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S Waldegger

Publications and source records attributed to S Waldegger.

At least 37 records · Page 2Linked to original sources
PubMed1998

Genomic organization and chromosomal localization of the human SGK protein kinase gene.

The SGK protein kinase is a novel member of the serine/threonine protein kinase family. Its corresponding gene belongs to the group of immediate-early genes. SGK transcription is controlled by cell volume alterations in different cell lines. To analyze the genomic structure and chromosomal location of the SGK gene, a human P1 clone was isolated by screening a human genomic library with a SGK cDNA probe. This clone was confirmed to encode the authentic SGK gene by the detection of exon-intron structures and the correspondence between the nucleotide sequences of exons and human cDNA. Using this P1 clone as a probe for fluorescence in situ hybridization, a single chromosomal locus for SGK was assigned to band 6q23, a region frequently affected by deletion in various human neoplasms.

Cell Line↗
PubMed1998

Cloning of sgk serine-threonine protein kinase from shark rectal gland – a gene induced by hypertonicity and secretagogues.

Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

Amino Acid Sequence↗
PubMed1998

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes.

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.

Animals↗
PubMed1998

Mechanisms and clinical significance of cell volume regulation.

A wide variety of factors challenge constancy of cell volume. Alterations of cell volume activate diverse cell volume regulatory mechanisms including ion transport, osmolyte accumulation, metabolism and expression of appropriate genes. A wealth of cellular signalling pathways link cell volume to the respective regulatory mechanisms. Cell volume emerges as a pathophysiologically important parameter in several diseases including diabetes mellitus, uraemia, hepatic insufficiency and hypercatabolic states. The role of altered cell volume in disease is a challenge which requires more experimental research and clinical investigation.

Animals↗
PubMed1998

Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine.

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.

Amantadine↗
PubMed1998

Functional significance of cell volume regulatory mechanisms.

To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.

Animals↗
PubMed1998

Na(+)-dependent and -independent amino acid transport systems in immortalized human kidney epithelial cells derived from the proximal tubule.

In the proximal tubule Na(+)-dependent (SDAT) and Na(+)-independent (SIAT) amino acid (AA) transporters are present. The effects of neutral, basic, and acidic AA on membrane voltage (Vm) of immortalized human kidney epithelial (IHKE-1) cells derived from the proximal tubule were examined using the slow whole-cell patch-clamp technique. In the presence of Na+ AA depolarized Vm in a concentration-dependent manner (0.05-5 mM) with Asp = Arg = Glu = 2Cys < Pro = Leu < Phe = AIB = Ala = Pro = Asn < Gly. In the absence of extracellular Na+ a decreased depolarization was seen with most neutral AA (Ala, Pro, Asn, Gly, Phe, and Leu), and the depolarization was increased with Asp, Glu, Arg, and 2Cys (1 mM each). In the absence of Na+ and a reduction in Cl- (5 mM) the depolarization by Arg was reduced. Unlike that predicted for transport by system b0,+ which exchanges neutral against dibasic amino acids, Leu does not hyperpolarize but depolarize Vm of IHKE-1 cells in the absence of extracellular Na+. After removal of Na+ (0 mM) and a reduction in Cl- (5 mM) in the extracellular solution, Leu or Glu hyperpolarized Vm, indicating that IHKE-1 cells possess two different SIAT systems, one Cl(-)-dependent and similar to system b0,+ and one novel Cl(-)-dependent system, which might be a Cl-/AA exchanger and can be blocked by the Cl(-)-channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (10 microM) and 4,4'-diisothiocyanostibene-2,2'-disulfonic acid (50 microM). B system-related AA transporters might be responsible for the C(-)-independent SIAT, since we were able to detect its signal by Northern blot analysis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Osmotically induced conductance and capacitance changes in in vitro perfused rectal gland tubules of Squalus acanthias.

The rectal gland of Squalus acanthias is critically involved in the homeostasis of NaCl and water metabolism and hence in overall osmoregulation. In the present study, we have examined the acute responses of rectal gland slices and in vitro perfused rectal gland tubule (RGT) cells to the exposure to dilute and hypertonic peritubule solutions. Five series were performed. (i) With changes in osmolality, Western blots to monitor tyrosine, threonine and serine phosphorylation in rectal gland slices did not reveal clear-cut changes in phosphorylation patterns. All other series were performed in in vitro perfused RGT. (ii) Relative cell volume was estimated by fura-2 fluorescence using the emission at the isosbestic excitation wavelength of 360 nm. Hypotonic solution (-100 mmol/l NaCl) reduced fura-2 fluorescence by 16% and hypertonic solution (+100 mmol/l NaCl) had the opposite effect (+12%). (iii) Transepithelial resistance was increased markedly by hypotonic solution, probably by cell swelling, and the opposite was seen with hypertonic solutions. (iv) Whole-cell patch clamp experiments indicated that hypotonic solution hyperpolarized the cells, and increased membrane conductance and membrane capacitance. The latter two changes correlated significantly with each other. Hypertonic solution had the opposite effect. (v) Measurements of the fura-2 fluorescence ratio (340/380 nm) revealed that hypotonic solution (-NaCl) increased cytosolic Ca2+ activtiy ([Ca2+]i). Hypertonic solution had no detectable effect on [Ca2+]i. These data indicate that RGT cells are swollen by removal of NaCl from the bath solution. This causes an increase in [Ca2+]i and a predominant increase in K+ conductance and hyperpolarization. Urea apparently permeates these cells quite well and its addition (+U) or its removal (-U) had only moderate osmotic effects. The removal of urea and replacement by mannitol produced effects similar to those seen with hypertonic NaCl solution.

Animals↗
PubMed1997

Effects of bradykinin on NIH 3T3 fibroblasts pretreated with lithium. Mimicking events of Ha-ras oncogene expression.

As shown previously, expression of Ha-ras oncogene in NIH 3T3 fibroblasts (+ ras cells) increases cellular concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and enhances bradykinin induced Ca2+ entry [1-3]. These cells respond to low concentrations of serum or bradykinin with sustained oscillations of the cell membrane potential due to pulsatile release of calcium from internal stores and subsequent activation of calcium sensitive K+ channels [1]. Furthermore Ha-ras oncogene expression leads to depolymerization of the actin filament network and delayed increase of cell volume [4-6]. Pretreatment of the same cells not expressing the oncogene (-ras cells) with Li+ similarly increases Ins(1,4,5)P3 and Ins(1,3,4,5)P4 [2]. As shown in the present study, -ras cells pretreated with Li+ similar to Ha-ras oncogene expressing cells respond to bradykinin with sustained oscillations of cell membrane potential, depolymerization of the actin filament network and increase of cell volume. The oscillations of the cell membrane potential and the depolymerization of the actin cytoskeleton can be inhibited by the calcium channel blocker lanthanum and the bradykinin induced increase of cell volume is inhibited by HOE 694, pointing to involvement of Na+/H+ exchange. The data indicate a close functional linkage of the calcium oscillations, cytoskeletal rearrangement and activation of the Na+/H+ exchanger. Thus, Li+ pretreatment mimicks crucial cellular events triggered by expression of the Ha-ras oncogene. However, unlike in cells expressing the Ha-ras oncogene, Li+ pretreatment alone does not allow for growth factor-independent proliferation of the cells.

3T3 Cells↗
PubMed1997

Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume.

Hepatic metabolism and gene expression are among other regulatory mechanisms controlled by the cellular hydration state, which changes rapidly in response to anisotonicity, concentrative substrate uptake, oxidative stress, and under the influence of hormones such as insulin and glucagon. Differential screening for cell volume sensitive transcripts in a human hepatoma cell line revealed a gene for a putative serine/threonine kinase, h-sgk, which has 98% sequence identity to a serum- and glucocorticoid regulated kinase, sgk, cloned from a rat mammary tumor cell line. h-sgk transcript levels were strongly altered during anisotonic and isotonic cell volume changes. Within 30 min h-sgk RNA was, independent of de novo protein synthesis, induced upon cell shrinkage and, due to a complete stop in h-sgk transcription, reduced upon cell swelling. Comparable changes of sgk transcript levels were observed in a renal epithelial cell line. h-sgk mRNA was detected in all human tissues tested, with the highest levels in pancreas, liver, and heart. The putative serine/threonine protein kinase h-sgk may provide a functional link between the cellular hydration state and metabolic control.

Animals↗
PubMed1997

An intracellular trafficking defect in type I cystinuria rBAT mutants M467T and M467K.

The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.

Amino Acid Transport Systems, Basic↗
PubMed1997

Effect of cellular hydration on protein metabolism.

In the past few years, the paramount importance of cell volume for the regulation of cell function, including protein metabolism, has been recognized. Among many other effects, cell swelling inhibits proteolysis and stimulates protein synthesis, whereas cell shrinkage stimulates proteolysis and inhibits protein synthesis. Moreover, cell swelling and cell shrinkage influence the expression of a number of genes, including carriers, enzymes, and signaling molecules. Hormones exploit the influence of cell volume on metabolism to exert their effects. Insulin swells hepatocytes by activation of Na-/H+ exchange and Na+,K+,2Cl- cotransport, while glucagon shrinks hepatocytes by activation of ion channels. The effects of these hormones on hepatic proteolysis completely depend on their influence on cell volume. The effects of cell volume are mediated in part by alterations of lysosomal pH, which modifies the activity of acidic lysosomal proteases. Transforming growth factor-beta 1, as other growth factors, activates the Na+/H+ exchanger, swells cells, leads to lysosomal alkalinization, inhibits proteolysis and may thus contribute to renal hypertrophy in chronic renal disease. Moreover, a decrease in cell volume correlates with catabolic states in a variety of diseases.

Animals↗
PubMed1996

Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1.

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.

Animals↗
PubMed1996

Specific blockade of slowly activating I(sK) channels by chromanols -- impact on the role of I(sK) channels in epithelia.

Chromanols, which were recently shown to inhibit cAMP-mediated Cl- secretion in colon crypts via a blockade of a cAMP-activated K+ conductance, were analyzed for their effects on distinct cloned K+ channels expressed in Xenopus oocytes. The lead chromanol 293B specifically inhibited I(sK) channels with an IC50 of 7 micromol/l without affecting the delayed rectifier Kv1.1 or the inward rectifier Kir2.1. Moreover, several other chromanols displayed the same rank order of potency for I(sK) inhibition as demonstrated in colon crypts. Finally, we tested the effects of the previously described I(sK) blocker azimilide on cAMP mediated Cl- secretion in rat colon crypts. Similar to 293B azimilide inhibited the forskolin induced Cl- secretion. These data suggest that I(sK) protein induced K+ conductances are the targets for the chromanol 293B and its analogues, and azimilide.

Animals↗
PubMed1996

Monoamine neurotransmitter transport mediated by the polyspecific cation transporter rOCT1.

The polyspecific cation transporter rOCT,1 which is localized in the basolateral membrane of rat renal proximal tubules and in sinusoidal membranes of hepatocytes, was analyzed for transport of monoamine neurotransmitters. In voltage-clamp experiments with rOCT1-expressing Xenopus oocytes, superfusion with dopamine, serotonin, noradrenaline, histamine and the permanent cation acetylcholine induced saturable inwardly directed currents with apparent Km values ranging from 20 to 100 microM. Transport of dopamine was also demonstrated by uptake measurements in oocytes and in the mammalian cell line (HEK 293) which was permanently transfected with rOCT1. The high uptake rates measured in rOCT1-expressing oocytes and in transfected HEK 293 cells suggest that rOCT1 is a high capacity transporter which mediates the first step in the excretion of monoamine neurotransmitters.

Acetylcholine↗