Summary. K +, Rb +, or Cs + complexes of valinomycin form ion pair complexes with picric acid and trinitrobenzenesulfonate (TNBS). The formation of a picrate-K +-valinomycin complex is supported by spectral evidence. These complexes have zero net charge and readily permeate the intact erythrocyte membrane. The K+-valinomycin complex has been used to convert the nonpenetrating TNBS into a penetrating covalent probe, making it as useful vectorial probe to measure accessible amino groups of proteins and phospholipids on both sides of the erythrocyte membrane.The enhanced transport of TNBS into the cell by valinomycin is dependent on external K § in the medium. The entry of TNBS into the cell is manifested by an increased labeling of hemoglobin and membrane phosphatidylethanolamine (PE).Stilbeneisothiocyanatedisulfonate (SITS) and anilinonaphthalenesulfonate (ANS) inhibit both the basal and K +-valinomycin stimulated labeling of PE and hemoglobin by TNBS. The data suggest two independent effects of ANS and SITS, one mediated by an inhibition of the anion transport protein and another by the incorporation of these hydrobic anions into the cell membrane with an increase in negative charge on the membrane which leads to an inhibition of TNBS permeation into the cell by electrostatic repulsion.In order to determine the asymmetry of amino-phospholipids in cell membranes, two different chemical probes have been used. One probe readily penetrates the membrane and labels aminophospholipids on both sides of the membrane. The second probe does not penetrate the membrane to any significant extent and labels aminophospholipids on the exterior surface only. These two probes must have different chemical properties since one penetrates and one does not penetrate the cell. Indeed the penetrating probe is usually nonionic and hydrophobic whereas the nonpenetrating probe is anionic. This creates inherent difficulties in chemical reactivity and solubility of the probes which opens to question a direct comparison of the degree of labeling of membrane components by these two probes. If a method were developed

BA‐induced diarrhea affects ⅓ of patients with chronic intestinal inflammation, but the underlying mechanisms remain to be elucidated. We previously reported a yin/yang in BA action, with dihydroxy, chenodeoxycholic acid (CDCA), disrupting TJs and its monohydroxy derivative, lithocholic acid (LCA) attenuating it. We hypothesize that structural differences account for the varied actions, namely the presence of the 7‐OH in CDCA, which can serve as a hydrogen bond donor. Recently, we used a fluorescein amine‐tagged CDCA (CDCA‐FA), to show that apical CDCA‐FA travels paracellularly to the basolateral surface (BLS), and like CDCA alters reactive oxygen species‐dependent TJ permeability (FASEB J’ 18:32, 747.19). We predict that, in contrast, LCA‐FA will not alter TJ or access BLS. We studied the structural basis for the yin/yang in BA action on TJ function, by synthesizing: 1. a fluorescein amine‐tagged LCA and 2. a 7, methoxy CDCA (CDCA‐Me).CDCA‐FA and LCA‐FA were synthesized by protecting their alcohol(s), converting‐COOH to an acid chloride, adding the fluorescein amine tag to form an amide, and removing the protecting groups. They were purified by column chromatography and the structure confirmed by NMR and mass spectrometry. We have synthesized CDCA‐Me, structurally similar to LCA, by protecting ‐ COOH of CDCA as a methyl ester, protecting 3‐OH as a silyl ether, converting 7‐OH to a–OCH3 using methyl triflate, and removing the protecting groups. The yield of pure CDCA‐Me is being optimized so its biological effects can be tested.Confluent T84 cells (TransEpithelial Resistance, TER >1000Ωcm2), were treated apically with DMSO (CTRL), 500μM CDCA‐FA, 50μM LCA‐FA ± 500μM CDCA/CDCA‐FA for 0.5–18 H. Cell viability was measured by propidium iodide staining, fluorescence microscopy and Image J analysis. TJ function was assessed by examining: a. pore function measured as TER; b. leak function measured as apparent permeability of CDCA‐FA (Papp CDCA‐FA) vs. LCA‐FA (Papp LCA‐FA) or FITC‐10kDa dextran flux across the monolayer.Exposure (18H) to 50μM LCA‐FA, like LCA, did not alter cell viability nor change CDCA‐FA's effect on cell death (% cell death, CTRL: 7±3 vs LCA‐FA: 9±4; CDCA‐FA: 17±5 vs. CDCA‐FA+ LCA‐FA: 18±6, n=5). Also similar to LCA, LCA‐FA alone or ± CDCA‐FA did not alter pore function (TER) over time (Ωcm2; t=18H; CTRL: 731 ± 10; LCA‐FA: 761 ± 157, CDCA‐FA: 96 ± 12*; CDCA‐FA+LCA‐FA: 118±24*, n=4; *p<0.05 vs CTRL). However, similar to LCA, LCA‐FA attenuated CDCA‐FA's permeability (18 H, Papp LCA‐FA cm/sec, LCA‐FA: 6±3; Papp CDCA‐FA, CDCA‐FA: 57±2; CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA‐FA: 21±5; p<0.05, n>3). Thus, addition of the fluorescent tag did not alter the function of BAs. In summary, CDCA, but not LCA, moves paracellularly to the BLS whereas LCA limits CDCA movement to protect barrier integrity. We postulate that the 7‐OH group in CDCA as a hydrogen bond donor is critical in its role in disrupting barrier function and triggering inflammation and our structure/function studies will guide new therapeutic strategies.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR, APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Artemisinin (ART), a common anti‐malarial drug, and its analogs are also useful as repurposed anti‐cancer drugs, but their properties are not fully elucidated. We previously synthesized novel trioxane (DMR) and dioxazanine (HSM) ART analogs and showed that: a. at low doses (5–10 μM), HSM > than DMR in inducing apoptosis in human colon and lung cancer cells, but not in normal lung cells; b. The actions involved reactive oxygen species (ROS) (Faseb J, ′18:32, 616.2). TfR expression is increased in cancer vs. normal cells. Thus, we hypothesize that, in cancer cells, TfR increases [Fe2+]i which acts on the endoperoxide to produce ROS and induce apoptosis. We examined the mechanism of action of DMR/HSM in inducing apoptosis in human breast, lung, and colon cancer cell lines as compared to normal cell lines. To increase the efficacy of the drug, we also designed and synthesized a 3rd novel analog with dual dioxazanine pharmacophores, PMW.Confluent normal (Breast: MCF10A, Lung: A549) and cancer (Breast: MCF7, Colon: T84, Lung: A549) cells were treated (18 H) with 1–50μM of DMR, HSM or PMW±Deferoxamine (DFO, iron chelator; 1–25 μM). Cells were stained with FITC‐Annexin V (apoptosis), propidium iodide (cell death) or CellRox Green (mitochondrial/nuclear ROS), imaged and quantified by flow cytometry and/or microscopy (Image J). Normal and cancer cell lysates (30 μg) were subjected to SDS‐PAGE and immunoblotting with polyclonal TfR antibody or anti‐GAPDH (control).As in other cancer cells, both DMR and HSM (5 – 50μM) induced apoptosis in breast cancer, but not normal, cells. In contrast to A549 lung cells, where HSM was more effective than DMR, in MCF7 breast cells, 50μM DMR was more efficient (~40%) in inducing apoptosis than HSM (10%). This dictates the development of novel tissue‐specific analogs. The new analog, PMW (1 – 50μM, 18H) did not induce apoptosis in the cancer cells. The larger size of PMW may have slowed entry, suggesting longer incubations.Image J analysis (mean pixel intensity; 18 H) of CellRox+ lung cells (n=3) showed that HSM dose‐dependently increased ROS in cancer A549: (DMSO: 1.5±0.2; HSM, 5 μM: 19±2; 10 μM: 48±4; 50 μM: 59±2); but not normal BEAS2B: (DMSO: 1.0±0.3; HSM, 5 μM: 1.8±1; 10 μM: 2.2±0.4; 50 μM: 2.9±2). Similarly, in breast cancer cells (n=3), DMR dose‐dependently increased ROS in MCF7 (DMSO: 1.0±0.3; DMR, 5 μM: 12±2; 10 μM: 51±5; 50 μM: 42±3); but not in MCF10A (DMSO: 1.0±0.2; DMR, 5 μM: 1.6±1; 10 μM: 3.1±1; 50 μM: 2.5±1).Immunoblotting detected a distinct 94 kDa TfR protein in colon, lung, and breast cancer, but not normal cells. The effect of DMR/HSM±DFO on apoptosis was examined to study the role of TfR. Albeit showing an inhibitory trend, DFO did not significantly alter ART analogs actions (% Annexin V+ cells,18 H: A549: HSM,10μM: 46±4; HSM+5μM DFO: 38±6; HSM +10μM DFO: 33±9; MCF7: DMR,10μM: 36±4; DMR+5μM DFO: 31±7; DMR+10μM DFO: 29±5; n≥3).Synthesizing novel ART‐analogs with improved pharmacokinetics to specifically target cancer cells and elucidating their mechanism of action will help develop new alternatives to treat cancer.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS; Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dysfunction of mucosal immune response and tight junctions (TJ) play an important role in the pathogenesis of inflammatory bowel diseases (IBD) and diarrhea. We reported that excess BA, namely chenodeoxycholic acid (CDCA; 500μM) induced PiC release and altered T84 TJ by increasing ROS. The effect on leak function of TJ was attenuated by CDCA's derivative, lithocholic acid (LCA, 50 μM) and ROS inhibitors (Physiol Rep, ′17, 5: e13294). In patients with IBD, PiC upregulate inducible nitric oxide synthase (iNOS). Thus, we hypothesize a role for RNS in mucosal damage and studied the involvement of oxidative/nitrosative stress in BA‐induced TJ dysfunction and cytokine release in T84 cells.We previously described the synthesis of fluorescein amine‐tagged CDCA (CDCA‐FA) and its use to track transepithelial (TE) BA movement. Confluent T84 cells (TE Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA‐FA, 50μM LCA, CDCA+LCA, ± PiC ([ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), ± 50μM L‐NAME ( L‐NG‐Nitroarginine methyl ester, NOS inhibitor), ± 1mM NAC, (N‐acetyl cysteine, ROS scavenger), for 0.5–18 H. [NO2/NO3] was measured by the Griess assay. We examined the role of RNS/ROS in BA action by studying the effect of BAs±L‐NAME± NAC on: a. Apoptosis (Annexin V, Flow cytometry); b. Paracellular permeability (Pore function as TER; Leak function as TE CDCA‐FA movement); and c. IL‐8 release (ELISA).CDCA (18 H), but not LCA, increased [NO2/NO3] 3‐fold and this was enhanced by PiC (μmol/mg protein; DMSO: 12±1; CDCA: 36±1; LCA: 16± 4, PiC: 18±1; PiC+CDCA: 55±2, n=3). LCA decreased CDCA±PiC‐induced [NO2/NO3] (CDCA+LCA: 25±1; PiC+LCA: 13±2, PiC+CDCA+LCA: 27±4; p<0.05). Inhibiting RNS did not alter CDCA‐induced apoptosis. In pore function, L‐NAME reduced the initial rate of CDCA‐FA‐induced decrease in TER (Ωcm2/sec; 1H CDCA‐FA: 12±1; CDCA‐FA+L‐NAME: 7±1, n>3; p<0.05 ), with no statistical difference at 4 and 18H. L‐NAME altered leak function, reducing CDCA‐FA flux by ~30% at 18 H, (Apparent permeability: Papp ×10−9 cm/sec: CDCA‐FA: 57±2; CDCA‐FA+L‐NAME: 30±1; p<0.05, n=3). LCA decreased CDCA‐FA flux by ~58%, and LCA+L‐NAME completely attenuated it (CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA+L‐NAME: 3±1; p<0.05, n>3). NAC+LCA caused only a ~85% decrease in CDCA‐FA flux (CDCA‐FA+LCA+NAC: 8±2).Like CDCA, CDCA‐FA±PiC stimulated IL‐8 release, which was decreased by LCA. Inhibiting RNS, but not ROS, caused ~40% reduction in CDCA‐induced IL‐8 release (ng/ml; CDCA: 4.6±0.5; CDCA+L‐NAME: 2.7±0.1; CDCA+NAC: 4.2±0.3; n=3), suggesting a role for NO in BA‐induced inflammatory process.We demonstrate a novel role for RNS in CDCA‐induced TJ dysfunction in T84 cells. Inhibiting RNS in the presence of LCA reverses CDCA action on leak function and reduces PiC release. Equally important, its roles overlap with but are distinct from ROS. Understanding the role of ROS/RNS in BA action can lead to novel therapeutic strategies for IBD.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.