Computational protocol: Arachidonic Acid–Induced Dilation in Human Coronary Arterioles: Convergence of Signaling Mechanisms on Endothelial TRPV4‐Mediated Ca2+ Entry

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Protocol publication

[…] Single‐channel currents were recorded with an Axopatch 200B or an EPC‐7 amplifier as previously described. The pipette solution contained (in mmol/L) 140 NaCl, 1 MgCl2, and 10 HEPES (pH 7.4 with CsOH). The bath solution for both cell‐attached and inside‐out patches was composed of (in mmol/L) 135 KCl, 0.8 CaCl2, 1 MgCl2, 5 glucose, 5 EGTA, and 5 HEPES. The pH of the bath solution was adjusted to 7.3 with CsOH (final Cs+ concentration of 4 mmol/L) and KOH. Channel currents were recorded for at least 3 to 6 minutes under control conditions and after treatment with 4α‐PDD (1 μmol/L) or AA (1 μmol/L). All chemicals were applied by bath perfusion at the rate of 1 mL/min. Single‐channel currents were sampled at 3 to 5 kHz and filtered at 1 kHz and analyzed using pClamp 10 software. Experiments were performed at room temperature. [...] Vessels from each patient were examined with vehicle (control) and then with a pharmacological inhibitor. This experimental design allowed for paired analysis, thereby minimizing potential effects of other factors such as underlying diseases, age, and sex. Statistical comparisons of the percent of vasodilation under different treatments were performed by 2‐way (factor) repeated‐measures ANOVA (factor 1, doses; factor 2, different treatments such as vehicle or an inhibitor), followed by the Holm‐Sidak test for pairwise comparisons or comparisons versus a control. When no interaction between dose and treatment was detected, the overall dose responses between control and inhibitor groups were tested; for nonparallel profiles with a significant interaction between dose and treatment, responses to vehicle and an inhibitor were compared at each individual dose. For all other nonvascular experiments, significant differences between mean values were evaluated by paired or unpaired t tests or 1‐way ANOVA followed by the Holm‐Sidak multiple comparison test, where appropriate. In addition, a nonparametric test (Wilcoxon–Mann–Whitney rank sum test) was used for some 2‐sample experiments with a small sample size. All procedures were performed using the statistical analysis programs provided in SigmaPlot, version 12. P values <0.05 weres considered statistically significant. Data are presented as mean±SEM. For all vessel function studies, n indicates the number of patients. […]

Pipeline specifications

Software tools pCLAMP, SigmaPlot
Applications Miscellaneous, Patch-clamp
Organisms Homo sapiens
Diseases Amino Acid Metabolism, Inborn Errors