A BASIC program is presented which facilitates the formulation of biologically relevant chemical solutions containing specified free concentrations of as many as three divalent metal cations (Ca2+, Mg2+ and the choice of a third divalent cation) at any pH in the presence of as many as three ligands (EGTA, ATP and GTP). The program uses the law of mass action and the absolute stability (association) constants found in the literature to calculate the total concentration of divalent metal cation needed to achieve a desired free concentration. The user enters the pH, the concentrations of the ligands used and the desired free concentrations of the divalent cations. This program was developed for use in a wide range of biological applications, particularly the rapid design of solutions which mimic certain aspects of intracellular fluid.

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of SnRb, [nH]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K + and CIcurrents were generated by imposing transmural ion gradients, and cytosolic free Ca ~+ was manipulated by means of a Ca~ § buffer system in the mucosal bathing solution. Raising the cytosolic free Ca ~ § concentration from the nanomolar to the micromolar range activated basolateral conductances for K + and CI-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca~ § increases in the K § and CI-conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca~+-activated conductive pathways for K § and CI-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca~ § channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.

A B S T R A C T The role of intracellular pH as a modulator of basolateral K ÷ and Clconductances in epithelial cells was studied using digitonin-permeabilized colonic cell layers so that cytosolic pH could be clamped at specific values, while basolateral K * and Ci-conductances were activated by stepwise increases in intracellular free Ca 2+. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to intracellular Ca z+, but changing extracellular pH had no effect. Maximal K + and CI-currents activated by Ca 2+ were not affected by changes in intracellular pH, suggesting that protons do not alter the conduction properties of the channels. Hill analysis of the Ca 2+ activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K~/~ for Ca 2+ activation. In the absence of Ca z+, changes in intracellular pH did not have a significant effect on the basolateral K + and C1-conductances. These results are consistent with the notion that changes in cytosolic pH can modulate basolateral conductances by modifying the action of calcium, perhaps by acting at or near the activation site to provide a mechanism of variable "gain control."

The effects of HgCl2 on ion transport were investigated using isolated sheets of flounder urinary bladder, a model epithelium that is capable of electrically silent NaCl absorption and electrogenic K secretion. Exposure of the mucosal surface of the bladder to submicromolar doses of HgCl2 reduced K secretion, but the effect was not due to blockade of apical K channels. Rather, the effects of HgCl2 were virtually identical to those seen with experimental maneuvers that blocked the thiazide-sensitive NaCl cotransporter in the apical membrane, e.g., hydrochlorothiazide, Cl-free solutions, and Na-free solutions. Mucosal HgCl2 also blocked 22Na absorption, suggesting that the effect of the metal was mediated by blockade of NaCl entry. The effects of HgCl2 had a rapid onset and were readily reversed by washing, suggesting a noncovalent binding reaction. The abundance of polyanionic Hg complexes in salt solutions prompts the speculation that one of these may bind to the Cl-binding site on the cotransporter, thereby blocking it. The results provide the first evidence that the thiazide-sensitive NaCl cotransporter is a specific site of action for inorganic mercury.

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