Prevalence of malnutrition in children under five years' age in District Tharparkar Sindh, Pakistan.

OBJECTIVE: To investigate the prevalence of malnutrition in children aged <5 years, and find out the risk factors associated with malnutrition in a rural setting. METHODS: The survey-based cross-sectional study was conducted from October 2017 to March 2018 in four Tehsils of district Tharparkar, Sindh, Pakistan, and comprised children of either gender aged <5 years who were randomly selected and assessed for weight and height using the World Health Organisation Anthro-2007 tool to obtain Z-score. Data was analysed using SPPS Version 18. RESULTS: Of the 597 subjects, 299(50.1%) were girls and 298(49.9%) were boys. Overall, 219(36.7%) were aged 12-23 months and 63(10.5%) were aged 48-59 months. Stunting was found in 485(81.1%) subjects, wasting 112(18.2%) and 342(57.3%) were underweight. The causes of malnutrition included age 6-11 months, number of siblings, monthly income <6000 rupees and duration of breast feeding <12 months (p<0.05). CONCLUSIONS: Higher prevalence of malnutrition was found in children aged <5 years in district Tharparkar.

OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel.

BACKGROUND/AIMS: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. METHODS: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+x0394;899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active T185EOSR1, catalytically inactive D164AOSR1, constitutively active T233ESPAK or catalytically inactive D212ASPAK. K+ channel activity was measured utilizing dual electrode voltage clamp. RESULTS: BK channel activity in BKM513I+x0394;899-903 expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by T185EOSR1 and T233ESPAK, but not by D164AOSR1 or D212ASPAK. CONCLUSIONS: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

ß-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2.

BACKGROUND/AIMS: ß-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a ß-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of ß-Klotho protein on the peptide transporters PEPT1 and PEPT2. METHODS: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM)-induced inward current (IGly) taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of ß-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/-) and corresponding wild type (kl+/+) mice. RESULTS: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble ß-Klotho protein. As shown for PEPT1, ß-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of ß-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. CONCLUSION: ß-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

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 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 Large Conductance Voltage-and Ca2+-Activated K+ Channels by the Janus Kinase JAK3.

BACKGROUND/AIMS: Janus kinase 3 (JAK3), a tyrosine kinase contributing to the regulation of cell proliferation and apoptosis of lymphocytes and tumour cells, has been shown to modify the expression and function of several ion channels and transport proteins. Channels involved in the regulation of cell proliferation include the large conductance voltage- and Ca(2+)-activated K(+) channel BK. The present study explored whether JAK3 modifies BK channel protein abundance and current. METHODS: cRNA encoding Ca(2+)-insensitive BK channel (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive (K851A)JAK3. Voltage gated K(+ )channel activity was measured utilizing dual electrode voltage clamp. Moreover, BK channel protein abundance was determined utilizing flow cytometry in CD19(+) B lymphocyte cell membranes from mice lacking functional JAK3 (jak3(-/-)) and corresponding wild-type mice (jak3(+/+)). RESULTS: BK activity in BK(M513I+Δ899-903) expressing oocytes was slightly but significantly decreased by coexpression of wild-type JAK3 and of (A568V)JAK3, but not by coexpression of (K851A)JAK3. The BK channel protein abundance in the cell membrane was significantly higher in jak3(-/-) than in jak3(+/+) B lymphocytes. The decline of conductance in BK and JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing BK with JAK3 and oocytes expressing BK alone, indicating that JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. CONCLUSION: JAK3 is a weak negative regulator of membrane BK protein abundance and activity.

SPAK and OSR1 sensitivity of voltage-gated K+ channel Kv1.5.

SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) are potent regulators of several transporters and ion channels. The kinases are under regulation of with-no-K(Lys) (WNK) kinases. The present study explored whether SPAK and/or OSR1 modify the expression and/or activity of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. cRNA encoding Kv1.5 was injected into Xenopus 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. Voltage-gated K(+) channel activity was quantified utilizing dual electrode voltage clamp and Kv1.5 channel protein abundance in the cell membrane utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type SPAK and (T233E)SPAK, but not by (T233A)SPAK and (D212A)SPAK. Similarly, Kv1.5 activity and Kv1.5-HA protein abundance were significantly down-regulated by wild-type OSR1 and (T185E)OSR1, but not by (T185A)OSR1 and (D164A)OSR1. Both, SPAK and OSR1 decrease cell membrane Kv1.5 protein abundance and activity.

Regulation of Voltage-Gated K+ Channel Kv1.5 by the Janus Kinase JAK3.

The tyrosine kinase Janus kinase 3 (JAK3) participates in the regulation of cell proliferation and apoptosis. The kinase further influences ion channels and transport proteins. The present study explored whether JAK3 contributes to the regulation of the voltage-gated K(+) channel Kv1.5, which participates in the regulation of diverse functions including atrial cardiac action potential and tumor cell proliferation. To this end, cRNA encoding Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active (A568V)JAK3, or inactive (K851A)JAK3. Voltage-gated K(+) channel activity was measured utilizing dual electrode voltage clamp, and Kv1.5 channel protein abundance in the cell membrane was quantified utilizing chemiluminescence of Kv1.5 containing an extracellular hemagglutinin epitope (Kv1.5-HA). As a result, Kv1.5 activity and Kv1.5-HA protein abundance were significantly decreased by wild-type JAK3 and (A568V)JAK3, but not by (K851A)JAK3. Inhibition of Kv1.5 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 (A568V)JAK3, suggesting that (A568V)JAK3 did not accelerate Kv1.5 protein retrieval from the cell membrane. A 24 h treatment with ouabain (100 µM) significantly decreased the voltage-gated current in oocytes expressing Kv1.5 without or with (A568V)JAK3 and dissipated the difference between oocytes expressing Kv1.5 alone and oocytes expressing Kv1.5 with (A568V)JAK3. In conclusion, JAK3 contributes to the regulation of membrane Kv1.5 protein abundance and activity, an effect sensitive to ouabain and thus possibly involving Na(+)/K(+) ATPase activity.

Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1.

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by (T233E)SPAK and (T185E)OSR1, but not by (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.

Up-regulation of Kv1.3 channels by janus kinase 2.

The janus-activated kinase 2 JAK2 participates in the signalling of several hormones including interferon, a powerful regulator of lymphocyte function. Lymphocyte activity and survival depend on the activity of the voltage-gated K(+) channel KCNA3 (Kv1.3). The present study thus explored whether JAK2 modifies the activity of voltage-gated K(+) channel KCNA3. To this end, cRNA encoding KCNA3 was injected in Xenopus oocytes with or without additional injection of cRNA encoding wild-type human JAK2, human inactive (K882E)JAK2 mutant, or human gain-of-function (V617F)JAK2 mutant. KCNA3-dependent depolarization-induced current was determined utilizing dual-electrode voltage clamp, and protein KCNA3 abundance in the cell membrane was quantified by chemiluminescence. Moreover, the effect of interferon-γ on voltage-gated K(+) current was determined by patch clamp in mainly KCNA3-expressing Jurkat T cells with or without prior treatment with JAK2 inhibitor AG490 (40 µM). As a result, KCNA3 channel activity and protein abundance were up-regulated by coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. The effect of JAK2 coexpression was reversed by AG490 treatment. In human Jurkat T lymphoma cells, voltage-gated K(+) current was up-regulated by interferon-γ and down-regulated by AG490 (40 µM). In conclusion, JAK2 participates in the signalling, regulating the voltage-gated K(+) channel KCNA3.

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.

Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho.

BACKGROUND/AIMS: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH)2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting ß-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3) and EAAT2 (SLC1A2), Na+ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. METHODS: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM)-induced inward current (IGlu) taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml) in the absence and presence of ß-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM). RESULTS: IGlu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes IGlu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. CONCLUSION: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

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.

Stimulation of erythrocyte cell membrane scrambling by gedunin.

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 Ca(2+)-activity ([Ca(2+)]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 [Ca(2+)]i. RESULTS: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca(2+)]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 Ca(2+). CONCLUSION: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca(2+) entry.

Downregulation of chloride channel ClC-2 by Janus kinase 3.

Janus kinase-3 (JAK3) fosters proliferation and counteracts apoptosis of lymphocytes and tumor cells. The gain of function mutation (A572V)JAK3 has been discovered in acute megakaryoplastic leukemia. JAK3 is inactivated by replacement of lysine by alanine in the catalytic subunit ((K855A)JAK3). Regulation of cell proliferation and apoptosis involves altered activity of Cl(-) channels. The present study, thus, explored whether JAK3 modifies the function of the small conductance Cl(-) channel ClC-2. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild-type JAK3, (A568V)JAK3 or (K851A)JAK3, and the Cl(-) channel activity determined by dual-electrode voltage clamp. Channel protein abundance in the cell membrane was determined utilizing chemiluminescence. As a result, expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK3 or (A568V)JAK3, but not by coexpression of (K851A)JAK3. Exposure of the oocytes expressing ClC-2 together with (A568V)JAK3 to the JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) increased the conductance. Coexpression of (A568V)JAK3 decreased the ClC-2 protein abundance in the cell membrane of ClC-2 expressing oocytes. The decline of conductance in ClC-2 and (A568V)JAK3 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing ClC-2 with (A568V)JAK3 and oocytes expressing ClC-2 alone, indicating that (A568V)JAK3 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK3 downregulates ClC-2 activity and thus counteracts Cl(-) exit-an effect possibly influencing cell proliferation and apoptosis.

Upregulation of excitatory amino acid transporters by coexpression of Janus kinase 3.

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3(A572V) is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3(K855A). Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3(A568V) or inactive JAK3(K851A), and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3(-/-)) and from corresponding wild-type mice (jak3(+/+)). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (Ig), which was significantly increased following coexpression of JAK3. Ig in oocytes expressing EAAT3 was further increased by JAK3(A568V) but not by JAK3(K851A). Ig in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal Ig and significantly reduced the glutamate concentration required for half maximal Ig (Km). Intestinal electrogenic glutamate transport was significantly lower in jak3(-/-) than in jak3(+/+) mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.

Regulation of ClC-2 activity by SPAK and OSR1.

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 (GCl), 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 GCl 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.

Downregulation of peptide transporters PEPT1 and PEPT2 by oxidative stress responsive kinase OSR1.

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 (T185A)OSR1, constitutively active (T185E)OSR1, and catalytically inactive (D164A)OSR1. 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 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 (T185E)OSR1, but not of (D164A)OSR1 or (T185A)OSR1. 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.