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P Deetjen

Publications and source records attributed to P Deetjen.

At least 19 recordsLinked to original sources
PubMed2001

P2Y(2) receptor-mediated inhibition of amiloride-sensitive short circuit current in M-1 mouse cortical collecting duct cells.

Extracellular nucleotides modulate renal ion transport. Our previous results in M-1 cortical collecting duct cells indicate that luminal and basolateral ATP via P2Y2 receptors stimulate luminal Ca2+-activated Cl- channels and inhibit Na+ transport. Here we address the mechanism of ATP-mediated inhibition of Na+ transport. M-1 cells had a transepithelial voltage (V(te)) of -31.4 +/- 1.3 mV and a transepithelial resistance (R(te)) of 1151 +/- 28 Omegacm(2). The amiloride-sensitive short circuit current (I(sc)) was -28.0 +/- 1.1 microA/cm2. The ATP-mediated activation of Cl- channels was inhibited when cytosolic Ca2+ increases were blocked with cyclopiazonic acid (CPA). Without CPA the ATP-induced [Ca2+](i) increase was paralleled by a rapid and transient R(te) decrease (297 +/- 51 Omegacm(2)). In the presence of CPA, basolateral ATP led to an R(te) increase by 144 +/- 17 Omegacm(2) and decreased V(te) from -31 +/- 2.6 to -26.6 +/- 2.5 mV. Isc dropped from -28.6 +/- 2.4 to -21.6 +/- 1.9 microA/cm2. Similar effects were observed with luminal ATP. In the presence of amiloride, ATP was without effect. This reflects ATP-mediated inhibition of Na+ absorption. Lowering [Ca2+](i) by removal of extracellular Ca2+ did not alter the ATP effect. PKC inhibition or activation were without effect. Na+ absorption was activated by pH(i) alkalinization and inhibited by pH(i) acidification. ATP slightly acidified M-1 cells by 0.05 +/- 0.005 pH units, quantitatively not explaining the ATP-induced effect. In summary this indicates that extracellular ATP via luminal and basolateral P2Y2 receptors inhibits Na+ absorption. This effect is not mediated via [Ca2+](i), does not involve PKC and is to a small part mediated via intracellular acidification.

Adenosine Triphosphate↗
PubMed2001

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(-)/ HCO3(-) 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.

Amino Acid Sequence↗
PubMed2000

The promoter for constitutive expression of the human ICln gene CLNS1A.

The ICln protein is expressed ubiquitously in mammals. Experiments designed to knock down the ICln protein in NIH 3T3 fibroblasts as well as in epithelial cells led to the conclusion that this protein is crucially involved in volume regulation after cytoplasmic swelling. Reconstitution of the ICln protein in lipid bilayers revealed the ion channel nature of ICln. Here we describe a new human promoter sequence, composed of 89 nucleotides, which is responsible for a highly constitutive expression of the ICln protein. The promoter sequence lacks a TATA box, and the transcription can be effected at multiple sites. In addition to the starting sites, upstream sequence elements are mandatory for an efficient transcription of the ICln gene (CLNS1A). These new nucleotide elements were defined by site-directed mutagenesis.

Animals↗
PubMed2000

Functional reconstitution of ICln in lipid bilayers.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

3T3 Cells↗
PubMed1999

ICln, an ion channel-forming protein associated with cell volume regulation.

It is not resolved whether the anionic channel involved in volume regulation after cell swelling comprises one or more subunits. Moreover, it remains to be determined which of the different proteins cloned so far, for which an involvement in cell volume regulation has been postulated, is the ideal candidate. In this review, we consider the role of the ICln protein, cloned from MDCK cells, in cell volume regulation.

Animals↗
PubMed1998

Characterization of the human gene coding for the swelling-dependent chloride channel ICln at position 11q13.5-14.1 (CLNS1A) and further characterization of the chromosome 6 (CLNS1B) localization.

Expression cloning revealed a chloride channel (ICln) that we found to be fundamental for the regulatory volume decrease in a variety of cells. The chromosomal localization of the human ICln-gene showed two loci, one at chromosome 11 in position q13.5-q14.1, termed CLNS1A, and a second one at chromosome 6 at position p12.1-q13, termed CLNS1B. In this study, we offer a detailed characterization of the CLNS1A gene and provide the exact position (6p12) and sequence data of CLNS1B, an intronless gene 91.3% homologous to the coding region of CLNS1A.

Amino Acid Sequence↗
PubMed1996

Chromosomal localization of the genes (CLNS1A and CLNS1B) coding for the swelling-dependent chloride channel ICln.

ICln is a cloned chloride channel paramount for regulatory volume decrease. Two different loci that carry the coding region for ICln were identified in the human genome. By PCR strategies an intronless copy of the gene was located on chromosome 6 at position 6p12.1-6q13 (CLNS1B). By fluorescence in situ hybridization a copy carrying introns with a putative length of 19 kb was located at chromosome 11 on position 11q13.5-q14.1 (CLNS1A). The characterization and chromosomal localization of the ICln gene offer the opportunity to study the regulatory sites of this gene in greater detail and could be helpful in establishing linkages between ICln and potential human diseases.

Chloride Channels↗
PubMed1996

The lysosomal Ca2+ pool in MDCK cells can be released by ins(1,4,5)P3-dependent hormones or thapsigargin but does not activate store-operated Ca2+ entry.

In several cell types, Ca2+ release from intracellular Ca2+ stores by Ins(1,4,5)P3 elicits Ca2+ influx from the extracellular space into the cytoplasm, termed store-operated Ca2+ entry (SOCE). In MDCK cells, the Ins(1,4,5)P3-sensitive Ca2+ store giving rise to SOCE essentially overlaps with the thapsigargin (TG)-sensitive store. Recent evidence suggests that in MDCK cells lysosomes form a Ca2+ pool that is functionally coupled with the Ins(1,4,5)P3-sensitive Ca2+ store: Ca2+ can be selectively released from lysosomes by glycyl-L-phenylalanine naphthylamide, an agent inducing lysosomal swelling with subsequent and reversible permeabilization of the vesicular membranes. This compartment is also depleted by Ins(1,4,5)P3-dependent agonists or TG, indicating that it is part of a larger, Ins(1,4,5)P3-sensitive Ca2+ pool. Here we show that whereas SOCE is triggered by Ca2+ release from the entire Ins(1,4,5)P3-sensitive Ca2+ pool, selective Ca2+ release from lysosomes alone is unable to trigger SOCE. This finding is consistent with measurements of the store-operated cation current, a direct parameter for store-operated Ca2+ and Na+ entry into MDCK cells. Hence it is proposed that the Ins(1,4,5)P3-sensitive Ca2+ pool is composed of different intracellular compartments that do not uniformly stimulate Ca2+ entry into the cell.

Animals↗
PubMed1996

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. Moreover, 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.

3T3 Cells↗
PubMed1996

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.

Adenosine Triphosphate↗
PubMed1996

Blockade of swelling-induced chloride channels by phenol derivatives.

1. In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ICln, a protein isolated from a cDNA library derived from Madin Darby canine Kidney (MDCK) cells. ICln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2. RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3. RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4. The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5. The binding site for the two phenol derivatives onto ICln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the ICln channel protein.

3T3 Cells↗
PubMed1996

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.

Animals↗
PubMed1995

Ultrapure polymerized bovine hemoglobin (UPPBHb) improves integrity of the isolated perfused rat kidney (IPRK): effects on function and structure.

To test the oxyphoretic properties and potential nephrotoxic side-effects of polymerized hemoglobin solutions, isolated rat kidneys were perfused in a recirculating system for 180 min. Group I was perfused with a substrate enriched Ringer solution containing hydroxyethylstarch (HES) to produce isoncotic conditions. In group II HES was substituted in part by UPPBHb (34 g/l) with a high portion of low molecular weight molecules (= UPPBHb1). In group III 34 g/l of UPPBHb containing an increased fraction of high molecular weight polymers (= UPPBHb2) was used. Only UPPBHb2-perfused kidneys showed a reduced renal perfusate flow (RPF, 13.3 +/- 1.1 ml/min g kw), when compared to HES-perfused controls (15.5 +/- 0.8) and UPPBHb1 (15.1 +/- 1.2). Glomerular filtration rate (GFR) was significantly higher in UPPBHb1-perfused kidneys (902 +/- 107 vs 633 +/- 55 microliters/min g kw for HES). This difference became even more pronounced in the third hour of perfusion (474 +/- 125 vs. 103 +/- 33). In contrast, UPPBHb2 produced low initial GFR levels of 385 +/- 25, which had only a minor tendency to decline with time. Parallel to GFR, absolute reabsorption of sodium (TNa) andoxygen consumption (QO2) showed values of 110 +/- 16 and 5.46 +/- 0.33 mumol/min g kw in UPPBHb1-kidneys vs 83 +/- 6 and 5.09 +/- 0.27 in controls and vs 53 +/- 4 and 3.66 +/- 0.12 in UPPBHb2-kidneys. Fractional excretion of sodium (FENa), of potassium (FEK), and of water (FEH2O) in UPPBHb1 and UPPBHb2-perfused kidneys were not significantly different from HES-perfused controls at any time of perfusion. Urinary flow rate (UFR) was similar in UPPBHb1- and HES-kidneys. Nevertheless, control kidneys tended to render oliguric during the third hour of perfusion (UFR 19.9 +/- 4.1 microliters/min g kw), whereas UPPBHb1 preserved urinary flow in a better way (83.7 +/- 32.4). UFR of UPPBHb2-kidneys was significantly reduced initially (30.2 +/- 5.1 vs. 105 +/- 33 for HES), but increased steadily up to 67 +/- 23. In the UPPBHb1 and HES group, all functional parameters determined declined dramatically within the third hour of perfusion, whereas UPPBHb2 produced functional stability. The in vivo reaction pattern of renal autoregulation was better preserved in UPPBHb-perfused kidneys than in HES-perfused controls: 74 +/- 6 vs. 59 +/- 5 vs. 42 +/- 4% (of full autoregulatory response) for UPPBHb1, UPPBHb2, and HES kidneys, respectively. Light- and electron microscopic analysis revealed major alterations only for the outer medulla of HES-kidneys.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Renal hemodynamic response to intravenous and oral amino acids in animals.

Oral or parenteral application of amino acids leads to marked hyperfiltration and increased of renal plasma flow. Amino acids stimulate the release of glucagon, which increases hepatic production and release of cyclic adenosine monophosphate (cAMP). In the kidney, the combined effect of cAMP and glucagon increases glomerular filtration rate (GFR), possibly by reducing NaCl concentration at the macula densa and depression of the tubuloglomerular feedback. Vasopressin-dependent urea recycling and delivery to the thick ascending limb could similarly reduce NaCl concentration at the macula densa. Beyond that, amino acids may trigger a hepatorenal reflex or directly interfere with renal function. Mechanisms invoked include dopamine from renal nerves, prostaglandins, nitric oxide (NO), and angiotensin II. At this point, it is not clear to which extent the described mechanisms participate in, permit, or fully account for the hyperfiltrative effect of amino acids.

Administration, Oral↗
PubMed1990

Damaging effects of high energy shock waves on cultured Madin Darby canine kidney (MDCK) cells.

Shock wave lithotripsy (ESWL) has become an almost non-invasive standard treatment modality for urolithiasis. Several investigations, however, demonstrated that ESWL is not completely free of side effects. Among others alteration of renal tubular function has been reported. To study the effect of shock waves on tubular cells directly an in-vitro model with cultured Madin Darby Canine Kidney (MDCK) cells was established. Suspensions of MDCK cells (7 groups of 6 containers each) were exposed to 0, 16, 32, 64, 128, 256 shock waves (Dornier HM4, 18 kV). Before and 0, 1, 3, 6, 9, 12, 24 h after ESWL the following parameters were measured in the nutrient medium: lactate dehydroxygenase (LDH), glutamate oxalacetate transaminase (GOT), electrolytes. LDH and GOT increased depending on the number of shock waves indicating a membrane damage of MDCK cells. The MDCK model seems suitable for further studies on the effect of shock waves on renal tubular cells.

Animals↗
PubMed1989

[The remarkable accomplishments of the kidney--from molecular mechanisms to organ function].

The kidneys control chemical composition and volume of the extracellular fluid. Total extracellular volume passes about 10 times per day through the kidneys. Only 1% is excreted including metabolic waste products and excess intake by nutrition. While the proximal tubules are adapted to mass transport, the distal tublar cells are specialized to selectively retain or reject water and electrolytes, controlled by exrenal hormonal factors. For these purposes an optimal adjustment of morphologic, encymatic, physico-chemical and molecular features has developed in the course of evolution. With modern experimental techniques it is possible to demonstrate the mode of operation of single protein molecules in the cell membranes as sites of active or passive transport processes. This enables us to describe the basic mechanism of kidney function and to better understand the sequence of events leading to pathological disturbances of this organ.

Aldosterone↗