Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca²⁺]i in mesenteric microvessels of pregnant rats

Abstract

Background and purpose: Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ET(A)R), but could activate ET(B)R in the endothelium and release vasodilator substances. However, the roles of ET(B)R in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear.

Experimental approach: Pressurized mesenteric microvessels from pregnant and virgin Sprague-Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca²⁺]i.

Key results: High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [Ca²⁺]i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca²⁺]i. Pretreatment with the ET(B)R antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [Ca²⁺]i, and was more potent in pregnant than in virgin rats. ET-1 + ET(A)R antagonist BQ-123, and the ET(B)R agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [Ca²⁺]i, suggesting up-regulated ET(B)R-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor Nω-nitro-L-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ET(B)R in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ET(B)R.

Conclusions and implications: The enhanced ET(B)R-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.

Figure 1

Effect of KCl (51 mM) (A, B) and PHE (10⁻⁵ M) (C, D) on vasoconstriction and [Ca²⁺]_i in mesenteric microvessels of virgin and pregnant (Preg) rats. Images of fura-2 loaded microvessels from virgin and pregnant rats were taken at rest and after stimulation with KCl or PHE. Simultaneous measurements of microvessel diameter, fura-2340 and 380 nm fluorescence signal, and 340/380 ratio were recorded. Cumulative KCl or PHE-induced vasoconstriction, 340/380 ratio, and vasoconstriction over Δ 340/340 ratio in mesenteric microvessels of virgin and pregnant rats were presented as means ± SEM, n = 8–12 different rats per group. # Measurements during stimulation with PHE (C and D) are significantly different (P < 0.05) from corresponding KCl measurements (A and B).

Figure 2

Effect of ET-1 (10⁻⁷ M) on vasoconstriction and [Ca²⁺]_i in mesenteric microvessels of virgin and pregnant rats. Simultaneous measurements of ET-1-induced changes in diameter (A, B) and fura-2 340/380 fluorescence ratio (C, D) were recorded in isolated microvessels from virgin (A, C) and pregnant rats (B, D). Cumulative ET-1-induced vasoconstriction, [Ca²⁺]_i, and vasoconstriction per Δ 340/340 ratio in mesenteric microvessels of virgin (E, G) and pregnant rats (F, H) were presented as means ± SEM, n = 8–12 different rats per group. * Measurements in pregnant rats (F and H) are significantly different (P < 0.05) from corresponding measurements in virgin rats (E and G).

Figure 3

Effect of ET_BR blockade on ET-1-induced constriction and [Ca²⁺]_i. Mesenteric microvessels of virgin and pregnant rats were either non-treated or pretreated with BQ-788 (10⁻⁶ M), then stimulated with ET-1 (10⁻⁷ M), and vasoconstriction and [Ca²⁺]_i measurements were recorded in virgin (A, C) and pregnant rats (B, D). Line graphs represent means ± SEM, n = 6 different rats per group. # Measurements in the presence of BQ-788 are significantly different (P < 0.05) from corresponding control measurements in the absence of BQ-788.

Figure 4

Effect of ACh (10⁻⁵ M) on endothelium-dependent vasodilation, and SNP (10⁻⁵ M) on endothelium-independent VSM relaxation and [Ca²⁺]_i in mesenteric microvessels of virgin (A, C) and pregnant rats (B, D). Simultaneous measurements of microvessel diameter and fura-2 340/380 fluorescence ratio were recorded, and cumulative ACh- and SNP-induced time-dependent changes in microvascular relaxation and [Ca²⁺]_i were presented. Concentration–relaxation curves to ACh (10⁻⁹–10⁻⁵ M) (E) and SNP (10⁻⁹–10⁻⁵ M) (F) were also constructed in mesenteric microvessels of virgin and pregnant rats, and the pD2 value was measured. Line graphs represent means ± SEM of vasodilation, n = 8–12 different rats per group. *P < 0.05, pregnant versus virgin.

Figure 5

Effect of ET_BR-mediated activation. Microvessels of virgin and pregnant rats were pretreated with the ET_AR antagonist BQ-123 (10⁻⁶ M) to block ET_AR-mediated vasoconstriction. Microvessels were then preconstricted with PHE (6 × 10⁻⁶ M), then stimulated with increasing concentrations of ET-1 (10⁻¹¹–10⁻⁷ M), and the % relaxation of PHE contraction (A) and underlying [Ca²⁺]_i (B) was recorded. Concentration–relaxation curves (C, E) and underlying [Ca²⁺]_i (D, F) in response to the ET_BR agonist S6c (10⁻¹¹–10⁻⁷ M) (C, D) and IRL-1620 (10⁻¹¹–10⁻⁷ M) (E, F) were compared in microvessels of virgin and pregnant rats. Line graphs represent means ± SEM of vasodilation and [Ca²⁺]_i, n = 8–12 different rats per group. *P < 0.05, pregnant versus virgin.

Figure 6

Effect of blocking NO, PGI₂ and EDHF in mesenteric microvessels of virgin and pregnant rats. Mesenteric microvessels from virgin (A) and pregnant rats (B, C, D) were preconstricted with PHE (6 × 10⁻⁶ M) and concentration–relaxation curves in response to ACh (10⁻⁹–10⁻⁵ M) (A, B), S6c (10⁻¹¹–10⁻⁷ M) (C), and IRL-1620 (10⁻¹¹–10⁻⁷ M) (D), and in the absence and presence of L-NAME (3 × 10⁻⁴ M) and INDO (10⁻⁶ M) and TEA (30 mM) or in endothelium-denuded microvessels (−Endo) were recorded. Line graphs represent means ± SEM of vasodilation, n = 8–12 different rats per group. −Endo experiments were n = 4 rats per group. *P < 0.05 versus control measurements in the absence of blockers. ^#P < 0.05 versus measurements in the presence of L-NAME + INDO.

Figure 7

Western blot analysis of ET_AR and ET_BR. Mesenteric microvessels with (+) and without (−) endothelium of both virgin and pregnant rats were homogenized and prepared for Western blot analysis of protein levels of ET_AR (A) and ET_BR (B). Bar graphs represent means ± SEM. n = 3 samples from pooled mesenteric arteries from 4 different rats per group. *P < 0.05 pregnant versus virgin. ^#P < 0.05 −Endothelium versus +Endothelium.