Journal: Cells

Article Title: Altered Ca 2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

doi: 10.3390/cells11010049

Figure Lengend Snippet: PV RBCs show increased Gárdos channel activity. ( A ) CCCP method analysis of PV and CT RBC membrane potential changes upon 100 µM NS309 addition. At the end of the experiment, cells were lysed with 3M NaCl 1% Triton X lysis solution to obtain the zero membrane potential (pHi = pHo) for absolute calculation of membrane potential. CT (mean—black line; SD—grey) and PV (mean—red line; SD—pink) RBCs. Data are displayed as mean with 95% confidence interval; CT ( n = 6) and PV ( n = 8). ( B ) Cell volume assay on Gárdos activity; 0.05% RBCs suspension was prepared in PBS with a final concertation of 0.2% BSA, 1 mM CaCl 2 , and 100 µM NS309. RBC size was measured using CASY before and 2.5, 5, 7.5, and 10 min after 100 μM NS309 addition. Mean with SD, n = 6, Mann–Whitney test. ( C – E ) Patch-clamp analysis. Upon cell catch external solution was added to the wells followed by 10 µM NS3623, 10 µM NS309, 5 µM TRAM-34, and 30 µM GdCl 3 . Currents were measured at room temperature applying −100 to +80 mV ramp voltage protocol for 300 ms, at a holding potential of −30 mV. The cell response was measured in pA at +80 mV. Statistical analysis of the currents at +80 mV in NS309 and TRAM-34 responding cells in ( C ) CT and PV RBCs ( n = 17; n = 48, respectively), ( D ) BaF3 EpoR JAK2 WT ( n = 20) and BaF3 EpoR JAK2 V617F ( n = 48), and ( E ) HEL ( n = 61) and HEL cells treated with 0.3 µM ruxolitinib for 24 h ( n = 63). Cell was considered responsive if it displayed at least a 20% current change. The left panel represents the cell current upon the addition of NS3623 (baseline), NS309, and TRAM-34. The central panel displays NS309-induced current increase, while the right panels represent TRAM-34-induced current decrease. The data are presented as median and box plots (25–75%) with whiskers (10–90%). Mann–Whitney test or Wilcoxon test, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns—not significant.

Article Snippet: Three minutes after RBC addition, 100 µM of NS309 was added (a compound which shifts Ca 2+ sensitivity of Gárdos of one order of magnitude [ ] (Alomone labs, Jerusalem, Israel) followed by RBC lysis 8 min after RBC addition using 3M NaCl 1% Triton solution (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Activity Assay, Lysis, MANN-WHITNEY, Patch Clamp

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Chemical structures of AP14145 (left) and NS8593 (right).

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Bar graph depicting the number of convulsions triggered by the administration of the vehicle, 10 mg·kg−1 AP14145 and 10 mg·kg−1 NS8593.

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques:

Journal: British Journal of Pharmacology

Article Title: A new negative allosteric modulator, AP14145, for the study of small conductance calcium‐activated potassium (K Ca 2) channels

doi: 10.1111/bph.14043

Figure Lengend Snippet: Calculated physicochemical properties and plasma protein binding of NS8593 and AP14145

Article Snippet: NS8593 and NS 309 was supplied by NeuroSearch A/S, Ballerup, Denmark.

Techniques: Protein Binding

Journal: British Journal of Pharmacology

Article Title: Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

doi: 10.1111/j.1476-5381.2011.01447.x

Figure Lengend Snippet: Effects of openers of KCa2.1-2.3 and KCa3.1 in eGFP-positive astrocytes patch clamped in the whole-cell configuration. (A) Typical (time-matched) control time-course indicating currents at the 0 mV point of voltage ramps plotted as a function of time. (B) Typical time-course showing an increase in outward current ∼7 min after application of NS309 [arrowheads indicate time points of the current-voltage relationships shown in (C)]. (C) Current-voltage relationship (1 s voltage ramp) showing selected full traces from the cell described in (B) under control conditions and after exposure to NS309. (D) Typical time course showing lack of induction of current by CyPPA [arrowheads indicate the time points at which the current-voltage relationships shown in (E) were obtained under control conditions and after exposure to CyPPA]. (F) Mean (±SEM) currents at 0 mV normalized to initial control current level. In the presence of 200 nM [Ca2+]i NS309 (500 nM; n = 8) evoked a significant current increase whereas CyPPA (30 µM) was without effect (n = 9) (one-way anova with post hoc Bonferroni's multiple comparison test ***P < 0.0001).

Article Snippet: NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) and CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine) were synthesized at the Department of Medicinal Chemistry at Boehringer Ingelheim (Biberach, Germany) according to the methods described in Strøbaek et al . (2004) and Hougaard et al . (2007) , respectively.

Techniques: Control, Comparison