Anti-Diarrheal Mechanism of the Traditional Remedy Uzara via Reduction of Active Chloride Secretion

BACKGROUND AND PURPOSE: The root extract of the African Uzara plant is used in traditional medicine as anti-diarrheal drug. It is known to act via inhibition of intestinal motility, but malabsorptive or antisecretory mechanisms are unknown yet. EXPERIMENTAL APPROACH: HT-29/B6 cells and human colonic biopsies were studied in Ussing experiments in vitro. Uzara was tested on basal as well as on forskolin- or cholera toxin-induced Cl(-) secretion by measuring short-circuit current (I(SC)) and tracer fluxes of (22)Na(+) and (36)Cl(-). Para- and transcellular resistances were determined by two-path impedance spectroscopy. Enzymatic activity of the Na(+)/K(+)-ATPase and intracellular cAMP levels (ELISA) were measured. KEY RESULTS: In HT-29/B6 cells, Uzara inhibited forskolin- as well as cholera toxin-induced I(SC) within 60 minutes indicating reduced active chloride secretion. Similar results were obtained in human colonic biopsies pre-stimulated with forskolin. In HT-29/B6, the effect of Uzara on the forskolin-induced I(SC) was time- and dose-dependent. Analyses of the cellular mechanisms of this Uzara effect revealed inhibition of the Na(+)/K(+)-ATPase, a decrease in forskolin-induced cAMP production and a decrease in paracellular resistance. Tracer flux experiments indicate that the dominant effect is the inhibition of the Na(+)/K(+)-ATPase. CONCLUSION AND IMPLICATIONS: Uzara exerts anti-diarrheal effects via inhibition of active chloride secretion. This inhibition is mainly due to an inhibition of the Na(+)/K(+)-ATPase and to a lesser extent to a decrease in intracellular cAMP responses and paracellular resistance. The results imply that Uzara is suitable for treating acute secretory diarrhea

Inhibitory effect of Uzara on the Na⁺/K⁺-ATPase enzymatic activity in membrane preparations from HT-29/B6 cells.

<p>In cell culture experiments, monolayers were treated with 50 µ µg/ml Uzara for 90 min. Then, cells were homogenized and a crude membrane fraction was prepared. Herein, the Na⁺/K⁺-ATPase activity was measured (n = 5). In a further set of experiments, membrane fractions of untreated HT-29/B6 cell monolayers were prepared and subsequently treated with 50 µg/ml Uzara which was added for 1 h to the enzyme activity assay (n = 8). Both conditions revealed a direct inhibitory activity on the ion pump. Values are expressed as percentage of control activity. Basal activity of the Na⁺/K⁺-ATPase was between 36 and 73 nmol P_i/(min·mg protein). Data are expressed as mean ± s.e.m. Asterisks indicate difference versus control: ***P<0.001.</p

Effects of DIDS on forskolin-induced I_SC in the absence and presence of Uzara or ouabain.

<p>Experiment in HCO₃⁻ free solutions equilibrated with 100% O₂. Application of 10 µM forskolin induced an I_SC that was reduced in the presence of Uzara (50 µg/ml) and ouabain (100 µM). Under all three conditions, application of DIDS (100 µ µM) caused a pronounced transient increase in I_SC, presumably due to an increase in intracellular Ca²⁺. Similar to the carbachol-induced I_SC, the current amplitude was reduced in the presence of Uzara and ouabain. I_SC in the presence of DIDS then rapidly decreased, if preparations were pre-incubated with Uzara (light blue) or ouabain (pink). This effect is attributed to additional Na⁺ loading of the cells. Application of DIDS before the addition of Uzara (blue) had no such accelerating effect.</p

Influence of Uzara and forskolin on Na⁺ and Cl⁻ fluxes in HT-29/B6 cells.

<p>J_Na and J_Cl, unidirectional Na⁺ and Cl⁻ fluxes; ^ms, from mucosal to serosal side; ^sm, from serosal to mucosal side; all fluxes are expressed as µmol·h⁻¹·cm⁻².</p><p>J^net, net fluxes (J^ms-J^sm).</p><p>J_res, residual fluxes (I_SC-J_Na^net+J_Cl^net, usually assumed to reflect bicarbonate secretion).</p><p>I_SC, short-circuit current (for comparison expressed in µmol·h⁻¹·cm⁻² of monovalent cations).</p><p>R^t, transepithelial resistance (Ω·cm²).</p><p>All values represent means ± SEM.</p><p>*, P versus control <0.05.</p><p>#, P versus control <0.001.</p><p>§, P versus forskolin alone <0.001.</p

Inhibitory effect of Uzara on the forskolin-induced I_SC rise in HT-29/B6 cells.

<p>I_SC was measured on HT-29/B6 monolayers mounted in Ussing chambers. In A and B, Uzara (50 µg/ml) was added on the basolateral side either 30 minutes prior (A) or 30 minutes after (B) basolateral addition of 10 µM forskolin. In C, different Uzara concentrations were applied basolaterally 30 minutes after forskolin stimulation. The percentage of the inhibitory activity of Uzara to the forskolin-induced I_SC rise was calculated after an incubation time of 1 hour. An IC₅₀ of 17 µg/ml and a Hill coefficient of 1.45 were determined from a Hill plot diagram and used to calculate the continuous line. All values are expressed as mean ± s.e.m. (n = 5-7). D: Transepithelial resistance measurements over 48 hours after application of either ethanol (control: final concentration 0.05%) or 50 µg/ml Uzara (final ethanol concentration 0.054%). With the exception of the resistances 24 hours after the application of uzara, none of the resistance values in the presence of uzara differed significantly from the corresponding control value. (means ± s.e.m. n = 12).</p

Inhibitory effect of Uzara and ouabain on the nystatin-induced I_SC rise in HT-29/B6 cells.

<p>The effects of Uzara and ouabain, a specific inhibitor of the Na⁺/K⁺-ATPase, were analyzed on the rise in I_SC induced by nystatin. Nystatin (15 µg/ml) which perforates cell membranes was applied to the mucosal compartment 30 min prior to basolateral addition of 50 µg/ml Uzara or 100 µM ouabain. I_SC was measured in HT-29/B6 cell monolayers mounted in Ussing chambers. The nystatin-induced rise in I_SC was diminished by Uzara as well as by ouabain. Data are expressed as mean ± s.e.m. (n = 4).</p

Inhibitory effect of Uzara on the forskolin-mediated I_SC rise in human colonbiopsy specimens.

<p>Delta I_SC (ΔI_SC) was determined in human colon mounted in Ussing chambers. Forskolin (10 µM) was added to the basolateral side 15 min prior to basolateral addition of 50 µg/ml Uzara. Also in colonic biopsies, Uzara diminished the forskolin-induced rise in I_SC. Data are expressed as mean ± s.e.m. (n = 5).</p

Inhibitory effect of Uzara on the forskolin-induced accumulation of intracellular cAMP in HT-29/B6 cells.

<p>HT-29/B6 monolayers were incubated basolaterally with 50 µg/ml Uzara 30 min prior to basolateral forskolin (10 µM) addition. After an incubation time of 1 h, cell lysates were prepared and the content of cAMP was assayed with EIA. The data shows that forskolin increased the intracellular cAMP level, whereas Uzara partly blocked this rise in cAMP. Values are expressed as mean ± s.e.m. (n = 5-6). Asterisks indicate difference versus control: ***P<0.001.</p

Effects of Uzara, ouabain and forskolin on R^para and R^trans in HT-29/B6 cells.

<p>The influence of Uzara and ouabain on R^para and R^trans with and without forskolin was determined by two-path impedance spectroscopy in HT-29/B6 cell monolayers mounted in Ussing chambers. Forskolin reduced R^trans, whereas R^para remained unaltered. The co-incubation of Uzara with forskolin caused no change of R^trans compared to forskolin alone, whereas R^para was clearly reduced in the co-incubation group. Similarly, compared to forskolin alone, ouabain together with forskolin had no effect on R^trans. The slight reduction of R^para observed under these conditions did not reach significance. Data are expressed as mean ± s.e.m. (n = 5-6). Untreated control values set as 1. Asterisk indicates a difference compared to forskolin: **P<0.01. Hash keys mean no statistical significance compared to forskolin alone.</p