Dennis E. Epps

Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells

The steady-state and dynamic fluorescence properties of the membrane potential-sensitive bis-oxonol dye Dibac4(3) were characterized in vitro using model ligand systems and in vivo in A10 smooth muscle cells by fluorescence microscopy in conjunction with the ACAS imaging system. In the latter system the dye responds to potassium ion-induced jumps in membrane potential with changes in its fluorescence intensity, which follow pseudo-first-order kinetics. The relationship between the magnitude of the changes and the corresponding rate constants excludes the possibility that a simple, one-step equilibrium between extracellular and cytoplasmic dye would be sufficient to account for this phenomenon. The necessity of invoking an additional step suggested that the redistribution of the free dye between the cytoplasm and the exocellular medium is rapid and that the slow step associated with the fluorescence changes may be the interaction of the dye with proteins in the cytoplasm, along the lines proposed by Bräuner et al. (Biochim. Biophys. Acta 771 (1984), 208, 216). The interaction of the dye with BSA and with egg lecithin SUVs was studied as a model for the in vivo phenomenon. The dependence of fluorescence intensity changes on the concentrations of the reagents shows the formation of a reversible dye/albumin complex with a 2/1-stoichiometry, with Kd = 0.03 +/- 0.01 microM and a reversible adsorption to the SUVs with Kd 0.45 +/- 0.08 microM. The fluorescence lifetime of the dye in solution, < 100 ps, results in a high solution steady-state anisotropy. The latter decreases considerably upon binding to BSA, SUVs and A10 cells concomitant with a large increase in the lifetime. With such a short lifetime of the free dye, selective gating of the excitation source or the photodetector should eliminate the high background of the unbound dye and thereby enhance the sensitivity of the system.

Kinetic analysis of the free-radical-induced lipid peroxidation in human erythrocyte membranes: Evaluation of potential antioxidants using cis-parinaric acid to monitor peroxidation

cis-Parinaric acid (PnA), cis-trans-trans-cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (epsilon = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radicals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78, 517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10-15 times more potent than the aminosteroids evaluated (see Table 1).

Human polymorphonuclear neutrophil activation with arachidonic acid

1 The capacity of arachidonic acid (AA) to stimulate granule exocytosis from human polymorphonuclear neutrophils (PMNs) was investigated. 2 AA induced the selected extracellular release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12 binding protein) granule constituents from human PMNs in a time‐and concentration‐dependent manner. 3 Cytochalasin B (CB) enhanced but was not required for PMN activation with AA. 4 Although extracellular calcium had no effect on granule exocytosis, AA did stimulate the mobilization of intracellular sequestered Ca2+ which resulted in an increase in cytosolic‐free Ca2+ ([Ca2+]i) as reflected by increased fluorescence of Fura‐2‐treated cells. 5 AA stimulated Ca2+/phospholipid‐dependent protein kinase C (PK‐C) activity in PMNs. 6 4,4′‐Diisothiocyano‐2,2′‐disulphonic acid stilbene (DIDS), an anion channel blocker, caused a concentration‐dependent inhibition of granule enzyme release. 7 Activation of PMNs with AA was unaffected by the lipoxygenase/cyclo‐oxygenase inhibitors, 5,8,11, 14‐eicosatetraynoic acid (ETYA) and benoxaprofen, a lipoxygenase inhibitor, 6, 9, deepoxy‐6,9‐(phenylimino)‐Δ6,8‐prostaglandin 11 (piriprost potassium) or a pure cyclo‐oxygenase inhibitor, flurbiprofen. 8 These data define the properties of AA as a secretory stimulus for human PMNs.

Spontaneous Aggregation and Cytotoxicity of the β-Amyloid Aβ1–40: A Kinetic Model

The time dependency of the spontaneous aggregation of the fibrillogenic β-Amyloid peptide, Aβ1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Aβ antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 μM amyloid peptide. The anti–Aβ antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length Aβ was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the Aβ1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of Aβ1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of Aβ1–40in vitro and possibly in vivo.

A slowly formed transient conformer of Aβ1–40 is toxic to inward channels of dissociated hippocampal and cortical neurons of rats

The mechanism by which amyloid peptide (Abeta(1-40)) produces effects on neurotransmission is currently unresolved. In initial experiments, using the patch-clamp technique, we found that 11.5 microM of preaggregated Abeta(1-40) altered the hippocampal neuron resting membrane potential and inhibited action potential firing. To identify the toxic species, the effects of Abeta(1-40) on sodium (I(Na)), calcium (I(Ca)), and potassium (I(K)) currents in hippocampal neurons were examined as a function of peptide aggregation state in a specially designed miniature recording chamber. Aggregation reactions were induced by constant shaking, starting with 50 microM monomeric peptide. At 10- to 30-min intervals, the ionic currents were examined on a single neuron suspended in control saline and then in a 100-microl sample of the aggregating peptide. We found that samples of the peptide taken 60-120 min into the aggregation process contained species that exhibited maximal inhibitory effects over a broad potential range in the rank ordering of I(Na) > I(Ca). I(K) was inhibited only slightly at depolarized potentials. Inhibition of APF through blockade of these channels would inhibit normal neuronal activity and directly contribute to cognitive dysfunction. In previous studies on SH-EP cells, we showed that neither monomeric nor fibrillar peptide had significant effect on cell viability except during exposure to the 60-120 minute aggregation product when cell death was recorded. Our kinetic model demonstrated that the toxic species was a slowly formed transient conformer (activated monomer), which was the only aggregating species that passed through a maximum concentration during aggregation. This species amounted to only a small fraction of the total amount of aggregating peptide. We conclude that, for both the native neurons in the present study as well as SH-EP1 cells, the activated monomeric conformer of the peptide is the toxic species.

Kinetic evaluation of lipophilic inhibitors of lipid peroxidation in DLPC liposomes

The authors have developed a kinetic method that allows one to obtain relative reactivity constants for lipophilic antioxidants in free radical systems. Two experimental model systems were developed: (a) a methanolic solution using AMVN as the free radical initiator and linoleic acid as the substrate, and (b) a multilamellar vesicle system composed of dilinoleoylphosphatidylcholine and AAPH as the substrate and the initiator, respectively. The use of these two systems allows researchers not only to determine the intrinsic reactivity of a potential antioxidant, but also to evaluate its potency in a membranous system where the contribution of the physical properties of the antioxidant to the inhibition of lipid peroxidation is important. These results show that all antioxidants tested acted in these systems as free radical scavengers, and they validate the synergism between intrinsic scavenging ability and membrane affinity and/or membrane-modifying physical properties in the inhibition of lipid peroxidation.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

An experimental method for the determination of enzyme-competitive inhibitor dissociation constants from displacement curves: application to human renin using fluorescence energy transfer to a synthetic dansylated inhibitor peptide.

We developed a facile procedure for the determination of enzyme-competitive inhibitor dissociation constants over a wide range of potencies at any ratios of enzyme, labeled ligand, and inhibitor. The assay uses displacement curves and a fluorescent-labeled ligand to allow experimental determination of dissociation constants (Kds) of inhibitors of human renin, a highly specific enzyme, for which numerous high affinity (up to 100 pM) inhibitors have been synthesized. The procedure involves binding a dansylated competitive inhibitor, U80215, followed by its displacement by an unlabeled inhibitor of renin. Binding of U80215 is monitored by fluorescent energy transfer from the renin tryptophans to the dansyl moiety; displacement of U80215 by an unlabeled inhibitor is monitored by a reversal of this process. The procedures may be used to determine the potencies of unlabeled inhibitors up to 100 pM affinities and to determine kinetic binding constants. The concepts described should also be useful in other protein/ligand systems.

Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.

The constituent tryptophans and bisANS as fluorescent probes of the active site and of a secondary binding site of stromelysin-1 (MMP-3)

The active site of the catalytic domain of stromelysin-1 (matrix metalloproteinase-3, MMP-3) was probed by fluorescence quenching, lifetime, and polarization of its three intrinsic tryptophans and by the environmentally sensitive fluorescent reporter molecule bisANS. Wavelength-dependent acrylamide quenching identified three distinct emitting tryptophan species, only one of which changes its emission and fluorescence lifetime upon binding of the competitive inhibitor Batimastat. Significant changes in the tryptophan fluorescence polarization occur upon binding by any of the three hydroxamate inhibitors Batimastat, CAS108383-58-0, and Celltech CT1418, all of which bind in the P2′-P3′ region of the active site. In contrast, the inhibitor CGS27023A, which is t hought to bind in the P1-P1′ region, does not induce any change in tryptophan fluorescence polarization. The use of the fluorescent probe bisANS revealed the existence of an auxiliary binding site extrinsic to the catalytic cleft. BisANS acts as a competitive inhibitor of stromelysin with a dissociation constant ofKi=22 µM. In addition to this binding to the active site, it also binds to the auxiliary site with a dissociation constant of 3.40±0.17 µM. The auxiliary site is open, hydrophobic, and near the fluorescing tryptophans. The binding of bisANS to the auxiliary site is greatly enhanced by Batimastat, but not by the other competitive inhibitors tested.