Biomedical subjects

Florian Lang

Publications and source records attributed to Florian Lang.

At least 91 records · Page 5Linked to original sources

PubMed2005

Influence of standard haemodialysis treatment on transcription of human serum- and glucocorticoid-inducible kinase SGK1 and taurine transporter TAUT in blood leukocytes.

BACKGROUND: Standard haemodialysis (HD) rapidly alters osmolality and composition of extracellular fluid and, thus, challenges cell volume constancy. Cell volume-sensitive genes upregulated by osmotic cell shrinkage include those encoding for taurine transporter TAUT as well as for serum- and glucocorticoid-inducible kinase SGK1. METHODS: Six HD patients were haemodialysed for 4 h with high-flux dialysers. Blood was drawn from the arterial section of the fistula immediately prior to start of HD and subsequently after 60, 120 and 240 min of HD treatment and, in addition, 120 min after HD treatment. Taurine plasma concentrations ([taurine]p) and erythrocytic taurine content ([taurine]e) were determined by high-performance liquid chromatography. SGK1 and TAUT transcript levels in leukocytes were quantified by real-time polymerase chain reaction. RESULTS: The [taurine]p was significantly higher in HD patients before HD treatment when compared with healthy controls and it decreased significantly during 4 h of HD. The ratio of SGK1/GAPDH and of TAUT/GAPDH transcript levels increased significantly by 50% or 27%, respectively, during HD. CONCLUSIONS: Standard HD treatment decreases plasma taurine concentration and upregulates leukocyte SGK1 and TAUT transcription. As SGK1 is a potent regulator of ion channels and transporters in nervous system, heart muscle and epithelial cells, the deranged regulation of SGK1 may contribute to acute side effects of HD treatment.

Glucocorticoids↗

PubMed2005

Altered expression of renal apical plasma membrane Na+ transporters in the early phase of genetic hypertension.

The present study explores whether the development of hypertension in the Milan strain of rats (MHS) rats is preceded or paralleled by alterations of mRNA and/or protein levels of the major luminal Na+ transporters. MHS rats were studied at 23-25 days after birth; age-matched Milan normotensive (MNS) rats were used as controls. The glomerular filtration rate (GFR), measured by inulin clearance, was higher in MHS than in MNS rats, while the mean blood pressure was not different in the two strains of animals indicating that the MHS rats were still in the prehypertensive state. Type 3 sodium/hydrogen exchanger (NHE3), bumetanide-sensitive sodium-potassium-2 chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC) and alpha-ENaC mRNA abundances were quantified by competitive PCR. In MHS compared with MNS, mRNA abundance was unchanged for NHE3 in proximal tubules, higher for NKCC2 in medullary thick ascending limbs of Henle's loops (TAL) and lower for NCC in distal convoluted tubules (DCT) and for alpha-ENaC along collecting ducts (CD). Western blot experiments revealed 1) unchanged NHE3; 2) a significant increase in NKCC2 in the outer medulla; 3) a significant decrease in NCC in the renal cortex and of alpha-ENaC in both the renal cortex and outer medulla, whereas beta- and gamma-ENaC remained unchanged. These data indicate that, in MHS rats, there is a strong upregulation of NKCC2 along the TAL associated with increased GFR, robust inhibition of NCC cotransporter along the DCT and modest downregulation of alpha-ENaC along the CD. The interplay of the various Na+ transporters may well explain why, at this age, the rats are still in the prehypertensive state.

Animals↗

PubMed2005

Surgical management of laryngotracheal stenosis in adults.

The purpose was to evaluate the outcome following the surgical management of a consecutive series of 26 adult patients with laryngotracheal stenosis of varied etiologies in a tertiary care center. Of the 83 patients who underwent surgery for laryngotracheal stenosis in the Department of Otorhinolaryngology and Head and Neck Surgery, University Hospital of Lausanne, Switzerland, between 1995 and 2003, 26 patients were adults (> or = 16 years) and formed the group that was the focus of this study. The stenosis involved the trachea (20), subglottis (1), subglottis and trachea (2), glottis and subglottis (1) and glottis, subglottis and trachea (2). The etiology of the stenosis was post-intubation injury ( n = 20), infiltration of the trachea by thyroid tumor ( n = 3), seeding from a laryngeal tumor at the site of the tracheostoma ( n = 1), idiopathic progressive subglottic stenosis ( n = 1) and external laryngeal trauma ( n = 1). Of the patients, 20 underwent tracheal resection and end-to-end anastomosis, and 5 patients had partial cricotracheal resection and thyrotracheal anastomosis. The length of resection varied from 1.5 to 6 cm, with a median length of 3.4 cm. Eighteen patients were extubated in the operating room, and six patients were extubated during a period of 12 to 72 h after surgery. Two patients were decannulated at 12 and 18 months, respectively. One patient, who developed anastomotic dehiscence 10 days after surgery, underwent revision surgery with a good outcome. On long-term outcome assessment, 15 patients achieved excellent results, 7 patients had a good result and 4 patients died of causes unrelated to surgery (mean follow-up period of 3.6 years). No patient showed evidence of restenosis. The excellent functional results of cricotracheal/tracheal resection and primary anastomosis in this series confirm the efficacy and reliability of this approach towards the management of laryngotracheal stenosis of varied etiologies. Similar to data in the literature, post-intubation injury was the leading cause of stenosis in our series. A resection length of up to 6 cm with laryngeal release procedures (when necessary) was found to be technically feasible.

Adolescent↗

PubMed2005

Mutation of colocalized residues of the pore helix and transmembrane segments S5 and S6 disrupt deactivation and modify inactivation of KCNQ1 K+ channels.

KCNQ1 (Kv 7.1) alpha-subunits and KCNE1 beta-subunits co-assemble to form channels that conduct the slow delayed rectifier K+ current (IKs) in the heart. Mutations in either subunit cause long QT syndrome (LQTS), an inherited disorder of cardiac repolarization. Here, the functional consequences of the LQTS-associated missense mutation V310I and several nearby residues were determined. Val310 is located at the base of the pore helix of KCNQ1, two residues below the TIGYG signature sequence that defines the K+ selectivity filter. Channels were heterologously expressed in Xenopus laevis oocytes and currents were recorded using the two-microelectrode voltage-clamp technique. V310I KCNQ1 reduced IKs amplitude when co-expressed with wild-type KCNQ1 and KCNE1 subunits. Val310 was also mutated to Gly, Ala or Leu to explore the importance of amino acid side chain volume at this position. Like V310I, V310L KCNQ1 channels gated normally. Unexpectedly, V310G and V310A KCNQ1 channels inactivated strongly and did not close normally in response to membrane hyperpolarization. Based on a homology model of the KCNQ1 channel pore, we speculate that the side group of residue 310 can interact with specific residues in the S5 and S6 domains to alter channel gating. When volume of the side chain is small, the stability of the closed state is disrupted and the extent of channel inactivation is enhanced. We mutated putative interacting residues in S5 and S6 and found that mutant Leu273 and Phe340 channels also can disrupt close states and modify inactivation. Together these findings indicate the importance of a putative pore helix-S5-S6 interaction for normal KCNQ1 channel deactivation and confirm its role in KCNQ1 inactivation. Disturbance of these interactions might underly LQTS associated with KCNQ1 mutant channels.

Amino Acid Sequence↗

PubMed2005

A novel potassium channel in lymphocyte mitochondria.

The margatoxin-sensitive Kv1.3 is the major potassium channel in the plasma membrane of T lymphocytes. Electron microscopy, patch clamp, and immunological studies identified the potassium channel Kv1.3, thought to be localized exclusively in the cell membrane, in the inner mitochondrial membrane of T lymphocytes. Patch clamp of mitoplasts and mitochondrial membrane potential measurements disclose the functional expression of a mitochondrial margatoxin-sensitive potassium channel. To identify unambiguously the mitochondrial localization of Kv1.3, we employed a genetic model and stably transfected CTLL-2 cells, which are genetically deficient for this channel, with Kv1.3. Mitochondria isolated from Kv1.3-reconstituted CTLL-2 expressed the channel protein and displayed an activity, which was identical to that observed in Jurkat mitochondria, whereas mitochondria of mock-transfected cells lacked a channel with the characteristics of Kv1.3. Our data provide the first molecular identification of a mitochondrial potassium conductance.

Blotting, Western↗

PubMed2005

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1.

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.

Animals↗

PubMed2005

Renal tubular transport and the genetic basis of hypertensive disease.

Several monogenic hypertensive disorders are caused by genetic mutations leading to the deranged function and/or regulation of renal tubular NaCl transport, such as mutations of the renal epithelial Na+ channel (ENaC) in Liddle syndrome, of the kinase WNK1 (with no K) in Gordon syndrome, and of the mineralocorticoid receptor, or of 11beta-hydroxysteroid dehydrogenase. Moreover, excessive formation of aldosterone in glucocorticoid-remediable hypertension leads to severe hypertension. Conversely, impaired function of the Na+,K+,2Cl- cotransporter (NKCC2), the renal outer medullary K+ channel (ROMK1), and the renal epithelial Cl- channel ClCKb/Barttin causes Bartter syndrome and defective Na+,Cl+ cotransporter (NCCT) Gitelman syndrome, salt-wasting disorders with hypotension. These monogenic disorders are rare, but illustrate the significance of renal tubular transport in blood pressure regulation. There is little doubt, however, that deranged renal salt reabsorption significantly contributes to essential hypertension polymorphisms of several genes participating in the regulation of renal Na+ transport have been shown to be associated with blood pressure and prevalence of hypertension. Two common genes will be discussed in more detail. The first encodes the renal Cl- channel ClCKb. A gain-of-function mutation of ClCKb, increasing channel activity by 7- to 20-fold is found in approximately 20% of unselected Caucasians and 40% of an unselected African population. The second common gene variant (prevalence, 3%-5% in unselected Caucasians), to be discussed in more detail, affects the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated by mineralocorticoids and enhancing the activity of ENaC, ROMK, and Na+/K+ATPase. Both gene variants are associated with slightly increased blood pressure. SGK1 further stimulates the glucose transporter SGLT1, and the SGK1 gene variant correlates, in addition, with increased body mass index.

Animals↗

PubMed2005

Partial cricotracheal resection for pediatric subglottic stenosis: long-term outcome in 57 patients.

OBJECTIVE: We sought to assess the long-term outcome of 57 pediatric patients who underwent partial cricotracheal resection for subglottic stenosis. METHODS: Eighty-one pediatric partial cricotracheal resections were performed in our tertiary care institution between 1978 and 2004. Fifty-seven patients had a minimal follow-up time of 1 year and were included in this study. Evaluation was based on the last laryngotracheal endoscopy, the responses to a questionnaire, and a retrospective review of the patient's data. The following parameters were analyzed: decannulation rates, breathing, voice quality, and deglutition. RESULTS: A single-stage partial cricotracheal resection was performed in 38 patients, and a double-stage procedure was performed in 19 patients. Sixteen patients underwent an extended partial cricotracheal resection (ie, partial cricotracheal resection combined with another open procedure). At a median follow-up time of 5.1 years, the decannulation rates after a single- or double-stage procedure were 97.4% and 95%, respectively. Two patients remained tracheotomy dependent. One patient had moderate exertional dyspnea, and all other patients had no exertional dyspnea. Voice quality was found to improve after surgical intervention for 1 +/- 1.34 grade dysphonia (P < .0001) according to the adapted GRBAS grading system (Grade, Roughness, Breathiness, Asthenia, and Strain). CONCLUSIONS: Partial cricotracheal resection provides good results for grades III and IV subglottic stenosis as primary or salvage operations. The procedure has no deleterious effects on laryngeal growth and function. The quality of voice significantly improves after surgical intervention but largely depends on the preoperative condition.

Adolescent↗

PubMed2005

Patch-clamp analysis of the "new permeability pathways" in malaria-infected erythrocytes.

The intraerythrocytic amplification of the malaria parasite Plasmodium falciparum induces new pathways of solute permeability in the host cell's membrane. These pathways play a pivotal role in parasite development by supplying the parasite with nutrients, disposing of the parasite's metabolic waste and organic osmolytes, and adapting the host's electrolyte composition to the parasite's needs. The "new permeability pathways" allow the fast electrogenic diffusion of ions and thus can be analyzed by patch-clamp single-channel or whole-cell recording. By employing these techniques, several ion-channel types with different electrophysiological profiles have been identified in P. falciparum-infected erythrocytes; they have also been identified in noninfected cells. This review discusses a possible contribution of these channels to the new permeability pathways on the one hand and their supposed functions in noninfected erythrocytes on the other.

Adenosine Triphosphate↗

PubMed2005

New insights into the role of serum- and glucocorticoid-inducible kinase SGK1 in the regulation of renal function and blood pressure.

PURPOSE OF REVIEW: The serum and glucocorticoid inducible kinase 1 (SGK1) is induced in the aldosterone sensitive distal nephron (ASDN) where it may stimulate Na reabsorption, partly by inhibiting ubiquitin ligase Nedd4-2-mediated retrieval of epithelial Na+ channel ENaC from the luminal membrane. We describe recent advances in our understanding of SGK1 function in the regulation of renal function and blood pressure. RECENT FINDINGS: Thiazolidinediones, i.e. activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), upregulate SGK1 and ENaC mRNA expression and increase cell-surface expression of ENaC alpha in a human cortical-collecting-duct cell line. cAMP/protein kinase A can induce phosphorylation and inhibition of Nedd4-2-independent of SGK1. Part of ENaC stimulation by SGK1 appears dependent on a SGK1 consensus motif in ENaC alpha and independent of Nedd4-2. SGK1-dependent upregulation of Na+ reabsorption in ASDN contributes to upregulation of renal K+ excretion. In oocytes, SGK1 activates various renal transport proteins including Na+/glucose cotransporter SGLT1, Na+-coupled dicarboxylate transporter NaDC-1, epithelial Ca+ channel TRPV5, renal outer medullary K+ channel ROMK and voltage gated K+ channels KCNE1/KCNQ1 and Kv1.3. A variant of the SGK1 gene associates with increased blood pressure and body mass index. SUMMARY: PPAR gamma activators may increase renal Na reabsorption by stimulating SGK1 and ENaC. Nedd4-2 integrates influences of cAMP/protein kinase A and SGK1. SGK1 can activate ENaC in part directly and independent of Nedd4-2. K+ homeostasis requires SGK1-dependent Na+ reabsorption in ASDN. SGK1 may affect renal transport mechanisms beyond Na+ reabsorption and K+ secretion in ASDN. Polymorphisms of SGK1 may be relevant to the pathophysiology of hypertension and other diseases.

Blood Pressure↗

PubMed2005

Intracellular multiplication of Legionella pneumophila depends on host cell amino acid transporter SLC1A5.

The infectious agent of Legionnaires' disease, Legionella (L) pneumophila, multiplies intracellularly in eukaryotic cells. This study has been performed to explore the nutrient requirements of L. pneumophila during intracellular replication. In human monocytes, bacterial replication rate was reduced by 76% in defined medium lacking L-cysteine, L-glutamine or L-serine. SLC1A5 (hATB(0,+)), a neutral amino acid transporter, was upregulated in the host cells after infection with L. pneumophila. Inhibition of SLC1A5 by BCH, a competitive inhibitor of amino acid uptake as well as siRNA silencing of the slc1a5 gene blocked intracellular multiplication of L. pneumophila without compromising viability of host cells. These observations suggest that replication of L. pneumophila depends on the function of host cell SLC1A5.

Amino Acid Transport System ASC↗

PubMed2005

Stimulation of erythrocyte phosphatidylserine exposure by lead ions.

Pb+ intoxication causes anemia that is partially due to a decreased life span of circulating erythrocytes. As shown recently, a Ca(2+)-sensitive erythrocyte scramblase is activated by osmotic shock, oxidative stress, and/or energy depletion, leading to exposure of phosphatidylserine at the erythrocyte surface. Because macrophages are equipped with phosphatidylserine receptors, they bind, engulf, and degrade phosphatidylserine-exposing cells. The present experiments were performed to explore whether Pb+ ions trigger phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated on the basis of annexin binding as determined using fluorescence-activated cell sorting (FACS) analysis. Exposure to Pb+ ions [> or =0.1 microM Pb(NO3)2] significantly increased annexin binding. This effect was paralleled by erythrocyte shrinkage, which was apparent on the basis of the decrease in forward scatter in FACS analysis. The effect of Pb+ ions on cell volume was virtually abolished, and the effect of Pb+ ions on annexin binding was blunted after increase of extracellular K+ concentration. Moreover, both effects of Pb+ ions were partially prevented in the presence of clotrimazole (10 microM), an inhibitor of the Ca(2+)-sensitive K+ channels in the erythrocyte cell membrane. Whole cell patch-clamp experiments disclosed a significant activation of a K(+)-selective conductance after Pb+ ion exposure, an effect requiring higher (10 microM) concentrations, however. In conclusion, Pb+ ions activate erythrocyte K+ channels, leading to erythrocyte shrinkage, and also activate the erythrocyte scramblase, leading to phosphatidylserine exposure. The effect could well contribute to the reported decreased life span of circulating erythrocytes during Pb+ intoxication.

Annexins↗

PubMed2005

Role of Sgk1 in salt and potassium homeostasis.

Aldosterone plays a pivotal role in NaCl and K(+) homeostasis by stimulation of Na(+) reabsorption and K(+) secretion in the aldosterone-sensitive distal nephron (ASDN). Recent studies demonstrated that the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by aldosterone in the ASDN and that polymorphisms of the kinase associate with arterial blood pressure in normotensive subjects. This review discusses the role of Sgk1 in NaCl and K(+) homeostasis as evidenced by in vivo studies, including those in Sgk1-deficient mice. The studies indicate that Sgk1 is not absolutely required for Na(+) reabsorption and K(+) secretion in the ASDN. On a standard NaCl and K(+) diet, modestly enhanced plasma aldosterone concentrations appear sufficient to establish a compensated phenotype in the absence of Sgk1. The kinase is necessary, however, for upregulation of transcellular Na(+) reabsorption in the ASDN. This may involve Sgk1-mediated stimulation of basolateral Na(+)-K(+)-ATPase as well as retention of epithelial Na(+) channel, ENaC, in the apical membrane. Such an upregulation is a prerequisite for adequate adaptation of 1) renal NaCl reabsorption during restricted dietary NaCl intake, as well as 2) K(+) secretion in response to enhanced K(+) intake. Thus gain-of-function mutations of Sgk1 are expected to result in renal NaCl retention and enhanced K(+) secretion. Further studies are required to elucidate renal and nonrenal aldosterone-induced effects of Sgk1, the role of other Sgk1 activators, as well as the link of Sgk1 polymorphisms to arterial hypertension in humans.

Aldosterone↗

PubMed2005

SGK1 as a determinant of kidney function and salt intake in response to mineralocorticoid excess.

Mineralocorticoids modify salt balance by both stimulating salt intake and inhibiting salt loss. Renal salt retention is accomplished by upregulation of reabsorption, an effect partially mediated by serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the contribution of SGK1 to the regulation of renal function, salt intake, and blood pressure during mineralocorticoid excess. DOCA/1% NaCl treatment increased blood pressure and creatinine clearance to a similar extent in SGK1-deficient sgk1(-/-) and wild-type sgk1(+/+) mice but led to more pronounced increase of proteinuria in sgk1(+/+) mice (by 474 +/- 89%) than in sgk1(-/-) mice (by 154 +/- 31%). DOCA/1% NaCl treatment led to significant increase of kidney weight (by 24%) and to hypokalemia (from 3.9 +/- 0.1 to 2.7 +/- 0.1 mmol/l) only in sgk1(+/+) mice. The treatment enhanced renal Na(+) excretion significantly more in sgk1(+/+) mice (from 3 +/- 1 to 134 +/- 32 micromol.24 h(-1).g body wt(-1)) than in sgk1(-/-) mice (from 4 +/- 1 to 49 +/- 8 micromol.24 h(-1).g body wt(-1)), pointing to SGK1-dependent stimulation of salt intake. With access to two drinking bottles containing 1% NaCl or water, DOCA treatment did not significantly affect water intake in either genotype but increased 1% NaCl intake in sgk1(+/+) mice (within 9 days from 3.5 +/- 0.9 to 16.5 +/- 2.4 ml/day) consistent with DOCA-induced salt appetite. This response was significantly attenuated in sgk1(-/-) mice (from 2.6 +/- 0.6 to 5.9 +/- 0.9 ml/day). Thus SGK1 contributes to the stimulation of salt intake, kidney growth, proteinuria, and renal K(+) excretion during mineralocorticoid excess.

Animals↗

PubMed2005

Requirement of PDZ domains for the stimulation of the epithelial Ca2+ channel TRPV5 by the NHE regulating factor NHERF2 and the serum and glucocorticoid inducible kinase SGK1.

Renal calcium reabsorption involves the epithelial calcium channel ECaC1 (TRPV5) which is tightly regulated by 1,25(OH)2D3. As shown recently, TRPV5 is activated by the serum and glucocorticoid inducible kinase SGK1, a kinase transcriptionally upregulated by 1,25(OH)2D3. This stimulatory effect is due to enhanced TRPV5 abundance in the plasma membrane and requires the presence of the scaffold protein NHERF2 (sodium hydrogen exchanger regulating factor 2). The present study aims to define the molecular requirements for the interaction of TRPV5 with SGK1 and NHERF2. Pull-down experiments and overlay assays revealed that the TRPV5 C-tail interacts in a Ca2+-independent manner with NHERF2. Deletion of the second but not of the first PDZ domain in NHERF2 abrogates the stimulating effect of SGK1/NHERF2 on TRPV5 protein abundance in the plasma membrane as quantified by chemiluminescence and electrophysiology. Thus, the second PDZ domain in NHERF2 is required for stabilization at or TRPV5 targeting to the plasma membrane. The experiments demonstrate the significance of SGK1 and NHERF2 as TRPV5 modulators which are likely to participate in the regulation of calcium homeostasis by 1,25(OH)2D3.

Animals↗

PubMed2005

Mechanisms of suicidal erythrocyte death.

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure ('eryptosis') mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in 'eryptosis' may similarly contribute to apoptosis of nucleated cells.

Animals↗

PubMed2005

Effect of sphingosine on Ca2+ entry and mitochondrial potential of Jurkat T cells--interaction with Bcl2.

Triggers of Jurkat T cell apoptosis include sphingosine and ceramide. Sphingosine and ceramide further inhibit capacitative Ca2+ entry (ICRAC), an effect leading to inactivation but not death of Jurkat T cells. Mitochondria are key organelles in the machinery leading to apoptosis and on the other hand have been shown to participate in the regulation of Ca2+ entry. The present experiments were performed to explore whether treatment of Jurkat T cells with sphingosine leads to apoptosis and reduced Ca2+ entry and whether those effects are sensitive to expression of the antiapoptotic protein Bcl2, localized in the outer mitochondrial membrane. Exposure of Jurkat T cells to 10 microM spingosine was according to DiOC6 fluorescence followed by mitochondrial depolarization and according to Fura-red/Fluo-3 fluorescence followed by decreased capacitative Ca2+ entry. Mitochondrial depolarization was significantly delayed in cells overexpressing wild type Bcl2 or Bcl2 targeted to the mitochondrial membrane, whereas no significant influence on mitochondrial depolarization was observed in cells expressing Bcl2 lacking the membrane targeting motif or Bcl2 targeted to the endoplasmatic reticulum. In contrast to mitochondrial potential, the blunting of capacitative Ca2+ entry following sphingosine treatment was not sensitive to mitochondrial Bcl2 expression. In conclusion sphingosine exposure leads to both, mitochondrial depolarization and inhibition of capacitative Ca2+ entry. Mitochondrial Bcl2 reverses the effect on mitochondria but not on Ca2+ entry and thus leads to dissociation of those two sequelae of sphingosine treatment.

Apoptosis↗

PubMed2005

Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 microM Dex for 4-5.5 h or 50 microM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.

Animals↗