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 (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.
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 (spaktg/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 spaktg/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 (Igly-gly). Coexpression of OSR1 significantly decreased Igly-gly in both PEPT1 and PEPT2 expressing oocytes. The effect of OSR1 coexpression on Igly-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 Igly-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.
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