Journal: PLoS ONE

Article Title: Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

doi: 10.1371/journal.pone.0078727

Figure Lengend Snippet: A: Representative recording of a B19 neuron pretreated with IbTX (300 nM) and after addition of NOC7 (100 µM). Note that NOC7 after IbTX caused a sustained depolarization with similar initial and plateau amplitudes. B: Quantification of the initial depolarization showing that the amplitude of membrane depolarization was significantly reduced in the NOC7 after IbTX group compared to NOC7 by itself. C: Quantification of the plateau depolarization in response to treatment shown in A. IbTX pretreatment did not affect the depolarizing effect of NO during the plateau phase.

Article Snippet: Diethylamine NONOate (DEA/NO, Calbiochem), cadmium chloride (CdCl 2 , Sigma), iberiotoxin (IbTX, Sigma), apamin (Alomone labs), and NG-nitro-L-arginine methyl ester (L-NAME, Calbiochem), were dissolved in distilled H 2 O to make 100 mM, 1 M, 200 µM, 1 mM, 1 M stock solutions, respectively.

Techniques:

Journal: PLoS ONE

Article Title: Nitric Oxide Regulates Neuronal Activity via Calcium-Activated Potassium Channels

doi: 10.1371/journal.pone.0078727

Figure Lengend Snippet: Elevation of NO by NO donors, such as NOC7 or DEA/NO, inhibits two types of Ca 2+ -activated K + channels in Helisoma B19 neurons. Apamin-sensitive SK channels contribute to part of the initial effect of NO and are fully responsible for its long-lasting effect on membrane depolarization, whereas IbTX-sensitive BK channels only partially contribute to the initial depolarization. Voltage-gated Ca 2+ channels do not participate in the depolarizing effect of extrinsically applied NO. The mechanism(s) by which NO inhibits these ion channels is presently unknown (indicated by dotted lines). Inhibitors used are indicated in gray.

Article Snippet: Diethylamine NONOate (DEA/NO, Calbiochem), cadmium chloride (CdCl 2 , Sigma), iberiotoxin (IbTX, Sigma), apamin (Alomone labs), and NG-nitro-L-arginine methyl ester (L-NAME, Calbiochem), were dissolved in distilled H 2 O to make 100 mM, 1 M, 200 µM, 1 mM, 1 M stock solutions, respectively.

Techniques:

Journal: PLoS ONE

Article Title: Zinc pyrithione activates K + channels and hyperpolarizes the membrane of rat pulmonary artery smooth muscle cells

doi: 10.1371/journal.pone.0192699

Figure Lengend Snippet: A-B Original records of PASMC membrane potential before and after applying 10μM ZnPy (ZP) in the absence ( A ) and presence ( B ) of 10μM glibencamide, with and without the addition of 1μM XE991. C . Mean (±SEM) membrane potentials measured before and after applying ZnPy in the absence (n = 14) or presence (n = 12) of glibenclamide or glibenclamide plus XE991 (n = 12). C = control, W = wash. *p<0.05, **p<0.01, ***p<0.001 by repeated measures ANOVA with Tukey’s multiple comparisons test (glibenclamide present) or paired t-test (glibenclamide absent). D-F . Records of membrane potential during responses to ZnPy before and after exposing cells to 10mM TEA ( D ), 1μM paxilline ( E ) or 50nM iberiotoxin ( F ). G . Mean (±SEM) membrane potentials measured from the recordings in D-F. N = 10 (TEA), 15 (paxilline) and 12 (iberiotoxin). **p<0.01, ***p<0.001 by repeated measures ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Iberiotoxin, paxilline and XE991 (10,10-bis(4Pyridinylmethyl)-9(10H)-anthracenone) dihydrochloride were from Tocris (Bristol, UK).

Techniques: Membrane, Control

Journal: PLoS ONE

Article Title: Zinc pyrithione activates K + channels and hyperpolarizes the membrane of rat pulmonary artery smooth muscle cells

doi: 10.1371/journal.pone.0192699

Figure Lengend Snippet: Upper traces show currents activated by a voltage step from -80 to 40mV before and after exposure to 10μM ZnPy, under control conditions or in the presence of 10mM TEA (A) , 1μM paxilline (Pax, B ) or 50nM iberiotoxin (IbTx, C ). Each trace is the average of records from 6 (TEA and iberiotoxin) or 8 (paxilline) cells. Lower panels show the ZnPy-induced currents, obtained by digitally subtracting records in the absence of ZnPy from those in its presence, either in control conditions (unblocked) or after exposure to a BK Ca blocker. The iberiotoxin-sensitive component in C , shown as the difference between the unblocked and iberiotoxin-inhibited currents, is superimposed by a double exponential function (red) that best fits the activation time course, with τ 1 = 6.2ms and τ 2 = 48ms: the fast component contributed 75.3% of the current.

Article Snippet: Iberiotoxin, paxilline and XE991 (10,10-bis(4Pyridinylmethyl)-9(10H)-anthracenone) dihydrochloride were from Tocris (Bristol, UK).

Techniques: Control, Activation Assay

Journal: International Journal of Applied and Basic Medical Research

Article Title: Avanafil Inhibits the Contractility of the Isolated Caprine Detrusor Muscle

doi: 10.4103/ijabmr.IJABMR_339_18

Figure Lengend Snippet: Representative traces from the study: (a) Contractile effect of 80 mM potassium chloride before (left side) and after (right side) addition of 30 μM avanafil. (b) Contractile effect of 80 mM potassium chloride before (left side) and after (right side) addition of 100 nM iberiotoxin and 30 μM avanafil

Article Snippet: Iberiotoxin (Santa Cruz Biotechnology) was dissolved in double-distilled water to give a 4 μM stock solution.

Techniques:

Journal:

Article Title: O 2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase

doi:

Figure Lengend Snippet: EM and IK are similar in resistance PASMCs from wild-type and CGD mice (A) Resting EM is similar in wild-type (filled bar) and CGD (empty bar) PASMCs. (B) Normoxic current density is similar in wild-type and CGD PASMCs. (C and D) In PASMCs from CGD mice, 4-AP, a Kv channel inhibitor, reduces IK at negative potentials (−10 mV, C, ∗, P < 0.05). Tetraethylammonium (TEA) inhibits IK only at positive potentials, where KCa channels are active (∗, P < 0.05, D). (E and F) Four minutes of acute hypoxia inhibits IK (n = 4 cells/group, ∗, P < 0.01). (G and H) In separate experiments, 4 min of acute hypoxia inhibits IK even after specifically inhibiting large conductance KCa channels with IBTX. This finding suggests the hypoxia-sensitive current is at least partially Kv current (†, ∗ P < 0.05 IBTX and hypoxia inhibit IK, respectively).

Article Snippet: All drugs were from Sigma except IBTX (Peptides International) and DPI (Research Biochemicals, Natick, MA).

Techniques:

Journal:

Article Title: Distinct contributions of small and large conductance Ca 2+ -activated K + channels to rat Purkinje neuron function

doi: 10.1113/jphysiol.2002.027854

Figure Lengend Snippet: A-C, recordings from three different Purkinje neurons show how spontaneous firing was affected by 200 nm apamin, 100 nm iberiotoxin or the combination of the two blockers. The same scale bar applies to each set of traces. D, ISI distributions and fits are shown for the traces in A-C. E, spontaneous firing frequency plotted as a function of recording time for each experiment. •, the traces shown in A-C. Black horizontal bars show when toxins were present in the bath solution. Response delays partly reflect the time needed for residual control solution to empty out of the perfusion tubing and inline solution heater. F, the effects of specific KCa channel blockers on the firing frequency are summarized. Black bars show the baseline frequency for each group (means ±s.e.m.), while hatched bars show the frequency in the presence of the blocker. The firing frequency increased from 29 ± 5 to 133 ± 37 Hz with the application of apamin (n = 7 cells), from 29 ± 5 to 50 ± 12 Hz with iberiotoxin (n = 6), from 27 ± 4 to 63 ± 14 Hz with 1 μm paxilline (n = 5) and from 22 ± 3 to 313 ± 37 Hz when both iberiotoxin and apamin were applied together (n = 5). For the control group, the firing rate increased from 41 ± 16 initially to 47 ± 19 Hz after 10 min of control recording time (n = 6). Apamin had a significant effect compared to the control (P < 0.05), and the simultaneous application of iberiotoxin and apamin differed significantly from both the control group (P < 0.0001) and from the apamin group (P < 0.01). When applied alone, neither iberiotoxin nor paxilline had a statistically significant effect compared to the control group (P > 0.5).

Article Snippet: Channel blockers Iberiotoxin, ω-Aga-IVA and SNX-482 were obtained from Peptides International, Inc. (Louisville, KY, USA).

Techniques: Control

Journal:

Article Title: Distinct contributions of small and large conductance Ca 2+ -activated K + channels to rat Purkinje neuron function

doi: 10.1113/jphysiol.2002.027854

Figure Lengend Snippet: A-D, spontaneous Na+ spikes were recorded in ACSF (thin traces), then in the presence of one or more channel blockers (thick traces). Each trace is an average of 10 action potentials except the thick trace in panel D, which shows a single spike burst characteristic of spontaneous activity in this condition. E, spontaneous Na+ spikes were recorded in ACSF while 10 mm BAPTA diffused into the cell from the pipette. The thin trace is an average of 10 action potentials recorded immediately after establishing the whole-cell configuration. The thick trace is a representative action potential burst recorded 3 min later. F, Na+ spike AHP amplitude was quantified as the difference between the initial voltage and the voltage 1 ms after the peak. G, the degree to which each treatment reduced the Na+ spike AHP amplitude is summarized for all groups (means ±s.e.m.). Apamin did not significantly affect the AHP, reducing it by only 2 ± 5 % (n = 5 cells). Iberiotoxin reduced the AHP by 53 ± 6 % (n = 7), paxilline by 57 ± 6 % (n = 5), cadmium by 71 ± 14 % (n = 5), BAPTA by 148 ± 29 % (n = 5) and the combination of iberiotoxin and apamin reduced the Na+ spike AHP by 106 ± 17 % (n = 5). All treatments except apamin differed significantly (P < 0.05) from the control, in which the AHP decreased in amplitude by 0 ± 9 % during 10 min in ACSF (n = 7).

Article Snippet: Channel blockers Iberiotoxin, ω-Aga-IVA and SNX-482 were obtained from Peptides International, Inc. (Louisville, KY, USA).

Techniques: Activity Assay, Transferring, Control

Journal:

Article Title: Distinct contributions of small and large conductance Ca 2+ -activated K + channels to rat Purkinje neuron function

doi: 10.1113/jphysiol.2002.027854

Figure Lengend Snippet: A, three traces from the same cell show Ca2+ spikes evoked under control conditions (left trace), in the presence of apamin (middle trace) and in the combined presence of iberiotoxin and apamin (right trace). Spikes were evoked by applying a 2 nA depolarizing current step for 3 s. B, (upper panel) five sequential Ca2+ spikes from each trace in A (spikes indicated by brackets) are shown aligned to peak time. Control traces are black, apamin traces are blue and iberiotoxin + apamin traces are green. The lower panel shows one trace from each condition on an expanded time scale. By 3 ms after the peak of the spike (dashed line), the iberiotoxin + apamin trace was 20 mV more depolarized than the apamin-only trace. C, iberiotoxin by itself (100 nm, red traces) increased the peak amplitude of Ca2+ spikes and reduced the AHP compared to control (black traces), but the subsequent addition of apamin to the iberiotoxin-containing bath (green traces) further attenuated the AHP (same scale as the upper traces in B.) D1, for all cells, Ca2+ spike AHP amplitudes were measured as the difference between the membrane potential 3 ms before the peak and the membrane potential 3 ms after the peak (when control spikes typically reached minimum voltages). Measurements are compared before and after the addition of a blocker or, in the case of the control group, before and after a 10 min time period. The effect of the different treatments is expressed as the change in AHP amplitude as a percentage of the initial AHP amplitude (100 × (treatment – control)/control). For the control group, the AHP amplitude changed by 0 ± 4 % (n = 5) over the 10 min recording period. Apamin increased the AHP amplitude by 10 ± 9 % (n = 4), but this did not differ significantly from the control group (P > 0.3). Iberiotoxin reduced the AHP amplitude by 31 ± 4 % (n = 5), while the combination of iberiotoxin and apamin reduced the AHP by 45 ± 14 % (n = 4), and both of these differed significantly from the control group (P < 0.05). D2, when BK channels were first blocked with iberiotoxin, the subsequent addition of apamin caused a further reduction of the AHP amplitude by 18 ± 5 % (n = 3) compared to iberiotoxin alone (green and black hatched bar), which was a significant effect (P < 0.05).

Article Snippet: Channel blockers Iberiotoxin, ω-Aga-IVA and SNX-482 were obtained from Peptides International, Inc. (Louisville, KY, USA).

Techniques: Control, Membrane

Journal: Scientific Reports

Article Title: The Gatekeepers in the Mouse Ophthalmic Artery: Endothelium-Dependent Mechanisms of Cholinergic Vasodilation

doi: 10.1038/srep20322

Figure Lengend Snippet: ( A ) Combined blocking with Apa and ChTX elicited total attenuation of endothelium-dependent vasodilation. ( B ) The combination blocking of the KCa channels with their respective specific blockers, Apa, TRAM-34 and IbTX, conferred negligible inhibitory effects on ACh-induced vasodilation. Values are expressed as mean ± s.e.m [n = 5–6 per group; *** P < 0.0001, L-NAME and Indomethacin vs L-NAME and Indomethacin and blocker(s)].

Article Snippet: The following drugs were used in this experiment: N ω -nitro L-arginine methyl ester (L-NAME), indomethacin, acetylcholine hydrochloride (ACh), phenylephrine, 1H-(1, 2, 4) oxadiazole (4, 3-alpha) quinoxaline-1-one (ODQ), catalase, baicalein 18 alpha-glycyrrhetinic acid (18α-GA), ouabain, glibenclamide, barium chloride (BaCl 2 ), and psora-4 [5-(4-Phenylbutoxy)psoralen] (all purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany), 17-octadecynoic acid (17-ODYA) and 1-[(2-chlorophenyl) Fdiphenylmethyl]-1H-pyrazole (TRAM-34) (Tocris Bioscience, Bristol, UK), iberiotoxin (IbTX), charybdotoxin (ChTX) and apamin (AnaSpec Inc., Fremont, CA, USA), margatoxin (MgTX), maurotoxin (MTX), α- and β-dendrotoxin (α- and β- DTX) (Alomone Labs, Jerusalem, Israel).

Techniques: Blocking Assay