Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.

<p>Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC₅₀ value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.</p

Effects of TRAM-34 on rat CYP Activity.

<p>Recombinant enzyme CYP2C6 (A), CYP2C11 (B), CYP1A2r (C) and CYP2B1 (D), substrate and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Percent control enzyme activity (ordinate) is plotted versus the log of inhibitor concentration (abscissa). All TRAM-34 and clotrimazole data points (A and B) represent the mean (±SEM) of 3 experiments performed in triplicates. Other clotrimazole data represent the mean of duplicates (C) or triplicates (D) from a single experiment. TRAM-34 IC₅₀ values were determined by non-linear regression and are shown in parentheses. Control enzyme activities were (mean ± SEM, n = 3 experiments each) 1.65±0.34 (<b>A</b>), 0.14±0.02 (<b>B</b>), 0.68±0.11 (<b>C</b>) and 6.13±0.7 (<b>D</b>) min^–1. In this and subsequent figures, error bars represent SEM of measurements, but, due to the small variability, are not always visible.</p

Effects of TRAM-34 on human CYP Activity.

<p>Recombinant enzyme CYP2C19 (A), CYP19A1h (B), CYP1A2h (C) and CYP2B6 (D), substrate and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Percent control enzyme activity (ordinate) is plotted versus the log of inhibitor concentration (abscissa). All TRAM-34 (<b>A–D</b>) and fluvoxamine (<b>C</b>) data points represent the mean (±SEM) of 3 experiments performed in triplicates. Data from the other inhibitors (A, B and D) represent the mean (±SEM) of triplicates from a single experiment. TRAM-34 IC₅₀ values were determined by non-linear regression and are shown in parentheses. Control enzyme activities were (mean ± SEM, n = 3 experiments) 0.30±0.03 (<b>A</b>), 0.13±0.003 (<b>B</b>), 4.26±0.09 (<b>C</b>) and 3.81±0.32 (<b>D</b>) min⁻¹.</p

Conditions for CYP Assays.

<p>Conditions used for the present CYP assays are summarized. All assays used fluorescence plate reader methods except where noted otherwise.</p>a<p>See text for regenerating system compositions.</p>b<p>LC-MS/MS methods used for this assay only.</p

Effect of the glutamate transporter inhibitor dl-TBOA on hypoosmotic medium induced amino acid release in the cortex and glutamate transporter reversal in cultured astrocytes.

<p>(a–b) Microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 500 µM dl-TBOA, given 20 minutes prior to and during one hour hypoosmotic medium perfusion. The data represent average dialysate levels of glutamate (a), aspartate (b) ±SEM from 4 rats. ** p<0.01 HYPO vs. HYPO+TBOA. (c) DL-TBOA effectively prevented reversal of glutamate transporter in cultured astrocytes. Cultured astrocytes were superfused for one hour with 1 mM ouabain and additionally for 20 min high [KCl] (100 mM) plus ouabain to induce glutamate transporter reversal. 300 µM dl-TBOA was given 10 minutes prior to and during the high [KCl] perfusion in the presence of ouabain. The data are the average values ±SEM for three experiments in each group. ** p<0.01 KCl vs. KCl+TBOA.</p

Assessment of connexin hemichannel involvement in air-stimulated ATP release.

<p>Uptake of the connexin permeable dye lucifer yellow (LY) was measured to confirm that connexin hemichannels open in response to air exposure and are blocked by the same drugs that prevent ATP release. Cells were pre-incubated with ATP release inhibitors using the same procedure as for the pharmacological analysis assays. LY (500 µM final concentration) was added in the media after air exposure or a change of half the media (control). Sixty minutes later, cultures were rinsed twice with media containing 1-octanol (2 mM) to remove residual LY, but minimize leakage of absorbed LY. LY levels in the cell lysates were then measured. Air exposure significantly increased LY uptake compared to control and the same drugs that inhibited air-stimulated ATP release prevented this increase in LY uptake. This indicates that connexin hemichannels are open during air-stimulated ATP release. Values represent 8 or 9 wells per group +/− SEM, * <i>p</i><0.05 vs. control.</p

Effect of H₂O₂ on hypoosmotic medium induced amino acid release in the cortex.

<p>(a–c) Two microdialysis probes implanted on opposite sides of the cortex were perfused with hypoosmotic medium in the presence or absence of 1 mM H₂O₂ given 20 minutes prior to and during one-hour hypoosmotic medium perfusion. The data represent the average dialysate levels ±SEM of glutamate (a), aspartate (b) and taurine (c) from 9 rats. ** p<0.01 HYPO vs. HYPO+H₂O₂. In separate experiments, rats were perfused with 1 mM H₂O₂ alone (N = 5).</p

Pharmacological analysis of the air-stimulated ATP release mechanism. A. propidium iodide (PI) labeling.

<p>PI labeling was measured to ensure that air exposure and drugs used in inhibition experiments did not compromise cell membranes. Only pretreatment with carbenoxolone significantly increased PI labeling and this remained below 3% of cells. All values represent averages +/− SEM of 6 dishes per group, * <i>p</i><0.05 vs. control. <b>To determine the mechanism of air-stimulated ATP release</b>, keratinocytes were pre-incubated for 10 minutes with known inhibitors of ATP release pathways before air exposure. ATP levels in the media were assayed just prior to air exposure to determine any effects of the drugs themselves on baseline ATP release (<b>B</b>, pre-air) and 7 minutes after air exposure (<b>C</b>, post-air) to measure the ability of drugs to inhibit air-stimulated ATP release. <b>B</b>. Pre-air ATP levels were significantly lower in differentiated cultures, but similar for most drug treatment groups. <b>C</b>. In both proliferating and differentiated cultures, pre-incubation with the connexin hemichannel blockers 1-octanol and carbenoxolone largely abolished air-stimulated ATP release. Glibenclamide and verapamil, drugs traditionally used as ABC transporter blockers, also significantly inhibited air-stimulated ATP release. Post-air ATP levels are expressed as percent of air alone. Values represent 12 (drug treatment) or 24 (air alone) dishes. Error bars are +/− SEM, *,⁺<i>p</i><0.05 vs. vehicle or air alone for proliferating and differentiated cells, respectively. <b>Inset. Schematic showing potential keratinocyte ATP release pathways and the major targets of inhibitors used.</b> Secondary targets discussed in the text are also shown in parentheses. Red dots and arrows represent regulatory ATP binding sites and routes of release, respectively. <b>Release Pathways.</b> P2X7: P2X7 ATP receptors, Px1/2: pannexins-1 & 2, Cx43: connexin-43, VRAC: volume-regulated anion channels (unknown molecular identity) and the ATP binding cassette (ABC) transporters CFTR, MDR1 and MRP1.</p

Effect of hypoosmotic or isoosmotic low [NaCl] medium on amino acid levels measured in the rat cortex <i>in vivo</i>.

<p>(a–c) Microdialysis probes, implanted in the rat frontoparietal cortex, were perfused with hypoosmotic medium (−95 mM NaCl, −65% osmolarity) or isoosmotic low NaCl medium (−95 mM NaCl +167 mM mannitol) for one hour. In these experiments, the rat brain was perfused with both the hypoosmotic and isoosmotic medium on opposite sides of the cortex. The data represent average dialysate levels of glutamate (a), aspartate (b), and taurine (c) ±SEM from 5 rats. ** p<0.01, hypoosmotic vs. isoosmotic low [NaCl], repeated measures ANOVA. (d–e) In several experiments dialysate levels of glutamine (d, N = 5), and asparagine (e, n = 3) were additionally measured on the “hypoosmotic” side of the brain.</p

Dependence of taurine and glutamate uptake on extracellular [Na⁺] in cultured astrocytes.

<p>Taurine and glutamate transport rates were measured in primary astrocyte cultures using [³H]taurine and d-[³H]aspartate. Extracellular concentrations of amino acids were adjusted to 10 µM using unlabeled taurine or l-glutamate. To compare glutamate versus taurine uptake, the values were normalized to uptake levels under basal conditions ([Na⁺]_o = 135 mM). Note that under basal conditions absolute d-[³H]aspartate uptake rate (nmols/mg protein) was ∼5-fold higher compared to taurine. Data are the mean values ±SEM of three experiments from each group.</p