Understanding Bidirectional Water Transport across Bronchial Epithelial Cell Monolayers: A Microfluidic Approach.

Deciphering the dynamics of water transport across bronchial epithelial cell monolayers is pivotal for unraveling respiratory physiology and pathology. In this study, we employ an advanced microfluidic system to explore bidirectional water transport across 16HBE14σ bronchial epithelial cells. Previous experiments unveiled electroneutral multiple ion transport, with chloride ions utilizing transcellular pathways and sodium ions navigating both paracellular and transcellular routes. Unexpectedly, under isoosmotic conditions, rapid bidirectional movement of Na+ and Cl- was observed, leading to the hypothesis of a substantial transport of isoosmotic solution (145 mM NaCl) across cell monolayers. To validate this conjecture, we introduce an innovative microfluidic device, offering a 500-fold sensitivity improvement in quantifying fluid flow. This system enables the direct measurement of minuscule fluid volumes traversing cell monolayers with unprecedented precision. Our results challenge conventional models, indicating a self-regulating mechanism governing water transport that involves the CFTR channel and anion exchangers. In healthy subjects, equilibrium is achieved at an apical potential of Δφap = -30 mV, while subjects with cystic fibrosis exhibit modulation by an anion exchanger, reaching equilibrium at [Cl] = 67 mM in the airway surface liquid. This nuanced electrochemical basis for bidirectional water transport in bronchial epithelia sheds light on physiological intricacies and introduces a novel perspective for understanding respiratory conditions.

Special Issue "Advances in Artificial and Biological Membranes: Mechanisms of Ionic Sensitivity, Ion-Sensor Designs, and Applications for Ion Measurement".

Ion sensors, conventionally known as ion-selective membrane electrodes, were devised 100 years ago with the invention of a pH electrode with a glass membrane (in 1906 Cremer, in 1909 Haber and Klemensiewicz) [...].

Precipitation of Inorganic Salts in Mitochondrial Matrix.

In the mitochondrial matrix there are insoluble, osmotically inactive complexes that maintain constant pH and calcium concentration. In the present paper we examine the properties of insoluble calcium and magnesium salts, namely phosphates, carbonates and polyphosphates which might play this role. We find that non-stoichiometric, magnesium-rich carbonated apatite, with very low crystallinity, precipitates in the matrix under physiological conditions. Precipitated salt acts as pH buffer, and hence can contribute in maintaining ATP production in ischemic conditions, delaying irreversible damages to heart and brain cells after stroke.

Measurement of Multi Ion Transport through Human Bronchial Epithelial Cell Line Provides an Insight into the Mechanism of Defective Water Transport in Cystic Fibrosis.

We measured concentration changes of sodium, potassium, chloride ions, pH and the transepithelial potential difference by means of ion-selective electrodes, which were placed on both sides of a human bronchial epithelial 16HBE14σ cell line grown on a porous support in the presence of ion channel blockers. We found that, in the isosmotic transepithelial concentration gradient of either sodium or chloride ions, there is an electroneutral transport of the isosmotic solution of sodium chloride in both directions across the cell monolayer. The transepithelial potential difference is below 3 mV. Potassium and pH change plays a minor role in ion transport. Based on our measurements, we hypothesize that in a healthy bronchial epithelium, there is a dynamic balance between water absorption and secretion. Water absorption is caused by the action of two exchangers, Na/H and Cl/HCO3, secreting weakly dissociated carbonic acid in exchange for well dissociated NaCl and water. The water secretion phase is triggered by an apical low volume-dependent factor opening the Cystic Fibrosis Transmembrane Regulator CFTR channel and secreting anions that are accompanied by paracellular sodium and water transport.

New ISE-Based Apparatus for Na+, K+, Cl-, pH and Transepithelial Potential Difference Real-Time Simultaneous Measurements of Ion Transport across Epithelial Cells Monolayer⁻Advantages and Pitfalls.

Cystic Fibrosis (CF) is the most common fatal human genetic disease, which is caused by a defect in an anion channel protein (CFTR) that affects ion and water transport across the epithelium. We devised an apparatus to enable the measurement of concentration changes of sodium, potassium, chloride, pH, and transepithelial potential difference by means of ion-selective electrodes that were placed on both sides of a 16HBE14σ human bronchial epithelial cell line that was grown on a porous support. Using flat miniaturized ISE electrodes allows for reducing the medium volume adjacent to cells to approximately 20 µL and detecting changes in ion concentrations that are caused by transport through the cell layer. In contrast to classic electrochemical measurements, in our experiments neither the calibration of electrodes nor the interpretation of results is simple. The calibration solutions might affect cell physiology, the medium composition might change the direction of actions of the membrane channels and transporters, and water flow that might trigger or cut off the transport pathways accompanies the transport of ions. We found that there is an electroneutral transport of sodium chloride in both directions of the cell monolayer in the isosmotic transepithelial concentration gradient of sodium or chloride ions. The ions and water are transported as an isosmotic solution of 145 mM of NaCl.

Calcium phosphate buffer formed in the mitochondrial matrix during preconditioning supports ΔpH formation and ischemic ATP production and prolongs cell survival -A hypothesis.

Ischemic preconditioning makes cells less sensitive to oxygen deprivation. A similar effect can be achieved by increasing the calcium concentration and applying potassium channel openers. A hypothetical mechanism of preconditioning is presented. In the mitochondrial matrix, there is a calcium hydroxide buffer consisting of a few insoluble calcium phosphate minerals. During ischemia, calcium ions stored in the matrix buffer start to leak out, forming an electric potential difference, while hydroxyl ions remain in the matrix, maintaining its pH and the matrix volume. Preconditioning factors increase the matrix buffer capacity. Production of ATP during ischemia might be the relic of a pre-endosymbiotic past.

Hydrocortisone inhibition of wild-type and αD200Q nicotinic acetylcholine receptors.

Short-term treatment with large doses of corticosteroids can result in acute weakness of muscles in processes that have not yet been fully characterized. Corticosteroids have been shown to exert direct inhibitory action on the muscle-type nicotinic acetylcholine receptor (AChR), and therefore can promote pharmacological muscle denervation. The mechanism of hydrocortisone (HC) blockage of AChR has not been fully established yet. It is uncommon for an electrically neutral molecule, for example, HC, to induce voltage-dependent changes in AChR kinetics. Our experiments aimed to determine the source of voltage-dependency in HC action. Wild-type (WT) and αD200Q receptors were transiently expressed in HEK293 cells. Recordings were performed in either the presence or absence of HC. We showed that the D-to-Q substitution is capable of suppressing the voltage dependency in the HC-induced block. We conclude that the distance between αD200 and the agonist-binding site depends on the membrane potential. The voltage-dependent changes of the αD200 position have not been considered yet. To our knowledge, the ability to induce voltage-dependency in blocker action has not been shown previously for an amino acid located outside the transmembrane portion of the receptor. Possible mechanisms of HC block (allosteric and knocking) in WT and αD200Q receptors are discussed.

Multi-electrode system for measurement of transmembrane ion-fluxes through living epithelial cells.

Cystic Fibrosis (CF) is the most common fatal human genetic disease. It is caused by the defect in a single anion channel protein which affects ion and water transport across the epithelial tissue. A flat multi-electrode platform of diameter 12mm, allowing for measurement of four ions: sodium, potassium, hydrogen and chloride by exchangeable/replaceable ion-selective electrodes is described. The measurement is possible owing to the architecture of the platform which accommodates all the electrodes and inlets/outlets. The platform fits to the cup and operates in a small volume of the solution bathing the living epithelial cell layer (membrane) deposited on a porous support of the cup, which allows for effective monitoring of ion concentration changes. By applying two multi-electrode platforms, it is possible to measure the ion transmembrane fluxes. The inlet and outlet tubes in the platforms allow for on-fly change of the calibrants, ion-concentration changes and ion channel blockers. Using different ion-concentration gradients and blockers of ion-transporting molecules we show for the first time that sodium ions flow from the basolateral to apical face of the cell monolayer via a paracellular route and return also via a transcellular one, while chloride anions are transported back and forth exclusively via a transcellular route.

Measurement of ion fluxes across epithelia.

Epithelial tissues line all wet surfaces of vertebrate bodies. Their major function is directional transport of ions and water. Cells forming an epithelial layer are bound together by a tight junction that forms a barrier to ion flux. Ions and water are transported via specialized molecules. The presence of a defect in a single ion channel molecule leads to cystic fibrosis - the most common, fatal, human genetic disease. The paper describes ion transport data obtained by means of different experimental techniques. Special attention is given to radiochemical tracers, transepithelial resistance determination, open circuit potential and short circuit current measurements, the nasal potential difference in healthy and cystic fibrosis patients, the use of ion selective electrodes, and electrochemical mapping of the cell membrane surface. The effect of different activators and blockers of ion transport molecules on measured parameters are also discussed.

Restoration of the response of the middle cerebral artery of the rat to acidosis in hyposmotic hyponatremia by the opener of large-conductance calcium sensitive potassium channels (BKCa).

Hyposmotic hyponatremia (the decrease of extracellular concentration of sodium ions from 145 to 121 mM and the decrease of hyposmolality from 300 to 250 mOsm/kg H2O) impairs response of the middle cerebral artery (MCA) to acetylcholine and NO donor (S-nitroso-N-acetyl-DL-penicillamine). Since acidosis activates a similar intracellular signaling pathway, the present study was designed to verify the hypothesis that the response of the MCA to acidosis is impaired during acute hyposmotic hyponatremia due to abnormal NO-related signal transduction in vascular smooth muscle cells. Studies performed on isolated, cannulated, and pressurized rat MCA revealed that hyposmotic hyponatremia impaired the response of the MCA to acidosis and this was associated with hyposmolality rather than with decreased sodium ion concentration. Response to acidosis was restored by the BKCa but not by the KATP channel activator. Patch-clamp electrophysiology performed on myocytes freshly isolated from MCAs, demonstrated that hyposmotic hyponatremia does not affect BKCa currents but decreases the voltage-dependency of the activation of the BKCa channels in the presence of a specific opener of these channels. Our study suggests that reduced sensitivity of BKCa channels in the MCA to agonists results in the lack of response of this artery to acidosis during acute hyposmotic hyponatremia.

[What we don't know about mitochondrial potassium channels?] / Czego nie wiemy o mitochondrialnych kanalach potasowych?

In the current work the authors present the most interesting, yet not fully understood issues regarding origin, function and pharmacology of the mitochondrial potassium channels. There are eight potassium channels known to contribute to the potassium permeability of the inner mitochondrial membrane: ATP-regulated channel, calcium-regulated channels of large, intermediate and small conductance, voltage-regulated Kv1.3 and Kv7.4 channels, two-pore-domain TASK-3 channel and SLO2 channel. The primary function of the mitochondrial potassium channels is regulation of the mitochondrial membrane potential. Additionally, mitochondrial potassium channels alter cellular respiration, regulation of the mitochondrial volume and ROS synthesis. However, mechanisms underlying these processes are not fully understood yet. In this work, the authors not only present available knowledge about this topic, but also put certain hypotheses that may set the direction for the future research on these proteins.

Multielectrode bisensor system for time-resolved monitoring of ion transport across an epithelial cell layer.

An ion-selective multielectrode bisensor system is designed to ensure reliable real-time concentration measurements of sodium, potassium, chloride, and pH in a small volume of biological liquid bathing a living human bronchial epithelial cell monolayer. The bisensor system allows the monitoring of major ions, which are simultaneously transported through the epithelia in both directions.

Ion transporting proteins of human bronchial epithelium.

The electrolyte transport system across human airway epithelium followed by water movement is essential for the normal mucociliary clearance that allows the maintenance of the aseptic condition of the respiratory tract. The function of epithelial cells is to control and regulate ionic composition and volume of fluids in the airways. Various types of proteins taking part in assuring effective ions and water transport in apical and basolateral membranes of the airway epithelium have been found (e.g., CFTR, ENaC, CaCC, ORCC, potassium channels, NaKATPase, aquaporins). The paper reviews the current state of the art in the field of ion channels, transporters, and other signaling proteins identified in the human bronchial epithelium.

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria.

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Effect of selected NAD+ analogues on mitochondria activity and proliferation of endothelial EA.hy926 cells.

The aim of the study was to examine the effect of 1-methylnicotinamide (MNA) and 1-methyl-3-nitropyridine (MNP) on mitochondria activity and proliferation of endothelial EA.hy926 cells. The activity of MNA was also referred to nicotinamide (NAM) being MNA metabolic precursor. NAM and MNA used at high concentrations (up to 1 mM) had no effect on mitochondria metabolism and proliferation of EA.hy926 cells. It could be related to the fact that these compounds hardly cross the cell membrane. It supports the results of our previous study suggesting that anti-inflammatory and anti-thrombotic effects of MNA could be associated with its ability to bind to glycosaminoglycans, especially heparins, located on the endothelium membrane without entering into target cells. In contrast, MNP caused substantial changes in mitochondria activity and proliferation of EA.hy926 cells. This compound used at low concentrations (below 100 microM) blocked the cell cycle of EA.hy926 cells in G1 phase and was very effective in inhibiting cell growth (IC50=13.8+/-2.4 microM). At higher concentrations (0.1-1 mM) MNP caused a significant reduction of cell survival. The observed effects of MNP could be related, at least in part, to its ability to influence the ATP and NAD+ intracellular levels. MNP caused also important changes in Ca2+ intracellular concentration, significant decrease in inner mitochondrial membrane potential and high increase in mitochondrial respiration of EA.hy926 cells. The observed effects of MNP may be related in part to its cellular metabolites detected after 45 min incubation with 250 microM MNP.

Pharmacology of mitochondrial potassium channels: dark side of the field.

Mitochondrial potassium channels play an important role in cytoprotection. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off-target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. Moreover, understanding their side-effects should facilitate the design of a specific mitochondrial channel opener with cytoprotective properties. In this review, we discuss the complex interactions of potassium channel inhibitors and activators with cellular structures.

The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

Single channel studies of the ATP-regulated potassium channel in brain mitochondria.

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 +/- 15 pS. The activity of this channel was inhibited by ATP/Mg(2+) and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.

Calcium ions regulate K⁺ uptake into brain mitochondria: the evidence for a novel potassium channel.

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca(2+) additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca(2+) effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK(Ca) channel). Furthermore, NS1619 - a BK(Ca) channel opener - induced potassium ion-specific effects on brain mitochondria similar to those induced by Ca(2+). These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-beta(4) subunit (of the BK(Ca) channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of beta(4) subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK(Ca) channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.

Potassium currents in human myogenic cells from healthy and congenital myotonic dystrophy foetuses.

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K(+) (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K(+) channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.