Bicarbonate presence in the bathing media doubles Na+ and fluid transepithelial transport and in parallel significantly increases Na+ and Cl- intracellular concentrations and contents, decreases K+ cell concentration without changing its amount, and causes a large cell swelling. Na+ and Cl- lumen-to-cell influxes are significantly enhanced, Na+ more so than Cl-. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO3(-)-ATPase in the brush border. The stimulation goes together with a large increase in a Na+-dependent H+ secretion into the lumen. All of these data suggests that HCO3- both activates Na+--Cl- cotransport and H+--Na+ countertransport at the luminal barrier. Thiocyanate inhibits Na+ and fluid transepithelial transport without affecting H+ secretion and HCO3(-)-dependent Na+ influx. It reduces Na+ and Cl- conentrations and contents, increases the same parameters for K+, causes a cell shrinking, and abolishes the lumen-to-cell Cl- influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na+-dependent and HCO3(-)-activated. Thus SCN- is likely to compete for the Cl- site on the cotransport carrier and to be slowly transferred by the cotransport system itself.

The reported experiments demonstrate that in rabbit gallbladder epithelium: a) the mucosa-cell Na+ and Cl- influxes (45 sec long) are reduced in Cl- -free and Na+ -free bathing solutions respectively; transconcentration effects are observed. b) Cell Cl- labelling through the luminal membrane, with 36Cl- (1 hr incubation), is nearly abolished if a Na+ -free bathing solution is used; but cell Na+ labelling with 22Na+ in the lumen (1 hr incubation) does not change if a Cl- -free bathing solution is employed. c) Cell Na+ and Cl- labelling through the basolateral membrane (1 hr incubation) is negligible; some Cl- labelling is obtained only when the intracellular electrical potential is artificially reduced. d) SO 2 4- is found to abolish net water transport, to cross the epithelium, but not to enter the cell. On the basis of these data a paracellular pathway for net Cl- transport is ruled out, a Na+ and Cl- cotransport through the cell luminal barrier and a Na+ active extrusion through the basolateral membrane are suggested. No definitive conclusion about Cl- extrusion is possible.

Summary. When amphotericin B is added tQ the medium bathing the luminal side of a rabbit gallbladder preparation, a serosa positive transmural p.d. (+2 to + 8 mV) arises in a few minutes.Some authors have suggested [16] that the antibiotic would reduce tight-junction selectivity and the negative p.d. due to the backdiffusion of Na + salts from the lateral spaces: then the opposite positive p.d., created by a hypothetical electrogenic Na + pump, would be revealed. Against such an explanation, the experiments reported here show that, in parallel with the transepithelial p.d. changes, after the antibiotic addition, the luminal membrane potential is largely depolarized and the ratio between the mucosal and serosal cell resistance decreases. Moreover, the dependence on K + of the luminal membrane potential is strongly reduced. Ten minutes after the antibiotic addition, modifications of cell water, of cell ion concentrations and contents and of net water transport begin to be observed. Conversely, during the first 10-rain period of treatment, no alteration in tightjunction selectivity is detectable by imposing dilution potentials across the tissue; by tracer technique a significant decrease in tight-junction selectivity is observed only 30 rain after treatment.Choline substitution for Na + completely abolishes amphotericin B effects, whereas C1 replacement by SO 2-does not affect the polyene action. As a conclusion, the primary action of the antibiotic consists of an increase of Na + conductance at the luminal cell barrier. Only a small fraction of the actual emf variation is measured across the whole epithelium because of the shunt in tight junctions.Rabbit gallbladder develops only a slightly negative (serosa negative) transmural potential when bathed on both sides by Krebs-Henseleit solution [5,9,10]. This is in contrast with the majority of the absorbing epithelia which exhibit a positive transmural p.d. of some mV [2,13,17,19]. Some years ago we found that in rabbit gallbladder a positive