Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: List of used drugs and their pharmacological characteristics .

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: (A) Representative confocal micrographs of rat right atrium and SA node sections showing immunofluorescence labeling for K IR 3.1 (GIRK1), K Ca 2.2 (SK2), K Ca 2.3 (SK3), and Ca V α 2 δ−1 channel subunits (first and third columns); the corresponding differential interference contrast (DIC) images are also shown for comparison (second and fourth columns). Last row shows cross-reactivity of secondary antibodies, Alexa Fluor 488 anti-rabbit (AF488 anti-Rb), and Alexa Fluor 568 anti-goat (AF568 anti-Gt), in which primary antibodies were omitted (see Table ). During documentation the settings on the confocal microscope were adjusted appropriately to show immunoreactivity for sections containing both primary and secondary antibodies and these settings were maintained when documenting cross-reactivity of secondary antibodies ran in parallel to minimize biases during capture and printing of digital images. White arrows indicate SA node arteries. Similar results were obtained in five additional experiments. Horizontal bar = 30 μm. (B) Tridimensional surface modeling representing immunoreactivity of images depicted in panel (A) . Color bar represents relative fluorescence intensity map. (C) Graph depicting semi-quantitative analysis of K IR 3.1, K Ca 2.2, K Ca 2.3, and Ca V α 2 δ−1 expression in right atria and SA node; the ordinates are immunofluorescence intensity ratio between K IR 3.1, K Ca 2.2, K Ca 2.3, and Ca V α 2 δ−1 staining in paired samples from the right atrium and SA node keeping the image acquisition settings constant. Positive and negative values indicate staining predominance in contractile myocardium and SA node of the right atrium, respectively. Values are mean ± SEM; at least 3 microscopic fields were analyzed per section of the right atrium and SA node obtained from three to five rats.

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Immunofluorescence, Labeling, Microscopy, Fluorescence, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: Effects of adenosine A 1 receptor activation on whole-cell voltage-clamp recordings in rat atrial myocytes. (Ai) Representative currents following a set of voltage pulses (260 ms), covering a wide range of potentials, with incremental depolarization steps (10 mV) from −130 to +60 mV (holding voltage −70 mV) (see inset). (Aii) Corresponding current density-voltage relationship showing three different components in terms of voltage dependence: a strong inward rectifier, an inward “hump,” and a delayed outward current. Data are expressed as mean ± SEM of four animals; recordings from three to five isolated atrial cardiomyocytes were averaged per experimental animal. (B) Representative current traces from a triple set of double depolarizing pulses: one first step to −40, −10, and +20 mV, lasting 50 ms, followed by a second pulse to +40 mV, lasting 750-ms (see inset). One can notice a larger outward current at +40 mV when preceded by a prepulse to −10 mV, which suggests a Ca 2+ -dependent current component. Panels (C,D) show current-voltage relationships obtained from currents recorded following a set of voltage steps (−130 to 0 mV, 10 mV steps, holding voltage −70 mV, 260 ms duration each) in the absence (Control) and in the presence of R-PIA (300 nM, Ci ) and tertiapin Q (300 nM, Di ) with or without R-PIA (300 nM). Data are expressed as mean ± SEM of three animals; recordings from four to five isolated atrial cardiomyocytes were averaged per experimental animal. Panel (Cii) shown are typical recording traces showing that activation of the adenosine A 1 receptor with R-PIA (300 nM) increases the Ca 2+ -dependent outward current obtained following application of the double-pulse protocol consisting of one pre-pulse of 50 ms duration to −10 mV immediately followed by a second pulse to +40 mV lasting 750-ms (see inset). This experiment was repeated using four cardiomyocytes isolated from three different animals (right-hand side panel); * P < 0.01 (paired Student's t -test) represent significant differences from control. (Ciii) Refers to normalized values of slope conductance (calculated with measurements of −120 to −80 mV) in cells obtained from three different animals in the absence (Control) and in the presence of R-PIA (300 nM). Panel (Dii) shows similar experiments as (Ciii) , but in this case tertiapin Q (300 nM) with or without R-PIA (300 nM) was used instead of R-PIA alone. Error bars represent SEM of three animals. * , # P < 0.05 (unpaired Student's t -test with Welch's correction) represent significant differences from control or from tertiapin Q alone, respectively.

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Activation Assay, Isolation

Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: Activation of adenosine A 1 receptors inhibits Ca 2+ -activated outward K Ca 2/SK current in isolated rat atrial myocytes when inwardly rectifying GIRK/K IR 3 channels are blocked with tertiapin Q. (Ai) Current-voltage relationship showing strong inward rectification obtained from currents recorded following a set of voltage steps (−130 to 0 mV, 10 mV steps, holding voltage −70 mV, 260 ms duration each) in the absence (Control) and in the presence of tertiapin Q (300 nM), apamin (30 nM) and R-PIA (300 nM), applied cumulatively (see the inset for representative currents). Data are expressed as mean ± SEM of three different animals; recordings from three to five isolated atrial cardiomyocytes were averaged per experimental animal. (Aii) Bar-graphs representing pooled data from three different animals in which the average slope conductance of the inward current was normalized to the maximum obtained in the control situation, without test drugs. In panel (B) shown are representative whole-cell voltage-clamp recording traces from double depolarization protocol held at holding potential of −70 mV using the same cell as in (Ai) . Currents were elicited by a single 10 ms depolarizing pulse from −70 to −10 mV followed by a 750-ms pulse to +40 mV (see inset in panel Bi ). Panel (Bi) shows the effects on Ca 2+ -activated outward K Ca 2/SK currents of cumulative applications of tertiapin Q (300 nM), apamin (30 nM), and R-PIA (300 nM). Sodium currents were truncated to facilitate visualization of effects. (Bii) Bar-graphs representing pooled data from three similar experiments in which peak current was normalized to the maximum current obtained in the absence of added drugs (Control). All experiments were performed in the presence of E4031 (10 μM) to prevent rapid delayed rectifier potassium currents operated by hERG channels from being activated. Error bars represent SEM of three animals. * , # P < 0.05 (unpaired Student's t -test with Welch's correction) represent significant differences from the control or from tertiapin Q alone, respectively; ns, not significant.

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Activation Assay, Isolation

Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: Concentration-response curves of the Ca v 1 (L-type) channel inhibitor, verapamil (0.03–10 μM), on the spontaneously beating rat atria in the absence (Control) and in the presence of apamin (30 nM, A,B) and tertiapin Q (300 nM, C,D), which selectively block K Ca 2/SK and GIRK/K IR channels, respectively . Drug applications followed the protocol depicted in Figure . The ordinates are percentage of variation of spontaneous contraction rate (chronotropic effect, A,C ) and mechanical tension (inotropic effect, B,D ) as compared to baseline values obtained before application of verapamil. The data are expressed as mean ± SEM from an n number of individual experiments.

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Concentration Assay, Blocking Assay

Journal: Frontiers in Pharmacology

Article Title: Ion Fluxes through K Ca 2 (SK) and Ca v 1 (L-type) Channels Contribute to Chronoselectivity of Adenosine A 1 Receptor-Mediated Actions in Spontaneously Beating Rat Atria

doi: 10.3389/fphar.2016.00045

Figure Lengend Snippet: Concentration-response curves of the K Ca 2/SK channel blocker, apamin (0.003–1 μM), on the spontaneously beating rat atria in the absence (Control), and in the presence of verapamil (1 μM) . Drug applications followed the protocol depicted in Figure . The ordinates are percentage of variation of spontaneous contraction rate (chronotropic effect, A ) and mechanical tension (inotropic effect, B ) as compared to baseline values obtained before application of apamin. The data are expressed as mean ± SEM from an n number of individual experiments. * P < 0.05 compared with the effect of apamin in the absence of verapamil.

Article Snippet: Small-conductance Ca 2+ -activated K + channel (K Ca 2.2) , AP10032PU-N , Goat (gt) , 1:400 , Acris antibodies.

Techniques: Concentration Assay