the junction. We chose

to use two agents that are selective for gap junctions

and inhibit transmission across gap junctions com-

posed of various subunit connexins: 18a-glycyrrhetinic

acid and its water-soluble analogue, carbenoxolone.

These agents have been shown to be selective and

potent in blocking the function of gap junctions

composed of a variety of connexins in a variety of

tissues and cell types reversibly.30±56 Our objective then

was to use pharmacological tools to evaluate require-

ments for gap junction function in canine gastrointes-

tinal muscle and neuromuscular activity.

MATERIALS AND METHODS

Tissue preparation

Mongrel dogs of either sex were euthanized with

an intravenous overdose of sodium pentobarbital

(100 mg kg±1), according to a protocol approved by the

McMaster University Animal Care Committee and

following the guidelines of the Canadian Council on

Animal Care. The abdomen was opened along the

midline, segments of lower oesophagus, ileum and

colon were excised and immediately put into oxygen-

ated Krebs±Ringer solution at 24 °C having the fol-

lowing composition (in mmol L±1): 115.0 NaCl2, 4.6

KCl, 1.2 MgSO4, 22.0 NaHCO3, 1.6 NaH2PO4, 2.5

CaCl2 and 11.0 glucose. The gastro-oesophageal junc-

tion was removed and opened along the greater curva-

ture. After careful removal of the mucosa by ®ne

dissection, the thickened ring of muscle, the lower

oesophageal sphincter (LOS), was removed. Other tis-

sues, ileum and proximal colon, were opened along the

mesenteric border. Mucosae were removed by ®ne

dissection, leaving the muscularis externa. In the case

of the ileum, this tissue was either used as such or, in

some cases, further dissected by removing the longi-

tudinal muscle and myenteric plexus. In the colon, the

whole thickness was used with or without removal of

the submuscular plexus. The ef®cacy of these proce-

dures to remove nerve plexuses was assessed by

examination of whole mounts after staining of nerves

and ICC by methylene blue.

In vitro studies

In all cases circular muscle strips were prepared by

cutting tissues into multiple strips of 15 ´ 2 mm.

These were tied with ®ne thread at both ends and

mounted vertically in 5 mL organ baths, bathed in

Krebs±Ringer solution at 37 °C and oxygenated with

95% O2 and 5% CO2. Strips were tied at the bottom to

an electrode holder, passed through concentric Pt

electrodes and tied at top to a force displacement

transducer (Grass FT OC3; Grass Instruments, Quincy,

MA, USA4 ). Tensions were recorded on Beckman R611

Dynagraphs (Beckman Instruments, Fulterton, CA,

USA). Electrodes were stimulated from a Grass 88

stimulator set as 40 V cm±1, 5 pp and 0.3 ms pulse

298 Ó 2001 Blackwell Science Ltd

E. E. Daniel et al. Neurogastroenterology and Motility

duration, which gives near maximal relaxation of LOS

by activation of enteric nerves.

LOS strips had 2 g of tension applied and equilibrated

for 1 h, during which the muscle strips contracted and

spontaneously developed tone. Active tension was the

difference between the observed tension and that

obtained at the end of the experiment when Ca2+-free

Ringer solution with 1 mM EGTA was applied. Relaxa-

tion responses to electrical ®eld stimulation (EFS) were

executed until reproducible responses were obtained.

Strips in which relaxation to EFS left 75% or more of

initial tone were not used. Increasing concentrations of

inhibitors of gap junction function were then added

cumulatively at 20-min intervals and effects on tone

and on nadirs of EFS induced relaxations measured. As

several agents that inhibit function of gap junctions also

lowered tone to the level of the nadir of relaxation, we

added carbachol (10±6 mol L±1) to restore tone so that

relaxation could be tested. At the end of each experi-

ment, sodium nitroprusside (SNP) at 10±4 mol L±1 was

added, followed by Ca2+-free Ringer solution with

1 mmol L±1 EGTA to ensure that no tone was present

and that the tissues were capable of relaxation.

Ileal and colonic circular muscle strips were moun-

ted similarly with 2 and 3 g of tension, respectively,

levels shown to produce optimal contraction. Ileal and

colonic strips did not usually develop signi®cant active

tension. They were stimulated repetitively with

60 mmol L±1 KCl (added hypertonically) with inter-

mittent washing until stable responses were achieved.

Then tetrodoxin (TTX; 10±6 mol L±1) and NG-nitro-L-

arginine (L-NOARG; 1 or 3 ´ 10±4 mol L±1) were added.

After con®rming that nerve functions were blocked,

ionomycin (10±6 mol L)1) or carbachol (10±6 mol L±1)

was added to induce regular phasic activity. Phasic

activity was followed for 20±30 min to ensure that it

was persistent and increasing concentration of inhibi-

tors of gap junction function were then added cumu-

latively at 20-min intervals. One or more tissues were

left as a time control to ensure the stability of phasic

activity throughout the experiment. Measurements

were made during 3- or 5-min intervals at the end of

each period of exposure (see examples in Fig. 1). At the

end of each experiment, sodium nitroprusside (SNP)

was added followed by Ca2+-free Ringer solution with

1 mmol L)1 EGTA to ensure that no tone was present

and that the tissues were responsive.

Patch-clamp techniques

The LOS was dissected as described above and strips

were cut into 1±2 mm2 square pieces and placed in the

dissociation solution.

Cell isolation

Cells were dissociated in a solution of (in mmol L±1):

0.25 EDTA, 125 NaCl, 4.8 KCl, 1 CaCl2, 1 MgCl2, 10

HEPES and 10 glucose for 30 min. An enzyme solution

containing papain (130 mg mL±1), (±)-1,4-dithio-l-thre-

itol (L-DTT, 15.4 mg mL±1)5 , bovine serum albumin

(BSA; 100 mg mL±1) and Sigma-Aldrich (Canada, Oak-

ville ON, Canada) collagenase blend H (occasionally F)

was added to the tissue pieces for 30±60 min. After

incubation the enzyme solution was decanted off and

the tissue pieces were rinsed in enzyme-free dissoci-

ation solution. Single cells were gently mechanically

agitated with siliconized Pasteur pipettes to disperse

and isolate single smooth muscle cells. Cells used in

this study were patch clamped at room temperature

(22±24 °C) usually within 8 h of isolation.

Patch-clamp methodology

Cells from the suspension were placed in a glass-bot-

tomed dish. Within 30 min cells adhered to the dish.

The cells were then washed by perfusion with Ca2+-

containing external solution containing (in mmol L±1):

Figure 1 This ®gure contains representative tracings fromrecordings of phasic activities of whole thickness circularstrips of colonic (left) and small intestinal (right) muscle.These show the activities of the strips during the controlinterval after TTX (10±6 mol L±1) and L-NOARG (3 ´10)4 mol L±1) and the intervals which were measured afterexposure for 20 min to increasing concentrations of carbe-noxolone, added cumulatively. The traces show approxi-mately 1 min of recording of colonic and 5 min of recording ofintestinal activity. The amplitudes of contraction in controlstrips were approximately 3 g for colon and 4 g for intestine.


Volume 13, Number 4, August 2001 Gap junctions and coupling, neurotransmission in GI tract

140 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES and

5.5 glucose, pH adjusted to 7.35 with NaOH. Patch

electrodes were made using borosilicate glass capillary

tubes using a Flaming Brown micropipette puller

(Sutter Instruments Inc., Novato,6 CA, USA). After

polishing using a microforge (7 Narishige MF-83; East

Meadow, NY, USA) and ®lling, pipettes had resistances

of 2±5 MW. High Ca2+ pipette solution contained (in

mmol L±1): 2.5 CaCl2, 140 KCl, 1 MgCl2, 10 HEPES, 4

Na-ATP, 0.3 EGTA, to obtain free Ca2+ of 8 lmol L±1.

CaCl2, KCl, and EGTA levels were adjusted to obtain

50 nmol L±1 and 200 nmol L±1 free Ca2+ levels as cal-

culated using8 MAX Chelator software (version 6.72) by

Bers et al.57

A standardized stimulation protocol was used to

evoke currents from isolated smooth muscle cells,

which were studied without leak subtraction. All cells

had access resistance < 25 MW. Cell capacitances

averaged 67.6 pF (n � 3), similar to our usual ®nding,

for canine LOS cells. Cells were held at ±50 mV and

subsequently depolarized in seven cumulative steps,

each 250 ms duration, of 20 mV. Current/voltage

curves were constructed using the maximum current

values measured at t� 200 ms in the pulse. Mem-

brane currents were recorded with an Axopatch 1C

voltage clamp ampli®er, ®ltered with a 0.3-db Bessel

®lter at 1 kHz and recorded online using pclamp 5.5b

software (Axon Instruments, Union City, CA, USA)9 .

Drugs used

The following drugs were obtained from Sigma-Aldrich:

carbenoxolone, 18a-glycyrrhetinic acid, ionomycin,

octanol, hepanol, L-NOARG, SNP and EGTA. All but

18a-glycyrrhetinic [made 10±2 mol L±1 in dimethyl

sulphoxide (DMSO)] and ionomycin (100% ethanol)

were dissolved in H2O. Controls for the DMSO and

alcohol contents at the highest concentrations used

were run. DMSO at the highest concentration caused

small reductions in tone or contraction amplitude.

TTX was obtained from Alomone Labs, Jerusalem,

Israel.

Data analysis

Tone and relaxation nadirs were measured in LOS in

each strip. Each was compared to the control values as

100% and to the observed initial nadir of relaxation.

Changes after different concentrations of inhibitor

were evaluated using Dunnett's multiple comparisons

test unless only one concentration was used. Paired

comparisons were then made. In circular muscle strips

of ileum and colon, measurements were made and

compared to control values of amplitude as 100% and

to initial frequencies in that strip. Changes with con-

centrations of inhibitor were evaluated using Dun-

nett's multiple comparisons test unless only one

concentration was used. Concentration-dependent

responses were evaluated using linear correlation

coef®cients. All statistical tests were carried out using

Prism 3 software (Prism Software, Lake Forrest, CA,

USA)10 .

RESULTS

Effects of 18a-glycyrrhetinic acidon membrane currents in LOS

As shown in Fig. 2, 50 lmol L±1 18a-glycyrrhetinic had

no effects on depolarization-induced currents in cells

isolated from canine LOS. This suggests that this lipid-

soluble agent has little effect on ion channels.

Relaxations of LOS to nerve stimulation

LOS tissues rapidly developed stable tone and there-

after reproducible relaxation to electrical ®eld stimu-

lation (40 V cm±1; 5 pp; 0.3 ms pulse duration).

Subsequent addition of carbenoxolone (10±5 increasing

to 10±4 mol L±1) did not markedly increase the level at

Figure 2 Effect of 18a-glycyrrhetinic acid (50 lmol L±1) oncurrents induced by depolarizing in 20 mV steps of 25 msduration from a holding potential of ±50 mV. Pipette [Ca2+]was 200 nmol L±1, resembling the normal intracellular levelin these cells. LOS cells demonstrate no inward currentsunder these conditions. 18a-Glycyrrhetinic acid had no sig-ni®cant effect on outward currents under these conditions.Studies with octanol or heptanol were frustrated by loss of sealwithin 2±3 min n� 5 ±j± Control, 18a GRA (50 lM) (after15 min).



which the nadir of relaxation occurred (Fig. 3). The

nadir was signi®cantly higher at 3 ´ 10±5 mol L±1 and

at 10±4 mol L±1, but not at 10±5 mol L±1. In no case was

the reduction in relaxation greater than 16%. At

3 ´ 10±5 mol L±1 and 10±4 mol L±1 carbenoxolone, the

amplitude of active tension was also signi®cantly

inhibited. In the latter case, carbachol (10±5 mol L±1)

was required to restore tone above the nadir. Control

experiments without carbenoxolone showed that car-

bachol increased tone signi®cantly (to 125.4 � 13.3%

of control; n� 5) but did not interfere with relaxation

to the control nadir (to 48.79 � 12.94% before and

48.80 � 8.49% afterward). Moreover, application of a

series of other known inhibitors of gap junction

function, including 18a-glycyrrhetinic acid, also failed

to inhibit relaxations signi®cantly, as summarized in

Table 1.

Phasic contraction of colon

The strips of colon circular muscle contracted phasi-

cally at about 5 min±1 when nerve function was

blocked with TTX (10±6 mol L±1) and L-NOARG

(3 ´ 10±4 mol L±1) in some cases. When ionomycin

(10±6 mol L±1) was added, all strips contracted phasi-

cally and those that contracted previously continued to

do so with increased amplitude. Application of

increasing concentrations (10±5±10±4 mol L±1) of car-

benoxolone had no signi®cant effects on the frequency

of contractions until a concentration of 10±4 mol L±1

Figure 3 (a) Effects of increasing concen-trations of carbenoxolone on the level ofrelaxation of LOS strips in response toelectrical ®eld stimulation (EFS) at40 V cm±2, 3 pp, 0.3 ms. This intensity ofEFS causes near maximal relaxation.Different concentrations of carbenoxo-lone were applied for 35 min in differentstrips (so that a different control nadirwas required for each concentration).n� 6 for 10±5 mol L±1, n�9 for 3 ´ 10±5

mol L±1, and n� 5 for 10±4 mol L±1. (b)Effects of increasing concentrations ofcarbenoxolone on the level of tone in theLOS strips shown in Fig. 2a. n-valuessame as Fig. 2a.

Table 1 Effects of inhibition of gapjunction conductance on relaxation tostimulation of enteric inhibitory nerves incanine LOS (mean � SEM)*

Effect onEFS* residual tone [% initial]

Agent (n) tone [% initial] Control After inhibitor

Heptanol (3 mmol L)1) (5) 72.0 � 2.6 27.5 � 5.6 20.0 � 2.4Octanol (1 mmol L)1) (2) 88.0 28.7 26.12,3-Butanedione monoxime

(5 mmol L)1) (4)68.0 � 11.0 27.3 � 4.3 19.0 � 3.7

18a-Glycyrrhetinic acid(0.3 mmol L)1) (5)

92.0 � 7.5 18.2 � 2.2 19.7 � 2.4

* Electrical ®eld stimulation of nerves (40 V cm)2; 0.3 ms pulse duration; 3 pulses/s). P £ 0.01 where tone less than initial tone; no other signi®cant difference.



reduced them signi®cantly by about 55%. However, all

concentrations of carbenoxolone markedly and signi-

®cantly reduced the amplitude of contractions (Fig. 4).

One possible cause of the reduction in frequency at

10±4 mol L±1 carbenoxolone was the marked reduction

in amplitude, which may have left some contractions

below our threshold for acceptance (1 mm on the

record). In one record, no contractions were visible.

Similar results were obtained with 18a-glycyrrhetinic

acid, the lipid-soluble analogue of carbenoxolone (not

shown). Attempts to initiate regular phasic contrac-

tions after removal of the submuscular plexus and its

network of ICC were ineffective despite application of

carbachol and other stimulants. Slow tonic contrac-

tions with small phasic activity superimposed occurred

at about 1 per min.

Phasic contractions of ileum

Phasic contractile activity was initiated in ileal cir-

cular muscle strips by the same procedures as in the

colon. However, in some experiments, carbachol

(10±6 mol L±1) was used after nerve inactivation

instead of ionomycin to initiate contractile activity.

Furthermore, the ileum produced phasic activity even

after removal of the longitudinal muscle and myenteric

plexus with its accompanying ICC network. With

full-thickness circular muscle strips, as shown in

Fig. 5, carbenoxolone from 10±5 to 10±4 mol L±1 had no

signi®cant effect on frequency, except that there was

a concentration-dependent tendency towards increase.

In contrast, there were signi®cant decreases in am-

plitude of contraction at 3 ´ 10±4 and 10±5 mol L±1 car-

benoxolone as well as a concentration-dependent

decrease in amplitude. As shown in Fig. 6, when

18a-glycyrrhetinic acid was used at 3 ´ 10±5 and

10±4 mol L±1 concentrations, there were signi®cant

dose-dependent increases in frequency, and decreases

in amplitude, of spontaneous contractions. DMSO

time controls (with the concentrations of DMSO used

in the experiments) run in parallel with these studies

revealed no changes associated with the solubilizing

agent (results not shown). In addition, in studies

conducted with carbachol instead of ionomycin as

the stimulant to phasic activity, carbenoxolone

(10±5±10±4 mol L±1) had no signi®cant effect on

frequency of contractions, but all concentrations

Figure 4 (a) Effects of successive increases of concentrationsof carbenoxolone for 20 min on the frequencies of sponta-neous contractions in strips of colonic circular muscle. Asigni®cant decrease was observed only at 10±4 mol L±1. n�5.(b) Effects of successive increases of concentration of carbe-noxolone for 20 min on the amplitudes of spontaneous con-tractions in strips of colonic circular muscle. Amplitudeexpressed as percentage change from control. A large andsigni®cant decrease was observed at all concentrations. n�5.

Figure 5 (a) Effects of successive increases of concentration ofcarbenoxolone for 20 min on the frequencies of spontaneouscontractions in strips of intestinal circular muscle. No signi-®cant decrease was observed. n� 4. (b) Effects of successiveincreases of concentration of carbenoxolone for 20 min on theamplitudes of spontaneous contractions in strips of intestinalcircular muscle. Amplitude expressed as mm of chart paper atmaximum contraction. A signi®cant decrease was observed atall concentrations except 10±5 mol L±1. n� 4.



signi®cantly reduced their amplitude (results not

shown).

When the longitudinal muscle and myenteric plexus,

with its ICC network, were removed, ionomycin still

produced regular spontaneous contractions as expected

from the ability of the ICC network of the deep mus-

cular plexus to function as a secondary pacemaker.16

Carbenoxolone (10±5±10±4 mol L±1) had no effect on the

frequencies of contractions while the amplitudes

decreased with concentration, signi®cantly at

10±4 mol L±1 (Fig. 7). Substitution of carbachol for

ionomycin to drive contractions or of 18a-glycyrrheti-

nic acid to inhibit gap junctions was not tried in these

experiments.

DISCUSSION

The major ®ndings of this study were that well-estab-

lished inhibitors of gap junction function did not

abolish or consistently affect pacemaking by ICC in

canine intestine. Furthermore, they did not abolish the

ability of stimulation of inhibitory nerves to mediate

relaxation of the LOS, as might have been expected if

ICC mediate this response.24,25 In other systems such

as cultured cells,33±36,44,48,50±56 isolated blood ves-

sels,38±43,46,49 isolated pancreatic islets,37 and perfused

liver,45 10±5±3 ´ 10±5 mol L±1 of 18a-glycyrrhetinic acid

or carbenoxolone was usually suf®cient to abolish

coupling through gap junctions. These studies used a

variety of techniques, electrical or dye coupling, spread

of Ca2+ waves and functional coupling45 to evaluate

gap junctional coupling. However, in our study, these

concentrations hardly affected and 10±4 mol L±1 of

either agent failed to abolish, spontaneous phasic

activity. Because gap junctions have been assumed to

couple the pacemaking of ICC networks to the driving

of circular muscle contractions (based on structural

®ndings and without functional evidence), and to

mediate ICC-dependent nerve relaxations, this raises

serious questions about whether the results have

other interpretations and, if not, what might be the

implications.

Figure 6 (a) Effects of successive increases of concentration of18a-glycyrrhetinic for 20 min on the frequencies of sponta-neous contractions in strips of intestinal circular muscle. Both3 ´ 10±5 and 10±4 mol L±1 caused increased frequencies.n� 5. (b) Effects of successive increases of concentration of18a-glycyrrhetinic for 20 min on the amplitudes of sponta-neous contractions in strips of intestinal circular muscle.Amplitude expressed as mm of chart paper at maximumcontraction. At 10±4 mol L±1 there was a signi®cant decrease.n� 5.

Figure 7 (a) Effects of successive increases of concentration ofcarbenoxolone for 20 min on the frequencies of spontaneouscontractions in strips of intestinal circular muscle from whichthe outer longitudinal muscle and myenteric plexus had beenremoved. No signi®cant decrease was observed. n� 5.(b) Effects of successive increases of concentration of carbe-noxolone for 20 min on the amplitudes of spontaneous con-tractions in strips of intestinal circular muscle from which theouter longitudinal muscle and myenteric plexus had beenremoved. Amplitude expressed as mm of chart paper atmaximum contraction. No signi®cant decrease was observedexcept at 10±4 mol L±1. n�5.



De®nitive evidence to support or reject that coupling

through gap junctions does or does not mediate the

pacemaking activity of the ICC networks in the small

intestine and colon could be derived if simultaneous

microelectrode recordings were made of slow waves in

ICC and in adjacent circular muscle before and after

addition of agents such as 18 a-glycyrrhetinic acid.

However, that would be very dif®cult technically and

would require extensive dissection of intestinal tissues

to allow access to the myenteric plexus. It would be

somewhat easier to access the colon submuscular

plexus and its ICC with microelectrodes. Direct evi-

dence that a recording was from an ICC imbedded in a

nerve plexus is also complex to obtain. Our results,

suggesting that gap-junction coupling is not required,

do motivate attempting such studies.

The most obvious alternate interpretation of our

results is that these inhibitors of gap junction function

were ineffective in our tissues. However, this seems

unlikely given that many previous studies have dem-

onstrated their ability to block gap junctions in a

variety of tissues and cell types and containing gap

junctions composed of multiple connexins.30±56 We

have shown that canine gap junctions contain Cx43

and Cx40 between circular muscle cells and multiple

connexins, including Cx43, 40 and 45 in gap junctions

associated with ICC networks.58

Alternatively, it might be argued that the gap-

junction inhibitors failed to penetrate the relevant gap

junctions; i.e. those connecting ICC networks to

circular muscle of the intestine or gap junctions

connecting intramuscular ICC to LOS cells. This, too,

is unlikely at least in colon, as the submuscular plexus

network of ICC is located at the internal border of

circular muscle exposed by removing the mucosa and

is directly exposed to bath chemicals. In the intestine,

the ICC networks in the myenteric and deep muscular

plexuses are enclosed more deeply by smooth muscle

cells within the muscle wall. However, 18a-glycyrrh-

etinic acid, which is lipid-soluble, had effects similar to

its water-soluble analogue, carbenoxolone. In LOS, a

series of inhibitors of gap-junction function, several of

which are lipid-soluble, failed to inhibit the ability of

nerve stimulation to relax the muscle, thus it seems

unlikely that gap junctions were protected by their

inaccessibility. The total exposure time to these

inhibitors was 60 min, as the concentration of inhibi-

tors was increased cumulatively without washing.

This allowed more than adequate time for diffusion of

inhibitors to the ICC plexuses in these muscle strips.

It might be argued that the contractions of circular

muscle depended on coupling between cells mediated

by gap junctions. Contractions might then have been

expected to decrease in amplitude if these gap junc-

tions were inhibited. This happened in all cases and

was nearly complete in colon, which has few structural

gap junctions in circular muscle,6,8 but which appears

to be well coupled.13,14 In addition, residual contrac-

tions after inhibition of gap junctions, when ionomy-

cin or carbachol was present, may have been driven by

these agents acting directly on smooth muscle cells.

Finally, it might be argued that our procedures to ini-

tiate spontaneous phasic contractions (TTX,

L-NOARG, ionomycin or carbachol) imbued the

smooth muscle with independent pacemaking activity;

i.e. activity no longer requiring input from ICC net-

works. This too seems unlikely. When we removed the

submuscular plexus from colon circular muscle, we

were unable to initiate regular activity in the residual

circular muscle with any of our procedures. Instead,

irregular, infrequent (< 1 min±1) prolonged tonic/phasic

contractions occurred. While removal of the myenteric

plexus and its ICC network in the ileum did not pre-

clude contractions in the residual circular muscle at

frequencies similar to those in intact strips, this was

very likely the result of the pacemaking from the ICC

network of the deep muscular plexus, shown previ-

ously to provide a secondary pacemaking system.15,16

However, the need in many experiments to stimulate

phasic activity with pharmacological tools, such as

carbachol or ionomycin, allows the possibility that

circular muscle of intestine acquires pacemaking

activity, and is no longer dependent on ICC coupling.

Alternate ways for coupling to occur, which do not

require gap junctions, have been proposed. These

include ®eld coupling,59,60 accumulation of K+

between cells,60,61 and coupling by stretch, mediated

by peg-and-socket connections between cells.61±66

Perhaps the most intriguing alternate possibility is the

peg-and-socket joint, a coupling structures described

recently by Thuneberg and colleagues after primary

OsO4 ®xation. In the mouse intestine, these connec-

tions are one-way between both ICC of the myenteric

plexus or of the deep muscular plexus, in that the ICC

are the acceptors of `pegs' from adjacent smooth mus-

cle cells.61±65 The hypothesis6512 of this group is that

`pegs are stretch sensors of the smooth muscle cells,

and as such provide for a coupling between the smooth

muscle cells, this perhaps being the major type of cel-

lular coupling in the longitudinal muscle layer, which

seems to be devoid of gap junctions'. In view of the one-

way relation between smooth muscle cells and ICC,

they further postulate that `the coupling between the

pacemakers and smooth muscle is effected similarly:

depolarization of the smooth muscle membrane

follows from stretch-activation of pegs tightly locked



in sockets of a spontaneously and rhythmically con-

tractile pacemaker network'.

This hypothesis is dif®cult to test directly because

we cannot observe these peg-and-socket joints in vivo.

Thuneberg and Peters62 have assembled a large body of

supportive evidence by examining the distribution of

these joints after ®xation following various physiolo-

gical manoeuvres. This theory might explain the con-

tinued pacemaking activity observed in terms of phasic

contractions after inhibition of gap junctions. More-

over, many cells, including smooth muscle, have been

shown to have mechano-sensitive ion channels.67±70

Finally, in colon, which has gap junctions con-

necting ICC to muscle more accessible than other

tissues, there was a signi®cant decrease in frequency

after exposure to 10±4 mol L±1 carbenoxolone. There-

fore, the possibility exists that gap junctions do play

some role in coupling ICC pacemaking to circular

muscle contractions. Further steps must be taken to

evaluate the actions of our pharmacological tools on

gap junction function at a cellular level. This might

be done by using thin slabs of colon13,14 or ileal

muscle15±17 and testing the effects on slow waves and

inhibitory junction potentials of carbenoxolone and

18a-glycyrrhetinic acid. In a study of ileal slabs, car-

ried out previously11 with octanol, which affects var-

ious other ion channels as well as gap junctions, we

reduced and slowed but did not eliminate slow waves.

Elimination of slow waves by carbenoxolone and

18a-glycyrrhetinic acid would suggest that these

agents were effective and raise the possibility that the

occurrence of spontaneous contractile activity and

associated stretch eliminates the need for coupling by

gap junctions.

REFERENCES

1 Daniel EE, Robinson K, Duchon G, Henderson RM. Thepossible role of close contacts (nexuses) in the propagationof control electrical activity in the stomach and smallintestine. Dig Dis Sci 1971; 16: 611±22.

2 Henderson RM, Duchon G, Daniel EE. Cell contacts induodenal smooth muscle layers. Am J Physiol 1971; 221:564±74.

3 Duchon G, Henderson R, Daniel EE. Circular muscle lay-ers in the small intestine. In: Daniel EE. ed. Proceedings ofthe Fourth International Symposium on GI Motility.Vancouver, Canada: Mitchell Press, 1974: 635±46.

4 Daniel EE, Daniel VP, Duchon G et al. Is the nexusnecessary for cell-to-cell coupling in smooth muscle?J Memb Biol 1976; 28: 207±39.

5 Daniel EE, Sakai Y, Fox JET, Posey-Daniel V. Structuralbases for function of circular muscle of canine corpus. CanJ Physiol Pharmacol 1984; 62: 1304±14.

6 Berezin I, Huizinga JD, Daniel EE. Interstitial cells of Cajalin canine colon: a special communication network at the

inner border of the circular muscle. J Comp Neurol 1988;273: 42±51.

7 Daniel EE, Berezin I, Allescher HD, Manaka H, Posey-Daniel V. Morphology of the canine pyloric sphincter inrelation to function. Can J Physiol Pharmacol 1989; 67:1560±73.

8 Berezin I, Huizinga JD, Daniel EE. Structural characteri-zation and interconnections of interstitial cells of Cajal inmyenteric plexus and circular muscle of canine colon. CanJ Physiol Pharmacol 1990; 68: 1419±31.

9 Daniel EE, Berezin I. Interstitial cells of Cajal: are theymajor players in control of gastrointestinal motility?J Gastrointest Motil 1992; 4: 1±24.

10 Berezin I, Daniel EE, Huizinga JD. Ultrastructure ofinterstitial cells of Cajal in the canine distal esophagus.Can J Physiol Pharmacol 1994; 72: 1049±59.

11 Daniel EE, Wang YF, Cayabyab F. Role of gap junctions instructural arrangements of interstitial cells of Cajal andcanine ileal smooth muscle. Am J Physiol 1998; 274:G1125±41.

12 Daniel EE, Wang YF. Gap junctions in intestinal smoothmuscle and interstitial cells of Cajal. Microsc Res Tech1999; 47: 309±20.

13 Smith TK, Reed JB, Sanders KM. Origin and propagation ofelectrical slow waves in circular muscle of canine colon.Am J Physiol 1987; 252: C215±4.

14 Smith TK, Sanders KM. Interaction of two electricalpacemakers in muscularis of canine proximal colon. Am JPhysiol 1987; 252: C290±9.

15 Hara Y, Kubota M, Szurszeweski JH. Electrophysiology ofsmooth muscle on the small intestine of some mammals.J Physiol (Lond) 1986; 372: 510±20.

16 Jimenez M, Cayabyab FS, Vergara P, Daniel EE. Hetero-geneity in electrical activity of the canine ileal circularmuscle: interaction of two pacemakers. Neurogastro-enterol Motil 1996; 8: 339±50.

17 Cayabyab FS, Jimenez M, Vergara P, de Bruin H, Daniel EE.In¯uence of nitric oxide and vasoactive intestinal peptideon the spontaneous and triggered electrical and mechanicalactivities of the canine ileum. Can J Physiol Pharmacol1997; 75: 383±97.

18 Cheung DW, Daniel EE. Comparative study of the smoothmuscle layers of the rabbit duodenum. J Physiol (Lond)1980; 309: 13±27.

19 Ward SM, Burns AJ, Torihashi S, Sanders KM. Mutation ofthe proto-oncogene c-kit blocks development of interstitialcells and electrical rhythmicity in murine intestine.J Physiol (Lond) 1994; 480: 91±7.13 .

20 Sanders KM. A case for interstitial cells of Cajal aspacemakers and mediators of neurotransmission inthe gastrointestinal tract. Gastroenterology 1996; 111:492±515.

21 Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkel-sen HB, Bernstein A. W/kit gene required for interstitialcells of Cajal and for intestinal pacemaker activity. Nature1995; 373: 347±9.

22 Thuneberg L. Interstitial cell of Cajal: intestinal pacema-ker cells? Adv Anat Embryol Cell Biol 1982; 71: 1±130.

23 Sanders KM, Ordog T, Koh SD, Torihashi S, Ward SM.Development and plasticity of interstitial cell of Cajal.Neurogastroenterol Motil 1999; 11: 311±88.

24 Burns AJ, Lomax AE, Torihashi S, Sanders KM, Ward SM.Interstitial cells of Cajal mediate inhibitory neurotrans-



mission in the stomach. Proc Nat Acad Sci USA 1996; 93:12008±13.

25 Ward SM, Morris G, Reese L, Wang X-Y, Sanders KM.Interstitial cells of Cajal mediate inhibitory neurotrans-mission in the lower esophageal and pyloric sphincters.Gastroenterology 1998; 115: 314±28.

26 Ward SM, Beckett EA, Wang X, Baker F, Khoyi M, SandersKM. Interstitial cells of Cajal mediate cholinergic neuro-transmission from enteric motor neurons. J Neurosci 2000;20: 1393±403.

27 Daniel EE, Posey-Daniel V. Neuromuscular structures inopossum esophagus: role of interstitial cells of Cajal. Am JPhysiol 1984; 246: G305±15.

28 Berezin I, Allescher H-D, Daniel EE. Ultra-structurallocalization of VIP-immunoreactivity in canine distaloesophagus. J Neurocytol 1987; 16: 749±57.

29 Allescher H-D, Berezin I, Jury J, Daniel EE. Characteristicsof canine lower esophageal sphincter: a new electrophysi-ological tool. Am J Physiol 1988; 255: G441±3.

30 Davidson JS, Baumgarten IM, Harley EH. Reversible inhi-bition of intercellular junctional communication by glyc-yrrhetinic acid. Biochem Biophys Res Commun 1986; 134:29±36.

31 Davidson JS, Baumgarten IM. Glycyrrhetinic acid deriva-tives: a novel class of inhibitors of gap-junctional inter-cellular communication. Structure±activity relationships.J Pharmacol Exp Ther 1988; 246: 1104±7.

32 Cohen KD, Berg BR, Sarelius IH. Remote arteriolar dila-tions in response to muscle contraction under capillaries.Am J Physiol Heart Circ Physiol 2000; 278: H1916±23.

33 Robe PA, Princen F, Martin D et al. Pharmacologicalmodulation of the bystander effect in the herpes simplexvirus thymidine kinase/ganciclovir gene therapy system:effects of dibutyryl adenosine 3¢,5¢-cyclic monophosphate,alpha-glycyrrhetinic acid, and cytosine arabinoside.Biochem Pharmacol 2000; 60: 241±9.

34 Oviedo-Orta E, Hoy T, Evans WH. Intercellular commu-nication in the immune system: differential expression ofconnexin40 and 43, and perturbation of gap junctionchannel functions in peripheral blood and tonsil humanlymphocyte subpopulations. Immunology 2000; 99:578±90.

35 Donahue HJ, Li Z, Zhou Z, Yellowley CE. Differentiationof human fetal osteoblastic cells and gap junctional inter-cellular communication. Am J Physiol Cell Mol Physiol2000; 278: C315±22.

36 Velasco A, Tabernero A, Granda B, Medina JM. ATP-sen-sitive potassium channel regulates astrocytic gap junctionpermeability by a Ca2+-independent mechanisms. J Neu-rochem 2000; 74: 1249±56.

37 Jonkers FC, Jonas JC, Gilon P, Henquin JC. In¯uence ofcell number on the characteristics and synchrony of Ca2+

oscillations in clusters of mouse pancreatic islet cells.J Physiol (Lond) 1999; 520: 839±49.

38 Hill CE, Eade J, Sandow SL. Mechanisms underlyingspontaneous rhythmical contractions in irideal arteriolesof the rat. J Physiol (Lond) 1999; 521: 507±16.

39 Fukuta H, Koshita M, Yamamoto Y, Suzuki H. Inhibitionof the endothelium-dependent relaxation by 18-beta-glyc-yrrhetinic acid in the guinea-pig aorta. Japan J Physiol1999; 49: 267±74.

40 Fujimoto S, Ikegami Y, Isaka M, Kato T, Nishimura K,Itoh ?tul> T. K+ channel blockers and cytochrome P450

inhibitors on acetylcholine-induced, endothelium-dependent relaxation in rabbit mesenteric artery. Eur JPharmacol 1999; 384: 7±15.

41 Chaytor AT, Martin PE, Evans WH, Randall MD, Grif®thTM. The endothelial component of cannabinoid-inducedrelaxation in rabbit mesenteric artery depends on gapjunctional communication. J Physiol (Lond) 1999; 520:539±50.

42 Kagota S, Yamaguchi Y, Nakamura K, Kunitomo M.Characterization of nitric oxide- and prostaglandin-inde-pendent relaxation in response to acetylcholine in rabbitrenal artery. Clin Exp Pharmacol Physiol 1999; 26: 790±6.

43 Grif®th TM, Taylor HJ. Cyclic AMP mediates EDHF-typerelaxations of rabbit jugular vein. Biochem Biophys ResCommun 1999; 263: 52±7.

44 Guo Y, Martinez-Williams C, Gilbert KA, Rannels DE.Inhibition of gap junction communication in alveolar epi-thelial cells by 18-alpha-glycyrrhetinic acid. Am J Physiol1999; 276: L1018±26.

45 Nathanson MH, Rios-Velez L, Burgstahler AD, MennoneA. Communication via gap junctions modulates bilesecretion in the isolated perfused rat liver. Gastroentero-logy 1999; 116: 1176±83.

46 Yamamoto Y, Imaeda K, Suzuki H. Endothelium-depend-ent hyperpolarization and intercellular electrical couplingin guinea-pig mesenteric arterioles. J Physiol (Lond) 1999;514: 505±13.

47 Vance MM, Wiley LM. Gap junction intercellular com-munication mediates the competitive cell proliferationdisadvantage of irradiated mouse preimplantation embryosin aggregation chimeras. Radiat Res 1999; 152: 544±51.

48 Le AC, Musil LS. Normal differentiation of cultured lenscells after inhibition of gap junction-mediated intercellularcommunication. Dev Biol 1998; 204: 80±96.

49 Taylor HJ, Chaytor AT, Evans WH, Grif®th TM. Inhibitionof the gap junctional component of endothelium-depend-ent relaxations in rabbit iliac artery by 18-alpha glycyrrh-etinic acid. Br J Pharmacol 1998; 125: 1±3.

50 Lee MJ, Rhee SK. Heteromeric gap junction channels in rathepatocytes in which the expression of connexin 26 isinduced. Mol Cells 1998; 8: 295±300.

51 Hirata K, Nathanson MH, Sears ML. Novel paracrine sig-naling mechanism in the ocular ciliary epithelium. ProcNatl Acad Sci USA 1998; 95: 8381±6.

52 Eugenin EA, Gonzalez H, Saez CG, Saez JC. Gap junctionalcommunication coordinates vasopressin-induced glyco-genolysis in rat hepatocytes. Am J Physiol 1998; 274:G1109±G1116.

53 Spanakis SG, Petridou S, Masur SK. Functional gap junc-tions in corneal ®broblasts and myo®broblasts. InvestOphthalmol Vis Sci 1998; 39: 1320±8.

54 Grandolfo M, Calabrese A, D'Andrea P. Mechanism ofmechanically induced intercellular calcium waves in rab-bit articular chondrocytes and in HIG-82 synovial cells.J Bone Miner Res 1998; 13: 443±53.

55 Tordjmann T, Berthon B, Claret M, Combettes L. Coordi-nated intercellular calcium waves induced by noradrenal-ine in rat hepatocytes: dual control by gap junctionpermeability and agonist. EMBO J 1997; 16: 5398±407.

56 Frame MK, de Feijter AW. Propagation of mechanicallyinduced intercellular calcium waves via gap junctions andATP receptors in rat liver epithelial cells. Exp Cell Res1997; 230: 197±207.



57 Bers DM, Patton CW, Nuccitelli R. A practical guide to thepreparation of Ca2+ buffers. Methods Cell Biol 1994; 40:4±28.

58 Wang YF, Daniel EE. Do multiple connexins make up gapjunctions in canine gastrointestinal tract. Neurogastroen-terol Motil 2000; 12: 497 (Abstract).

59 Vigmond EJ, Bardakjian BL. The effect of morphologicalinterdigitation on ®eld coupling between smooth musclecells. IEEE Trans Biomed Eng 1995; 42: 162±71.

60 Sperelakis N, LoBracco B Jr, Mann JE, Marschall R.Potassium accumulation in intercellular junctions com-bined with electric ®eld stimulation for propagation incardiac muscle. Innov Tech Biol Methods 1985; 6: 25±43.

61 Vigmond EJ, Bardakjian BL, Thuneberg L, Huizinga JD.Intercellular coupling mediated by potassium accumula-tion in peg-and-socket junctions. IEEE Trans Biomed Eng2000; 20: in press.

62 Thuneberg L, Peters S. Toward a concept of stretch-coupling in smooth muscle. 1. Anatomy of intestinalsegmentation and sleeve contraction. Anat Rec 2000; 262:110±24.

63 Thuneberg L, Peters S. Formation of peg and socket junc-tions. Neurogastroenterol Motil 2000; 12: 294 (Abstract).

64 Thuneberg L, Rahnamai MA, Riazi H. The peg-and-socketjunction: an alternative to the gap junction in couplingsmooth muscle cells. Neurogastroenterol Motil 1998; 10:479 (Abstract).

65 Thuneberg L. One hundred years of interstitial cells ofCajal. Microscop Res Tech 1999; 47: 223±38.

66 Farrugia G, Holm AN, Rich A, Sarr MG, Szurszewski JH,Rae JL. A mechanosensitive calcium channel in humanintestinal smooth muscle cells. Gastroenterology 1999; 11:900±5.14

67 Waniishi Y, Inoue R, Ito Y. Preferential potentiation byhypotonic cell swelling of muscarinic cation current inguinea pig ileum. Am J Physiol 1997; 272: C240±53.

68 Ko KS, McCulloch CA. Partners in protection: interde-pendence of cytoskeleton and plasma membrane in adap-tations to applied forces. J Memb Biol 2000; 174: 85±95.

69 McBride TA, Stockert BW, Gorin FA, Carlsen RC. Stretch-activated ion channels contribute to membrane depolar-ization after eccentric contractions. J Appl Physiol 2000;88: 91±101.

70 Ghazi A, Berrier C, Ajouz B, Besnard M. Mechanosensitiveion channels and their mode of activation. Biochimie 1998;80: 357±62.



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