Role of E-type prostaglandin receptor EP3 in the vasoconstrictor activity evoked by prostacyclin in thromboxane-prostanoid receptor deficient mice

Abstract

Prostacyclin, also termed as prostaglandin I₂ (PGI₂), evokes contraction in vessels with limited expression of the prostacyclin receptor. Although the thromboxane-prostanoid receptor (TP) is proposed to mediate such a response of PGI₂, other unknown receptor(s) might also be involved. TP knockout (TP^-/-) mice were thus designed and used to test the hypothesis. Vessels, which normally show contraction to PGI₂, were isolated for functional and biochemical analyses. Here, we showed that the contractile response evoked by PGI₂ was indeed only partially abolished in the abdominal aorta of TP^-/- mice. Interestingly, further antagonizing the E-type prostaglandin receptor EP3 removed the remaining contractile activity, resulting in relaxation evoked by PGI₂ in such vessels of TP^-/- mice. These results suggest that EP3 along with TP contributes to vasoconstrictor responses evoked by PGI₂, and hence imply a novel mechanism for endothelial cyclooxygenase metabolites (which consist mainly of PGI₂) in regulating vascular functions.

Figure 1. Mutation in TP^−/− mice and phenotype.

(a) mutation in the TP locus. Top: WT DNA sequencing showing the surrounding sequences and the fragment to be deleted (underlined) in TP^−/− mice. Bottom left: sequencing of mutated DNA showing the deletion of 22 bp DNA fragment in TP^−/− mice. The bar separates the upper and down stream sequences of the deleted fragment. Bottom right: partial sequences of TP mRNA transcripts or those of proteins to be translated in WT (upper) and TP^−/− mice (lower). (b) RT-PCR showing the expressions of un-mutated mRNAs in WT and TP^−/− mouse aortas. Bands were visualized with a SYBR Safe DNA gel stain (Thermo Scientific) and the image was captured by an electrophoresis imaging cabinet (Universal Hood II; Bio-rad, Hercules, CA, USA). M: 100 bp ladder size marker (Thermo Scientific). (c) bleeding time in TP^−/− and WT mice. Values are expressed as mean ± SEM; n = 5; **P < 0.01.

Figure 2. Responses to U46619 and PGs in L-NAME-treated WT and TP^−/− abdominal aortas.

(a,b) comparison of contractions evoked by the TP agonist U46619 (a), and PGI₂ or PGF_2α (b) in WT and TP^−/− vessels. (c,d) contraction to PGE₂ (c) or PGD₂ (d) in WT or TP^−/− mice and that of TP^−/− vessels treated with the TP antagonist SQ29548 (10 μM; +SQ) or the EP3 antagonist L798106 (1 μM; +L). Values are expressed as mean ± SEM; n = 5 for each. **P < 0.01 vs. the value of WT mice; ⁺⁺P < 0.01 vs. TP^−/− mice.

Figure 3. Effect of EP3 antagonism on the response to PGI₂ or PGE₂ in PE-pre-contracted TP^−/− abdominal aortas.

(a) summaries (n = 5 for each) of responses (top) to 1 μM PGI₂ and forces of PE-evoked contractions (pre-force; bottom) in control L-NAME-treated TP^−/− vessels or those additionally with the EP3 antagonist L798106 (1 μM; +L) or the EP1 antagonist SC19220 (10 μM; +SC). (b) representative traces with summarized values showing the control response to PGI₂ in endothelium-denuded TP^−/− vessels [TP^−/−/EC (−)] or that with L798106 (+L). *P < 0.05. (c) summaries (n = 5 for each) of responses (top) to 0.1 μM PGE₂ and forces of PE-evoked contractions (bottom) as in (a). **P < 0.01 vs. control value of TP^−/− vessels. In (a–c), *P < 0.05 or **P < 0.01 vs. control value of TP^−/− vessels. Data are expressed as mean ± SEM. (d) representative traces showing the response to PGE₂ in L-NAME-treated TP^−/− vessels (TP^−/−) or that obtained with L798106 (+L).

Figure 4. Responses to ACh in L-NAME-treated TP^−/− abdominal aortas or those of WT mice with TP inhibited.

(a) representative traces with summarized values showing responses to ACh (10 μM) under baseline conditions in WT (top) and TP^−/− (bottom) vessels. P < 0.01 vs. WT vessels (b,c) representative traces (b) and/or summaries of time-courses of responses to ACh (c top) along with forces of PE-evoked contractions (pre-force; c bottom) in precontracted TP^−/− vessels or those obtained with the non-selective COX inhibitor indomethacin (10 μM; +IND), with the EP3 antagonist L798106 (1 μM; +L) or with both L789106 and the IP antagonist CAY10441 (1 μM; +L/CAY). (d) time-courses of responses to ACh (top) and forces of PE-evoked contractions (pre-force; bottom) in precontracted WT vessels in the presence of the TP antagonist SQ29548 (WT/SQ) or in those additionally treated with L798106 (1 μM; +L) or both L798106 and indomethacin (+L/IND). In (c and d) **P < 0.01 vs. TP^−/− or WT/SQ; ⁺⁺P < 0.01 vs. TP^−/−/L or +L. Data were expressed as mean ± SEM (n = 5 for each).

Figure 5. Effect of TP^−/− on COX products and IP, EP3 and FP mRNA levels.

(a–c) summaries of the PGI₂ metabolite 6-keto-PGF_1α (a), PGE₂ (b), and the TxA₂ metabolite TxB₂ (c) in TP^−/− and WT aortas under the basal and ACh (10 μM)-stimulated conditions. (d) real-time PCR detection of IP, EP3 and FP mRNAs in TP^−/− and WT aortas. The level of mRNAs was normalized by that of β-actin with the average value of WT assumed as 1.0. *P < 0.05 and **P < 0.01; NS: not significant. Data are expressed as mean ± SEM (n = 6 for each).

Figure 6. Effects of EP3 antagonism on vasoconstrictor responses in L-NAME-treated WT vessels.

(a) control (CTL) responses evoked by PGI₂ under baseline conditions in the abdominal aorta (AAo), and that obtained with the EP3 antagonist L798106 (1 μM; +L) (b) responses (top) to PGI₂ (1 μM) or AA (3 μM) and forces of PE-evoked contractions (pre-force; bottom) in pre-contracted AAo treated with the TP antagonist SQ29548 (10 μM; WT/SQ) or those additionally with L798106 (1 μM; +SQ/L) or both L789106 and the IP antagonist CAY10441 (1 μM; +SQ/L/CAY). **or ⁺⁺P < 0.01 vs. WT/SQ or +SQ/L, respectively. (c) effect of L789106 (1 μM; +L) or SQ29548 (10 μM; +SQ) on the contraction to PGE₂ (10 μM; PGE₂) or ACh (10 μM) in AAo. (d) effect of L789106 (1 μM; +L) on contraction to 1 or 10 μM PGI₂ in carotid (CA) and renal arteries (CA), respectively. In (a,c and d) *P < 0.05, and **P < 0.01 vs. WT control (WT/CTL). Data are expressed as mean ± SEM (n = 5 for each).