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.

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 (I_gly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. Results: No appreciable I_gly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes I_gly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, I_gly-gly without significantly modifying the concentration required for halfmaximal I_gly-gly (K_M). Following disruption of carrier insertion with brefeldin A (5 µM) I_gly-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, I_gly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased I_gly-gly in mouse intestine. Conclusion: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.

Background/Aims: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored whether gedunin stimulates eryptosis. Methods: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca²⁺]_i. Results: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca²⁺]_i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca²⁺. Conclusion: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca²⁺ entry.

Background/Aims: SPAK (STE20-related proline/alanine-rich kinase) is a powerful regulator of renal tubular ion transport and blood pressure. Moreover, SPAK contributes to the regulation of cell volume. Little is known, however, about a role of SPAK in the regulation or organic solutes. The present study thus addressed the influence of SPAK on the peptide transporters PEPT1 and PEPT2. Methods: To this end, cRNA encoding PEPT1 or PEPT2 were injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type, SPAK, WNK1 insensitive inactive ^T233ASPAK, constitutively active ^T233ESPAK, and catalytically inactive ^D212ASPAK. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp and PEPT2 protein abundance in the cell membrane by chemiluminescence. Intestinal electrogenic peptide transport was estimated from peptide induced current in Ussing chamber experiments of jejunal segments isolated from gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak^tg/tg) and respective wild-type mice (spak^+/+). Results: In PEPT1 and in PEPT2 expressing oocytes, but not in oocytes injected with water, the dipeptide gly-gly (2 mM) generated an inward current, which was significantly decreased following coexpression of SPAK. The effect of SPAK on PEPT1 was mimicked by ^T233ESPAK, but not by ^D212ASPAK or ^T233ASPAK. SPAK decreased maximal peptide induced current of PEPT1. Moreover, SPAK decreased carrier protein abundance in the cell membrane of PEPT2 expressing oocytes. In intestinal segments gly-gly generated a current, which was significantly higher in spak^tg/tg than in spak^+/+ mice. Conclusion: SPAK is a powerful regulator of peptide transporters PEPT1 and PEPT2.

Background/Aims: OSR1 (oxidative-stress-responsive kinase 1) participates in the regulation of renal tubular ion transport, cell volume and blood pressure. Whether OSR1 contributes to the regulation of organic solute transport remained; however, elusive. The present study thus explored the OSR1 sensitivity of the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 were injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type OSR1, WNK1 insensitive inactive ^T185AOSR1, constitutively active ^T185EOSR1, and catalytically inactive ^D164AOSR1. Electrogenic peptide (glycine-glycine) transport was determined by dual electrode voltage clamp, the abundance of hemagglutinin-tagged PEPT2 (PEPT2-HA) by chemiluminescence. Results: In Xenopus oocytes injected with cRNA encoding PEPT1 or PEPT2, but not in oocytes injected with water, the dipeptide gly-gly (2 mM) generated an appreciable inward current (I_gly-gly). Coexpression of OSR1 significantly decreased I_gly-gly in both PEPT1 and PEPT2 expressing oocytes. The effect of OSR1 coexpression on I_gly-gly in PEPT1 expressing oocytes was mimicked by coexpression of ^T185EOSR1, but not of ^D164AOSR1 or ^T185AOSR1. Kinetic analysis revealed that coexpression of OSR1 decreased maximal I_gly-gly. OSR1 further decreased the PEPT2-HA protein abundance in the cell membrane. Conclusion: OSR1 has the capacity to downregulate the peptide transporters PEPT1 and PEPT2 by decreasing the carrier protein abundance in the cell membrane.

Background/Aims: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are powerful regulators of diverse transport processes. Both kinases are activated by cell shrinkage and participate in stimulation of regulatory cell volume increase (RVI). Execution of RVI involves inhibition of Cl^- channels. The present study explored whether SPAK and/or OSR1 regulate the activity of the Cl^- channel ClC-2. Methods: To this end, ClC-2 was expressed in Xenopus laevis oocytes with or without additional expression of wild type SPAK, constitutively active SPAK^T233E, WNK1 insensitive inactive SPAK^T233A, catalytically inactive SPAK^D212A, wild type OSR1, constitutively active OSR1^T185E, WNK1 insensitive inactive OSR1^T185A, and catalytically inactive OSR1^D164A. Cl^- channel activity was determined by dual electrode voltage clamp. Results: Expression of ClC-2 was followed by the appearance of a conductance (G_Cl), which was significantly decreased following coexpression of wild-type SPAK, SPAK^T233E, wild type OSR1 or OSR1^T185E, but not by coexpression of SPAK^T233A, SPAK^D212A, OSR1^T185A, or OSR1^D164A. Inhibition of ClC-2 insertion by brefeldin A (5 μM) resulted in a decline of G_Cl which was similar in the absence and presence of SPAK or OSR1, suggesting that SPAK and OSR1 did not accelerate the retrieval of ClC-2 protein from the cell membrane. Conclusion: SPAK and OSR1 are powerful negative regulators of the cell volume regulatory Cl^- channel ClC-2.

Background/Aims: The kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) participate in the regulation of the NaCl cotransporter NCC and the Na⁺,K⁺,2Cl^- cotransporter NKCC2. The kinases are regulated by WNK (with-no-K[Lys]) kinases. Mutations of genes encoding WNK kinases underly Gordon's syndrome, a monogenic disease leading to hypertension and hyperkalemia. WNK kinases further regulate the renal outer medullary K⁺ channel ROMK1. The present study explored, whether SPAK and/or OSR1 have similarly the potential to modify the activity of ROMK1. Methods: ROMK1 was expressed in Xenopus oocytes with or without additional expression of wild-type SPAK, constitutively active ^T233ESPAK, catalytically inactive ^D212ASPAK, wild-type OSR1, constitutively active ^T185EOSR1 and catalytically inactive ^D164AOSR1. Channel activity was determined utilizing dual electrode voltage clamp and ROMK1 protein abundance in the cell membrane utilizing chemiluminescence of ROMK1 containing an extracellular hemagglutinin epitope (ROMK1-HA). Results: ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type SPAK and ^T233ESPAK, but not by ^D212ASPAK. Similarly, ROMK1 activity and ROMK1-HA protein abundance were significantly down-regulated by wild-type OSR1 and ^T185EOSR1, but not by ^D164AOSR1. Conclusion: ROMK1 protein abundance and activity are down-regulated by SPAK and OSR1.

Background: The creatine transporter CreaT (SLC6A8), a Na⁺,Cl^- coupled transporter is expressed in diverse tissues including the brain. Genetic defects of SLC6A8 result in mental retardation with seizures. The present study explored the regulation of CreaT by Janus kinase JAK3, which is expressed in a variety of tissues including the brain and participates in the regulation of cell survival and differentiation of neuronal precursor cells. Methods: CreaT was expressed in Xenopus laevis oocytes with or without wild-type JAK3, constitutively active ^A568VJAK3 and inactive ^K851AJAK3. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. Results: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes co-expression of JAK3 or ^A568VJAK3, but not co-expression of ^K851AJAK3 was followed by a significant decrease of creatine induced current. According to kinetic analysis JAK3 significantly decreased the maximal creatine transport rate. In CreaT and JAK3 expressing oocytes the creatine induced current was significantly increased by JAK3 inhibitor WHI-P154 (22 µM). Conclusion: JAK3 is a powerful negative regulator of the creatine transporter CreaT.