K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport

<p>Abstract</p> <p>Background</p> <p>Lung epithelial Na⁺channels (ENaC) are regulated by cell Ca²⁺signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K⁺channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K⁺channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport.</p> <p>Methods</p> <p>Verapamil-induced depression of heterologously expressed human αβγ ENaC in <it>Xenopus </it>oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and <it>in vivo </it>alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca²⁺signal in H441 cells was analyzed using Fluo 4AM.</p> <p>Results</p> <p>The rate of <it>in vivo </it>AFC was reduced significantly (40.6 ± 6.3% of control, <it>P </it>< 0.05, n = 12) in mice intratracheally administrated verapamil. K_Ca3.1(1-EBIO) and K_ATP(minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca²⁺signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca²⁺in αβγ ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, K_V(pyrithione-Na), K _Ca3.1(1-EBIO), and K_ATP(minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na⁺and K⁺transport pathways.</p> <p>Conclusions</p> <p>Our observations demonstrate that K⁺channel openers are capable of rescuing reduced vectorial Na⁺transport across lung epithelial cells with impaired Ca²⁺signal.</p

Action potential characterization of human induced pluripotent stem cell-derived cardiomyocytes using automated patch-clamp technology.

Abstract Recent progress in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) research led to high-purity preparations of human cardiomyocytes (CMs) differentiated from these two sources-suitable for tissue regeneration, in vitro models of disease, and cardiac safety pharmacology screening. We performed a detailed characterization of the effects of nifedipine, cisapride, and tetrodotoxin (TTX) on Cor.4U(®) human iPSC-CM, using automated whole-cell patch-clamp recordings with the CytoPatch(™) 2 equipment, within a complex assay combining multiple voltage-clamp and current-clamp protocols in a well-defined sequence, and quantitative analysis of several action potential (AP) parameters. We retrieved three electrical phenotypes based on AP shape: ventricular, atrial/nodal, and S-type (with ventricular-like depolarization and lack of plateau). To suppress spontaneous firing, present in many cells, we injected continuously faint hyperpolarizing currents of -10 or -20 pA. We defined quality criteria (both seal and membrane resistance over 1 GΩ), and focused our study on cells with ventricular-like AP. Nifedipine induced marked decreases in AP duration (APD): APD90 (49.8% and 40.8% of control values at 1 and 10 μM, respectively), APD50 (16.1% and 12%); cisapride 0.1 μM increased APD90 to 176.2%; and tetrodotoxin 10 μM decreased maximum slope of phase to 33.3% of control, peak depolarization potential to 76.3% of control, and shortened APD90 on average to 80.4%. These results prove feasibility of automated voltage- and current-clamp recordings on human iPSC-CM and their potential use for in-depth drug evaluation and proarrhythmic liability assessment, as well as for diagnosis and pharmacology tests for cardiac channelopathy patients

Raman micro-spectroscopy as a viable tool to monitor and estimate the ionic transport in epithelial cells

The typical response to the lowering of plasma Na-vertical bar concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na+ concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na+ current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone