RXFP1 Receptor Activation by Relaxin-2 Induces Vascular Relaxation in Mice via a Gαi2-Protein/PI3Kß/γ/Nitric Oxide-Coupled Pathway

Abstract

Background: Relaxins are small peptide hormones, which are novel candidate molecules that play important roles in cardiometablic syndrome. Relaxins are structurally related to the insulin hormone superfamily, which provide vasodilatory effects by activation of G-protein-coupled relaxin receptors (RXFPs) and stimulation of endogenous nitric oxide (NO) generation. Recently, relaxin could be demonstrated to activate G_i proteins and phosphoinositide 3-kinase (PI3K) pathways in cultured endothelial cells in vitro. However, the contribution of the G_i-PI3K pathway and their individual components in relaxin-dependent relaxation of intact arteries remains elusive. Methods: We used Gα_i2- (Gnai2^-/-) and Gα_i3-deficient (Gnai3^-/-) mice, pharmacological tools and wire myography to study G-protein-coupled signaling pathways involved in relaxation of mouse isolated mesenteric arteries by relaxins. Human relaxin-1, relaxin-2, and relaxin-3 were tested. Results: Relaxin-2 (∼50% relaxation at 10^-11 M) was the most potent vasodilatory relaxin in mouse mesenteric arteries, compared to relaxin-1 and relaxin-3. The vasodilatory effects of relaxin-2 were inhibited by removal of the endothelium or treatment of the vessels with N (G)-nitro-L-arginine methyl ester (L-NAME, endothelial nitric oxide synthase (eNOS) inhibitor) or simazine (RXFP1 inhibitor). The vasodilatory effects of relaxin-2 were absent in arteries of mice treated with pertussis toxin (PTX). They were also absent in arteries isolated from Gnai2^-/- mice, but not from Gnai3^-/- mice. The effects were not affected by FR900359 (Gα_q protein inhibitor) or PI-103 (PI3Kα inhibitor), but inhibited by TGX-221 (PI3Kβ inhibitor) or AS-252424 (PI3Kγ inhibitor). Simazine did not influence the anti-contractile effect of perivascular adipose tissue. Conclusion: Our data indicate that relaxin-2 produces endothelium- and NO-dependent relaxation of mouse mesenteric arteries by activation of RXFP1 coupled to G_i2-PI3K-eNOS pathway. Targeting vasodilatory G_i-protein-coupled RXFP1 pathways may provide promising opportunities for drug discovery in endothelial dysfunction and cardiometabolic disease.

Keywords: ADRF; NO; RXFP1 receptor; endothelial Gαi2; perivascular-adipose tissue; relaxin-2; serelaxin.

FIGURE 1

Effects of relaxins on mesenteric arteries. (A) Original representative recording for relaxin-2 induced relaxation with (left) and without endothelium (right). (B) Original representative recording for relaxin-1 induced relaxation. (C) Original representative recording for relaxin-3 induced relaxation. PE, phenylephrine. ACh, acetylcholine. For numbers of experiments, see Figure 2.

FIGURE 2

Summary data for relaxations induced by relaxins. (A) Summary data for % relaxation (PE) by 100 pM relaxin-1 (n = 6 rings out of 5 mice), 100 pM relaxin-2 (n = 8 out of six mice), 100 pM relaxin-3 (n = 6 out of five mice), vehicle control in vessels with endothelium (n = 6 out of four mice), and vehicle control in vessels without endothelium (n = 6 out of four mice) and for % relaxation relative to ACh (100%) response. Relaxin-1 (n = 6 out of five mice), relaxin-2 (n = 8 out of six mice), relaxin-3 (n = 6 out of five mice), and vehicle control in vessels with endothelium (n = 6 out of four mice). (B) Summary data for % relaxation (PE) by 10 pM relaxin-1 (n = 6 rings out of four mice), 10 pM relaxin-2 (n = 7 out of four mice), 10 pM relaxin-3 (n = 6 out of four mice), vehicle control in vessels with endothelium (n = 6 out of four mice), and vehicle control in vessels without endothelium (n = 6 out of four mice) and for % relaxation relative to ACh (100%) response. Relaxin-1 (n = 6 out of four mice), relaxin-2 (n = 7 out of four mice), relaxin-3 (n = 6 out of four mice), and vehicle control in vessels with endothelium (n = 6 out of four mice). ^∗P < 0.05 using one-way ANOVA followed by Bonferroni multiple comparisons test; n.s., not significant.

FIGURE 3

Effects of simazine on relaxin-2 induced relaxations. (A) Original representative recording on relaxin-2 induced relaxation in the presence of 100 nM simazine. (B) Summary data for relaxin-2 induced relaxation in the presence of 100 nM simazine (n = 9 out of six mice). ^∗P < 0.05 using one-way ANOVA followed by Bonferroni multiple comparisons test; n.s., not significant.

FIGURE 4

Effects of FR900359 and L-NAME on acetylcholine and relaxin-2 induced relaxations. (A) Summary data for relaxin-2 induced relaxationsin arteries in the presence of L-NAME (100 μM, 30 min) (, n = 6 out of four mice), in the presence of FR900359 (100 nM, 30 min) (, n = 6 out of four mice), and in non-treated arteries (control group) (∙, n = 6 out of four mice). (B) Summary data for ACh-induced relaxations in arteries in the presence of L-NAME (100 μM) (, n = 6 out of four mice), in the presence of FR900359 (100 nM) (, n = 6 out of four mice), and non-treated arteries (control group) (, n = 6 out of four mice). ^∗P < 0.05 for relaxin-2 + L-NAME vs. control or ACh + L-NAME vs. non-treated vessels or ACh + FR900359 vs. non-treated vessels; repeated-measures two-way ANOVA, followed by Bonferroni post hoc test.

FIGURE 5

Effects of treatment of mice with pertussis toxin (PTX) or sham (0.9% NaCl) on relaxin-2 relaxations. (A) Original representative recording of relaxin-2 induced relaxations in arteries of PTX treated mice. (B) Original representative recording of relaxin-2 induced relaxations in arteries of 0.9% NaCl treated (control) mice. (C) Summary data of relaxin-2 induced relaxation. (D) Summary data of ACh induced relaxation. PTX group; n = 11 out of eight mice. 0.9% NaCl group; n = 7 out of four mice. ^∗P < 0.05 using unpaired t-test; n.s., not significant.

FIGURE 6

Vasorelaxant effects of relaxin-2 in Gα_i2 deficient (Gnai2^-/-), Gα_i3 deficient (Gnai3^-/-), and control arteries. (A,B) Original representative recordings. (C) Summary of data. Gα_i2 deficient arteries; n = 9 out of five mice. Control; n = 9 out of four mice. (D) Summary of data. Gα_i3 deficient arteries; n = 6 out of five mice. Control arteries; n = 7 out of four mice. ^∗P < 0.05 using unpaired t-test.; n.s., not significant.

FIGURE 7

Effects of PI3K inhibitors on relaxin-2 induced relaxation. (A) Original representative recording. (B) Summary of data. Relaxin-2-induced relaxation in the presence of 100 μM L-NAME (n = 6 out of four mice), 100 nM AS-252424 (n = 7 out of four mice), 100 nM TGX-221 (n = 7 out of four mice), or 100 nM PI-103 (n = 6 out of four mice). ^∗P < 0.05 using one-way ANOVA followed by Bonferroni multiple comparisons test; n.s., not significant.

FIGURE 8

Effects of simazine on phenylephrine (PE)-dependent contractions in the presence (+) or absence (-) of perivascular adipose tissue (PVAT). Summary data for PE induced contractions in (+) PVAT (∙, n = 8 out of four mice) or (-) PVAT (, n = 8 out of four mice) rings. Incubation of the rings with simazine (100 nM, 30 min) had no effects on contractions caused by PE in (+) PVAT (∙, n = 8 out of four mice) and (-) PVAT (, n = 9 out of 4 mice) arterial rings (P > 0.05 each). ^∗P < 0.05 for (-) PVAT vs. (+) PVAT or (-) PVAT + Simazine vs. (+) PVAT + Simazine; repeated-measures two-way ANOVA, followed by Bonferroni post hoc test.

FIGURE 9

Proposed vasodilatory pathways caused by relaxin-2. Relaxin-2 activates RXFP1 (blocked by simazine), which leads to G_i2 activation and dissociation of Gα_i2 and βγ subunits (blocked by PTX). Gβγ subunits in turn activate PI3Kβ (blocked by TGX-221) and PI3Kγ (blocked by AS-252424) to initiate eNOS activation and NO release (blocked by L-NAME) to cause relaxation. PI3Kα (blocked by PI-103) seems not to be involved in this pathway. On the other hand, ACh binds to muscarinic M3 receptors coupled to G_q/11 (blocked by FR900359) to produce eNOS/NO dependent arterial relaxation.