Big conductance calcium-activated potassium channel openers control spasticity without sedation.

BACKGROUND AND PURPOSE: Our initial aim was to generate cannabinoid agents that control spasticity, occurring as a consequence of multiple sclerosis (MS), whilst avoiding the sedative side effects associated with cannabis. VSN16R was synthesized as an anandamide (endocannabinoid) analogue in an anti-metabolite approach to identify drugs that target spasticity. EXPERIMENTAL APPROACH: Following the initial chemistry, a variety of biochemical, pharmacological and electrophysiological approaches, using isolated cells, tissue-based assays and in vivo animal models, were used to demonstrate the activity, efficacy, pharmacokinetics and mechanism of action of VSN16R. Toxicological and safety studies were performed in animals and humans. KEY RESULTS: VSN16R had nanomolar activity in tissue-based, functional assays and dose-dependently inhibited spasticity in a mouse experimental encephalomyelitis model of MS. This effect occurred with over 1000-fold therapeutic window, without affecting normal muscle tone. Efficacy was achieved at plasma levels that are feasible and safe in humans. VSN16R did not bind to known CB1 /CB2 /GPPR55 cannabinoid-related receptors in receptor-based assays but acted on a vascular cannabinoid target. This was identified as the major neuronal form of the big conductance, calcium-activated potassium (BKCa ) channel. Drug-induced opening of neuronal BKCa channels induced membrane hyperpolarization, limiting excessive neural-excitability and controlling spasticity. CONCLUSIONS AND IMPLICATIONS: We identified the neuronal form of the BKCa channel as the target for VSN16R and demonstrated that its activation alleviates neuronal excitability and spasticity in an experimental model of MS, revealing a novel mechanism to control spasticity. VSN16R is a potential, safe and selective ligand for controlling neural hyper-excitability in spasticity

Lysosomal acid lipase regulates VLDL synthesis and insulin sensitivity in mice

AIMS/HYPOTHESIS: Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal (-/-) ) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). METHODS: We studied metabolic adaptations in Lal (-/-) mice. RESULTS: Despite loss of adipose tissue, Lal (-/-) mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [(3)H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal (-/-) mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal (-/-) mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal (-/-) mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal (-/-) mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal (-/-) mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. CONCLUSIONS/INTERPRETATION: Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Lal (-/-) mice. We conclude that decreased plasma VLDL production enhances glucose uptake into skeletal muscle to compensate for the lack of energy supply

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca2+-activated K+ channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca2+ and voltage-gated potassium (BKCa) channels. Here, we explored the effect of LPI on intermediate-conductance Ca2+-activated K+ (IKCa) channels using excised inside-out patches from endothelial cells. The functional expression of IKCa was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IKCa channels with a slope conductance of 39 pS in symmetric K+ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IKCa single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IKCa single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature

Bildung über den Äther

Es wird ein historisch-medienpädagogischer Überblick über die Bedeutung des Mediums Radio als Bildungsmittel und sein Einsatz im Unterricht bzw. für ein lebenslanges Lernen gegeben. Neben der Betrachtung der historischen Entwicklung des Schulfunks in Österreich, wir auch auf "Neue Medien", insbesondere den Podcast als konsequente Weiterentwicklung des "Bildungsradios" eingegangen. Ein Ästhetischer Vergleich zwischen Schulfunk und Podcast, sowie zwei Interviews zum Thema "Podcast im Unterricht", runden diese Arbeit ab

Calcium signaling in ß-cell physiology and pathology: a revisit

Pancreatic beta (β) cell dysfunction results in compromised insulin release and, thus, failed regulation of blood glucose levels. This forms the backbone of the development of diabetes mellitus (DM), a disease that affects a significant portion of the global adult population. Physiological calcium (Ca2+) signaling has been found to be vital for the proper insulin-releasing function of β-cells. Calcium dysregulation events can have a dramatic effect on the proper functioning of the pancreatic β-cells. The current review discusses the role of calcium signaling in health and disease in pancreatic β-cells and provides an in-depth look into the potential role of alterations in β-cell Ca2+ homeostasis and signaling in the development of diabetes and highlights recent work that introduced the current theories on the connection between calcium and the onset of diabetes

Probing subcellular iron availability with genetically encoded nitric oxide biosensors

Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.Vifor Pharm

The Multicellular Effects of VDAC1 N-Terminal-Derived Peptide

The mitochondrial voltage-dependent anion channel-1 (VDAC1) protein functions in a variety of mitochondria-linked physiological and pathological processes, including metabolism and cell signaling, as well as in mitochondria-mediated apoptosis. VDAC1 interacts with about 150 proteins to regulate the integration of mitochondrial functions with other cellular activities. Recently, we developed VDAC1-based peptides that have multiple effects on cancer cells and tumors including apoptosis induction. Here, we designed several cell-penetrating VDAC1 N-terminal-derived peptides with the goal of identifying the shortest peptide with improved cellular stability and activity. We identified the D-Δ(1-18)N-Ter-Antp comprising the VDAC1 N-terminal region (19-26 amino acids) fused to the Antp, a cell-penetrating peptide. We demonstrated that this peptide induced apoptosis, autophagy, senescence, cell volume enlargement, and the refusion of divided daughter cells into a single cell, it was responsible for reorganization of actin and tubulin filaments, and increased cell adhesion. In addition, the peptide induced alterations in the expression of proteins associated with cell metabolism, signaling, and division, such as enhancing the expression of nuclear factor kappa B and decreasing the expression of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha. These cellular effects may result from the peptide interfering with VDAC1 interaction with its interacting proteins, thereby blocking multiple mitochondrial/VDAC1 pathways associated with cell functions. The results of this study further support the role of VDAC1 as a mitochondrial gatekeeper protein in controlling a variety of cell functions via interaction with associated proteins

Endothelial dysfunction and vascular disease

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G i (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G q (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients. © 2008 Scandinavian Physiological Society.postprin