Proliferation of SH-SY5Y neuroblastoma cells on confined spaces.

BACKGROUND: Microfluidics offers precise drug delivery and continuous monitoring of cell functions, which is crucial for studying the effects of toxins and drugs. Ensuring proper cell growth in these space-constrained systems is essential for obtaining consistent results comparable to standard Petri dishes. NEW METHOD: We investigated the proliferation of SH-SY5Y cells on circular polycarbonate chambers with varying surface areas. SH-SY5Y cells were chosen for their relevance in neurodegenerative disease research. RESULTS: Our study demonstrates a correlation between the chamber surface area and SH-SY5Y cell growth rates. Cells cultured in chambers larger than 10 mm in diameter exhibited growth comparable to standard 60-mm dishes. In contrast, smaller chambers significantly impeded growth, even at identical seeding densities. Similar patterns were observed for HeLaGFP cells, while 16HBE14σ cells proliferated efficiently regardless of chamber size. Additionally, SH-SY5Y cells were studied in a 12-mm diameter sealed chamber to assess growth under restricted gas exchange conditions. COMPARISON WITH EXISTING METHODS: Our findings underscore the limitations of small chamber sizes in microfluidic systems for SH-SY5Y cells, an issue not typically addressed by conventional methods. CONCLUSIONS: SH-SY5Y cell growth is highly sensitive to spatial constraints, with markedly reduced proliferation in chambers smaller than 10 mm. This highlights the need to carefully consider chamber size in microfluidic experiments to achieve cell growth rates comparable to standard culture dishes. The study also shows that while SH-SY5Y and HeLaGFP cells are affected by chamber size, 16HBE14σ cells are not. These insights are vital for designing effective microfluidic systems for bioengineering research.

Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Membrane lytic activity of antibacterial ionenes, critical role of phosphatidylcholine (PC) and cardiolipin (CL).

Understanding the mechanism by which an antibacterial agent interacts with a model membrane provides vital information for better design of future antibiotics. In this study, we investigated two antibacterial polymers, hydrophilic C0-T-p and hydrophobic C8-T-p ionenes, known for their potent antimicrobial activity and ability to disrupt the integrity of lipid bilayers. Our hypothesize is that the composition of a lipid bilayer alters the mechanism of ionenes action, potentially providing an explanation for the observed differences in their bioactivity and selectivity. Calcein release experiments utilizing a range of liposomes to examine the impact of (i) cardiolipin (CL) to phosphatidylglycerol (PG) ratio, (ii) overall vesicle charge, and (iii) phosphatidylethanolamine (PE) to phosphatidylcholine (PC) ratio on the activity of ionenes were performed. Additionally, polymer-bilayer interactions were also investigated through vesicle fusion assay and the black lipid membrane (BLM) technique The activity of C0-T-p is strongly influenced by the amount of cardiolipin, while the activity of C8-T-p primarily depends on the overall vesicle charge. Consequently, C0-T-p acts through interactions with CL, whereas C8-T-p modifies the bulk properties of the membrane in a less-specific manner. Moreover, the presence of a small amount of PC in the membrane makes the vesicle resistant to permeabilization by tested molecules. Intriguingly, more hydrophilic C0-T-p retains higher membrane activity compared to the hydrophobic C8-T-p. However, both ionenes induce vesicle fusion and increase lipid bilayer ion permeability.

Potassium Channels, Glucose Metabolism and Glycosylation in Cancer Cells.

Potassium channels emerge as one of the crucial groups of proteins that shape the biology of cancer cells. Their involvement in processes like cell growth, migration, or electric signaling, seems obvious. However, the relationship between the function of K+ channels, glucose metabolism, and cancer glycome appears much more intriguing. Among the typical hallmarks of cancer, one can mention the switch to aerobic glycolysis as the most favorable mechanism for glucose metabolism and glycome alterations. This review outlines the interconnections between the expression and activity of potassium channels, carbohydrate metabolism, and altered glycosylation in cancer cells, which have not been broadly discussed in the literature hitherto. Moreover, we propose the potential mediators for the described relations (e.g., enzymes, microRNAs) and the novel promising directions (e.g., glycans-orinented drugs) for further research.

Effect of Quercetin on mitoBKCa Channel and Mitochondrial Function in Human Bronchial Epithelial Cells Exposed to Particulate Matter.

Particulate matter (PM) exposure increases reactive oxygen species (ROS) levels. It can lead to inflammatory responses and damage of the mitochondria thus inducing cell death. Recently, it has been shown that potassium channels (mitoK) located in the inner mitochondrial membrane are involved in cytoprotection, and one of the mechanisms involves ROS. To verify the cytoprotective role of mitoBKCa, we performed a series of experiments using a patch-clamp, transepithelial electrical resistance assessment (TEER), mitochondrial respiration measurements, fluorescence methods for the ROS level and mitochondrial membrane potential assessment, and cell viability measurements. In the human bronchial epithelial cell model (16HBE14σ), PM < 4 µm in diameter (SRM-PM4.0) was used. We observed that PM decreased TEER of HBE cell monolayers. The effect was partially abolished by quercetin, a mitoBKCa opener. Consequently, quercetin decreased the mitochondrial membrane potential and increased mitochondrial respiration. The reduction of PM-induced ROS level occurs both on cellular and mitochondrial level. Additionally, quercetin restores HBE cell viability after PM administration. The incubation of cells with PM substantially reduced the mitochondrial function. Isorhamnetin had no effect on TEER, the mitoBKCa activity, respiratory rate, or mitochondrial membrane potential. Obtained results indicate that PM has an adverse effect on HBE cells at the cellular and mitochondrial level. Quercetin is able to limit the deleterious effect of PM on barrier function of airway epithelial cells. We show that the effect in HBE cells involves mitoBKCa channel-activation. However, quercetin's mechanism of action is not exclusively determined by modulation of the channel activity.

The Distribution and Role of the CFTR Protein in the Intracellular Compartments.

Cystic fibrosis is a hereditary disease that mainly affects secretory organs in humans. It is caused by mutations in the gene encoding CFTR with the most common phenylalanine deletion at position 508. CFTR is an anion channel mainly conducting Cl- across the apical membranes of many different epithelial cells, the impairment of which causes dysregulation of epithelial fluid secretion and thickening of the mucus. This, in turn, leads to the dysfunction of organs such as the lungs, pancreas, kidney and liver. The CFTR protein is mainly localized in the plasma membrane; however, there is a growing body of evidence that it is also present in the intracellular organelles such as the endosomes, lysosomes, phagosomes and mitochondria. Dysfunction of the CFTR protein affects not only the ion transport across the epithelial tissues, but also has an impact on the proper functioning of the intracellular compartments. The review aims to provide a summary of the present state of knowledge regarding CFTR localization and function in intracellular compartments, the physiological role of this localization and the consequences of protein dysfunction at cellular, epithelial and organ levels. An in-depth understanding of intracellular processes involved in CFTR impairment may reveal novel opportunities in pharmacological agents of cystic fibrosis.

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent "On-Off" switching DNA aptamer.

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 µM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies. Graphical abstract.

SERCA, complex I of the respiratory chain and ATP-synthase inhibition are involved in pleiotropic effects of NS1619 on endothelial cells.

A large conductance potassium (BKCa) channel opener, NS1619 (1,3-dihydro-1- [2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one), is well known for its protective effects against ischemia-reperfusion injury; however, the exact mode of its action remains unclear. The aim of this study was to characterize the effect of NS1619 on endothelial cells. The endothelial cell line EA.hy926, guinea pig hearts and submitochondrial particles isolated from the heart were used. In the isolated guinea pig hearts, which were perfused using the Langendorff technique, NS1619 caused a dose-dependent increase in coronary flow that was inhibited by L-NAME. In EA.hy926 cells, NS1619 also caused a dose-dependent increase in the intracellular calcium ion concentration [Ca(2+)]i, as measured using the FURA-2 fluorescent probe. Moreover, NS1619 decreased the oxygen consumption rate in EA.hy926 cells, as assessed using a Clark-type oxygen electrode. However, when NS1619 was applied in the presence of oligomycin, the oxygen consumption increased. NS1619 also decreased the mitochondrial membrane potential, as measured using a JC-1 fluorescent probe in the presence and absence of oligomycin. Additionally, the application of NS1619 to submitochondrial particles inhibited ATP synthase. In summary, NS1619 has pleiotropic actions on EA.hy926 cells and acts not only as an opener of the BKCa channel in EA.hy926 cells but also as an inhibitor of the respiratory chain component, sarcoplasmic reticulum ATPase, which leads to the release of Ca(2+) from the endoplasmic reticulum. Furthermore, NS1619 has the oligomycin-like property of inhibiting mitochondrial ATP synthase.

[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.

Mitochondrial mechanisms of endothelial dysfunction.

Endothelial cells play an important physiological role in vascular homeostasis. They are also the first barrier that separates blood from deeper layers of blood vessels and extravascular tissues. Thus, they are exposed to various physiological blood components as well as challenged by pathological stimuli, which may exert harmful effects on the vascular system by stimulation of excessive generation of reactive oxygen species (ROS). The major sources of ROS are NADPH oxidase and mitochondrial respiratory chain complexes. Modulation of mitochondrial energy metabolism in endothelial cells is thought to be a promising target for therapy in various cardiovascular diseases. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial ROS generation and can antagonise oxidative stress-induced endothelial dysfunction. Several studies have revealed the important role of UCP2 in hyperglycaemia-induced modifications of mitochondrial function in endothelial cells. Additionally, potassium fluxes through the inner mitochondrial membrane, which are involved in ROS synthesis, affect the mitochondrial volume and change both the mitochondrial membrane potential and the transport of calcium into the mitochondria. In this review, we concentrate on the mitochondrial role in the cytoprotection phenomena of endothelial cells.

The potassium channel opener CGS7184 activates Ca²âº release from the endoplasmic reticulum.

CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate) is a synthetic large-conductance Ca(2+)-activated potassium (BK(Ca)) channel opener. The existing literature suggests that potassium channels are involved in cardioprotection, particularly during ischemia-reperfusion events. However, the cellular mechanisms mediating the effects of CGS7184 remain unclear. In the present study, we investigated the effect of the BK(Ca) channel opener CGS7184 on Ca(2+) homeostasis in H9C2 and C2C12 cell lines, Ca(2+) uptake by isolated sarcoplasmic reticulum (SR) vesicles, SR Ca(2+)-ATPase (SERCA) activity, and single-channel properties of the ryanodine receptor calcium release channel (RYR2) when incorporated into a planar lipid bilayer. The effects of CGS7184 on calcium homeostasis in C2C12 and H9C2 cell lines were measured with a Fura-2 fluorescent indicator. The BK(Ca) channel opener CGS7184, when added to the H9C2 and C2C12 cells, caused a concentration-dependent release of calcium from internal stores. Calcium accumulation by the SR vesicles isolated from cardiac and skeletal muscle was inhibited by CGS7184 with a half-maximal inhibition value of 0.45 ± 0.04 µM and 0.37 ± 0.03 µM, respectively. The results of the present study indicate that the BK(Ca) channel opener CGS7184 modulates cytosolic Ca(2+) concentration in H9C2 and C1C12 cells due to its interaction with the endoplasmic reticulum (ER). CGS7184 approximately doubled the opening probability of RYR2 channels; however, the compound seemed to most strongly affect channels with a higher control activity. These results strongly suggest that the BK(Ca) channel opener CGS7184 affects intracellular calcium homeostasis by interacting with the sarcoplasmic reticulum RYR2 channels.

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.

Large-conductance K+ channel opener CGS7184 as a regulator of endothelial cell function.

Large-conductance Ca(2+)-activated potassium (BK(Ca)) channels are present in endothelium, but their regulatory role remains uncharacterized. The aim of the present study was to investigate the pharmacological effects of the BK(Ca) channel opener ethyl-1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate (CGS7184) on endothelium in the aorta and coronary circulation, particularly with regard to nitric oxide (NO)-dependent regulation of vascular tone, as well as effects of CGS7184 on NO production, calcium homeostasis, and mitochondrial function in cultured endothelial cells. The vasorelaxant action of CGS7184 was studied in coronary circulation and in the aorta using isolated perfused guinea pig heart and rat aortic rings, respectively. The effects of CGS7184 on calcium homeostasis, mitochondrial membrane potential, NO production, and mitochondrial respiration were tested in cultures of EA.hy 926 endothelial cells. The BK(Ca) channel opener CGS7184 caused a concentration-dependent (0.03-3 microM) relaxation of the rat aorta and coronary vasodilatation in the isolated guinea pig heart. Both responses were profoundly inhibited by the nitric oxide (NO) synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) (100 microM). CGS7184 (5 microM) also increased basal NO production in EA.hy 926 cells by approximately two-fold. Moreover, CGS7184 induced a concentration-dependent (0.1-10 microM) elevation in intracellular calcium concentration. Interestingly, CGS7184 affected mitochondrial function by causing mitochondrial potential depolarization and an increase in oxygen consumption in EA.hy 926 endothelial cells. The BK(Ca) channel opener CGS7184 activates NOS pathways and modulates mitochondrial function in the endothelium. Both effects may be triggered by the CGS7184-induced modulation of intracellular Ca(2+) homeostasis in EA.hy 926 endothelial cells.