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Aim: The purpose of the present research was a presentation of case report of Robertsonian translocation composed of homologous chromosomes 21q;21q and reproductive risk found in the family affected by this type oftranslocation., Methods: Cytogenetic diagnosis has been done on chromosome preparations of lymphocytes cultured from peripheral blood by Moorhead method., Results: Analyses of cytogenetic diagnosis was performed on the couple who has been through 10 spontaneous miscarriages and two additional births with Down syndrome. The woman had Robertsonian translocation between homologous chromosomes 21: 45XX,der(21;21)(q10;q10), and there was no change in her phenotype, whereas her husband had a normal phenotype and karyotype: 46, XY. Their first child with Down syndrome symptoms did not undergo the cytogenetic analysis. By cytogenetic analysis it was discovered that their second child has Trisomy 21 with Robertsonian translocations between homologous chromosomes 21: 46,XY,+21,der(21;21) (q10;q10)mat, and that he inherited it from his mother., Conclusion: Chromosomal aberration that our patient suffered from and that is presented in this paper has caused spontaneous miscarriages and birth of children with Down syndrome. Based on cytogenetic analysis in prenatal diagnosis and genetic consultation of affected family with Robertsonian translocation 21q;21q, it is unlikely to select healthy offspring by a parent with that aberration.

The Tau-tubulin-kinase 2 (TTBK2) is a serine/threonine kinase expressed in various tissues including tumors. Up-regulation of TTBK2 increases resistance of tumor cells against antiangiogenic treatment and confers cell survival. Tumor cell survival critically depends on cellular uptake of glucose, which is partially accomplished by SGLT1 (SLC5A1) mediated Na(+)-coupled glucose transport. The present study explored whether TTBK2 participates in the regulation of SGLT1 activity. To this end, electrogenic glucose transport was determined in Xenopus oocytes expressing SGLT1 with or without wild-type TTBK2, truncated TTBK2([1-450]) or kinase inactive mutants TTBK2-KD and TTBK2-KD([1-450]). TTBK2, but not TTBK2([1-450]), TTBK2-KD or TTBK2-KD([1-450]), increased membrane carrier protein abundance and electrogenic glucose transport capacity in SGLT1-expressing Xenopus oocytes. Thus TTBK2 is a completely novel regulator of Na(+)-coupled glucose transport., (Copyright © 2012 S. Karger AG, Basel.)

Insulin and growth factors activate the phosphatidylinositide-3-kinase pathway, leading to stimulation of several kinases including serum- and glucocorticoid-inducible kinase isoform SGK3, a transport regulating kinase. Here, we explored the contribution of SGK3 to the regulation of renal tubular phosphate transport. Coexpression of SGK3 and sodium-phosphate cotransporter IIa significantly enhanced the phosphate-induced current in Xenopus oocytes. In sgk3 knockout and wild-type mice on a standard diet, fluid intake, glomerular filtration and urine flow rates, and urinary calcium ion excretion were similar. However, fractional urinary phosphate excretion was slightly but significantly larger in the knockout than in wild-type mice. Plasma calcium ion, phosphate concentration, and plasma parathyroid hormone levels were not significantly different between the two genotypes, but plasma calcitriol and fibroblast growth factor 23 concentrations were significantly lower in the knockout than in wild-type mice. Moreover, bone density was significantly lower in the knockouts than in wild-type mice. Histological analysis of the femur did not show any differences in cortical bone but there was slightly less prominent trabecular bone in sgk3 knockout mice. Thus, SGK3 has a subtle but significant role in the regulation of renal tubular phosphate transport and bone density.

Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The inward rectifier K(+) channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen-Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKβ1+AMPKγ1), of the constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2(S795A) lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2(S795A) was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2., (Copyright © 2011 Elsevier Inc. All rights reserved.)

JAK2 (Janus kinase-2) overactivity contributes to survival of tumor cells and the (V617F)JAK2 mutant is found in the majority of myeloproliferative diseases. Tumor cell survival depends on availability of glucose. Concentrative cellular glucose uptake is accomplished by Na(+) coupled glucose transport through SGLT1 (SLC5A1), which may operate against a chemical glucose gradient and may thus be effective even at low extracellular glucose concentrations. The present study thus explored whether JAK2 activates SGLT1. To this end, SGLT1 was expressed in Xenopus oocytes with or without wild type JAK2, (V617F)JAK2 or inactive (K882E)JAK2 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of glucose to the extracellular bath generated a current (I(g)), which was significantly increased following coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. Kinetic analysis revealed that coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The stimulating effect of JAK2 expression was abrogated by preincubation with the JAK2 inhibitor AG490. Chemiluminescence analysis revealed that JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I(g) during inhibition of carrier insertion by brefeldin A was similar in the absence and presence of JAK2. Thus, JAK2 fosters insertion rather than inhibiting retrieval of carrier protein into the cell membrane. In conclusion, JAK2 upregulates SGLT1 activity which may play a role in the effect of JAK2 during ischemia and malignancy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa-expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3(KI) mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3(KI) animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3(KI) mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3(KI) mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss., (Copyright © 2011 by the American Society of Nephrology)

Connexins provide intercellular connections that allow passage of ions and small organic molecules. They clamp the cell membrane potential and cellular ion composition to that of neighboring cells. The cell membrane potential and ion composition of an energy-depleted cell could thus be maintained despite its compromised Na(+)/K(+) activity. By the same token, however, the breakdown of ion gradients in that cell imposes an additional challenge to the neighboring cells, which may jeopardize their survival. Thus, timely closure of connexins may be critically important for the survival of those cells. Energy depletion stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase that senses energy depletion and stimulates several cellular mechanisms to enhance energy production and to limit energy utilization. The present study explored whether AMPK regulates connexin 26. To this end, cRNA encoding connexin 26 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1β1γ1), of the constitutively active (γR70Q)AMPK (α1β1γ1[R70Q]) or of the inactive mutant (αK45R)AMPK (α1[K45R]β1γ1). Connexin 26 activity was determined in dual-electrode voltage-clamp experiments. Moreover, connexin 26 abundance was determined in the oocyte cell membrane by chemiluminescence and confocal microscopy. As a result, connexin 26-mediated current and connexin 26 protein abundance were significantly decreased by coexpression of (γR70Q)AMPK and, to a lower extent, by wild-type AMPK but not by (αK45R)AMPK. In conclusion, AMPK is a potent regulator of connexin 26.

The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.

Tumor cells utilize preferably glucose for energy production. They accomplish cellular glucose uptake in part through Na(+)-coupled glucose transport mediated by SGLT1 (SLC5A1). This study explored the possibility that the human papillomavirus 18 E6 protein HPV18 E6 (E6) participates in the stimulation of SGLT1 activity. E6 is one of the two major oncoproteins of high-risk human papillomaviruses, which are the causative agent for cervical carcinoma. According to Western blotting, SGLT1 is expressed in the HPV18-positive cervical carcinoma cell line HeLa. To explore whether E6 affects SGLT1 activity, SGLT1 was expressed in Xenopus oocytes with and without E6 and electrogenic glucose transport determined by dual electrode voltage clamp. In SGLT1-expressing oocytes, but not in oocytes injected with water or expressing E6 alone, glucose triggered a current (I(g)). I(g) was significantly increased by coexpression of E6 but not by coexpression of E2. According to chemiluminescence and confocal microscopy, coexpression of E6 significantly increased the SGLT1 protein abundance in the cell membrane. The decay of I(g) following inhibition of carrier insertion by Brefeldine A (5 μM) was not significantly affected E6 coexpression. Accrodingly, E6 was not effective by increasing carrier protein stability in the membrane. In conclusion, HPV18 E6 oncoprotein participates in the upregulation of SGLT1., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Klotho, a transmembrane protein, protease and hormone has been shown to exert a profound effect on phosphate metabolism. Klotho overexpression lowers and Klotho deficiency increases the plasma phosphate concentration, effects in part attributed to an inhibitory effect of Klotho on the formation of 1,25-dihydroxycholecalciferol (1,25(OH) (2)D(3)), the active form of Vitamin D. Beyond that Klotho has been shown to decrease renal tubular phosphate transport more directly. The influence of Klotho on the plasma phosphate concentration contributes to the profound effect of Klotho on ageing and life span. The present study explored whether Klotho influences the major renal tubular (NaPi-IIa) and the major intestinal (NaPi-IIb) phosphate transporters. For functional analysis NaPi-IIa or NaPi-IIb were expressed in Xenopus oocytes both, without or with additional coexpression of Klotho and electrogenic phosphate transport was estimated from the phosphate-induced current (Ip). According to RT-PCR Klotho is expressed in the murine kidney and intestine. Coexpression of Klotho decreased Ip in both NaPi-IIa- and NaPi-IIb-expressing oocytes. Klotho decreased the maximal Ip without appreciably affecting the concentration required for halfmaximal Ip. Treatment of NaPi-IIa- or NaPi-IIb-expressing oocytes with Klotho protein similarly decreased Ip. In conclusion, Klotho down regulates both, renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters., (Copyright © 2011 S. Karger AG, Basel.)

The Janus-activated kinase-2 JAK2 is involved in the signaling of leptin and erythropoietin receptors and mediates neuroprotective effects of the hormones. In theory, JAK2 could be effective through modulation of the glutamate transporters, carriers accounting for the clearance of glutamate released during neurotransmission. The present study thus elucidated the effect of JAK2 on the glutamate transporters EAAT1, EAAT2, EAAT3 and EAAT4. To this end, cRNA encoding the carriers was injected into Xenopus oocytes with or without cRNA encoding JAK2 and glutamate transport was estimated from glutamate induced current (I(glu)). I(glu) was observed in Xenopus oocytes expressing EAAT1 or EAAT2 or EAAT3 or EAAT4, but not in water injected oocytes. Coexpression of JAK2 resulted in an increase of I(glu) by 83% (EAAT1), 67% (EAAT2), 42% (EAAT3) and 126% (EAAT4). As shown for EAAT4 expressing Xenopus oocytes, the effect of JAK2 was mimicked by gain of function mutation (V617F)JAK2 but not by the inactive mutant (K882E)JAK2. Incubation with JAK2 inhibitor AG490 (40 μM) resulted in a gradual decrease of I(glu) by 53%, 79% and 92% within 3, 6 and 24 hours. Confocal microscopy and chemiluminescence analysis revealed that JAK2 coexpression increased EAAT4 protein abundance in the cell membrane. Disruption of transcription did not appreciably modify the up-regulation of I(glu) in EAAT4 expressing oocytes. The decay of I(glu) following inhibition of carrier insertion with brefeldin A was similar in oocytes expressing EAAT4 + JAK2 and oocytes expressing EAAT4 alone, indicating that JAK2 did not appreciably affect carrier retrieval from the membrane. In conclusion, JAK2 is a novel powerful regulator of glutamate transporters and thus participates in the protection against excitotoxicity., (Copyright © 2011 S. Karger AG, Basel.)

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.

β-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. β-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that β-catenin influences membrane transport. To this end, β-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of β-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na(+)/K(+)-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of β-catenin on the endogenous Na(+)/K(+)-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of β-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of β-catenin expression. The stimulating effect of β-catenin on both Na(+)/K(+) ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of β-catenin, i.e. the regulation of transport., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na+-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active (gammaR70Q)AMPK (alpha1beta1gamma1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I(g)), which was half maximal (K(M)) at approximately 650 microM glucose concentration. Coexpression of (gammaR70Q)AMPK did not affect K(M) but significantly enhanced the maximal current (approximately 1.7 fold). Coexpression of wild type AMPK or the kinase dead (alphaK45R)AMPK mutant (alpha1(K45R)beta1gamma1) did not appreciably affect I(g). According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 microM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na+-coupled glucose transport.

The serum- and glucocorticoid-inducible kinase SGK1 and the protein kinase PKB/Akt presumably phosphorylate and, by this means, activate the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3), which has in turn been shown to regulate transporters and channels. SGK1-regulated channels include the Ca(2+) channel TRPV6, which is expressed in a variety of epithelial and nonepithelial cells including tumor cells. SGK1 and protein kinase B PKB/Akt foster tumor growth. The present study thus explored whether TRPV6 is regulated by PIKfyve. TRPV6 was expressed in Xenopus laevis oocytes with or without additional coexpression of constitutively active (S422D)SGK1, constitutively active (T308D,S473D)PKB, wild-type PIKfyve, and (S318A)PIKfyve lacking the SGK1 phosphorylation site. TRPV6 activity was determined from the current (I(Ca)) resulting from TRPV6-induced Ca(2+) entry and subsequent activation of Ca(2+)-sensitive endogenous Cl(-) channels. TRPV6 protein abundance in the cell membrane was determined utilizing immunohistochemistry and Western blotting. In TRPV6-expressing oocytes I(H) was increased by coexpression of (S422D)SGK1 and by (T308D,S473D)PKB. Coexpression of wild-type PIKfyve further increased I(H) in TRPV6 + (S422D)SGK1-expressing oocytes but did not significantly modify I(Ca) in oocytes expressing TRPV6 alone. (S318A)PIKfyve failed to significantly modify I(Ca) in the presence and absence of (S422D)SGK1. (S422D)SGK1 increased the TRPV6 protein abundance in the cell membrane, an effect augmented by additional expression of wild-type PIKfyve. We conclude that PIKfyve participates in the regulation of TRPV6.

The neutral amino acid transporter SLC6A19 (B(0)AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo., (Copyright 2010 S. Karger AG, Basel.)

Glycogen synthase kinase 3 GSK3β participates in a wide variety of functions including regulation of glucose metabolism. It is ubiquitously expressed including epithelial tissues. However, whether GSK3β participates in the regulation of epithelial transport is not known. The present study thus explored whether GSK3β influences the Na(+)-coupled transport of glucose. To this end, SGLT1 was expressed in Xenopus oocytes with or without GSK3β and glucose-induced current (I(g)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing SGLT1 but not in water-injected oocytes glucose induced an inwardly directed I(g), which was significantly enhanced by coexpression of GSK3β. According to chemiluminescence and confocal microscopy, GSK3β increased the SGLT1 protein abundance in the oocyte cell membrane. To explore whether GSK3β sensitivity of SGLT1 participates in the regulation of electrogenic intestinal glucose transport, Ussing chamber experiments were performed in intestinal segments from gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,β (gsk3(KI)), in which the serine of the PKB/SGK phosphorylation site was replaced by alanine, and from wild type mice (gsk3(WT)). The glucose-induced current was significantly larger in gsk3(KI) than in gsk3(WT) mice. The present observations reveal a novel function of GSK3, i.e. the stimulation of Na(+)-coupled glucose transport., (Copyright © 2010 S. Karger AG, Basel.)

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of ENaC-mediated sodium transport and is involved in the transduction of growth-factor-dependent cell survival and proliferation. The identification of molecular partners for Sgk1 is crucial for the understanding of its mechanisms of action. We performed a yeast two-hybrid screening based on a human kidney cDNA library to identify molecular partners of Sgk1. As a result the screening revealed a specific interaction between Sgk1 and a 60 kDa Lysophospholipase (LysoLP). LysoLP is a poorly characterized enzyme that, based on sequence analysis, might possess lysophospholipase and asparaginase activities. We demonstrate that LysoLP has indeed a lysophospholipase activity and affects metabolic functions related to cell proliferation and regulation of membrane channels. Moreover we demonstrate in the Xenopus oocyte expression system that LysoLP downregulates basal and Sgk1-dependent ENaC activity. In conclusion LysoLP may represent a new player in the regulation of ENaC and Sgk1-dependent signaling., (Copyright © 2010 S. Karger AG, Basel.)

Mutations in GJB2, a gene encoding the gap junction protein connexin 26 (Cx26), are a major cause for inherited and sporadic non-syndromic hearing loss, albeit with highly variable clinical effects. To determine new mutations and their frequencies in a Southern Egyptian population restriction fragment length polymorphism, gene sequencing, and single strand conformational polymorphism revealed only 2 mutations for GJB2: c.35delG and p.I71N. The allelic frequency of the c.35delG mutation was 8.7% (found in 27 out of 310 investigated alleles) resulting in a relatively low carrier frequency (1.6%) in Upper Egypt. The new mutation, a substitution of isoleucin (I) (a non-polar amino acid) by the polar amino acid asparagin (N), was localized within the conserved Cx26 structure. The functional significance of p.I71N was tested by injection of cRNA into Xenopus laevis oocytes. Cx26 hemi-channel activity was measured by depolarization activated conductance in non-coupled oocytes. As a result, the p.I71N mutated channel was non-functional. The study discloses a novel, functionally relevant GJB2 mutation and defines the contribution of Cx26 alterations to the hearing loss in the Southern Egyptian population., (Copyright © 2010 S. Karger AG, Basel.)

According to recent in vitro experiments, the peptide transporter PepT2 is stimulated by the serum- and glucocorticoid-inducible kinase SGK1. The present study explored the contribution of SGK1 to the regulation of electrogenic intestinal peptide transport. Intestinal PepT1 was expressed in Xenopus oocytes, and peptide transport was determined by dual electrode voltage clamping. Peptide transport in intestinal segments was determined utilizing Ussing chamber. Cytosolic pH (pH( i )) was determined by BCECF fluorescence and Na(+)/H(+) exchanger activity was estimated from Na(+)-dependent pH recovery (pH ( i )) following an ammonium pulse. In PepT1-expressing Xenopus oocytes, coexpression of SGK1 enhanced electrogenic peptide transport. Intestinal transport and pH( i ) of untreated mice were similar in SGK1 knockout mice (sgk1 ( -/- )) and their wild-type littermates (sgk1 ( +/+ )). Glucocorticoid treatment (4 days 10 microg/g body weight (bw)/day dexamethasone) increased peptide transport in sgk1 ( +/+ ) but not in sgk1 (-/-) mice. Irrespective of dexamethasone treatment, luminal peptide (5 mM glycyl-glycine) led to a similar early decrease of pH( i ) in sgk1 (-/-) and sgk1 (+/+) mice, but to a more profound and sustained decline of pH( i ) in sgk1 (-/-) than in sgk1 ( +/+ ) mice. In the presence and absence of glycyl-glycine, pH ( i ) was significantly enhanced by dexamethasone treatment in sgk1 ( +/+ ) mice, an effect significantly blunted in sgk1 ( -/- ) mice. During sustained exposure to glycyl-glycine, pH ( i ) was significantly larger in sgk1 (+/+) mice than in sgk1 (-/-) mice, irrespective of dexamethasone treatment. In conclusion, basal intestinal peptide transport does not require stimulation by SGK1. Glucocorticoid treatment stimulates both Na(+)/H(+) exchanger activity and peptide transport, effects partially dependent on SGK1. Moreover, chronic exposure to glycyl-glycine stimulates Na(+)/H(+) exchanger activity, an effect again involving SGK1.

Introduction: According to previous observations, the gene encoding the phosphatidylinositol-4-phosphate 5-kinase II alpha (PIP5K2A) is associated with schizophrenia. Specifically, the mutation (N251S)PIP5K2A has been discovered in schizophrenic patients but not in healthy individuals. A defect of the excitatory amino acid transporter EAAT3 has similarly been implicated in the development of schizophrenia. The present study thus explored whether PIP5K2A is involved in the regulation of EAAT3 activity., Materials and Methods: EAAT3 was expressed in Xenopus oocytes either without or with PIP5K2A, and EAAT3 transporter activity was estimated from the glutamate (2-mM)-induced current (I(glu)) in dual electrode voltage clamp experiments. EAAT3 protein abundance in the cell membrane was estimated by Western blotting and confocal microscopy., Results: In EAAT3-expressing oocytes, I(glu) was enhanced by coexpression of wild type PIP5K2A. Coexpression of the schizophrenia-associated mutant (N251S)PIP5K2A significantly decreased I(glu) in oocytes expressing EAAT3 with or without additional expression of wild type PIP5K2A. Thus, (N251S)PIP5K2A exerts a dominant inhibitory effect., Discussion: Membrane abundance of EAAT3 was increased by wild type PIP5K2A and decreased by (N251S)PIP5K2A in both EAAT3-expressing oocytes and human embryonic kidney cells. The present observations disclose a novel mechanism of EAAT3 regulation, which may contribute to the deranged regulation of excitability in schizophrenic patients.

Owing to its antibiotic activity, silver is used for water purification, wound care and a wide variety of implants. Silver metal and silver compounds ionize in solution, and silver ions interfere with the function of a wide variety of proteins. In mammalian cells, silver ions may trigger apoptosis by stimulation of cytochrome c release from mitochondria. The present study explored the effect of AgNO3 on eryptosis, the suicidal death of erythrocytes, cells devoid of mitochondria. Similar to apoptosis of nucleated cells, eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by energy depletion, cellular depletion of nitric oxide (NO) and activation of protein kinase C (PKC). Phosphatidylserine exposure was determined by annexin V-binding, cell volume by forward scatter, cellular ATP by a luciferin-luciferase assay kit, and hemolysis by photometry. A 48 h exposure to AgNO3 (> or =100 nm) but not to NaNO3 significantly enhanced the percentage of annexin V-binding cells, slightly but significantly decreased forward scatter and significantly decreased cytosolic ATP. Furthermore, inhibition of PKC by staurosporine and donation of NO by sodium nitroprusside significantly blunted silver-induced eryptosis. In conclusion, AgNO3 triggers cell membrane scrambling, an effect attributed to ATP depletion, PKC activation and decrease of cellular NO., (Copyright 2009 John Wiley & Sons, Ltd.)

AMP-activated protein kinase (AMPK), an energy-sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca(2+) activity ([Ca(2+)](i)) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca(2+)](i) (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence-activated cell sorting (FACS) analysis. Glucose removal increased [Ca(2+)](i), decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 microM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca(2+)](i) by Ca(2+) ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM). As compared with erythrocytes from wild-type littermates (ampk(+/+)), erythrocytes from AMPKalpha1-deficient mice (ampk(-/-)) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk(-/-) mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.

The Na(+),glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (I(glu)). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I(glu) in EAAT2 expressing oocytes. I(glu) was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K127N)SGK1 did not significantly alter I(glu) in EAAT2 expressing oocytes and significantly decreased I(glu) in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on I(glu) was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S318A)PIKfyve). Furthermore, additional coexpression of (S318A)PIKfyve virtually abolished I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed participates in the regulation of EAAT2., (2009 S. Karger AG, Basel.)

The suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. Erythrocyte cell membrane scrambling is stimulated by increase of cytosolic Ca2+ concentration ([Ca2+](i)) and formation of ceramide. Phosphatidylserine (PS) exposing cells are rapidly cleared from circulating blood. Ca2+ entry and/or ceramide formation and thus eryptosis are triggered by lead, mercury, aluminium, and copper ions. The present study explored whether eryptosis could be similarly triggered by exposure to gold. To this end, erythrocytes from healthy volunteers were exposed to AuCl and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), [Ca2+](i) (Fluo3-dependent fluorescence), and ceramide formation (anti-ceramide-FITC fluorescence) were determined by flow cytometry. Exposure of erythrocytes to low concentrations of AuCl (> or =0.75microg/ml) increased [Ca2+](i) but did not affect ceramide formation. AuCl at concentrations > or =0.5microg/ml significantly increased the number of PS exposing erythrocytes and decreased forward scatter at low concentrations of AuCl pointing to cell shrinkage. Aurothiomalate (> or =1microg/ml), a gold containing drug effective against rheumatoid arthritis, similarly triggered PS exposure of erythrocytes. The present observations disclose a novel action of gold, which may well contribute to side effects during treatment with gold preparations.

Selenium is an essential element incorporated into selenoproteins. Selenium deficiency may predispose to immune deficiency, mood disorders, and cancer. On the other hand, excessive environmental exposure to selenite may cause a variety of disorders including anemia. At least in theory, the anemia could result from accelerated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by an increase in the cytosolic Ca(2+) concentration and by ceramide. The present experiments explored, whether high concentrations of selenite stimulate eryptosis. According to Fluo3 fluorescence, selenite (>or=200 microg/l sodium selenite) within 48 hours significantly increased the cytosolic Ca(2+) concentration in human erythrocytes. According to binding of selective fluorescent antibodies, selenite (>or= 200 microg/l) significantly increased ceramide formation. Annexin V-binding demonstrated that selenite (>or=200 microg/l) significantly increased phosphatidylserine exposure of erythrocytes. Forward scatter analysis further revealed that selenite (>or=200 microg/l) significantly decreased cell volume. In contrast to selenite, selenate failed to trigger eryptosis. In conclusion, selenite triggers suicidal erythrocyte death at least partially by increasing the cytosolic Ca(2+) concentration and by stimulating the formation of ceramide. The present study discloses novel cellular effects of this essential nutrient., (Copyright 2008 S. Karger AG, Basel.)

Cadmium ions are known to trigger apoptosis. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. Cellular mechanisms known to trigger cell membrane phospholipid scrambling include increased cytosolic Ca(2+) activity and activation of a sphingomyelinase with formation of ceramide. The present experiments were performed to explore whether cadmium ions (Cd(2+)) trigger phosphatidylserine exposure of erythrocytes and to possibly identify underlying mechanisms. Phosphatidylserine exposure was estimated from annexin V-binding as determined in fluorescence activated cell sorting (FACS) analysis. Exposure to Cd(2+) (>or= 5.5 microM Cd(2+)) indeed significantly increased annexin V-binding. This effect was paralleled by erythrocyte shrinkage as apparent from the decrease of forward scatter in FACS analysis. According to Fluo3 fluorescence, Cd(2+) increased the entry of Ca(2+) into erythrocytes. According to antibody binding, Cd(2+) did not stimulate the formation of ceramide. In the nominal absence of extracellular Ca(2+) and in the presence of cation channel inhibitor amiloride the effects of Cd(2+) on erythrocyte phosphatidylserine exposure and forward scatter were blunted. In conclusion, in human erythrocytes Cd(2+) stimulates entry of Ca(2+), which activates Ca(2+)-sensitive K(+) channels leading to erythrocyte shrinkage and triggers Ca(2+)-sensitive erythrocyte membrane scrambling leading to phosphatidylserine exposure., (Copyright 2008 S. Karger AG, Basel.)

Vanadium, a trace element, as vanadate (VO4(3-)) is known to interfere with a wide variety of enzymes including Ca2+ ATPase and Na+/+ ATPase. VO4(3-) is excreted mainly via the kidney. In renal insufficiency, the impaired VO4(3-) excretion leads to VO4(3-) accumulation in blood.The present study explored the effect of VO4(3-) on eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Stimulators of eryptosis include an increase of the cytosolic Ca2+ concentration. Erythrocyte Ca2+ activity was estimated from Fluo-3 fluorescence, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to VO4(3-) increased cytosolic Ca2+ concentration, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. In conclusion, VO4(3-) induces eryptosis at least partially through increase of cytosolic Ca2+ concentration, an effect presumably contributing to the development of anemia in chronic renal failure., (Copyright 2008 S. Karger AG, Basel.)