sucrose fluxes and junctional water flow across necturus gall bladder epithelium

Sucrose Fluxes and Junctional Water Flow across Necturus Gall Bladder EpitheliumAuthor(s): A. E. Hill and Bruria S. HillSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 200, No.1139 (Feb. 23, 1978), pp. 163-174Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/77333 .

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Proc. R. Soc. Lond. B. 200, 163-174 (1978)

Printed in Great Britain

Sucrose fluxes and junctional water flow across Necturus gall bladder epithelium

BY A. E. HILL AND BRURIA S. HILL

The Physiological Laboratory, Cambridge CB2 3EG, U.K.

(Communicated by R. D. Keynes, F.R.S. - Received 17 May 1977)

[Plate 1]

When sucrose is present in a luminal solution bathing Necturus gall bladder epithelium, the secreted fluid is also found to contain sucrose at more than 0.9 of the luminal concentration. A first-order convection- diffusion equation has been used to calculate the emergent sucrose concentration, by using values for the dimensions of the paracellular system and assuming that the sucrose flow is extracellular. The results indicate that most of the sucrose flow across the junctions must be convective in nature, and the size of the junctional water flow required amounts to most of the overall epithelial water flow, i.e. during secretion the fluid is generated by junctional flow and not by standing-gradient osmosis over the membrane lining the interspaces.

The junctional complexes at the luminal side of the interspaces are not zonulae occludentes but parallel alignments of the cell membranes with a constant (peak to peak) spacing of 14.2 nm. As the reflexion coefficient of these junctions for salt is unknown, but could be low, it is by no means certain that the junctional water flow is driven by osmosis.

INTRODUCTION

It has recently been argued that both in Necturus proximal tubular epithelium (Sackin & Boulpaep I975) and Necturus gall bladder epithelium (Hill & Hill I978),

the transported fluid crosses into the interspaces by way of the junctions and not primarily across the cells and their lining membranes. The latter transcellular fluid pathway is required in standing-gradient osmotic theory (Diamond & Bossert I967). In this theory the precise separation of the interspace membranes is very critical in achieving a high degree of coupling between the flow of salt and water in the interspaces. Although the electron micrographs of gall bladder indicate that the interspace widths do not approach the minute dimensions required theoretically, there is always the possibility that for one reason or another the interspaces are more distended after the fixation procedure than before; what is therefore required is a different way of approaching the problem.

It has been observed many times that extracellular markers, such as sucrose, cross leaky epithelia, presumably by the junctions. Smulders & Wright (1971)

concluded that sucrose crossed gall bladder via aqueous channels because the [ 163 ]



164 A. E. Hill and B. S. Hill

reflexion coefficient (presumably at the cell membrane) was unity, but the acti- vation energy for permeation was equal to that in free solution; Perez-Gonzalez & Whittembury (I974) observed increased permeation of sucrose and other non- electrolytes during treatment of proximal tubular epithelium with osmotic gradients, although kidney cells are not permeated by these compounds; and finally Berry & Boulpaep (I975) measured an apparent effect of fluid transfer on sucrose fluxes across Necturus proximal tubule, and concluded that there was an interaction between the flows of water and sucrose crossing the junctions. In tight epithelia such as frog skin and toad skin, the anomalous transport of sucrose observed when the outer medium is made hypertonic has also been explained by interaction between water and sucrose flows along the junctions and interspaces (Ussing I966; Ussing & Johansen I969).

The purpose of the experiments described in this paper was to measure the transport of sucrose from the gall bladder lumen into the secreted fluid during normal secretory activity of the tissue, and then to calculate the magnitude of the water flow across the junctions which is required to explain the results. Initially, the water flow is considered to occur by standing-gradient osmosis from the cells into the interspaces, and the sucrose diffuses across the junctions and enters the flow stream; then the effect of diverting an increasing fraction of the water flow via the junctions is examined. To carry out this calculation with any accuracy the dimensions of the junctions are required, and these have to be obtained by quantitative transmission electron microscopy. The results of such a study, which is being extended to other leaky epithelia, show that where the zonulae occludentes are expected to be located, Necturus gall bladder epithelial cells do not contain junctions which bear comparison with the true 'tight' junctions of frog skin and toad urinary bladder, and are devoid of any lateral contacts or 'kisses'

(Diamond I974).

METHODS

Necturus gall bladders were perfused at their luminal side with aerated saline (50 mosmolar) as described in a companion paper (Hill & Hill I978), and the secretion collected in a closed chamber after 12 h. C14-sucrose in trace amounts (0.001 mm) but high specific activity was added to the perfusate, and the secretion collected during 12-14 h; aliquot portions of both the perfusate and secreted fluid were then compared by scintillation assay, and the ratio of counts determined. The concentration of sucrose in the secretion, expressed as a fraction of that in the perfusate, is called the 'specific secreted concentration'. 113-insulin and C14-urea were also used in a few experiments, although the results are not used as a basis for calculation in this paper.

Tissue was fixed for electron microscopy by perfusing the bladder with 1 %0 glutaraldehyde and post-fixing and staining as described in a companion paper (Hill & Hill I977). Junctions were viewed at high magnification when it could be seen that they were slits formed by a parallel alignment of two opposing cell



Proc.. R. Soc. Lond. B, volume 200 Hill & Hill, plate 1

FIGURE 1. A Necturu8 gall bladder junction in normal saline. There is usually a prominent desmosome between the junction (arrows) and the start of the interspace system. The junctions are constant in structure and quite unlike typical zonulae occludentes which have a central line of membrane fusion. Magn. x 112500.

(Facing p. 165)



Sucrose flow across Necturus gall bladder 165

membranes; there were no areas of membrane fusion to be seen, and no bridging structures or 'studs' spanning the intermembrane space (figure 1, plate 1). Occasionally the intermembrane space appeared to be indistinct in places, but on tilting the section with a goniometer attachment to the microscope stage, these could be clearly seen to be regions where the junctions were twisting in the plane of the section; the indistinct regions then disappeared and the intermembrane space could be plainly resolved again, at a specific angle of tilt.

-4-Q

Ca

0

0 20 40 60 distance/nm

FIGURE 2. A single densitometer scan of a junction as shown in figure 1. The bimolecular leaflets are visible as double peaks, and the intermembrane space is approximately equal to the thickness of a cell membrane.

Several junctions without apparent twist were chosen for scanning on a double- beam microdensitometer. The junictions were scanned six times at six different depths and the scans digitized; they were then aligned by choosing as origin the density minima of the intermembrane space, which was quite sharp in all scans, and subsequently attenuated, summed, and plotted by computer. In figure 2 is shown a single scan in which two membrane leaflets were resolved in each peak, but these were not always seen so clearly and were always lost when the scans were averaged.

The overall lengths of the junctions were also measured directly from enlarged prints of the junctional regions.




RESULTS

The specific secreted concentration of sucrose (N = 9) was 0.92 (s.e.m. 0.09). The variation in the secretion rate was considerable, ranging from about 3 to 25 tl fluid cm-2 epithelium, h-1. The6 sac preparations had a surface area of between 1.5 and 2.0 cm2. It thus appears that sucrose can permeate the junctional complexes of Necturus gall bladder with relative ease, during normal secretion. Both urea and inulin crossed the junctions at a high rate, but with lower specific secreted concentrations than sucrose, inulin being retarded with respect to urea. Occasionally sucrose, urea, and mannitol have been used in experiments at high concentration in the luminal perfusate, and they were found at high concentration in the secretion; the osmolarity of the bathing solutions in the experiments was quite low (50 mos/l), and it appears that this may raise the permeability of the paracellular route (Perez-Gonzalez & Whittembury 1974), leaving the fluid transfer intact. This finding is in contradistinction to that of Diamond (i964) who found little transport of sugars in rabbit gall bladder during secretion.

The specific secreted concentration however, has to be multiplied by a factor which takes into account the dilution effect of the 'cold' solution held in the dead space of the corium at the moment when 14C sucrose is added to the perfusion fluid. The thickness of the corium in Necturus gall bladder is between 75 and 80 ,um, and the area of the epithelium 1.5 and 2.0 cm2. During the 10-12 h of an experiment, about 0.2 cm3 of solution was collected, and therefore from 6 to 9 0 of this secretion was contributed by sucrose-free solution from the corium. The dilution factor is thus somewhere between 1.06 and 1.09, and the true specific secreted concentration lies between the values of 0.975 and 1.00.

The mean intermembrane distance from one cell membrane to the other across the junctions (peak to peak) was 14.2 nm (s.e.m. 0.7, N -6) which represents the combined results of 36 scans of 6 junctions. The effective intermembrane space must therefore represent 14.2 nm minus the width of two membrane semi-thick- nesses, i.e. minus the width of a total unit membrane thickness; if one takes this to lie between 6 and 8 nm, the intermembrane space comes out to be 6-8 nm in width, and these values have been used to calculate the sucrose fluxes in this paper.

The length of the junctions was taken as 0.7 gtm (0.72, s.e.m. 0.04) and the linear extent of the junction parallel to the luminal surface has been estimated from scanning electron micrographs to be 12.7 m (s.e.m. 0.06, N = 4) per cm2 epithelium (Hill & Hill I978). From electron micrographs the length of the intercellular spaces was estimated to be 35 gtm, a value which is somewhat greater than the cell height; the interspace width could obviously be a matter open to discussion and is very difficult to measure for it varies considerably from region to region. Measurement of the width of intercellular spaces at twenty points along their length gave a spectrum of values from 0.02 to 0.5 ,um. The smaller values represent the minimum separation displayed by cell membranes in contact, while the larger




values represent local expansions of the spaces which are quite extensive in their distribution. This spread of values has been considered below, in relation to both standing-gradient equilibration and the sucrose fluxes along the interspace. The value of all the relevant paracellular dimensions are collected in table 1.

TABLE 1. DIMENSIONS OF THE PARACELLULAR SYSTEM

length of junctions, n/aim 0.7 width of junctions, d/nm 6-8 linear extent of junction per cm2 mucosal surface, 1/m 12.7 width of interspaces, r/pim 0.02-0.5 length of interspaces, L/jm 35

DISCUSSION

Diffusion - convection in the paracellular pathway The nature of the problem is outlined in figure 3. The junction is treated as a short

quasi-homogeneous slit, over which the sucrose flux js is given in terms of the diffusion coefficient D by

Js = D(Cp-CO)dl

where jw is the water flow through the system per square centimetre epithelium, c is the average concentration in the system, and o- is the reflexion coefficient (Kedem & Katchalsky 1958). The product of the junction width, d, and the linear extent of junction per square centimetre of epithelium, 1, defines the cross- sectional area of the junction, while D/n is its effective permeability coefficient. When jw is zero, sucrose crosses the junction purely by diffusion, from a luminal concentration CQ to a concentration CO at the beginning of the intercellular space.

Along the interspace, of length L, sucrose flows by both convection and diffusion

s= vxcx-D(dCldx)xrl, (2)

where r is the interspace width, cX is the concentration and vx is the fluid velocity at any point x between 0 and L. If v is known as a function of x, then the equations can be solved in the following way: jw is a fraction of the total water flow Jw across 1 cm2 epithelium, and is set to a particular value between 0 and Jw; a value of CO is chosen, say 0.9 Cp, and the sucrose flux is calculated from equation (1) with o- = 0; the secreted concentration CL is then calculated as

CL = is/jw (3)

because at the end of the interspace dCldx must be zero; with this boundary condition (C at x = L) and a suitable expression for v as a function of distance x down the interspace, equation (2) is integrated to give a concentration profile and hence CO. This will in general be different from the value of Co initially chosen, and so the whole process is repeated with a new value of CO until the




junction itrpc

[> ci d ri

n~~~~

.U Lc

O CL 0 2

distance along paracellular pathway FIGURE 3. The geometry of the paracellular pathway with an explanation of some of the

symbols used in equations 1-3. The average concentration c in the junction is taken to be the mean of Cp and Co. CL is the specific secreted concentration of sucrose. I is the linear extent of interspace normal to the page (not shown).

08 /

04-

0 0.2 0.4 0.6 0.8 1.0 distance along interspace

FIGUR:E 4. The velocity profile along an interspace as described by equation (4). The flow is relative to that which emerges from the interspace, the length of which is dimension- less. (a) even distribution of pumping over the interspace; (b) confinement of pumping to the first 10 % of the interspace; (e) similar to (b) but with half the water entering the interspace via the junction by an unspecified mechanism.




two values match. The value of CL from the final integration is the specific secreted concentration, if CQ = 1.0. The effects on the specific secreted concentration CL, of varying the fraction of water j,4Jw flowing across the junction can then be in- vestigated, along with variations in any other parameter such as r.

Velocity profiles in the interspaces

Initially therefore, it is necessary to arrive at an expression for the velocity profile down the interspace. If water is entering the channel by standing-gradient osmosis then the velocity profile is predetermined: it is given by the equation

N?j ii'r2 d?ir dv _r2 IdV\2 Cr ~~~ 11=0 4 +iP dz3- p dx2- 27 dxz: P(dxz

=? 4

in which osmotically active solute at a concentration C in the cells is pumped at a rate N over the lateral intercellular membranes whose osmotic permeability is P, into the interspace of width 2 r (Diamond & Bossert I967). A study of the osmolarity of the secreted fluid by the Necturus gall bladder shows that it is isotonic with the perfusion fluid to within 1 % (Hill & Hill I978); equation (4) can therefore be solved for the case where the emergent osmolarity is 1.01 C, at any particular value of N. The osmotic permeability required to do this depends upon the value of C and r chosen, and the interspace length, but this does not concern us here for the moment. The result shows that although the magnitude of the velocity depends upon the dimensions of the system, the shape of the velocity curve does not; when dv/dx is zero at the end of the interspace, as must occur in any system such as the 'unilateral' gall bladder preparation which is bathed by its own secretion, all the curves are isomorphs. Diamond & Bossert (i967) considered the case where pumping of salt was confined to the first tenth of the interspace, and the solution of this gives a curve that is somewhat more hyperbolic than the one obtaining when salt is pumped along the whole interspace, as might be expected. The two curves in a normalized form are shown in figure 4a and b. If the salt pumping is confined to an even shorter initial length of the interspace the velocity profile is initially very slightly steeper than curve (b) in figure 4, quite apart from the improbability of such a situation.

For a given water flow leaving the interspaces, the velocity at any depth of the interspace can therefore be described; other velocity profiles could be sought, but to be initially steeper in form they would require that the osmotic permeability of the lateral interspace membranes be both discontinuous and very high. This point will be discussed below. For the purpose of the calculations the curves have been fitted by fourth-order polynomials, and the shape of curve (b), figure 4, for instance, is identical to that calculated by Diamond & Bossert (I967). When a fraction jw of the total water flow is made to cross the junction the profile of the velocity curve is attenuated but begins at a higher initial velocity, figure 4, curve (c). This profile in fact reflects the osmotic equilibration properties of the interspace downstream from the junction.




Specific secreted concentration of sucrose

In figure 5 are plotted curves showing the effect of diverting water flow via the junction on the specific secreted concentration of sucrose, by using the expression for the velocity profile from figure 4, curve (a) in which pumping is evenly distributed along the whole interspace membranes. The interspace width is 0.02 gm.

?0.8 -

0 20 40 60 80 100 junctional water flow as a percentage of the total

FIGURE 5. The specific secreted concentration as a function of the fraction of water crossing the junctions. Total water flow rate = 2.78 x 10-6 cm3 s-1 cmn2 epithelium, r = 0.02 gm, d = 6 nm; (a) free diffusion coefficient for sucrose in the junctions; (b) one-tenth free diffusion coefficient for sucrose in the junction. Solute pumping in the interspaces evenly distributed.

The two curves represent sets of solutions obtained by using two different values for the diffusion coefficient of sucrose in the junction, corresponding to free diffusion and one-tenth of the free diffusion value. It is apparent that to reach the values observed for the specific secreted concentration, the fraction of water crossing the junction must be between 90 and 100 % of the water crossing the epithelium.

The junctions are unlikely to be simple open slits, if for no other reason than that there must be some structural entity that apparently fixes the intermembrane distance at this fairly constant value. The only measurements that can shed light on this are those of the electrical conductance of the junctions. The paracellular pathway in 1 cm2 of Necturus gall bladder has a resistance of approximately 300 Q, (Fromter I972) but it is not clear how much of this is contributed by the inter-

spaces, and how much by the junctions. Rough estimates of the interspace resistance put its contribution as high as 100 Q but it is probably less; if the

junction width is 6 nm and its length is 0.7 gm, then the total junctional resistance

per square centimetre epithelium should be 9 Q if the junction is filled with 0.1 m NaCl solution. This could be reconciled with the observed resistance if only about 5 % of the cross-sectional area of the junction is available for diffusion. In




view of this, a diffusion coefficient of one tenth that in free solution (0.52 x 10-5

cm2/s) in the junction is a reasonable estimate, while a free solution value would be unrealistic. Figure 5 therefore shows that an interspace width of 0.02 Am, which would be the dimensions required to produce a quasi-isotonic solution by standing-gradient osmosis in the interspaces, leads to the conclusion that most of the water crosses the junctions.

1.0 a

2o0.8 - c

0.6

() 20 40 60 80 100 junctional water flow as a percentage of the total

FIGURE 6. The specific secreted concentrations as a function of the fraction of water crossing the junctions. Total water flow rate = 2.78 x O-6 cm3 S-' cm-2 epithelium, r = 0.5 ,m; solute pumping in the interspaces confined to one-tenth of the length. (a), (b), d 8 nm and 6 nm, respectively, sucrose diffusion coefficient in the junctions that of free solution; (c), (d), d = 8 nm and 6 nm, respectively, sucrose diffusion coefficient in the junctions one-tenth that of free solution.

Figure 6 shows the same relation as figure 5 but with different values of some of the parameters. The curves are for velocity profiles in the case where pumping of salt is confined to the first tenth of the interspace. Again a substantial fraction of the water can be seen to flow across the junctions, although the interspace width is now 0.5 ,um. Where the fraction of water traversing the junctions is high, it can be seen that changes in junction width have only a small effect, and this is due to the fact that diffusion of solute across the junction is contributing little to the flow.

Figure 7 shows the effect of varying the water flow rate. This is a variable which showed quite a spectrum of values, i.e. the secretion rate showed an eightfold variation within the bladders used. The actual variation in specific secreted concentration was reasonably small however, and if we discount the possibility that the bladders all secreted at the same rate but for different periods of time, then it must be apparent that where figure 7 is concerned, the junction water flow must have been high or else the variation in the specific secreted sucrose concentration would have been considerable.




Junctional, fow and coupling efficiency

A standing-gradient system can be characterized by stating its coupling efficiency; this is quite simply the fractional equilibration produced by the system. For example, if an amount of salt S is pumped into the interspace then 100 % equilibration is achieved when an amount of water is drawn osmotically into the

1.0 ..

08 -/

0 20 40 60 80 100 junctional water flow as a percentage of the total

FIGURE 7. The specific secreted concentration as a function of junctional water flow. The overall water flow is shown as (a) 1.0 x 10-6, (b) 2.78 x 10-6, and (C) 7.0 x 10-6 cm3 s-i

cm2 epithelium, while the solute pumping in the interspaces is evenly distributed. r 0.02 ,um, d = 60 nm, sucrose diffusion coefficient in the junctions equal to that of free solution.

interspace, equal to S/C where C is the basal concentration bathing the membranes; in reality the amount will be SIC, where C, is the concentration of fluid emerging from the interspace, and so the coupling efficiency of the interspace is C/Ct. For the Necturus gall bladder the overall coupling efficiency of the paracellular pathway (junction and interspace) is 1/1.01, i.e. 0.9901. We can partition the water flow into a fraction crossing the junctions y, and a fraction crossing the interspace membranes (1 - y). When x is the coupling efficiency of the interspace then

Y+(1-y)x- 0.9901

which has been plotted in figure 8. The interesting point of this exercise is to show how critical is the dependence of junctional water flow upon coupling efficiency in the interspace. Unless the interspace efficiency is very high indeed, water flow is practically all junctional; at an interspace coupling efficiency of 0.9 for example, 91 % of the water flow would have to be junctional to explain the observed fluid flow. This has a direct consequence for leaky epithelia: the difference between a coupling efficiency of 0.9 and 0.99 is fairly marginal in terms of the widths of




interspaces in various secretory epithelia, while at the same time these widths are undoubtedly varying with osmolarity and flow rate; consequently, any detailed discussion as to the precise dimensions of interspaces is not likely to rule out the possibility of substantial junctional water flow in these systems.

0l98

.0.94\

0

Co\

0.92

0.90 L 0 20 40 60 80 100

junctional water flow as a percentage of the total

FIGURE 8. The junctional water flow required to compensate for lower efficiencies of interspace coupling. The maximum efficienicy is 0.99 if the secreted fluid is produced solely by standing gradients in the interspaces of Necturus gall bladder epithelium.

The inability of standing-gradient osmosis to explain fluid production at low osmolarities in the Necturus gall bladder (Hill & Hill I978), is complemented in this paper by the demonstration that a study of sucrose entrainment in the fluid secretion requires that between 90 and 100 % of the water flow be across the junctions.

This is not envisaged in standing-gradient theory, with respect to which Diamond (1971) has stated 'we do not know whether some water as well flows directly across the tight junctions, though sequential equilibration along the lateral spaces via the cells would still be essential for obtaining an isotonic transported fluid'.

In both Necturus proximal tubule (Sackin & Boulpaep I975), and rabbit corneal endothelium (Lim & Fischbarg 1976; Fischbarg I977) it has been shown that standing-gradient osmotic theory fails to explain the production of an isotonic secretion, and consequently water must be flowing over the junctions. This was first mooted by Whittembury (I967).

Junctional reflexion coefflcients It has been assumed that osmosis is responsible for the juncltional flow, although

a consideration of the junctional route in terms of osmotic permeabilities shows that junctional osmosis is unlikely to account for isotonic fluid secretion (Hill 1975); the hydraulic conductivities of junctions may be quite high, but unless




their reflexion coefficients are high too, the osmotic permeabilities will be low. In this study, the reflexion coefficient of sucrose can be calculated from equation (1) when the diffusion coefficient in the junctions is known; assuming a restricted diffusion coefficient of one-tenth that of free solution, and a specific secreted concentration of 0.98, the reflexion coefficient comes out to be 0.05.

Obviously the reflexion coefficient of the junctions for NaCl is now the single most important unknown parameter in the theory of salt-water coupling in leaky epithelia, and its magnitude will decide whether it is possible to achieve isotonic secretion by osmotic flow across the junctions.

We should like to thank Professor R. D. Keynes, F.R.S., for his help and for extending departmental facilities to B.S. H., and Dr G. Whittembury for discussion and criticism of the results.

REFERENCES

Berry, C. A. & Boulpaep, E. L. 1975 Nonelectrolyte permeability of the paracellular pathway in Necturus proximal tubule. Am. J. Physiol. 228, 581-595.

Diamond, J. M. I964 The mechanism of isotonic water transport. J. gen. Physiol. 48, 15-42. Diamond, J. M. I97I Water-solute coupling and ion selectivity in epithelia. Phil. Trans. R

Soc. Lond. B 262, 141-151. Diamond, J. M. 1974 Tight and leaky junctions of epithelia: a perspective on kisses in the

dark. Fedn Proc. 33, 2220-2224. Diamond, J. M. & Bossert, W. H. I967 Standing gradient osmotic flow: a mechanism for

coupling of salt and water transport in epithelia. J. gen. Physiol. 50, 2061-2083. Fischbarg, J. 1977 Fluid transport by corneal endothelium. Proceedings of the III Inter-

national Conference on Comparative Physiology. (ed. K. Schmidt-Nielsen, L. Bolis & S. H. P. Maldnell.) Cambridge University Press (in the press).

Fromter, E. F. 1972 The route of passive ion movement through the epithelium of Necturus gallbladder. J. Membrane Biol. 8, 259-301.

Hill, A. E. 1975 Solute-solvent coupling in epithelia: contribution of the junctional pathway to fluid production. Proc. R. Soc. Lond. B 191, 537-547.

Hill, B. S. & Hill, A. E. 1978 Fluid transfer by Necturus gall bladder epithelium as a function of osmolarity. Proc. R. Soc. Lond. B 200, 151-162.

Kedem, 0. & Katchalsky, A. 1958 Thermodynamic analysis of the permeability of biological membranes to non-electrolytes. Biochim. Biophys. Acta 27, 229-246.

Lim, J. J. & Fischbarg, J. 1976 Standing gradient osmotic flow. Examination of its validity using an analytical method. Biochimi, Biophys. Acta 443, 339-347.

Perez-Gonzalez, M. & Whittembury, G. 1974 Widening of the paracellular pathway in the kidney tubule by a transtubular osmotic gradient. Pflugers Archiv 351, 1-12.

Sackin, H. & Boulpaep, E. I. 1975 Models for coupling of salt and water transport: proximal tubular reabsorption in Necturus kidney. J. gen. Physiol. 66, 671-733.

Smulders, A. P. & Wright, E. M. 197I The magnitude of non-electrolyte selectivity in the gall bladder epithelium. J. Membrane Biol. 5, 297-318.

Ussing, H. H. I966 Anomalous transport of electrolytes and sucrose through isolated frog skin induced by hypertonicity of the outside bathing solution. Ann. N. Y. Acad. Sci. 137, 543-555.

Ussing, H. H. & Johansen, B. I969 Anomalous transport of sucrose and urea in toad skin. Nephron 6, 317-328.

Whittembury, G. I967 Sobre los mecanismos de absorcion en el tubo proximal del rinon. Acta cient. venez. Suppl. 3, 71-83.



sucrose fluxes and junctional water flow across necturus gall bladder epithelium

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