Hepatoprotective action of prostaglandin A2 analogs under CCl4-induced liver injury in vitro

Aim: The cytoprotective effects of six novel synthetic prostaglandin A(2) analogs against carbon tetrachloride (CCl(4)) as a toxic agent were studied with isolated rat liver hepatocytes in vitro. Results: It was found that hepatocytes treatment with CCl(4) induced: (i) a significant increase of lactic dehydrogenase (LDH) release from cytoplasm; (ii) leakage of glutamate dehydrogenase (GDH) and acid phosphatase from mitochondria and lysosomes, respectively; (iii) 10-fold increase of trien conjugates formation; and (iv) a reduction of free SH-groups by 50%. Prostanoids U-26, U-9 and U-34 decreased cytotoxic index of CCl(4) on average by 1.5-2.0 times and were more effective than PGI(2), the well-known hepatoprotector of prostanoids type. The protective action of the prostanoids was not a cAMP- or Ca(2+)-dependent process. However, prostanoids U-26, U-9 and U-34 normalized intracellular content of SH-groups, reduced trien conjugates formation by 60-80% and strongly prevented enzyme leakage through cellular membranes. They were also able to inhibit CCl(4) effects via decreasing cytochrome P(450)2E1 activity. Conclusion: The results obtained demonstrate that prostanoids provide cytoprotective effects on liver hepatocytes through the prevention of lipid peroxidation of the plasma and the cellular membranes and maintenance of their barrier function.     

AMP-activated protein kinase regulates hERG potassium channel

Besides their role in cardiac repolarization, human ether-a-go-go-related gene potassium (hERG) channels are expressed in several tumor cells including rhabdomyosarcoma cells. The channels foster cell proliferation. Ubiquitously expressed AMP-dependent protein kinase (AMPK) is a serine-/threonine kinase, stimulating energy-generating and inhibiting energy-consuming processes thereby helping cells survive periods of energy depletion. AMPK has previously been shown to regulate Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Ca²⁺ channels and K⁺ channels. The present study tested whether AMPK regulates hERG channel activity. Wild type AMPK (α1β1γ1), constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), or catalytically inactive ^αK45RAMPK (α1(K45R)β1γ1) were expressed in Xenopus oocytes with hERG. Tail currents were determined as a measure of hERG channel activity by two-electrode-voltage clamp. hERG membrane abundance was quantified by chemiluminescence and visualized by immunocytochemistry and confocal microscopy. Moreover, hERG currents were measured in RD rhabdomyosarcoma cells after pharmacological modification of AMPK activity using the patch clamp technique. Coexpression of wild-type AMPK and of constitutively active ^γR70QAMPK significantly downregulated the tail currents in hERG-expressing Xenopus oocytes. Pharmacological activation of AMPK with AICAR or with phenformin inhibited hERG currents in Xenopus oocytes, an effect abrogated by AMPK inhibitor compound C. ^γR70QAMPK enhanced the Nedd4-2-dependent downregulation of hERG currents. Coexpression of constitutively active ^γR70QAMPK decreased membrane expression of hERG in Xenopus oocytes. Compound C significantly enhanced whereas AICAR tended to inhibit hERG currents in RD rhabdomyosarcoma cells. AMPK is a powerful regulator of hERG-mediated currents in both, Xenopus oocytes and RD rhabdomyosarcoma cells. AMPK-dependent regulation of hERG may be particularly relevant in cardiac hypertrophy and tumor growth.     

Downregulation of the renal outer medullary K+ channel ROMK by the AMP-activated protein kinase

The 5′-adenosine monophosphate-activated serine/threonine protein kinase (AMPK) is stimulated by energy depletion, increase in cytosolic Ca²⁺ activity, oxidative stress, and nitric oxide. AMPK participates in the regulation of the epithelial Na⁺ channel ENaC and the voltage-gated K⁺ channel KCNE1/KCNQ1. It is partially effective by decreasing PIP₂ formation through the PI3K pathway. The present study explored whether AMPK regulates the renal outer medullary K⁺ channel ROMK. To this end, cRNA encoding ROMK was injected into Xenopus oocytes with and without additional injection of constitutively active AMPK^γR70Q (AMPK^α1-HA+AMPK^β1-Flag+AMPKγ1^R70Q), or of inactive AMPK^αK45R (AMPK^α1K45R+AMPK^β1-Flag+AMPK^γ1-HA), and the current determined utilizing two-electrode voltage-clamp and single channel patch clamp. ROMK protein abundance was measured utilizing chemiluminescence in Xenopus oocytes and western blot in whole kidney tissue. Moreover, renal Na⁺ and K⁺ excretion were determined in AMPK^α1-deficient mice (ampk ^?/? ) and wild-type mice (ampk ^+/+ ) prior to and following an acute K⁺ load (111 mM KCl, 30 mM NaHCO₃, 4.7 mM NaCl, and 2.25 g/dl BSA) at a rate of 500 μl/h. As a result, coexpression of AMPK^γR70Q but not of AMPK^αK45R significantly decreased the current in ROMK1-expressing Xenopus oocytes. Injection of phosphatidylinositol PI_(4,5)P₂ significantly increased the current in ROMK1-expressing Xenopus oocytes, an effect reversed in the presence of AMPK^γR70Q. Under control conditions, no significant differences between ampk ^?/? and ampk ^+/+ mice were observed in glomerular filtration rate (GFR), urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentrations as well as absolute and fractional Na⁺ and K⁺ excretion. Following an acute K⁺ load, GFR, urinary flow rate, serum aldosterone, plasma Na⁺, and K⁺ concentration were again similar in both genotypes, but renal absolute and fractional Na⁺ and K⁺ excretion were higher in ampk ^?/? than in ampk ^+/+ mice. According to micropuncture following a K⁺ load, delivery of Na⁺ to the early distal tubule but not delivery of K⁺ to late proximal and early distal tubules was increased in ampk ^?/? mice. The upregulation of renal ROMK1 protein expression by acute K⁺ load was more pronounced in ampk ^?/? than in ampk ^+/+ mice. In conclusion, AMPK downregulates ROMK, an effect compromising the ability of the kidney to excrete K⁺ following an acute K⁺ load.     

Reactive oxygen species initiate a metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression

Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH₂ associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation.     

Early Surgery in Prone Position for Associated Injuries in Patients Undergoing Non-operative Management for Splenic and Liver Injuries

Background

In patients undergoing non-operative management (NOM) of blunt splenic and/or liver injuries, no data exist on the safety of same-admission surgery in prone position for concomitant injuries.

Methods

Retrospective study including adult trauma patients with blunt splenic/liver injuries and attempted NOM from 01/2009 to 06/2015 was conducted. Patient and injury characteristics as well as outcomes [failed (f)NOM, mortality] of patients with/without surgery in prone position were compared (‘prone’ vs. ‘non-prone’ group).

Results

A total of 244 patients with blunt splenic/liver injury and attempted NOM were included. Forty patients (16.4%) underwent surgery in prone position on median post-injury day 2.0 [interquartile range (IQR) 3.0]. Surgery in prone position was mostly performed for associated spinal or pelvic injuries. The ISS was significantly higher, and the proportion of patients with high-grade injuries (OIS?≥?3) was significantly less frequent in the ‘prone? group (30.0?±?14.5 vs. 23.9?±?13.2, p?=?0.009 and 27.5 vs. 53.9%, p?=?0.002). In-hospital mortality as well as NOM failure rates were not significantly different between the ‘prone’ and ‘non-prone? group (2.5 vs. 2.9%, p?=?1.000; 0.0 vs. 4.4%, p?=?0.362). Eleven patients with high-grade injuries were operated in prone position at median day 3 (IQR 3.0). None of these patients failed NOM. However, one patient with a grade IV splenic injury required immediate splenectomy after being operated in right-sided position on the day of admission.

Conclusion

In this single-center analysis, surgery in prone position was performed in a substantial number of patients with splenic/liver injuries without increasing the fNOM rate. However, caution should be used in patients with grade IV/V splenic injuries.

     

Apoptosis and proliferation differences between CD34+ and CD34- leukemic subpopulations in childhood acute leukemia

In view of the clinical and biological significance of leukemic heterogeneity we studied the efficacy of spontaneous apoptosis and cell cycle distribution in CD34+ and CD34 - leukemic subpopulations. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) leukemic samples with CD34 heterogeneous expression were separated into CD34+ and CD34 - fractions using fluorescence activated cell sorting. Cell cycle distribution, and apoptosis of the sorted subpopulations were estimated. CD34+ leukemic subpopulations had lower ability to apoptosis than that of CD34 - fractions in 6 out of 8 ALL samples and in 4 out of 5 AML samples. CD34+ fractions showed a higher percentage of proliferating cells compared to CD34 - cells in T-lineage ALL. These differences may lead to a more resistant phenotype of one of the subpopulations and reappearance this population in relapse.     

CD34+ leukemic subpopulation predominantly displays lower spontaneous apoptosis and has higher expression levels of Bcl-2 and MDR1 genes than CD34− cells in childhood AML

In view of obscure clinical and biological significance of leukemic cells heterogeneity, we studied the efficacy of apoptosis, proliferation, and expression levels of the Bcl-2, MDR1, LRP, and BCRP genes in sorted CD34+ and CD34− subpopulations of childhood AML leukemic samples. In five out of nine cases, CD34+ cells were less sensitive to spontaneous apoptosis and had from 1.2- to 5.0-fold higher expression levels of Bcl-2 (eight of ten) and from 1.5- to 28.7-fold higher expression levels of MDR1 (eight of ten). The expression levels of the LRP gene were from 1.1- to 1.8-fold higher in CD34+ subpopulations (five of ten cases), and the expression levels of the BCRP gene were from 1.1- to 22.4-fold higher in CD34+ leukemic cells (six of ten). In all M4 cases, the expression levels of LRP were higher in the CD34− subpopulation. Significant differences in the patterns of genes expression between patients do not allow us to conclude that the CD34+ fractions have more resistant phenotype than the CD34− subpopulations. Nevertheless, distinctions between CD34+ and CD34− cells may lead to different chemosensitivities between leukemic subpopulations in vivo and may determine the alteration of the leukemic immunophenotype during treatment and in relapse.     

SYK regulates B-cell migration by phosphorylation of the F-actin interacting protein SWAP-70

B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.