EFFECT OF INSULIN ON HYDROGEN-PEROXIDE PRODUCTION BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES - STUDIES WITH MONOCLONAL ANTIINSULIN RECEPTOR ANTIBODIES, AND AN AGONIST AND AN INHIBITOR OF PROTEIN-KINASE-C

This study evaluated the effect of insulin on the respiratory burst of human polymorphonuclear leukocytes (PMNLs) and the signalling pathways involved in this process, especially the involvement of protein kinase C (PKC). Isolated human PMNLs from healthy volunteers were incubated with different concentrations of insulin (10(-10)-10(-7) mol/l) and for different durations of incubation (5-90 min). The intracellular production of hydrogen peroxide (H2O2) was detected employing a previously validated flow cytometric assay using 2',7'-dichlorofluorescein-diacetate (DCFH-DA) as a marker for H2O2 production. Specificity of insulin action was verified using an insulin antagonist (the monoclonal antibody MA-10). To identify the signalling pathway involved, we used: (a) monoclonal antibody MA-5, directed against the alpha-subunit of the insulin receptor, that partially mimics insulin without activating tyrosine kinase; (b) H7, an inhibitor of PKC involved in O-2- production in PMNLs, and (c) phorbol myristate acetate (PMA) that binds and stimulates PKC. Insulin caused a dose- and time-dependent stimulation of H2O2 release by human PMNLs. The effect of insulin was blocked by MA-10. The actions of insulin and PMA on H2O2 release were additive, whereas the actions of MA-5 and PMA were not. H7 partially inhibited the H2O2 production stimulated by insulin and completely inhibited MA-5 action. We conclude that insulin stimulates, in a dose- and time-related manner, the respiratory burst of human PMNLs. PKC activation can only partially account for the intracellular mechanisms involved in this process

L-type Ca2+ current as the predominant pathway of Ca2+ entry during INa activation in β-stimulated cardiac myocytes

In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after β-adrenergic stimulation.To clearly distinguish between Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum the Ca2+ store was blocked with 0.1 μm thapsigargin and 10 μm ryanodine. Omitting Na+ from the pipette filling solution minimized Ca2+ entry via Na+-Ca2+ exchange.Individual guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellular Ca2+ concentration was simultaneously recorded with a laser-scanning confocal microscope using fluo-3 as a Ca2+ indicator.Ca2+ entry pathways were discriminated using pharmacological blockers under control conditions and during β-adrenergic stimulation with 1 μm isoproterenol (isoprenaline) in the bathing solution or 100 μm cAMP in the patch-clamp pipette.Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded during L-type Ca2+ current activation but, more interestingly, also during Na+ current (INa) activation. The Ca2+ influx signal arising from L-type Ca2+ current activation was usually blocked by 50 μm Cd2+. However, the Ca2+ influx signal elicited by the Na+ current activation protocol was only curtailed to 56.4 ± 28.2% by 100 μm Ni2+ but was reduced to 17.9 ± 15.1% by 50 μm Cd2+ and consistently eliminated by 5 mm Ni2+.The pronounced Cd2+ and moderate Ni2+ sensitivity of the Ca2+ influx signals suggested that the predominant source of Ca2+ influx during the Na+ current activation – before and during β-adrenergic stimulation – was a spurious activation of the L-type Ca2+ current, presumably due to voltage escape during Na+ current activation.Calculations based on the relationship between Ca2+ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca2+ concentration changes as small as 5.4 ± 0.7 nm. Thus, we can estimate an upper limit for the Ca2+ permeability of the phosphorylated TTX-sensitive Na+ channels which is less than 0.04:1 for Ca2+ ions flowing through Na+ channels via the proposed ‘slip-mode’ Ca2+ conductance. Therefore the slip-mode Ca2+ conductance of Na+ channels does not contribute noticeably to the Ca2+ signals observed in our experiments

Abnormal intrastore calcium signaling in chronic heart failure

Diminished Ca release from the sarcoplasmic reticulum (SR) is an important contributor to the impaired contractility of the failing heart. Despite extensive effort, the underlying causes of abnormal SR Ca release in heart failure (HF) remain unknown. We used a combination of simultaneous imaging of cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes and recordings from single-ryanodine receptor (RyR) channels reconstituted into lipid bilayers to investigate alterations in intracellular Ca handling in an experimental model of chronic HF. We found that diastolic free [Ca] inside the SR was dramatically reduced because of a Ca leak across the SR membrane, mediated by spontaneous local release events (Ca sparks), in HF myocytes. Additionally, the magnitudes of intrastore Ca depletion signals during global and focal Ca release events were blunted, and [Ca](SR) recovery was slowed after global but not focal Ca release in HF myocytes. At the single-RyR level, the sensitivity of RyRs to activation by luminal Ca was greatly enhanced, providing a molecular mechanism for the maintained potentiation of Ca sparks (and increased Ca leak) at reduced intra-SR [Ca] in HF. This work shows that the diminished SR Ca release characteristic of failing myocardium could be explained by increased sensitivity of RyRs to luminal Ca, leading to enhanced spark-mediated SR Ca leak and reduced intra-SR [Ca]

XPO1 mutations identify early‐stage CLL characterized by shorter time to first treatment and enhanced BCR signalling

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-& kappa;B signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL