GPER Regulates Endothelin-Dependent Vascular Tone and Intracellular Calcium

Abstract

Aims

An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca²⁺]_i) is essential for endothelin-1 (ET-1)-induced vasoconstriction. Based on previous findings that activation of the G protein-coupled estrogen receptor (GPER) inhibits vasoconstriction in response to ET-1 and regulates [Ca²⁺]_i in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca²⁺]_i and constriction of intact arteries.

Main methods

Pressurized carotid arteries of GPER-deficient (GPER⁰) and wildtype (WT) mice were loaded with the calcium indicator fura 2-AM. Arteries were stimulated with the GPER-selective agonist G-1 or solvent followed by exposure to ET-1. Changes in arterial diameter and VSMC [Ca²⁺]_i were recorded simultaneously. Vascular gene expression levels of ET_A and ET_B receptors were determined by qPCR.

Key findings

ET-1-dependent vasoconstriction was increased in arteries from GPER⁰ compared to arteries from WT mice. Despite the more potent vasoconstriction to ET-1, GPER deficiency was associated with a marked reduction in the ET-1-stimulated VSMC [Ca²⁺]_i increase, suggesting an increase in myofilament force sensitivity to [Ca²⁺]_i. Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca²⁺]_i responses to ET-1, and expression levels of ET_A or ET_B receptor were unaffected by GPER deficiency.

Significance

These results demonstrate that endogenous GPER inhibits ET-1-induced vasoconstriction, an effect that may be associated with reduced VSMC Ca²⁺ sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system.

Introduction

Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor known [1] that regulates basal vascular tone, and contributes to the development of vascular diseases, such as arterial hypertension and atherosclerosis through stimulation of cell growth and inflammation [2]. ET-1 is the predominant isoform of the endothelin peptide family, which acts in an autocrine and paracrine manner via G protein-coupled endothelin receptors ET_A and ET_B [2, 3]. Whereas ET_A receptors mediate vasoconstriction and cell proliferation, ET_B receptors primarily facilitate the release of nitric oxide and ET-1 clearance [2, 3]. In vascular smooth muscle cells (VSMC), ET_A receptor activation by ET-1 induces an increase in intracellular VSMC calcium concentration (VSMC [Ca²⁺]_i), which is the intracellular messenger that triggers subsequent vasoconstriction [3, 4].

Natural estrogens, such as 17β-estradiol, have been implicated in protection from cardiovascular diseases, including arterial hypertension and atherosclerosis [5, 6]. 17β-Estradiol inhibits ET-1-dependent vasoconstriction in several vascular beds [7–11], possibly via inhibition of Ca²⁺ influx and/or stimulation of Ca²⁺ efflux in VSMC [7, 12]. 17β-Estradiol is a non-selective activator of estrogen receptors ERα and ERβ, which mainly function as ligand-activated transcription factors [13]. More recently, 17β-estradiol has also been shown to bind to and activate the G protein-coupled estrogen receptor GPER (previously termed GPR30), an intracellular seven-transmembrane spanning receptor [14]. GPER is expressed throughout the vascular system of humans and animals of both sexes [13, 15], and animals lacking the GPER gene display increased endothelium-dependent contractility [16]. Moreover, the GPER-selective agonist G-1 [17] induces acute vasodilation and indirectly inhibits the response to several vasoconstrictors including ET-1 [13, 15, 18–20]. Other GPER agonists such as selective estrogen receptor modulators (SERMs) including raloxifene, the selective estrogen receptor downregulator (SERD) fulvestrant, or the phytoestrogen genistein also inhibit vasoconstriction to ET-1 [13, 15, 19, 21, 22]. Although selective GPER activation regulates [Ca²⁺]_i in VSMC cells [18] and other cell types [13], it is unknown whether and to what extent endogenous GPER modulates the VSMC [Ca²⁺]_i response to vasoconstrictors like ET-1 in intact arteries. To test the hypothesis that endogenous GPER inhibits ET-1-induced arterial constriction by regulating changes in VSMC [Ca²⁺]_i, we simultaneously measured changes in vascular tone and VSMC [Ca²⁺]_i in response to ET-1 in arteries from wildtype (WT) and GPER-deficient (GPER⁰) mice. In addition, we determined the acute effects of selective GPER activation by G-1 on vascular responses.

Materials and Methods

Materials

Fura 2-AM (fura 2-acetoxymethyl ester) and pluronic acid were from Invitrogen (Carlsbad, CA, USA), and ET-1 was from Enzo Life Sciences (Farmingdale, NY, USA). G-1 was synthesized as described [17] (provided by Jeffery Arterburn, New Mexico State University, Las Cruces, NM, USA), and dissolved in ethanol. G-1 and ET-1 were diluted in bicarbonate buffered physiological saline solution (PSS, composition in mmol/L: 129.8 NaCl, 5.4 KCl, 0.83 MgSO₄, 0.43 NaH₂PO₄, 19 NaHCO₃, 1.8 CaCl₂, and 5.5 glucose) to the concentrations required before use.

Animals

Male C57Bl6 (3 months of age, The Jackson Laboratory, Bar Harbor, ME) and GPER⁰ mice (Proctor & Gamble, Cincinnati, OH, provided by Jan S. Rosenbaum) were housed at the animal research facility of the University of New Mexico Health Sciences Center. GPER⁰ mice were bred as described [23] and backcrossed 10 generations onto C57Bl6 mice. Animals were maintained under controlled temperature of 22–23 °C on a 12h light, 12h dark cycle and fed normal chow ad libitum. All procedures were approved by and carried out in accordance with institutional policies and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolated Vessel Preparation and Experimental Setup

Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (2.2 mg/g BW) and exsanguinated by cardiac puncture. Common carotid arteries were excised, immediately placed into PSS and carefully dissected from adherent tissue. A subset of carotid arteries was snap-frozen in liquid nitrogen and stored at −80 °C until further analysis. For functional experiments, carotid arteries were transferred to a vessel chamber (Living Systems, St. Albans, VT, USA), cannulated on glass micropipettes at each end, and secured with silk ligatures. Arteries were pressurized to 90 mmHg with PSS using a servo-controlled peristaltic pump (Living Systems), and superfused with PSS (37 °C, pH 7.4, oxygenated with 21% O₂, 5% CO₂, and balanced N₂) at a rate of 5 mL per minute as described [24]. To avoid flow-dependent changes in vessel diameter, experiments were performed with the distal cannula closed. Arterial segments that did not maintain pressure were discarded.

Measurement of Carotid Artery VSMC [Ca²⁺]_i and Constriction

Following a 30 min equilibration period, pressurized arteries were incubated for 45 min with 2 μmol/L fura 2-AM and 0.05% pluronic acid in 4 mL HEPES-PSS (composition in mmol/L: 134 NaCl, 6 KCl, 1 MgCl, 10 HEPES, 2 CaCl₂, 0.026 EDTA, and 10 glucose; pH 7.4) in the dark at room temperature [24]. After incubation, arteries were washed with oxygenated PSS (37 °C) for 15 min to remove excess dye. Arteries were then stimulated with the GPER-selective agonist G-1 (3 μmol/L) [17] or solvent (ethanol 0.1% vol/vol) for 20 min as described [18, 19], followed by exposure to ET-1 (10 nmol/L) as the constricting agonist. Some arteries were subsequently incubated in Ca²⁺ free PSS to determine maximal diameter, which did not differ from inner diameter before stimulation with ET-1 (data not shown). Fura 2-AM-loaded vessels were alternately excited at 340 nm and 380 nm at a frequency of 1 Hz with a HyperSwitch dual-excitation light source (IonOptix, Milton, MA, USA), and the respective 510 nm emissions collected with a photomultiplier tube (F₃₄₀/F₃₈₀). Background-subtracted F₃₄₀/F₃₈₀ emission ratios as a measure of relative VSMC [Ca²⁺]_i and inner arterial diameter from bright-field images were continuously calculated with IonWizard software (Version 5.0, IonOptix) throughout the experiment as described [24].

Quantitative Real-time Polymerase Chain Reaction (qPCR)

Frozen carotid arteries were disrupted and homogenized using a rotor-stator homogenizer, and total RNA was extracted using the silica-based RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA, USA). RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). qPCR was performed using TaqMan gene expression assays on a 7500 FAST real-time PCR System (Applied Biosystems). The following sets of primers were used: 5′-GAAGGACTGGTGGCTCTTTG-3′ (forward) and 5′-CTTCTCGACGCTGTTTGAGG-3′ (reverse) for amplification of a specific cDNA fragment encoding for murine ET_A receptor (GenBank ID: BC008277); 5′-CGGTATGCAGATTGCTTTGA-3′ (forward) and 5′-CACCTGTGTGGATTGCTCTG-3′ (reverse) for amplification of a specific cDNA fragment encoding for murine ET_B receptor (GenBank ID: BC026553); 5′-TTCACCACCATGGAGAAGGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGA-3′ (reverse) for amplification of a specific cDNA fragment encoding for murine GAPDH (GenBank ID: NM_008084), which served as house-keeping control. Gene expression was calculated using the 2^−ΔΔC(T) method [25].

Statistical Analyses

Vasoconstriction was calculated as percent reduction of the maximal internal arterial diameter. Data was analyzed using the Student’s t-test or two-way repeated-measures ANOVA followed by Bonferroni post hoc analysis where appropriate (Prism version 5.0 for Macintosh, GraphPad Software, San Diego, CA, USA). Values are shown as mean±SEM of independent experiments. A P<0.05 value was considered statistically significant.

Results

Effect of GPER Deficiency on ET-1-Induced Vasoconstriction

The vasoconstrictor response to ET-1 was significantly greater in arteries from GPER⁰ mice (31.8±1.5% vs. 24.0±2.9%, n=5, P<0.05 vs. WT; Figs. 1 and 2). However, the kinetics of vasoconstriction, measured as the time to maximal constriction, were delayed by approximately 90 seconds in GPER⁰ arteries (506±26 vs. 413±22 seconds, n=5, P<0.03 vs. WT; Fig. 2C). Acute selective GPER activation by G-1 had no effect on ET-1-induced vasoconstriction (26.7±1.6% vs. 24.0±2.9%, n=5–7, P=n.s. vs. solvent).

Fig. 1.

Original recordings of vasoconstrictor responses (measured as change in arterial diameter, top) and VSMC [Ca²⁺]_i (given as relative F₃₄₀/F₃₈₀ ratio, bottom) of carotid arteries from WT and GPER⁰ mice following exposure to endothelin-1 (10 nmol/L, arrows). Changes in arterial diameter and VSMC [Ca²⁺]_i were recorded simultaneously.

Fig. 2.

Endothelin-1-induced constriction in carotid arteries of WT and GPER⁰ mice. Time-dependent vasoconstriction to endothelin-1 (10 nmol/L, A), maximal responses (B), and time until maximal constriction (C) are shown. Vasoconstriction was calculated as the percent reduction of the maximal internal arterial diameter. Values are means±SEM; n=5. *P<0.05 vs. WT.

GPER-Dependent Regulation of ET-1-Stimulated VSMC [Ca²⁺]_i Increase

Despite the greater vasoconstrictor response to ET-1, GPER deficiency was associated with a 40% reduction in the ET-1-stimulated VSMC [Ca²⁺]_i increase (0.097±0.015 vs. 0.162±0.018, n=7–8, P<0.02 vs. WT; Figs. 1 and 3A). Steady-state VSMC [Ca²⁺]_i did not differ between WT and GPER⁰ mice (0.579±0.012 vs. 0.561±0.008, n=7–8, P=n.s., Fig. 3A). Moreover, acute selective GPER activation by G-1 had no effect on the calcium response evoked by ET-1 in WT arteries (0.749±0.039 vs. 0.740±0.021, n=7, P=n.s. vs. solvent, Fig. 3A). In addition to the overall inhibition of ET-1-stimulated VSMC [Ca²⁺]_i, time-dependent VSMC [Ca²⁺]_i responses were delayed 1.4-fold in GPER⁰ arteries independent of treatment with G-1 or solvent (66.7±6.8 vs. 48.6±2.4 seconds for maximal VSMC [Ca²⁺]_i increase, n=14, P<0.02 vs. WT; Fig. 3B).

Fig. 3.

Changes in VSMC [Ca²⁺]_i (A) and time to maximal VSMC [Ca²⁺]_i increase (B) in response to endothelin-1 (ET-1, 10 nmol/L). Carotid arteries of WT and GPER⁰ mice were pretreated with the GPER-selective agonist G-1 (3 μmol/L) or solvent (ethanol, EtOH 0.1% vol/vol) for 20 min. Values are means±SEM; n=6–8. *P<0.05 vs. WT; †P<0.05 vs. baseline.

Effect of GPER Deficiency on Endothelin Receptor Expression

In view of the greater vasoconstrictor response to ET-1 in GPER deficient animals, we evaluated steady-state ET_A and ET_B receptor mRNA expression levels in carotid arteries of WT and GPER⁰ mice. GPER deficiency affected neither vascular gene expression of ET_A receptor nor ET_B receptor (Table 1), indicating that the more potent response to ET-1 in GPER⁰ mice is not due to changes in receptor expression levels. Moreover, vascular gene expression levels of ET_A receptor were 5.7-fold higher than ET_B receptor (P<0.001; Table 1).

Table 1.

Gene expression levels of ET_A and ET_B receptors in carotid arteries of WT and GPER⁰ mice

Gene	WT	GPER0
ETA receptor	304±54	336±18
ETB receptor	53±9*	59±13*

Values are calculated using the 2^−ΔΔC(T) method [25] and expressed as arbitrary units (mean±SEM); n=5–6.

^*P<0.001 vs. ET_A receptor.

Discussion

This study has identified endogenous GPER as a regulator of ET-1-induced vasoconstriction and the associated changes in VSMC [Ca²⁺]_i in intact arteries. GPER deficiency results in increased contractility despite a pronounced reduction in ET-1-stimulated VSMC [Ca²⁺]_i, suggesting an increase in myofilament force sensitivity to [Ca²⁺]_i in arteries lacking GPER.

We have previously reported that the GPER agonists G-1 and ICI 182,780 acutely inhibit isometric contractions to ET-1 in porcine coronary arteries [19]. The present study extends these findings showing that in murine carotid arteries, the presence of endogenous GPER is sufficient to inhibit vasoconstriction to ET-1 without affecting ET_A or ET_B receptor gene expression levels. Similarly, we recently reported that GPER deletion increases endothelium-dependent contractility [16]. In carotid arteries used in the present study, the GPER-selective agonist G-1 had no acute effect on ET-1-induced constriction, indicating differential effects of chronic basal activity and acute activation of GPER. Moreover, differences in responsiveness to G-1 between species and vascular beds may explain its varied effects on ET-1-induced contraction in murine carotid and porcine coronary arteries [19]. Similarly, while estrogen acutely inhibits vasoconstriction to ET-1 in several vascular beds of different species [7–10], this effect might not apply to all arteries [26].

We have previously shown in isolated VSMC that direct administration of the GPER-selective agonist G-1 acutely increases [Ca²⁺]_i, while pretreatment with G-1 inhibits serotonin-mediated Ca²⁺ increases [18]. Moreover, it has been demonstrated that the non-selective ER agonist 17β-estradiol acutely inhibits ET-1-stimulated [Ca²⁺]_i increases in coronary artery VSMC from female pigs by stimulating Ca²⁺ efflux [12]. The present study now demonstrates that GPER modulates ET-1-stimulated VSMC [Ca²⁺]_i in intact arteries providing further evidence that endogenous GPER is specifically involved in the regulation of VSMC [Ca²⁺]_i and the subsequent constrictor response to ET-1.

Ca²⁺ is a principal intracellular messenger that triggers cross-bridge cycling between actin filaments and myosin resulting in VSMC contraction [27]. However, vasoconstrictors such as ET-1 may increase myosin regulatory light chain (MLC) phosphorylation and VSMC force under certain conditions while [Ca²⁺]_i is clamped, a phenomenon referred to as Ca²⁺ sensitization [3, 28]. Our observation that GPER deficiency is associated with greater ET-1-induced vasoconstriction despite a markedly reduced increase in VSMC [Ca²⁺]_i suggests an increase in myofilament force sensitivity to even low levels of [Ca²⁺]_i. Interestingly, the lack of GPER considerably delayed the contractile response to ET-1, which could reflect the time required for activation of pathways involved in regulating MLC phosphorylation [28]. Endogenous estrogens have previously been implicated in reducing the sensitivity of contractile proteins to Ca²⁺: In basilar arteries of male and ovariectomized female rats, reducing MLC phosphorylation by inhibition of Rho-associated kinase (ROK) results in a stronger dilator response compared to females with intact ovaries or receiving 17β-estradiol replacement [29]. Moreover, greater serotonin-dependent contractions in aorta from male compared to female mice are abolished after ROK inhibition [30]. Based on the recognition of GPER as a potential mediator of the Ca²⁺ desensitizing effects of estrogens, future studies should address whether and through which mechanisms GPER regulates MLC phosphorylation independent of changes in VSMC [Ca²⁺]_i.

The present study was performed in arteries from male mice, indicating that GPER, a receptor for a hormone traditionally implicated in female biology, also affects the male vasculature. Indeed, GPER is expressed in the cardiovascular system of both females and males [13, 15], with GPER-mediated acute relaxation of carotid arteries observed in male rodents [18, 20]. However, basal intrinsic activity of GPER in the absence of a ligand, which has been observed for many other G protein-coupled receptors [31], may be sufficient to mediate vascular effects. Furthermore, local conversion from androgen precursors into estrogens by the enzyme aromatase within the vascular wall could yield sufficient tissue concentrations to mediate relevant physiological effects [13, 15]. In addition, genetic deletion of the classical ERα and ERβ has adverse effects on vascular tone in males [32–34]. Taken together, these findings indicate a role for multiple estrogen receptors in male vascular physiology. Nevertheless, future studies should address potential sex differences in GPER-dependent regulation of ET-1-induced VSMC [Ca²⁺]_i and vasoconstriction.

Conclusions

GPER-mediated inhibition of the ET-1-stimulated VSMC [Ca²⁺]_i increases represents a newly identified mechanism that may contribute to the observed inhibition of ET-1-dependent vasoconstriction. Tonic inhibition of ET-1-induced vasoconstriction by endogenous GPER may contribute to the protective effects of estrogens or other GPER agonists such as SERMs [13, 15] in the pathogenesis of cardiovascular diseases [5, 6].