Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 1. LTB4 inhibits L-type calcium channel currents in A7r5 cells. (A) Representative current traces show the nifedipine sensitive L-type channel currents in A7r5 cells with/without LTB4 incubation for 12 h (middle and left). 100 nM LTB4 inhibited L-type calcium channels in a time dependent manner; Histogram plots of mean ± S.E.M. of L-type calcium channel current density normalized by control (1 h to 24 h, n = 30∼72, right). *P < 0.05 compared to control using a one-way ANOVA test. (B) Current-voltage (I-V) currents obtained from A7r5 cells with/without LTB4 for 12 h (left); I-V relationship curves generated by peak current density at each test voltage (right). Data were presented as means ± S.E.M. from 70 cells. (C) Statistical analysis of the inhibitory effects of various concentrations of LTB4 (1∼100 nM) on L-type calcium channels (n = 46∼80, *P < 0.05 compared to control using a one-way ANOVA followed by Tukey's test). © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 2. The effects of LTB4 on the steady-state activation /inactivation properties and the mRNA/protein expression of L-type calcium channels in A7r5 cells. (A) 100 nm LTB4 did not alter the steady-state activation property of L-type calcium channel currents. The current traces show the voltage-dependent L-type calcium channel current activation curves in the absence (top left) and presence (bottom left) of 100 nM LTB4. The normalized data points were fitted using the Boltzmann equation (right). (B) 100 nM LTB4 did not alter the steady-state activation kinetics of L-type calcium channel currents (right); Representative currents with/without LTB4 shown to the left. (C) 100 nM LTB4 reduced mRNA expression of CaV1.2 channels in A7r5 cells at various time points (1∼12 hour, left, *P < 0.05 compared to control using a one-way ANOVA test); 12 hour treatment with 100 nM LTB4 significantly reduced protein expression of CaV1.2 channels (right); A representative image (Upper) and the statistical analysis of Western blot from 5 independent experiments (*P < 0.05 compared to control, t test , lower). © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 3. LTB4 inhibition of L-type channel currents is mediated by the BLT1 receptor. (A) The BLT1 receptor antagonist LY29311 (5 µM) abrogated the LTB4 inhibition of CaV1.2 channels. Representative current trances were elicited by a test pulse at +10 mV from holding at −50 mV (top); Bar graph plots of means ± S.E.M. of normalized CaV1.2 current density (bottom). (B) The BLT2 receptor antagonist LY255283 (5 µM) did not change the inhibitory effect of LTB4 on CaV1.2 channels. Currents show LTB4 effect on CaV1.2 currents (top); Statistical analysis of the effects of LY255283 on the LTB4 induced inhibition of CaV1.2 channels (bottom). *P < 0.05 compared to control , †P < 0.05 compared to LY255283 alone by ANOVA. © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 4. The LTB4 inhibition of CaV1.2 channel currents is mediated by the TAK1 kinase pathway. (A) (5Z)-7-Oxozeaenol, an inhibitor of TAK1 kinase, abrogated the LTB4 inhibition of CaV1.2 channel currents (right). Sample currents show (5Z)-7-Oxozeaenol effect on the LTB4 induced inhibition of CaV1.2 currents (left). (B). The Src kinase inhibitor PP2 did not alter the inhibitory effect of LTB4 on CaV1.2 channels (right). Sample currents show 10µM PP2 effect on the LTB4 induced inhibition of CaV1.2 currents (left) (C) LY4920002, an inhibitor of PI3K did not change the LTB4 inhibition of CaV1.2 channel currents(right). Sample currents show 10 µM LY4920002 effect on the LTB4 induced inhibition of CaV1.2 currents (left). *P < 0.05 compared to control, †P < 0.05 compared to PP2/ LY4920002 alone by ANOVA. © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 5. The LTB4 inhibition of L-type channel currents is mediated by the TAK1-p38 signaling pathway. (A) The p38 specific inhibitor SB203580 abrogated the LTB4 inhibition of L-type channel currents in A7r5 cells (right). Sample currents show 10 µM SB203580 effect on the LTB4 induced inhibition of CaV1.2 currents (left). (B) The NF-κB pathway inhibitor U0126 did not change the LTB4 inhibition of CaV1.2 channel currents(right). Sample currents show 10 µM U0126 effect on the LTB4 induced inhibition of CaV1.2 currents (left). (C) JNK inhibitor Sp600125 did not alter the LTB4 inhibition of CaV1.2 channel currents(right). Sample currents show 20 µM Sp600125 effect on the LTB4 induced inhibition of CaV1.2 currents (left). *P < 0.05 compared to control , †P < 0.05 compared to U0126/ Sp600125 alone by ANOVA. © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

Leukotriene B₄ Inhibits L-Type Calcium Channels via p38 Signaling Pathway in Vascular Smooth Muscle Cells Cell Physiol Biochem 2015;37:1903-1913 - DOI:10.1159/000438551 Fig. 6. LTB4 increases p38 activity in A7r5 cells, and blockage of p38 abrogated the LTB4 induced decrease of protein expression of CaV1.2 channels. (A) LTB4 increased phosphorylation of p38 in A7r5 cells in a time-dependent manner. A representative mage (upper) and the statistical analysis of Western blot (n = 3∼8, *P < 0.05 compared to control by ANOVA, lower). (B) The p38 inhibitor SB203580 abrogated the LTB4 induced decrease of CaV1.2 protein expression. A representative image (upper) and the statistical analysis of Western blot from 6 independent experiments (*P < 0.05 compared to control , †P < 0.05 compared to LTB4 alone by ANOVA, lower). © 2015 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0