Active and passive properties of rabbit descending colon: A microelectrode and nystatin study

Abstract

The electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K⁺ activity (K⁺) was measured using liquid ion exchanger microelectrodes ((K⁺)=76±2mm) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K₂SO₄ Ringer's solution which was designed to mimic intracellular K⁺ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (R _s =691±63 Ω cm² and 770±247 Ω cm², respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705±123 V cm² and basolateral membrane resistance was 95±14 V cm². To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringer's solution followed by a series of changes in which all or part of the Na⁺ was replaced by equimolar amounts of K⁺. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows:P_Na/P_K=.08 andP_Cl/P_K=.07 (uncorrected for shunt) andP_Na/P_K=.04 andP_Cl/P_K=.06 (basolateral membrane alone). In a second set of experiments, evidence for an electrogenic Na⁺ pump in the basolateral membrane is presented. A small ouabain-sensitive potential could be generated in the nystatin-treated colon in the absence of chemical or electrical gradients by mucosal, but not serosal, addition of NaCl. We conclude that this electrogenic pump may contribute to the basolateral membrane potential; however, the primary source of this potential is “passive”: specifically, a potassium gradient which is maintained by an “active” transport process. An appendix compares the results of nystatin experiments to amiloride experiments which were conducted separately on the same tissues. The purpose of this comparison was to develop a comprehensive model of colonic transport. The analysis reveals a leak conductance in the apical membrane and the presence of an amiloride-insensitive conductance pathway.