60kDa Lysophospholipase, a New Sgk1 Molecular Partner Involved in the Regulation of ENaC

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

Proton Pump Inhibitors Intake and Iron and Vitamin B12 Status: A Prospective Comparative Study with a Follow up of 12 Months

BACKGROUND: Proton pump inhibitors (PPIs) represent the most widely prescribed antisecretory agents, but their prolonged use, may influence iron and vitamin B12 status, which could have important implications for clinical practice.AIM: We undertook this study aiming to investigate the association between PPIs use for 12 months and potential changes in iron and vitamin B12 status, as well as whether this potential association varies among four specific PPI drugs used in the study.METHODS: A total of 250 adult subjects were recruited into this study, of which 200 subjects were PPIs users while 50 subjects belonged to the control group. Serum iron, ferritin, vitamin B12, and homocysteine (Hcy) levels were measured before the start of the study and after 12 months. Mann - Whitney U test and Kruskal - Wallis test was used to compare the baseline characteristics of the study groups, while Wilcoxon test was used to analyse post - pre differences.RESULTS: Statistical analysis showed significant changes within PPIs group and specific PPIs subgroups between the two-time points in serum ferritin and vitamin B12 levels, respectively, while no significant changes in serum iron and homocysteine levels were shown. However, subsequent diagnosis of hypoferremia and hypovitaminosis B12 in the whole study sample at 12 months was established in only 3.8% and 2.9% of the subjects, respectively.CONCLUSION: PPIs use for 12 months did not result in clinically significant iron and/or vitamin B12 deficiency; thus, these findings argue routine screening under normal circumstances, although monitoring in elderly and malnourished may be of precious value

Die AMP aktiviert protein Kinase in der Regulation von natriumgekoppelten trensportern (SGLT1, EAAT3 & 4) und eryptosis

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 Ca2+ activity ([Ca2+]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. [Ca2+]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 [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 myM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ 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. AMP-activated protein kinase, a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilisation. 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 gammaR70QAMPK (alpha1beta1gamma1 (R70Q)) alone, the addition of glucose to the extracellular bath generated a current (Ig), which was halfmaximal (KM) at = 650 µM glucose concentration. Coexpression of gammaR70QAMPK did not affect KM but significantly enhanced the maximal current (=1.7 fold). Coexpression of wild type AMPK or the kinase dead alphaK45RAMPK mutant (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 µM) enhanced the SGLT1 protein abundance in the cell membrane of Caco-2 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 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, 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 (Ig), which was half maximal (KM) at 74 µM (EAAT3) or at 4 µM (EAAT4) glutamate. Coexpression of constitutively active gammaR70QAMPK or of wild type AMPK did not affect KM but significantly decreased the maximal Ig in both EAAT3-(by 34%) and EAAT4-(by 49%) expressing oocytes. Coexpression of the inactive mutant alphaK45R AMPK (alpha1 (K45R) beta1gamma1) did not appreciably affect Ig. According to confocal microscopy and chemiluminescence coexpression of gammaR70QAMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK downregulates Na+-coupled glutamate transport.Die AMP-aktivierte Proteinkinase (AMPK) ist ein Enzym, das den Energiehaushalt der Zelle reguliert: AMPK erhöht die intrazelluläre ATP Konzentration, indem es energieverbrauchende Prozesse bremst und energieproduzierende fördert. Im Falle erschöpfter Energiereserven starten Erythrozyten ihr Apoptose-Programm, auch Eryptose genannt. Initiiert durch eine Zunahme der zytosolischen Ca2+-Konzentration ([Ca2+]i) kommt es zu Zellschrumpfung und der Exposition von Phosphatidylserin (PS) auf der Außenseite der Erythrozytenmembran. Die vorliegende Studie untersuchte, welche Rolle die AMPK in der Regulation der Eryptosis spielt. Die Expression von AMPK in den Erythrocyten wurde mittels der Western Blot Methode und Konfokalmikroskopie nachgewiesen. Die intrazelluläre Ca2+-Konzentration ([Ca2+]i), das Zellvolumen der Erythrozyten sowie Phosphatidylserin auf der Außenseite der Membran wurden mittels Durchflusszytometrie (FACS) ermittelt. Glukosemangel im Extrazellulärmedium führte zu einer Erhöhung von [Ca2+]i, verkleinerte das Zellvolumen und erhöhte den Anteil von PS in der äußeren Membran. Versuchsreihen mit dem AMPK-Inhibitor Compound C (20 µM) ergaben, dass bei Anwesenheit von Glukose keine signifikante Veränderung der Eryptoserate eintritt. Hingegen verstärkte Compound C bei Glucoseentzug die Eryptose deutlich. Das Ca2+-Ionophor Ionomycin führt zu einer Erhöhung von [Ca2+]i, welche wiederum die Eryptose auslöst. Dieser Effekt wurde durch den AMPK-Aktivator 5-Aminoimidazol-4-carboxamid-1-beta-D-ribofuranosid (AICAR; 1 mM) abgeschwächt. Verglichen mit Erythrozyten des Wild-Typs (ampk+/+), waren Erythrozyten von AMPK&alpha1- defizienten Mäusen (ampk-/-) signifikant anfälliger für Energiemangel bedingte Eryptose. Die ampk-/--Mäuse hatten trotz exzessiver Retikulozytose eine Anämie und litten unter schwerer Splenomegalie, was auf einen erhöhten Erythrozytenumsatz hinweist. Diese Beobachtungen zeigen, dass AMPK die Lebensdauer von zirkulierenden Erythrozyten beeinflusst. AMP-aktivierte Protein Kinase, eine Serin/Threonin-Kinase, wird durch Energiemangel aktiviert. Sie stimuliert die Energieprodukion und hemmt den Energieverbrauch. Dieser Effekt der AMPK wurde insofern für die GLUT-Familie der erleichterten Glucosetransporter nachgewiesen, als die Aktivierung der AMPK zu einem erhöhten Glukoseeinstrom druch GLUT-Transporter führt. Die vorliegende Studie untersuchte die Möglichkeit, ob die AMPK eventuell den Na+ gekoppelten Glukosetransport durch SGLT1 (SLC5A1) reguliert. Dazu wurde die cRNA von SGLT1 in Xenopus laevis-Oozyten mit und ohne AMPK exprimiert. Der elektrogene Symport von Na+/Glucose wurde mithilfe der Zwei-Elektroden-Spannungsklemme (DEVC) gemessen. In SGLT1- exprimierenden Oozyten konnte durch die Zugabe von Glukose zum extrazellulären Bad ein Strom (Ig) generiert werden. Dieser erreichte seine halbmaximale Stärke bei einer Konzentration von (KM) = 650 µM Glucose. Bei Oozyten, in die lediglich Wasser oder die konstitutiv aktive gammaR70QAMPK (alpha1beta1gamma1(R70Q)) injiziert wurde, ließ sich dieser Strom durch Glukosezugabe nicht erzeugen. Die Koexpression von gammaR70QAMPK hatte keinen Einfluss auf KM, erhöhte aber signifikant den maximalen Strom Ig max (= 1.7 fach). Die Koexpression des AMPK-Wildtyps oder der inaktivierten alphaK45RAMPK Mutante (alpha1(K45R)beta1gamma1) wirkte sich auf Ig nicht messbar aus. AICAR (1 mM), Phenformin (1 mM) und A-769662 (10 µM) erhöhten die SGLT1-Expression in der Zellmembran von Caco-2 Zellen, wie durch Konfokalmikroskopie und Western Blotting gezeigt werden konnte. Dies legt nahe, dass die AMPK-Aktivität die Membrantranslokation von SGLT1 erhöhen könnte. Diese Beobachtungen deuten auf eine Rolle der AMPK für die Regulation des Na+- gekoppelten Glukosetransports. Die Glutamattransporter EAAT3 und EAAT4 werden in Neuronen exprimiert. Durch die Aufnahme von Glutamat und Aspartat in die Neuronen dienen die beiden Transporter der Entfernung anregender Transmitter aus dem Extrazellulärraum. In einer Sauerstoffmangelsituation sammelt sich Glutamat im Extrazellulärraum an und kann damit exzitotoxisch wirken. Diese Studie erforschte die Möglichkeit der Regulation von EAAT3 und/oder EAAT4 durch die AMPK. Hierzu wurde die cRNA von EAAT3 oder EAAT4 sowohl mit als auch ohne cRNA der AMPK in Xenopus Oozyten exprimiert. Der elektrogene Na+/Glutamat Kotransport wurde elektrophysiologisch gemessen. In EAAT3- und in EAAT4- exprimierenden Oozyten generierte Glutamat einen Strom (Ig), welcher seine halbmaximale Stärke bei Glutamatkonzentrationen von (KM EAAT3) = 74µM (EAAT3) beziehungsweise (KM EAAT4) = 4µM (EAAT4) erreichte. Die Koexpression der konstitutiv aktiven gammaR70QAMPK oder der Wildtyp-AMPK hatte keinen Einfluss auf KM, aber reduzierte den maximalen Strom Ig: in Oozyten mit EAAT3 um 34% und Oozyten mit EAAT4 um 49%. Die Koexpression der inaktiven Mutante alphaK45RAMPK (alpha1 (K45R) beta1gamma1) hatte keinen erkennbaren Einfluss auf Ig. Unter dem Einfluss von gammaR70QAMPK oder des Wildtyps der AMPK wurden deutlich weniger EAAT3- und EAAT4-Transporter in die Membran eingebaut. Dies wurde mit Konfokalmikroskopie und Chemilumineszenz nachgewiesen. Die Untersuchungen zeigten, dass die AMPK den Na+-gekoppelten Glutamat Transport reduziert

Insight into the Mechanisms Underlying the Tracheorelaxant Properties of the Sideritis raeseri Extract

Sideritis raeseri Boiss. and Heldr. (Lamiaceae), known as “mountain tea,” is a native plant from the Mediterranean region, which is widely used in traditional medicine. This study evaluates the effects of the ethanol extract of Sideritis raeseri (SR) on airway smooth muscle activity and identifies the underlying mechanism. The S. raeseri extract (SRE) was extracted from air-dried parts of the shoot system of SR. The SRE (0.3–2 mg/mL) was tested in isolated rabbit tracheal rings, suspended in the organ bath, filled with Krebs solution, and bubbled with the carbogen mixture (95% O2/5% CO2) under a resting tension of 1 g in 37°C. In in vitro experiments, the SRE relaxed against acetylcholine-induced constriction in tracheal rings. Furthermore, SRE inhibited Ca2+-induced contractions in carbachol (CCh, 1 μM) as well as in the K+-depolarized trachea (80 mM). Our findings showed the NO/cGMP involvement in tracheorelaxant effects of SR. To this end, the effect of the SRE was potentiated by bradykinin (nitric oxide (NO) synthase activator, 100 nM), whereas it was inhibited by ODQ (inhibitor of NO-sensitive guanylyl cyclase, 10 μM) and L-NAME (NO synthase inhibitor, 100 μM), as well as indomethacin (cyclooxygenase inhibitor, 10 μM). These data suggest that the tracheorelaxant effect of the SRE is mediated at least partly by NO/cyclic guanosine monophosphate and cyclooxygenase-1-prostaglandin E2-dependent signaling. These findings indicate that the SRE may be used in various respiratory disorders

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 (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1-HA), of inactive AMPKαK45R (AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA) or of constitutively active AMPKγR70Q (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q). 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 (Ip), which was significantly decreased by coexpression of wild-type AMPK and of AMPKγR70Q but not of AMPKαK45R. 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