We determined the site of kininogenase secretion in the nephron by performing stop-flow studies in dogs. Kininogenase activity, inulin, sodium, and potassium were measured in the same fractions. The highest kininogenase concentration was found in the fractions with the lowest sodium concentration corresponding to urine samples that must have been trapped in the distal nephron. Kininogenases in these urine fractions were 5-17 times higher than in fractions from the proximal nephron or from the free-flow sample. The ratio of kininogenase increase to inulin increase was always higher than 3.0, thus indicating that the increase in kininogenase concentration was mainly due to secretion and not to water reabsorption. When a peak of potassium was present, it occurred 1-2 ml before the peak of kininogenases. We concluded that kininogenases are secreted into the urine at the level of the distal nephron by either the tubule itself or by a specialized structure located at this part of the nephron, such as the macula densa.

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The mammalian kidney bumetanide-sensitive Na+-K+-2Cl− and thiazide-sensitive Na+-Cl− cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na+-K+-2Cl− and Na+-Cl− cotransporters to design and study chimeric proteins in which the NH2-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na+-K+-2Cl− cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH2-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na+-Cl− cotransporter.

In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP. The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added. It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.