J Physiol-1967-Cofré-177-90

J. Physiol. (1967), 188, pp. 177-190 177With 3 text-figure8Printed in Great Britain

ACTIVE SODIUM TRANSPORT BY THE COLONOF BUFO MARINUS: STIMULATION BY ALDOSTERONE AND

ANTIDIURETIC HORMONE

BY G. COFRI* AND J. CRABBIFrom the Section on Endocrinology, Laboratory ofExperimental Surgery, University Clinics St Pierre,

Louvain, Belgium

(Received 1 February 1966)

SUMMMARY

1. The isolated colon of Bufo marinus transports sodium actively fromthe mucosal (lumen) to the serosal side, and this transport is expressedquantitatively by the short-circuit current.

2. Upon dilution of sodium in Ringer solution on the mucosal side ofthe preparation, short-circuit current remained a fair expression of sodiumtransport from mucosa to serosa.

3. In view of this, the relation between short-circuit current anddilution of sodium of the luminal side was examined. This relation wascurvilinear, which suggests the intervention of a saturable step in thetransfer of sodium from lumen to serosal surface of colon.

4. The relation between short-circuit current on the one hand, and theamount of sodium drawn from the luminal side and recovered in the mem-brane ('active sodium transport pool') on the other hand, appeared (almost)linear instead. This is meant to indicate that the 'pump' operates far fromcapacity. Hence, the observed saturation of sodium transport, when con-centration of sodium on the mucosal side was increased, probably occursat the mucosal border of the preparation.

5. After treatment with aldosterone, the 'active sodium transportpool' and short-circuit current increased to the same extent, from whichit is inferred that the hormone merely allows sodium easier access to the'pump' which would react in proportion. Consequently, no direct influenceof aldosterone on the 'pump' proper need be postulated.

6. Upon exposure of the colon to antidiuretic hormone, there were(modest) increases of short-circuit current and of osmotic water flow acrossthe wall of the organ.

* Recipient of a fellowship from the O.C.D. (Belgium). Present address: Laboratorio deFisiologia, Universidad de Chile, Casilla 147, Santiago, Chili.

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18. COFRfl AND J. CRABBJ

INTRODUCTION

In mammals at least, the colon shares with the distal part(s) of the renaltubule the property of establishing and maintaining steep concentrationgradients for sodium between lumen and serosal surface so that excreta canbe practically devoid of sodium. The isolated amphibian skin and bladder,adopted as biological models for the study of some aspects of renal tubularfunction, behave in an analogous fashion. The colon (Ussing & Andersen,1955), like the amphibian skin (Ussing & Zerahn, 1951) and bladder(Leaf, Anderson & Page, 1958) is constituted by a sheet of epithelial cellscapable of active sodium transport from the outside to the inside. In Bufomarinus, sodium transport by the colon (Cofre6 & Crabbe, 1965), the skin(Crabbe, 1964a) and the bladder (Crabbe, 1961; Porter & Edelman, 1964;Sharp & Leaf, 1964) is increased by aldosterone.

It was thought that a relation might exist between the efficiency of thesodium-retaining properties of these structures and the responsiveness ofthe latter to aldosterone. Therefore, sodium transport by the colon ofB. marinus was examined as to some of its characteristics.With dilute sodium on the outside, i.e. on the luminal side, the behaviour

of the isolated colon resembled that of the skin and bladder in that thesodium transport activity of the membranes reached a maximum wellbefore sodium concentration outside was brought back to normal. Onthe basis of additional experiments, this early saturation is ascribed toproperties of the outer membrane of the epithelial cells of the colon.

It is therefore proposed that aldosterone stimulates sodium transportacross toad colon by increasing the permeability of this outer membranefor sodium rather than by exerting a direct effect on the sodium 'pump'.

In addition, the influence of antidiuretic hormone on sodium transportand osmotic water flux across the isolated toad colon was examined.

METHODSToads, Bufo marinus, of either sex, were used after a few days spent half-immersed in tap

water or dilute saline. After pithing, the abdomen was opened and the colon was excised,slit longitudinally along its dorsal surface and gently cleaned of its contents. It was thenstretched as a diaphragm between conical lucite chambers (inner diameter: 16 or 20 mm,according to the size of the organ) and incubated asrecommendedbyUssing& Zerahn (1951),both surfaces being exposed to aerated frog Ringer solution (NaCl: 115 mM; KHCO3: 2-5 mM;CaC12: 1 mM).Only those preparations with a d.c. resistance exceeding that of the circuit were taken

into consideration, lest errors arise in short-circuiting (see Appendix).About half of the experiments to be reported were performed on colons stripped of their

inner, chiefly muscular, layers which were discarded; this dissection resulted in an appre-ciable thinning without undue interference with the electrical properties of the organ (seebelow) provided it was performed patiently while keeping the membrane moistened.

178




HORMONES AND SODIUM TRANSPORT 179Whereas the weight of whole colon (n = 20) averaged 81.1 mg/cm2+ 62 (s.E. of mean), itamounted to 21-6 mg/cm2 + 2-2 (s.x. of mean) after stripping (n = 40).

For experiments with radioisotopes of sodium, 24Na, as 24NaCl, was obtained from C.E.N.(Mol, Belgium) and 22Na, as 22NaCl, from the Radiochemical Centre (Amersham, England).[J4C]inulin, as the carboxyl derivative (specific activity: 1x6 ftc/mg), was purchased fromNew England Nuclear Corp. (Boston, U.S.A.) and recrystallized before use.

Experiments were carried out on unstripped colon as described by Ussing & Zerahn (1951)in order to correlate short-circuit current and net sodium transport. Upon stabilization ofthe short-circuit current, 1 wuc 22Na was introduced in the solution on the mucosal (outer)side of the incubated short-circuited colon, while 50 ,tc 24Na were added on the other side.Thirty minutes later, sampling was begun and proceeded at 30 min intervals for 2j hr.After appropriate dilution of the samples, the latter were counted with a well-type crystalcounter; 24Na was measured immediately, the samples being disposed so that correctionsfor decay could be dispensed with. 22Na was counted 3 weeks later. Enough counts weresecured for both radioisotopes to bring the statistical errors to 2 % or less.

The determination of the 'active sodium transport pool' was attempted on stripped colonaccording to a method described previously (Crabbe & De Weer, 1965). In short, the Ringersolution on the mucosal side was diluted 1 to 4 with sodium-free Ringer solution (MgCl2: 57*5mm; KHCO3:2*5 mM; CaCl2: 1 mM; sucrose: 57-5 mM;) the mixture contained in addition,to 24Na, [14C]inulin in order to allow for determination of the extracellular space on themucosal surface of the tissue. The solution on the serosal side was pure Ringer's. Twentyminutes approximately after beginning the incubation, sodium flux was determined for a30 min period, the preparation being short-circuited. Thereafter, the set-up was dismantledas quickly as possible, the membrane was blotted, weighed wet and reweighed after dryingfor 16 hr at 95 IC. 24Na was counted immediately, 14C was counted 3 weeks later on analiquot of the tissue eluate.A series of incubations was conducted on skin and colon with Ringer solution on the

mucosal surface diluted to a varying extent with the solution in which sodium was replacedby magnesium. After about 1 hr of incubation, appropriate dilution of sodium was achievedand maintained for 45 min, whereupon the solution was replaced by pure Ringersolution. The short-circuit current during the experimental period was expressed relative tothe current averaged for the periods immediately before and after the period of exposureto dilute sodium.

Sodium concentration was determined in every case by flame photometry.Experiments aimed at an investigation of the effect of antidiuretic hormone on osmotic

water flow were carried out as follows. Ringer solution diluted 1 to 5 with distilled waterwas introduced in the lumen of unstripped colons tied at both ends and immersed in Ringersolution; the weight of the preparations was recorded every 15 min for at least 2 hr. Thecolon, filled with 5 ml. solution, measured usually 3-4 cm in length, the surface of thecylinder of such a height and volume was estimated at 20 cm2. When the colon was strippedas described, osmotic flow was determined by sequential spectrophotometric determinationsof the concentration of human haemoglobin introduced on the serosal side of the incubationchambers, the mucosal surface being exposed to Ringer solution diluted 1 to 10 with distilledwater (Curran & Solomon, 1957).(+ )-Aldosterone (Aldocorten CIBA) was used in vitro at a concentration of 10-5 M; when

it was administered to the animal, a dose of 10-25 ltg diluted to 0-25 ml. with Ringersolution was injected subcutaneously at least 4 hr before sacrifice. Whenever used, Pitressin(aqueous, Parke, Davis and Co) was added in amounts such that final concentrations were100-200 m-u.Iml. Both hormones, when used in vitro, were added to the serosal side of thepreparation only.

The data were analysed statistically according to Snedecor (1956); ratios were convertedto their decimal logarithms for such analyses.




180 G. COFRJ AND J. CRABBED

RESULTS

Short-circuit current as an expression of net sodiumtransport by the isolated colon of the toad

In four instances the colon (unstripped) was prepared as described andexposed to 22Na on the mucosal side and to 24Na on the opposite (serosal)side. Valid data were obtained for sixteen bidirectional flux periods.As can be seen from Table 1, the agreement was satisfactory between

net sodium flux as calculated from short-circuit current and flux measuredisotopically, since the mean ratio, given at the far right of the table, is notsignificantly different from 100 % (P > 0.3).

TABLE 1. Equivalence between isotopic and electrical measurements of sodium flux acrossthe isolated colon of Bufo marinus (means+ S.E. of mean)

Unidirectional sodium flux*A_ Net sodium flux*

Mucosa Serosa A Equivalenceto serosa to mucosa Measured Calculatedt A-B

(A) (B) (A-B) (C) c xl001-73+0-16 0-67 + 0-10 1-06+0-09 1-14+0-10 93-0

* In /,u-equiv. Na+/30 min. (5-9-100-7)t Calculated from short-circuit current, on the basis of 100 ,uA = 1-865 /u-equiv. Na+/30

mm.

TABLE 2. Relationship between mucosa-to-serosa sodium flux and short-circuit currentwhen toad colons were exposed to modified Ringer solution on the mucosal side

Mean sodium Mucosa-to-Number concentration serosa Net

State of on the sodium sodiuim Equivalenceof experi- mucosal side flux* fluxt 100 x (1)

colon ments (m-equiv/l.) (1) (2) (2)Unstripped 10 16-8 0-76+0-11 0-74+0-14 111-0

(102.3-120.3)Stripped 30 27-9 1-00+ 0-13 0-87 + 0-10 106-9

* In /s-equiv Na+/30 min (means+ S.E. of mean). (100.5113.7)t Calculated from short-circuit current, on the basis of 100 ,A = 1-865 ,u-equiv Na+/30

min (means + S.E. of mean).

When sodium was diluted on the mucosal side of the unstripped colonthe mucosa-to-serosa sodium flux was but slightly in excess of net sodiumtransport calculated from short-circuit current (Cofre & Crabbe, 1965)despite the asymmetry thereby introduced in the incubation system. Thisconclusion also applies to experiments using stripped colon: with bothtypes of preparation, the difference between the mean ratio given at thefar right of Table 2 and 100 % was statistically insignificant (P > 0.2).




HORMONES AND SODIUM TRANSPORT

Short-circuit current in relation to sodium concentrationon the mucosal side

During transit through the colon, faecal sodium concentration can bedrastically reduced. This may be due to the persistence of a high rate of netsodium transport from mucosal to serosal surface of the colon even at lowconcentration of sodium in the lumen of the organ. Since short-circuitcurrent still reflected net sodium flux after dilution of sodium on themucosal side, as shown in Table 2, an attempt was made to evaluate towhat extent short-circuit current would be depressed as a consequence.In sixty instances, sodium concentration on the mucosal side of the

colon was correlated with the change in short-circuit current resulting fromdilution of Ringer fluid with sodium-free solution. Fifty analogus experi-ments were performed with the ventral skin of the toad, a biologicalmembrane which, like the colon, can transport sodium inwards despiteunfavourable concentration gradients for this ion.The data were arranged according to the sodium concentrations on the

outside, and they were averaged for groups of six in the case of the colon,five in the case of the skin.As seen in Fig. 1, toad skin and colon behaved similarly: upon dilution

of sodium in the fluid outside, short-circuit current hardly decreasedunless sodium concentration was less than about one third of the controlvalue in the case of the colon, and less than one sixth when the skin wasexamined. The results suggest that sodium transport across these mem-branes proceeds with a high efficiency at low sodium concentrations out-side, and reaches saturation early, well before sodium concentrations arebrought back to 115 m-equiv./l.

Response of the colon to aldosteroneEnhancement ofsodium transport across the intact toad colon caused by

aldosterone has already been reported (CofreA& Crabbe, 1965). Afterstripping ofthe colon the stimulating effect of aldosterone injected into theanimal could still be demonstrated (Table 3). There was a slight decreaseof the current observed when the data obtained with stripped colon werecompared with the data for the unstripped colon. This is likely to be theconsequence of dilution of sodium in the Ringer fluid on the mucosal sideof stripped colon. The d.c. resistance of the membranes, stripped ornot, increased under the influence of aldosterone which influenced trans-membrane potential relatively more than short-circuit current (Table 3);the reason for this is unknown.

In additional experiments, the colon was exposed to aldosterone invitro. To this effect, colons of large toads were stripped and divided so as to

181




182 G. COFRt AND J. CRABBJ

allow incubation of paired fragments in ten instances. An equal number ofincubations had been carried out with paired unstripped preparations(Cofre & Crabbe, 1965). Table 4 summarizes all results.

Colon Skin

+ 100 I I0~~~~~~~~~~~~~~~~~~~~

0 25 so 0 25 50

[Nal (m-equiv)Fig. 1. Variations in the short-circuit current as a consequence of dilution ofsodium on the outside of toad skin and colon.

Each vertical line represents the mean (±+S.E. of mean) of six experiments inthe case of the colon, of five in the case of the skin. The current is expressed as afraction of the current read when the membrane is exposed to undiluted Ringer.The interrupted straight lines describe what the relationship between sodium

current and sodiujm concentration outside would be ff the former variable weredirectly proportional to the latter one.

In the case of the colon, when sodium concentration in Ringer on the outsidewas 60-80 m-equiv/1. the current was 0-96 (n = 8); this value is not given on thegraph for reasons of symmetry.

TABLE 3. Stiimulation of active sodium transport across toad colon afterinjection of aldosterone to the animal (means+ S.iE. of mean)

Treated withUntreated aldosterone

State of , A- r Icolon n IcA/CM2 mV n ,uA/cM2 mV

UnLstripped* 20 21-5 +1-4 5-8 + 0 3 15 42-1 +3 0 23-6 +1-2Strippedt 8 15-8 + 18 5-2 +1-3 8 30-1 +2-4 17-0 +3-1

*Ringer fluid on the outside.t Modified Ringer fluid on the outside (28 m-equiv Na/l.)

The influence of aldosterone on sodium transport by the isolated colonappears only after 60-90 min of incubation. Therefore the effect of aldo-sterone, present at the uniform concentration of 10-5 M on the serosal side,was exQamined by comparing the change in short-circuit current between





the first and the third hour of incubation for the untreated tissue, withthis change for the matched, treated preparation in twenty pairs of mem-branes (Crabbe, 1964b). Analysed in this way, the stimulating effect ofaldosterone amounted to 5-3 ,tA/cm2 + 1-6 (S.E. mean difference) (P < 0-01).

TARLE 4. Stimulation of active sodium transport by the isolated toadcolon incubated in the presence of aldosterone (means + S.E. of mean)

Short-circuit current (ptA/cm2) Transmembrane potential (mV)A A

Treatment First hour* Last hour First hour* Last hourWhole colon (n = 10)

None 20-0+2-6 15-4+1F4 4-5+0 7 4 0+0 5Aldosterone 20-9+2-1 23-0 + 2-7 7 0+004 8*1+0*6

Stripped colon (n = 10)None 24-0 + 3-6 13-9+ 2*1 7-3+1*5 5-8 +1*4Aldosterone 22-0 + 2-2 15-8 + 1-6 7-6 + 0-6 70 + 0-8

* There was no systematic difference between the upper and lower halves of the pre-parations. Care was, nevertheless, taken to expose to the hormone alternatively upper andlower halves.

'Active sodium transport pool' in toad colonThe tendency toward rapid restoration of normal short-circuit current

across the colon as the concentration of sodium outside was increasedmight be ascribed to saturable permeability barrier on the mucosal sideof the cell layer(s) responsible for active sodium transport, or/and tosaturation of the sodium 'pump'. The assumption is made that theepithelium of the colon resembles the toad bladder (Frazier, 1962), in thatthe 'pump' is located at or near the serosal border of the cells.The method of investigation recently adopted for the toad bladder

(Crabbe & De Weer, 1965) was applied to the stripped colon. An attemptwas made to relate the amount of sodium drawn from the mucosal sideinto the membrane to the rate of transmembrane sodium transport, insixteen instances. Eight preparations were obtained from toads givenaldosterone-so as to stimulate sodium transport-while eight came fromuntreated animals. The size of this sodium pool averaged 43 n-equiv foruntreated membranes, and 66 n-equiv for those stimulated by aldosterone.Mean short-circuit current, expressed in term of net sodium transport,averaged 0-79 and 1-38 ,u-equiv/30 min, respectively. Thus, sodium trans-port and pool increased under the influence of aldosterone by more than50 % as a mean.

Considering as a first approximation that all points belong to one popu-lation, it appears when the individual data are plotted (Fig. 2), that thesize of the sodium pool might show a tendency to increase faster than therate of sodium transport.Data concerning the inulin space on the mucosal side, total sodium

183




184 G. COFRJ AND J. CRABBflcontent of the tissue and hydration of the latter are summarized in Table 5.Treatment of the toad with aldosterone failed to bring about significantchanges in these variables.

- 150._> _ o

0

0~~~~~~

o 100

o 0

0S o0e 0

o 00o

'cz~~~~o0 05 10 1.5 20

Sodium transport (/z-equiv/30 min)

Fig. 2. Relationship between 'active sodium transport pool' and short-circuitcurrent expressed as net sodium transport.The open dots stand for values obtained with (stripped) colon from untreated

toads; the black dots correspond to (stripped) colons from toads treated withaldosterone.The sixteen points fit a regression line obeying the equation

Y = -3-0+52-9 X (r = 0.62).

TABLE 5. Inulin space, sodium content and tissue water for the isolated(stripped) colon (means + S.E. of mean)

Treated withUntreated aldosterone(n = 8) (n = 8)

Tissue water (ml./100 g dry residue) 480-4+ 30-4 512-4+22-5Inulin space on the mucosal side (in 4-6+ 1.1 4-8+ 1-4per cent tissue water*)Sodium content (,u-equiv/ml. tissue 100.5+ 6-1 97-2 + 2-0water)

* As was found with the toad bladder (Crabbe & De Weer, 1965), expression of inulinon this basis results in a smaller dispersion about the mean than when the area incubatedwas used as a reference.

Influence of antidiuretic hormone on the toad colonSodium transport by toad bladder and skin is stimulated by aldosterone

and by antidiuretic hormone; in addition the latter increases the per-meability of these membranes to water. It was therefore thought of in-terest to examine whether toad colon, just shown to react to aldosterone,would also respond to antidiuretic hormone.




HORBONES AND SODIUM TRANSPORT 185(a) The effect of antidiuretic hormone on short-circuit current was

assessed by carrying out ten paired incubations on stripped colon. FromFig. 3 it appears that there was indeed a discrete but lasting stimulation.After 1 hr of contact of stripped colon with antidiuretic hormone, short-circuit current had increased by an average of 8 1 tA, contrasting with amean decrease of 11 1tuA in matched, untreated colon. The hormonaleffect thus amounted at that time to 19-2 ,tA ± 6-4 (s.E. mean difference)(P < 0.02). This response was not enhanced when the membranes wereexposed to antidiuretic hormone after 1 hr of incubation only.

60Vasopressi n

50[0-.~

30 _O_

.1 0 o

C)C)

C)

20

n=10

10_

-30 -15 0 +15 +30 +45 +60Time (min)

Fig. 3. Stimulation of short-circuit current upon addition of antidiuretic hormoneto the solution bathing the inner surface of (stripped) toad colon.The dotted lines stand for matched preparations before addition of the hormone,

and for the untreated preparations thereafter. The continuous line corresponds tothe paired treated colon. Diameter of incubation chambers was 3-14 cm2.

(b) It was thought worth while to examine whether the action of anti-diuretic hormone on sodium transport by the colon of the toad wasassociated with a facilitation of water transfer across the preparation. Theunstripped organ was first studied by the 'bag' technique (see Methods) intwenty instances, from which it appeared that the osmotic water flow in-creased from 3-9 ,ul./cm2.hr + 0 4 in base line conditions to 5 0 /ul./cm2.hr+ 0-7 during the hour which followed addition of the octapeptide (100m-u./ml.). This rise was barely significant statistically (P < 0.05), whenthe analysis relied upon comparison of osmotic flow after and before anti-diuretic hormone (mean A: +25-3 %; S.E. of mean A: + 12-44-- + 39.4).




G. COFRJa AND J. CRABBI

Because of the possible interference of the layers interposed between theepithelium of the unstripped colon and the solution bathing the serosalside, eight additional experiments were carried out on stripped membranes.The rate of dilution of haemoglobin on the serosal side, by water drivenosmotically from the mucosal side, was established before and afteraddition of antidiuretic hormone. Light transmission across aliquotsfrom the serosal side increased from a mean of 0-423 to a mean of 0*4491 hr later, during the control period. It further increased to 0'465 duringthe hour of exposure to antidiuretic hormone (200 m-u./ml.). There thuswas no evidence that the effect obtained with the 'bag' method, usingintact colon, was unduly modest owing to supportive structures locatedbehind the mucosa.

DISCUSSION

Ussing & Andersen (1955) were the first to demonstrate that there is anactive transport of sodium across toad (Bufo bufo) colon, and they showedthat this process can be measured by short-circuit current technique.These findings were confirmed in the case of the isolated frog colon(Cooperstein & Hogben, 1959) and for the isolated colon of B. marinus, asreported here.When the colon of B. marinus was incubated in Ringer solution, the

sodium flux outward, from serosa to mucosa, amounted on the averageto one third of the flux in the other direction; this is comparable withvalues obtained with the amphibian colon and the toad bladder (Leaf et al.1958). Of interest is that there was a fair agreement between short-circuitcurrent and sodium flux inward, from mucosa to serosa, when sodium wasdiluted on the mucosal side; this suggests that, under such circumstancesthe flux of sodium in the opposite direction decreases in amplitude.Dilution of sodium in the solution outside has actually been found toexert such an influence in the case of frog skin (Kirschner, 1955).

Unlike the small intestine, the colon can establish and maintain sizeableconcentration gradients for sodium between its lumen (mucosal side) andthe serosal surface. This might be due to a decrease of the sodium fluxtoward the lumen and also to efficient 'pumping' of sodium inward atlow concentrations of this ion in the lumen. Now, when sodium concen-tration is increased on that (mucosal) side, 'pumping' does not reactproportionately: we are dealing, it seems, with a mechanism complyingwith saturation kinetics as is shown by the relationship between short-circuit current across the colon and the concentration of sodium in thelumen. The same kind of relationship was obtained when toad skin (Fig. 1),frog skin (Cereijido, Herrera, Flanigan & Curran, 1964) and toad bladder(Frazier, Dempsey & Leaf, 1962) were examined.

186





The situation is quite different in the case of the small intestine as foundwith the rat (Asano, 1964) and the rabbit (Schultz & Zalusky, 1964) since,for these preparations, there is a direct proportionality between sodiumconcentration in the lumen and sodium transport inward.The reaction of the sodium transport mechanism(s) to dilution of

sodium on the luminal (mucosal) side of the colon of B. marinus, could beattributed to an early saturation of structures allowing penetration ofsodium into the cells and/or to a limited capacity of the 'pumps'. Byanalogy with the frog skin (Koefoed-Johnsen & Ussing, 1958) and toadbladder (Frazier, 1962), the first step is thought to take place at the outer,or mucosal border of the epithelial cells lining the lumen of the colon whilethe 'pump' would be located at or near the serosal, or inner border of thesecells. That the 'pump' probably operates far from capacity in the experi-mental conditions chosen can be inferred from the fact that the rate ofsodium transport increased (almost) as fast as the size of the 'activesodium transport pool' when the relationship between both variables wasexamined.

It is implied that the 'active sodium transport pool' is located at leastin part within the tissue, between the outer permeability barrier and thesodium 'pump'. Backing this assumption are the facts that, under theinfluence of cyanide (Crabbe, 1965) and ouabain (J. Crabbe6 & P. De Weer,unpublished), the 'active sodium transport pool' increased in the toadbladder while sodium transport by the membrane decreased.

Since there was a proportionality between the 'active sodium transportpool' in toad colon tissue and sodium transport by this organ, one has toconsider the possibility that the limiting factor responsible for the earlysaturation of sodium transport, as the concentrations of sodium on theoutside are increased, operates at the site of entrance of sodium in the cellsof the colon mucosa.

Stripping of the colon was required in order to determine exactly the'active sodium transport pool'; this pool was disproportionately large(664 n-equiv Na) in the single case of measurement carried out on an un-stripped preparation. It is therefore possible that inadequate stripping isthe reason for the large size of the pool in one of the other preparations(Fig. 2). The inner layers of the colon probably interfere with diffusion ofthe ion inward; it actually took less time for steady state conditions to bereached during studies relating radiosodium flux to short-circuit current,when stripped colon was used.The adrenal cortex is known to exert an influence on the efficiency with

which sodium is withdrawn from the lumen of the colon (Ross & Spencer,1954; Moll & Koczorek, 1962). Aldosterone in particular enhances sodiumabsorption by the colon (Holtz, Fink, Zintel & Grouse, 1965; Levitan &

187




1. COFRfl AND J. CRABBJ9Ingelfinger, 1965; Wrong, Metcalfe & Gibson, 1965). The demonstrationof a direct stimulating action of this hormone on toad colon (Cofre6 &Crabbe, 1965) has now been extended to the case of stripped colon pre-parations. The characteristics of this stimulation are analogous to what hasbeen found with toad bladder since there was a latency period of about1 hr between addition of the steroid to the incubating fluid and the detec-tion of a hormonal effect; this hormonal effect, in vivo and in vitro,amounted to about 50 % of the base line activity after 3-4 hr. Furthermore,as in the case of the toad bladder, the size of the 'active sodium transportpool' and the short-circuit current increased almost proportionally fortoad colon stimulated by aldosterone. It thus is conceivable that toadcolon, as toad bladder, reacts to aldosterone because this steroid hormoneallows sodium easier access to the 'pump' that would operate faster as amere consequence of increased availability of sodium originating from themucosal side.

This is why vasopressin was assayed on toad colon; this polypeptide isalso capable of stimulation of active sodium transport by the toad bladder,through action at the mucosal surface of the membrane presumably(CrabbeA& De Weer, 1965; Frazier et al. 1962). A modest effecton sodiumtransport by the isolated colon was observed: furthermore, there was asmall increase of the permeability to water. Ussing & Andersen (1955)mentioned that sodium transport by toad colon reacts by an increase toantidiuretic hormone; so did Aulsebrook (1961) who used the isolated ratcolon. Uranga (1958) has concluded that antidiuretic hormone did notinfluence the movement of water across the wall of the intestinal tract ofthe living toad. The method used might explain the discrepancy betweenher findings and ours: if antidiuretic hormone acted only on the colon-where its effect is not very marked, as reported here-and not on the smallintestine, the failure to detect a hormonal effect might be due to hetero-geneity of the preparation selected.

APPENDIX

When the membrane is short-circuited

is EmI M (1)where Isc, is the current required to reduce the transmembrane potentialEM to nil, and RM is the d.c. resistance of the membrane. RM has beenfound to hold a constant value, at least between I = 0 and I = IS, inthe case of toad colon.

188




HORMONES AND SODIUM TRANSPORT 189

During incubation in a circuit such as devised by Ussing & Zerahn(1951), one has

ISCIC -E,,,I+x (2)R41+Rc~

where x is the complement to EM required for RC, the d.c. resistance of thecircuit, to be taken into account during short-circuiting.From (1) and (2), it follows that x = EM(RclRM).Thus, for the membrane to be short-circuited, current should pass to an

extent such that the balance position on the electrometer is exceeded bythis value x.RC is obtained from measurements in the absence of the membrane; the

smaller RM is relative to RC, the larger x will be, with unavoidable in-accuracies liable to interfere with the precise determination of Isce.

This study was carried out with the financial support of the 'Fonds de la RechercheScientifique Medicale', Belgium (Grant no. 555). Aldosterone was kindly supplied by CIBA,Belgium, and Pitressin by Parke, Davis and Co., Belgium.

It is a pleasure to thank here Dr Fischer, Warner Laboratories and the Belgian Embassyat Rio de Janeiro, Brazil, for their invaluable assistance in the shipment of Bufo marinus;Dr E. J. Ross who consented to read and correct the manuscript; and Dr B. Andersen whotaught one of us (J.C.) how to isolate the colon mucosa by stripping.

REFERENCES

ASANO, T. (1964). Metabolic disturbances and short-circuit current across intestinal wall ofrat. Am. J. Physiol. 207, 415-422.

AuLSEBIROOE, K. A. (1961). Effect of vasopressin on sodium transfer by rat colon in vitro.Endrocrinology 68, 1063-1065.

CEREIJDO, M., HERRERA, F. C., FLANIGAN, W. J. & CuRRAN, P. F. (1964). The influence ofNa concentration on Na transport across frog skin. J. gen. Physiol. 47, 879-893.

CoFRA, G. & CRABBE', J. (1965). Stimulation by aldosterone of active sodium transport bythe isolated colon of the toad, Bufo marinus. Nature, Lond. 207, 1299-1300.

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