Palmitic Acid Modulates Microglial Cell Response to Metabolic Endotoxemia in an In Vitro Study.

Metabolic endotoxemia (ME) is characterized by a 2-3-fold increase in blood endotoxin levels and low-grade systemic inflammation without apparent infection. ME is usually accompanied by metabolic syndrome, characterized by central obesity and hyperlipidemia. According to numerous studies, ME may lead to functional brain disorders, including cognitive decline, depression, and dementia. In the current in vitro study, we aimed to determine the direct and indirect impact of endotoxin (LPS) and palmitic acid (PA), representing saturated fatty acids, on the inflammatory and oxidative stress response in the human microglial HMC3 cells unstimulated and stimulated with IFNγ. The study's results revealed that direct HMC3 cell exposition to endotoxin and PA increased inflammatory response measured as levels of IL-6 and MCP-1 released into the medium and PGE2 levels in cell lysates. Moreover, direct HMC3 cell treatment with PA and LPS induced oxidative stress, i.e., ROS and COX-2 production and lipid peroxidation. On the contrary, an indirect effect of LPS and PA on microglial cells, assessed as the impact of macrophage metabolites, was much lower regarding the inflammatory response, although still associated with oxidative stress. Interestingly, IFNγ had a protective effect on microglial cells, reducing the production of pro-inflammatory mediators and oxidative stress in HMC3 cells treated directly and indirectly with LPS and PA.

Co-Application of Statin and Flavonoids as an Effective Strategy to Reduce the Activity of Voltage-Gated Potassium Channels Kv1.3 and Induce Apoptosis in Human Leukemic T Cell Line Jurkat.

Voltage-gated potassium channels of the Kv1.3 type are considered a potential new molecular target in several pathologies, including some cancer disorders and COVID-19. Lipophilic non-toxic organic inhibitors of Kv1.3 channels, such as statins and flavonoids, may have clinical applications in supporting the therapy of some cancer diseases, such as breast, pancreas, and lung cancer; melanoma; or chronic lymphocytic leukemia. This study focuses on the influence of the co-application of statins-simvastatin (SIM) or mevastatin (MEV)-with flavonoids 8-prenylnaringenin (8-PN), 6-prenylnarigenin (6-PN), xanthohumol (XANT), acacetin (ACAC), or chrysin on the activity of Kv1.3 channels, viability, and the apoptosis of cancer cells in the human T cell line Jurkat. We showed that the inhibitory effect of co-application of the statins with flavonoids was significantly more potent than the effects exerted by each compound applied alone. Combinations of simvastatin with chrysin, as well as mevastatin with 8-prenylnaringenin, seem to be the most promising. We also found that these results correlate with an increased ability of the statin-flavonoid combination to reduce viability and induce apoptosis in cancer cells compared to single compounds. Our findings suggest that the co-application of statins and flavonoids at low concentrations may increase the effectiveness and safety of cancer therapy. Thus, the simultaneous application of statins and flavonoids may be a new and promising anticancer strategy.

Statins as inhibitors of voltage-gated potassium channels Kv1.3 in cancer cells.

Voltage-gated potassium channels are integral membrane proteins selectively permeable for potassium ions and activated upon change of membrane potential. Voltage-gated potassium channels of the Kv1.3 type were discovered both in plasma membrane and in inner mitochondrial membrane (mito Kv1.3 channels). For some time Kv1.3 channels located both in plasma membrane and in mitochondria are considered as a potentially new molecular target in several pathologies including some cancer disorders. Lipophilic nontoxic organic inhibitors of Kv1.3 channels may potentially find a clinical application to support therapy of some cancer diseases such as breast, pancreas and lung cancer, melanoma or chronic lymphocytic leukaemia (B-CLL). Inhibition of T lymphocyte Kv1.3 channels may be also important in treatment of chronic and acute respiratory diseases including severe pulmonary complication in corona virus disease Covid 19, however further studies are needed to confirm this supposition. Statins are small-molecule organic compounds, which are lipophilic and are widely used in treatment of hypercholesterolemia and atherosclerosis. Electrophysiological studies performed in our laboratory showed that statins: pravastatin, mevastatin and simvastatin are effective inhibitors of Kv1.3 channels in cancer cells of human T cell line Jurkat. We showed that application of the statins in the concentration range from 1.5 µM to 50 µM inhibited the channels in a concentration-dependent manner. The inhibitory effect was the most potent in case of simvastatin and the least potent in case of pravastatin. The inhibition was partially irreversible in case of simvastatin and fully reversible in case of pravastatin and mevastatin. It was accompanied by a significant acceleration of the current inactivation rate without any significant change of the activation rate. Mechanism of the inhibition is probably complex, including a direct interaction with the channel protein and perturbation of lipid bilayer structure, leading to stabilization of the inactivated state of the channels.

Voltage-Gated Potassium Channel Kv1.3 as a Target in Therapy of Cancer.

Voltage-gated potassium channel Kv1.3 is an integral membrane protein, which is selectively permeable for potassium ions and is activated upon a change of membrane potential. Channel activation enables transportation of potassium ions down their electrochemical gradient. Kv1.3 channel is expressed in many cell types, both normal and cancer. Activity of the channel plays an important role in cell proliferation and apoptosis. Inhibition of Kv1.3 channel may be beneficial in therapy of several diseases including some cancer disorders. This review focuses on Kv1.3 channel as a new potentially attractive molecular target in cancer therapy. In the first part, changes in the channel expression in selected cancer disorders are described. Then, the role of the channel activity in cancer cell proliferation and apoptosis is presented. Finally, it is shown that some low molecular weight organic inhibitors of the channel including selected biologically active plant-derived polycyclic compounds may selectively induce apoptosis of Kv1.3-expressing cancer cells while sparing normal cells and healthy organs. These compounds may be promising candidates for putative application in therapy of some cancer disorders, such as melanoma, pancreatic ductal adenocarcinoma (PDAC), or B-type chronic lymphocytic leukemia (B-CLL).

The Influence of 6-Prenylnaringenin and Selected Non-prenylated Flavonoids on the Activity of Kv1.3 Channels in Human Jurkat T Cells.

The influence of a prenylated flavonoid-6-prenylnaringenin (6-PR) and selected non-prenylated flavonoids: acacetin, chrysin, baicalein, wogonin, and luteolin on the activity of voltage-gated potassium channels Kv1.3 was investigated in human leukemic Jurkat T cells. Electrophysiological measurements were accompanied by studies on the cytotoxic effect of the examined compounds on Jurkat T cells. Electrophysiological studies were performed using the whole-cell patch-clamp technique. Cell viability was determined using the MTT assay. 6-PR inhibited Kv1.3 channels in Jurkat T cells in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 5.76 µM. Among non-prenylated flavonoids, acacetin and chrysin inhibited Kv1.3 channels in Jurkat T cells when applied at the concentration of 30 µM, whereas baicalein, wogonin, and luteolin were ineffective at this concentration. The inhibitory effects of acacetin and chrysin on Kv1.3 channels were significantly less potent than the inhibition caused by 6-PR. All tested compounds inhibited growth of Jurkat T cells in a concentration-dependent manner. Wogonin and chrysin were the most cytotoxic flavonoids tested, whereas baicalein and 6-PR were the least cytotoxic compounds. In accordance to our hypothesis the prenylated flavonoid (6-PR) was much more effective inhibitor of Kv1.3 channels than non-prenylated compounds selected for this study. The inhibition of Kv1.3 channels by 6-PR, acacetin, and chrysin was not related to cytotoxicity of these compounds. The channels' inhibition might be involved in anti-proliferative and pro-apoptotic effects of 6-PR, acacetin and chrysin observed in cancer cell lines expressing these channels.

Voltage-Gated Potassium Channels Kv1.3--Potentially New Molecular Target in Cancer Diagnostics and Therapy.

Voltage-gated potassium channels, Kv1.3, which were discovered in 1984, are integral membrane proteins which are activated ("open") upon change of the cell membrane potential, enabling a passive flux of potassium ions across the cell membrane. The channels are expressed in many different tissues, both normal and cancer. Since 2005 it has been known that the channels are expressed not only in the plasma membrane, but also in the inner mitochondrial membrane. The activity of Kv1.3 channels plays an important role, among others, in setting the cell resting membrane potential, cell proliferation, apoptosis and volume regulation. For some years, these channels have been considered a potentially new molecular target in both the diagnostics and therapy of some cancer diseases. This review article focuses on: 1) changes of expression of the channels in cancer disorders with special regard to correlations between the channels' expression and stage of the disease, 2) influence of inhibitors of Kv1.3 channels on proliferation and apoptosis of cancer cells, 3) possible future applications of Kv1.3 channels' inhibitors in therapy of some cancer diseases. In the last section, the results of studies performed in our Laboratory of Bioelectricity on the influence of selected biologically active plant-derived compounds from the groups of flavonoids and stilbenes and their natural and synthetic derivatives on the activity of Kv1.3 channels in normal and cancer cells are reviewed. A possible application of some compounds from these groups to support therapy of cancer diseases, such as breast, colon and lymph node cancer, and melanoma or chronic lymphocytic leukemia (B-CLL), is announced.

Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 µM for xanthohumol and about 7.8 µM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 µM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents' activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

The influence of 8-prenylnaringenin on the activity of voltage-gated Kv1.3 potassium channels in human Jurkat T cells.

Using the whole-cell patch-clamp technique, we investigated the influence of 8-prenylnaringenin on the activity of the voltage-gated Kv1.3 potassium channels in the human leukemic T lymphocyte cell line Jurkat. 8-prenylnaringenin is a potent plant-derived phytoestrogen that has been found to inhibit cancer cell proliferation. The results show that it inhibited the Kv1.3 channels in a concentration-dependent manner. Complete inhibition occurred at concentrations higher than 10 µM. The inhibitory effect of 8-prenylnaringenin was reversible. It was accompanied by a significant acceleration of channel inactivation without any pronounced change in the activation rate. Of the naringenin derivatives tested to date, 8-prenylnaringenin is the most potent inhibitor of the Kv1.3 channels. The potency of the inhibition may be due to the presence of a prenyl group in the molecule of this flavonoid. The inhibition of the Kv1.3 channels might be involved in the antiproliferative and pro-apoptotic effects of 8-prenylnaringenin that have been observed in cancer cell lines expressing these channels.

Influence of specific immunotherapy on the activity of human T lymphocyte Kv1.3 voltage-gated potassium channels in insect venom allergic patients.

Kv1.3 channels play an important role in T lymphocytes function. CD4(+) and CD4(+)CD25(+) T cells are two broad categories of T cells that are critically involved in the immunoresponse to allergens and that are also a major target for allergen immunotherapy. The aim of the study was to evaluate the effects of venom immunotherapy (VIT) on the activity of Kv1.3. channels on noncultured subsets: CD4(+) and CD4(+)CD25(+) T cells of insect venom allergic patients. Eleven patients with allergic reactions to bee or wasp venoms participated in the study. The patients were provided VIT according to the ultrarush protocol. CD4(+) and CD4(+)CD25(+) T cells were isolated from peripheral blood mononuclear cells of VIT-treated patients by an immunomagnetic method. We used the whole-cell patch clamp technique to investigate the whole potassium chord conductance (gK) of Kv1.3. channels in CD4(+) and CD4(+)CD25(+) T cells of venom-sensitive patients before and during the course of VIT. The conductance of Kv1.3. channels on CD4(+)CD25(+) T cells decreased during the course of VIT. On day 0 it was 0.054 ± 0.07 [nS], and on day 70 it was 0.008 ± 0.09 [nS] (P = 0.03). The observed decrease of the gK of the Kv1.3 channels in the subpopulation of activated T cells may contribute to T cell tolerance and functional unresponsiveness of these cells to allergen in the early stages of VIT.

The influence of protons and zinc ions on the steady-state inactivation of Kv1.3 potassium channels.

Using the whole-cell patch-clamp technique, we investigated the influence of extracellular pH and zinc ions (Zn(2+)) on the steady-state inactivation of Kv1.3 channels expressed in human lymphocytes. The obtained data showed that lowering the extracellular pH from 7.35 to 6.8 shifted the inactivation midpoint (V(i)) by 17.4 +/- 1.12 mV (n = 6) towards positive membrane potentials. This shift was statistically significant (p < 0.05). Applying 100 microM Zn(2+) at pH 6.8 further shifted the Vi value by 16.55 +/- 1.80 mV (n = 6) towards positive membrane potentials. This shift was also statistically significant (p < 0.05). The total shift of the Vi by protons and Zn(2+) was 33.95 +/- 1.90 mV (n = 6), which was significantly higher (p < 0.05) than the shift caused by Zn(2+) alone. The Zn(2+)-induced shift of the V(i) at pH 6.8 was almost identical to the shift at pH = 7.35. Thus, the proton-and Zn(2+)-induced shifts of the V(i) value were additive. The steady-state inactivation curves as a function of membrane voltage were compared with the functions of the steady-state activation. The total shift of the steady-state inactivation was almost identical to the total shift of the steady-state activation (32.01 +/- 2.10 mV, n = 10). As a result, the "windows" of membrane potentials in which the channels can be active under physiological conditions were also markedly shifted towards positive membrane potentials. The values of membrane voltage and the normalised chord conductance corresponding to the points of intersection of the curves of steady-state activation and inactivation were also calculated. The possible physiological significance of the observed modulatory effects is discussed herein.

Inhibition of the activity of human lymphocyte Kv1.3 potassium channels by resveratrol.

The whole-cell patch-clamp technique was applied to study the modulatory effect of resveratrol on voltage-gated potassium channel Kv1.3 expressed in human lymphocytes. Results demonstrate that application of resveratrol in the concentration range 1-200 muM: inhibited the channel activity in a concentration-dependent manner to about 18% of the control value. The half-blocking concentration of resveratrol was 40.9 microM: , whereas the Hill coefficient was 1.05. The inhibition was time-dependent and slowly reversible. The inhibitory effect of resveratrol was correlated in time with a significant slowing of the current activation, whereas the inactivation rate remained unaffected upon application of resveratrol. The inhibition of Kv1.3 channels was voltage-independent. The steady-state activation of the currents remained unchanged upon resveratrol application. The magnitude of the inhibitory effect of resveratrol was not altered when resveratrol was coapplied with genistein. The possible mechanism of the inhibitory effect and its significance for biological activity of resveratrol are discussed.

The influence of zinc on the modulatory effect of sphingosylphosphorylcholine on Kv1.3 channels in human T lymphocytes.

In the present study, the whole-cell patch-clamp technique was applied to investigate the influence of co-application of zinc ions and sphingosylphosphorylcholine (SPC) on the SPC-induced shift of the activation midpoint and slowing of activation kinetics of Kv1.3 channels in human T lymphocytes. The results obtained provided evidence that the effects exerted by SPC and Zn were not additive. The shift was significantly diminished in a concentration-dependent manner upon co-application of 10 microM SPC and Zn in the concentration range 10-300 microM. However, the shift was not abolished in the presence of 100 and 300 microM of Zn co-applied with SPC. It was shown that the extent of the shift upon SPC and Zn co-application was similar to the shift observed for Zn applied without SPC. The slowing of the activation kinetics was also diminished upon SPC and Zn co-application; however, no clear dependence on concentration was observed. Moreover, the slowing was not abolished in the presence of 100 and 300 microM of Zn. It was shown that the slowing of the activation kinetics upon Zn and SPC co-application was primarily due to the effect exerted by SPC. The steepness of the voltage dependence of steady-state activation of the channels was not changed upon SPC and Zn co-application. Possible mechanisms underlying the observed phenomena and their possible physiological significance are discussed.

The voltage- and time-dependent blocking effect of trifluoperazine on T lymphocyte Kv1.3 channels.

Phenothiazines are well-known calmodulin inhibitors that interact with many receptors and channels including a variety of potassium channels. In this study, we report a blocking effect of trifluoperazine (TFP) on voltage-gated Kv1.3 channels expressed in human T lymphocytes. Application of TFP in the concentration range from 1 to 20 microM reduced the current amplitude to about a half of the control value. The currents were blocked to less than 0.05 of the control value at 50 microM TFP concentration. The blocking effect was accompanied by a substantial increase in the current inactivation rate, whereas the activation rate and the steady-state activation and inactivation were not changed significantly. The blocking effect of TFP was voltage dependent being most potent at +60mV and least potent at -20mV. The blocking effect of TFP on the currents and the recovery from block was time dependent. Other calmodulin antagonists: tamoxifen (TMX) and thioridazine also inhibited the channels at micromolar concentrations. The effects exerted by TMX and thioridazine resembled the inhibitory effect of TFP. The blocking effect of thioridazine was time dependent and appeared to be more potent that the inhibition by TFP and TMX. TFP, TMX and thioridazine inhibited the activity of Kv1.3 channels only when applied extracellularly. The inhibitory effect of all the compounds was reversible. The possible physiological significance of the current inhibition is discussed.

Inhibition of the activity of T lymphocyte Kv1.3 channels by extracellular zinc.

The inhibitory effect of zinc on voltage-gated Kv1.3 channels in human T lymphocytes was investigated using the "whole-cell" patch-clamp technique. Application of 10 and 20 microM Zn caused a concentration-dependent shift of activation midpoint of the whole-cell currents from -19.65+/-1.03 mV (mean+/-SE) under control conditions to 9.84+/-0.66 mV upon application of 20 microM Zn. This effect was saturated at zinc concentrations higher than 20 microM. The activation rate was considerably slower, whereas the deactivation rate was not significantly affected by Zn. Inactivation midpoint was shifted from -53.06+/-0.44 mV under control conditions to -36.05+/-0.48 mV in the presence of 100 microM Zn. Inactivation rate was not significantly affected upon Zn treatment. Whole-cell potassium currents were reduced to about 70% of their control values with no clear concentration dependence in the zinc concentration range from 10 to 100 microM. When raising the zinc concentration to levels above 100 microM, a concentration-dependent inhibition of the whole-cell currents appeared additionally to the changes in channel gating. The channels were half-blocked at the zinc concentration of 346+/-40 microM and the Hill slope coefficient was 1.89+/-0.21. The inhibitory effect of zinc was not complete at micromolar concentrations and was saturated at concentrations higher than 1mM. This inhibitory effect was not accompanied by any further modification in the shift of the activation and inactivation midpoints nor by a slowing of the channel activation rate. The inhibitory effect of zinc was significantly diminished in the presence of 150 mM K(+) in the extracellular solution, whereas the zinc-induced shift of the activation threshold and slowing of the activation kinetics remained unchanged when raising extracellular potassium concentration. It is suggested that zinc acts on two independent binding sites on the channels. Binding to one site that is saturated at concentrations higher than 20 microM affects the channel gating. Binding to another site at concentrations higher than 100 microM inhibits the currents without affecting the channel gating.

[Patch-clamp technique in investigations of potassium channel activation in T lymphocytes]. / Technika patch-clamp w badaniach modulacji aktywnosci kanalów potasowych w limfocytach T.

This review focuses on results of patch-clamp studies on modulation of T lymphocyte potassium channel activity by physiologically relevant factors. In the preface the patch-clamp technique is briefly presented and basic properties of potassium channels in T lymphocytes are characterised. The paper contains an overview of the data on modulatory effects of extracellular and intracellular pH, temperature, extracellular potassium, extracellular divalent and trivalent metal cations, channel phosphorylation processes and membrane lipid metabolities on potassium channel activity. Some still unresolved problems in that area are indicated.

The influence of membrane lipid metabolites on lymphocyte potassium channel activity.

In the present study, the whole-cell patch-clamp technique was applied to elucidate modulatory effects of high-density lipoproteins (HDL), sphingosine (SPH), sphingosine-1-phosphate (SPP), lysophosphatidic acid (LPA) and sphingosyl- phosphorylcholine (SPC) on the activity of Kv1.3 channels in human T lymphocytes (TL). Obtained data provide evidence that application of SPC at micromolar concentrations shifts the channel activation midpoint by about 20 mV towards positive membrane potentials. This effect occurs in a concentration-dependent manner and is saturated at SPC concentrations higher than 10 micro M. The shift of channel activation midpoint is accompanied by a pronounced slowing of the activation kinetics. The modulatory effect of SPC is clearly voltage-dependent, being most potent at -20 mV and least potent at +60 mV. The steady-state inactivation curve is also shifted by about 20 mV towards positive membrane potentials. The kinetics of channel inactivation and deactivation (closure) remain unaffected upon SPC treatment. In contrast, application of HDL (250 micro g/ml), SPH (50 and 100 micro M), SPP (10 micro M) and LPA (10 and 36 micro M) does not exert any modulatory effect on the channel activity. The effect of SPC on Kv1.3 channel gating resembles the effect exerted by extracellular zinc at the concentration of 10 micro M. It is concluded that the effect of SPC is specific and may be due to the presence of a choline residue in SPC molecules. The possible mechanism and the physiological significance of this modulatory effect on Kv1.3 channels are discussed.