Ahmad Almilaji

Fluoxetine induced suicidal erythrocyte death.

The antidepressant fluoxetine inhibits ceramide producing acid sphingomyelinase. Ceramide is in turn known to trigger eryptosis the suicidal death of erythrocytes characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Ceramide is effective through sensitizing the erythrocytes to the pro-eryptotic effect of increased cytosolic Ca2+ activity ([Ca2+]i). In nucleated cells, fluoxetine could either inhibit or stimulate suicidal death or apoptosis. The present study tested whether fluoxetine influences eryptosis. To this end cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release and [Ca2+]i from Fluo-3 fluorescence intensity. As a result, a 48 h exposure of erythrocytes to fluoxetine (≥25 µM) significantly decreased forward scatter, increased annexin V binding and enhanced [Ca2+]i. The effect on annexin V binding was significantly blunted, but not abolished, in the absence of extracellular Ca2+. In conclusion, fluoxetine stimulates eryptosis, an effect at least in part due to increase of cytosolic Ca2+ activity.

PIKfyve sensitivity of hERG channels.

Background/Aims: Human ether-a-go-go (hERG) channels contribute to cardiac repolarization and participate in the regulation of tumor cell proliferation. Mutations in hERG channels may cause long QT syndrome and sudden cardiac death due to ventricular arrhythmias. HERG channel activity is up-regulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1 and SGK3. Related kinases are protein kinase B (PKB/Akt) isoforms. SGK´s and PKB/Akt´s activate phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which in turn up-regulates several carriers and channels. An effect of PIKfyve on hERG channels, has, however, never been shown. The present study thus explored the putative influence of PIKfyve on hERG channel expression and activity. Methods: hERG channels were expressed in Xenopus oocytes with or without PIKfyve and/or PKB, expression of endogenous and injected hERG quantified by RT-PCR, and hERG channel activity determined utilizing dual electrode voltage clamp. Moreover, hERG protein abundance in the cell membrane was visualized utilizing specific antibody binding and subsequent confocal microscopy and quantified by chemiluminescence. Results: Coexpression of wild type PIKfyve increased hERG channel activity in hERG-expressing Xenopus oocytes. hERG channel activity was further increased by coexpression of PKB, an effect augmented by additional coexpression of PIKfyve, but not by additional coexpression of PKB/Akt-resistant PIKfyve mutant PIKfyveS318A. Coexpression of PIKfyve increased hERG channel protein abundance in the cell membrane. Inhibition of hERG channel insertion into the cell membrane by Brefeldin A (5 µM) resulted in a decline of current, which was similar in Xenopus oocytes expressing hERG together with PIKfyve and in Xenopus oocytes expressing hERG alone. Conclusion: hERG is up-regulated by PIKfyve, which is in turn activated by PKB/Akt.

OSR1-sensitive renal tubular phosphate reabsorption

Background: The oxidative stress-responsive kinase 1 (OSR1) participates in the WNK-(with no K) kinase dependent regulation of renal salt excretion and blood pressure. Little is known, however, about the role of OSR1 in the regulation of further renal transport systems. The present study analyzed the effect of OSR1 on NaPiIIa, the major renal tubular phosphate transporter. Methods: Immunohistochemistry and confocal microscopy were employed to determine renal localization of OSR1 and NaPiIIa. To elucidate the effect of OSR on NaPiIIa activity, cRNA encoding NaPiIIa was injected into Xenopus oocytes with or without additional injection of cRNA encoding OSR1, and phosphate transport was estimated from phosphateinduced currents determined with dual electrode voltage clamp. To elucidate the in vivo significance of OSR1 serum phosphate and hormone concentrations as well as urinary phosphate output of mice carrying one allele of WNK-resistant OSR1 (osr1tg/+) were compared to the respective values of wild type mice (osr1+/+). Results: NaPiIIa and OSR1 were both expressed in proximal renal tubule cells. Coexpression of OSR1 significantly up-regulated phosphate-induced currents in NaPiIIa-expressing Xenopus oocytes. Despite decreased serum phosphate concentration urinary phosphate excretion was significantly increased and NaPiIIa protein abundance in the brush border membrane significantly reduced in osr1tg/+ mice as compared to osr1+/+ mice. Serum PTH and calcitriol levels were similar in osr1tg/+ mice and in osr1+/+ mice, serum FGF23 concentration was, however, significantly higher in osr1tg/+ mice than in osr1+/+ mice. Conclusions: OSR1 is expressed in proximal renal tubules and participates in the regulation of FGF23 release and renal tubular phosphate transport.

Upregulation of Peptide Transporters PEPT1 and PEPT2 by Janus Kinase JAK2

Background/Aims: Janus-activated kinase-2 JAK2 participates in the signaling of several hormones including growth hormone, fosters tumor growth and modifies the activity of several Na+ coupled nutrient transporters. Peptide uptake into intestinal and tumor cells is accomplished by electrogenic peptide transporters PEPT1 and PEPT2. The present study thus explored whether JAK2 contributes to the regulation of PEPT1 and PEPT2 activity. Methods: cRNA encoding either PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active V617FJAK2 or inactive K882EJAK2. The current created by the dipeptide glycine-glycine (Igly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. Results: No appreciable Igly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes Igly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, Igly-gly without significantly modifying the concentration required for halfmaximal Igly-gly (KM). Following disruption of carrier insertion with brefeldin A (5 µM) Igly-gly declined similarly fast in Xenopus oocytes expressing PEPT1 with JAK2 and in Xenopus oocytes expressing PEPT1 alone. In oocytes expressing both, PEPT1 and V617FJAK2, Igly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased Igly-gly in mouse intestine. Conclusion: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.

Upregulation of Na + ,Cl - -Coupled Betaine/ γ-Amino-Butyric Acid Transporter BGT1 by Tau Tubulin Kinase 2

Background/Aims: The serine/threonine kinase Tau-tubulin-kinase 2 (TTBK2) is expressed in various tissues including kidney, liver and brain. Loss of function mutations of TTBK2 lead to autosomal dominant spinocerebellar ataxia type 11 (SCA11). Cell survival is fostered by cellular accumulation of organic osmolytes. Carriers accomplishing cellular accumulation of organic osmolytes include the Na+, Cl--coupled betaine/γ-amino-butyric acid transporter BGT1. The present study explored whether TTBK2 participates in the regulation of BGT1 activity. Methods: Electrogenic transport of GABA was determined in Xenopus oocytes expressing BGT1 with or without wild-type TTBK2, truncated TTBK2[1-450] or kinase inactive mutants TTBK2- KD and TTBK2[1-450]-KD. Results: Coexpression of wild-type TTBK2, but not of TTBK2[1-450], TTBK2-KD or TTBK2[1-450]-KD, increased electrogenic GABA transport. Wildtype TTBK2 increased the maximal transport rate without significantly modifying affinity of the carrier. Coexpression of wild-type TTBK2 significantly delayed the decline of transport following inhibition of carrier insertion with brefeldin A, indicating that wild-type TTBK2 increased carrier stability in the cell membrane. Conclusion: Tau-tubulin-kinase 2 TTBK2 is a powerful stimulator of the osmolyte and GABA transporter BGT1.

Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K+ induced pump current (Ipump) as well as ouabain-inhibited current (Iouabain) both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased Ipump in human microvascular endothelial cells (HMEC). Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine.

Upregulation of the Creatine Transporter Slc6A8 by Klotho

Background/Aims: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. β-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. Methods: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. Results: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by β-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. Conclusion: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring β-glucuronidase activity of Klotho protein.

Regulation of the Voltage Gated K+ Channel Kv1.3 by Recombinant Human Klotho Protein

Background/Aims: Klotho, a protein mainly produced in the kidney and released into circulating blood, contributes to the negative regulation of 1,25(OH)2D3 formation and is thus a powerful regulator of mineral metabolism. As β-glucuronidase, alpha Klotho protein further regulates the stability of several carriers and channels in the plasma membrane and thus regulates channel and transporter activity. Accordingly, alpha Klotho protein participates in the regulation of diverse functions seemingly unrelated to mineral metabolism including lymphocyte function. The present study explored the impact of alpha Klotho protein on the voltage gated K+ channel Kv1.3. Methods: cRNA encoding Kv1.3 (KCNA3) was injected into Xenopus oocytes and depolarization induced outward current in Kv1.3 expressing Xenopus oocytes determined utilizing dual electrode voltage clamp. Experiments were performed without or with prior treatment with recombinant human Klotho protein (50 ng/ml, 24 hours) in the absence or presence of a β-glucuronidase inhibitor D-saccharic acid-1,4-lactone (DSAL, 10 µM). Moreover, the voltage gated K+ current was determined in Jcam lymphoma cells by whole cell patch clamp following 24 hours incubation without or with recombinant human Klotho protein (50 ng/ml, 24 hours). Kv1.3 protein abundance in Jcam cells was determined utilising fluorescent antibodies in flow cytometry. Results: In Kv1.3 expressing Xenopus oocytes the Kv1.3 currents and the protein abundance of Kv1.3 were both significantly enhanced after treatment with recombinant human Klotho protein (50 ng/ml, 24 hours), an effect reversed by presence of DSAL. Moreover, treatment with recombinant human Klotho protein increased Kv currents and Kv1.3 protein abundance in Jcam cells. Conclusion: Alpha Klotho protein enhances Kv1.3 channel abundance and Kv1.3 currents in the plasma membrane, an effect depending on its β-glucuronidase activity.

Down-Regulation of the Na + -Coupled Phosphate Transporter NaPi-IIa by AMP- Activated Protein Kinase

Background/Aims: The Na⁺-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na⁺ gradient across the apical cell membrane, which is maintained by Na⁺ extrusion across the basolateral cell membrane through the Na⁺/K⁺ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na⁺ accumulation and K⁺ loss with eventual decrease of cell membrane potential, Cl^- entry and cell swelling. Upon energy depletion, early inhibition of Na⁺-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK^α1-HA+AMPK^β1-Flag+AMPK^γ1-HA), of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA) or of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (I_p), which was significantly decreased by coexpression of wild-type AMPK and of AMPK^γR70Q but not of AMPK^αK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

Akt2-and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca²⁺ signaling. We thus explored whether Akt2 regulates DC Ca²⁺ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2^-/-) and their wild type littermates (akt2^+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca²⁺ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2^+/+ but not akt2^-/- DCs. CCL21-induced increase in cytosolic Ca²⁺ concentration, thapsigargin-induced release of Ca²⁺ from intracellular stores with subsequent store-operated Ca²⁺ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release as well as Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity were all significantly lower in mature akt2^-/- than in mature akt2^+/+ DCs. Transcript levels of IP₃ receptor IP₃R2 and of IP₃R2 regulating transcription factor ETS1 were significantly higher in akt2^+/+ than in akt2^-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2^+/+ and to a lower extent in akt2^-/- DCs. Following maturation, protein abundance of IP₃R2 and ETS1 were similarly higher in akt2^+/+ than in akt2^-/- DCs. The IP₃R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2^+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP₃R2 mRNA abundance, thapsigargin- and ATP-induced Ca²⁺ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP₃R2 transcription.