Quercetin protects cardiomyoblasts against hypertonic cytotoxicity by abolishing intracellular Ca2+ elevations and mitochondrial depolarisation.

BACKGROUND AND AIM: Osmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. EXPERIMENTAL PROCEDURE: Protective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. KEY RESULTS: Quercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. CONCLUSIONS AND IMPLICATIONS: Quercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.

Purification and Application of Genetically Encoded Potassium Ion Indicators for Quantification of Potassium Ion Concentrations within Biological Samples.

Vital cells maintain a steep potassium ion (K+ ) gradient across the plasma membrane. Intracellular potassium ion concentrations ([K+ ]) and especially the [K+ ] within the extracellular matrix are strictly regulated, the latter within a narrow range of ∼3.5 to 5.0 mM. Alterations of the extracellular K+ homeostasis are associated with severe pathological alterations and systemic diseases including hypo- or hypertension, heart rate alterations, heart failure, neuronal damage or abnormal skeleton muscle function. In higher eukaryotic organisms, the maintenance of the extracellular [K+ ] is mainly achieved by the kidney, responsible for K+ excretion and reabsorption. Thus, renal dysfunctions are typically associated with alterations in serum- or plasma [K+ ]. Generally, [K+ ] quantifications within bodily fluids are performed using ion selective electrodes. However, tracking such alterations in experimental models such as mice features several difficulties, mainly due to the small blood volume of these animals, hampering the repetitive collection of sample volumes required for measurements using ion selective electrodes. We have recently developed highly sensitive, genetically encoded potassium ion indicators, the GEPIIs, applicable for in vitro determinations of [K+ ]. In addition to the determination of [K+ ] within bodily fluids, GEPIIs proved suitable for the real-time visualization of cell viability over time and the exact determination of the number of dead cells. © 2019 The Authors.

C16 ceramide is crucial for triacylglycerol-induced apoptosis in macrophages.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca(2+) from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl-/- and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl-/- macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl-/- macrophages, FB1 treatment rescued Atgl-/- macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl-/- macrophages by activation of the mitochondrial apoptosis pathway.

Evidence for a receptor-activated Ca2+ entry pathway independent from Ca2) store depletion in endothelial cells.

Ca(2+) entry in endothelial cells is a key signaling event as it prolongs the Ca(2+) signal activated by a receptor agonist, and thus allows an adequate production of a variety of compounds. The possible routes that lead to Ca(2+) entry in non-excitable cells include the receptor-activated Ca(2+) entry (RACE), which requires the presence of an agonist to be activated, and the store-operated Ca(2+) entry (SOCE) pathway, whose activation requires the depletion of the ER Ca(2+) store. However, the relative importance of these two influx pathways during physiological stimulation is not known. In the present study we experimentally differentiated these two types of influxes and determined under which circumstances they are activated. We show that La(3+) (at 10 microM) is a discriminating compound that efficiently blocks SOCE but is almost without effect on histamine-induced Ca(2+) entry (RACE). In line with this, histamine does not induce massive store depletion when performed in the presence of extracellular Ca(2+). In addition, inhibition of mitochondrial respiration significantly reduces SOCE but modestly affects RACE. Thus, agonist-induced Ca(2+) entry is insensitive to La(3+), and only modestly affected by mitochondrial depolarization. These data shows that agonist relies almost exclusively on RACE for sustained Ca(2+) signaling in endothelial cells.

Ca2+ refilling of the endoplasmic reticulum is largely preserved albeit reduced Ca2+ entry in endothelial cells.

In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP(3). A correlation of the kinetics of ER Ca2+refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+entry.

Expression of the Kdp ATPase is consistent with regulation by turgor pressure.

The kdpFABC operon of Escherichia coli encodes the four protein subunits of the Kdp K+ transport system. Kdp is expressed when growth is limited by the availability of K+. Expression of Kdp is dependent on the products of the adjacent kdpDE operon, which encodes a pair of two-component regulators. Studies with kdp-lac fusions led to the suggestion that change in turgor pressure acts as the signal to express Kdp (L. A. Laimins, D. B. Rhoads, and W. Epstein, Proc. Natl. Acad. Sci. USA 78:464-468, 1981). More recently, effects of compatible solutes, among others, have been interpreted as inconsistent with the turgor model (H. Asha and J. Gowrishankar, J. Bacteriol. 175:4528-4537, 1993). We re-examined the effects of compatible solutes and of medium pH on expression of Kdp in studies in which growth rate was also measured. In all cases, Kdp expression correlated with the K+ concentration when growth began to slow. Making the reasonable but currently untestable assumptions that the reduction in growth rate by K+ limitation is due to a reduction in turgor and that addition of betaine does not increase turgor, we concluded that all of the data on Kdp expression are consistent with control by turgor pressure.

Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with ricin toxoid vaccine encapsulated in polymeric microspheres.

Intranasal (i.n.) immunization with ricin toxoid (RT) vaccine encapsulated in poly (lactide-co-glycolide) microspheres (RT-PLG-Ms) and poly (L-lactide) microspheres (RT-PLA-Ms) stimulated systemic and mucosal immune responses and protected mice from aerosolized ricin intoxication. High titers of anti-ricin IgG2a were stimulated in the serum of mice with one or two doses of RT-Ms 6 weeks postimmunization. However, in the lungs, no IgG2a or total IgG was elicited either with RT-Ms or with aqueous RT. At 6 weeks postimmunization, a single dose of the RT-Ms stimulated secretory IgA (sIgA) in the lungs of four of six mice, but a second immunizing dose did not enhance the stimulation. A single dose of aqueous RT vaccine failed to stimulate sIgA in the lungs, while, a second dose induced sIgA in 50% of the mice. One or two i.n. doses of RT-Ms protected most of the mice against lethal aerosol-delivered ricin toxin 6 weeks postimmunization. In contrast, protection was absent or marginal after one or two doses of aqueous RT vaccine. In both studies, the protection against lethal aerosol challenge was significantly better with one dose of RT-Ms than with two doses of aqueous vaccine, which may be attributed to the induction of sIgA in the lungs and the serum. Duration of the IgG2a and IgA in the serum, particularly that of IgG2a was much longer after the administration of RT-Ms than after the aqueous vaccine. The geometric mean IgG2a titers stimulated with two doses of RT-Ms remained high during 40 weeks postimmunization and were up to 25 times higher than the titers induced with aqueous RT vaccine. After 6 weeks, the IgG2a induced by two doses of aqueous vaccine was no longer detectable. Persistence of antibody response was predictive of efficacy. At 1 year postimmunization with two doses of RT-Ms, 100% of mice were protected against lethal ricin challenge. However, at the same time no protection was afforded by two doses of aqueous RT. The results of the present study consistently demonstrated the advantages of microencapsulated RT vaccine to stimulate effective and long-lasting protection by i.n. administration.

Characterization of the non-specific humoral and cellular antiviral immunity stimulated by the chloroform-methanol residue (CMR) fraction of Coxiella burnetii.

Modulation of the immune response by the chloroform-methanol residue (CMR) of phase I Coxiella burnetii whole cell was studied in Rift Valley fever virus-infected, or in naive endotoxin-non-responder C3H/HeJ mice. A single dose of CMR completely protected the mice from viral infection. Treating virus-infected mice with antibodies directed against interferons alpha/beta (IFN-alpha beta) and gamma (IFN-gamma) eliminated the CMR-induced protection. CMR stimulated the production of high levels of IFN-alpha/beta and 2'-5'-oligoadenylate synthetase activities in sera of the CMR-treated mice. IFN-gamma was present in supernatants of cultured spleen cells of CMR-treated, virus-infected mice, but not in their serum. Priming mice with CMR optimized the release of INF-gamma, interleukin-1 alpha (IL-1 alpha) and IL-6 from splenocytes in vitro. When stimulated in vitro, IL-2 and granulocyte-macrophage stimulating factor (GM-CSF) did not require in vivo priming for release from cultured spleen cells. Fluorescence-assisted cytometry of CMR-treated mouse spleen cells showed there was a CMR-dependent increase in the percentage of T-cells and Ia-positive T-cells. There also was a biphasic increase in the ratio between Th (L3T4) and Ts (Lyt2) cells. Biological activities stimulated by CMR indicate that CMR is a potent immunostimulant, which may modulate specific and non-specific antiviral responses.

Dependence of ricin toxoid vaccine efficacy on the structure of poly(lactide-co-glycolide) microparticle carriers.

Biodegradable microparticles made of poly(lactide-co-glycolide) (PLG) were used for protracted and pulsed-release of the incorporated ricin toxoid (RT) vaccine to reduce the multiple immunization doses and the time required to induce complete protection against lethal aerosol-borne ricin challenge. The release rate of RT encapsulated in PLG microparticles was controlled by polymer selection and varying the preparation procedures, which allowed us to control microparticle size and the distribution of the vaccine in the polymeric matrix. PLG-microparticles in which RT vaccine was distributed heterogeneously in small pockets stimulated a rapid antibody response which was independent of the polymeric composition of the carriers. PLG-microparticles in which RT vaccine was distributed homogeneously throughout the polymeric matrix induced a slower antibody response, which depended on the polymeric composition of the carriers. Administration of RT in homogeneous microparticles made from 50/50 PLG or 100% polylactide stimulated two distinct anti-ricin IgG peaks, while RT in heterogeneous microparticles stimulated identical IgG peaks. An early (3 weeks) and long-lasting (1 year or longer) anti-ricin antibody response was evoked by a single administration of encapsulated RT vaccine when prepared by the above-mentioned conditions. In contrast, three administrations of the aqueous RT were required to stimulate similar antibody response. Reduction of immunization time from 6 to 4 weeks was achieved with RT encapsulated in small homogeneous microparticles but not with homogeneous large microparticles. These results demonstrated the usefulness of biodegradable microparticles to improve the efficacy of immunization with RT vaccine and probably many other vaccines as well.

The effect of sucralfate on the steady-state serum concentrations of phenytoin.

The effect of sucralfate on steady-state phenytoin concentrations was evaluated in 6 normal volunteers. The subjects took phenytoin at bedtime until steady-state was confirmed (8 to 9 days) and then took in addition sucralfate 1g four times daily for a further 7 days. Serum phenytoin concentrations were measured on days 1, 3, and 7 of sucralfate administration. Measured phenytoin concentrations were 10.9 +/- 3.0 mcg/ml and 11.0 +/- 3.1 mcg/ml before sucralfate and 10.8 +/- 3.1 mcg/ml, 11.4 +/- 3.2 mcg/ml, and 11.1 +/- 2.5 mcg/ml during sucralfate administration (p greater than 0.05; ANOVA). These results suggest there is not a significant drug interaction between sucralfate and phenytoin.