ANALYSIS OF MALPIGHIAN TUBULE ION TRANSPORT BY MEASUREMENT OF ION CONCENTRATION GRADIENTS IN EXTRACELLULAR UNSTIRRED LAYERS

Abstract

The pH and concentrations of K+ and Cl- in the unstirred layer (USL) associated with the basolateral surfaces of Malpighian tubules of Rhodnius prolixus and Drosophila melanogaster were measured with extracellular ion-selective microelectrodes. When stimulated with 5 hydroxytryptamine (5HT) in vitro, the upper Malpighian tubule of R. prolixus 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, [C1-]USL) were above those in the bathing saline, consistent with accumulation of K+ and Clin the USL during 5HT-stimulated KC1 reabsorption. [K+]USL exceeded [K+]BATH 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 potassium 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 5HT-stimulated secretion of K+ from bath to lumen. Changes in [K+]USL during 5HT-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 with isolated tubules. It is suggested 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 5HTstimulated upper and lower tubules; contributions to USL acidification are discussed. When stimulated with leukokinin (LKI) and cAMP in vitro, the main segment of the D. melanogaster MT secretes Na+, K+, and Cl". Concentrations of [K+] in the USL of the main segment were lower than those in the bathing saline at both stellate cell sites and principal cell sites, consistent with depletion of K+ in the USL during LKI/cAMPstimulated secretion. [K+]USL at sites near stellate cells was significantly lower (p<0.05) than at sites near principal cells. Reasons for this non-uniformity are discussed. [K+]USL also exceeded [K+]BATH at principal cell sites in both the proximal and distal regions of MT, consistent with LKI/cAMP-stimulated reabsorption of K+. Studies using a Cl--sThe pH and concentrations of K+ and Cl- in the unstirred layer (USL) associated with the basolateral surfaces of Malpighian tubules of Rhodnius prolixus and Drosophila melanogaster were measured with extracellular ion-selective microelectrodes. When stimulated with 5-hydroxytryptamine (5HT) in vitro, the upper Malpighian tubule of R. prolixus 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, [C1-]USL) were above those in the bathing saline, consistent with accumulation of K+ and Clin the USL during 5HT-stimulated KC1 reabsorption. [K+]USL exceeded [K+]BATH 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 potassium 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 5HT-stimulated secretion of K+ from bath to lumen. Changes in [K+]USL during 5HT-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 with isolated tubules. It is suggested 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 5HTstimulated upper and lower tubules; contributions to USL acidification are discussed. When stimulated with leukokinin (LKI) and cAMP in vitro, the main segment of the D. melanogaster MT secretes Na+, K+, and Cl". Concentrations of [K+] in the USL of the main segment were lower than those in the bathing saline at both stellate cell sites and principal cell sites, consistent with depletion of K+ in the USL during LKI/cAMPstimulated secretion. [K+]USL at sites near stellate cells was significantly lower (p<0.05) than at sites near principal cells. Reasons for this non-uniformity are discussed. [K+]USL also exceeded [K+]BATH at principal cell sites in both the proximal and distal regions of MT, consistent with LKI/cAMP-stimulated reabsorption of K+. Studies using a Cl--selective vibrating microelectrode were inconclusive. The results also show that techniques described in this thesis 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.elective vibrating microelectrode were inconclusive. The results also show that techniques described in this thesis 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.