Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (Ile), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of Ile following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells

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

Tannic Acid Induced Suicidal Erythrocyte Death

Background: The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca2+-activity ([Ca2+]i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca2+ by ceramide. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fuorescence and ceramide utilizing fluorescent antibodies. Results: A 48 h treatment with tannic acid was followed by significant decrease of forward scatter (≥ 1 µg/ml) and significant increase of annexin-V-binding (≥ 10 µg/ml). Tannic acid did not significantly modify [Ca2+]i (up to 50 µM) but significantly increased ceramide formation (50 µM). The annexin-V-binding following tannic acid treatment (50 µM) was significantly blunted in the nominal absence of extracellular Ca2+. Conclusions: Tannic acid stimulates eryptosis, an effect at least partially due to ceramide formation with subsequent sensitization of erythrocytes to cytosolic Ca2+

Up-regulation of hERG K⁺ channels by B-RAF.

Human ether-a-go-go related-gene K⁺ channels (hERG) participate in the regulation of tumor cell proliferation and apoptosis. HERG channel activity is up-regulated by growth factors. Kinases sensitive to growth factor signaling include the serine/threonine protein kinase B-RAF. The present study thus explored whether B-RAF influences hERG channel expression and activity. To this end, hERG channels were expressed in Xenopus oocytes with or without wild-type B-RAF, hERG channel activity was determined utilizing dual-electrode voltage clamp and hERG protein abundance in the cell membrane was analyzed utilizing confocal microscopy as well as chemiluminescence. Moreover, in rhabdomyosarcoma RD cells the effect of B-RAF inhibitor PLX-4720 on hERG-mediated current was quantified by whole-cell patch clamp and hERG cell surface protein abundance by utilizing biotinylation of cell surface proteins as well as flow cytometry. As a result, co-expression of wild-type B-RAF in hERG-expressing Xenopus oocytes significantly increased hERG channel activity and hERG channel protein abundance in the cell membrane. Treatment for 24 hours of B-RAF and hERG-expressing Xenopus oocytes with B-RAF inhibitor PLX-4720 (10 µM) significantly decreased hERG-mediated current and hERG cell surface expression. Similarly, in rhabdomyosarcoma RD cells, treatment for 24 hours with B-RAF inhibitor PLX-4720 significantly decreased hERG cell membrane protein abundance and hERG-mediated current. In conclusion, B-RAF is a powerful regulator of hERG channel activity and cell surface hERG protein abundance

Up-Regulation of K ir

Background/Aims: The serum & glucocorticoid inducible kinase SGK3, an ubiquitously expressed serine/threonine kinase, regulates a variety of ion channels. It has previously been shown that SGK3 upregulates the outwardly rectifying K+ channel KV11.1, which is expressed in cardiomyocytes. Cardiomyocytes further express the inward rectifier K+ channel Kir2.1, which contributes to maintenance of resting cell membrane potential. Loss-of-function mutations of KCNJ2 encoding Kir2.1 result in Andersen-Tawil syndrome with periodic paralysis, cardiac arrhythmia and dysmorphic features. The present study explored whether SGK3 participates in the regulation of Kir2.1. Methods: cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild type SGK3, constitutively active S419DSGK3 or inactive K191NSGK3. Kir2.1 activity was determined by two-electrode voltage-clamp and Kir2.1 protein abundance in the cell membrane by immunostaining and subsequent confocal imaging or by chemiluminescence. Results: Injection of 10 ng cRNA encoding wild type SGK3 and S419DSGK3, but not K191NSGK3 significantly enhanced Kir2.1-mediated currents. SGK inhibitor EMD638683 (50 µM) abrogated S419DSGK3-induced up-regulation of Kir2.1. Moreover, wild type SGK3 enhanced the channel protein abundance in the cell membrane. The decay of Kir2.1-mediated currents following inhibition of channel insertion into the cell membrane by brefeldin A (5 µM) was similar in oocytes coexpressing Kir2.1 and SGK3 as in oocytes expressing Kir2.1 alone, suggesting that SGK3 influences channel insertion into rather than channel retrieval from the cell membrane. Conclusions: SGK3 is a novel regulator of Kir2.1

25-Hydroxyvitamin D 3

Background: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. Methods: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). Results: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. Conclusion: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor