Nathan R. Tykocki, PhD, BinXi Wu, BS, William F

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Divergent signaling mechanisms for venous versus arterial contraction as revealed by endothelin-1 Nathan R. Tykocki, PhD, BinXi Wu, BS, William F. Jackson, PhD, Stephanie W. Watts, PhD Journal of Vascular Surgery Volume 62, Issue 3, Pages 721-733 (September 2015) DOI: 10.1016/j.jvs.2014.03.010 Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 1 Effects of the phospholipase C (PLC) inhibitor U-73122 (1 μM) on endothelin-1 (ET-1)-induced contraction in aorta and vena cava. Measurement of ET-1-induced contraction in rat aorta and vena cava in the presence or absence of 1 μM U-73122 (A and B) or its inactive analogue U-73343 (1 μM) (C and D). Vehicle (0.01% dimethyl sulfoxide [DMSO]) or antagonists were incubated with tissue for 1 hour before agonist exposure. Points represent mean ± standard error of the mean for the number of animals indicated in parentheses. NE, Norepinephrine; PE, phenylephrine. *P < .05 vs vehicle. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 2 Effects of the phospholipase C (PLC) inhibitor U-73122 (10 μM) on endothelin-1 (ET-1)-induced contraction in aorta and vena cava. Measurement of ET-1-induced contraction in rat aorta and vena cava exposed to vehicle and 10 μM U-73122 (A and B) or its inactive analogue U-73343 (10 μM) (C and D). Vehicle (0.1% dimethyl sulfoxide [DMSO]) or antagonists were incubated with tissue for 1 hour before agonist exposure. Points represent mean ± standard error of the mean for the number of animals indicated in parentheses. NE, Norepinephrine; PE, phenylephrine. *P < .05 vs vehicle. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 3 Representative Western blot analysis of inositol trisphosphate receptor (IP3R) protein expression. A-C, Representative Western blot analysis of IP3 receptor protein expression from 50-μg whole-tissue homogenate from rat aorta (RA) and vena cava (RVC). Homogenates from rat brain (Br) and liver (L) are also included as controls. Blots were probed with antibodies against IP3R-1 (A), IP3R-2 (B), and IP3R-3 (C) as well as β-actin (loading control). Representative of more than three experiments for each receptor. D and E, Summary bar graphs of IP3 receptor densitometry in aorta (D) and vena cava (E), normalized to β-actin (loading control). *P < .05 (one-way analysis of variance and Tukey post hoc comparison). n.s., Not significant. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 4 Representative immunohistochemical staining for each of the three inositol trisphosphate receptor (IP3R) subtypes in freshly dissociated smooth muscle cells from rat aorta. A-C, Red fluorescence indicates the presence of punctate fluorescent staining (inset) for IP3R-1 (A), IP3R-2 (B), and IP3R-3 (C) protein. D-F, Green fluorescence indicates staining for smooth muscle α-actin. G-I, Overlay of IP3R (red), smooth muscle α-actin (green), and DAPI nuclear stain (blue). J-L, Negative controls, in which primary antibodies were absent. Representative of three experiments. N.P., No primary antibody. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 5 Representative immunohistochemical staining for each of the three inositol trisphosphate receptor (IP3R) subtypes in freshly dissociated smooth muscle cells from rat vena cava. A-C, Red fluorescence indicates the presence of punctate fluorescent staining (inset) for IP3R-1 (A), IP3R-2 (B), and IP3R-3 (C) protein. D-F, Green fluorescence indicates staining for smooth muscle α-actin. G-I, Overlay of IP3R (red), smooth muscle α-actin (green), and DAPI nuclear stain (blue). J-L, Negative controls, in which primary antibodies were absent. Representative of three experiments. N.P., No primary antibody. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 6 Rat aorta and vena cava contract to the membrane-permeable inositol trisphosphate (IP3) analogue Bt-IP3, and effects of 2-aminoethoxydiphenylborane (2-APB) on endothelin-1 (ET-1)-induced contraction. A and B, Representative tracings of rat aorta (A) and vena cava (B) contraction during exposure to Bt-IP3, a membrane-permeable analogue of IP3. Shown are responses from tissues incubated with vehicle (0.1% dimethyl sulfoxide [DMSO]) (A and B, left) and 10 μM Bt-IP3 (A and B, right). Representative of seven experiments. C and D, Summary bar graphs of contraction in aorta and vena cava to 10 μM Bt-IP3. Black bars represent maximal contraction to 10 μM Bt-IP3. White bars represent maximum contraction to vehicle (1% DMSO/0.1% Pluronic). Bars represent mean ± standard error of the mean for the number of animals indicated in parentheses. E and F, Contractile response to increasing concentrations of ET-1 in rat aorta (E) and vena cava (F), in the presence or absence of the IP3 receptor antagonist 2-APB (100 μM). Vehicle or antagonists were incubated with tissue for 1 hour before ET-1 exposure. NE, Norepinephrine; PE, phenylephrine. *P < .05 vs vehicle. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 7 1-Oleoyl-2-acetyl-sn-glycerol (OAG)-induced contraction in aorta and vena cava and protein kinase C (PKC) dependence. A and B, Representative tracings of aorta and vena cava contractions to increasing concentrations of OAG (0.01-100 μM, marked by arrows). C and D, Measurement of OAG-induced contraction in aorta and vena cava. E, Summary bar graph representing relaxation of OAG-induced contraction in aorta and vena cava in the presence of chelerythrine (10 μM). Black bars represent maximal contraction to OAG (100 μM). White bars represent maximum contraction to OAG after addition of chelerythrine (10 μM). F and G, Contractile responses to increasing concentrations of endothelin-1 (ET-1) in rat aorta (F) and vena cava (G) in the presence or absence of the PKC antagonist chelerythrine (10 μM). Vehicle (0.1% dimethyl sulfoxide [DMSO]) or antagonists were incubated with tissue for 1 hour before ET-1 exposure. In C-F, points and bars represent mean ± standard error of the mean for the number of animals indicated in parentheses. Chel, Chelerythrine; NE, norepinephrine; PE, phenylephrine. *P < .05 vs vehicle. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 8 Cartoon depicting differences in arterial vs venous endothelin-1 (ET-1) signaling. In aorta, ET-1-induced contraction is largely dependent on phospholipase C (PLC) activation and IP3-mediated Ca2+ release. In vena cava, however, IP3 receptors are minimally involved in ET-1 signaling. Instead, diacylglycerol (DAG), either directly or by activating protein kinase C (PKC), may increase the influx of calcium through calcium-permeable ion channels and initiate contraction. However, the exact mechanisms by which DAG and protein kinase can influence contraction remain relatively unknown and may be independent of calcium channel activation. CC, Ca2+-permeable ion channel; ER, endoplasmic reticulum; ETR, endothlin-1 receptor; IP3, 1,4,5-inositol trisphosphate; PIP2, phosphatidylinositol bisphosphate; RyR, ryanodine receptor; SERCA, smooth endoplasmic reticulum Ca2+ adenosinetriphosphatase. Journal of Vascular Surgery 2015 62, 721-733DOI: (10.1016/j.jvs.2014.03.010) Copyright © 2015 Society for Vascular Surgery Terms and Conditions