J. Physiol. (1979), 290, pp. 331-350 With 5 text-figureM Printed in Great Britain

331

THE EFFECTS OF THEOPHYLLINE AND CHOLERAGEN ON SODIUM AND CHLORIDE ION MOVEMENTS WITHIN ISOLATED RABBIT ILEUM

BY R. J. NAFTALIN AND N. L. SIMMONS* From the Department of Physiology, King's College, Strand, London WC2R 2LS

(Received 14 June 1978) SUMMARY

1. Theophylline (10 mM) and choleragen change the direction of net Cl- movements across rabbit ileum, in the short-circuit current condition, from absorption to secretion. The specific activity ratio R of C1- tracers within the tissue coming from mucosal and serosal solutions respectively is increased, which is consistent with an increase in Cl- exchange flux across the mucosal border. 2. Net Na+ movement is also changed from net absorption to secretion by theophylline and choleragen; the specific activity ratio R of Na+ tracers is raised by theophylline. Because of the large paracellular component to transepithelial Na+ movements, an increase in Na+ exchange flux across the mucosal border is not detected. 3. 2,4,6-Triaminopyrimidine (20 mM) which has been previously shown to block paracellular Na+ movements, blocks both the theophylline and choleragen-dependent reversal of net Na+ movement by preventing the decrease in m-s Na flux. The theophylline-dependent increase in the ratio R of Na+ is still present, and is consistent with an increase in Na+ exchange flux across the mucosal border - unmasked by removal of the paracellular flux components. 4. Ouabain (0.1 mM) abolishes net absorption of Na+ and Cl- in control and net secretion of Na+ and Cl- in theophylline-treated tissue. Ouabain does not affect the theophylline-dependent increase in Cl- exchange across the mucosal border. 5. Replacement of Ringer Cl- with SO'- or Na+ by choline prevents the effects of theophylline and choleragen on Na+ and C1- fluxes respectively. 6. Ethacrynate (0 1 mM) prevents the theophylline-dependent effects on net Na+ movement. Raising ethacrynate to 0-2 mm abolishes the effects of theophylline on Cl- exchange. An interpretation of these results is that theophylline and choleragen raise the Cl- permeability of the brush border. This increases NaCl leakage from the hypertonic lateral intercellular space into the mucosal solution thereby causing secretion. The selective action of triaminopyrimidine and ethacrynate (0.1 mM) on Na+ flux indicates that Na+ and Cl- move via separate transport pathways across the mucosal border. * Present

address: Department of Physiology, St Andrews University, St Andrews, Fife.

0022-3751/79/3260-8531 $01.30 © 1979 The Physiological Society

332

R. J. NAFTALIN AND N. L. SIMMONS INTRODUCTION

Both theophylline and choleragen act upon the small intestine to change it from a tissue which absorbs fluid and electrolyte into one where fluid and electrolytes are actively secreted into the luminal, or mucosal bathing fluid. (For a recent review see Hendrix & Paulk, 1977.) It is considered that theophylline and choleragen-induced secretion results from a neutral NaCl secretary process which may be triggered by raised intracellular levels of cyclic AMP (Powell, Binder & Curran, 1972, 1973; Nellans, Frizzell & Schultz, 1973, 1974). However, Nellans et al. (1973, 1974) consider that net secretion of NaCl also arises from an inhibition by cyclicAMP of a brushborder influx pathway for NaCl. An alternative view proposed by Field (1971) and Field, Fromm, Al-Awqati & Greenough (1972) is that raised levels of intracellular cyclicAMP result in an active electrogenic Cl- secretion which is accompanied by passive Na+ movements. The evidence for this view is that in the presence of theophylline, choleragen, or exogenous cyclicAMP an increase in the transepithelial p.d., short-circuit current and also flux measurements, in the short-circuit current condition, indicate that net Cl- secretion but no net Na+ secretion occurs. Replacement of Ringer Cl- by S02-, or replacement of Ringer Na+ by choline prevents theophylline or choleragen from inducing changes in net Na+ or Cl- movements respectively (Nellans et al. 1973, 1974), thus indicating that both ions are required before the secretary process is observed. Furthermore, Powell et al. (1973) and Nellans et al. (1973, 1974) have shown that the reduction in net Na+ absorption is equal to the change in net Cl- flux following addition of theophylline. This 1: 1 stoichiometry in the theophylline-dependent change in Na+ and Cl- net movements, whilst being compatible with Field's view that Cl- is actively secreted, is more consistent with the view that the secretary process is neutral. In a previous paper (Simmons & Naftalin, 1976b), we showed that estimates of the steady-state Na+ movements across the mucosal and serosal borders of rabbit ileum could be obtained by using a double label isotope method which measures the bidirectional-transepithelial fluxes across rabbit ileum simultaneously and the specific activity ratio within the tissue during steady-state flux of isotopes originating from the mucosal and serosal bathing solutions. In order to eliminate paracellular Na+ movements, 2,4,6-triaminopyrimidine, which has been shown to block paracellular Na+ movements in a variety of tissues (Moreno, 1975) was used; this double label technique can be adapted to estimate the steady-state unidirectional fluxes of Clacross the mucosal and serosal borders of rabbit ileum using 36Cl and 82Br, or 87Br as tracers of Cl- movements. These methods have been used to determine the effects of theophylline and choleragen on the fluxes of Na+ and Cl- via the paracellular pathway and across the mucosal and serosal borders. Some of the results in this and the next paper have been presented (Naftalin & Simmons, 1976; Holman, Naftalin & Simmons, 1977) to the Physiological Society and were the substance of a communication given at the first meeting of the European Intestinal Transport Group at Arolla, Switzerland 1977.

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333

METHODS

materialss 2,4,6-Triamninopyrimuidine was obtained from Aldrich Chemicals, Co. Inc., Milwaukee, U.S.A., ethiacrynic acid from M.ercke, Sharpe & Dohbme. Hoddesdon, Herts and cholera enterotoxin from Schwartz MaIm Biochlemicals, York House, Empire Wray, Weembley, London. All other reagents used were of Analar grade. 2tNa, 24Na, 36CI and 82Br were obtained as aqueous isotonic solutions in NaCl from the Radiochemical Centre, Amnersham. "7Br was obtained from the M.R.C. cyclotron unit at Royal PostGraduate Hospital, Hammersmith.

Ani mals-Ringer solutions Male white New Zealand rabbits 2-3 kg in weight were used. Animals were killed by I.v. injection of Neinbutal. Preparation of ileum stripped of its serosa and external muscle layers is as previously described (Simmons & Naftalin, 1976b). Ringer comprised 140 min-NaCl, 10 mrM-KHCO3, 0 4 mnM-KH2PO4, 2-4 mM-K2HPO4, 1-2 mmCaCl2 and 1-2 mm-MgCl2. All solutions were gassed with 95 00 02: 5 00 C02 to pH 74. Zero Na Ringer was obtained by replacement of NaCl by choline Cl; zero Cl- Ringer was achieved by replacement of NaCl, CaCl2 by their sulphate salts, mannitol was used to maintain isosmolarity. The base 2,4,6-triaininopyrimidine was neutralized before being used (Simmons & Naftalin, 1976 b). Flux measurements and voltage clamp The procedure which was used for bidirectional flux measurements under voltage clamp has been described previously (Simmons & Naftalin, 1976b). Bidirectional Na fluxes were determined using 22Na and 24Na as tracers for mucosa-serosa (m-s) and serosa-mucosa (s-m) fluxes respectively. The method of Wood & Tomlinson (1974) for the measurement of bidirectional Cl fluxes has been adapted for use in rabbit ileumn. The method involves the use of 82Br (or 77Br) and 36CI as tracers for Cl-. That 82Br (or "7Br) may be routinely used as a tracer for Cl- is evident from the data contained in Tables 1 and 2 (Tomlinson & Wood, 1972). Table 1 shows the results of Cl- fluxes determined in experiments in which 36Cl and 82Br (or 77Br) were used simultaneously to estimate Cl- flux. It can be seen that flux calculated using either Br- isotope is consistently larger than flux calculated using 36CI tracer. This result is similar to that found by Wood and Tomlinson (1974). The ratio of Br/36C1 calculated fluxes does not differ between mucosa to serosa flux (J13) either for 82Br or 77Br. These ratios were subsequently used to correct the Br-estimated Cl- flux. Compartments 1, 2, and 3 refer to the mucosal, cell and serosal compartments respectively. Fluxes were calculated from the bidirectional fluxes J13, J31 and the ratio of isotope specific activities (R) in the cellular compartment, by the following relationships (Naftalin & Curran, 1974): J= J31 R + J13, J21 =J31 ( + R) J23 J13(1 + 1/R), J32 = J31 + J13/R. The ratio R (specific activity of isotope originating from the mucosal solution: specific activity of isotope originating from the serosal solution, contained within the tissue fluid) is determined following incubation to measure transmnural bidirectional fluxes. Tissues were briefly washed in ice-cold isotonic, wash media (choline Cl, or Na isethionate) and blotted to free tissue of adhering bathing solution containing isotope activity. In experiments with theophylline this measurement is made 1 hr 20 min following removal of the ileum from the animal; with experiments involving cholera enterotoxin, the time lag is greater (2 hr 20 min) which leads to the necessity of 1 hr preincubation with choleragen (see below).

Isotope counting 22Na and 24Na activities were determined as previously described (Simmons & Naftalin, 197Gb). 82Br was counted by its y emissions using an Ecko y counter. 36CI was counted from its

R. J. NAFTALIN AND N. L. SIMMONS

334

fl-emissions in a Packard Tricarb scintillation counter following decay of 82Br activity to insignificant levels (1 month). MCl activity was corrected for differing amounts of quench by the external standard channels ratio method. 82Br activity was corrected for decay during the initial counting period. Table 2 shows that the ratio of Br-/Cl--calculated flux is also invariant at three levels of transepithelial potential difference. This finding is consistent with the view that the ratio of Br-/Cl- calculated fluxes is similar in both the transcellular and paracellular pathways. The absolute magnitude of change in MCl-flux is small over the range of transepithelial p.d. used, in agreement with the known ion selectivity of the shunt pathway (Wright & Diamond, 1977; Frizzell & Schultz, 1972). An additional precaution in the use of 82Br (or 77Br) as a tracer for Cl- was routinely applied; this involved undertaking experiments in which the experimental conditions were run at least in duplicate, thus allowing estimations at unidirectional flux (either mucosa to serosa, or serosa to mucosa) by both M6Cl and a Br isotope. Variation in the control relationship between Br and Cl-calculated flux could therefore be detected if present. No such variation was observed. TABLE 1. 6Cl and 82Br tracer fluxes of Cl- determined simultaneously across rabbit ileum in the short-circuit current condition, together with the estimated tissue concentration of Cl from the 36CI and 82Br distribution ratios between tissue and bathing solution. M6CI and 77Br tracer fluxes of Cl- determined simultaneously across rabbit ileum in the short-circuit condition. *Note tissue Cl- is consistent with the estimated concentration of Cl- within rabbit ileum using X-ray microprobe microanalysis (Gupta et al. 1978). No significant difference between Br/Cl tracer ratios is noted when m-s and s-m fluxes are compared or between fluxes measured with 77Br and 82Br Isotope A

I_

I

n

36Cl

82Br

Ratio of 82Br/M"Cl flux

Mucosa-serosa Cl flux (CUMole cm-2 hr-1) Serosa-mucosa Cl flux (Qsmole cm hrcl)

6

9-33 ± 0-48

10-81± 0-69

1-15+0-03

6

6-23 ± 0-38

7-55 + 0-37

Tissue Cl- (mrnole/ litre tissue water) B Mucosa-serosa Cl flux moleoe cm- hr-1) Serosa-mucosa Cl flux

12

37-40 ± 5-31

MCl

37-91 + 4-42 77Br

1-21 + 0-03 Ratio of tissue Cl as estimated by 82Br or MCl 1-03 ± 0-03 Ratio of 77Br/6Cl flux

6

7-97±0-57

9-21 + 0-48

1-18 + 0-04

5

7-68± 0-84

8-90 ± 1-03

1-20 ± 0-03

A

*

TABLE 2. Ratio of 82Br: MCl mucosa-serosal fluxes determined simultaneously across control tissue at three levels of clamping potential. All errors are expressed as s.E. of mean. The Ringer NaCI = 140 mm. No significant difference in the 82Br: "Cl flux ratio at any level is apparent m-s Cl flux (Crmole cm-2 hr-1) ~~A

..

Isotope

Trans-epithelial potential (mV) 0 +10 (serosal anode) -10 (serosal cathode)

Ratio of

82Br/3Cl

n

MCl

3 3

10-72 ± 0 35 11-40± 0-56

14-17 + 1-18

1-29 ± 0-09 1-24 + 0-06

3

9-32±0-70

12-14 ± 0-84

1-30 ± 0-07

82Br 13-90±+ 0 70

'I

ION SECRETION BY RABBIT ILEUM

335

Using 36Cl or 82Br distribution as tracers for Cl- in tissue water gave no significant difference (Table 1).

Unidirectional flux calculations Unidirectional flux calculations across the mucosal and serosal borders of rabbit ileum were as previously described (Naftalin & Curran, 1974; Simmons & Naftalin, 1976b). "7Br was counted together with "Cl by its f + emissions using the Tricarb Spectrometer Adequate discrimination between 77Br and 36CI activities was obtained. 77Br activity was corrected for cross-over of 36CI activity, decay during the counting period, and for varying quench between samples. 36Cl activity was corrected for cross-over of 77Br activity and for quench. In two experiments 77Br activity was left to decay. The results of counting 36CI activity in this fashion were identical to the results obtained previously using the dual-label counting technique.

Statistics Variation in results is expressed as the standard error of the mean (s.E. of mean). Significance of differences were tested using a two-tailed Students test (unpaired means solution). One-tailed tests and paired tests were used where appropriate.

RESULTS

(A) Effects of 10 mM-theophylline (i) Bidirectional transepithelial Na and Cl fluxes Table 3 shows the effect of 10 mM-theophylline on the bidirectional Na+ and Clfluxes in the short-circuit current conditions. Theophylline reverses the net C1absorption to net secretion (P < 0-001). This change in the direction of net Cl- flux is primarily the result of a reduction in m-s Cl flux (J31) (P < 0-001). Concurrently there is a small, but non-significant, increase in s-m Cl- flux, J3.. Net Na+ absorption is reduced from 1-82 urmole cm-2 hr-1 to - 0-47 /tmole cm-2 hr-1 by theophylline. This change in net Na+ flux is due to a reduction in m-s Na+ flux by theophylline. These results are in accord with those of other workers (Field, 1971; Field et al. 1972; Powell et al. 1973; Nellans et al. 1974). The reductions in m-s Na and Cl fluxes are however equivalent as reported previously by Nellans et al. (1974).

(ii) The tissue isotope ratio R: unidirectional Cl- and Na+fluxes across the mucosal and serosal boundaries The effects of 10 mM-theophylline upon the tissue isotope specific ratio R and upon the calculated unidirectional ion fluxes (see Methods) are as shown in Table 4. Theophylline causes the ratio R for both Na (P < 0.05) and Cl- (P < 0-001) to increase significantly. It was shown previously for Na+ (Simmons & Naftalin, 1976b) that the ratio of Na within the whole tissue reflects accurately the ratio R within epithelial cells and submucosal layers. Table 5 demonstrates the homogeneity of the distribution of R between the epithelial and submucosal layers for both Na+ and Cl-. No significant regional differences in the ratio R are observed. However, with theophylline present, there are increases in the ratio R for both Na+ and Cl- within all compartments. This view may be quantified by estimating the unidirectional fluxes across the mucosal and serosal borders using the relationships defined in Methods. In tissue

336

R. J. NAFTALIN AND N. L. SIMMONS

TABLE 3. Unidirectional m-s, s-m transepithelial fluxes of C1- and Na+ across rabbit ileum incubated in the short-circuit current condition. Each flux determination is the mean value of two successive 30 min periods following 20 min preincubation with tracer. Jlt of Na+ and Clare determined from the absolute difference between J._ and J.m, The statistical significance of the results is estimated using Student's t test

/Sequiv cm-2 hr-1 I

n

Na fluxes Control Triaminopyrimidine (20 mM) Theophylline (10 mM) Triaminopyrimidine +

22 18 34 18

~

J_, 8-59 + 0-47 5-46 + 0-28*** 5.54+ 036*** 6-76 + 0.57*

theophylline 4 6-02 + 0.48* Ouabain (0.1 mM) 5-52+ 077* 4 Ouabain + theophylline 6-40 + 0.73* 5 Sulphate Ringer 7-10 + 0-85 6 Sulphate Ringer + theophylline Cl fluxes 23 8-33 ± 0-42 Control 8-43 + 0-64 15 Triaminopyrimidine 5-97 ± 0-57*** 11 Theophylline 5 7-63 ± 0-61 Triaminopyrimidine + theophylline 11 5-54± 043*** Ouabain 6-87 + 1-23 7 Ouabain + theophylline 7-35 + 0-55 5 Choline Ringer 8-70+ 1-32 3 Choline Ringer+ theophylline Test V8. control: * P < 0-05, ** P < 0-01,

~

A

Js-m

Jnet

1-82 + 0-35 6-76 0-45 4-61 + 0.25*** 0-84 + 0.23* - 0-47+ 015*** 6-02 + 0-31 0-90 + 0-32* 5-90 0- 30

5-93 0-04

4-88 + 0.5* 4-03 + 0.38** 6-04 ± 0-79

0-32 + 0.31* 0-64 + 0.47* 2-38 ± 0-48 1-03 ± 0-36

6-55 0-44 7-24 ± 0-48 7-48 0-53 9-67 ± 0-69**

1-78 ± 0-34 1-19 ± 0-37 - 1-51 + 0-46*** - 2-02 + 0.67***

5-67 + 0-53 5-94 ± 0-71 6-91 ± 0-65 8-03 ± 1-90

- 0-12+

044*** 0-92 + 0.94* 0-43 + 0-75 0-67 ± 0-77

*** P < 0-001.

treated with 10 mM-theophylline it is estimated that the unidirectional exchange of Cl across the mucosal border is significantly increased (P < 0-05 for J12, P < 0-001 for J21) compared to control tissue. Cl- fluxes across the serosal border are significantly reduced following treatment with theophylline (Table 4). No significant change in Na+ exchange across the mucosal boundary is observed comparing theophylline treated with control tissue. This may reflect the high conductance of the tight-junction to Na+ (see below). (B) Effects of 2,4,6-triaminopyrimidine on the theophylline-dependent changes in electrolyte fluxes (1) Bidirectional ion fluxes Examination of Table 3 shows that 20 mM-triaminopyrimidine has no significant effects on m-s, s-m or net Cl- fluxes either in the presence, or absence of 10 mMtheophylline. This is consistent with the known action of triaminopyrimidine, namely to decrease the paracellular Na+ conductance by blocking Na+ movement across the tight-junctions, without having any effect on transcellular ion transport or anion movements (Moreno, 1975; Simmons & Naftalin, 1976a).

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The effects of theophylline and choleragen on sodium and chloride ion movements within isolated rabbit ileum.

J. Physiol. (1979), 290, pp. 331-350 With 5 text-figureM Printed in Great Britain 331 THE EFFECTS OF THEOPHYLLINE AND CHOLERAGEN ON SODIUM AND CHLOR...
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