Intestinal Transport

The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (α-, β-, and γ-subunits of Enac and Cftr), transporters ( Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na+/H+ exchanger (NHE) regulatory factor ( Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, γ Enac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the sub...

The intestinal transport of nutrients exhibits distinct diurnal rhythmicity, and the enterocytes harbor a circadian clock. However, temporal regulation of the genes involved in colonic ion transport, i.e., ion transporters and channels operating in absorption and secretion, remains poorly understood. To address this issue, we assessed the 24-h profiles of expression of genes encoding the sodium pump (subunits Atp1a1 and Atp1b1), channels (␣-, ␤-, and ␥-subunits of Enac and Cftr), transporters (Dra, Ae1, Nkcc1, Kcc1, and Nhe3), and the Na ϩ /H ϩ exchanger (NHE) regulatory factor (Nherf1) in rat colonic mucosa. Furthermore, we investigated temporal changes in the spatial localization of the clock genes Per1, Per2, and Bmal1 and the genes encoding ion transporters and channels along the crypt axis. In rats fed ad libitum, the expression of Atp1a1, ␥Enac, Dra, Ae1, Nhe3, and Nherf1 showed circadian variation with maximal expression at circadian time 12, i.e., at the beginning of the subjective night. The peak ␥Enac expression coincided with the rise in plasma aldosterone. Restricted feeding phase advanced the expression of Dra, Ae1, Nherf, and ␥Enac and decreased expression of Atp1a1. The genes Atp1b1, Cftr, ␣Enac, ␤Enac, Nkcc1, and Kcc1 did not show any diurnal variations in mRNA levels. A low-salt diet upregulated the expression of ␤Enac and ␥Enac during the subjective night but did not affect expression of ␣Enac. Similarly, colonic electrogenic Na ϩ transport was much higher during the subjective night than the subjective day. These findings indicate that the transporters and channels operating in NaCl absorption undergo diurnal regulation and suggest a role of an intestinal clock in the coordination of colonic NaCl absorption.

Cystic fibrosis (CF) is frequently associated with progressive loss of exocrine pancreas function, leading to incomplete digestion and absorption of dietary fat. Supplementing patients with pancreatic lipase reduces fat excretion, but it does not completely correct fat malabsorption, indicating that additional pathological processes affect lipolysis and/or uptake of lipolytic products. To delineate the role of such (post) lipolytic processes in CF-related fat malabsorption, we assessed fat absorption, lipolysis, and fatty acid uptake in two murine CF models by measuring fecal fat excretion and uptake of oleate- and triolein-derived lipid. Pancreatic and biliary function was investigated by determining lipase secretion and biliary bile salt (BS) secretion, respectively. A marked increase in fecal fat excretion was observed in cftr null mice but not in homozygous ΔF508 mice. Fecal BS loss was enhanced in both CF models, but biliary BS secretion rates were similar. Uptake of free fatty...

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA-gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA-gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal infl ammation. Our fi ndings strongly implicate CD71 in the pathogenesis of CD.

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune...

A net absorption of oxalate and chloride was observed when isolated, shortcircuited segments of rat colon were bathed by a calcium-containing buffer. Removal of calcium promoted a twofold decrease in transmural resistance, while the net chloride flux was reduced and the net oxalate transport abolished. It was concluded that net oxalate absorption was not observed in previous studies (employing calcium-free buffers) because calcium is required to maintain the integrity of the conductive pathways across colonic epithelia. * We have also consistently observed that there is a net mucosal-to-serosal transport of oxalate across the isolated, short-circuited rabbit colon in the presence of calcium. A detailed consideration of these observations will be presented elsewhere.

Secondary hyperaldosteronism enhances the rate of K secretion in distal colon, at least in part, through the stimulation of Na+-K+-CI-cotransport across the basolateral membrane. To maintain a constant intracellular C1-activity an increase in C1-transport out of the cell must be assumed. We explored, under amiloride lo-' M and short circuited conditions, conductive pathways for C1-exit in the distal colon of K+-adapted rats by means of a putative C1-channel blocker, NPPB (5-nitro-2(3-phenylpropylamino-benzoate. Results prior to NPPB showed an increase in J C P after K+ loading from 5.84 f 0.66 to 8.33 f0.86 and JClsm from 4.77 f 0.55 to 8.16 f 0.96 pEq h-' cm12 (P < 0.001), when compared with controls. Net fluxes were not different between groups. Luminal NPPB in K+ adaptation resulted in a decrease of JCP", from 7.85-t 1.5 to 6.69 f 1.5 pEq h-' cm-' (P < 0.05). There were no changes in both unidirectional CI-fluxes in controls under luminal NPPB and in potential difference (V) and short-circuit current (Isc) under any condition. Finally, K' adaptation resulted in an increase of luminal cyclic AMP (CAMP) concentration (0.09k0.02 to 0.20 f 0.03 pmol 100 PI-', P < 0.05), when compared with control rats. The data may suggest a transcellular recycling of CI-and an activated NPPB inhibitable serosal to mucosal CI-pathway on luminal membrane in the K+ adapted state, possibly mediated by an increase in CAMP production.

who were all vital in the completion of this work. I also thank my family for their untiring support and guidance throughout my life, as well as all of my friends, who have made the journey so wonderful along the way. Finally, I would like to thank the National Institutes of Health and the Cockrell School of Engineering for their funding of my research and education. vi

Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA–gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA–gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune...

The objectives of this study were to determine the effect of an increase in diet fermentability on 1) the rate and extent to which short-chain fatty acid (SCFA) absorption pathways adapt relative to changes in Na(+) transport, 2) the epithelial surface area (SA), and 3) the barrier function of the bovine ruminal epithelium. Twenty-five Holstein steer calves were assigned to either the control diet (CON; 91.5% hay and 8.5% supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain (G), and 8.5% supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 days (G21). All calves were fed at 2.25% body weight at 0800. Calves were killed (at 1000), and ruminal tissue was collected to determine the rate and pathway of SCFA transport, Na(+) transport and barrier function in Ussing chambers. Tissue was also collected for SA measurement and gene expression. Mean reticular pH decreased from 6.90 for CON to 6.59 for G7 and then increased (quadratic P &lt; 0.001). While effective SA of th...

The diarrhea observed in patients with cholera is known to be related to secretion of water and electrolytes into the intestinal lumen. However, the exact mechanisms involved in these secretory processes have remained unclear. Although it is clear that purified toxin acts on epithelial cell metabolism, its activity on Na + transport across intestinal mucosa is equivocal: reported either to prevent net Na + absorption or to cause net secretion of Na + from serosa to mucosa. Since total transmural Na + fluxes across "leaky" epithelia involve very significant movement via a paracellular shunt pathway, we studied the effects of cholera toxin on the cellular and paracellular pathways of Na + movement. Unidirectional Na + fluxes were examined as functions of applied potential in control tissues and in tissues from the same animal treated with purified cholera toxin. Treatment of rabbit ileum in vitro with toxin stimulated the cellular component of serosa-to-mucosa Na ÷ flux (from 2.41~0.49/~equiv./h per cm z under control conditions to 4.71±0.43 #equiv./h per cm z after treatment with toxin, P < 0.01). The effect of cholera toxin on Na + movement through the cells from mucosa to serosa appeared to be insignificant. Finally, a marked decrease in the Na + permeability (P < 0.01) and no detectable significant changes in transference number for Na + of the paracellular shunt pathway were observed following treatment with cholera toxin. These results provide direct evidence for the hypothesis that purified cholera toxin stimulates active sodium secretion but has minimal effect on sodium absorption.

POWELL, DON W., HENRY J. BINDER, AND PETER F. CURRAN. Active electrolyte secretion stimulated by choleragen in rabbit ileum in vitro. Am. J. Physiol. 225(4): 781-787. 1973.-Purified cholera toxin added to the mucosal solution of rabbit ileum in vitro caused changes in ion fluxes and electrical properties after a 50-to 60-min lag period. Potential difference and short-circuit current increased and tissue conductance decreased.

Two differently oriented approaches in intestinal physiology can be distinguished. One, mainly based on in vitro experiments, seeks explanations at the level of the epithelium itself. The other, mainly based on in vivo experiments, looks for explanations at the level of regulatory nervous and endocrine mechanisms and their interaction. These two approaches complement each other.

Recent in vitro studies in human colon have demonstrated marked segmental differences in electrogenic Na transport. In the present study, the Na channel blocker amiloride was used further to characterise basal and aldosterone-induced electrogenic Na transport in isolated human distal and proximal colon. Bathed in NaC1 Ringer solution, distal and proximal colon exhibited similar basal electrical properties, but the amiloride-sensitive short-circuit current (Isc) was 200% greater in the distal than in the proximal segment. Bathed in choline-C1 Ringer solution, total Isc decreased by 97% in distal colon and by 88% in proximal colon, indicating that Na dependent transport process(es) account almost entirely for the I~c in both segments. Substituting Na2SO, for NaC1 Ringer solution (i) increased amiloride-sensitive Is~ by 56% (p < 0.01) in distal colon but had no effect on amiloride-sensitive Is~ in proximal colon, and (ii) decreased amiloride-insensitive Is~ in distal and proximal colon by 52% (p < 0.05) and 81% (p < 0.001) respectively. After the addition of nystatin to the apical membrane, the relationship between total I~o and mucosal Na concentration indicated that the activity of the basolateral membrane Na pump was similar in both colonic segments. In a further series of experiments, exposure of distal colon to I ~tmol/1 aldosterone for 5 h increased total I~c by 52% (p < 0.05), which reflected stimulation of its amiloride-sensitive component; in contrast, aldosterone had no effect on proximal colon. These results indicate that Na-dependent electrogenic processes (with electrogenic Na transport predominant) are present throughout human colon, but there is marked segmental variability in the Na conductive properties of the apical membrane. Apical Na entry in distal colon occurs mainly through 'classical' amiloride and aldosterone-sensitive Na channels. In contrast, the predominant apical Na entry mechanism in proximal colon is an amiloride and aldosterone-insensitive pathway.

A B S T R A C T The effect of aspirin on normal and cholera toxin-stimulated electrolyte transport has been investigated in vitro, because this drug appears to inhibit cholera toxin-induced intestinal secretion in in vivo animal models. In the Ussing chamber, 10 mM aspirin decreased the control rabbit ileal potential difference and short-circuit current by 50% and increased conductance by 28%. Bidirectional electrolyte flux determinations showed that aspirin significantly increased both Na and Cl absorption and reduced the residual flux (which probably represents HCOs secretion) to zero. This effect of aspirin appears to be identical to that reported by others with catecholamines as determined with similar techniques. However, a-adrenergic blockers did not prevent the electrical effects of aspirin, suggesting that aspirin does not have its effect through release of tissue stores of catecholamines. In the presence of aspirin, cholera toxin increased the potential difference and short-circuit current, and decreased the conductance of rabbit ileum in a fashion qualitatively similar to control tissues. However, aspirin reversed cholera toxin-stimulated Na transport from secretion to absorption, inhibited cholera toxin, induced Cl secretion by 58%, and partially, but not significantly, inhibited HCOs secretion. Thus, the inhibitory effect of aspirin on cholera toxin-induced electrolyte secretion appears to be due to aspirin-stimulated Na and This work was presented in part at the American Gas-Cl absorption. Although aspirin reduced tissue cyclic AMP concentrations in normal and cholera toxin-stimulated ileum, it also inhibited the electrolyte secretion induced by exogenous cyclic AMP. Thus, if aspirin's stimulatory effect on sodium and anion absorption in normal tissue and its inhibitory effect on cholera toxin-stimulated sodium and anion secretion involves a cyclic AMPmediated system, the effect must be at a step distal to cyclic AMP production or degradation. The exact mechanism of aspirin's effect on normal and cholera toxininduced electrolyte transport, and its possible usefulness in the treatment of cholera diarrhea, remains to be determined.

The effect of cholera toxin (CT) and Escherichia coli heat-stable enterotoxin (ST) on the ileum and colon was examined in vivo in the rat in an attempt to clarify the effects of enterotoxins on colonic mucosa and to determine if these effects were influenced by short-chain fatty acids (SCFA). Both CT and ST induced similar changes in water and elec trolyte fluxes, and the magnitude of these changes in loops of colon was similar to that observed in loops of ileum. The addition of luminal SCFA, acetate, propionate and butyrate did not influence the effect of either toxin in loops of ileum. However, in loops of colon exposed to CT, luminal butyrate (40 mM ) largely reversed the effect of CT by converting net water secretion (mean ± SE, -363 ± 154 nTcm"2-min_l) to net water absorption (470 ± 194 nl-cm-2-min_1) and by significantly reducing the net secretion of sodium ions. In loops of colon exposed to ST, similar effects were observed although net water secretion (-784 ± 114 nl-cm-2-min-') was only partially reversed by butyrate (-318 ± 102 nl"cm-2*min-1). In contrast to butyrate, acetate and propionate did not influence changes in colonic fluxes of water and sodium induced by enterotoxins. Oxidation of butyrate and glucose was observed to be depressed in colonocytes pre-exposed to CT but not to ST. In this model, colonic secretion induced by enterotoxins is similar to that observed in the ileum but differs from ileal secretion in its modulation by luminal butyrate.

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Net K-transport was investigated in three different segments of rabbit proximal and distal colon. In vivo studies used a dialysis method, and in vitro studies an everted sac preparation. Results from both methods are in good agreement and show net K-absorption in the two segments of the proximal colon, and net K-secretion in the distal colon. K-absorption in the proximal colon seems to be an active mechanism, because K is transported against an electrical potential gradient. The present study is a comparative approach to demonstrate the heterogeneity of an organ function, and represents the first direct comparison of K-transport in various segments of rabbit colon under in vitro and in vivo conditions.

1. The epithelia of the hen lower intestine show a Na-channel, Na-cotransport, chloride cells, and chloride absorption and secretion. 2. The short circuit current is affected by low salt levels, amiloride, glucose, lysine, leucine, galactose, ouabain, bumetanide, aldosterone, dexamethasone and spironolactone. 3. The properties of the different sodium and chloride channels are described.

The second part of rabbit proximal colon was investigated in vitro under short circuit conditions. Unidirectional sodium and chloride fluxes were measured during the soft faeces period and during the hard faeces period. Rabbit late proximal colon has a potential difference (psi mS) of 4 mV, a tissue conductance (GT) of 10-11 mS/cm2 and a short circuit current (Isc) of 1.5 mueq/cm2 X hr. Under control conditions sodium (2.65 mueq/cm2 X hr) and chloride (0.67 mueq/cm2 X hr) are absorbed. Ouabain abolished psi ms,Isc and the net sodium flux totally, whereas 0.1 mM amiloride only slightly decreased the net sodium flux. No differences in electrical properties and Na,Cl-fluxes were found between the faeces periods. Removal of sodium abolished psi ms and Isc totally, and a high potassium solution depolarized the preparation (psi ms = 0). A linear current-voltage relation characterizes the tissue as an ohmic resistor between -40 and +50 mV, and reveals a slope conductance of 14 mS/cm2 under...

The effect ofthe Iaxative component rhein on electrical properties and Cl transpoft was investigated in partially stripped epithelial sheets of rabbit distal colon under shortcircuit conditions. Whereas mucosal rhein had no effect, serosally applied rhein stimulated electrogenic Cl secretion in a dose-dependent manner with a half-maximal stimulation at 0.05 mmol/1. The Stimulation was partially reversible by serosal bumetanide. Indometacin preincubation inhibited the effect of rhein, but did not prevent theophylline-induced Cl secretion. The results suggest a prostaglandin-mediated effect of rhein on the apical Cl conductance of the colonic cells.

The regulation of sodium and chloride transport in hen coprodeuro by mineralocorticoids was investigated with isolated epithelia under short-circuit conditions . Unidirectional ftuxes of Na and Cl were measured by isotopes and modulated by amiloride , theophylline and bumetanide. Hens were maintained either on low-NaCI diet (LS) or on high-NaCI diet (HS). Plasma aldosterone (PA) Ievels of these groups were measured with radioimmunoassay. A group of HS hens received injections of aldosterone on a 6-hr schedule before experiments. Another group of LS hens was resalinated , and experiments carried out on a 24hr interval.

The mammalian colon secretes HCOf and in vivo studies have frequently demonstrated that HCO3 secretion is lumen Cl-dependent. However, in prior studies usingisolated perfused rat distal colonic crypts we observed minimal HCO3 absorption in the basal state and cAMP stimulated lumen Cl-independent HCO3 secretion via an apical membrane anion channel (Gastroenterology 118:101, 2000). To determine whether HCO3 secretion from surface cells represents a similar or different process we employed a pH star method to determine HCOf secretion across intact stripped colonic mucosa from rat distal colon. Luminal HCO3 concentration was determined using bi-burette pH stat technique in stripped rat colonic mucosa. In the absence of bath HCO3, HCOf secretion was minimal (0.1 +_ 0.01 IJ~eq/cm2/ h). In contrast, HCO3 secretion (5.6-0.031zeq/cm2/h) was seen in the presence of bath HCOf (basal secretion). The data presented in this table indicates the presence of three distinct HCOf secretory processes.Basal HCOf secretion is lumen Cl-dependent, inhibited by DIDS, an anion exchange inhibitor, but not by NPPB, a non-specific anion channel blocker, indicating a role for an apical membrane C1-HCOf exchange. Addition of bntyrate also stimulated HCOf secretion which is Cl-independent but was not inhibited by either DIDS or NPPB indicating a role for an apical membrane SCFA-HCOf exchange that is independent of C1-HCOf exchange. HCOf secretion is also present following the addition of 8-Br-cAMP but with different characteristics: lumen Cl-independent, DIDS-insensitive but inhibited by NPPB, indicating a requirement for an apical membrane anion channel(s). This HCOf secretion was only partially inhibited by a CFTR inhibitor 100 IxM glibendamide (3.3+-0.031zeq/cm2/h). Conclusions: At least three distinct HCOf secretory processes are present in the rat distal colon: 1) a basal, Cl-dependent HCO3 secretion via C1-HCOf exchange in surface cells, 2) butyrate-stimulated, Cl-independent HCOf secretion via SCFA-HCOf exchange and 3) a cAMP activated, Cl-independent HCOf secretion via anion channel(s) that may be present in both surface and crypt cells.

This communication summaries a series of observations of the transport function of the crypt of the rat distal colon. Development of methods to study both 22 Na uptake by apical membrane vesicles prepared from crypt cells and intracellular pH i (pH i ), fluid movement (Jv), and bicarbonate secretion during microperfusion of the crypt has led to the identification of (1) a novel Cl-dependent Na-H exchange (Cl-NHE) that most likely represents the coupling of a Cl channel to a Na-H exchange isoform that has not as yet been identified and (2) bicarbonate secretion that appears to be most consistent with HCO 3 uptake across the basolateral membrane by a mechanism that is closely linked to Cl transport and its movement across the apical membrane via an anion channel. Na-dependent fluid absorption is the constitutive transport process in the crypt, while fluid secretion is regulated by one or more neurohumoral agonists. Cl-NHE is responsible for both the recovery/regulation of pHi in crypt cells to an acid load and fluid absorption.

The purpose of the present study was to investigate whether active Cl − secretion in the pigmented rabbit conjunctiva was subject to cAMP, Ca# + and protein kinase C (PKC) modulation. The excised pigmented rabbit conjunctivas were mounted in the modified Ussing-type chambers for measurement of unidirectional $'Cl fluxes under the open-circuit condition and of the short-circuit current (I sc ), potential difference, and transconjunctival electrical resistance. The results indicate that Cl − secretion across the conjunctiva was abolished by mucosal application of 1 m N-phenylanthranilic acid and was reduced by 40 % by serosal application of 10 µ bumetanide. Net Cl − flux was stimulated by 133 % by 1 m 8-Br cAMP, 107 % by 10 µ A23187, and 87 % by 1 µ phorbol 12-myristate-13-acetate (PMA), suggesting that cAMP, Ca# + , and PKC all modulated active Cl − secretion, respectively. There existed a linear correlation between measured changes in net Cl − flux and observed changes in I sc (r# l 0n99). The serial treatment of the conjunctiva with (a) 1 m 8-Br cAMP and 10 µ A23187 and (b) 10 µ A23187 and 1 µ PMA resulted in sequence-independent, additive stimulation of I sc . In the case of 1 m 8-Br cAMP and 1 µ PMA, additive stimulation of I sc was observed only when 1 m 8-Br cAMP was added prior to 1 µ PMA. These results suggest that a given pharmacological agent may affect more than one channel type and that there might be a possible connection among the channels at the signal transduction level. In summary, Cl − appears to enter the pigmented rabbit conjunctiva from the serosal fluid via Na + -(K + )-2Cl − cotransport process and exit to the mucosal fluid via channels, resulting in active Cl − secretion. Active Cl − secretion in the pigmented rabbit conjunctiva appears to be modulated by cAMP, Ca# + , and PKC.

The purpose of the present study was to investigate whether active Cl − secretion in the pigmented rabbit conjunctiva was subject to cAMP, Ca# + and protein kinase C (PKC) modulation. The excised pigmented rabbit conjunctivas were mounted in the modified Ussing-type chambers for measurement of unidirectional $'Cl fluxes under the open-circuit condition and of the short-circuit current (I sc ), potential difference, and transconjunctival electrical resistance. The results indicate that Cl − secretion across the conjunctiva was abolished by mucosal application of 1 m N-phenylanthranilic acid and was reduced by 40 % by serosal application of 10 µ bumetanide. Net Cl − flux was stimulated by 133 % by 1 m 8-Br cAMP, 107 % by 10 µ A23187, and 87 % by 1 µ phorbol 12-myristate-13-acetate (PMA), suggesting that cAMP, Ca# + , and PKC all modulated active Cl − secretion, respectively. There existed a linear correlation between measured changes in net Cl − flux and observed changes in I sc (r# l 0n99). The serial treatment of the conjunctiva with (a) 1 m 8-Br cAMP and 10 µ A23187 and (b) 10 µ A23187 and 1 µ PMA resulted in sequence-independent, additive stimulation of I sc . In the case of 1 m 8-Br cAMP and 1 µ PMA, additive stimulation of I sc was observed only when 1 m 8-Br cAMP was added prior to 1 µ PMA. These results suggest that a given pharmacological agent may affect more than one channel type and that there might be a possible connection among the channels at the signal transduction level. In summary, Cl − appears to enter the pigmented rabbit conjunctiva from the serosal fluid via Na + -(K + )-2Cl − cotransport process and exit to the mucosal fluid via channels, resulting in active Cl − secretion. Active Cl − secretion in the pigmented rabbit conjunctiva appears to be modulated by cAMP, Ca# + , and PKC.

Summary Addition of BaCl2 to the solution bathing colons of normal lizards did not alter the absorptive Na+ flux, but did reverse the net absorption of Cl 2 to become net secretion. Cl2 secretion resulted from an increase in its serosal-to-mucosal movement and was positively correlated to an increase in short-circuit current. Ba2+-induced short-circuit current was dependent on the presence

Pulmonary oedema results from elevated transvascular pressure gradients, as occur in heart failure, or from increased vascular permeability to solutes, as occurs in the premature and adult respiratory distress syndromes. It is important to study mechanisms involved in pulmonary oedema clearance, as little is gained if one removes the cause of the oedema and the lungs cannot clear the alveolar fluid. Indeed, adults with cardiogenic or non-cardiogenic oedema survive if they have active absorption of airspace fluid, yet die if they show no evidence of active fluid clearance ). The Na + and fluid transport pathways required to clear alveolar pulmonary oedema are incompletely understood and the specific effects of pulmonary oedema fluid itself on the ion and fluid transporting ability of the distal lung are unknown.

The effects of the diuretic drug ozolinone on electrogenic Cl− secretion by rabbit colonic mucosa were investigated in vitro. Electrical properties and unidirectional Cl− fluxes were measured in stripped preparations mounted in Ussing-type chambers. After abolition of electrogenic Na+ absorption by amiloride (10−4 mol/l) on the mucosal side electrogenic Cl− secretion was induced by addition of PGE1 (10−6mol/l, serosal side) and theophylline (10−2mol/l, both sides). Under these conditions, the monitored short-circuit current (Isc) equals the amount of Cl− secreted as evidenced by determination of unidirectional Cl− fluxes. After establishing a stable Cl− secretion its sensitivity to the enantiomers of the diuretic was studied. Only levorotatory (-)-ozolinone, but not the dextrorotatory (+)form, inhibited Cl− secretion on serosal application. This effect was fully accounted for by a reduction in the serosal-to-mucosal Cl− fluxes (J sm Cl ). It was readily reversible and concentration-dependent with a K i value of 6×10−4mol/l, but absent when the drug was added to the mucosal side. The results are in agreement with the hypothesis that loop diuretics inhibit a coupled NaCl entry mechanism across the baso-lateral membrane into colonic epithelial cells. This mechanism is though to account for Cl− influx into the cells.