J. Physiol. (1967), 188, pp.

177-190 With 3 text-figure8 Printed in Great Britain

177

ACTIVE SODIUM TRANSPORT BY THE COLON OF BUFO MARINUS: STIMULATION BY ALDOSTERONE AND ANTIDIURETIC HORMONE

BY G. COFRI* AND J. CRABBI From the Section on Endocrinology, Laboratory of Experimental Surgery, University Clinics St Pierre, Louvain, Belgium

(Received 1 February 1966)

SUMMMARY

1. The isolated colon of Bufo marinus transports sodium actively from the mucosal (lumen) to the serosal side, and this transport is expressed quantitatively by the short-circuit current. 2. Upon dilution of sodium in Ringer solution on the mucosal side of the preparation, short-circuit current remained a fair expression of sodium transport from mucosa to serosa. 3. In view of this, the relation between short-circuit current and dilution of sodium of the luminal side was examined. This relation was curvilinear, which suggests the intervention of a saturable step in the transfer of sodium from lumen to serosal surface of colon. 4. The relation between short-circuit current on the one hand, and the amount of sodium drawn from the luminal side and recovered in the membrane ('active sodium transport pool') on the other hand, appeared (almost) linear instead. This is meant to indicate that the 'pump' operates far from capacity. Hence, the observed saturation of sodium transport, when concentration of sodium on the mucosal side was increased, probably occurs at the mucosal border of the preparation. 5. After treatment with aldosterone, the 'active sodium transport pool' and short-circuit current increased to the same extent, from which it is inferred that the hormone merely allows sodium easier access to the ' pump' which would react in proportion. Consequently, no direct influence of 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 across the wall of the organ.
* Recipient of a fellowship from the O.C.D. (Belgium). Present address: Laboratorio de Fisiologia, Universidad de Chile, Casilla 147, Santiago, Chili.

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178

18. COFRfl AND J. CRABBJ

INTRODUCTION

In mammals at least, the colon shares with the distal part(s) of the renal tubule the property of establishing and maintaining steep concentration gradients for sodium between lumen and serosal surface so that excreta can be practically devoid of sodium. The isolated amphibian skin and bladder, adopted as biological models for the study of some aspects of renal tubular function, 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 cells capable of active sodium transport from the outside to the inside. In Bufo marinus, 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 the sodium-retaining properties of these structures and the responsiveness of the latter to aldosterone. Therefore, sodium transport by the colon of B. 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 the sodium transport activity of the membranes reached a maximum well before sodium concentration outside was brought back to normal. On the basis of additional experiments, this early saturation is ascribed to properties of the outer membrane of the epithelial cells of the colon. It is therefore proposed that aldosterone stimulates sodium transport across toad colon by increasing the permeability of this outer membrane for sodium rather than by exerting a direct effect on the sodium 'pump'. In addition, the influence of antidiuretic hormone on sodium transport and osmotic water flux across the isolated toad colon was examined.
METHODS Toads, 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 then stretched as a diaphragm between conical lucite chambers (inner diameter: 16 or 20 mm, according to the size of the organ) and incubated as recommended by Ussing& 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 appreciable thinning without undue interference with the electrical properties of the organ (see below) provided it was performed patiently while keeping the membrane moistened.

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HORMONES AND SODIUM TRANSPORT

179

Whereas the weight of whole colon (n = 20) averaged 81.1 mg/cm2+ 62 (s.E. of mean), it amounted 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 from New 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 of the 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 crystal counter; 24Na was measured immediately, the samples being disposed so that corrections for decay could be dispensed with. 22Na was counted 3 weeks later. Enough counts were secured for both radioisotopes to bring the statistical errors to 2 % or less. The determination of the 'active sodium transport pool' was attempted on stripped colon according to a method described previously (Crabbe & De Weer, 1965). In short, the Ringer solution on the mucosal side was diluted 1 to 4 with sodium-free Ringer solution (MgCl2: 57*5 mm; 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 the mucosal surface of the tissue. The solution on the serosal side was pure Ringer's. Twenty minutes approximately after beginning the incubation, sodium flux was determined for a 30 min period, the preparation being short-circuited. Thereafter, the set-up was dismantled as quickly as possible, the membrane was blotted, weighed wet and reweighed after drying for 16 hr at 95 IC. 24Na was counted immediately, 14C was counted 3 weeks later on an aliquot 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 replaced by magnesium. After about 1 hr of incubation, appropriate dilution of sodium was achieved and maintained for 45 min, whereupon the solution was replaced by pure Ringer solution. The short-circuit current during the experimental period was expressed relative to the current averaged for the periods immediately before and after the period of exposure to 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 water was introduced in the lumen of unstripped colons tied at both ends and immersed in Ringer solution; the weight of the preparations was recorded every 15 min for at least 2 hr. The colon, filled with 5 ml. solution, measured usually 3-4 cm in length, the surface of the cylinder of such a height and volume was estimated at 20 cm2. When the colon was stripped as described, osmotic flow was determined by sequential spectrophotometric determinations of the concentration of human haemoglobin introduced on the serosal side of the incubation chambers, the mucosal surface being exposed to Ringer solution diluted 1 to 10 with distilled water (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 Ringer solution 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 were 100-200 m-u.Iml. Both hormones, when used in vitro, were added to the serosal side of the preparation only. The data were analysed statistically according to Snedecor (1956); ratios were converted to their decimal logarithms for such analyses.

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180

G. COFRJ AND J. CRABBED

RESULTS

Short-circuit current as an expression of net sodium transport by the isolated colon of the toad In four instances the colon (unstripped) was prepared as described and exposed 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 measured isotopically, since the mean ratio, given at the far right of the table, is not significantly different from 100 % (P > 0.3).
TABLE 1. Equivalence between isotopic and electrical measurements of sodium flux across the isolated colon of Bufo marinus (means+ S.E. of mean)

Unidirectional sodium flux*

A_

Mucosa to serosa (A) 1-73+0-16

* In /,u-equiv.

Serosa to mucosa

Net sodium flux*

A

Measured

(B) 0-67 + 0-10

Calculatedt

Equivalence

(A-B) 1-06+0-09

(C) 1-14+0-10

t Calculated from short-circuit current,

mm.

Na+/30 min.

A-B c xl00 93-0 (5-9-100-7)

on the basis of 100 ,uA = 1-865 /u-equiv.

Na+/30

TABLE 2. Relationship between mucosa-to-serosa sodium flux and short-circuit current when toad colons were exposed to modified Ringer solution on the mucosal side
State of colon
Number of experiments 10
Mean sodium concentration on the mucosal side

Mucosa-toserosa sodium
flux* (1) 0-76+0-11

sodiuim
fluxt (2) 0-74+0-14

Net

Unstripped

(m-equiv/l.) 16-8

Equivalence 100 x (1)

(2) 111-0

min

(102.3-120.3) 30 27-9 Stripped 1-00+ 0-13 0-87 + 0-10 106-9 (100.5113.7) * In /s-equiv Na+/30 min (means + S.E. of mean). t Calculated from short-circuit current, on the basis of 100 ,A = 1-865 ,u-equiv Na+/30

(means + S.E. of mean).

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

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HORMONES AND SODIUM TRANSPORT

181

Short-circuit current in relation to sodium concentration on the mucosal side During transit through the colon, faecal sodium concentration can be drastically reduced. This may be due to the persistence of a high rate of net sodium transport from mucosal to serosal surface of the colon even at low concentration of sodium in the lumen of the organ. Since short-circuit current still reflected net sodium flux after dilution of sodium on the mucosal side, as shown in Table 2, an attempt was made to evaluate to what 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 from dilution of Ringer fluid with sodium-free solution. Fifty analogus experiments were performed with the ventral skin of the toad, a biological membrane which, like the colon, can transport sodium inwards despite unfavourable 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 decreased unless sodium concentration was less than about one third of the control value in the case of the colon, and less than one sixth when the skin was examined. The results suggest that sodium transport across these membranes proceeds with a high efficiency at low sodium concentrations outside, and reaches saturation early, well before sodium concentrations are brought back to 115 m-equiv./l.
Response of the colon to aldosterone Enhancement of sodium transport across the intact toad colon caused by & Crabbe, 1965). After aldosterone has already been reported (CofreA stripping of the colon the stimulating effect of aldosterone injected into the animal could still be demonstrated (Table 3). There was a slight decrease of the current observed when the data obtained with stripped colon were compared with the data for the unstripped colon. This is likely to be the consequence of dilution of sodium in the Ringer fluid on the mucosal side of stripped colon. The d.c. resistance of the membranes, stripped or not, increased under the influence of aldosterone which influenced transmembrane potential relatively more than short-circuit current (Table 3); the reason for this is unknown. In additional experiments, the colon was exposed to aldosterone in vitro. To this effect, colons of large toads were stripped and divided so as to
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182 G. COFRt AND J. CRABBJ allow incubation of paired fragments in ten instances. An equal number of incubations had been carried out with paired unstripped preparations (Cofre & Crabbe, 1965). Table 4 summarizes all results.
Colon

Skin

100
0~~~~~~~~~~~~~~~~~~~~

25

so

25

50

[Nal (m-equiv)
Fig. 1. Variations in the short-circuit current as a consequence of dilution of sodium on the outside of toad skin and colon.
Each vertical line represents the mean (+S.E. of mean) of six experiments in the case of the colon, of five in the case of the skin. The current is expressed as a fraction 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 were directly proportional to the latter one. In the case of the colon, when sodium concentration in Ringer on the outside was 60-80 m-equiv/1. the current was 0-96 (n = 8); this value is not given on the graph for reasons of symmetry.
TABLE 3. Stiimulation of active sodium transport across toad colon after injection of aldosterone to the animal (means + S.iE. of mean) Treated with Untreated aldosterone State of , AI colon n mV n IcA/CM2 mV ,uA/cM2 20 21-5 +1-4 5-8 + 0 3 UnLstripped* 15 42-1 +3 0 23-6 +1-2 8 15-8 + 18 5-2 +1-3 Strippedt 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.)
r

The influence of aldosterone on sodium transport by the isolated colon appears only after 60-90 min of incubation. Therefore the effect of aldosterone, present at the uniform concentration of 10-5 M on the serosal side, was exQamined by comparing the change in short-circuit current between
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183 the first and the third hour of incubation for the untreated tissue, with this change for the matched, treated preparation in twenty pairs of membranes (Crabbe, 1964b). Analysed in this way, the stimulating effect of aldosterone 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 toad colon incubated in the presence of aldosterone (means + S.E. of mean) Short-circuit current (ptA/cm2)
A

HORMONES AND SODIUM TRANSPORT

Transmembrane potential (mV)

A

Treatment
None Aldosterone

First hour*
20-0+2-6 20-9+2-1

Last hour Whole colon (n = 10)

First hour*

Last hour
4 0+0 5

15-4+1F4
23-0 + 2-7

4-5+0 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*4 Aldosterone 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 preparations. Care was, nevertheless, taken to expose to the hormone alternatively upper and lower halves.

'Active sodium transport pool' in toad colon The tendency toward rapid restoration of normal short-circuit current across the colon as the concentration of sodium outside was increased might be ascribed to saturable permeability barrier on the mucosal side of the cell layer(s) responsible for active sodium transport, or/and to saturation of the sodium 'pump'. The assumption is made that the epithelium of the colon resembles the toad bladder (Frazier, 1962), in that the '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 attempt was made to relate the amount of sodium drawn from the mucosal side into the membrane to the rate of transmembrane sodium transport, in sixteen instances. Eight preparations were obtained from toads given aldosterone-so as to stimulate sodium transport-while eight came from untreated animals. The size of this sodium pool averaged 43 n-equiv for untreated 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 transport and pool increased under the influence of aldosterone by more than
50

% 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 the size of the sodium pool might show a tendency to increase faster than the rate of sodium transport. Data concerning the inulin space on the mucosal side, total sodium
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184 G. COFRJ AND J. CRABBfl content of the tissue and hydration of the latter are summarized in Table 5. Treatment of the toad with aldosterone failed to bring about significant changes in these variables.
-

._>
0

150

o 100
0~~~~~~

S e
o

0 o0

00o

'cz
0

20 1.5 10 Sodium transport (/z-equiv/30 min) Fig. 2. Relationship between 'active sodium transport pool' and short-circuit current 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 with aldosterone. 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 with Untreated aldosterone (n = 8) (n = 8) 512-4+22-5 480-4+ 30-4 Tissue water (ml./100 g dry residue) 4-6+ 1.1 4-8+ 1-4 Inulin space on the mucosal side (in per cent tissue water*) 100.5+ 6-1 97-2 + 2-0 Sodium content (,u-equiv/ml. tissue water)
* As was found with the toad bladder (Crabbe & De Weer, 1965), expression of inulin on this basis results in a smaller dispersion about the mean than when the area incubated was used as a reference.

~~~~o05

Influence of antidiuretic hormone on the toad colon

Sodium transport by toad bladder and skin is stimulated by aldosterone and by antidiuretic hormone; in addition the latter increases the permeability of these membranes to water. It was therefore thought of interest to examine whether toad colon, just shown to react to aldosterone, would also respond to antidiuretic hormone.
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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. From Fig. 3 it appears that there was indeed a discrete but lasting stimulation. After 1 hr of contact of stripped colon with antidiuretic hormone, shortcircuit current had increased by an average of 8 1 tA, contrasting with a mean decrease of 11 1tuA in matched, untreated colon. The hormonal effect 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 were exposed to antidiuretic hormone after 1 hr of incubation only.
60

Vasopressi n

50[
0-.~

C)
C)

30

_O_

C)

0 .1 20
n=10

10_

+45 +30 +15 0 +60 Time (min) Fig. 3. Stimulation of short-circuit current upon addition of antidiuretic hormone to 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 to the paired treated colon. Diameter of incubation chambers was 3-14 cm2.

-30

-15

(b) It was thought worth while to examine whether the action of antidiuretic hormone on sodium transport by the colon of the toad was associated with a facilitation of water transfer across the preparation. The unstripped organ was first studied by the 'bag' technique (see Methods) in twenty instances, from which it appeared that the osmotic water flow increased 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 (100 m-u./ml.). This rise was barely significant statistically (P < 0.05), when the analysis relied upon comparison of osmotic flow after and before antidiuretic hormone (mean A: + 25-3 %; S.E. of mean A: + 12-44-- + 39.4).
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186 G. COFRJa AND J. CRABBI Because of the possible interference of the layers interposed between the epithelium of the unstripped colon and the solution bathing the serosal side, eight additional experiments were carried out on stripped membranes. The rate of dilution of haemoglobin on the serosal side, by water driven osmotically from the mucosal side, was established before and after addition of antidiuretic hormone. Light transmission across aliquots from the serosal side increased from a mean of 0-423 to a mean of 0*449 1 hr later, during the control period. It further increased to 0'465 during the hour of exposure to antidiuretic hormone (200 m-u./ml.). There thus was no evidence that the effect obtained with the 'bag' method, using intact colon, was unduly modest owing to supportive structures located behind the mucosa.
DISCUSSION

Ussing & Andersen (1955) were the first to demonstrate that there is an active transport of sodium across toad (Bufo bufo) colon, and they showed that 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, as reported here. When the colon of B. marinus was incubated in Ringer solution, the sodium flux outward, from serosa to mucosa, amounted on the average to one third of the flux in the other direction; this is comparable with values obtained with the amphibian colon and the toad bladder (Leaf et al. 1958). Of interest is that there was a fair agreement between short-circuit current and sodium flux inward, from mucosa to serosa, when sodium was diluted on the mucosal side; this suggests that, under such circumstances the flux of sodium in the opposite direction decreases in amplitude. Dilution of sodium in the solution outside has actually been found to exert such an influence in the case of frog skin (Kirschner, 1955). Unlike the small intestine, the colon can establish and maintain sizeable concentration gradients for sodium between its lumen (mucosal side) and the serosal surface. This might be due to a decrease of the sodium flux toward the lumen and also to efficient 'pumping' of sodium inward at low concentrations of this ion in the lumen. Now, when sodium concentration is increased on that (mucosal) side, 'pumping' does not react proportionately: we are dealing, it seems, with a mechanism complying with saturation kinetics as is shown by the relationship between shortcircuit current across the colon and the concentration of sodium in the lumen. 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.
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187 HORMONES AND SODIUM TRANSPORT The situation is quite different in the case of the small intestine as found with the rat (Asano, 1964) and the rabbit (Schultz & Zalusky, 1964) since, for these preparations, there is a direct proportionality between sodium concentration 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 be attributed to an early saturation of structures allowing penetration of sodium into the cells and/or to a limited capacity of the 'pumps'. By analogy with the frog skin (Koefoed-Johnsen & Ussing, 1958) and toad bladder (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 while the 'pump' would be located at or near the serosal, or inner border of these cells. That the 'pump' probably operates far from capacity in the experimental conditions chosen can be inferred from the fact that the rate of sodium transport increased (almost) as fast as the size of the 'active sodium transport pool' when the relationship between both variables was examined. It is implied that the 'active sodium transport pool' is located at least in part within the tissue, between the outer permeability barrier and the sodium 'pump'. Backing this assumption are the facts that, under the influence of cyanide (Crabbe, 1965) and ouabain (J. Crabbe6 & P. De Weer, unpublished), the 'active sodium transport pool' increased in the toad bladder while sodium transport by the membrane decreased. Since there was a proportionality between the 'active sodium transport pool' in toad colon tissue and sodium transport by this organ, one has to consider the possibility that the limiting factor responsible for the early saturation of sodium transport, as the concentrations of sodium on the outside are increased, operates at the site of entrance of sodium in the cells of 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 unstripped preparation. It is therefore possible that inadequate stripping is the 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 of the ion inward; it actually took less time for steady state conditions to be reached 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 sodium absorption by the colon (Holtz, Fink, Zintel & Grouse, 1965; Levitan &
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1. COFRfl AND J. CRABBJ9 Ingelfinger, 1965; Wrong, Metcalfe & Gibson, 1965). The demonstration of a direct stimulating action of this hormone on toad colon (Cofre6 & Crabbe, 1965) has now been extended to the case of stripped colon preparations. The characteristics of this stimulation are analogous to what has been found with toad bladder since there was a latency period of about 1 hr between addition of the steroid to the incubating fluid and the detection 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 transport pool' and the short-circuit current increased almost proportionally for toad colon stimulated by aldosterone. It thus is conceivable that toad colon, as toad bladder, reacts to aldosterone because this steroid hormone allows sodium easier access to the 'pump' that would operate faster as a mere consequence of increased availability of sodium originating from the mucosal side. This is why vasopressin was assayed on toad colon; this polypeptide is also capable of stimulation of active sodium transport by the toad bladder, through action at the mucosal surface of the membrane presumably & De Weer, 1965; Frazier et al. 1962). A modest effecton sodium (CrabbeA transport by the isolated colon was observed: furthermore, there was a small increase of the permeability to water. Ussing & Andersen (1955) mentioned that sodium transport by toad colon reacts by an increase to antidiuretic hormone; so did Aulsebrook (1961) who used the isolated rat colon. Uranga (1958) has concluded that antidiuretic hormone did not influence the movement of water across the wall of the intestinal tract of the living toad. The method used might explain the discrepancy between her findings and ours: if antidiuretic hormone acted only on the colonwhere its effect is not very marked, as reported here-and not on the small intestine, the failure to detect a hormonal effect might be due to heterogeneity of the preparation selected.
188

APPENDIX

When the membrane is short-circuited

is I

Em M

(1 )

where Isc, is the current required to reduce the transmembrane potential EM to nil, and RM is the d.c. resistance of the membrane. RM has been found to hold a constant value, at least between I = 0 and I = IS, in the case of toad colon.
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189 HORMONES AND SODIUM TRANSPORT During incubation in a circuit such as devised by Ussing & Zerahn (1951), one has (2) ISCIC -E,,,I+x

R41+Rc~

where x is the complement to EM required for RC, the d.c. resistance of the circuit, 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 by this 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 inaccuracies liable to interfere with the precise determination of Isce.
This study was carried out with the financial support of the 'Fonds de la Recherche Scientifique 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 Embassy at 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 who taught one of us (J.C.) how to isolate the colon mucosa by stripping.

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