Use of FRET-Sensor 'Mermaid' to Detect Subtle Changes in Membrane Potential of Primary Mouse PASMCs.

Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.

Bioabsorbable WE43 Mg alloy wires modified by continuous plasma-electrolytic oxidation for implant applications. Part I: Processing, microstructure and mechanical properties.

In our work, a novel processing strategy for the continuous fabrication and surface modification of wires from Magnesium alloy WE43 by means of plasma-electrolytic oxidation (PEO) is presented. In the first step, wires with a strong basal texture and small grain size (≈ 1 µm) were manufactured by combined cold drawing and in-line stress-relief heat treatment steps that optimized the mechanical properties (in terms of strength and ductility) by means of annealing. In a second step, and to the best of our knowledge for the first time ever, the wires were continuously surface-modified with a novel plasma electrolytic oxidation process, which was able to create a homogeneous porous oxide layer made of MgO and Mg3(PO4)2 on the wire surface. While the oxide layer slightly diminished the tensile properties, the strength of the surface-modified wires could be maintained close to 300 MPa with a strain-to-failure ≈ 8 %. Furthermore, the thickness of the oxide layer could be controlled by immersion time within the electrolytic bath and was adjusted to realize a thicknesses of ≈ 8 µm, which could be obtained in <20 s. Our experiments showed that the chemical composition, morphology and porosity of the oxide layer could be tailored by changing electrical parameters. The combined cold drawing and heat treatment process with additional continuous plasma electrolytic oxidation processing can be upscaled to produce a novel generation of bioabsorbable Mg wires with optimized mechanical, degradation and biological performance for use in biomedical applications.

Phospholipase C epsilon (PLCε) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca(2+) concentration [Ca(2+) ]i . Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca(2+) influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCß and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes.