Journal:

Article Title: Tachykinin-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK 2 receptors

doi: 10.1038/sj.bjp.0703708

Figure Lengend Snippet: Concentration-response curves for (a) the NK1 receptor-selective agonist, [Sar9,Met(O2)]-SP (Sar; n=3), (b) the NK2 receptor-selective agonists, ([Nle10]-NKA(4–10) (Nle; n=14), and GR 64,349 (n=5) and (c) the NK3 receptor-selective agonists, [MePhe7]-NKB (MePhe; n=4) and senktide (n=3). Vertical lines show s.e.mean. Error bars that are not shown lie within the dimensions of the symbol in this and all other figures.

Article Snippet: Drugs and solutions [Sar 9 ,Met(O 2 ) 11 ]-SP, NKA, [Nle 10 ]-NKA(4–10), [MePhe 7 ]-NKB, senktide, amastatin and DL -thiorphan were purchased from Auspep (Melbourne, Australia).

Techniques: Concentration Assay

Journal:

Article Title: Tachykinin-induced contraction of the guinea-pig isolated oesophageal mucosa is mediated by NK 2 receptors

doi: 10.1038/sj.bjp.0703708

Figure Lengend Snippet: Concentration-response curves for the NK2 receptor-selective agonists (a) GR 64,349 alone (n=5) and in the presence of the NK2 receptor-selective antagonist, GR 159,897 (n=5), and (b) [Nle10]-NKA(4–10) (Nle) alone (n=14) and in the presence of the NK2 receptor-selective antagonist, SR 48,968 (n=4 for all concentrations, except for 30 nM, where n=3).

Article Snippet: Drugs and solutions [Sar 9 ,Met(O 2 ) 11 ]-SP, NKA, [Nle 10 ]-NKA(4–10), [MePhe 7 ]-NKB, senktide, amastatin and DL -thiorphan were purchased from Auspep (Melbourne, Australia).

Techniques: Concentration Assay

Journal: Circulation Research

Article Title: Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

doi: 10.1161/CIRCRESAHA.119.315641

Figure Lengend Snippet: The NKA (Na + , K + -ATPase) and Na + -Ca 2+ exchanger (NCX) are robustly expressed within the t-tubules of both ventricular and atrial cardiomyocytes. A , Immunolabeling in intact tissue and isolated ventricular cells showed a robust expression of the α 1 NKA isoform in both the surface membrane and t-tubules, while the α 2 isoform was preferentially expressed in t-tubules. B , In atrial cells, NKA α 1 was expressed in both the surface membrane and, when present, the t-tubules. NKA α 2 expression was low in atrial cells, never present in t-tubules, and appeared limited to the intercalated disks. NCX (right) was robustly expressed in both the surface membrane and t-tubules of ventricular and atrial cells. C , In ventricular cells, preferential blockade of NKA α 2 with 0.3 µmol/L ouabain inhibited the biphasic response of Ca 2+ transients to hypokalemia (5 of 5 cells). In atrial cells, both biphasic (7 cells) and monophasic responses (3 cells) continued to be observed in the presence of low-dose ouabain, in agreement with limited α 2 expression in these cells. Scale bars in ( A ) and ( B )=10 µm in zoomed-out images, 2 µm in enlargements.

Article Snippet: The following primary and secondary antibodies were used at the indicated dilutions: NCX (Swant, R3F1, 1:100), NKA-α1 (Merck Millipore, 05-369, 1:100), NKA-α2 (Merck Millipore, 07-674, 1:100), F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 488, Thermo Fisher, A-11017, 1:200), and F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 546, Thermo Fisher, A-11071, 1:200).

Techniques: Immunolabeling, Isolation, Expressing, Membrane

Journal: Circulation Research

Article Title: Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

doi: 10.1161/CIRCRESAHA.119.315641

Figure Lengend Snippet: Decreased Na + , K + -ATPase (NKA) activity during hypokalemia inhibits Ca 2+ extrusion by t-tubular Na + -Ca 2+ exchanger (NCX), elevating sarcoplasmic reticulum (SR) Ca 2+ content. A , NKA activity was measured during hyperpolarizing voltage ramps and calculated as the K + -sensitive current (see Online Figure I for protocol and representative traces). Hypokalemia induced a modest and similar reduction in NKA current in ventricular myocytes and atrial cells, regardless of the presence of t-tubules (n cells =10, 7, 9; n hearts =4, 4, 5 in ventricular, tubulated atrial, untubulated atrial cells). B , SBFI experiments revealed no change in global cytosolic [Na + ] during the protocol (n cells =12, 8; n hearts =4, 4 for ventricular, atrial cells). C , However, tubulated cells showed slowed Ca 2+ removal by NCX during hypokalemia, as indicated by the declining phase of Ca 2+ transients stimulated in the continuous presence of 10 mmol/L caffeine (change in tau values shown at right, n cells =8, 16; n hearts =3, 5, in ventricular, tubulated atrial cells, P =0.016, 0.011 vs K o =5.0 by Wilcoxon signed-rank test). No change in Ca 2+ removal rate was observed in untubulated atrial cells (n cells =16; n hearts =5; P =0.912). Transient magnitude during K o =2.7+caffeine: F/F 0 =1.46±0.07, 1.66±0.12, 1.57±0.08 in ventricular, tubulated, and atrial cells. D , Ventricular cells and tubulated atrial cells exhibited increased SR Ca 2+ content during hypokalemia, as assessed by the magnitude of caffeine-induced Ca 2+ release (n cells =8, 7, 7; n hearts =6, 6, 5 in ventricular, tubulated atrial, untubulated atrial cells; representative traces illustrated in Online Figure VI). Statistics: ( A ): 2-way repeated measures ANOVA with Bonferroni correction (see Online Table II for full results); ( B ): Kruskal-Wallis test (difference in medians: P =1.000, 1.000 in ventricular, atrial cells); ( C ): Kruskal-Wallis test with Dunn correction (difference in medians: P =0.023); ( D ): paired t -test. * P <0.05 vs K o =5.0.

Article Snippet: The following primary and secondary antibodies were used at the indicated dilutions: NCX (Swant, R3F1, 1:100), NKA-α1 (Merck Millipore, 05-369, 1:100), NKA-α2 (Merck Millipore, 07-674, 1:100), F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 488, Thermo Fisher, A-11017, 1:200), and F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 546, Thermo Fisher, A-11071, 1:200).

Techniques: Activity Assay

Journal: Circulation Research

Article Title: Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

doi: 10.1161/CIRCRESAHA.119.315641

Figure Lengend Snippet: Close Na + , K + -ATPase (NKA)-Na + -Ca 2+ exchanger (NCX) crosstalk drives the biphasic Ca 2+ transient response to hypokalemia . A mathematical model of the rat ventricular cardiomyocyte was employed to investigate the contribution of various ion channels and transporters to changes in cellular [Na + ], SR Ca 2+ content, and Ca 2+ transients. A , With baseline model settings (blue lines), reducing [K + ] o from 5.0 to 2.7 mmol/L triggered a modest accumulation of intracellular [Na + ], and a biphasic response of SR Ca 2+ content and Ca 2+ transients which occurred over a markedly longer time course than that observed experimentally. To simulate greater crosstalk between NCX and NKA, the fraction of NCX operating in reverse mode (NCX rev ) was increased in a step-wise manner, which produced an augmented and accelerated biphasic response (see also enlargement of the first 1000 s in [ B ], presented normalized to [K + ] o =5.0). Removing the NCX rev contribution prevented any secondary rise in Ca 2+ transients, in resemblance to experiments in untubulated cells. C , Sensitivity analyses were performed to examine the effects of modulating individual ion fluxes±5%. Reducing t-tubule-associated fluxes such as I Na (g Na ) or the number of L-type-RyR units (LCC-RYR) did not inhibit the biphasic response, while changing NCX and NKA fluxes had proportionally opposite effects. D , In addition to augmenting the biphasic response to hypokalemia, increasing NCX rev slowed Ca 2+ transient decay at steady state.

Article Snippet: The following primary and secondary antibodies were used at the indicated dilutions: NCX (Swant, R3F1, 1:100), NKA-α1 (Merck Millipore, 05-369, 1:100), NKA-α2 (Merck Millipore, 07-674, 1:100), F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 488, Thermo Fisher, A-11017, 1:200), and F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 546, Thermo Fisher, A-11071, 1:200).

Techniques: Produced

Journal: Circulation Research

Article Title: Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

doi: 10.1161/CIRCRESAHA.119.315641

Figure Lengend Snippet: Schematic overview . Distinct mechanisms were observed to promote arrhythmogenesis in ventricular and atrial cardiomyocytes. DAD indicates delayed afterdepolarization; EAD, early afterdepolarization; NCX, Na + -Ca 2 + exchanger; and NKA, Na + , K + -ATPase.

Article Snippet: The following primary and secondary antibodies were used at the indicated dilutions: NCX (Swant, R3F1, 1:100), NKA-α1 (Merck Millipore, 05-369, 1:100), NKA-α2 (Merck Millipore, 07-674, 1:100), F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 488, Thermo Fisher, A-11017, 1:200), and F(ab’)2-goat anti-mouse IgG (H+L) secondary antibody (Alexa Fluor 546, Thermo Fisher, A-11071, 1:200).

Techniques: