Chairs: A. Assinger (Vienna, Austria); N.D. Ullrich (Heidelberg, Germany)
|Synthetic Peptides restore the Epithelial Sodium Channel Function in Pseudohypoaldosteronism Type 1B Mutants
1 University of Vienna, Department of Pharmacology and Toxicology, Vienna, Austria
2 APEPTICO GmbH, , Vienna, Austria
3 Augusta University, Vascular Biology Center, Medical College of Georgia, Augusta, United States
Abstract text :
Pseudohypoaldosteronism type 1B (PHA1B) is a rare, life-threatening, salt-wasting syndrome, which presents in the first days of life with failure to thrive, vomiting, dehydration, low blood pressure; hyperkalemia, hyponatremia and metabolic acidosis suggest hypoaldosteronism, but plasma aldosterone and renin activity are high. This end-organ resistance to aldosterone is caused by various loss-of-function mutations in the amiloride-sensitive epithelial sodium channel (ENaC). Synthetic peptides (e.g. solnatide) mimicking the lectin-like domain (TIP) of the human tumor necrosis factor (TNF) has been shown to activate current through wildtype (WT) ENaC and ENaC carrying point mutations associated with PHA1B. In addition other types of mutations, which were described in PHA1B patients, were created in vitro and expressed heterologously in HEK-293 cells. The channel activity was studied using Patch-clamp technique and the expression using Western blots of biotinylated surface proteins. The membrane abundance varied considerably among the mutants; some were higher, others lower than WT, but all observed mutant ENaCs were at least present at the cell membrane, in our expression system. Nonetheless the current density of all tested mutations was decreased compared to WT likely to be the cause of the disease. Regardless of the type of mutation the peptides were able to restore the channel function of mutant ENaC to current density levels of WT or even higher. Our findings suggest that the synthetic peptides solnatide and its congener represent a promising new strategy to treat PHA1B, which hitherto has been treated only symptomatically.
|Different modulation of ion currents in hippocampal pyramidal neurons and NG108-15 cell line by delta opioid receptor antagonist naltrindole
1 Centre of Biosciences SAS, Institute of Molecular Physiology and Genetics, Bratislava, Slovakia
2 Biomedical Research Center SAS, Institute of Experimental Endocrinology, Bratislava, Slovakia
Abstract text :
Naltrindole (NTI) represents a highly potent, selective non-peptide antagonist of delta opioid receptors (DOR). Part of its effects on neuronal excitability may be mediated by an effect on activity of voltage dependent ion currents. We compared effect of an acute application of NTI on ion currents in rat hippocampal pyramidal neurons, which express all subtypes of opioid receptors, and differentiated NG108-15 cells, which predominantly express DOR.
Hippocampal neurons were isolated from newborn Wistar rats and maintained in a primary culture up to two weeks. Measurements were done at Day 9-12 in vitro. Differentiation of NG108-15 cells cultured in a serum-free Dulbecco"s modified Eagle"s medium was induced by an addition of 1 mM dbcAMP, and 1x N2 supplement for 7-11 days. Ion currents were measured by a whole-cell patch clamp. Concentration of NTI was 10 µM.
NTI significantly inhibited sodium current in hippocampal neurons but did not affect it in NG108-15 cells. Similarly, calcium currents were inhibited in hippocampal neurons but not in NG108-15 cells. In contrast, NTI significantly inhibited both transient and sustained potassium current in NG108-15 cells. In hippocampal neurons, inhibition of potassium currents was less prominent and was not statistically significant. Inhibition of individual ion currents developed slowly allowing us to presume that it was mediated by activation of intracellular signaling pathway rather that by direct interaction of NTI with the channel protein.
In conclusion, inhibitory effects of NTI in hippocampal neurons and in NG108-15 cells are complementary suggesting that they may be mediated through different signaling pathways including an interaction with different subtypes of opioid receptors.
|Investigation of the extracellular Ca2+ entry in mouse pancreatic ductal cells
1 University of Szeged, First Department of Medicine, Szeged, Hungary
2 Univ. of Pécs, Szeged, Institute for Transl. Med. &1st Dep. of Medicine, MTA-SZTE Transl. Gastroenterology Research Group, Szeged, Szeged, Hungary
Abstract text :
Introduction: Acute pancreatitis (AP) is the most common inflammatory disorder in the gastrointestinal tract with an overall mortality of 20-30% in severe cases. The treatment of AP is not resolved yet, urging the identification of novel drug targets. Toxic cellular Ca2 overload was highlighted as a key event in pancreatic acinar and ductal cells during the pathogenesis of AP. In addition, the inhibition of Orai1 in pancreatic acinar cells markedly decreased the Ca2 toxicity and the severity of AP. However, We have no information regarding the role of Orai1 in pancreatic ductal physiology or pathophysiology.
Methods: Wild type FVB/N mice were used for the isolation of pancreatic ductal fragments. The intracellular pH and Ca2 level of the pancreatic ductal cells (PDC) were measured by microfluorimetry. The effect of selective Orai1 inhibitors provided by CalciMedica was evaluated.
Results: The tested compounds dose-dependently inhibited Ca2 influx during the carbachol induced Ca2 signal in PDC. Inhibition was complete at a concentration of 10 µM (CM-B: 99.87%, CM-C: 95.29%). Next, endoplasmic reticulum Ca2 stores were depleted with cyclopiazonic acid and the inhibition of store-operated Ca2 entry (SOCE) was investigated after the re-addition of extracellular Ca2 . Under these conditions CM-B and CM-C significantly, but not completely, decreased SOCE in PDC (55.96% and 55.03% respectively). The removal of extracellular Na to abolish activity of the Na /Ca2 exchanger had no effect on the inhibition of SOCE by CM-B or CM-C. We also showed that the inhibition of Orai1 has no effect on the basal secretion of HCO3- by PDC, which is the main physiological function of these cells.
Conclusions: We showed that Orai1 has a significant role in the Ca2 signaling of PDC. In the next step we will evaluate the pathophysiological relevance of the channel.
|BLOCKAGE OF EXOSOME GENERATION REDUCES TAU PROTEIN CAUSED NEURONAL LOSS AND MICROGLIA PROLIFERATION
1 Lithuanian University of Health Sciences, Neuroscience Institute , Kaunas, Lithuania
2 Vilnius University , Institute of Biotechnology, Vilnius, Lithuania
Abstract text :
Progressive neurodegeneration is associated with tau modifications leading to neurotoxicity and brain cell death. In addition, various abnormal tau monomers and aggregates accumulate in interstitial and cerebrospinal fluids. Extracellular tau might be toxic to brain cells. There is a growing evidence that exosomes may contribute to cell-to-cell transmission of pathogenic tau protein. Exosome uptake might be regulated by various factors such as protein kinase C (PKC) that controls sphingomyelinase activity involved in exosome generation. The aim of this study was to investigate toxic effect of extracellular tau on brain cell culture and whether exosome and PKC inhibitors may suppress neurotoxicity of exogenous tau (2N4R isoform). Cultures of rat cerebellar granule cells (CGC) were treated with various type of monomeric or aggregated recombinant 2N4R tau plus/minus 1 µM Ro31-8220 (PKC inhibitor) and 13 µM GW4869 (exosome inhibitor) for 48 hours. Neuronal and microglial cells densities and viability were evaluated by fluorescence microscopy. Our results showed that tau protein 2N4R isoform at micromolar concentration slightly decreased viability of neurons, but caused significant neuronal loss (~70 %) and microglia proliferation (~200 %) in CGC. Neuronal loss and microglia proliferation were completely prevented by GW4869 that inhibits neutral sphingomyelinase. PKC inhibitor also blocked tau neurotoxicity and microglial proliferation in cell culture. 2N4R had similar effects independently of the preparation method. Our data suggest that neutral sphingomyelinase and PKC activation is required for 2N4R induced neuron necrosis, loss and microglia proliferation in CGC.
|TRPA1 and TRPV1 photosensitization by 7-dehydrocholesterol – connections to the Smith-Lemli-Opitz syndrome
1 Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Austria
2 Friedrich-Alexander University Erlangen-Nuremberg, Institute of Physiology and Pathophysiology, Erlangen, Germany
3 Faculty of Biology, University of Bucharest, Department of Anatomy, Physiology and Biophysics, Bucharest, Romania
Abstract text :
Question: Low activity of the 7-Dehydrocholesterol (7DHC) reducing enzyme by means of a loss of function mutation can lead to a 1000-fold increase in 7DHC plasma levels. This is the pathophysiological basis of the autosomal recessive Smith-Lemli-Opitz syndrome, characterized by congenital abnormalities, retardation, and exceptional hypersensitivity when exposed to sunlight. Focusing on the TRPA1 and TRPV1 ion channels, we investigated how elevated 7DHC levels cause a sensitivity to UVA light.
Methods: Calcium-based microfluorimetric assessment of the photosensitisation caused by 7DHC treatment and UVA light exposure was performed on transfected HEK293t cells and mouse DRG neurons. 7DHC-generated currents were recorded using the whole-cell patch clamp technique on transfected HEK293t cells. CGRP release from the isolated mouse trachea served as an index of 7DHC- and light-induced neuronal activation.
Results: Human TRPA1-transfected HEK293t cells exhibited significant calcium transients upon 7DHC and UVA exposure, as opposed to untransfected cells. 7DHC and UVA exposure alone activated cells, but the combination had a supra-additive effect. TRPV1 transfected cells lead to lower responses compared to TRPA1 in identical experiments. Considerable photosensitization occurred after 1-15 hours of 7DHC preexposure in cells expressing TRPA1 or TRPV1. TRPA1 antagonist A-967079 and TRPV1 antagonist BCTC significantly reduced 7DHC and UVA-induced responses. DRG neurons exposed to 7DHC for 4 hours yielded similar results and confirmatory evidence was obtained from the respective TRP channel knockout mice. Tissue CGRP release was also significantly increased due to 7DHC and light.
Conclusions: TRPA1 and, to a lesser extent, TRPV1 mediate 7DHC induced photosensitization to UVA light exposure.