J. Physiol. (1979), 290, pp. 351-366 With 1 text-figure Printed in Great Britain
351
FLUID MOVEMENTS ACROSS RABBIT ILEUM COUPLED TO PASSIVE PARACELLULAR ION MOVEMENTS
BY G. D. HOLMAN* AND R. J. NAFTALIN From the Department of Physiology, King's College, Strand, London WC2R 2LS
(Received 14 June 1978) SUMMARY
1. Theophylline (10 mM) and choleragen (1 x 10-6 g ml.-') abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mm) and ethacrynate (0X1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0-1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P < 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0-1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl + ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer+ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-' Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choieragen-treated tissue gives rise to net fluid secretion. Present address: Department of Biochemistry, School of Biological Sciences, University of Bath, Bath. *
0022-3751/79/3270.0532 $01.50 © 1979 The Physiological Soeiety
352
G. D. HOLMAN AND R. J. NAFTALIN INTRODUCTION
Huss & Marsh (1975) and Sackin & Boulpaep (1975) have suggested that the presence of hypertonic fluid within the lateral intercellular spaces may cause net fluid and electrolyte uptake across the tight-junctions of transporting epithelia. Experimental results consistent with such amplification of both net solute and solvent movements via the paracellular pathway has been obtained from work with Necturws proximal tubules (Berry & Boulpaep, 1975) and rabbit ileum (Simmons & Naftalin, 1976b). Amplification of solvent flow across the paracellular pathway implies that the hypertonic solute within the lateral intercellular space can exert an osmotic pressure across the tight-junction and the reflexion coefficient of the junction towards the major solute species within the lateral intercellular space is above zero. Yet it has been repeatedly observed that the paracellular pathways of loose epithelia, as well as having a high electrical conductivity also are permeable to substances of relatively high molecular weight, e.g. sucrose, inulin and polyethylene glycol (Wright, Smulders & Tormey, 1972; Berry & Boulpaep, 1975; Munck & Rasmussen, 1977; for review, see Ussing, Erlij & Lassen, 1974), which suggests that the permselectivity of the tight-junction is not based entirely on the size of the solute. There is considerable evidence to support the view that the tight-junction in loose epithelia are cationselective (Fromter, 1972; Fromter & Diamond, 1972; Frizzell & Schultz, 1972; Moreno & Diamond, 1975). A recent study of ion concentration gradients within the lateral intercellular spaces of actively absorbing rabbit ileum has revealed a steep increase in salt concentration from the junctional end towards the middle of the lateral intercellular space which indicates that there is a substantial rate of fluid flow into the lateral intercellular space via the tight-junction. It follows that the solute within it must exert an osmotic pressure across the junction and hence that the reflexion coefficient of the junction towards NaCl is above zero (Gupta, Hall & Naftalin, 1978). In the previous paper (Naftalin & Simmons, 1979), it was shown that triaminopyrimidine, an agent which blocks monovalent cation transport via the tightjunction (Moreno, 1975), prevents theophylline and choleragen from inhibiting net Na+ absorption across rabbit ileum. From this, we deduced that the tight-junction might have a role in secretion as well as in absorption. The view that the paracellular pathway is an important factor in intestinal electrolyte secretion is supported by Powell's (1974) findings that cyclic AMP, theophylline and choleragen all increase the passive permeability of the tissue more to Clthan to Na+. Since passive transepithelial electrolyte movements are considered to be mainly paracellular (Frizzell & Schultz, 1972; Desjeux, Thai & Curran, 1974; Simmons & Naftalin, 1976b), Powell's finding implies that the permselectivity properties of the paracellular pathway are affected by intestinal secretagogues. In control rabbit ileum, the tight-junction is cation-selective (Frizzell & Schultz, 1972); hence increase in anion conductance should increase the total electrical conductance across the tight-junction (see the following paper); will increase the permeability of the junctions to neutral NaCl movement and also should reduce the reflexion coefficient of the junctions to salt (see Discussion). This salt leakage could also induce fluid secretion as a consequence of electro-
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 353 osmotic coupling of solute and fluid movement within the tight-junction (see Discussion). Electro-osmotic fluid secretion is dependent on an asymmetrical distribution of NaCl across the junction normally maintained by Na-pump activity. A simple test of the validity of this mode of secretion is to determine the effects of reversal of the normally existing concentration gradients across the junction on net fluid movements across the tissue. It is to be expected that if the junction in the presence of theophylline, or choleragen, has a negative reflexion coefficient, that fluid will move from the side containing hypertonic NaCi towards the isotonic solution. If the Na pump is inhibited with ouabain, so that hypertonic NaCl is no longer sequestered within the lateral intercellular space and hypertonic NaCl is now placed in the mucosal bathing solution and isotonic NaCl Ringer in the serosal solution; in control tissues, net fluid flow will be from the serosal towards the mucosal solution, whereas with theophylline, or choleragen present, flow in the opposite direction would be predicted. This paper will describe the results of this test and also the effects of modifiers of theophylline and choleragen-induced net electrolyte secretion on active and passive movements across rabbit ileum in vitro. METHODS
(1) Water flux in transporting tissue Rabbit ileum was everted and made in sacs using the standard Wilson-Wiseman technique (Wilson & Wiseman, 1954). The sacs were approximately 3-0 cm long and weighed approximately 1 g. Serosal solution (1 ml.) was placed within the sacs. After 30 min preincubation in 15 ml. mucosal bathing solution maintained at 37 0C and gassed with 95 % 02: 5 % C02, the sacs were carefully blotted to remove surface water and weighed with their contents on a torsion balance to the nearest milligram and then returned to the incubation flask. Two more weighings at 30 min intervals were made (or at 30 min intervals for the next 2-5 hr in the choleragen experiment), and the water flux was calculated as follows:
(wt. sac at time, t1 - wt. sac at initial time to) x 60 (wet wt. of sacs-contents) (t1-to)(min) (2) Water fluxes in ouabain poisoned tissue Several different experiments were carried out with sacs prepared and incubated as above.
(3) Water flux in the presence of a NaCi gradient The mucosal solution contained hypertonic (200 mM) NaCl Ringer solution + 0.1 mM-ouabain and drugs indicated in the Results section. The serosal solution contained NaCl (140 mM) Ringer solution + 0-1 mM-ouabain and the drugs indicated in the Results section. After 30 min incubation an initial weighing was made. This was followed by another after a further 30 min incubation. The water flux was calculated from the weight difference between the two readings and the wet weight of the transporting tissue, less contents. Various substitutions were made using the ions indicated in the section describing the solutions. The ion gradient was established by using hypertonic mucosal and isotonic (serosal) solutions containing 0*1 mM-ouabain. In another series of experiments water flux across the unstripped epithelium was measured with isotonic mannitol (280 mm) Ringer in the serosal solution. The mucosal solutions contained 200 min-NaCl Ringer + 0-1 mi-ouabain and drugs indicated in the Results section. The serosal solution contained isotonic mannitol Ringer + 0 1 mM-ouabain and the drugs indicated in the Results section. Generally three weighings were made during I2
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each incubation. All the waters fluxes g-1 wet of tissue were converted to flux cm-2 by multiplying by 23-7 (see below).
(4) Net unidirectional Na+ and Cl- fluxes acro88 tissue treated with 0-1 mM-ouabain These flux determinations were carried out in the Ussing-type chambers. The bathing solutions on the mucosal and serosal surfaces of the tissue were maintained at 37 0C using a circulating water-bath as described previously (Naftalin & Holman, 1974). 177cm2 serosal surface area of tissue was exposed to the solutions. The weight of exposed tissue was approximately 0-075 g. This figure was used to convert water flux g-1 wet weight of tissue to water flux per cm-2. 7 ml. solution was added to the mucosal and serosal chambers and gassed with 95 % 02, 5 % Co2. After 30 min incubation, samples were taken for radioactivity counting from both solutions. Two further samples were taken at 30 min intervals thereafter. The mucosal bathing solution contained hypertonic electrolyte solution + 0- 1 mM-ouabain, the serosal solution contained isotonic mannitol Ringer + 0. 1 mM-ouabain. The drugs were added, as indicated in Results, in equal concentrations to the mucosal and serosal bathing solution, either 22Na or 6Cl, obtained from the Radiochemical Centre, Amersham, were added to the mucosal solution at the beginning of incubation. Samples of the radioactive solutions were counted using a Packard Tribarb liquid scintilation counter using 0-25 % PPO in toluene/ triton x 100 (1: 1) w/v as scintillant.
(5) Solutions NaCl Ringer: both hypertonic (200 mM) and isotonic (140 mM) NaCl Ringer contained 10 mMKHCO3, 2-4 mM-K2HPO4, 0-4 mM-KH2PO4, 1-2 mM-CaSO4, 1-2 mM-MgSO4. Mannitol-Ringer: this contained 280 mM-mannitol, 10 mM-KHCO3, 2-4 K2HPO4, 0-4 mM-KHPO4, 1.2 MM-CaSO4 and 1-2 mM-MgSO4. Isethionate Ringer: 140 mM-Na isethionate replaces 140 mM-NaCl. (5) Materials
2,4,6-Triaminopyrimidine was obtained from Aldrich Chemicals, Co. Inc., Milwaukee, U.S.A., ethacrynate from Merck, Sharpe & Dohme, Hoddesdon, Herts. All other materials were obtained as Analar grade, from B.D.H., Poole, Dorset, or Sigma Chemicals Ltd., Fancy Rd., Poole, Dorset. Choleragen was purchased from Schwartz Mann, Biochemicals, York House, Empire Way, Wembley, London. RESULTS
A (i) Reversal of active fluid absorption induced by theophylline and choleragen in everted sacs of rabbit ileum 10 mM-theophylline, when added to both the mucosal and serosal solutions, prevents net fluid absorption and occasionally causes net fluid secretion. However, on averaging the data, no net flux in either direction is obtained (Table 1). The action of theophylline can be detected during 1 hr exposure of the intestinal sacs to the drug, whereas the effect of 1 Fug ml.-' pure choleragen is not observed until 90 min of incubation has elapsed. This is consistent with the time course for the effect of the toxin on electrolyte movements (Naftalin & Simmons, 1979). During this necessarily prolonged incubation, the fluid absorption in control falls to zero. Secretion, considered as a decrement in sac weight, begins in choleragen-treated tissue after 90 min
(Fig. 1). (ii) Effects of triaminopyrimidine on fluid movements in sacs of rabbit ileum 20 mM-triaminopyrimidine, which blocks paracellular cation movements (Moreno, 1975; Simmons & Naftalin, 1976b), reduces the rate of fluid absorption in control tissue within the first hour by approximately 50 %, when it is added to both mucosal
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 355 and serosal solutions (P < 0.005). However, no decline in the rate of fluid absorption in tissues treated with triaminopyrimdine is seen during 2-5 hr incubation (Fig. 1). The decrease in the rate of fluid absorption after 90 min incubation in controls may be due to spontaneous changes in the permselectivity of the paracellular shunt. When triaminopyrimidine is present together with either theophylline or choleragen, no significant difference in the rate of fluid absorption is observed in comparison to absorption in sacs exposed to triaminopyrimidine alone (Table 1 and Fig. 1). Thus, triaminopyrimidine prevents fluid secretion induced by theophylline, or choleragen in rabbit small intestine, just as it prevents the effects of these agents on net Na+ movements (see previous paper, Naftalin & Simmons, 1979). TABiE 1. Fluid transfer measured across everted sacs of rabbit ileum in which serosal and mucosal bathing solutions are identical. Control conditions have isotonic Ringer as the bathing solution
Jnv
Condition Control Theophylline (10 mM) Triaminopyrimidine (20 mM) Triaminopyrimidine + theophylline Isethionate Ringer Isethionate + theophylline Ethacrynate (0.1 mm) Ethacrynate + theophylline Ouabain (0 1 mm) Ouabain + theophylline
(,Ul cm-2 hr-1 +
Significance,
Significance,
cf. control (1) P
of. theophylline (2) P < 0*001
s.E. of mean) 19-01 + 2-54 1-22+ 1-72 8-50+ 1-01
11
8 8
< 0-001
9-36 ± 2-28
5
< 0-005
13-68 + 0-72 12-36 + 2-12
3
n.s.
3
n.s.
< 0-01 < 0-01
13-61 ± 2-39
9
n.s.
< 0-001
13-25+ 1-86
3
n.s.
< 0-01
1.44 + 0-68
6 5
< 0-001
n.s. n.s.
-1-11+ 1-02
n
< 0-005
< 0-001
< 0-01
05
E~Ig
~Tap, Tap+ Ch
L
0Q2 0-
01
yA
60
Cholera
90
180 150 120 Time (min) Fig. 1. The time-dependent changes in net fluid movements across sacs of everted rabbit ileum expressed as the total weight increment of the sac per gram wet weight
of transporting tissue. * *-, Control (C) (6); *-*, triaminopyrimidine (20 mM) present in both mucosal and serosal bathing solutions (3); 0-0O choleragen (1 jig ml.-I) present in the Ringer (4); 0r-O- choleragen (1 jig ml.-') +triaminopyrimidine (20 mM) present in the bathing solutions (4). Numbers in parentheses refer to number of experiments. 12-2
356
G. D. HOLMAN AND R. J. NAFTALIN
(iii) Effects of 0.1 mM-ethacrynate on fluid movements in rabbit ileum Carpenter, Curlin & Greenough (1969) have shown that ethacrynate prevents cholera toxin-induced fluid secretion in dog intestine in vivo and Al-Awqati, Field & Greenough (1974) have found that 0-1 mM-ethacrynate (a concentration too low to inhibit the Na+ pump) prevents net secretion of Cl- and Na+ induced by choleragen, cyclic AMP or theophylline. These findings were confirmed in the previous paper (Naftalin & Simmons, 1979). In Table 1 it can be seen that 0.1 mM-ethacrynate prevents the theophyllineinduced reduction of net fluid absorption by rabbit ileum, confirming the earlier result of Carpenter et al. (1969).
(iv) The effects of replacing Ringer Cl- on the theophylline-induced fluid secretion in rabbit ileum In the previous paper, it was shown that with Cl- absent from the bathing solution, both theophylline and choleragen fail to reduce net Na+ absorption. Fluid absorption is also unaffected by theophylline when isethionate entirely replaces Ringer Cl(Table 1). B (1) Modification of osmotically induced fluid movements across rabbit ileum It is well known that the paracellular pathway is the major route for electrical potential-induced movements of ions (Frizzell & Schultz, 1972) across intestine and also other loose epithelia (Moreno & Diamond, 1975). Since theophylline and choleragen have been shown to increase the passive mobility of Cl- within the paracellular pathway (Powell, 1974 and the following paper) it is of interest to determine whether these agents also effect osmotically induced fluid movements across rabbit ileum. The method adopted here to observe these passive effects involves inhibition of all active fluid movements by adding 0.1 mM-ouabain to both mucosal and serosal bathing solutions. This concentration is sufficient to abolish the hypertonicity of the extracellular compartment within the tissue (Simmons & Naftalin, 1976a; Gupta et al. 1978) and to abolish both active absorption and secretion of fluid (Table 1). The everted sacs of rabbit ileum were filled with isotonic solution and placed in hypertonic NaCl Ringer as described in Methods. In controls it is observed that following inhibition of active absorption by ouabain, fluid moves from the isotonic serosal solution into the hypertonic mucosal solution (Table 2a), i.e. net passive secretion is observed. But with 10 mM-theophylline present in both mucosal and serosal solutions, there is a significant (P < 0-001) net passive absorption, i.e. fluid moves passively from the hypertonic solution into the isotonic solution. With 1 jtg ml.-' choleragen net passive secretion is reduced (P < 0.005). Theophylline does not induce passive fluid absorption when Ringer Cl- is replaced by isethionate, or when triaminopyrimidine is present in both mucosal and serosal bathing solutions, or when Na+ is replaced by choline, or when 0 mM-ethacrynate is present in both bathing solutions. In fact, passive secretion is observed in these conditions. However when Na+ is replaced by Li+, theophylline abolishes net passive secretion (Table 2a). -
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 357 These results indicate that a mobile anion and cation pair are required for theophylline-dependent reversal of net fluid movement. Substitution of either a large organic anion, e.g. isethionate, or an immobile cation, e.g. choline, for Cl- and Na+ respectively, prevents the theophylline-induced reversal of osmotic flow. Since triaminopyrimidine prevents the theophylline-induced reversal of osmotic fluid movements, it can be deduced that the cation-selective channels within the tight-junction must be involved in control of the direction of fluid movement across the epithelium. TABLEz 2. (a) Net passive flux of fluid across everted sacs of rabbit ileum from hypertonic mucosal solutions containing 200 mm-NaCl salt solution into isotonic salt (Ringer) solution in the serosal solution. Ouabain (0.1 mM) is present in both solutions. + ve flux means net flux from mucosal to serosal side of the tissue and -ve flux is net flux from serosal-mucosal side Significance P Jr , (,Ul. cm-9 hr-L of. theophylline cf. control n Condition s.E. of mean) < 0-001 12 Control -4-03±1*02 < 0-001 12 Theophylline (10mm) +3-26±0.72 n.s. 6 -3.43 ± 127 Triaminopyrimidine (20 mM) < 0 005 n.s. 6 -1'95 ± 1-06 Triaminopyrimidine + theophylline < 0-025 4 - 8.73 + 0-72 Isethionate Ringer
351
FLUID MOVEMENTS ACROSS RABBIT ILEUM COUPLED TO PASSIVE PARACELLULAR ION MOVEMENTS
BY G. D. HOLMAN* AND R. J. NAFTALIN From the Department of Physiology, King's College, Strand, London WC2R 2LS
(Received 14 June 1978) SUMMARY
1. Theophylline (10 mM) and choleragen (1 x 10-6 g ml.-') abolish net fluid absorption by everted sacs of rabbit ileum. Triaminopyrimidine (20 mm) and ethacrynate (0X1 mM) prevent this inhibition of net fluid movement. Replacing Ringer Cl- with isethionate prevents the theophylline-dependent decrease in fluid absorption also. 2. Ouabain (0-1 mM) abolishes net fluid movements in both control and theophylline-treated tissue. 3. With ouabain present, hypertonic NaCl (200 mM) in the mucosal solution causes net fluid secretion (serosal-mucosal flux). With theophylline added to both the mucosal and serosal solution, net fluid absorption (mucosal-serosal flux) is observed (P < 0.001). Triaminopyrimidine (20 mM), or ethacrynate (0-1 mM), or replacement of Ringer Na+ with choline, or Ringer Cl- with isethionate all prevent the theophylline-induced reversal of osmotic flow. 4. Theophylline increases passive net flux of Na+ and Cl- from mucosal solution containing hypertonic (200 mM) NaCl + ouabain (0.1 mM) across sheets of ileum into serosal solution containing mannitol Ringer+ouabain. The increased passive Na+ flux is blocked by triaminopyrimidine and the increased Na+ and Cl- fluxes are blocked by ethacrynate (0.1 mM). 5. The suggested route of increased NaCl leakage is via the paracellular pathway as it is inhibited by triaminopyrimidine. The increase, itself, is a consequence of the increased passive permeability of the mucosal border to Cl-, induced by theophylline or choleragen. Water is apparently electro-osmotically coupled to the paracellular Na+ leakage (100 mole water mole-' Na+), hence increased passive leakage reverses osmotic flow. In active tissue the lateral intercellular space contains hypertonic NaCl, and hence increased leakage of NaCl across the tight-junction in theophylline or choieragen-treated tissue gives rise to net fluid secretion. Present address: Department of Biochemistry, School of Biological Sciences, University of Bath, Bath. *
0022-3751/79/3270.0532 $01.50 © 1979 The Physiological Soeiety
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G. D. HOLMAN AND R. J. NAFTALIN INTRODUCTION
Huss & Marsh (1975) and Sackin & Boulpaep (1975) have suggested that the presence of hypertonic fluid within the lateral intercellular spaces may cause net fluid and electrolyte uptake across the tight-junctions of transporting epithelia. Experimental results consistent with such amplification of both net solute and solvent movements via the paracellular pathway has been obtained from work with Necturws proximal tubules (Berry & Boulpaep, 1975) and rabbit ileum (Simmons & Naftalin, 1976b). Amplification of solvent flow across the paracellular pathway implies that the hypertonic solute within the lateral intercellular space can exert an osmotic pressure across the tight-junction and the reflexion coefficient of the junction towards the major solute species within the lateral intercellular space is above zero. Yet it has been repeatedly observed that the paracellular pathways of loose epithelia, as well as having a high electrical conductivity also are permeable to substances of relatively high molecular weight, e.g. sucrose, inulin and polyethylene glycol (Wright, Smulders & Tormey, 1972; Berry & Boulpaep, 1975; Munck & Rasmussen, 1977; for review, see Ussing, Erlij & Lassen, 1974), which suggests that the permselectivity of the tight-junction is not based entirely on the size of the solute. There is considerable evidence to support the view that the tight-junction in loose epithelia are cationselective (Fromter, 1972; Fromter & Diamond, 1972; Frizzell & Schultz, 1972; Moreno & Diamond, 1975). A recent study of ion concentration gradients within the lateral intercellular spaces of actively absorbing rabbit ileum has revealed a steep increase in salt concentration from the junctional end towards the middle of the lateral intercellular space which indicates that there is a substantial rate of fluid flow into the lateral intercellular space via the tight-junction. It follows that the solute within it must exert an osmotic pressure across the junction and hence that the reflexion coefficient of the junction towards NaCl is above zero (Gupta, Hall & Naftalin, 1978). In the previous paper (Naftalin & Simmons, 1979), it was shown that triaminopyrimidine, an agent which blocks monovalent cation transport via the tightjunction (Moreno, 1975), prevents theophylline and choleragen from inhibiting net Na+ absorption across rabbit ileum. From this, we deduced that the tight-junction might have a role in secretion as well as in absorption. The view that the paracellular pathway is an important factor in intestinal electrolyte secretion is supported by Powell's (1974) findings that cyclic AMP, theophylline and choleragen all increase the passive permeability of the tissue more to Clthan to Na+. Since passive transepithelial electrolyte movements are considered to be mainly paracellular (Frizzell & Schultz, 1972; Desjeux, Thai & Curran, 1974; Simmons & Naftalin, 1976b), Powell's finding implies that the permselectivity properties of the paracellular pathway are affected by intestinal secretagogues. In control rabbit ileum, the tight-junction is cation-selective (Frizzell & Schultz, 1972); hence increase in anion conductance should increase the total electrical conductance across the tight-junction (see the following paper); will increase the permeability of the junctions to neutral NaCl movement and also should reduce the reflexion coefficient of the junctions to salt (see Discussion). This salt leakage could also induce fluid secretion as a consequence of electro-
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 353 osmotic coupling of solute and fluid movement within the tight-junction (see Discussion). Electro-osmotic fluid secretion is dependent on an asymmetrical distribution of NaCl across the junction normally maintained by Na-pump activity. A simple test of the validity of this mode of secretion is to determine the effects of reversal of the normally existing concentration gradients across the junction on net fluid movements across the tissue. It is to be expected that if the junction in the presence of theophylline, or choleragen, has a negative reflexion coefficient, that fluid will move from the side containing hypertonic NaCi towards the isotonic solution. If the Na pump is inhibited with ouabain, so that hypertonic NaCl is no longer sequestered within the lateral intercellular space and hypertonic NaCl is now placed in the mucosal bathing solution and isotonic NaCl Ringer in the serosal solution; in control tissues, net fluid flow will be from the serosal towards the mucosal solution, whereas with theophylline, or choleragen present, flow in the opposite direction would be predicted. This paper will describe the results of this test and also the effects of modifiers of theophylline and choleragen-induced net electrolyte secretion on active and passive movements across rabbit ileum in vitro. METHODS
(1) Water flux in transporting tissue Rabbit ileum was everted and made in sacs using the standard Wilson-Wiseman technique (Wilson & Wiseman, 1954). The sacs were approximately 3-0 cm long and weighed approximately 1 g. Serosal solution (1 ml.) was placed within the sacs. After 30 min preincubation in 15 ml. mucosal bathing solution maintained at 37 0C and gassed with 95 % 02: 5 % C02, the sacs were carefully blotted to remove surface water and weighed with their contents on a torsion balance to the nearest milligram and then returned to the incubation flask. Two more weighings at 30 min intervals were made (or at 30 min intervals for the next 2-5 hr in the choleragen experiment), and the water flux was calculated as follows:
(wt. sac at time, t1 - wt. sac at initial time to) x 60 (wet wt. of sacs-contents) (t1-to)(min) (2) Water fluxes in ouabain poisoned tissue Several different experiments were carried out with sacs prepared and incubated as above.
(3) Water flux in the presence of a NaCi gradient The mucosal solution contained hypertonic (200 mM) NaCl Ringer solution + 0.1 mM-ouabain and drugs indicated in the Results section. The serosal solution contained NaCl (140 mM) Ringer solution + 0-1 mM-ouabain and the drugs indicated in the Results section. After 30 min incubation an initial weighing was made. This was followed by another after a further 30 min incubation. The water flux was calculated from the weight difference between the two readings and the wet weight of the transporting tissue, less contents. Various substitutions were made using the ions indicated in the section describing the solutions. The ion gradient was established by using hypertonic mucosal and isotonic (serosal) solutions containing 0*1 mM-ouabain. In another series of experiments water flux across the unstripped epithelium was measured with isotonic mannitol (280 mm) Ringer in the serosal solution. The mucosal solutions contained 200 min-NaCl Ringer + 0-1 mi-ouabain and drugs indicated in the Results section. The serosal solution contained isotonic mannitol Ringer + 0 1 mM-ouabain and the drugs indicated in the Results section. Generally three weighings were made during I2
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G. D. HOLMAN AND R. J. NAFTALIN
each incubation. All the waters fluxes g-1 wet of tissue were converted to flux cm-2 by multiplying by 23-7 (see below).
(4) Net unidirectional Na+ and Cl- fluxes acro88 tissue treated with 0-1 mM-ouabain These flux determinations were carried out in the Ussing-type chambers. The bathing solutions on the mucosal and serosal surfaces of the tissue were maintained at 37 0C using a circulating water-bath as described previously (Naftalin & Holman, 1974). 177cm2 serosal surface area of tissue was exposed to the solutions. The weight of exposed tissue was approximately 0-075 g. This figure was used to convert water flux g-1 wet weight of tissue to water flux per cm-2. 7 ml. solution was added to the mucosal and serosal chambers and gassed with 95 % 02, 5 % Co2. After 30 min incubation, samples were taken for radioactivity counting from both solutions. Two further samples were taken at 30 min intervals thereafter. The mucosal bathing solution contained hypertonic electrolyte solution + 0- 1 mM-ouabain, the serosal solution contained isotonic mannitol Ringer + 0. 1 mM-ouabain. The drugs were added, as indicated in Results, in equal concentrations to the mucosal and serosal bathing solution, either 22Na or 6Cl, obtained from the Radiochemical Centre, Amersham, were added to the mucosal solution at the beginning of incubation. Samples of the radioactive solutions were counted using a Packard Tribarb liquid scintilation counter using 0-25 % PPO in toluene/ triton x 100 (1: 1) w/v as scintillant.
(5) Solutions NaCl Ringer: both hypertonic (200 mM) and isotonic (140 mM) NaCl Ringer contained 10 mMKHCO3, 2-4 mM-K2HPO4, 0-4 mM-KH2PO4, 1-2 mM-CaSO4, 1-2 mM-MgSO4. Mannitol-Ringer: this contained 280 mM-mannitol, 10 mM-KHCO3, 2-4 K2HPO4, 0-4 mM-KHPO4, 1.2 MM-CaSO4 and 1-2 mM-MgSO4. Isethionate Ringer: 140 mM-Na isethionate replaces 140 mM-NaCl. (5) Materials
2,4,6-Triaminopyrimidine was obtained from Aldrich Chemicals, Co. Inc., Milwaukee, U.S.A., ethacrynate from Merck, Sharpe & Dohme, Hoddesdon, Herts. All other materials were obtained as Analar grade, from B.D.H., Poole, Dorset, or Sigma Chemicals Ltd., Fancy Rd., Poole, Dorset. Choleragen was purchased from Schwartz Mann, Biochemicals, York House, Empire Way, Wembley, London. RESULTS
A (i) Reversal of active fluid absorption induced by theophylline and choleragen in everted sacs of rabbit ileum 10 mM-theophylline, when added to both the mucosal and serosal solutions, prevents net fluid absorption and occasionally causes net fluid secretion. However, on averaging the data, no net flux in either direction is obtained (Table 1). The action of theophylline can be detected during 1 hr exposure of the intestinal sacs to the drug, whereas the effect of 1 Fug ml.-' pure choleragen is not observed until 90 min of incubation has elapsed. This is consistent with the time course for the effect of the toxin on electrolyte movements (Naftalin & Simmons, 1979). During this necessarily prolonged incubation, the fluid absorption in control falls to zero. Secretion, considered as a decrement in sac weight, begins in choleragen-treated tissue after 90 min
(Fig. 1). (ii) Effects of triaminopyrimidine on fluid movements in sacs of rabbit ileum 20 mM-triaminopyrimidine, which blocks paracellular cation movements (Moreno, 1975; Simmons & Naftalin, 1976b), reduces the rate of fluid absorption in control tissue within the first hour by approximately 50 %, when it is added to both mucosal
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 355 and serosal solutions (P < 0.005). However, no decline in the rate of fluid absorption in tissues treated with triaminopyrimdine is seen during 2-5 hr incubation (Fig. 1). The decrease in the rate of fluid absorption after 90 min incubation in controls may be due to spontaneous changes in the permselectivity of the paracellular shunt. When triaminopyrimidine is present together with either theophylline or choleragen, no significant difference in the rate of fluid absorption is observed in comparison to absorption in sacs exposed to triaminopyrimidine alone (Table 1 and Fig. 1). Thus, triaminopyrimidine prevents fluid secretion induced by theophylline, or choleragen in rabbit small intestine, just as it prevents the effects of these agents on net Na+ movements (see previous paper, Naftalin & Simmons, 1979). TABiE 1. Fluid transfer measured across everted sacs of rabbit ileum in which serosal and mucosal bathing solutions are identical. Control conditions have isotonic Ringer as the bathing solution
Jnv
Condition Control Theophylline (10 mM) Triaminopyrimidine (20 mM) Triaminopyrimidine + theophylline Isethionate Ringer Isethionate + theophylline Ethacrynate (0.1 mm) Ethacrynate + theophylline Ouabain (0 1 mm) Ouabain + theophylline
(,Ul cm-2 hr-1 +
Significance,
Significance,
cf. control (1) P
of. theophylline (2) P < 0*001
s.E. of mean) 19-01 + 2-54 1-22+ 1-72 8-50+ 1-01
11
8 8
< 0-001
9-36 ± 2-28
5
< 0-005
13-68 + 0-72 12-36 + 2-12
3
n.s.
3
n.s.
< 0-01 < 0-01
13-61 ± 2-39
9
n.s.
< 0-001
13-25+ 1-86
3
n.s.
< 0-01
1.44 + 0-68
6 5
< 0-001
n.s. n.s.
-1-11+ 1-02
n
< 0-005
< 0-001
< 0-01
05
E~Ig
~Tap, Tap+ Ch
L
0Q2 0-
01
yA
60
Cholera
90
180 150 120 Time (min) Fig. 1. The time-dependent changes in net fluid movements across sacs of everted rabbit ileum expressed as the total weight increment of the sac per gram wet weight
of transporting tissue. * *-, Control (C) (6); *-*, triaminopyrimidine (20 mM) present in both mucosal and serosal bathing solutions (3); 0-0O choleragen (1 jig ml.-I) present in the Ringer (4); 0r-O- choleragen (1 jig ml.-') +triaminopyrimidine (20 mM) present in the bathing solutions (4). Numbers in parentheses refer to number of experiments. 12-2
356
G. D. HOLMAN AND R. J. NAFTALIN
(iii) Effects of 0.1 mM-ethacrynate on fluid movements in rabbit ileum Carpenter, Curlin & Greenough (1969) have shown that ethacrynate prevents cholera toxin-induced fluid secretion in dog intestine in vivo and Al-Awqati, Field & Greenough (1974) have found that 0-1 mM-ethacrynate (a concentration too low to inhibit the Na+ pump) prevents net secretion of Cl- and Na+ induced by choleragen, cyclic AMP or theophylline. These findings were confirmed in the previous paper (Naftalin & Simmons, 1979). In Table 1 it can be seen that 0.1 mM-ethacrynate prevents the theophyllineinduced reduction of net fluid absorption by rabbit ileum, confirming the earlier result of Carpenter et al. (1969).
(iv) The effects of replacing Ringer Cl- on the theophylline-induced fluid secretion in rabbit ileum In the previous paper, it was shown that with Cl- absent from the bathing solution, both theophylline and choleragen fail to reduce net Na+ absorption. Fluid absorption is also unaffected by theophylline when isethionate entirely replaces Ringer Cl(Table 1). B (1) Modification of osmotically induced fluid movements across rabbit ileum It is well known that the paracellular pathway is the major route for electrical potential-induced movements of ions (Frizzell & Schultz, 1972) across intestine and also other loose epithelia (Moreno & Diamond, 1975). Since theophylline and choleragen have been shown to increase the passive mobility of Cl- within the paracellular pathway (Powell, 1974 and the following paper) it is of interest to determine whether these agents also effect osmotically induced fluid movements across rabbit ileum. The method adopted here to observe these passive effects involves inhibition of all active fluid movements by adding 0.1 mM-ouabain to both mucosal and serosal bathing solutions. This concentration is sufficient to abolish the hypertonicity of the extracellular compartment within the tissue (Simmons & Naftalin, 1976a; Gupta et al. 1978) and to abolish both active absorption and secretion of fluid (Table 1). The everted sacs of rabbit ileum were filled with isotonic solution and placed in hypertonic NaCl Ringer as described in Methods. In controls it is observed that following inhibition of active absorption by ouabain, fluid moves from the isotonic serosal solution into the hypertonic mucosal solution (Table 2a), i.e. net passive secretion is observed. But with 10 mM-theophylline present in both mucosal and serosal solutions, there is a significant (P < 0-001) net passive absorption, i.e. fluid moves passively from the hypertonic solution into the isotonic solution. With 1 jtg ml.-' choleragen net passive secretion is reduced (P < 0.005). Theophylline does not induce passive fluid absorption when Ringer Cl- is replaced by isethionate, or when triaminopyrimidine is present in both mucosal and serosal bathing solutions, or when Na+ is replaced by choline, or when 0 mM-ethacrynate is present in both bathing solutions. In fact, passive secretion is observed in these conditions. However when Na+ is replaced by Li+, theophylline abolishes net passive secretion (Table 2a). -
PASSIVE ION AND FLUID MOVEMENTS ACROSS ILEUM 357 These results indicate that a mobile anion and cation pair are required for theophylline-dependent reversal of net fluid movement. Substitution of either a large organic anion, e.g. isethionate, or an immobile cation, e.g. choline, for Cl- and Na+ respectively, prevents the theophylline-induced reversal of osmotic flow. Since triaminopyrimidine prevents the theophylline-induced reversal of osmotic fluid movements, it can be deduced that the cation-selective channels within the tight-junction must be involved in control of the direction of fluid movement across the epithelium. TABLEz 2. (a) Net passive flux of fluid across everted sacs of rabbit ileum from hypertonic mucosal solutions containing 200 mm-NaCl salt solution into isotonic salt (Ringer) solution in the serosal solution. Ouabain (0.1 mM) is present in both solutions. + ve flux means net flux from mucosal to serosal side of the tissue and -ve flux is net flux from serosal-mucosal side Significance P Jr , (,Ul. cm-9 hr-L of. theophylline cf. control n Condition s.E. of mean) < 0-001 12 Control -4-03±1*02 < 0-001 12 Theophylline (10mm) +3-26±0.72 n.s. 6 -3.43 ± 127 Triaminopyrimidine (20 mM) < 0 005 n.s. 6 -1'95 ± 1-06 Triaminopyrimidine + theophylline < 0-025 4 - 8.73 + 0-72 Isethionate Ringer
