the actions of digestive enzymes, and for mixing and propulsion

Journal of Physiology (1988), 402, pp. 555-564 555With 6 text-figures

Printed in Great Britain

CHLORIDE ION TRANSPORT INTO PIG JEJUNAL BRUSH-BORDERMEMBRANE VESICLES

BY G. W. FORSYTH AND S. E. GABRIELFrom the Department of Veterinary Physiological Sciences, University of

Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada

(Received 4 August 1987)

SUMMARY

1. This study was carried out to determine the types and activities of carrierproteins which transport the chloride ion in pig jejunal brush-border membranes,with an emphasis on studying the properties of chloride conductance activity invesicles prepared from these membranes.

2. Sodium-chloride co-transport activity was not detected in this tissue. Asodium-proton antiport with typical amiloride sensitivity was present. An anionexchanger linking chloride to hydroxyl or bicarbonate ions was also found in the pigjejunal brush-border membrane vesicles.

3. Chloride conductance activity in this system was specifically dependent on thebuffering agents used for vesicle preparation. Conductance activity could not bedemonstrated in vesicles prepared in imidazolium acetate or in HEPES-Tris buffers.HEPES-tetramethylammonium buffering of vesicles in the chloride uptake systemproduced a significant conductance response to a potassium gradient plusvalinomycin.

4. Chloride conductance showed saturable kinetics with respect to substrateconcentration, with a Michaelis-Menten constant (Km) of approximately 116 mm anda maximum velocity (Vmax) of 132 nmol (mg protein)-' min-'.

5. Preliminary screening of potential inhibitors of chloride conductance showedonly minimal inhibitor effects of SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-sulphonic acid), anthracene-9-carboxylate, N-phenylanthranilate and piretanide.

6. The conductance activity in pig jejunal vesicles appears to have stringent bufferrequirements, and to be relatively insensitive to the effects of reported conductanceinhibitors.

INTRODUCTION

The movement of the chloride anion across the intestinal brush-border membraneplays a major role in the physical and chemical operations of the gastrointestinaltract. Chloride exchange for bicarbonate in the proximal jejunum may be importantboth for the neutralization of gastric acidity, and for the net absorption of luminalchloride. Particularly in the proximal jejunum the net fluid flux is often operating inthe direction of fluid secretion to produce the proper consistency of the ingesta forthe actions of digestive enzymes, and for mixing and propulsion through the

5. W. FORSYTH AND S. E. GABRIEL

intestinal tract by peristalsis. This net fluid secretion is presumed to be driven by achloride secretory process involving conductive movement of chloride across theapical or brush-border membrane of the enterocyte. Chloride conductance is aregulated process which should maintain proper fluidity of the ingesta, but theregulation can be disturbed by external agents such as bacterial enterotoxins or themethyl xanthines (Naftalin & Simmons, 1978) or by the abnormal release ofendogenous secretory signals such as vasoactive intestinal peptide.

This study was designed to characterize the activity of the proteins mediatingchloride transport in pig jejunal brush-border membrane. Two separate mechanismshave been described to explain the electroneutral process of NaCl absorption. Atightly coupled Na+-Cl- co-transporter has been demonstrated in whole tissues,whole-cell microelectrode studies and rabbit ileal brush-border membrane vesicles(Nellans, Frizzell & Schultz, 1973; Frizzell, Field & Schultz, 1979; Fan, Faust &Powell, 1983). An alternative system involves dual exchange. Na+-H+ and Cl--OH- antiporters were found in brush-border membrane vesicles prepared fromseveral tissues including rabbit renal cortex (Warnock & Yee, 1981), rabbit ileum(Knickelbein, Aronson, Atherton & Dobbins, 1983; Knickelbein, Aronson, Schron,Seifter & Dobbins, 1985) and rat small intestine (Liedtke & Hopfer, 1982a, b).The activity of the electroneutral Na+-Cl- transport in pig jejunal brush-border

membrane vesicles was investigated. Conditions were established for the assay of anelectrogenic chloride conductance pathway in the pig jejunal vesicle system. Theproperties of this conductive pathway were characterized in terms of substrateaffinity, transport velocity and the actions of several inhibitors.

METHODS

Tissue source. Segments of proximal jejunum 50 cm in length, starting 15 cm distal from theligament of Trietz, were removed surgically from anaesthetized 15-17 kg weanling pigs of mixedLandrace-Yorkshire breeding. The segments were opened, rinsed in isotonic saline and scrapedwith a glass slide to remove the mucosal surface. The mucosal scrapings were suspended inhomogenizing buffer (vide infra) and frozen at -70 °C until used for vesicle preparation.

Vesicle preparation. Brush-border membrane vesicles were prepared by homogenization ofmucosal scrapings, differential centrifugation and divalent cation precipitation ofnon-membranousmaterial in a standard technique which produced a 15-fold enrichment of alkaline phosphataseactivity (Maenz & Forsyth, 1982). The vesicles were isolated in a buffer containing 300 mm-mannitol, 16 mM-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonate (HEPES) and 10 mM-Tris(pH 7-4). Vesicle protein concentration was assayed by Coomassie Blue binding according toBradford (1976).

Transport measurements. The transport of radiolabelled solutes into brush-border membranevesicles was measured using a vacuum filtration technique. Uptakes were started by mixing 0-2 mgbrush-border membrane vesicle protein contained in 15 ,1 with an equal volume of isotonic mediacontaining 36Cl, or another isotope of interest. Isotope uptake was stopped by removal of theuptake media via filtration on 0 45 ,um porosity mixed cellulose esters membrane filters, followedby rapid washing with isotonic MgSO4 solution. Filters were air dried and radioactivity measuredusing a Beckman LS3800 f-counter in the case of [36Cl]- and [3H]glucose, and an LKB 1282Compugamma y-counter for 22Na determinations.The dependence of Na+ and Cl- self-exchange on counter-ions was measured according to the

method of Liedtke & Hopfer (1982a). Vesicles were equilibrated with 100 mM-mannitol, 50 mM-HEPES-Tris (pH 7 5) and 150 mM-Na+ with gluconate to replace Cl, or with 150 mM-Cl- and K+or tetramethylammonium (TMA) to replace Na+. After complete equilibration of all species self-exchange rate measurements were initiated by addition of 22Na (2 ,uCi/ml) or 36C1 (1 #Ci/ml).

556

PIG JEJUNAL CHLORIDE CONDUCTANCE

Sodium-proton antiport activity was measured in vesicles equilibrated with 100 mM-mannitoland 50 mM-N-morpholinoethanesulphonate (MES)-Tris (pH 6 0). The proton gradient wasproduced by resuspending the equilibrated vesicles in 100 mM-sorbitol, 50 mMHEPES-Tris to afinal pH of 7-5 with added 1-0 mM-22Na-gluconate (2 0 ,uCi/ml). The non-gradient condition wasobtained by resuspending the vesicles in uptake buffer (pH 7 5) with 22Na addition. The effect ofamiloride concentrations from 10 mm to 10 /LM on the initial velocity of the Na+-H+ antiport wasdetermined.Anion exchange activity was measured in vesicles equilibrated with 100 mM-mannitol, 50 mm-

HEPES-Tris (pH 7 5), and either 100 mM-KHCO3 or 100 mM-potassium gluconate. Uptake bufferscontained 100 mM-mannitol, 90 mM-potassium gluconate, 10 mM-KCl (1 1sCi 36C1/ml), and either50 mM-HEPES-Tris (pH 7 5) or 50 mM-MES-Tris (pH 5 5).

Chloride conductance measurements were attempted under a variety of conditions. For mostdirect comparison we can cite 300 mM-mannitol, with any of imidazolium acetate, HEPES-Tris orHEPES-TMA as buffer systems present at 70 mm concentration and pH 7-5 for equilibratingbuffers. Uptake of Cl- would be initiated by dilution of equilibrated vesicles in an equal volume ofthe same buffer, with equiosmolar replacement of mannitol by 100 mM-potassium gluconate and10 mM-K36CI (1 1sCl/ml). For some conductance measurements the 300 mM-mannitol in theequilibrating buffer was replaced by 100 mM-mannitol and 100 mM-TMA gluconate, and for controlmeasurements by 100 mM-mannitol and 100 mM-potassium gluconate.

Variations in the K+ gradient were accomplished by replacing mannitol with potassiumgluconate in the uptake buffer. Variations in the Cl- gradient were produced by replacingpotassium gluconate in the uptake buffer with KCl.

Inhibitors were incubated with vesicles for a minimum of 5 min prior to initiating standardchloride conductance conditions. Uptake was stopped at 15 s by a 100-fold dilution with isotonicMgSO4 solution. All transport measurements are means of either nine or twelve individualdeterminations+ standard error of the mean, unless otherwise stated.

Kinetic parameters and th values were obtained by computer-assisted fitting to a rectangularhyperbola (Enzpack-Elsevier Biomedical Software). Student's paired t statistic was used todetermine levels of significance.

Materials. All chemicals were reagent grade or better and purchased from common commercialsources. Piretanide was a gift from Hoechst Canada Ltd (Varennes, Quebec). 22NaCl (11 Ci/mmol),and H36Cl (581 ,sCi/mmol), were both purchased from Amersham Canada (Oakville, Ontario).

RESULTS

Lack of coupled Na+-Cl- co-transportThe tightly coupled Na+-Cl- co-transport model reported for some epithelial

tissues suggests a dependence of each ion for the opposing counter-ion during atransport process, i.e. Na+ transport dependent on Cl- concentration. In pig jejunalvesicles buffered with HEPES-Tris the rate of Na+ self-exchange decreased slightlywhen the gluconate counter-ion was replaced by Cl- (gluconate ti 105 s, Cl- ti 140 s).Replacement of Na+ by K+ did not affect the Cl- self-exchange activity in pig jejunalbrush-border vesicles (Na+ t1 92 s, K+ ti 97 s). Similar results were obtained withvesicles buffered in imidazolium acetate (Fig. 1).

AnttiportsThe dual antiports, Na+-H+ and Cl--OH-, were investigated by applying proton

gradients across the vesicle membrane. An outwardly directed proton gradient wasused to establish conditions for Na+-H+ exchange (Fig. 2). The data indicate aninitial accumulation of Na+ ions above the final equilibrium concentration in thepresence of a pH gradient (pHi 6-0, pH. 75). Na+ uptake was linear for 15 s underthese conditions, and the initial rate of proton-dependent Na+ uptake was inhibited50% by IO0 x 10' M-amiloride.

557

G. W. FORSYTH AND S. E. GABRIEL

12 -;

8- * -

4-* 150 mM-Cl- y= 11-0867-0-0074x* 150 mM-Na8 y = 9-635-0 0004667x

0 50 100 150Na+ or Cl- (mM)

Fig. 1. The effect of substituting gluconate for C1- or TMA for Na+ on the self-exchangerates for Na+ and C1-. Vesicles were prepared in imidazolium acetate buffer containingadditionally ATP, AsO4 and F-. Regression lines indicate Na+ and Cl- dependence oncounter-ion concentration.

-4.C0

E

02E-

1a

z o

0 5 10Time (min)

-1 1-15 60

of an outwardly directed proton gradient on the rate ofjejunal brush-border vesicles.

Na+ uptake by

a)

a)

0)IV

E

C

E-6EQ

-V

0 1 2 3 4 5Time (min)

60

Fig. 3. Effect of an inwardly directed proton gradient on the rate of Cl- uptake by jejunalbrush-border vesicles. The gradients consisted of 100 mM-bicarbonate and a pH. of 5-5with a pH, of 7-5. External Cl- was 10 mm under all uptake conditions.

558

x0

8a)

4(a)

sx)CD

C.)

x

a1)a/)

Fig. 2. Effect

PIG JEJUNAL CHLORIDE CONDUCTANCE 559

The activity of the Cl--anion exchanger was then studied with a reversed,inwardly directed proton gradient, with and without intravesicular bicarbonate ions(Fig. 3). The rate of Cl- uptake increased in the presence of a proton gradient, andwas fastest with the combined proton-bicarbonate gradient condition. The absenceof any Cl- ion accumulation above equilibrium concentrations was in distinctcontrast to the overshoot observed with the Na+-H+ exchange system.

-a. Control14 - K+gradient

C

a 120.

E' 10

7

0 ~~~5"

43

00 22

0 20 40 60 80Time (min)

Fig. 4. Cl- conductance rates measured in vesicles pre-equilibrated in HEPES-TMA. Theinset shows initial velocity up to the 3 min transport measurement.

Basal conductance conditionsMaximal effects on vesicle permeability to K+ were obtained with 7 jug of

valinomycin per milligram of vesicle protein. This level of valinomycin was used inall conductance studies. Repeated attempts to demonstrate a stimulation of the rateof Cl- uptake by a K+ gradient plus valinomycin were not successful in vesicles whichwere prepared in HEPES-Tris buffer or in imidazolium acetate buffer. Changingfrom the hypertonic uptake media of Liedtke and Hopfer to equal osmolarity ofinternal and external solutions did not give a detectable conductance response invesicles prepared in these buffers. Other changes attempted in the vesicle preparationtechnique (substitution of Ca2+ for Mg2+ in the precipitation step) or in uptakeconditions (varying uptake temperature between 0 and 37 °C, or using potassiumacetate or sulphate to generate the K+ gradient) did not produce a conductanceresponse. The conductance channel in pig jejunal brush-border membrane vesiclesappeared to be in a closed or basal state under these conditions for Cl- uptake.

Conductance conditionsUtilizing a procedure for vesicle buffering employing HEPES-TMA according to

Knickelbein et al. (1985), we have been able to demonstrate a Cl--conductive pathwayin pig brush-border membrane vesicles (Fig. 4). The control condition was obtainedby pre-incubating vesicles in a solution containing 100 mm-K+ ions. At the onset ofthe uptake in this control condition the vesicles are resuspended in 100 mM-K+ ions


resulting in no net K+ gradient across the vesicle membrane and therefore nomembrane potential. For the test condition, vesicles without internal K+ ions wereadded to uptake media containing 100 mM-K+ in the presence of valinomycin (7 jug/mg protein). This condition produced a significantly faster rate of Cl- uptake incomparison to the control (t, of 35 vs. 75 s for the K+ gradient condition vs. the

10

a,0cl._

0)

E

ECma,

0.5J

0 1 2 3 4 5 6Time (min)

Fig. 5. The effect of the K+ gradient on the rate of Cl- uptake by jejunalbrush-border vesicles.

30

-

1E w

CLv, ._r

Q 0L0.C) Co

E

0 100 200 300Cl 1mM)

Fig. 6. The effect of Cl- concentration on the rate of Cl- uptake by jejunal brush-bordervesicles. Results are the means, and the standard errors, of the differences between theCl- uptake rate from the conductance and the non-conductance condition. The insetrepresents a Hanes-Woolf derivative plot of the kinetic data and indicates a Km of116 mm and a Vmax of 132 nmol (mg protein)-' min-'. S, substrate (Cl-) concentration; V,velocity.

control). The stimulatory effect of the K+ gradient on the rate of Cl- uptake waseliminated when K+ levels were allowed to equilibrate across the vesicle membranebefore Cl- addition, or when valinomycin was omitted from the vesicle suspension.

Chloride conductance propertiesConsistent with the definition of Cl- conductance, we were able to determine the

extent of the dependence of Cl- conductance on the magnitude of the K+ gradient(Fig. 5). The rate of Cl- uptake was increased by 100% over the control (no gradient)

560


condition when the K+ concentration gradient was imposed by 100 mM-externalK+, with no internal K+. There was no significant increase in conductance ratebeyond this gradient condition (in going from 100 to 150 mm external K+).The Cl- conductive activity measured in this system showed apparent saturation

at high Cl- concentrations, as the increase in initial uptake rates was less than theincrease in Cl- concentration (Fig. 6). Kinetic analysis indicated a half-maximaltransport rate corresponding to a Km value of 116 mm for Cl-, and a Vmax of 132 nmol(mg protein)-' min-1.

TABLE 1. Effects of reported conductance inhibitors on the kinetic parameters of theconductance protein

Km for Cl- VmaxInhibitor (mmol/1) (nmol mg-' min-')

None 116 1325 mM-SITS 165 1245 mM-NPA 150 985 mM-Piretanide 142 116

InhibitorsSeveral compounds which are reported to inhibit various Cl- transport processes

were tested for their potency against the porcine Cl- conductance protein. SITS,piretanide, N-phenylanthranilate and anthracene-9-carboxylate were tested at levelsranging from 1 to 25 mm. Maximal inhibition of K+ gradient-dependent Cl- uptakewas reached at the 5 mm level, although all inhibitors reduced Cl- uptake in both theK+ gradient as well as the control condition. The maximum effects of the inhibitorson the kinetic parameters of conductance activity are given in Table 1. Anthracene-9-carboxylate had no measurable inhibitory effect.

DISCUSSION

Data from the experiments measuring self-exchange rates point to the absence ofa tightly coupled Na'-Cl- co-transport process in pig jejeunal brush-bordermembrane. There was no detectable mutual dependence of Na+ and Cl- fortransmembranous exchange. The self-exchange rates measured in pig jejunal vesicleswere considerably lower than reported values for rat intestinal brush-border vesicles(Liedtke & Hopfer, 1982a). The tB values for Na+ exchange (112 s) and Cl- exchange(96 s) are consistent with much lower concentration or activity of membrane carriersthan in the rat system which had values of 18 and 12 s for Na+ and Cl- respectively.It is not clear if these differences reflect distinct membrane transport propertiesbetween the species, or variations in methodology affecting the tightness of sealingof the brush-border membrane into vesicles. The Na+Cl- co-transport activityreported by Fan et al. (1983) has aKm of 4-5 mm for Na+, and if present in the porcinevesicles, it apparently does not contribute significantly to ion transport underconditions of physiological ion concentration.The existence of a Na+-H+ antiport was shown by a substantial overshoot of

vesicle Na+ content induced by a proton gradient, and by the inhibition of the initial

561


rate of Na+ uptake by amiloride. The parallel antiport for Cl--HC03- was alsopresent. Stimulation of the anion exchange process by bicarbonate indicated somespecificity or preference for HC03- over OH-, as observed by Knickelbein et al.(1985) for rabbit ileum. The absence of an overshoot of Cl- uptake in the pig jejunalsystem would appear to indicate relatively less anion exchange activity in this tissuethan in the rabbit ileum. Other investigators have found varying levels of Cl-transport activity occurring via anion exchange (Frizzell et al. 1979; Leidtke &Hopfer, 1982 b). The low activity of anion exchange in the pig jejunal vesicle systemis probably an advantage in trying to characterize and quantify the Cl- conductanceactivity of this tissue.

Difficulties encountered in preliminary efforts to show Cl- conductance activity inthe system buffered with imidazolium acetate led us to attempt several variations ofthe protocol. The failure of this system to show a conductance response wasconsidered from another view point. Lack of measurable conductance activity couldbe an important observation relating to the physiological regulation of conductanceactivity. The basal state of conductance in vivo should be relatively low to preventexcessive Cl- loss down the large electrochemical gradient existing in the proximaljejunum. The maximal value for basal conductance could correspond to the serosalto mucosal Cl- flux (Jclm), but the true value is probably much lower, as most ofJcI can be accounted for as idling activity of the anion exchange carrier. Thisminimal basal in vivo rate of conductance may be preserved under some conditionsof vesicle preparation, leading to the lack of measurable Cl- conductance in responseto an imposed K+ gradient. The conductance rates usually measured in vitro (Liedtke& Hopfer, 1982b) could then be considered to correspond to activated conductance,analogous to an in vivo secretory state. According to this hypothesis, activation mayoccur spontaneously during vesicle preparation due to protein kinase activation orto dissociation of a regulatory component from the conductance channel. In supportof this hypothesis there are no reports of in vitro activation of Cl- conductance invesicle systems already showing substantial conductance activity. We also have beenunable to activate conductance above the levels measured in vesicles prepared inHEPES-TMA buffer. However, we do have results showing conductance activationfollowing cyclic AMP addition to vesicles prepared in the 'no conductance'imidazolium acetate condition (G. W. Forsyth & S. E. Gabriel, unpublished observa-tions).

Under physiological conditions the activity of the Na+-C1- co-transporter isregulated inversely with Cl- conductance activity. Vesicles with active conductanceisolated in HEPES-TMA could lack in vitro co-transport activity if this inverserelationship were maintained during vesicle isolation. The absence of detectable co-transport in vesicles prepared in HEPES-Tris or imidazolium acetate buffers (basalconductance state) suggests that the lack of counter-ion dependence observed in thisstudy was not due to physiological inhibition of the co-transporter.

Nellans et al. (1973), Liedtke & Hopfer (1982b), and Knickelbein et al. (1985) havedetected conductive pathways for Cl- in tissues with secretory capacity from variousspecies, but there is a paucity of information on the characteristics of the conductiveprocess. The properties found for Cl- conductance activity in pig jejunal brush-border vesicles were appropriate to the in vivo function of the ion transporter. If the

562


C1- transport activity were an electrogenic pathway, then it would be expected to bedependent on the magnitude of the K+ gradient as shown in Fig. 5. The C1- transportrate appeared to be maximal when uptake was driven by 100 mM-external K+. Theabsence of a Cl- 'overshoot' with maximal stimulation was surprising. It is clear,however, that the faster rate of Cl- uptake (ti of 35 s for the K+ gradient-dependenceCl- uptake vs. 75 s for the control condition) was evidence for a conductanceprocess.Measurements of initial rates of Cl- uptake with a range of Cl- concentrations from

20 to 250 mm suggested saturable kinetics with a substrate affinity constant, Km, ofapproximately 116 mm and a Vmax of 132 nmol (mg protein)-' min-'. These resultsare compatible with the presence of a protein-mediated conductance process, asopposed to passive ion leakiness in the vesicle membrane. The affinity constant isrelatively high, but it is suitable for the transport of an ion which is present atconcentrations of > 100 mm in the extracellular fluid.N-Phenylanthranilate (DiStefano, Wittner, Schlatter, Lang, Englert & Greger,

1985) and piretanide (Zeuthen, Ramos & Ellory, 1978) are reported as potentinhibitors of electrogenic Cl- conductance, but they were not effective inhibitors ofCl- conductance in pig jejunal brush-border vesicles. SITS had low inhibitoryactivity, similar to the 35% inhibition reported by Liedtke & Hopfer (1982b).Perhaps the vesicles isolated in HEPES-TMA buffer may lack some functional orregulatory part of the conductance channel involved in inhibitor recognition. Thispoint could be investigated by measuring inhibition of conductance in vesiclesisolated in the basal conductance state.

Technical assistance was ably provided by Eva Yonge. Financial support for this research camefrom the Medical Research Council of Canada and the Saskatchewan Agriculture DevelopmentFund.

REFERENCES

BRADFORD, M. (1976). A rapid and sensitive method for the quantitation of microgram quantitiesof protein using the principle of protein-dye binding. Analytical Biochemistry 72, 248-251.

DISTEFANO, A., WITTNER, M., SCHLATTER, E., LANG, H. J., ENGLERT, H. & GREGER, R. (1985).Diphenylamine-2-carboxylate, a blocker of the Cl conductive pathway in Cl transportingepithelia. Pfugers Archiv 405, S95-100.

FAN, C. C., FAUST, R. G. & POWELL, D. W. (1983). Coupled sodium-chloride transport by rabbitileal brush border membrane vesicles. American Journal of Physiology 244, G375-385.

FRIZZELL, R. A., FIELD, M. & SCHULTZ, S. G. (1979). Sodium-coupled chloride tranport byepithelial tissues. American Journal of Physiology 236, F1-8.

KNICKELBEIN, R., ARONSON, P. S., ATHERTON, W. & DoBBINS, J. W. (1983). Sodium and chloridetransport across rabbit ileal brush border. I. Evidence for Na-H exchange. American Journal ofPhysiology 245, G504-510.

KNICKLEBEIN, R., ARONSON, P. S., SCHRON, C. M., SEIFTER, J. & DOBBINS, J. W. (1985). Sodiumand chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange andcoupling. American Journal of Physiology 249, G236-245.

LIEDTKE, C. M. & HOPFER, U. (1982 a). Mechanism of Cl translocation across small intestinal brush-border membrane. I. Absence of Na-Cl cotransport. American Journal of Physiology 242,G263-271.

LIEDTKE, C. M. & HOPFER, U. (1982 b). Mechanism of Cl translocation across small intestinal brush-border membrane. II. Demonstration ofCl-OH exchange and Cl conductance. American Journalof Physiology 242, G272-280.

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564 G. W. FORSYTH AND S. E. GABRIEL

MAENZ, D. D. & FORSYTH, G. W. (1982). Ricinoleate and deoxycholate are calcium ionophores injejunal brush-border vesicles. Journal of Membrane Biology 70, 125-133.

NAFTALIN, R. J. & SIMMONS, N. L. (1978). The effects of theophylline and choleragen on sodiumand chloride ion movements within isolated rabbit ileum. Journal of Physiology 290, 331-350.

NELLANS, H. N., FRIZZELL, R. A. & SCHULTZ, S. G. (1973). Coupled sodium-chloride influx acrossthe brush border of rabbit ileum. American Journal of Physiology 225, 467-475.

WARNOCK, D. G. & YEE, V. J. (1981). Chloride uptake by brush-border membrane vesicles isolatedfrom rabbit renal cortex. Journal of Clinical Investigations 67, 103-115.

ZEUTHEN, T., RAMOS, M. & ELLORY, J. C. (1978). Inhibition of active chloride transport bypiretanide. Nature 273, 678-680.

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