Tigecycline Absorption Improved by Selected Excipients.

To investigate the effects of (2,6-di-O-methyl)-ß-cyclodextrin (DM-ß-CD), (2-hydroxypropyl)-ß-cyclodextrin (HP-ß-CD), tocopherol polyethylene glycol 1000 succinate (TPGS), sodium desoxycholate (SDOCH), trimethyl chitosan (TMC), and sodium caprate (C10) on the plasma concentration and the oral bioavailability of tigecycline in broiler chickens. To test the effects of the excipients on absorption of tigecycline, a tetracycline that is poorly absorbed from the gastrointestinal tract, broiler chickens were used as an animal model. Tigecycline (10 mg/kg body weight) was administered intravenously, orally, and orally with one of the excipients. Plasma samples were taken after administration. To measure tigecycline concentrations, high-performance liquid chromatography coupled with tandem mass spectrometry was used. Compartmental and non-compartmental analyses were used for pharmacokinetic analyses of mean plasma concentrations versus time. With the exception of sodium caprate, all the excipients significantly increased the area under the curve and bioavailability of tigecycline (p < 0.05). These parameters were approximately doubled by HP-ß-CD, TPGS, and SDOCH, with 95% confidence intervals (95% CIs) for the difference that included only increases of 1.5-fold or higher (bioavailability: control, 1.67%; HP-ß-CD, 3.24%; TPGS, 3.30%; and SDOCH, 3.24%). The increases in these parameters were smaller with DM-ß-CD and TMC (DM-ß-CD, 2.41%; TMC, 2.55%), and the 95% CIs ranged from close to no difference to nearly double the values in the control group. These results indicate that HP-ß-CD, TPGS, and SDOCH substantially increase the area under the curve and oral bioavailability of tigecycline. They suggest that DM-ß-CD and TMC may also substantially increase these parameters, but more research is needed for more precise estimates of their effects.

Inhibition of BKCa channels protects neonatal hearts against myocardial ischemia and reperfusion injury.

BKCa channels are large-conductance calcium and voltage-activated potassium channels that are heterogeneously expressed in a wide array of cells. Activation of BKCa channels present in mitochondria of adult ventricular cardiomyocytes is implicated in cardioprotection against ischemia-reperfusion (IR) injury. However, the BKCa channel's activity has never been detected in the plasma membrane of adult ventricular cardiomyocytes. In this study, we report the presence of the BKCa channel in the plasma membrane and mitochondria of neonatal murine and rodent cardiomyocytes, which protects the heart on inhibition but not activation. Furthermore, K+ currents measured in neonatal cardiomyocyte (NCM) was sensitive to iberiotoxin (IbTx), suggesting the presence of BKCa channels in the plasma membrane. Neonatal hearts subjected to IR when post-conditioned with NS1619 during reoxygenation increased the myocardial infarction whereas IbTx reduced the infarct size. In agreement, isolated NCM also presented increased apoptosis on treatment with NS1619 during hypoxia and reoxygenation, whereas IbTx reduced TUNEL-positive cells. In NCMs, activation of BKCa channels increased the intracellular reactive oxygen species post HR injury. Electrophysiological characterization of NCMs indicated that NS1619 increased the beat period, field, and action potential duration, and decreased the conduction velocity and spike amplitude. In contrast, IbTx had no impact on the electrophysiological properties of NCMs. Taken together, our data established that inhibition of plasma membrane BKCa channels in the NCM protects neonatal heart/cardiomyocytes from IR injury. Furthermore, the functional disparity observed towards the cardioprotective activity of BKCa channels in adults compared to neonatal heart could be attributed to their differential localization.

BKCa Channels as Targets for Cardioprotection.

The large-conductance calcium- and voltage-activated K+ channel (BKCa) are encoded by the Kcnma1 gene. They are ubiquitously expressed in neuronal, smooth muscle, astrocytes, and neuroendocrine cells where they are known to play an important role in physiological and pathological processes. They are usually localized to the plasma membrane of the majority of the cells with an exception of adult cardiomyocytes, where BKCa is known to localize to mitochondria. BKCa channels couple calcium and voltage responses in the cell, which places them as unique targets for a rapid physiological response. The expression and activity of BKCa have been linked to several cardiovascular, muscular, and neurological defects, making them a key therapeutic target. Specifically in the heart muscle, pharmacological and genetic activation of BKCa channels protect the heart from ischemia-reperfusion injury and also facilitate cardioprotection rendered by ischemic preconditioning. The mechanism involved in cardioprotection is assigned to the modulation of mitochondrial functions, such as regulation of mitochondrial calcium, reactive oxygen species, and membrane potential. Here, we review the progress made on BKCa channels and cardioprotection and explore their potential roles as therapeutic targets for preventing acute myocardial infarction.

Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation.

Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.

Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner.

Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca(2+) signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0-10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca(2+)-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca(2+)-overload-induced mitochondrial superoxide anion formation.

A-kinase anchoring protein 79/150 coordinates metabotropic glutamate receptor sensitization of peripheral sensory neurons.

Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-kinase anchoring protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity after group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP-dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature.

The Role of Janus Kinase 3 in the Regulation of Na⁺/K⁺ ATPase under Energy Depletion.

BACKGROUND/AIMS: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na(+)/K(+)-ATPase. The present study explored whether JAK3 regulates Na(+)/K(+)-ATPase in dendritic cells (DCs). METHODS: Ouabain (100 µM)-sensitive (Iouabain) and K(+)-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na(+)/K(+)-ATPase α1-subunit mRNA levels by RT-PCR, Na(+)/K(+)-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active (A568V)JAK3 or inactive (K851A)JAK3. RESULTS: Na(+)/K(+)-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3(-/-)DCs than in jak3(+/+)DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3(+/+) DCs but not in jak3(-/-)DCs. Cellular ATP was significantly lower in jak3(-/-)DCs than in jak3(+/+)DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3(-/-)DCs than in jak3(+/+)DCs and strongly blunted by ouabain in both jak3(+/+) and jak3(-/-)DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of (A568V)JAK3 but not of (K851A)JAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between (A568V)JAK3 and (K851A)JAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). CONCLUSIONS: JAK3 down-regulates Na(+)/K(+)-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

Divergence in endothelin-1- and bradykinin-activated store-operated calcium entry in afferent sensory neurons.

Endothelin-1 (ET-1) and bradykinin (BK) are endogenous peptides that signal through Gαq/11-protein coupled receptors (GPCRs) to produce nociceptor sensitization and pain. Both peptides activate phospholipase C to stimulate Ca(2+) accumulation, diacylglycerol production, and protein kinase C activation and are rapidly desensitized via a G-protein receptor kinase 2-dependent mechanism. However, ET-1 produces a greater response and longer lasting nocifensive behavior than BK in multiple models, indicating a potentially divergent signaling mechanism in primary afferent sensory neurons. Using cultured sensory neurons, we demonstrate significant differences in both Ca(2+) influx and Ca(2+) release from intracellular stores following ET-1 and BK treatments. As intracellular store depletion may contribute to the regulation of other signaling cascades downstream of GPCRs, we concentrated our investigation on store-operated Ca(2+) channels. Using pharmacological approaches, we identified transient receptor potential canonical channel 3 (TRPC3) as a dominant contributor to Ca(2+) influx subsequent to ET-1 treatment. On the other hand, BK treatment stimulated Orai1 activation, with only minor input from TRPC3. Taken together, data presented here suggest that ET-1 signaling targets TRPC3, generating a prolonged Ca(2+) signal that perpetuates nocifensive responses. In contrast, Orai1 dominates as the downstream target of BK receptor activation and results in transient intracellular Ca(2+) increases and abridged nocifensive responses.

ß-arrestin-2-biased agonism of delta opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) in primary sensory neurons.

Despite advances in understanding the signaling mechanisms involved in the development and maintenance of chronic pain, the pharmacologic treatment of chronic pain has seen little advancement. Agonists at the mu opioid receptor (MOPr) continue to be vital in the treatment of many forms of chronic pain, but side-effects limit their clinical utility and range from relatively mild, such as constipation, to major, such as addiction and dependence. Additionally, chronic activation of MOPr results in pain hypersensitivity known as opioid-induced hyperalgesia (OIH), and we have shown recently that recruitment of ß-arrestin2 to MOPr, away from transient potential vanilloid eceptor type 1 (TRPV1) in primary sensory neurons contributes to this phenomenon. The delta opioid receptor (DOPr) has become a promising target for the treatment of chronic pain, but little is known about the effects of chronic activation of DOPr on nociceptor sensitivity and OIH. Here we report that chronic activation of DOPr by the DOPr-selective agonist, SNC80, results in the sensitization of TRPV1 and behavioral signs of OIH via ß-arrestin2 recruitment to DOPr and away from TRPV1. Conversely, chronic treatment with ARM390, a DOPr-selective agonist that does not recruit ß-arrestin2, neither sensitized TRPV1 nor produced OIH. Interestingly, the effect of SNC80 to sensitize TRPV1 is species-dependent, as rats developed OIH but mice did not. Taken together, the reported data identify a novel side-effect of chronic administration of ß-arrestin2-biased DOPr agonists and highlight the importance of potential species-specific effects of DOPr agonists.

Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1) via ß-arrestin-2-mediated cross-talk.

The transient receptor potential family V1 channel (TRPV1) is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C). Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that ß-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of ß-arrestin2 sequestration by G-protein coupled receptors (GPCRs) on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in ß-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates ß-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven ß-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

1,25(OH)2 vitamin D3-dependent inhibition of platelet Ca2+ signaling and thrombus formation in klotho-deficient mice.

Platelets are activated by increased cytosolic Ca(2+) concentration ([Ca(2+)]i) following store-operated calcium entry (SOCE) accomplished by calcium-release-activated calcium (CRAC) channel moiety Orai1 and its regulator STIM1. In other cells, Ca(2+) transport is regulated by 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 formation is inhibited by klotho and excessive in klotho-deficient mice (kl/kl). The present study explored the effect of klotho deficiency on platelet Ca(2+) signaling and activation. Platelets and megakaryocytes isolated from WT and kl/kl-mice were analyzed by RT-PCR, Western blotting, confocal microscopy, Fura-2-fluorescence, patch clamp, flow cytometry, aggregometry, and flow chamber. STIM1/Orai1 transcript and protein levels, SOCE, agonist-induced [Ca(2+)]i increase, activation-dependent degranulation, integrin αIIbß3 activation and aggregation, and thrombus formation were significantly blunted in kl/kl platelets (by 27-90%). STIM1/Orai1 transcript and protein levels, as well as CRAC currents, were significantly reduced in kl/kl megakaryocytes (by 38-73%) and 1,25(OH)2D3-treated WT megakaryocytes. Nuclear NF-κB subunit p50/p65 abundance was significantly reduced in kl/kl-megakaryocytes (by 51-76%). Transfection with p50/p65 significantly increased STIM1/Orai1 transcript and protein levels in megakaryocytic MEG-01 cells (by 46-97%). Low-vitamin D diet (LVD) of kl/kl mice normalized plasma 1,25(OH)2D3 concentration and function of platelets and megakaryocytes. Klotho deficiency inhibits platelet Ca(2+) signaling and activation, an effect at least partially due to 1,25(OH)2D3-dependent down-regulation of NF-κB activity and STIM1/Orai1 expression in megakaryocytes.

Akt2- and ETS1-dependent IP3 receptor 2 expression in dendritic cell migration.

BACKGROUND/AIMS: The protein kinase Akt2/PKBß is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca(2+) signaling. We thus explored whether Akt2 regulates DC Ca(2+) signaling. METHODS: DCs were derived from bone marrow of Akt2-deficient mice (akt2(-/-)) and their wild type littermates (akt2(+/+)). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca(2+) concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. RESULTS: Upon maturation, chemokine CCL21 stimulated migration of akt2(+/+) but not akt2(-/-) DCs. CCL21-induced increase in cytosolic Ca(2+) concentration, thapsigargin-induced release of Ca(2+) from intracellular stores with subsequent store-operated Ca(2+) entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) release as well as Ca(2+) release-activated Ca(2+) (CRAC) channel activity were all significantly lower in mature akt2(-/-) than in mature akt2(+/+) DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2(+/+) than in akt2(-/-) DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2(+/+) and to a lower extent in akt2(-/-) DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2(+/+) than in akt2(-/-) DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2(+/+)DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca(2+) release, SOCE and CRAC channel activation, as well as DC migration. CONCLUSION: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Energy-sensitive regulation of Na+/K+-ATPase by Janus kinase 2.

Janus kinase 2 (JAK2) contributes to intracellular signaling of leptin and erythropoietin, hormones protecting cells during energy depletion. The present study explores whether JAK2 is activated by energy depletion and regulates Na(+)/K(+)-ATPase, the major energy-consuming pump. In Jurkat cells, JAK2 activity was determined by radioactive kinase assay, phosphorylated JAK2 detected by Western blotting, ATP levels measured by luciferase assay, as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance determined by real-time PCR and Western blotting, respectively. Ouabain-sensitive K(+)-induced currents (Ipump) were measured by whole cell patch clamp. Ipump was further determined by dual-electrode voltage clamp in Xenopus oocytes injected with cRNA-encoding JAK2, active (V617F)JAK2, or inactive (K882E)JAK2. As a result, in Jurkat T cells, JAK2 activity significantly increased following energy depletion by sodium azide (NaN3) or 2,4- dinitro phenol (DNP). DNP- and NaN3-induced decrease of cellular ATP was significantly augmented by JAK2 inhibitor AG490 and blunted by Na(+)/K(+)-ATPase inhibitor ouabain. DNP decreased and AG490 enhanced Ipump as well as Na(+)/K(+)-ATPase α1-subunit transcript and protein abundance. The α1-subunit transcript levels were also enhanced by signal transducer and activator of transcription-5 inhibitor CAS 285986-31-4. In Xenopus oocytes, Ipump was significantly decreased by expression of JAK2 and (V617F)JAK2 but not of (K882E)JAK2, effects again reversed by AG490. In (V617F)JAK2-expressing Xenopus oocytes, neither DNP nor NaN3 resulted in further decline of Ipump. In Xenopus oocytes, the effect of (V617F)JAK2 on Ipump was not prevented by inhibition of transcription with actinomycin. In conclusion, JAK2 is a novel energy-sensing kinase that curtails energy consumption by downregulating Na(+)/K(+)-ATPase expression and activity.

Sgk1-dependent stimulation of cardiac Na+/H+ exchanger Nhe1 by dexamethasone.

BACKGROUND/AIMS: The serum- and glucocorticoid-inducible kinase Sgk1 contributes to cardiac remodeling and development of heart failure, which is paralelled by Sgk1-dependent stimulation of the cardiac Na(+)/H(+) exchanger Nhe1. Glucocorticoids are powerful stimulators of Sgk1 expression and influence cardiac remodeling. The present study thus explored whether the glucocorticoid receptor agonist dexamethasone influenced cardiac Sgk1 expression, as well as activity, expression and phosphorylation at Ser(703) of the cardiac Na(+)/H(+) exchanger Nhe1. METHODS: Experiments were performed in HL-1 cardiomyocytes and gene targeted mice lacking functional Sgk1 (sgk1(-/-)) and respective wild type mice (sgk1(+/+)). Gene expression was determined by quantitative RT-PCR and Nhe1 phosphorylation was determined utilizing a specific antibody against a 14-3-3 binding motif at P-Ser(703), which represents a putative phosphorylation site recognition motif for Sgk1 and is involved in Nhe1 activation. Cytosolic pH (pHi) was determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Nhe activity by the Na(+)-dependent realkalinization after an ammonium pulse. RESULTS: Treatment of HL-1 cardiomyocytes with dexamethasone was followed by a significant increase in Sgk1 mRNA expression, parallelled by increased Na(+)/H(+) exchanger activity. Furthermore, dexamethasone significantly increased Nhe1 and Spp1 mRNA expression. The effects of dexamethasone were blunted by cotreatment of HL-1 cardiomyocytes with the Sgk1 inhibitor EMD638683. Cotreatment with Nhe1 inhibitor cariporide similarly prevented dexamethasone-stimulated Spp1 mRNA expression. In sgk1(+/+) mice, dexamethasone significantly increased cardiac Sgk1 mRNA levels. In sgk1(+/+) mice, but not in sgk1(-/-) mice, dexamethasone significantly increased cardiac Nhe1 mRNA expression and Nhe1 phosphorylation at Ser(703). Furthermore, cardiac Spp1, Ctgf, Nppa and Nppb mRNA levels were significantly increased in dexamethasone treated sgk1(+/+) mice, effects significantly blunted in sgk1(-/-) mice. CONCLUSIONS: Sgk1 is critically involved in the phosphorylation and activation of the cardiac Na(+)/H(+) exchanger Nhe1.

Down-regulation of Na/K+ atpase activity by human parvovirus B19 capsid protein VP1.

BACKGROUND/AIMS: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA) sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na(+)/K(+) ATPase. The present study explored whether VP1 modifies Na(+)/K(+) ATPase activity. METHODS: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A) and K(+) induced pump current (I(pump)) as well as ouabain-inhibited current (I(ouabain)) both reflecting Na(+)/K(+)-ATPase activity were determined by dual electrode voltage clamp. RESULTS: Injection of cRNA encoding VP1, but not of VP1(H153A) or water, was followed by a significant decrease of both, I(pump) and I(ouabain) in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours) but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM) and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml). According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml) similarly decreased I(pump) in human microvascular endothelial cells (HMEC). CONCLUSION: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na(+)/K(+) ATPase, an effect at least partially due to phospholipase A2 (PLA2) dependent formation of lysophosphatidylcholine.

Altered regulation of cytosolic Ca²âº concentration in dendritic cells from klotho hypomorphic mice.

The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²âº concentration ([Ca²âº]i). [Ca²âº]i is increased by store-operated Ca²âº entry and decreased by K⁺-independent (NCX) and K⁺-dependent (NCKX) Na⁺/Ca²âº exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D. Formation of 1,25(OH)2D3 is inhibited by the antiaging protein Klotho. Thus 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²âº]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²âº]i increase upon acute application of LPS (1 µg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 µM). CCL21-dependent migration was significantly less in klothohm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)2D3 the first 2 days after isolation from bone marrow. Feeding klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²âº]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Na⁺/Ca²âº-exchange activity, thus blunting the increase of [Ca²âº]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.

Expression and functional significance of the Ca(2+)-activated Cl(-) channel ANO6 in dendritic cells.

BACKGROUND/AIMS: Migration of dendritic cells (DCs), antigen presenting cells that link innate and adaptive immunity, is critical for initiation of immune responses. DC migration is controlled by the activity of different ion channels, which mediate Ca(2+) flux or set the membrane potential. Moreover, cell migration requires local volume changes at the leading and rear end of travelling cells, which might be mediated by the fluxes of osmotically active solutes, including Cl(-). The present study explored the functional expression, regulation and role of Cl(-) channels in mouse bone marrow-derived DCs. METHODS/RESULTS: In whole-cell patch clamp experiments we detected outwardly rectifying Cl(-) currents which were activated by elevation of cytosolic Ca(2+), triggered either by ionomycin in the presence of extracellular Ca(2+) or mobilization of Ca(2+) by IP(3) Most importantly, Ca(2+)-activated Cl(-) channels (CaCCs) were activated by CCL21 (75 ng/ml), an agonist of the chemokine receptor CCR7. The currents showed sensitivity to Cl(-) channel blockers such as tannic acid (10 µM), digallic acid (100 µM) and more specific CaCC blockers niflumic acid (300 µM) and AO1 (20 µM). According to RT-PCR and Western blot data, Anoctamin 6 (ANO6) is expressed in DCs. Knock-down of ANO6 with siRNA led to inhibition of CaCC currents in DCs. Moreover, chemokine-induced migration of both immature and LPS-matured DCs was reduced upon ANO6 knock-down. CONCLUSION: Our data identify ANO6 as a Ca(2+)-activated Cl(-) channel in mouse DCs, show its activation upon chemokine receptor ligation and establish an important role of ANO6 in chemokine-induced DC migration.

SGK3 regulates Ca(2+) entry and migration of dendritic cells.

BACKGROUND/AIMS: Dendritic cells (DCs) are antigen-presenting cells linking innate and adaptive immunity. DC maturation and migration are governed by alterations of cytosolic Ca(2+) concentrations ([Ca(2+)](i)). Ca(2+) entry is in part accomplished by store-operated Ca(2+) (SOC) channels consisting of the membrane pore-forming subunit Orai and the ER Ca(2+) sensing subunit STIM. Moreover, DC functions are under powerful regulation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses proinflammatory cytokine production but supports DC migration. Downstream targets of PI3K include serum- and glucocorticoid-inducible kinase isoform SGK3. The present study explored, whether SGK3 participates in the regulation of [Ca(2+)](i) and Ca(2+)-dependent functions of DCs, such as maturation and migration. METHODS/RESULTS: Experiments were performed with bone marrow derived DCs from gene targeted mice lacking SGK3 (sgk3(-/-)) and DCs from their wild type littermates (sgk3(+/+)). Maturation, phagocytosis and cytokine production were similar in sgk3(-/-) and sgk3(+/+) DCs. However, SOC entry triggered by intracellular Ca(2+) store depletion with the endosomal Ca(2+) ATPase inhibitor thapsigargin (1 µM) was significantly reduced in sgk3(-/-) compared to sgk3(+/+) DCs. Similarly, bacterial lipopolysaccharide (LPS, 1 µg/ml)- and chemokine CXCL12 (300 ng/ml)- induced increase in [Ca(2+)](i) was impaired in sgk3(-/-) DCs. Moreover, currents through SOC channels were reduced in sgk3(-/-) DCs. STIM2 transcript levels and protein abundance were significantly lower in sgk3(-/-) DCs than in sgk3(+/+) DCs, whereas Orai1, Orai2, STIM1 and TRPC1 transcript levels and/or protein abundance were similar in sgk3-/- and sgk3(+/+) DCs. Migration of both, immature DCs towards CXCL12 and LPS-matured DCs towards CCL21 was reduced in sgk3(-/-) as compared to sgk3(+/+) DCs. Migration of sgk3(+/+) DCs was further sensitive to SOC channel inhibitor 2-APB (50 µM) and to STIM1/STIM2 knock-down. CONCLUSION: SGK3 contributes to the regulation of store-operated Ca(2+) entry into and migration of dendritic cells, effects at least partially mediated through SGK3-dependent upregulation of STIM2 expression.

Lipopolysaccharide-sensitive H+ current in dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 µM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 µg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.

Enhanced Ca²âº entry and Na+/Ca²âº exchanger activity in dendritic cells from AMP-activated protein kinase-deficient mice.

In dendritic cells (DCs), chemotactic chemokines, such as CXCL12, rapidly increase cytosolic Ca(2+)concentrations ([Ca(2+)](i)) by triggering Ca(2+) release from intracellular stores followed by store-operated Ca(2+) (SOC) entry. Increase of [Ca(2+)](i) is blunted and terminated by Ca(2+) extrusion, accomplished by K(+)-independent Na(+)/Ca(2+) exchangers (NCXs) and K(+)-dependent Na(+)/Ca(2+) exchangers (NCKXs). Increased [Ca(2+)](i) activates energy-sensing AMP-activated protein kinase (AMPK), which suppresses proinflammatory responses of DCs and macrophages. The present study explored whether AMPK participates in the regulation of DC [Ca(2+)](i) and migration. DCs were isolated from AMPKα1-deficient (ampk(-/-)) mice and, as control, from their wild-type (ampk(+/+)) littermates. AMPKα1, Orai1-2, STIM1-2, and mitochondrial calcium uniporter protein expression was determined by Western blotting, [Ca(2+)](i) by Fura-2 fluorescence, SOC entry by inhibition of endosomal Ca(2+) ATPase with thapsigargin (1 µM), Na(+)/Ca(2+) exchanger activity from increase of [Ca(2+)](i), and respective whole-cell current in patch clamp following removal of extracellular Na(+). Migration was quantified utilizing transwell chambers. AMPKα1 protein is expressed in ampk(+/+) DCs but not in ampk(-/-) DCs. CXCL12 (300 ng/ml)-induced increase of [Ca(2+)](i), SOC entry, Orai 1 protein abundance, NCX, and NCKX were all significantly higher in ampk(-/-) DCs than in ampk(+/+) DCs. NCX and NCKX currents were similarly increased in ampk(-/-) DCs. Moreover, CXCL12 (50 ng/ml)-induced DC migration was enhanced in ampk(-/-) DCs. AMPK thus inhibits SOC entry, Na(+)/Ca(2+) exchangers, and migration of DCs.