Gut, 1986, 27, 999-1005

Alimentary tract and pancreas

Electrophysiology of the human colon: evidence ofsegmental heterogeneityG I SANDLE, N K WILLS, W ALLES AND H J BINDER

From the Departments of Internal Medicine and Physiology, Yale University School of Medicine, NewHaven, CT, USA

SUMMARY The electrical properties of epithelial cell membranes in human descending andascending colon were studied using microelectrodes and the Na channel blocker amiloride.Under control (pre-amiloride) conditions, the transepithelial electrical measurements in the twocolonic segments were similar. The mucosal addition of 0.1 mM amiloride to descending colontotally abolished the transepithelial voltage (V,) and short-circuit current (Isc), and significantlyincreased the total tissue resistance (Rt) by 19% (p<OO05). Intracellular recordings indescending colon obtained with microelectrodes revealed that the transepithelial effects ofamiloride reflected hyperpolarisation of the apical membrane and a significant increase in apicalmembrane resistance, changes which were consistent with amiloride-blockade of apical Nachannels and complete inhibition of electrogenic Na transport. An appreciable amiloride-insensitive conductance was also present in the apical membrane of the descending colon. Incontrast, the mucosal addition of 0* 1 mM amiloride to ascending colon decreased Vt by only 30%(p<002) and Isc by 43% (p<0-05), but had no significant effect on Rt. These results indicate that(i) electrogenic Na transport accounts totally for Isc in human descending colon in vitro, but onlypartly for Isc in human ascending colon, suggesting that Na transport in ascending colon ismediated primarily by electroneutral processes, and (ii) the apical membrane of humandescending colon contains an amiloride-sensitive conductance for Na in parallel with anamiloride-insensitive conductance, which may transport K.

Segmental differences in the colonic absorption ofNa and water have been shown in a variety ofspecies, including man.1-7 Perfusion studies in hu-man colon indicate that Na and water are absorbedmainly in the ascending and transverse colon,4 5while little or no electrolyte and water absorptionoccurs in the rectum.5 8 Dialysis studies have pro-vided similar results, with a gradual decrease in Naabsorption between the transverse colon and the

6rectum. Although recent in vitro studies haveestablished segmental differences in Na transportprocesses in rabbit7 9 10 and rat11-13 colon, similarstudies have not been done in human colon. Thosein vitro studies that have been carried out on humancolonic epithelium have concentrated exclusively onthe descending colon,1417 and confirmed that active(electrogenic) Na absorption accounts for the high(20-50 mV) transmucosal potential differenceAddress for correspondence: Dr G I Sandle, Department of Medicine, HopeHospital, Clinical Sciences Building, Eccles Old Road, Salford M6 8HD.

Received for publication 13 December 1985.

observed in vivo.1820 This potential difference isabolished by the mucosal addition of amiloride,'6which blocks Na channels in the apical membrane ofthe surface epithelial (Na transporting) cells.'7 Inthe present study we have used this property ofamiloride to compare the magnitude of electrogenicNa transport in human ascending and descendingcolon. The results suggest that different mechanismsfor Na transport may operate in the ascending anddescending segments of the colon in man.

In addition, we have investigated the electrophy-siology of human descending colon in more detailusing intracellular microelectrodes and amiloride.Our findings are consistent with a cellular model forNa and K transport which is similar to that proposedfor rabbit descending colon.2'

Methods

PATIENTSTissues were obtained from 11 patients (age 33-84

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years) undergoing resection of the ascending ordescending colon for carcinoma or diverticulardisease. In patients with carcinoma, tissue wasobtained at least 12 cm from the edge of the tumour.Segments of colon were placed immediately intooxygenated NaCl-Ringer solution at 37°C beforetransporting to the laboratory. The epithelial layerwas dissected free by opening the colon as a flatsheet, rinsing the luminal surface, and then remov-ing serosal muscle and fat using a razor blade withthe aid of a stereoscopic microscope, while the tissuewas bathed in oxygenated Ringer solution. Theisolated epithelium was then mounted betweenUssing-type chambers modified to minimise edgedamage, and allow temperature regulation andmicroelectrode impalements from the luminal (apic-al) side.22 Dissection and mounting of the tissue wasusually completed within 10-15 minutes after re-moval from the patient. The pH and temperature ofthe bathing solutions were maintained at 7-4 and37°C respectively, while being continuously stirredand gassed with 95% 02-5% C02. Tissue surfacearea was 1.0 cm.2

Tissues were normally bathed with a NaCl-Ringer solution containing (in mmol/l): Na 136-2; K7*0; Cl 121; Ca 2-0; Mg 1-2; HCO3 25; H2PO4 1-2;S04 1-2; and glucose 10-0. This solution wasidentical to that used in previous studies in rabbit21and rat23 descending colon and contained a slightlyhigher concentration of K than normally found inhuman plasma. In preliminary experiments, how-ever, this solution and a similar solution containingK 5-2 mmol/l gave identical transepithelial andmicroelectrode results, and therefore the solutioncontaining K 7-0 mmol/l was used throughout (seealso ref.l ).

Transepithelial voltage (Vt) was monitored withIM KCl-4% agar bridges placed 5 mm on either sideof the tissue, and attached to 3M KCI half-cells.Current pulses were passed across the epithelium viaAg-AgCl electrodes located at the back of eachchamber. Glass fibre filled microelectrodes (tipdiameter <0-5 ,um) were prepared with a horizontalpipette puller (Industrial Science Associates, Ridge-wood, NY), filled with 0-5 M KCI, and had tipresistances of 40-100Mfl in NaCl-Ringer solution.Cell impalements were carried out from the apicalside of the tissue, and microelectodes were posi-tioned with an accuracy of 1 [tm using a manuallyoperated hydraulic microdrive (Trent Wells, South-gate, CA). Membrane voltages were measuredwithin ±0- 1 mV with a high impedance electrometer(WP Instruments, New Haven, CT, Model 750),and microelectrodes were referenced to the serosalsolution such that basolateral membrane voltage(Vbl) was monitored directly.

Current pulses were passed using an Anapulsestimulator and stimulus isolation unit (WP Instru-ments, New Haven, CT, Models 302-T and 305,respectively). Measurements of voltage and currentwere recorded on a dual-beam oscilloscope (Tektro-nix, Beaverton, OR, Model D12), and a highfrequency response chart recorder (Gould, Cleve-land, OH, Model 2200). Apical membrane voltage(Va) was calculated as Va=Vt-Vbl, and the ratio ofthe changes in apical (AVa) and basolateral (AVbI)membrane voltages in response to the current pulsewas used to calculate the resistances ratio, a(a=ratio of the apical to basolateral membraneresistances, Ra/Rbl). Total tissue resistance (R,) anda were corrected for series resistance of the bathingsolution, as described previously.'0 Amiloride (a giftof Merck, Sharp and Dohme Research Laboratories,West Point, PA) was added to the mucosal bath to aconcentration of 071 mM by adding aliquots from a10 mM stock solution.

IMPALEMENT CRITERIAIn all tissues, three to five successful impalementswere done before and after the addition of amilor-ide, and average values of transepithelial andmicroelectrode measurements were calculated foreach tissue before obtaining mean values for thegroup. Impalements were judged to be acceptable ifthey met the following criteria: (i) Vbl reached asteady value within 10 seconds, (ii) Vbl and aremained stable throughout the impalement (gener-ally 30-75 seconds), (iii) the microelectrode tipresistance was unchanged by the impalement, and(iv) the microelectrode recorded the baseline vol-tage (VJ) upon withdrawal.2' 24Owing to the limitednumber of tissues, impalement damage could not beassessed by comparing impalements from themucosal side with those from the serosal side.Consequently, we cannot completely exclude thepossibility of some impalement damage to the apicalmembrane, and the values of membrane voltagesand conductances should be taken as estimates.

Results are expressed as mean ± SEM, andstatistical comparisons were undertaken using Stud-ent's t test for paired or unpaired data asappropriate.25

ResultsCOMPARISON OF TRANSEPITHELIALMEASUREMENTS IN DESCENDING AND ASCENDINGCOLONTable 1 presents values of V, Isc and Rt obtained in13 tissues from the descending colon, and fourtissues from the ascending colon. Under control(pre-amiloride) conditions, there were no significantdifferences in the transepithelial parameters be-

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Table 1 Comparison of transepithelial measurements andthe effects of0-1 mM amiloride in ascending anddescending colon

V, (mV) Isc (1A/cm2) R, (Q7.cm')

Ascending (n=4)Pre-amiloride -10±3 88±31 124+5Postamiloride -7±3 50±21 139+3p* <0-02 <0-05 NS

Descending (n= 13)Pre-amiloride -14±2 97±11 138±10Postamiloride -1+1 7+8 164+14p* <0-001 <0-001 <0-005

Results are expressed as mean±SEM. Tissues were bathed inNaCI-Ringer solution. Pre- and postamiloride measurements wereobtained when values of Vt (transepithelial voltage, mucosalsurface negative), Isc (short-circuit current) and R, (totalresistance) were constant. n is the number of tissues studied (fromtwo subjects for ascending colon and nine subjects for descendingcolon).*difference between pre- and postamiloride values.

tween the two colonic segments. Transepithelialvalues for human proximal colon have not beenreported previously, but Vt in the descending colon(-14±2 mV) was similar to the mean valuesreported in other studies.'4 15 17 R, in the descend-ing colon (138±10 ft.cm2) was higiher than thepreviously reported value of 96f.cm2 ; this differ-ence may reflect (i) our chamber design, whichminimised edge damage, or (ii) differences inmembrane area due to variations in the degree oftissue stretch, as other investigators15 have reportedvalues of Isc which were approximately twice thatobserved in the present study (97±11 RA/cm2), or(iii) possible differences between transport rates.

Despite the similarities between the basal trans-epithelial results in the descending and ascendingcolon, the two groups of tissues exhibited differentresponses to the mucosal addition of amiloride(Table 1). In the descending colon, amiloridecompletely abolished Vt and Isc (postamiloridevalues of Vt and Isc were not significantly differentfrom zero; p> 03 and p> 0*4, respectively) andsignificantly increased R, by 19% (p<0-005). Thesechanges indicate that electrogenic Na transport wasentirely responsible for the potential differenceacross this colonic segment. In contrast, the additionof amiloride to ascending colon decreased V, by30% (p<002) and Isc by 43% (p<005), but therewas no significant change in R,. Amiloride induceddecreases in V, and Isc in the descending colon(13±2 mV and 90±12 tLA/cm2, respectively) weresignificantly greater than those in the ascendingcolon (3±1 mV, p<005 and 38±12 ,uA/cmp<0-05, respectively). These data indicate that the

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ascending colon is only partially responsive toamiloride - that is, electrogenic Na transportaccounts for less than half of the short circuit currentand the remainder reflects the transport of otherions.

Transepithelial measurements obtained in de-scending colon after the addition of amiloride wereused to calculate the transepithelial electromotiveforce (EMF) and the shunt resistance (Rs=11Gs),which is assumed to represent a lumped resistanceprovided by the paracellular pathway - that is,intercellular tight junction and the lateralinterspace,2' 26 27 and any conductance in parallelcell types.2' According to the simple equivalentcircuit shown in Figure 1 (see ref.21), the Natransport process can be modelled as a battery(ENa=the transepithelial EMF) in series with theresistive pathway for Na (RNa=l/GNa). These arearranged in parallel with Rs. V, can therefore bedescribed by the equation:

ENa.GNatGNa+Gs

where ENa.GNa=Isc (the short circuit current) andGNa+Gs=Gt (the total conductance). By furthersubstitution, Gt=ENa (Isc)+Gs. It should benoted that the plot of Gt and Isc was linear (Fig. 2),consistent with the assumption that amiloridealtered only one circuit parameter - that is, GNa.

Mucosal solution

RN0

Cell RS

ENo

Serosal solutionFig. 1 Simple equivalent circuit describing Na transport:RNa=resistance of the Na transport pathway; R,=shuntresistance; ENa=the transepithelial EMF or Na battery.

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7,

6

5.

4.

80 120Isc (,pA/cm2)

Fig. 2 Typical experiment showing the effect ofamilorideon total tissue conductance (Gd and short-circuit current(Isc) in human descending colon. Values ofG, and Isc wereobtained before (uppermost data point) and atfive secondintervals after the addition ofamiloride to the mucosalsolution (final concentration 0.1 mM). For this tissue, linearregression analysis revealed ENa (slope- ) =85 mV and Gs(G, intercept)=4.9 mS/cm2 (r=0-998).

This plot gives an inverse slope of ENa and a G,intercept equal to G,. Using this approach, themean values of ENa and G, were 82±15 mV and5*2±0-4 mS/cm2 (Rs=197±15 a.cm2), respectively(n=6).

MICROELECTRODE MEASUREMENTS INDESCENDING COLONTable 2 presents values of Vbl, Va and a (=Ra/RbI)obtained during successful impalements in six tissuesfrom the descending colon. Under control condi-tions, the values of Vt and R, were similar to thosereported in Table 1. Vbl (-37±4 mV) and Va (20±3mV) were lower than reported for rat distal colon

Table 2 Effect of0-1 mM amiloride on microelectrodemeasurements in descending colon

V, (mV) R, (fl.cm2) Vhl (mV) V. (mV) a

Pre-amiloride -17±2 157±10 -37±4 20±3 2-0±0-9

Post-amiloride 1±2 189±13 -32±2 33±2 4-0±1-9

p* <0-005 <0-01 NS <0-025 <0-01

Results are expressed as mean±SEM. Tissues were bathed withNaCI-Ringer solution. Pre- and postamiloride measurements wereobtained when transepithelial measurements were constant.Vt=transepithelial voltage (mucosal surface negative); R,=totalresistance; VbI=basolateral membrane voltage (negative withrespect to serosa); Va=apical membrane voltage (positive withrespect to cell interior); a=membrane resistance ratio (calculatedas AV./AVbI; see Methods). Six tissues were studied.*difference between pre- and postamiloride values.

Sandle, Wills, Alles, and Binder

(Vbl =-50 mV; Va,=47 mV, refs" ') and rabbitdescending colon (Vbl=-52 mV; Va=32 mV,refs2t 29). The addition of amiloride completelydepolarised the epithelium (which mainly reflectedhyperpolarisation of the apical membrane(AVa=13±4 mV) as there was no significant changein Vbl), and increased Rt by 20% (p<001) and a by100% (p<0.01). These changes are generally consis-tent with amiloride blockade of Na conductivechannels in the apical membrane.

Discussion

COMPARISON OF TRANSEPITHELIAL ELECTRICALPROPERTIES OF DESCENDING AND ASCENDINGCOLONThe transepithelial electrical measurementsobtained in human descending colon in the presentstudy agree closely with those reportedpreviously. 14 15 17 Basal electrical measurements inhuman ascending colon, reported here for the firsttime. were similar to those in the descendingsegment. The most striking difference betweentissues from the two sites, however, was in theirresponse to amiloride. In descending colon, mucosaladdition of amiloride resulted in complete trans-epithelial depolarisation and a significant decreasein total tissue conductance (AGt 1-1+0-3 mS/cm2,p<0005). These changes are consistent with amil-oride blockade of apical Na channels, and show thatelectrogenic Na transport accounts for alll' oralmost all14 30 of the observed short circuit current inhuman descending colon. It should be noted,however, that Rask-Madsen and Hjelt16 found that75% of net Na absorption across human descendingcolon persisted in the presence of amiloride. Recentstudies by Sellin and DeSoignie31 in human descend-ing colon have shown similar rates of net Na and Clabsorption under short circuit conditions with netNa absorption almost three times greater than theshort circuit current; 0.1 mM amiloride decreasedthe short circuit current by 80% but inhibited net Naabsorption by only 50%. These studies and those ofRask-Madsen and Hjelt suggest that other amilorideinsensitive or electroneutral Na transport processesmay also operate in this epithelium.

In contrast with the descending colon, the addi-tion of amiloride to ascending colon produced asignificant but limited (30%) decrease in trans-epitheial voltage with a smaller and statisticallyinsignificant increase in total tissue resistance. Therelative insensitivity of the ascending colon toamiloride indicates that amiloride sensitive elec-trogenic Na transport is not the main source of theshort circuit current (and hence the transepithelialvoltage) in this segment. Although in vitro studies

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have shown that human transverse and descendingcolon have similar rates of net Na and Cl absorptionand short circuit currents, only 30% of the shortcircuit current in the transverse colon was amiloride-sensitive.31 It is possible that varying degrees ofamiloride sensitivity are partly related to variationsin Na balance and circulating aldosterone levelsbetween different patients. It seems likely, however,that the difference in amiloride sensitivity that we

have shown between the descending and ascendingcolon reflects true regional differences in amiloridesensitive Na transport rather than drug induced ortechnical artifact, as none of the patients hadreceived corticosteroids, diuretics, or cardiac glyco-sides before surgery, and care was taken to avoidtraumatised areas of resected colon. Althoughadditional studies are required to establish thenature of possible electroneutral Na transport pro-

cesses it is clear that amiloride sensitive electrogenicNa transport is present in all parts of the humancolon and is more marked in the descendingsegment.

MICROELECTRODE AND AMILORIDE STUDIES IN

DESCENDING COLONThe basal intracellular electrical properties of hu-man descending colon are qualitatively similar tothose found in rabbit descending colon"L - that is,apical membrane voltage (positive with respect tocell interior) is appreciably lower than the baso-lateral membrane voltage (negative with respect toserosal solution). The electrical basis for the baso-lateral membrane voltage in human descendingcolon is unclear, but it may reflect a K diffusionpotential across the basolateral membrane, as itdoes in rabbit descending colon;21 both colonicepithelia possess ouabain sensitive Na,K-ATPaseactivity in the basolateral membrane which isassumed to mediate cellular K uptake and maintaina high intracellular K concentration.The addition of amiloride to descending colon

resulted in marked hyperpolarisation of the apicalmembrane (AVa 13±4mV, p<0.025) without asignificant change in basolateral membrane voltage,and a 100% increase (p<001) in the membraneresistance ratio (a). These changes reflect amilorideinhibition of apical Na channels and a decrease inapical membrane conductance, and provide thecellular basis for the decreases in transepithelialvoltage and total tissue conductance outlined above.

Pre- and postamiloride microelectrode data wereused to obtain estimates of individual membraneconductances as previously described for otheramiloride sensitive epithelia.21 24 A number ofassumptions have been made in resolving total tissueconductance (Gt) into its apical (Ga), basolateral

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(Gbl) and shunt (Gj) components, and the values arepreliminary. First, the method assumes that amil-oride selectively decreases Ga without changing Gbland G,. Human colon was therefore modelled as asimple equivalent circuit, where Ra and RbI wereseries resistors arranged in parallel with the shuntresistance (Rs), and membrane conductances werecalculated by solving the simultaneous equations:Gt'=Gs+Gbi (1+a')-l and Gt"=Gs+Gbi (1+a')-lwhere Gt=lI/R Gs=I/Rs and Gbj=l/RbI' and super-scripts (') and (") denote values before and after theaddition of amiloride. If amiloride changes only onecircuit parameter-that is, GNa and therefore Ga' assuggested by the data in Figure 2, the drug shouldincrease VbI. The reason for our failure to detect arise in Vbl in the present study is unclear, but it maybe related to the necessity to obtain pre- andpostamiloride values of VbI from different cells.Second, it is assumed that the ratio of the changes inapical and basolateral membrane voltages that occurin response to the transepithelial current pulse isequal to the membrane resistance ratio (a=GbI/Ga).In relatively 'leaky' (low resistance) epithelia thisassumption may lead to underestimation of a32 33and a degree of error when calculating membraneconductances. Despite these limitations, as an initialstep in resolving the membrane properties of thisepithelium, we have calculated the inidividual mem-brane conductances, which are presented in Table 3.These results indicate that amiloride blockade ofapical Na channels resulted in a 50% decrease in theapical membrane conductance (Ga). An appreciableapical membrane conductance remained in thepresence of amiloride (3.0±0-8 mS/cm2), however,which could reflect conductive channels for ionsother than Na (vide infra).Our previous current fluctuation studies have

shown the presence of Na channels in the apicalmembrane of the human descending colon. 7 Ifamiloride sensitive Na channels form the mainconductive pathway in this membrane, amilorideshould, in theory, decrease Ga almost to zero andincrease a to an infinitely large value. The post-amiloride values of Ga (3-0±0-8 mS/cm2) and a

Table 3 Membrane conductances in descending colon

Ga (mS/cm2) Gb, (mSICn2) G, (mS/cm2)

Pre-amiloride 6-1±1-4 9-2±3-2 3-3±0-4Postamiloride 3-0±0-8 -p* <0.02Results are expressed as mean±SEM. Tissues were bathed inNaCi-Ringer solution. Ga. GbI and G,=apical, basolateral andshunt conductances, respectively. Six tissues were studied.*Difference between pre- and postamiloride value.

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(4-0±1.9) suggest, however, the presence of anappreciable apical conductance, even after theblockade of apical Na channels. Although part ofthis amiloride insensitive conductance may reflect asmall degree of impalement damage or distributedresistance effects, several observations suggest thatit mainly represents an apical conductance toanother ion(s) - for example, K. First, humandescending colon exhibits net K secretion undershort circuit conditions, indicating the presence ofan active (transcellular) K secretory process.15 30Second, our previous current fluctuation andmicroelectrode studies in human descending colonhave shown a K conductance in the apicalmembrane which may be mediated by K channels; aserosally directed K gradient across the colonproduced a Lorentzian component in the powerdensity spectrum which resembled signals fromapical K channels in rabbit colon. 17

We are grateful to the attending surgeons at Yale-New Haven Hospital without whom this work wouldnot have been possible. This work was supported byNational Institute of Arthritis, Diabetes, and Di-gestive and Kidney Diseases Grants AM-29962 (toN K Wills) and AM-18777 (to H J Binder). G ISandle was supported by a National Foundation forIleitis and Colitis Senior Research Fellowship.

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