Peter Deetjen

Salt and Water Balance

In warm-blooded animals water makes up about 60 percent of the body weight and exists as a solution of organic and mineral substances. This water is in constant exchange with the environment as a result of periodic uptake from the gut and continual loss through the skin, respiratory passages, and kidney. Within the body the water is distributed in several more or less discrete compartments whose contents are called the “body fluids.” The anatomical boundaries separating these compartments and the differences in the solutes present in each are of fundamental biological significance.

Methode zur Durchstrmung einzelner Nephronabschnitte

ZusammenfassungEs wird eine Mikroperfusionsapparatur beschrieben, mit der konstante Perfusionsraten zwischen 10 und 35 · 10−6 ml/min eingestellt werden können. Mit Hilfe dieser Apparatur können Teile eines Nephrons in situ kontinuierlich durchströmt werden. Die angegebene Technik erlaubt Tubulusperfusionen, ohne daß Beimischungen von Glomerulumfiltrat das Perfusat verändern, so daß Messungen lokaler, tubulärer Transportraten von Elektrolyten und Nichtelektrolyten oder Permeabilitäten von Wasser und gelösten Substanzen möglich sind.

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release

To test for a possible role of lysosomes in intracellular Ca2+ homeostasis, the effects of glycyl-L-phenylalanine-beta-naphthylamide (GPN), known to permeabilize these organelles by osmotic swelling, were studied in single MDCK cells. Fluorescence of acridine orange, rhodol green dextran, lysotracker green and FITC-dextran indicated that GPN (0.2 mmol/l) elicited a reversible permeabilization of lysosomes. Cytosolic Ca2+ ([Ca2+]i) as determined by Fura-2 fluorescence increased from 60 +/- 11 to 534 +/- 66 nmol/l (n = 41) in the presence of GPN. Whereas only a single intracellular Ca2+ release could be induced by GPN in a Ca(2+)-free perfusate, repetitive release could be evoked in Ca2+ containing solutions suggesting reuptake of Ca2+ into lysosomal stores. GPN-induced Ca2+ release was blunted after pretreatment with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, or repeated applications of ATP inducing Ca2+ release from inositol trisphosphate (InsP3) sensitive Ca2+ stores. The effect of ATP on Ca2+ release was, however, not abolished by preceding GPN treatment. GPN-induced Ca2+ release from lysosomes was independent of InsP3 formation or Ca(2+)-induced Ca2+ release, since it was unaffected by the phospholipase C inhibitor U-73, 122 or by caffeine and ruthenium red. These results suggest that Ca2+ largely accumulates in lysosomal vesicles. Moreover, these organelles seem to be part or functionally coupled with InsP3-sensitive Ca2+ stores.

Na+/H+Exchangers: Linking Osmotic Dysequilibrium to Modified Cell Function

The Na+/H+ exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na+ ions and extrusion of H+ ions, which are readily replenished from intracellular buffers. This will result in a net import of Na+. In many systems NHE operates in parallel to Cl–-/ HCO33– exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H+ ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na+/H+ exchanger, termed NHE1 - 6, have been cloned so far. NHE 1 - 5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Glucose Reabsorption in the Rat Kidney

ZusammenfassungMit Hilfe der Mikroperfusionstechnik wurde die Glucoseresorption an einzelnen proximalen Tubuli der Rattenniere in situ bei verschiedenen tubulären Perfusionsstromstärken untersucht.1. Bei normalen Glucosekonzentrationen im Plasma gibt es keine nennenswerte Rückdiffusion von Glucose aus dem peritubulären Raum in die Tubulusflüssigkeit, so daß bei der Bestimmung der Glucoseresorptionsrate dieser Faktor zu vernachlässigen ist.2. Eine Änderung der Stromstärke in den perfundierten proximalen Tubulussegmenten führt zu einer gleichsinnigen Änderung der Glucoseresorptionsrate.3. Es besteht dabei eine lineare Korrelation der Glucoseresorptionsrate zur Nettoresorption von Wasser.SummaryGlucose reabsorption was studied at different perfusion rates in the single perfused proximal nephron of the rat. At normal plasma glucose levels diffusion of glucose from peritubular blood to tubular fluid is negligible. Per unit tubular length an increment in glucose reabsorption can be demonstrated with increments in perfusion rate. There is a close correlation between this increment in glucose reabsorption and the net reabsorption of water.

Fluorescence-optical measurements of chloride movements in cells using the membrane-permeable dye diH-MEQ.

Fluorescence-optical measurements of the intracellular chloride concentration facilitate identification of chloride movements across the cell membrane of living cells. The two main dyes used for this purpose are 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-quinolyl acetoethyl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodide (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with the acetoxymethyl (AM) forms of other dyes for sensing intracellular ions. In addition, the original method, which described absolute concentration measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limitations of single wavelength excitation and emission dyes with respect to exact concentration measurements. More-over, to enhance the signal-to-noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux in the cells under investigation. We verified the method by using fibroblasts and activating ICln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN− the MEQ quench measured in NIH 3T3 fibroblasts is dramatically enhanced in hypotonically challenged cells compared with cells under isotonic conditions. Antisense oligodeoxynucleotides sensing ICln considerably impeded the swelling-induced chloride current (ICl) in NIH 3T3 fibroblasts. Accordingly, the chloride movement measured by the SCN− quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), two known blockers of chloride transport in the plasma membrane of a variety of cells. In conclusion, fluroscence-optical measurements using MEQ as the chloride-sensitive dye provide a reliable and easy-to-use method for measuring changes of the chloride flux across the cell membrane of living cells.

Insight into the Structure-Function Relation of Chloride Channels

Chloride channels are highly selective transport proteins ubiquitously expressed in eukaryotic cells. Biophysical methods allow discrimination between several different types of chloride channels with

ICln: a chloride channel paramount for cell volume regulation.

Cell volume regulation is a ubiquitous cell regulatory mechanism based on meticulously controlled ion transport mechanisms. Keeping the absolute volume constant seems to be of the highest priority for most cells and is achieved at the expense of altered intracellular ion concentrations. We have been able to demonstrate that ICln, a chloride channel cloned from epithelial cells, is paramount for the ability of swollen cells to regulate their volume back to that under resting conditions. A unique feature of ICln is the distinct sensitivity of these channels for nucleotides and nucleoside analogues added to the extracellular fluid. In addition, cromolyn sodium and nedocromil sodium, drugs used by patients with asthma, are able to impede the function of these channels.

Evidence for an amiloride sensitive Na+ pathway in the amphibian diluting segment induced by K+ adaptation

The effect of amiloride on cell membrane potentials and intracellular Na activity (Nai) was tested in early distal tubules of the isolated perfused kidney of control and of K-adapted (high-K diet)Amphiuma. Conventional and Na-sensitive liquid ion-exchanger microelectrodes were employed to measure the peritubular cell membrane potential (PDpt), the transepithelial potential difference (PDte) and the Na electrochemical gradient across the peritubular cell membrane (EptNa), in the absence and the presence of amiloride (1·10−4mol·1−1) in both groups of animals. Amiloride did not affect PDpt and EptNa in control animals but depolarized PDpt and EptNa by about 8 mV in K-adapted animals. Nai (11.0 ±0.6 mmol·l−1 in early distal cells of control animals) did not change significantly by this maneuver. However, Nai decreased to extremely low values (2.3±0.2 mmol·l−1) when the luminal cotransport system for Na, Cl and K was inhibited by the luminal application of furosemide (5·10−5 mol/l) and when the luminal cell membrane was exposed simultaneously to amiloride. The amiloride-induced effects on PDpt, EptNa and Nai occurred within seconds and were fully reversible.We conclude that high-K diet (K adaptation) induces an amiloride-sensitive pathway in the luminal cell membrane of early distal cells ofAmphiuma which exists in parallel with the furosemide-sensitive cotransport system located in this cell barrier. The results suggest a luminal amiloride-sensitive Na/H exchange mechanism which regulates the luminal K permeability.

Kinetics ofl-proline reabsorption in rat kidney studied by continuous microperfusion

AbstractRenal tubular reabsorption of3H and14C labelledl-proline was measured in vivo et situ by continuous microperfusion of single proximal tubules of the rat. The reabsorption is shown to be saturable. Passive diffusion plays a relatively small role in the reabsorption. A maximum possible permeability coefficient of 25 μm2·s−1 for proline was calculated. Two transport systems were found, one with a small affinity and a high capacity, the other with a very high affinity and a small capacity. The following values were estimated: