Analysis of epithelial transport by measurement of K+, Cl- and pH gradients in extracellular unstirred layers: Ion secretion and reabsorption by Malpighian tubules of Rhodnius prolixus

The pH and concentrations of K⁺ and Cl^- in the unstirred layer (USL) associated with the basolateral surfaces of the upper and lower Malpighian tubules of Rhodnius prolixus were measured using extracellular ion-selective microelectrodes. When stimulated with 5-hydroxytryptamine (5-HT) in vitro, the upper Malpighian tubule secretes Na⁺, K⁺, Cl^- and water at high rates; the lower Malpighian tubule reabsorbs K⁺ and Cl^- but not water. Concentrations of K⁺ and Cl^- in the unstirred layer of the lower Malpighian tubule ([K⁺]USL, [Cl^-]USL,) were greater than those in the bathing saline, consistent with the accumulation of K⁺ and Cl^- in the USL during 5-HT-stimulated KCl reabsorption. [K⁺]USL exceeded [K⁺]Bath by as much as 5.3-fold. Calculations of K⁺ flux based on measurements of [K⁺]USL at various distances from the tubule surface agreed well with flux calculated from the rate of fluid secretion and the change in K⁺ concentration of the secreted fluid during passage through the lower tubule. Concentrations of K⁺ in the unstirred layer of the upper Malpighian tubule were reduced relative to those in the bathing saline, consistent with depletion of K⁺ from the USL during 5-HT-stimulated secretion of K⁺ from bath to lumen. Changes in [K⁺]USL during 5-HT-stimulated K⁺ secretion from single upper Malpighian tubule cells could be resolved. Although differences between [K⁺]USL and [K⁺]Bath were apparent for upper and lower tubules in an in situ preparation, they were reduced relative to the differences measured using isolated tubules. We suggest that convective mixing of the fluids around the tubules by contractions of the midgut and hindgut reduces, but does not eliminate, differences between [K⁺]USL and [K⁺]Bath in situ. The USL was slightly acidic relative to the bath in 5-HT-stimulated upper and lower tubules; contributions to USL acidification are discussed. The results also show that the techniques described in this paper can resolve rapid and localized changes in ion transport across different regions of Malpighian tubules in response to stimulants or inhibitors of specific membrane transporters.