Guichun Han

Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells

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 (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-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 inhibiting NO synthase with 10 microM N(G)-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(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the 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.

Activation of G protein-coupled estrogen receptor induces endothelium- independent relaxation of coronary artery smooth muscle

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.

A novel transduction mechanism mediating dopamine-induced vascular relaxation: opening of BKCa channels by cyclic AMP-induced stimulation of the cyclic GMP-dependent protein kinase.

Dopamine dilates the coronary, renal and other vascular beds; however, the signaling pathway underlying this effect is unclear. In this study the signal-transduction process mediating dopamine-induced relaxation of porcine coronary arteries was investigated in isolated vessels and single arterial myocytes. Dopamine-induced relaxation of arteries was mediated through the DA- receptor and involved K+ efflux, and subsequent patch-clamp studies demonstrated that either dopamine or fenoldopam, a selective DA-1 agonist, increased the opening probability of the large-conductance, calcium- and voltage-activated K+ (BKCa) channel in coronary myocytes. Moreover, blockade of this channel by iberiotoxin prevented dopamine-induced coronary relaxation. Dopamine stimulation of BKCa channels was completely prevented by a DA-1-receptor antagonist, but was unaffected by propranolol. Furthermore, inhibiting adenylyl cyclase activity prevented stimulation of BKCa channel activity, whereas chlorophenylthio (CPT)-cyclic adenosine monophosphate (AMP), a membrane-permeable analog of cyclic AMP, mimicked the effects of dopamine. Interestingly, inhibiting the cyclic AMP-dependent protein kinase (PKA) did not affect the response to dopamine, whereas dopamine-induced channel activity was completely blocked by inhibiting the activity of the cyclic guanosine monophosphate (GMP)-dependent protein kinase (PKG). These findings demonstrate that activation of DA-1 receptors causes stimulation of BKCa channel activity by a mechanism involving cyclic AMP-dependent stimulation of PKG, but not PKA, and further suggest that this cross-reactivity mediates dopamine-induced coronary vasodilation.

Estrogen Receptor α Mediates Acute Potassium Channel Stimulation in Human Coronary Artery Smooth Muscle Cells

The pleiotropic effects of estrogen are mediated via stimulation of two estrogen receptor (ER) subtypes, ERα and ERβ. Although a number of studies have identified expression of one or both subtypes in estrogen target tissues, fewer studies have correlated ER expression with a functional role of these proteins in regulating cellular excitability. In the present study, we have combined cellular fluorescence, immunocytochemistry, and molecular expression techniques with single-channel patch-clamp studies to determine which ER mediates estrogen-stimulated potassium channel activity in human coronary artery smooth muscle cells (HCASMC). We had demonstrated previously that estrogen stimulates activity of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel in HCASMC via a nongenomic mechanism. We now demonstrate expression of both ERα and ERβ subtypes in HCASMC. Functionally, however, expression of ERα antisense plasmid abolished the acute effect of estrogen on these channels, whereas estrogen retained its ability to stimulate BKCa channels in cells transfected with only green fluorescence protein. In contrast, overexpression of ERα enhanced the stimulatory action of estrogen in HCASMC. Transfection with ERα antisense/sense plasmid did not alter ERβ expression. These findings indicate that the ERα isoform mediates estrogen-induced stimulation of BKCa channels in HCASMC and thereby provide evidence for a receptor-dependent signaling mechanism that can mediate estrogen-induced inhibition of cellular excitability.

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs). Methodology/Principal Findings The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10−8 to 10−5 M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium. Conclusion GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.

PGE2 action in human coronary artery smooth muscle: Role of potassium channels and signaling cross-talk

Cyclic AMP-stimulating agents are powerful vasodilators, but our knowledge of the signal transduction mechanisms of these agents, particularly in human arteries, is limited. We now report direct molecular effects of prostaglandin E2 (PGE2) on cultured human coronary artery smooth muscle cells (HCASMC). Patch-clamp studies revealed that 10 µM PGE2 opens a high-conductance (∼200 pS), calcium-stimulated potassium (BKCa) channel in intact HCASMC. In contrast, PGE2 had no direct effect on channels in cell-free patches, indicating involvement of a soluble second messenger. Enzyme immunoassay demonstrated that PGE2 enhances production of cAMP in HCASMC, but does not increase [cGMP]. Furthermore, forskolin, CPT-cAMP, or CPT-cGMP mimicked the stimulatory effect of PGE2 on BKCa channel activity. Interestingly, the response to PGE2 was unaffected by inhibiting the cAMP-dependent protein kinase, but was antagonized by inhibitors of the cGMP-dependent protein kinase (PKG). Furthermore, cAMP-stimulated PKG activity mimicked the effect of PGE2. These studies suggest a novel PGE2 action in human arteries: opening of BKCa channels via cAMP cross-activation of PKG in HCASMC. It is proposed that this signaling mechanism may mediate the vasodilatory response to cAMP-dependent agents in the human coronary and other vascular beds.

GPER: A novel target for non-genomic estrogen action in the cardiovascular system

A key to harnessing the enormous therapeutic potential of estrogens is understanding the diversity of estrogen receptors and their signaling mechanisms. In addition to the classic nuclear estrogen receptors (i.e., ERα and ERβ), over the past decade a novel G-protein-coupled estrogen receptor (GPER) has been discovered in cancer and other cell types. More recently, this non-genomic signaling mechanism has been found in blood vessels, and mediates vasodilatory responses to estrogen and estrogen-like agents; however, downstream signaling events involved acute estrogen action remain unclear. The purpose of this review is to discuss the latest knowledge concerning GPER modulation of cardiovascular function, with a particular emphasis upon how activation of this receptor could mediate acute estrogen effects in the heart and blood vessels (i.e., vascular tone, cell growth and differentiation, apoptosis, endothelial function, myocardial protection). Understanding the role of GPER in estrogen signaling may help resolve some of the controversies associated with estrogen and cardiovascular function. Moreover, a more thorough understanding of GPER function could also open significant opportunities for the development of new pharmacological strategies that would provide the cardiovascular benefits of estrogen while limiting the potentially dangerous side effects.

G protein-coupled estrogen receptor 1 mediates relaxation of coronary arteries via cAMP/PKA-dependent activation of MLCP

Activation of GPER exerts a protective effect in hypertension and ischemia-reperfusion models and relaxes arteries in vitro. However, our understanding of the mechanisms of GPER-mediated vascular regulation is far from complete. In the current study, we tested the hypothesis that GPER-induced relaxation of porcine coronary arteries is mediated via cAMP/PKA signaling. Our findings revealed that vascular relaxation to the selective GPER agonist G-1 (0.3-3 μM) was associated with increased cAMP production in a concentration-dependent manner. Furthermore, inhibition of adenylyl cyclase (AC) with SQ-22536 (100 μM) or of PKA activity with either Rp-8-CPT-cAMPS (5 μM) or PKI (5 μM) attenuated G-1-induced relaxation of coronary arteries preconstricted with PGF2α (1 μM). G-1 also increased PKA activity in cultured coronary artery smooth muscle cells (SMCs). To determine downstream signals of the cAMP/PKA cascade, we measured RhoA activity in cultured human and porcine coronary SMCs and myosin-light chain phosphatase (MLCP) activity in these artery rings by immunoblot analysis of phosphorylation of myosin-targeting subunit protein-1 (p-MYPT-1; the MLCP regulatory subunit). G-1 decreased PGF2α-induced p-MYPT-1, whereas Rp-8-CPT-cAMPS prevented this inhibitory effect of G-1. Similarly, G-1 inhibited PGF2α-induced phosphorylation of MLC in coronary SMCs, and this inhibitory effect was also reversed by Rp-8-CPT-cAMPS. RhoA activity was downregulated by G-1, whereas G36 (GPER antagonist) restored RhoA activity. Finally, FMP-API-1 (100 μM), an inhibitor of the interaction between PKA and A-kinase anchoring proteins (AKAPs), attenuated the effect of G-1 on coronary artery relaxation and p-MYPT-1. These findings demonstrate that localized cAMP/PKA signaling is involved in GPER-mediated coronary vasodilation by activating MLCP via inhibition of RhoA pathway.

Expression of GPR30, ERα and ERβ in endometrium during window of implantation in patients with polycystic ovary syndrome: a pilot study

Women with polycystic ovary syndrome (PCOS) exhibit a lower pregnancy rate, which may be related to decreased estrogen receptor (ER) expression or endometrial receptivity. We measured expression of ERα, ERβ and the novel G protein-coupled ER (GPR30) in endometrium during window of implantation (WOI) in PCOS patients. Fifteen Chinese women with PCOS were compared to 15 normal subjects. Serial trans-vaginal ultrasonic scanner (TVUS) examinations detected follicular development, and endometrial thickness and pattern were assessed via TVUS on the day of ovulation. GPR30 expression was detected in the cytoplasm of endometrial epithelial cells, and was significantly lower in the PCOS group (p < 0.05). ERα and ERβ expression was lower in the PCOS group, and was detected mainly in the nucleus of endometrial epithelial cells. There was no significant difference in endometrium thickness (p > 0.05), but there was a significant difference in the ultrasonic pattern (p < 0.05). Endometrial expression of GPR30, ERα and ERβ was decreased during WOI in PCOS patients, and was accompanied by poor endometrial receptivity, low pregnancy rate and higher spontaneous abortions. We propose that restored receptor expression might improve endometrial receptivity and help lower infertility associated with PCOS.

Essential Role of the 90-Kilodalton Heat Shock Protein in Mediating Nongenomic Estrogen Signaling in Coronary Artery Smooth Muscle

Under normal physiological conditions, estrogen is a coronary vasodilator, and this response involves production of NO from endothelial cells. In addition, estrogen also stimulates NO production in coronary artery smooth muscle (CASM); however, the molecular basis for this nongenomic effect of estrogen is unclear. The purpose of this study was to investigate a potential role for the 90-kDa heat shock protein (Hsp90) in estrogen-stimulated neuronal nitric-oxide synthase (nNOS) activity in coronary artery smooth muscle. 17β-Estradiol produced a concentration-dependent relaxation of endothelium-denuded porcine coronary arteries in vitro, and this response was attenuated by inhibiting Hsp90 function with 1 μM geldanamycin (GA) or 100 μg/ml radicicol (RAD). These inhibitors also prevented estrogen-stimulated NO production in human CASM cells and reversed the stimulatory effect of estrogen on calcium-activated potassium (BKCa) channels. These functional studies indicated a role for Hsp90 in coupling estrogen receptor activation to NOS stimulation in CASM. Furthermore, coimmunoprecipitation studies demonstrated that estrogen stimulates bimolecular interaction of immunoprecipitated nNOS with Hsp90 and that either GA or RAD could inhibit this association. Blocking estrogen receptors with ICI182780 (fulvestrant) also prevented this association. These findings indicate an essential role for Hsp90 in nongenomic estrogen signaling in CASM and further suggest that Hsp90 might represent a prospective therapeutic target to enhance estrogen-stimulated cardiovascular protection.