The G Protein-Coupled Estrogen Receptor 1 (GPER1/GPR30) Agonist G-1 Regulates Vascular Smooth Muscle Cell Ca2+ Handling (original) (raw)
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AJP: Endocrinology and Metabolism, 2011
Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERβ, estrogen may also stimulate G protein-coupled estrogen receptor 1 (GPER) in nonvascular tissue; however, a potential role for GPER in coronary arteries is unclear. The purpose of this study was to determine how GPER activity influenced coronary artery reactivity. In vitro isometric force recordings were performed on endothelium-denuded porcine arteries. These studies were augmented by RT-PCR and single-cell patch-clamp experiments. RT-PCR and immunoblot studies confirmed expression of GPER mRNA and protein, respectively, in smooth muscle from either porcine or human coronary arteries. G-1, a selective GPER agonist, produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro. This response was attenuated by G15, a GPER-selective antagonist, or by inhibiting large-conductance calcium-activated potassium (BK(Ca)) channels with iberiotoxin, but not by inhibiting NO signaling. Last, single-channel patch-clamp studies demonstrated that G-1 stimulates BK(Ca) channel activity in intact smooth muscle cells from either porcine or human coronary arteries but had no effect on channels isolated in excised membrane patches. In summary, GPER activation relaxes coronary artery smooth muscle by increasing potassium efflux via BK(Ca) channels and requires an intact cellular signaling mechanism. This novel action of estrogen-like compounds may help clarify some of the controversy surrounding the vascular effects of estrogens.
GPER regulates endothelin-dependent vascular tone and intracellular calcium
Life Sciences, 2012
Aims: An increase in intracellular vascular smooth muscle cell calcium concentration (VSMC [Ca 2+ ] 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 [Ca 2+ ] i in cultured VSMC, we investigated whether endogenous GPER regulates ET-1-induced changes in VSMC [Ca 2+ ] i and constriction of intact arteries. Main methods: Pressurized carotid arteries of GPER-deficient (GPER 0 ) 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 [Ca 2+ ] 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 GPER 0 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 [Ca 2+ ] i increase, suggesting an increase in myofilament force sensitivity to [Ca 2+ ] i . Activation of GPER by G-1 had no effect on vasoconstriction or VSMC [Ca 2+ ] 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 Ca 2+ sensitivity. This represents a potential mechanism through which GPER could contribute to protective effects of endogenous estrogen in the cardiovascular system.
Pharmacology Research & Perspectives
The G protein-coupled estrogen receptor (GPER) is a significant modulator of arterial contractility and blood flow. The GPER-specific activator, G-1, has been widely used to characterize GPER function in a variety of tissue types. Large conductance, calcium (Ca 2+)-activated K + (BK) channels are sensitive to 17b-estradiol (17b-E2) and estrogenic compounds (e.g., tamoxifen, ICI 182 780) that target estrogen receptors. The purpose of this study was to investigate the effects of G-1 on BK channel activation and function in cerebral arterial myocytes. Inside-out and perforated patch clamp were utilized to assess the effects of G-1 (50 nmolÁL À1-5 lmolÁL À1) on BK channel activation and currents in cerebral arterial myocytes. Pressurized artery myography was used to investigate the effects of G-1 on vasodilatory response and BK channel function of cerebral resistance size arteries. G-1 reduced BK channel activation in cerebral arterial myocytes through elevations in BK channel mean close times. Depressed BK channel activation following G-1 application resulted in attenuated physiological BK currents (transient BK currents). G-1 elicited vasodilation, but reduced BK channel function, in pressurized, endothelium-denuded cerebral arteries. These data suggest that G-1 directly suppresses BK channel activation and currents in cerebral arterial myocytes, BK channels being critically important in the regulation of myocyte membrane potential and arterial contractility. Thus, GPER-mediated vasodilation using G-1 to activate the receptor may underestimate the physiological function and relevance of GPER in the cardiovascular system.
GPER/GPR30 and Regulation of Vascular Tone and Blood Pressure
Immunology‚ Endocrine & Metabolic Agents in Medicinal Chemistry, 2011
Natural estrogens such as 17 -estradiol are endogenous vasodilators and have been implicated in the gender differences of hypertension. These hormones activate estrogen receptors ER and ER , which mediate part of estrogendependent vasodilation. In addition, a novel G protein-coupled estrogen-binding receptor termed GPER/GPR30 has been identified that is expressed in the cardiovascular system. Using knock-out animals or drugs selectively targeting GPER/GPR30, a significant role for this receptor as a mediator of acute estrogen-dependent vasodilation involving nitric oxide (NO) and blood pressure-lowering activity has been demonstrated. The accumulating evidence that GPER/GPR30 is responsible for control of vascular tone indicates that this receptor may represent a novel drug target for pharmacologic treatment of hypertension in postmenopausal women and possibly also men.
The G protein-coupled estrogen receptor GPER/GPR30 as a regulator of cardiovascular function
Vascular Pharmacology, 2011
Endogenous estrogens are important regulators of cardiovascular homeostasis in premenopausal women and interfere with the development of hypertension and coronary artery disease. These hormones act via three different estrogen receptors affecting both gene transcription and rapid signaling pathways in a complex interplay. In addition to the classical estrogen receptors ERα and ERβ, which are known mediators of estrogen-dependent vascular effects, a G protein-coupled estrogen receptor termed GPER that is expressed in the cardiovascular system has recently been identified. Endogenous human 17β-estradiol, selective estrogen receptor modulators (SERMs) including tamoxifen and raloxifene, and selective estrogen receptor downregulators (SERDs) such as ICI 182,780 are all agonists of GPER, which has been implicated in the regulation of vasomotor tone and protection from myocardial ischemia/reperfusion injury. As a result, understanding the individual role of ERα, ERβ, and GPER in cardiovascular function has become increasingly complex. With accumulating evidence that GPER is responsible for a variety of beneficial cardiovascular effects of estrogens, this receptor may represent a novel target to develop effective strategies for the treatment of cardiovascular diseases by tissue-specific, selective activation of estrogen-dependent molecular pathways devoid of side effects seen with conventional hormone therapy.
Cardiovascular Research, 2002
Objective: Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular / molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. Methods: Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. Results: Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.563 pS), calcium-and voltage-activated potassium (BK) channel was the Ca target of estrogen action. Channel activity was stimulated |15-fold by nanomolar concentrations of 17b-estradiol, and this stimulation was reversed .90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17b-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by G inhibiting NO synthase with 10 mM N-monomethyl-L-arginine. Conclusions: Our cellular and molecular studies identify the BK Ca channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK channel or a closely associated regulatory molecule, and further evidence suggests involvement of the Ca NO / cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.
Pharmacology, 2010
Coronary artery disease, which predominantly affects epicardial coronary arteries , represents the leading cause of death worldwide in women and men alike . Endogenous estrogens protect from development of coronary atherosclerosis in premenopausal women and are involved in the regulation of vascular tone and, thus, blood pressure [4] . These effects have mainly been attributed to activation of estrogen receptors (ER) ␣ and  [3] . Natural estrogen (17  -estradiol) acutely dilates human and porcine coronary arteries , and also inhibits responses to vasoconstrictors [6-9] . Using selective agonists for either ER ␣ or ER  , individual roles of these receptors mediating dilation of coronary arteries have been demonstrated .
Journal of Biological Chemistry, 2016
Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca 2؉ signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17-estradiol (E 2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca 2؉ and Ca 2؉-CaM indicated that E 2 also increases free Ca 2؉-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/ GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor ␣, the plasma membrane Ca 2؉-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E 2 substantially increases CaM binding to estrogen receptor ␣ and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-G ␥ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E 2 increases CaM-PMCA association, but the expected stimulation of Ca 2؉ efflux is reversed by E 2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca 2؉ signals and promote Ca 2؉-dependent CaM interactions with other CaM targets. Consequently, E 2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E 2 generates a feedforward loop via GPER/GPR30, which enhances Ca 2؉ /CaM signals and functional linkage in the endothelial CaM target network. Estrogen has numerous effects in the vascular endothelium that are linked to the protection of cardiovascular functions (1).
Deletion of G Protein-Coupled Estrogen Receptor Increases Endothelial Vasoconstriction
Hypertension, 2012
Endogenous estrogens mediate protective effects in the cardiovascular system, affecting both endothelium-dependent and -independent mechanisms. Previous studies have suggested that nonselective estrogen receptor agonists such as endogenous estrogens inhibit endotheliumdependent vasoconstriction; however, the role of estrogen receptors in this response has not yet been clarified. This study investigated whether the novel intracellular G protein-coupled estrogen receptor GPER regulates vascular reactivity in mice. Effects of chronic deficiency (using mice lacking the GPER gene) and acute inhibition (using the GPER-selective antagonist G15) on endothelium-dependent and -independent vascular reactivity, and the effects of GPER deficiency on vascular gene expression and structure were investigated. We found that chronic GPER deficiency is associated with increased endothelial prostanoid-mediated vasoconstriction, but had no effect on endothelial nitric oxide (NO) bioactivity, gene expression of endothelial NO synthase and thromboxane prostanoid (TP) receptor, or vascular structure. GPER deletion also increased TP receptor-mediated contraction. Acute GPER blockade enhanced endothelium-dependent contractions, and reduced endothelial NO bioactivity. Contractions to TP receptor activation were unaffected by G15. In conclusion, this study has identified GPER as the first estrogen receptor with inhibitory activity on endothelium-dependent contractility. These findings may be important for understanding and treating diseases associated with increased endothelial vasoconstrictor prostanoid activity such as hypertension and obesity.
Journal of Cardiovascular Pharmacology, 2011
Our studies in the mRen2.Lewis female rat, an angiotensin II-and estrogen-dependent model of hypertension, revealed that chronic activation of estrogen receptor GPR30 markedly reduces blood pressure in ovariectomized females. The present studies measured acute vasodilation to the selective GPR30 agonist G-1 and 17-β-estradiol (10 -9 to 10 -5.5 M) in isolated aortic rings and mesenteric arteries from intact mRen2.Lewis females. Maximal relaxation was greater in mesenteric vessels versus the aorta for both G-1 (47 ± 8% vs. 80 ± 5% of phenylephrine preconstriction, P < 0.001) and estradiol (42 ± 7% vs. 83 ± 4% of phenylephrine preconstriction, P < 0.001). The GPR30 antagonist G15 attenuated the response to both estradiol and G-1. Removal of the endothelium or pretreatment with L-NAME partially attenuated vasorelaxation. Responses were not altered in mesenteric vessels from ovariectomized females. Immunohistochemical analysis revealed GPR30 expression in mesenteric endothelial and smooth muscle cells, and smooth muscle expression was confirmed in cultured cells. We conclude that estradiol-induced relaxation in conduit and resistance vessels from mRen2.Lewis females may be mediated by the novel estrogen receptor GPR30. The direct vasodilatory response of G-1 in resistance vessels presents one mechanism for the reduction in blood pressure induced by chronic G-1 administration.