HT-29, an undifferentiated human colon cell line, is known to differentiate when cultured without glucose. This study aimed to characterize ion transport in the clone HT-29/B6, which was selected from HT-29 cells differentiated by glucose-free culture. HT-29/B6 cells seeded onto filter membranes grew as polarized monolayers, mainly consisting of mucus-forming cells and exhibiting high transepithelial resistance. Short-circuit current (Isc) of unstimulated HT-29/B6 monolayers in Ussing chambers was 0.1 +/- 0.01 mumol.h-1.cm-2, and conductance was 2.0 +/- 0.2 mS/cm2. Serosal forskolin (FSK; 10(-5) M) induced a sustained Isc of 1.9 +/- 0.1 mumol.h-1.cm-2, associated with a rise of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Isc was identified as Cl- secretion by tracer studies and by the inhibitory effects of serosal bumetanide and Ba2+. The Cl- channel blockers NPPB and DPC diminished FSK-induced Isc at respective doses of 3 x 10(-4) and 10(-3) M, being effective from either side of the monolayer. Cl- secretion could be triggered by vasoactive intestinal peptide (10(-8) M), prostaglandin E1 (10(-6) M), and dibutyryl cAMP (10(-3) M) as well. In conclusion, HT-29/B6 cells grow as polarized monolayers, forming mucus and secreting Cl- in response to secretagogues. This clone may not only serve as a model for investigation of cellular mechanisms of intestinal Cl- secretion but may also be helpful to elucidate the contribution of mucus cells to this process.

It has been possible to obtain in a mammalian epithelium of dietetically and surgically untreated animals a dose response of in vitro-added aldosterone (Aldo, 10(-10) to 10(-5) M) on electrogenic Na+ absorption (JeNa). JeNa was measured in the Ussing chamber on stripped rat late distal colon 8 h after in vitro addition of Aldo. Submaximal effects were obtained at 3 nM Aldo; after a lag time of 2 h, short-circuit current (Isc) increased to a maximum of 234 +/- 15 microA/cm2 and dropped after 0.1 mM amiloride to -18 +/- 3 microA/cm2, resulting in JeNa of 9.4 +/- 0.6 mumol.h-1 x cm-1. Net Na+ tracer fluxes and Isc exhibited parallel time courses, so that electroneutral Na+ transport was not induced in late distal colon by acute Aldo. A plot of JeNa vs. Na conductance revealed an electromotive force (ENa) of 126 +/- 1 mV for all Aldo concentrations tested. Kinetic data were as follows: Michaelis constant 1.2 nM, maximal velocity (Vmax) 10.5 mumol.h-1 x cm-2, and Hill coefficient 2.1. In contrast to the large effect in late distal colon, 3 nM Aldo caused JeNa of < 1 mumol.h-1 x cm-2 in early distal colon, proximal colon, and cecum. Antimineralocorticoid sensitivity and ENa did not vary with Aldo concentration or time of the experiment, consistent with a unique mechanism during the early and late response up to 8 h, as well as at mineralocorticoid and glucocorticoid Aldo concentrations. Acute Aldo in a range of 0.1-10 nM fully controls JeNa between zero and Vmax in late distal colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelial and subepithelial resistance of rat jejunum was measured in vitro by two independent methods. (i) Transepithelial AC impedance data were interpreted in terms of a simple parallel RpCp element (representing the epithelial cell layer) in series with an ohmic resistor RS (representing the subepithelial layers). (ii) In separate experiments, the tip of a microelectrode was positioned between epithelium and subepithelial layers and the respective resistances were obtained from DC-pulse voltage divider ratios between both structures. The total tissue resistance as measured in conventional Ussing-chamber experiments (49 +/- 4 Ohm X cm2, mean of both methods) was formed to 81 +/- 6% (40 +/- 3 Ohm X cm2) by subepithelial layers and to only 19 +/- 3% (9 +/- 1 Ohm X cm2) by the epithelial cell line. We conclude that rat jejunum is more conductive than assumed so far. In in vitro flux studies on intact jejunal sheets a pronounced back-diffusion of absorbed substances will lead to an underestimation of the true net transport capacity of this structure. This error averages about fivefold and will be found likewise in conventional short-circuit measurements.

Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

Functionally isolated segments of rat colon and rectum were perfused in situ in a closed loop system. Rectum was defined as the lower 25--35% of the length of large intestine (cecum excluded). Perfusion conditions were optimized at 0.5 ml.min-1 and 3 cm H2O luminal pressure. Variation of perfusion rate between 0.2 and 2 ml.min-1 did not influence net volume transport (JNV). Luminal distension following elevation of hydrostatic pressure to 18 cm H2O reversibly increased Jnv. Under control conditions Jnv and Na+-transport rates (JnNa) of colon were 2--3 times higher than those of rectum. In colon transepithelial electrical potential difference (psims) was time independent --12 mV (lumen negative) whereas rectal psims increased with time from --6 mV, reaching a plateau of --67 mV within 6 h. Amiloride 10(-4) mol.l-1 had no effect on psims, Jnv, and JnNa in colon but did slightly depress K+-secretion in colon descendens. In contrast, psims in rectum was dose-dependently depressed, being reversed to +7 mV at 10(-4) mol.l-1. Jnv and JnNa were decreased by half. Acetazolamide in addition to amiloride lowered the positive post-amiloride rectal psims by half. Adrenalectomy had no effect on colonic psims, but abolished psims of the rectum. A single dose of 40 microgram.kg-1 b.w. aldosterone during the experiment restored the typical time course of rectal psims, but did not affect psims in colon. It is concluded that aldosterone induces an amiloride-sensitive Na+-pathway only in rectum, but not in colon, and that colon and rectum differ basically in their transport properties, quantitatively as well as qualitatively, as do the kidney distal convoluted tubule and the cortical collecting duct.