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

Activation of the Na+-K+ pump by hyposmolality through tyrosine kinase-dependent Cl− conductance in Xenopus renal epithelial A6 cells

We studied the regulatory mechanism of Na+ transport by hyposmolality in renal epithelial A6 cells.Hyposmolality increased (1) Na+ absorption, which was detected as an amiloride-sensitive short-circuit current (INa), (2) Na+-K+ pump activity, (3) basolateral Cl− conductance (Gb,Cl), and (4) phosphorylation of tyrosine, suggesting an increase in activity of protein tyrosine kinase (PTK).A Cl− channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which abolished Gb,Cl, blocked the INa by inhibiting the Na+-K+ pump without any direct effect on amiloride-sensitive Na+ channels. Diminution of Gb,Cl by Cl− replacement with a less permeable anion, gluconate, also decreased the hyposmolality-increased Na+-K+ pump activity.The PTK inhibitors tyrphostin A23 and genistein induced diminution of the hyposmolality-stimulated Gb,Cl, which was associated with attenuation of the hyposmolality-increased Na+-K+ pump activity.Taken together, these observations suggest that: (1) hyposmolality activates PTK; (2) the activated PTK increases Gb,Cl; and (3) the PTK-increased Gb,Cl stimulates the Na+-K+ pump.This PTK-activated Gb,Cl-mediated signalling of hyposmolality is a novel pathway for stimulation of the Na+-K+ pump

Regulation of an amiloride-sensitive Na+-permeable channel by a β2-adrenergic agonist, cytosolic Ca2+ and Cl− in fetal rat alveolar epithelium

In cell-attached patches formed on the apical membrane of fetal alveolar epithelium, terbutaline (a specific β2-adrenergic agonist) increased the open probability (Po) of an amiloride-sensitive Na+-permeable non-selective cation (NSC) channel (control, 0.03 ± 0.04; terbutaline, 0.62 ± 0.18; n = 8, P < 0.00001) by increasing the mean open time 100-fold without any significant change in the mean closed time and without any change in the single channel conductance (control, 27.8 ± 2.3 pS; terbutaline, 28.2 ± 2.1 pS; n = 8).The Po of the unstimulated channel increased when the apical membrane was depolarized due to a decrease in the closing rate and an increase in the opening rate, while the Po of the terbutaline-stimulated channel did not depend on the membrane potential.Increased cytosolic [Ca2+] also increased the Po of the channel in a manner consistent with one Ca2+-binding site on the cytosolic surface of the channel. Terbutaline increased the sensitivity of the channel to cytosolic Ca2+ by shifting the concentration of cytosolic Ca2+ ([Ca2+]c) required for half-maximal activation to a lower [Ca2+]c value, leading to an increase in Po.An increase in the cytosolic Cl− concentration ([Cl−]c) decreased the Po of the channel consistent with two Cl−-binding sites by increasing the closing rate without any significant change in the opening rate. Terbutaline increased Po by reducing the effect of cytosolic Cl− to promote channel closing.Taken together, these observations indicate that terbutaline activates a Ca2+-activated, Cl−-inhibitable, amiloride-sensitive, Na+-permeable NSC channel in fetal rat alveolar epithelium in two ways: first, through an increase in Ca2+ sensitivity, and second, through a reduction in the effect of cytosolic Cl− to promote channel closing

Effect of pH and monovalent cations on the Raman spectrum of water: Basics revisited and application to measure concentration gradients at water/solid interface in Si3N4 biomaterial

The effect of hydrogen carbonate (HCO3-) and cations (Na+, K+) solvated in water were revisited according to high spectrally resolved Raman measurements. Water solutions with different bicarbonate concentrations or added with increasing amounts of monovalent cations were examined with respect to their Raman spectra both in the bulk state and at the solid/liquid interface with a silicon nitride (Si3N4) bioceramic. Spectroscopic calibrations confirmed that the Raman emission from OH-stretching in water is sensitive to molarity variations (in the order of tens of mM). The concentration gradient developed at the solid/liquid interface in cation-added solutions interacting with a Si3N4 surface was measured and found to be peculiar to individual cations. Local variation in pH was detected in ionic solutions interacting with Si3N4 samples, which might represent a useful property for Si3N4 in a number of biomedical applications. (C) 2015 Elsevier B.V. All rights reserved