Journal: British Journal of Pharmacology

Article Title: Comparative pharmacology of chemically distinct NADPH oxidase inhibitors

doi: 10.1111/j.1476-5381.2010.00920.x

Figure Lengend Snippet: NOX expression and NADPH oxidase activity in three different cell lines. (A) Reverse transcriptase PCR of the five human (NOX1–5) and four rodent (NOX1–4) NOX isoform mRNAs in the human colon epithelial cell line CaCo-2, the human leucoblast cell line HL-60 and the rat aortic smooth muscle cell line A7r5. All cell lines expressed more than one NOX subunit. For measurement of NADPH oxidase activity, CaCo-2 homogenates + substrate NADPH (B), PMA-treated HL-60 cells (C and E) and A7r5 homogenates + substrate NADPH (D) were incubated in the absence or presence of SOD (250 U·mL −1 ), CAT (1000 U·mL −1 ), oxypurinol (100 µM) or l -NAME (100 µM). ROS formation was measured by lucigenin (B and D), or L012 (B) chemiluminescence, or by cytochrome c reduction (E). In none of the cells, detectable ROS formation originated from NOS (inhibited by l -NAME) or XOD (inhibited by oxypurinol). Only in HL-60 cells, the assay signals were fully inhibited by SOD and thus due to superoxide. In CaCo-2 and A7r5 cell homogenates, even the combination of SOD and CAT only partially attenuated the assay signal, suggesting that only a fraction was due to superoxide and/or H 2 O 2 . In later experiments, only the SOD/CAT-inhibited signal component was considered as a measure of superoxide/H 2 O 2 formation in CaCo-2 and A7r5 cell homogenates. Data labelled ‘blank’ represent signals in the absence of cells or homogenate. Values represent means ± SEM of n ≥ 6 observations; *** P < 0.001, significantly different from control values.

Article Snippet: A7r5 cells (rat, smooth muscle embryonic aorta, ATCC-No. CRL 1444) were cultured in Dulbecco's modified Eagle's medium (Sigma, Deisenhofen, Germany) supplemented with 0.1% glucose, 10% heat-inactivated calf serum, 100 U·mL −1 penicillin, 100 μg·mL −1 streptomycin and 2 mM glutamine.

Techniques: Expressing, Activity Assay, Reverse Transcription, Incubation, Control

Journal: British Journal of Pharmacology

Article Title: Comparative pharmacology of chemically distinct NADPH oxidase inhibitors

doi: 10.1111/j.1476-5381.2010.00920.x

Figure Lengend Snippet: Maximal inhibition and potency of apocynin, AEBSF and VAS3947 to inhibit NADPH oxidase activity. Activity was measured in CaCo-2 (A) or A7r5 (D) cell homogenates as NADPH-induced and SOD/CAT-inhibitable lucigenin-derived chemiluminescence, or in DMSO-differentiated HL-60 cells as measured by L012-derived chemiluminescence (B) or cytochrome c reduction (C). The left panels depict maximal inhibition; the right panels concentration–response curves. Concentrations used for maximal inhibition were in CaCo-2 cell homogenates 1 mM apocynin, 1 mM AEBSF and 30 µM VAS3947 (A); in HL-60 cell-mediated L012 chemiluminescence 1 mM apocynin, 100 µM AEBSF and 10 µM VAS3947 (B); in HL-60 cell-mediated cytochrome c reduction 1 mM apocynin, 1 mM AEBSF and 10 µM VAS3947 (C); and in A7r5 cell homogenates 1 mM apocynin, 1 mM AEBSF and 30 µM VAS3947 (D). IC 50 values for VAS3947 were 12 µM (A), 2 µM (B), 1 µM (C) and 13 µM (D). Apocynin had a potency in the micromolar range (97 µM) only in the HL-60/L012 assay (B). AEBSF had the lowest potency (420 µM in B, 304 µM in C; 1.2 mM in D), and in the HL-60/L012 assay it even enhanced signal intensity at low micromolar concentrations (EC 50 = 30 µM). Values represent means ± SEM of n ≥ 5 experiments. * P < 0.05, *** P < 0.001, significantly different from control values.

Article Snippet: A7r5 cells (rat, smooth muscle embryonic aorta, ATCC-No. CRL 1444) were cultured in Dulbecco's modified Eagle's medium (Sigma, Deisenhofen, Germany) supplemented with 0.1% glucose, 10% heat-inactivated calf serum, 100 U·mL −1 penicillin, 100 μg·mL −1 streptomycin and 2 mM glutamine.

Techniques: Inhibition, Activity Assay, Derivative Assay, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Canonical Transient Receptor Potential 6 (TRPC6), a Redox-regulated Cation Channel *

doi: 10.1074/jbc.M109.093500

Figure Lengend Snippet: A–D , Ca 2+ imaging experiments, showing involvement of endogenous H 2 O 2 in AVP-stimulated Ca 2+ entry. A , representative traces, showing the AVP-evoked Ca 2+ response in HEK293T cells expressing TRPC6 and an empty vector ( T6 + vector ) or in cells co-transfected with TRPC6 and AVP V1R in the presence ( T6 + V1R + Catalase ) or absence ( T6 + V1R ) of PEG-catalase (250 units/ml for 30 min). Ca 2+ concentration in the bathing solution is indicated by the numbers inside the top open bar. Application of 100 n m AVP is indicated by the top solid bar . [Ca 2+ ] B indicates the Ca 2+ concentration in the bathing solution. B , summary data, showing Ca 2+ entry responses in different groups. *, p < 0.05, compared with T6 + vector; †, p < 0.05, compared with T6 + vector and T6 + V1R. C , effect of catalase on AVP-induced Ca 2+ release in cells co-transfected with TRPC6 and AVP V1R. D , effect of catalase on normalized Ca 2+ entry (ratio of Ca 2+ entry to Ca 2+ release) in response to AVP stimulation. *, p < 0.05, compared with T6 + V1R. B–D , n indicates the number of cells analyzed from 4 to 5 sets of transfection. E–G , in A7r5 cells. E , whole-cell patch clamp, showing inward currents in response to 100 μ m H 2 O 2 in A7r5 with ( siRNA-T6 ) and without ( Scramble ) knockdown of TRPC6 at a holding potential of −60 mV. The dashed lines indicate zero currents. Application and removal of H 2 O 2 were indicated by the vertical lines. F , Western blot, showing TRPC6 expression levels in A7r5 cells transfected with TRPC6 siRNA sequence ( siRNA-T6 ) and scrambled sequence ( Scramble ). Actin served as a loading control. UT , untransfected. G , summary data, showing the H 2 O 2 effect on whole-cell currents in A7r5 cells with ( SiRNA-T6 ) and without ( Scramble ) knockdown of TRPC6. The responses were measured by the difference between the membrane current before application of H 2 O 2 and the peak current after application of H 2 O 2 . The whole-cell currents were expressed as current density (pA/pF), normalized to the cell membrane capacitance. n indicates the number of cells analyzed from three sets of transfection. Asterisk indicates statistically significant difference compared with Scramble (Student's t test).

Article Snippet: Both human embryonic kidney 293 (HEK293T) cells and A7r5 vascular smooth muscle cells were purchased from ATCC (Manassas, VA) and were cultured in Dulbecco's modified Eagle's medium (Hyclone SH30021.01 for HE293T and ATCC 30–2002 for A7r5) with 25 m m HEPES, containing 10% fetal bovine serum and 100 units/ml penicillin/streptomycin.

Techniques: Imaging, Expressing, Plasmid Preparation, Transfection, Concentration Assay, Patch Clamp, Western Blot, Sequencing