The venoms of the lesser (Echiichthys vipera) and greater (Trachinus draco) weever fish- A review.

In comparison with other animal venoms, fish venoms remain relatively understudied. This is especially true for that of the lesser Echiichthys vipera and greater weever fish Trachinus draco which, apart from the isolation of their unique venom cytolysins, trachinine and dracotoxin, respectively, remain relatively uncharacterised. Envenomation reports mainly include mild symptoms consisting of nociception and inflammation. However, like most fish venoms, if the venom becomes systemic it causes cardiorespiratory and blood pressure changes. Although T. draco venom has not been studied since the 1990's, recent studies on E. vipera venom have discovered novel cytotoxic components on human cancer cells, but due to the scarcity of research on the molecular make-up of the venom, the molecule(s) causing this cytotoxicity remains unknown. This review analyses past studies on E. vipera and T. draco venom, the methods used in the , the venom constituents characterised, the reported symptoms of envenomation and compares these findings with those from other venomous Scorpaeniformes.

Reversine inhibits Colon Carcinoma Cell Migration by Targeting JNK1.

Colorectal cancer is one of the most commonly diagnosed cancers and the third most common cause of cancer-related death. Metastasis is the leading reason for the resultant mortality of these patients. Accordingly, development and characterization of novel anti-cancer drugs limiting colorectal tumor cell dissemination and metastasis are needed. In this study, we found that the small molecule Reversine reduces the migration potential of human colon carcinoma cells in vitro. A coupled kinase assay with bio-informatics approach identified the c-Jun N-terminal kinase (JNK) cascade as the main pathway inhibited by Reversine. Knockdown experiments and pharmacological inhibition identified JNK1 but not JNK2, as a downstream effector target in cancer cell migration. Xenograft experiments confirm the effect of JNK inhibition in the metastatic potential of colon cancer cells. These results highlight the impact of individual JNK isoforms in cancer cell metastasis and propose Reversine as a novel anti-cancer molecule for treatment of colon cancer patients.

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death.

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

Inhibition of Suicidal Erythrocyte Death by Reversine.

BACKGROUND/AIMS: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca2+ loading (30 minutes treatment with 1 µM Ca2+ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). RESULTS: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca2+ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca2+ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. CONCLUSIONS: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca2+ loading and oxidative stress.

Inhibition of Colon Carcinoma Cell Migration Following Treatment with Purified Venom from Lesser Weever Fish (Trachinus Vipera).

BACKGROUND: Injury by the sting of Lesser weever fish (Trachinus vipera) may lead to severe pain, edema or tissue necrosis. Cellular effects of the venom are still incompletely understood. Previous observations revealed that purified Lesser weever fish venom (LWFV) induces suicidal death of erythrocytes and HCT116 human colon carcinoma cells. The present study addressed the effect of the venom on colon carcinoma cell toxicity, shape and migration both in p53+/+ and/or p53-/- conditions. METHODS: Cells were exposed to medium without or with 500 µg/ ml LWFV. Cell shape, cell area and circularity were visualized and quantified by fluorescence microscopy. Cell volume, granularity and cells toxicity were assessed via the apoptotic parameters dissipation of mitochondrial inner transmembrane potential, phosphatidylserine surface exposure and cell membrane permeabilization were measured utilizing flow cytometry. Cell migration was evaluated using wound healing assay and two-dimensional migration assay. RESULTS: LWFV treatment was followed by a marked change of cell shape and size, significant decrease of cell area and circularity, significant impairment of cell migration, as well as induction of apoptosis after long exposition. CONCLUSIONS: LWFV exposure leads to cell shrinkage, increased granularity, apoptosis and impairment of cell migration, effects presumably contributing to LWFV-induced tissue injury.

Purified Lesser weever fish venom (Trachinus vipera) induces eryptosis, apoptosis and cell cycle arrest.

Accidents caused by the sting of Trachinus vipera (known as Lesser weever fish) are relatively common in shallow waters of the Mediterranean. Symptoms after the sting vary from severe pain to edema or even tissue necrosis in some cases. Here we show that purified Lesser weever fish venom induces eryptosis, the suicidal erythrocyte death, and apoptosis of human colon carcinoma cells. The venom leads to erythrocyte shrinkage, phosphatidylserine translocation and increased intracellular Ca2+, events typical for eryptosis. According to mitochondrial staining cancer cells dyed after the activation of the intrinsic apoptotic pathway. Trachinus vipera venom further causes cell cycle arrest.

Down-Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by Glycogen Synthase Kinase GSK3ß.

BACKGROUND: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium. METHODS: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. 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 GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt. CONCLUSION: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt.

SGK3 Sensitivity of Large-Conductance Ca2+-Activated K+ Channel.

BACKGROUND/AIMS: Large conductance Ca2+-activated K+ channels (maxi K+ channels or BK channels) are rapidly activated by increase of cytosolic Ca2+ activity. The channels participate in the regulation of diverse functions including neuronal excitation and cell volume. The BK channels may be modified by kinases. Channel regulating kinases include the serum & glucocorticoid inducible kinase 3 (SGK3). The present study explored whether SGK3 modifies the activity of BK channels. METHODS: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type SGK3, constitutively active S419DSGK3, or catalytically inactive K191NSGK3. K+ channel activity was measured utilizing dual electrode voltage clamp. RESULTS: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly increased by co-expression of SGK3 or active S419DSGK3, but not by coexpression of inactive K191NSGK3. CONCLUSION: SGK3 is a novel positive regulator of BK channels, and thus participates in the regulation of cell volume and excitability.

Stimulation of Suicidal Erythrocyte Death by the CDC25 Inhibitor NSC-95397.

BACKGROUND/AIMS: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (≥ 1 µM), significantly decreased forward scatter (≥ 2.5 µM), and significantly increased Fluo3-fluorescence (≥ 1 µM), DCFDA fluorescence (5 µM) and ceramide abundance (≥ 5 µM). The effect of NSC-95397 (5 µM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+ and by addition of the protein kinase C inhibitor staurosporine (1 µM). CONCLUSIONS: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca2+ and activation of staurosporine sensitive kinase(s).

Up-Regulation of the Large-Conductance Ca2+-Activated K+ Channel by Glycogen Synthase Kinase GSK3ß.

BACKGROUND/AIMS: The pleotropic functions of the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels) include regulation of neuronal excitation and cell volume. Kinases participating in those functions include the glycogen synthase kinase GSK3 ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by the antidepressant Lithium. The present study thus explored whether GSK3ß modifies the activity of BK channels. METHODS: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type GSK3ß, inactive K85RGSK3ß, or wild-type GSK3ß with wild-type PKB. K+ channel activity was measured utilizing dual electrode voltage clamp. RESULTS: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly increased by co-expression of GSK3ß, but not by co-expression of K85RGSK3ß. The effect of wild type GSK3ß was abrogated by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM). Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes expressing BK alone. CONCLUSION: GSK3ß may up-regulate BK channels, an effect disrupted by Lithium or additional expression of PKB and possibly participating in the regulation of cell volume and excitability.

SGK3 Sensitivity of Voltage Gated K+ Channel Kv1.5 (KCNA5).

BACKGROUND: The serum & glucocorticoid inducible kinase isoform SGK3 is a powerful regulator of several transporters, ion channels and the Na+/K+ ATPase. Targets of SGK3 include the ubiquitin ligase Nedd4-2, which is in turn a known regulator of the voltage gated K+ channel Kv1.5 (KCNA5). The present study thus explored whether SGK3 modifies the activity of the voltage gated K+ channel KCNA5, which participates in the regulation of diverse functions including atrial cardiac action potential, activity of vascular smooth muscle cells, insulin release and tumour cell proliferation. METHODS: cRNA encoding KCNA5 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild-type SGK3, constitutively active S419DSGK3, inactive K191NSGK3 and/or wild type Nedd4-2. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp. RESULTS: Voltage gated current in KCNA5 expressing Xenopus oocytes was significantly enhanced by wild-type SGK3 and S419DSGK3, but not by K191NSGK3. SGK3 was effective in the presence of ouabain (1 mM) and thus did not require Na+/K+ ATPase activity. Coexpression of Nedd4-2 decreased the voltage gated current in KCNA5 expressing Xenopus oocytes, an effect largely reversed by additional coexpression of SGK3. CONCLUSION: SGK3 is a positive regulator of KCNA5, which is at least partially effective by abrogating the effect of Nedd4-2.

Up-Regulation of Voltage Gated K+ Channels Kv1.3 and Kv1.5 by Protein Kinase PKB/Akt.

BACKGROUND: The voltage gated K+ channels Kv1.3 and Kv1.5 contribute to the orchestration of cell proliferation. Kinases participating in the regulation of cell proliferation include protein kinase B (PKB/Akt). The present study thus explored whether PKB/Akt modifies the abundance and function of Kv1.3 and Kv1.5. METHODS: Kv1.3 or Kv1.5 was expressed in Xenopus laevis oocytes with or without wild-type PKB/Akt, constitutively active T308D/S473DPKB/Akt or inactive T308A/S473APKB/Akt. The channel activity was quantified utilizing dual electrode voltage clamp. Moreover, HA-tagged Kv1.5 protein was determined utilizing chemiluminescence. RESULTS: Voltage gated K+ currents were observed in Kv1.3 or Kv1.5 expressing oocytes but not in water-injected oocytes or in oocytes expressing PKB/Akt alone. Co-expression of PKB/Akt or T308D/S473DPKB/Akt, but not co-expression of T308A/S473APKB/Akt significantly increased the voltage gated current in both Kv1.3 and Kv1.5 expressing oocytes. As shown for Kv1.5, co-expression of PKB/Akt enhanced the channel protein abundance in the cell membrane. In Kv1.5 expressing oocytes voltage gated current decreased following inhibition of carrier insertion by brefeldin A (5 µM) to similarly low values in the absence and presence of PKB/Akt, suggesting that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. CONCLUSION: PKB/Akt up-regulates both, Kv1.3 and Kv1.5 K+ channels.

Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3.

BACKGROUND/AIMS: Janus kinase 3 (JAK3), a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na+/K+ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K+ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. METHODS: cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing two electrode voltage clamp. RESULTS: KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and A568VJAK3, but not by K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM) but not by inhibition of transcription with actinomycin D (50 nM). Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM) resulted in a decline of the voltage gated current, which was similar in the absence and presence of A568VJAK3, suggesting that A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. CONCLUSION: JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154.

Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by the Janus Kinase JAK3.

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 A568V JAK3 and inactive K851A JAK3. 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 K851A JAK3 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.

SPAK and OSR1 Sensitive Kir2.1 K+ Channels.

BACKGROUND/AIMS: Kir2.1 (KCNJ2) channels are expressed in neurons, skeletal muscle and cardiac tissue and maintain the resting membrane potential. The activity of those channels is regulated by diverse signalling molecules. The present study explored whether Kir2.1 channels are sensitive to the transporter and channels regulating kinases SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are in turn regulated by WNK (with-no-K[Lys]) kinases. METHODS: cRNA encoding Kir2.1 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233E SPAK, WNK insensitive T233A SPAK, catalytically inactive D212A SPAK, wild-type OSR1, constitutively active T185E OSR1, WNK insensitive T185A OSR1 and catalytically inactive D164A OSR1. Inwardly rectifying K+ channel activity was quantified utilizing dual electrode voltage clamp and Kir2.1 channel protein abundance in the cell membrane was measured utilizing chemiluminescence of Kir2.1 containing an extracellular HA-tag epitope. RESULTS: Kir2.1 activity was significantly enhanced by wild-type SPAK and T233E SPAK, but not by T233A SPAK and D212A SPAK, as well as by wild-type OSR1 and T185E OSR1, but not by T185A OSR1 and D164A OSR1. As shown for SPAK, the kinases enhanced Kir2.1 protein abundance in the cell membrane. The difference of current and conductance between oocytes expressing Kir2.1 together with SPAK or OSR1 and oocytes expressing Kir2.1 alone was dissipated following a 24 hours inhibition of channel insertion into the cell membrane by brefeldin A (5 µM). CONCLUSIONS: SPAK and OSR1 are both stimulators of Kir2.1 activity. They are presumably effective by enhancing channel insertion into the cell membrane.

SPAK and OSR1 Sensitive Cell Membrane Protein Abundance and Activity of KCNQ1/E1 K+ Channels.

BACKGROUND/AIMS: KCNQ1/E1 channels are expressed in diverse tissues and serve a variety of functions including endolymph secretion in the inner ear, cardiac repolarization, epithelial transport and cell volume regulation. Kinases involved in regulation of epithelial transport and cell volume include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether KCNQ1/E1 channels are regulated by SPAK and/or OSR1. METHODS: cRNA encoding KCNQ1/E1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, wild-type OSR1, constitutively active T185EOSR1, WNK insensitive T185AOSR1 and catalytically inactive D164AOSR1. Voltage gated K+ channel activity was quantified utilizing dual electrode voltage clamp and KCNQ1/E1 channel protein abundance in the cell membrane utilizing chemiluminescence of KCNQ1/E1 containing an extracellular Flag tag epitope (KCNQ1-Flag/E1). RESULTS: KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly enhanced by wild-type SPAK and T233ESPAK, but not by T233ASPAK and D212ASPAK. Similarly, KCNQ1/E1 activity and KCNQ1-Flag/E1 protein abundance were significantly increased by wild-type OSR1 and T185EOSR1, but not by T185AOSR1 and D164AOSR1. CONCLUSIONS: SPAK and OSR1 participate in the regulation of KCNQ1/E1 protein abundance and activity.

Up-regulation of epithelial Na(+) channel ENaC by human parvovirus B19 capsid protein VP1.

BACKGROUND: Clinical disorders caused by parvovirus B19 (B19V) infection include endothelial dysfunction with cardiac ischemia. The virus is effective in part by lysophosphatidylcholine-producing phospholipase A2 (PLA2) activity of B19V capsid protein VP1. Mechanisms compromising endothelial function include up-regulation of amiloride sensitive epithelial Na(+)-channel ENaC leading to endothelial cell stiffness. Regulators of ENaC include ubiquitin-ligase Nedd4-2. The present study explored whether VP1 modifies ENaC-activity. METHODS: cRNA encoding ENaC was injected into Xenopus oocytes without or with cRNA encoding VP1. Experiments were made with or without coexpression of Nedd4-2. ENaC activity was estimated from amiloride (50 µM) sensitive current. RESULTS: Injection of cRNA encoding ENaC into Xenopus oocytes was followed by appearance of amiloride sensitive current, which was significantly enhanced by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on ENaC was mimicked by treatment of ENaC expressing oocytes with lysophosphatidylcholine (1 µg/ml). The effect of VP1 and lysophosphatidylcholine was not additive. ENaC activity was downregulated by Nedd4-2, an effect not reversed by VP1. CONCLUSIONS: The B19V capsid protein VP1 up-regulates ENaC, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.

Up-Regulation of Intestinal Phosphate Transporter NaPi-IIb (SLC34A2) by the Kinases SPAK and OSR1.

BACKGROUND/AIMS: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), kinases controlled by WNK (with-no-K[Lys] kinase), are powerful regulators of cellular ion transport and blood pressure. Observations in gene-targeted mice disclosed an impact of SPAK/OSR1 on phosphate metabolism. The present study thus tested whether SPAK and/or OSR1 contributes to the regulation of the intestinal Na(+)-coupled phosphate co-transporter NaPi-IIb (SLC34A2). METHODS: cRNA encoding NaPi-IIb was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The phosphate (1 mM)-induced inward current (I(Pi)) was taken as measure of phosphate transport. RESULTS: I(Pi) was observed in NaPi-IIb expressing oocytes but not in water injected oocytes, and was significantly increased by co-expression of SPAK, (T233E)SPAK, OSR1, (T185E)OSR1 or SPAK+OSR1, but not by co-expression of (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. SPAK and OSR1 both increased the maximal transport rate of the carrier. CONCLUSIONS: SPAK and OSR1 are powerful stimulators of the intestinal Na+-coupled phosphate co-transporter NaPi-IIb.

SPAK and OSR1 Sensitivity of Excitatory Amino Acid Transporter EAAT3.

BACKGROUND/AIMS: Kinases involved in the regulation of epithelial transport include SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1). SPAK and OSR1 are both regulated by WNK (with-no-K(Lys)) kinases. The present study explored whether SPAK and/or OSR1 influence the excitatory amino acid transporter EAAT3, which accomplishes glutamate and aspartate transport in kidney, intestine and brain. METHODS: cRNA encoding EAAT3 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Glutamate-induced current was taken as measure of electrogenic glutamate transport and was quantified utilizing dual electrode voltage clamp. Furthermore, Ussing chamber was employed to determine glutamate transport in the intestine from gene-targeted mice carrying WNK insensitive SPAK (spak(tg/tg)) and from corresponding wild-type mice (spak(+/+)). RESULTS: EAAT3 activity was significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. SPAK decreased maximal transport rate without affecting significantly affinity of the carrier. Similarly, EAAT3 activity was significantly downregulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Again OSR1 decreased maximal transport rate without affecting significantly affinity of the carrier. Intestinal electrogenic glutamate transport was significantly lower in spak(+/+) than in spak(tg/tg) mice. CONCLUSION: Both, SPAK and OSR1 are negative regulators of EAAT3 activity.

Negative regulation of the creatine transporter SLC6A8 by SPAK and OSR1.

BACKGROUND/AIMS: Transport regulation involves several kinases including SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), which are under control of WNK (with-no-K[Lys]) kinases. The present study explored whether SPAK and/or OSR1 participate in the regulation of the creatine transporter CreaT (SLC6A8), which accomplishes Na+ coupled cellular uptake of creatine in several tissues including kidney, intestine, heart, skeletal muscle and brain. METHODS: cRNA encoding SLC6A8 was injected into Xenopus laevis oocytes with or without additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 and catalytically inactive (D164A)OSR1. Transporter activity was determined from creatine (1 mM) induced current utilizing dual electrode voltage clamp. RESULTS: Coexpression of wild-type SPAK and of (T233E)SPAK, but not of (T233A)SPAK or of (D212A)SPAK was followed by a significant decrease of creatine induced current in SLC6A8 expressing oocytes. Coexpression of SPAK significantly decreased maximal transport rate. Coexpression of wild-type OSR1, (T185E)OSR1 and (T185A)OSR1 but not of (D164A)OSR1 significantly negatively regulated SLC6A8 activity. OSR1 again decreased significantly maximal transport rate. CONCLUSIONS: Both, SPAK and OSR1, are negative regulators of the creatine transporter SLC6A8.