Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes.

Lysosomes are degradative organelles that facilitate the removal and recycling of potentially cytotoxic materials and mediate a variety of other cellular processes, such as nutrient sensing, intracellular signaling, and lipid metabolism. Due to these central roles, lysosome dysfunction can lead to deleterious outcomes, including the accumulation of cytotoxic material, inflammation, and cell death. We previously reported that cationic amphiphilic drugs, such as imipramine, alter pH and lipid metabolism within macrophage lysosomes. Therefore, the ability for imipramine to induce changes to the lipid content of isolated macrophage lysosomes was investigated, focusing on sphingomyelin, cholesterol, and glycerophospholipid metabolism as these lipid classes have important roles in inflammation and disease. The lysosomes were isolated from control and imipramine-treated macrophages using density gradient ultracentrifugation, and mass spectrometry was used to measure the changes in their lipid composition. An unsupervised hierarchical cluster analysis revealed a clear differentiation between the imipramine-treated and control lysosomes. There was a significant overall increase in the abundance of specific lipids mostly composed of cholesterol esters, sphingomyelins, and phosphatidylcholines, while lysophosphatidylcholines and ceramides were overall decreased. These results support the conclusion that imipramine's ability to change the lysosomal pH inhibits multiple pH-sensitive enzymes in macrophage lysosomes.

Cholesterol content regulates silica-induced lysosomal membrane permeability.

Inhalation of crystalline silica has been well documented to cause pulmonary inflammation and lung disease such as silicosis. Respirable silica particles deposit in the lungs and are phagocytosed by alveolar macrophages. Subsequently, phagocytosed silica remains undegraded within lysosomes causing lysosomal damage known as phagolysosomal membrane permeability (LMP). LMP can trigger the assembly of the NLRP3 inflammasome resulting in release of inflammatory cytokines that contribute to disease. In order to better understand the mechanisms of LMP this study used murine bone marrow derived macrophages (BMdM) as a cellular model to investigate the mechanism of silica-induced LMP. Reduction of lysosomal cholesterol in bone marrow derived macrophages with 18:1 phosphatidylglycerol (DOPG) liposome treatment increased silica-induced LMP and IL-1ß release. Conversely, increasing lysosomal and cellular cholesterol with U18666A reduced IL-1ß release. Co-treatment of bone marrow derived macrophages with 18:1 phosphatidylglycerol and U18666A resulted in a significant reduction of the effects of U18666A on lysosomal cholesterol. Phosphatidylcholine 100-nm liposome model systems were used to examine the effects of silica particles on lipid membrane order. Time-resolved fluorescence anisotropy of the membrane probe, Di-4-ANEPPDHQ, was used to determine changes to membrane order. Silica increased lipid order that was attenuated by inclusion of cholesterol in the phosphatidylcholine liposomes. These results demonstrate that increased cholesterol can attenuate silica-induced membrane changes in liposomes and cell models, while decreasing cholesterol exacerbates silica-induced membrane changes. Selective manipulation of lysosomal cholesterol may be a way of attenuating lysosomal disruption and preventing silica-induced chronic inflammatory disease progression.

The Application of Fluorescence Anisotropy for Viscosity Measurements of Small Volume Biological Analytes.

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 µL and 100 µL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze-thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements' accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems.

Exposure to copper increases hypoxia sensitivity and decreases upper thermal tolerance of giant salmonfly nymphs (Pteronarcys californica).

Many aquatic insects are exposed to the dual stressors of heavy metal pollution and rising water temperatures from global warming. These stresses may interact and have stronger impacts on aquatic organisms if heavy metals interfere with the ability of these organisms to handle high temperatures. Here we focus on the effect of copper on upper thermal limits of giant salmonfly nymphs (Order: Plecoptera, Pteronarcys californica), a stonefly species which is common in parts of western North America. Experimental exposure to copper reduced upper thermal limits by âˆ¼ 10 °C in some cases and depressed the hypoxia tolerance (Pcrit) of nymphs by âˆ¼ 0.5 mg L-1 DO. These results suggest that copper inhibits the delivery of oxygen, which may explain, in part, the strong reductions in CTMAX that we report. Fluorescence microscopy of Cu-exposed individuals indicated high levels of copper in chloride cells but no clear evidence of damage to or high levels of copper on the gills themselves. Our study indicates that populations of aquatic insects from copper-polluted environments may be further at risk to future warming than those from uncontaminated environments.

Molecular recognition between membrane epitopes and nearly free surface silanols explains silica membranolytic activity.

Inhaled crystalline silica causes inflammatory lung diseases, but the mechanism for its unique activity compared to other oxides remains unclear, preventing the development of potential therapeutics. Here, the molecular recognition mechanism between membrane epitopes and "nearly free silanols" (NFS), a specific subgroup of surface silanols, is identified and proposed as a novel broad explanation for particle toxicity in general. Silica samples having different bulk and surface properties, specifically different amounts of NFS, are tested with a set of membrane systems of decreasing molecular complexity and different charge. The results demonstrate that NFS content is the primary determinant of membrane disruption causing red blood cell lysis and changes in lipid order in zwitterionic, but not in negatively charged liposomes. NFS-rich silica strongly and irreversibly adsorbs zwitterionic self-assembled phospholipid structures. This selective interaction is corroborated by density functional theory and supports the hypothesis that NFS recognize membrane epitopes that exhibit a positive quaternary amino and negative phosphate group. These new findings define a new paradigm for deciphering particle-biomembrane interactions that will support safer design of materials and what types of treatments might interrupt particle-biomembrane interactions.

Fluorescence lifetime imaging microscopy and time-resolved anisotropy of nanomaterial-induced changes to red blood cell membranes.

With the use of engineered nano-materials (ENM) becoming more prevalent, it is essential to determine potential human health impacts. Specifically, the effects on biological lipid membranes will be important for determining molecular events that may contribute to both toxicity and suitable biomedical applications. To better understand the mechanisms of ENM-induced hemolysis and membrane permeability, fluorescence lifetime imaging microscopy (FLIM) was performed on human red blood cells (RBC) exposed to titanium dioxide ENM, zinc oxide ENM, or micron-sized crystalline silica. In the FLIM images, changes in the intensity-weighted fluorescence lifetime of the lipophilic fluorescence probe Di-4-ANEPPDHQ were used to identify localized changes to membrane. Time-resolved fluorescence anisotropy and FLIM of RBC treated with methyl-ß-cyclodextrin was performed to aid in interpreting how changes to membrane order influence changes in the fluorescence lifetime of the probe. Treatment of RBC with methyl-ß-cyclodextrin caused an increase in the wobble-in-a-cone angle and shorter fluorescence lifetimes of di-4-ANEPPDHQ. Treatment of RBC with titanium dioxide caused a significant increase in fluorescence lifetime compared to non-treated samples, indicating increased membrane order. Crystalline silica also increased the fluorescence lifetime compared to control levels. In contrast, zinc oxide decreased the fluorescence lifetime, representing decreased membrane order. However, treatment with soluble zinc sulfate resulted in no significant change in fluorescence lifetime, indicating that the decrease in order of the RBC membranes caused by zinc oxide ENM was not due to zinc ions formed during potential dissolution of the nanoparticles. These results give insight into mechanisms for how these three materials might disrupt RBC membranes and membranes of other cells. The results also provide evidence for a direct correlation between the size, interaction-available surface area of the nano-material and cell membrane disruption.

Effects of titanium dioxide and zinc oxide nano-materials on lipid order in model membranes.

Engineered nano-materials (ENM) have been reported to affect lipid membrane permeability in cell models, but a mechanistic understanding of how these materials interact with biological membranes has not been described. To assess mechanisms of permeability, liposomes composed of DOPC, DOPS, or POPC, with or without cholesterol, were used as model membranes for measuring ENM-induced changes to lipid order to improve our understanding of ENM effects on membrane permeability. Liposomes were treated with either titanium dioxide (TiO2) or zinc oxide (ZnO) ENM, and changes to lipid order were measured by time-resolved fluorescence anisotropy of a lipophilic probe, Di-4-ANEPPDHQ. Both ENM increased lipid order in two lipid models differing in headgroup charge. TiO2 increased lipid order of POPC liposomes (neutral charge), while ZnO acted primarily on DOPS liposomes (negative charge). Addition of cholesterol to these models significantly increased lipid order while in some cases attenuated ENM-induced changes to lipid order. To assess the ability of ENM to induce membrane permeability, liposomes composed of the above lipids were assayed for membrane permeability by calcein leakage in response to ENM. Both ENM caused a dose-dependent increase in permeability in all liposome models tested, and the addition of cholesterol to the liposome models neither blocked nor reduced calcein leakage. Together, these experiments show that ENM increased permeability of small molecules (calcein) from model liposomes, and that the magnitude of the effect of ENM on lipid order depended on ENM surface charge, lipid head group charge and the presence of cholesterol in the membrane.

Using Time-Resolved Fluorescence Anisotropy of di-4-ANEPPDHQ and F2N12S to Analyze Lipid Packing Dynamics in Model Systems.

The fluorescence probes di-4-ANEPPDHQ and F2N12S have solvochromatic emission spectra and fluorescence lifetimes that are sensitive to order within the environment of lipid membranes. We show in this communication that the time-resolved fluorescence anisotropy of these probes, analyzed either by the wobble-in-a-cone model or by the model-independent order parameter S2, provides complementary information about dynamics and lipid packing in a variety of homogeneous lipid membranes systems.