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Biomedical subjects

Florian Lang

Publications and source records attributed to Florian Lang.

At least 73 records · Page 4Linked to original sources
PubMed2005

Inhibition of erythrocyte "apoptosis" by catecholamines.

Osmotic shock, oxidative stress and Cl- removal activate a non-selective Ca2+-permeable cation conductance in human erythrocytes. The entry of Ca2+ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl- enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the beta agonist isoproterenol (IC50 approx. 1 microM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca2+ activity following Cl- removal, an effect again significantly blunted by isoproterenol exposure (10 microM). Whole-cell patch-clamp experiments performed in Cl- free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 microM). Phenylephrine (IC50<10 microM), dobutamine (IC50 approx. 1 microM) and dopamine (IC50 approx. 3 microM) similarly inhibited the effect of Cl- removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl- removal-activated Ca2+ entry into erythrocytes, thus preventing increase of cytosolic Ca2+ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.

Annexin A5↗
PubMed2005

Reduced locomotion in the serum and glucocorticoid inducible kinase 3 knock out mouse.

The serum and glucocorticoid inducible kinase isoform SGK3 is expressed in the brain including hippocampal neurons. It is activated by phosphoinositide-3 (PI3) kinase and thus a putative target of neurotrophic factors. In vitro experiments pointed to the ability of SGK3 to regulate several transporters and ion channels including the AMPA receptor GluR1. In order to explore the in vivo functional significance of SGK3 in the regulation of spatial learning and exploratory behavior, we assessed the performance of SGK3 knockout mice (SGK3-/-) and their wild type littermates (SGK3+/+) in a place navigation task in the water-maze, radial maze in a battery of forced and free exploration tests, acoustic startle and a test for motoric coordination. According to water-maze and radial maze testing reference and working memory was intact in SGK3-/- mice. However, detailed analysis of swimming patterns of SGK3-/- mice in the water-maze revealed a deficit in precision and goal-directed navigation in space. SGK3-/- mice showed reduced exploratory activity, which was observed in several environments and increased centre field avoidance in the open-field. SGK3-/- mice further showed reduced darting behavior on open surfaces, indicating that the knock out may modify basic patterns of locomotion. In conclusion, lack of SGK3 leads to subtle behavioral defects which may result from deranged neuronal regulation of transporters and ion channels.

Analysis of Variance↗
PubMed2005

Glycogen metabolism in rat ependymal primary cultures: regulation by serotonin.

Ependymal primary cultures are a model for studying ependymal energy metabolism. Intracellular glycogen is built up in the cultures dependent on culture age and the presence of glucose and glutamate. This energy store is mobilized upon glucose withdrawal, stimulation with isoproterenol, forskolin or serotonin and after uncoupling of oxidative phosphorylation from ATP production. Serotonin regulates ependymal glycogen metabolism predominantly via 5-HT receptor (5-HTR) 7, which elicits an increase in the level of ependymal cyclic AMP. Although the most abundant mRNAs for serotonin receptors are those of 5-HTR 2B and 5-HTR 3A, ependymal cells in primary culture do not respond to serotonin with an increase in their concentration of cytosolic calcium ions. The mRNAs of 5-HTRs 1A, 6, 1B, 5B, 7, 1/2C and 5A are also detectable in order of decreasing abundance. The mRNAs for 5-HTRs 1D, 1F, 3B and 4 are absent from the cultured cells. The ability of serotonin to mobilize ependymal glycogen depends on the culture age and the time allowed for glycogen buildup. During glycogen buildup time, glutamate is consumed by the cells. An increased ability of 5-HT to mobilize ependymal glycogen stores is noticed after the depletion of glutamate from the glycogen buildup medium. In ependymal primary cultures, cilia are colocalized with glycogen phosphorylase isozyme BB, while the MM isoform is not expressed. It is known from the literature that an increase in the concentration of cytosolic cAMP in ependymal cells leads to a decrease in ciliary beat frequency. Therefore, the present data point towards a function for ependymal glycogen other than supplying energy for the movement of cilia.

Animals↗
PubMed2005

Stimulation of the creatine transporter SLC6A8 by the protein kinases SGK1 and SGK3.

Creatine binds phosphate thus serving energy storage. Cellular creatine uptake is accomplished by the Na+,Cl-, creatine transporter CreaT (SLC6A8). The present study explored the regulation of SLC6A8 by the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated during ischemia. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes creatine induced a current which was significantly enhanced by coexpression of wild type SGK1 and constitutively active (S422D)SGK1, but not inactive (K127N)SGK1. Kinetic analysis revealed that (S422D)SGK1 enhanced maximal current without significantly altering affinity. The effect of SGK1 was mimicked by the constitutively active isoform (S419D)SGK3 but not by inactive (K119N)SGK3, wild type isoform SGK2 or constitutively active related kinase (T308D,S473D)PKB. In conclusion, the kinases SGK1 and SGK3 increase SLC6A8 activity by increasing the maximal transport rate of the carrier. Deranged SGK1 and/or SGK3 dependent regulation of SLC6A8 may affect energy storage particularly in skeletal muscle, heart, and neurons.

Animals↗
PubMed2005

Stimulation of erythrocyte phosphatidylserine exposure by mercury ions.

The sequelae of mercury intoxication include induction of apoptosis. In nucleated cells, Hg2+-induced apoptosis involves mitochondrial damage. The present study has been performed to elucidate effects of Hg2+ in erythrocytes which lack mitochondria but are able to undergo apoptosis-like alterations of the cell membrane. Previous studies have documented that activation of a Ca2+-sensitive erythrocyte scramblase leads to exposure of phosphatidylserine at the erythrocyte surface, a typical feature of apoptotic cells. The erythrocyte scramblase is activated by osmotic shock, oxidative stress and/or energy depletion which increase cytosolic Ca2+ activity and/or activate a sphingomyelinase leading to formation of ceramide. Ceramide sensitizes the scramblase to Ca2+. The present experiments explored the effect of Hg2+ ions on erythrocytes. Phosphatidylserine exposure after mercury treatment was estimated from annexin binding as determined in FACS analysis. Exposure to Hg2+ (1 microM) indeed significantly increased annexin binding from 2.3+/-0.5% (control condition) to 23+/-6% (n=6). This effect was paralleled by activation of a clotrimazole-sensitive K+-selective conductance as measured by patch-clamp recordings and by transient cell shrinkage. Further experiments revealed also an increase of ceramide formation by approximately 66% (n=7) after challenge with mercury (1 microM). In conclusion, mercury ions activate a clotrimazole-sensitive K+-selective conductance leading to transient cell shrinkage. Moreover, Hg2+ increases ceramide formation. The observed mechanisms could similarly participate in the triggering of apoptosis in nucleated cells by Hg2+.

Annexins↗
PubMed2005

Effects of I(Ks) channel inhibitors in insulin-secreting INS-1 cells.

Potassium channels regulate insulin secretion. The closure of K(ATP) channels leads to membrane depolarisation, which triggers Ca(2+) influx and stimulates insulin secretion. The subsequent activation of K(+) channels terminates secretion. We examined whether KCNQ1 channels are expressed in pancreatic beta-cells and analysed their functional role. Using RT/PCR cellular mRNA of KCNQ1 but not of KCNE1 channels was detected in INS-1 cells. Effects of two sulfonamide analogues, 293B and HMR1556, inhibitors of KCNQ1 channels, were examined on voltage-activated outwardly rectifying K(+) currents using the patch-clamp method. It was found that 293B inhibited 60% of whole-cell outward currents induced by voltage pulses from -70 to +50 mV with a concentration for half-maximal inhibition (IC(50)) of 37 microM. The other sulfonamide analogue HMR1556 inhibited 48% of the outward current with an IC(50) of 7 microM. The chromanol 293B had no effect on tolbutamide-sensitive K(ATP) channels. Action potentials induced by current injections were broadened and after-repolarisation was attenuated by 293B. Insulin secretion in the presence but not in the absence of tolbutamide was significantly increased by 293B. These results suggest that 293B- and HMR1556-sensitive channels, probably in concert with other voltage-activated K(+) channels, influence action potential duration and frequency and thus insulin secretion.

4-Aminopyridine↗
PubMed2005

Characterization of mouse amino acid transporter B0AT1 (slc6a19).

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+-amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+-substrate-transporter] complex, which is consistent with the experimental data.

Amino Acid Transport Systems↗
PubMed2005

The role of the CO2 laser in the management of laryngotracheal stenosis: a survey of 100 cases.

Over the last decade, improvement of CO2 lasers with the microspot and ultrapulse technologies has broadened the indications for endoscopic CO2-laser resection of benign laryngotracheal stenosis (LTS). This article reviews 100 patients treated solely by endoscopic means for a LTS. There were 47 grade III, 41 grade II and 12 grade I stenoses according to the Myer-Cotton classification. The postoperative results show that the improvement to a nearly normal (>80% luminal size) airway declines from 92% (11/12 patients) for grade I to 46% (19/41 patients) for grade II and 13% (6/47 patients) for grade III stenoses. When compared to open surgery for more severe grades of stenosis (31 grade IV, 66 grade III and 3 grade II stenoses), the results of the endoscopy group is much less favorable: 36% of patients in the endoscopy group versus 76% of patients in the open surgery group were rehabilitated to a normal respiration without exertional dyspnea and 38% versus 5% patients remained tracheotomy dependent. However, if strict selection and therapeutic criteria are respected, a significant number of grade I and II stenoses, and to a lesser degree of grade III stenoses, can be improved to a nearly normal airway by endoscopic means only. The endoscopic treatment is potentially less invasive and risky and only needs a short hospital stay. To try this as a first treatment modality in a selected group of patients is worthwhile, provided that this endoscopic treatment is not repeated a second time, if the stenosis recurs to its initial grade after a primary CO2-laser treatment. Some guidelines for safe endoscopic treatment modalities with of the CO2 laser, dilatation and/or stenting are proposed.

Adolescent↗
PubMed2005

Long chain CoA esters as competitive antagonists of phosphatidylinositol 4,5-bisphosphate activation in Kir channels.

Long chain fatty acid esters of coenzyme A (LC-CoA) are potent activators of ATP-sensitive (K(ATP)) channels, and elevated levels have been implicated in the pathophysiology of type 2 diabetes. This stimulatory effect is thought to involve a mechanism similar to phosphatidylinositol 4,5-bisphosphate (PIP2), which activates all known inwardly rectifying potassium (Kir) channels. However, the effect of LC-CoA on other Kir channels has not been well characterized. In this study, we show that in contrast to their stimulatory effect on K(ATP) channels, LC-CoA (e.g. oleoyl-CoA) potently and reversibly inhibits all other Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir7.1). We also demonstrate that the inhibitory potency of the LC-CoA increases with the chain length of the fatty acid chain, while both its activatory and inhibitory effects critically depend on the presence of the 3'-ribose phosphate on the CoA group. Biochemical studies also demonstrate that PIP2 and LC-CoA bind with similar affinity to the C-terminal domains of Kir2.1 and Kir6.2 and that PIP2 binding can be competitively antagonized by LC-CoA, suggesting that the mechanism of LC-CoA inhibition involves displacement of PIP2. Furthermore, we demonstrate that in contrast to its stimulatory effect on K(ATP) channels, phosphatidylinositol 3,4-bisphosphate has an inhibitory effect on Kir1.1 and Kir2.1. These results demonstrate a bi-directional modulation of Kir channel activity by LC-CoA and phosphoinositides and suggest that changes in fatty acid metabolism (e.g. LC-CoA production) could have profound and widespread effects on cellular electrical activity.

Acyl Coenzyme A↗
PubMed2005

Decreased intestinal glucose transport in the sgk3-knockout mouse.

Xenopus oocyte coexpression experiments revealed the capacity of the serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) to up-regulate a variety of transport systems including the sodium-dependent glucose transporter SGLT1. The present study explored the functional significance of SGK3-dependent regulation of intestinal transport. To this end, experiments were performed in gene targeted mice lacking functional sgk3 (sgk3(-/-)) and their wild type littermates (sgk3(+/+)). Oral food intake and fecal dry weight were significantly larger in sgk3(-/-) than in sgk3(+/+) mice. Glucose-induced current (I(g)) in Ussing chamber as a measure of Na(+) coupled glucose transport was significantly smaller in sgk3(-/-) than in sgk3(+/+) mouse jejunal segments. Fasting plasma glucose concentrations were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. Intestinal electrogenic transport of phenylalanine, cysteine, glutamine and proline were not significantly different between sgk3(-/-) and sgk3(+/+) mice. In conclusion, SGK3 is required for adequate intestinal Na(+) coupled glucose transport and impaired glucose absorption may contribute to delayed growth and decreased plasma glucose concentrations of SGK3 deficient mice. The hypoglycemia might lead to enhanced food intake to compensate for impaired intestinal absorption.

Animals↗
PubMed2005

The serine/threonine kinases SGK1, 3 and PKB stimulate the amino acid transporter ASCT2.

The human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2/SLC1A5) plays an important role in the transport of neutral amino acids in epithelial cells. The serine and threonine kinases SGK1-3 and protein kinase B have been implicated in the regulation of several members of the SLC1 transporter family by enhancing their plasma membrane abundance. The present study explored whether those kinases modulate hASCT2. In Xenopus oocytes heterologously expressing hASCT2, coexpression of constitutively active (S422D)SGK1, (S419D)SGK3 or (T308DS473D)PKB upregulated the transporter activity. The stimulation requires the catalytical activity of the kinases since the inactive mutants (K127N)SGK1, (K191N)SGK3, and (T308AS473A)PKB failed to modulate the transporter. According to kinetic analysis and chemiluminescence assays, SGK1 and SGK3 modulate hASCT2 by enhancing the transporter abundance in the plasma membrane. As SGK1, 3 and PKB are activated by insulin and IGF1, the described mechanisms presumably participate in the hormonal stimulation of cellular amino acid uptake.

Amino Acid Transport System ASC↗
PubMed2005

Permselectivity and pH-dependence of Plasmodium falciparum-induced anion currents in human erythrocytes.

Intraerythrocytic survival of the malaria pathogen Plasmodium falciparum requires delivery of nutrients and disposal of waste products across the host erythrocyte membrane. Recent patch-clamp experiments have demonstrated inwardly and outwardly rectifying anion conductances in infected but not in control erythrocytes. A ClC-2-generated fraction of the inwardly rectifying current is activated by cell swelling and presumably subserves host cell volume regulation. In contrast, the outwardly rectifying current is insensitive to cell volume but allows the passage of lactate and is involved in the transport of nutrients. The present study was performed to characterize the permselectivity and pH sensitivity of the anion conductances using whole-cell recording. The outwardly rectifying and the inwardly rectifying currents exhibited permselectivities of Cl- > or = Br- approximately I- > SCN- and SCN- > I- > Br- > Cl-, respectively, as evident from the reversal potentials recorded under biionic conditions. While the inwardly rectifying current was not affected significantly by alterations of pH between 6.0 and 8.4, the outward rectifier was inhibited strongly by alkalinization to pH > or = 7.8. Fluxes of 14C-lactate and parasite growth were decreased markedly by the increase of bath pH, an effect that may at least in part be due to inhibition of the outward rectifier and subsequently impaired transport across the erythrocyte membrane.

Animals↗
PubMed2005

Activation of NHE3 by dexamethasone requires phosphorylation of NHE3 at Ser663 by SGK1.

Glucocorticoids stimulate Na+ absorption by activation of the epithelial Na+/H+ exchanger NHE3 in the kidney and intestine. It has been thought that glucocorticoid-induced activation of NHE3 is solely dependent on transcriptional induction of the NHE3 gene. While the transcriptional regulation remains an essential part of the chronic effect of glucocorticoids, a previous study by us identified the serum- and glucocorticoid-inducible kinase 1 (SGK1) as an important component of the activation of NHE3 by glucocorticoids. In this work, we have demonstrated phosphorylation of NHE3 by SGK1 as the key mechanism for the stimulation of the transport activity by glucocorticoids. By using in vitro SGK1 kinase assay and site-directed mutagenesis, we have identified Ser663 of NHE3 to be the major site of phosphorylation by SGK1. Ser663 is invariantly conserved in all NHE3 proteins from several species, and the mutation of Ser663 to Ala blocks the effect of dexamethasone, demonstrating the importance of phosphorylation at Ser663. We also show that phosphorylation of NHE3 precedes the changes in NHE3 activity, and the increased activity is associated with an increased amount of NHE3 proteins in the surface membrane. These data reveal that dexamethasone activates NHE3 activity by phosphorylating the NHE3 protein, which initiates trafficking of the protein into the plasma membrane.

Amino Acid Sequence↗
PubMed2005

Regulation of the excitatory amino acid transporter EAAT5 by the serum and glucocorticoid dependent kinases SGK1 and SGK3.

In the mammalian retina, glutamate re-uptake is mediated by the sodium dependent cotransport systems EAAT1-5 thus terminating neuronal excitation and preventing neuroexcitotoxicity. In retinal amacrine and ganglion cells, EAAT5 is colocalized with the serum and glucocorticoid inducible kinase SGK1, a serine/threonine kinase known to regulate transport. The study explored the possible regulation of EAAT5 by SGK1, its isoform SGK3, and the closely related protein kinase B. EAAT5 was coexpressed in Xenopus laevis oocytes with or without the respective kinases. Transport activity was quantified by electrophysiology and cell surface expression was determined by chemiluminescence. Both EAAT5 mediated currents and EAAT5 protein abundance at the cell surface were increased by a factor of 1.5-2 upon coexpression of SGK1 or SGK3 but not following coexpression of PKB. In conclusion, the kinases SGK1 and SGK3 increase EAAT5 activity by increasing cell surface abundance of the carrier.

Amino Acid Substitution↗
PubMed2005

Regulation of GluR1 abundance in murine hippocampal neurones by serum- and glucocorticoid-inducible kinase 3.

Phosphatidylinositol 3 kinase (PI3-kinase) is activated during and is required for hippocampal glutamate receptor-dependent long-term potentiation. It mediates the delivery of AMPA receptors to the neuronal surface. Among the downstream targets of PI3-kinase are three members of the serum- and glucocorticoid-inducible kinase family, SGK1, SGK2 and SGK3. In Xenopus oocytes expressing the AMPA subunit GluR1, we show that SGK3, and to a lesser extent SGK2, but not SGK1, increase glutamate-induced currents by increasing the abundance of GluR1 protein in the cell membrane. We further show Sgk3 mRNA expression in the hippocampus by RT-PCR and in situ hybridization. According to Western blotting, the hippocampal abundance of GluR1 is significantly lower in gene-targeted mice lacking SGK3 (Sgk3-/-) than in their wild-type littermates (Sgk3+/+). The present observations disclose a novel mechanism in the regulation of GluR1.

Animals↗
PubMed2005

Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression.

Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid-dependent genes include the serum- and glucocorticoid-inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3-kinase) pathway by growth factors such as insulin-like growth factor-1 (IGF1) or tumour growth factor beta (TGF-beta). Previously, a fourfold higher expression of SGK1 has been observed in fast-learning rats as compared with slow-learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 microg day-1 for 8-20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate-induced currents following treatment with dexamethasone (1 microm) and TGF-beta (1 microm). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate-induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids.

Animals↗
PubMed2005

Stimulation of erythrocyte ceramide formation by platelet-activating factor.

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.

Annexins↗