RESUMO
Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide and in the Western world, one-third of all deaths are attributed to CVD. A conspicuous characteristic of this healthcare epidemic is that most CVD is higher in men than in age-matched premenopausal women, yet reasons for these obvious sex differences remain poorly understood. Driven by clinical case and epidemiological studies and supported by animal experiments, a strong dogma emerged early on that testosterone (TES) exerts deleterious effects on cardiovascular health and exacerbates development of CVD and metabolic dysfunctions in men. In this review, earlier and more recent clinical and experimental animal evidence of cardiovascular and metabolic effects of androgens are discussed. The more recent evidence overwhelmingly suggests that it is progressive, age-dependent declines in TES levels in men that exacerbate CVD and metabolic dysfunctions, while TES exerts beneficial systemic hypotensive effects and protects against metabolic syndrome (MetS) and type2 diabetes mellitus (T2DM). Recent findings reveal existence of bi-directional modulation of glucose and fat homeostasis by TES in females vs males, such that age-dependent declines in TES levels in males and abnormal increases in normally low TES levels in females both result in similar dysfunction in glucose and fat homeostasis, resulting in development of MetS and T2DM, central risk factors for development of CVD, in men as well as women. These findings suggest that the long-held view that TES is detrimental to male health should be discarded in favor of the view that, at least in men, TES is beneficial to cardiovascular and metabolic health.
Assuntos
Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Síndrome Metabólica , Animais , Feminino , Masculino , Androgênios/metabolismo , Testosterona/metabolismo , GlucoseRESUMO
BACKGROUND: Hormone replacement therapy was found to be effective in cardiovascular protection only in younger women, not in older women. In this study, we tested whether G protein-coupled estrogen receptor 1 (GPER) activation improves vascular activities in response to ET-1 and ACh in aging rats. METHODS: Isometric tension study was applied on aortic rings isolated from young adult (5-7 months) and reproductive senescent middle-aged (10-12 months) female Sprague Dawley rats and age matched males. RESULTS: The aortic contractile response to ET-1 and the relaxation response to ACh were reduced in the female middle-aged rats compared to the female young adult rats. The presence of G-1, the GPER agonist, normalized the reduced vascular activities. Cyclooxygenase inhibitor, meclofenamate, blocked the increased constriction effect of G-1, but further enhanced relaxation effect of G-1. There was no significant difference in aortic reactivity to either ET-1 or ACh between the male middle-aged and young adult rats. The contractile response to ET-1 was not different within the same age of the two sex groups, but there was a remarkable difference in relaxation response to ACh between young adult females and males with better response in females. GPER activation greatly improved the aortic relaxation of both young adult and middle-aged females, but not the males. CONCLUSIONS: Endothelial dysfunction occurs earlier in males, but in females, dysfunction delays until middle age. GPER activation improves the vascular activities in females, but not males. It is promising to employ GPER as a potential drug target in cardiovascular disease in women.
Assuntos
Receptores de Estrogênio , Receptores Acoplados a Proteínas G , Idoso , Animais , Endotélio Vascular , Estrogênios/farmacologia , Feminino , Proteínas de Ligação ao GTP/farmacologia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
BACKGROUND: Acutely, testosterone (TES) and other androgens are efficacious vasodilators, both in vitro and in vivo; however, their long-term effects on arterial blood pressure (BP) remain unclear. It was hypothesized that endogenous androgens exert long-term anti-hypertensive effects on systemic BP through a combination of genomic and nongenomic effects to enhance vasodilation of the systemic vasculature. METHODS: The long-term effects of endogenous TES and exogenous TES replacement therapy (TRT) on BP were studied in intact (InT) and castrated (CsX) male Sprague-Dawley (SD) and testicular-feminized male (Tfm, androgen receptor defective) rats (12 weeks old). Systolic BP (tail-cuff plethysmography) was determined weekly for 15 weeks in InT-control and CsX rats. Some CsX-SD rats received androgen replacement therapy at 10-15 weeks with TES-enanthate (TRT; 1.75 mg/kg, 2x/week) or DHT-enanthate (DRT; 1.00 mg/kg. 2x/week) and a separate group of CsX-SD rats received losartan-potassium in drinking water (LST, 250 mg/L) for the entire 15 week period. Expression of renin, angiotensinogen (Agt), angiotensin converting enzyme (ACE), and angiotensin II type I receptor (AT1R) mRNA in kidney and aorta were determined by real-time PCR (rt-PCR) and plasma renin levels were determined by radioimmunoassay. RESULTS: There was a progressive rise in BP over 10 weeks in CsX (109 ± 3.3 vs. 143 ± 3.5 mmHg), while BP remained stable in InT-control (109 ± 3.0 vs. 113 ± 0.3). BP gradually declined to normal in CsX-TRT rats (113 ± 1.3), while BP remained elevated in CsX (140 ± 1.2) and normal in InT-control (113 ± 0.3). LST prevented the development of hypertension in CsX at 10 weeks (100 ± 1.5 in CsX + LST vs. 143 ± 3.5 in CsX). During the next 5 weeks with TES-RT, BP declined in CsX-TRT (113 ± 1.3) and remained lower in CsX + LST (99 ± 0.4). DHT-RT reduced BP in CxS to a similar extent. In Tfm, CsX resulted in a similar rise in BP (109 ± 0.7 vs. 139 ± 0.4 mmHg), but TRT reduced BP more rapidly and to a greater extent (106 ± 2.8). rt-PCR of the kidney revealed that CsX increased expression of mRNA for renin (92%), ACE (58%), and AT1R (80%) compared to InT, while TES RT normalized expression of renin, AT1R, and ACE mRNA to levels of InT rats. Plasma renin levels exhibited changes similar to those observed for renin mRNA expression. CONCLUSIONS: This is the first study to examine the long-term effects of endogenous and exogenous androgens on BP in male SD and Tfm rats. These data reveal that endogenous androgens (TES) exert anti-hypertensive effects that appear to involve non-genomic and possibly genomic mechanism(s), resulting in reductions in RAS expression in the kidney and enhanced systemic vasodilation.
Assuntos
Androgênios/metabolismo , Hipertensão/metabolismo , Sistema Renina-Angiotensina/fisiologia , Testosterona/análogos & derivados , Androgênios/farmacologia , Animais , Anti-Hipertensivos/uso terapêutico , Aorta/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Rim/metabolismo , Losartan/uso terapêutico , Masculino , Orquiectomia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Testosterona/farmacologiaRESUMO
Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gßγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.
Assuntos
Vasos Coronários/fisiologia , Receptores ErbB/genética , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Ciclopentanos/farmacologia , Flavonoides/farmacologia , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Cardiovasculares , Miócitos de Músculo Liso/metabolismo , Quinazolinas/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Suínos , Ativação Transcricional , Tirfostinas/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/genética , Vasodilatação/fisiologiaRESUMO
Dehydroepiandrosterone (DHEA), testosterone (TES) and its 5-reduced metabolites induce a nongenomic vasorelaxation in several vascular beds of mammals; similarly these hormones produce systemic hypotensive and antihypertensive responses in normotensive and hypertensive male rats. Thus, it was hypothesized that the antihypertensive response of androgens, whose levels are elevated during gestation, protect against gestational hypertension. An animal model of preeclampsia was induced in female Wistar rats using DOCA-salt-treated pregnant (PT) and normal pregnant (NP) rats. In vivo experiments in conscious rats revealed that bolus intravenous injections of DHEA, TES, 5α- or 5ß-dihydrotestosterone (-DHT) log -1.0 to 2.0µmolk-1min-1, produced substantial transient reductions in arterial blood pressure (BP), without significant changes in heart rate (HR). Mean arterial blood pressure (MAP) was reduced significantly in both groups. PT rats were more sensitive to the antihypertensive responses of androgens than NP. DHEA and 5ß-DHT were the most potent to reduce MAP: 66±07 and 69±2.0mmHg in PT but only 33±0.5 and 35±1.2mmHg in NP rats, respectively. In isolated aortas of PT and NP, the concentration-response curves to each androgen (0.1-100µM) indicated that KCl-induced pre-contraction is more sensitive to all androgens than phenylephrine (Phe) pre-contractions. Notably, 5ß-DHT is the greatest vasorelaxant with KCl-induced contraction than with Phe contraction of both groups, suggesting a preferential blockade on L-VOCCs. TES exhibited minor vasorelaxing effect of aortas pre-contracted with KCl, compared to its precursor DHEA and its 5-reduced metabolites. These data show that these androgens exert acute vasorelaxing effects in vitro and remarkably, reduce the BP in vivo in PT and NP at term pregnancy. Moreover, a deficit in feto-placental androgen production during pregnancy may trigger the development of preeclampsia or gestational hypertension.
Assuntos
Androgênios/farmacologia , Anti-Hipertensivos/farmacologia , Modelos Teóricos , Pré-Eclâmpsia/tratamento farmacológico , Vasodilatação/efeitos dos fármacos , Animais , Feminino , Gravidez , Ratos , Ratos WistarRESUMO
Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 µM), or ESI-09 (20 µM), or CE3F4 (100 µM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 µM); while concurrent administration of BFA and PKI (5 µM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 µM), induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 µM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 µM) treatment, but not by 007 (50 µM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.
Assuntos
Vasos Coronários/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ciclopentanos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hidrazonas/farmacologia , Isoxazóis/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Fosforilação/efeitos dos fármacos , Quinolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Tionucleotídeos/farmacologia , Proteínas rap1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Androgens are vasoactive steroids that induce acute vasodilation in a number of isolated vascular beds from different species, but the effects of these hormones on systemic blood pressure (BP) have been studied little. Although it has been reported that androgens exert systemic hypotensive effects through peripheral vasodilation in normotensive rats, there have not been any reports of systemic hypotensive effects of androgens in animals with hypertension. This study was designed to evaluate the acute effects of testosterone (TES) and its 5-reduced metabolites on systemic BP in hypertensive rats and to test the hypothesis that hypotestosteronemia may be involved in the pathogenesis of hypertension. Chronic, indwelling catheters were implanted in carotid artery and jugular vein of 18-21-week-old male spontaneously hypertensive rats (SHR) and normotensive-control Wistar-Kyoto (WKY) rats, for BP recording and drug administration, respectively. Bolus injections of TES, 5α- or 5ß-dihydrotestosterone (5α- and 5ß-DHT), were administrated cumulatively to conscious rats at doses of 0.1-100µmolkg-1min-1. 5ß-DHT was also administrated during the pressor effect of Bay K 8644, an L-type voltage-operated Ca2+ channel (L-VOCC) agonist. In separate experiments, BP of orchidectomized normotensive male WKY and Wistar rats, with or without androgen-replacement therapy, was evaluated weekly for 10 weeks by tail-cuff plethysmography. TES and its metabolites reduced BP in a dose-dependent manner, while heart rate was reduced with some androgens at the highest doses. The hypotensive effects of androgens were markedly greater in SHR, with a rank order potency of: 5ß-DHT>TES>5α-DHT. 5ß-DHT, the most potent antihypertensive androgen, abolished the pressor response to Bay K 8644 in SHR. TES deprivation by orchidectomy increased BP in normotensive WKY and Wistar rats, but this hypertension was prevented by TES replacement therapy. BP responses to androgens are androgen structure-dependent. These data indicate that: 1) androgens play a significant role in the control of BP and may contribute to the pathogenesis of hypertension; 2) blockade of L-VOCC is involved in the antihypertensive effects of androgens, which are non-genomically mediated; and 3) hypotestosteronemia may be a risk factor for hypertension.
Assuntos
Androgênios/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Estado de Consciência/efeitos dos fármacos , Hipertensão/patologia , Animais , Canais de Cálcio Tipo L/metabolismo , Di-Hidrotestosterona/farmacologia , Hipertensão/metabolismo , Masculino , Orquiectomia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Fatores de Risco , Testosterona/farmacologiaRESUMO
In the present study, interactions of age and estrogen in the modulation of cerebrovascular function were examined in small arteries <150 µM. The hypothesis tested was that age enhances deleterious effects of exogenous estrogen by augmenting constrictor prostanoid (CP)-potentiated reactivity of the female (F) cerebrovasculature. F Sprague-Dawley rats approximating key stages of "hormonal aging" in humans were studied: perimenopausal (mature multi-gravid, MA, cyclic, 5-6 mo of age) and postmenopausal (reproductively senescent, RS, acyclic 10-12 mo of age). Rats underwent bilateral ovariectomy and were given estrogen replacement therapy (E) or placebo (O) for 14-21 days. Vasopressin reactivity (VP, 10(-12)-10(-7) M) was measured in pressurized middle cerebral artery segments, alone or in the presence of COX-1- (SC560, 1 µM) or COX-2- (NS398, 10 µM) selective inhibitors. VP-stimulated release of prostacyclin (PGI2) and thromboxane (TXA2) were assessed by radioimmunoassay of 6-keto-PGF1α and TXB2 (stable metabolites). VP-induced vasoconstriction was attenuated in ovariectomized + estrogen-replaced, multigravid adult rats (5-6 mo; MAE) but potentiated in older ovariectomized + estrogen-replaced, reproductively senescent rats (12-14 mo; RSE). SC560 and NS398 reduced reactivity similarly in ovariectomized multigravid adult rats (5-6 mo; MAO) and ovariectomized reproductively senescent rat (12-14 mo; RSO). In MAE, reactivity to VP was reduced to a greater extent by SC560 than by NS398; however, in RSE, this effect was reversed. VP-stimulated PGI2 was increased by estrogen, yet reduced by age. VP-stimulated TXA2 was increased by estrogen and age in RSE but did not differ in MAO and RSO. Taken together, these data reveal that the vascular effects of estrogen are distinctly age-dependent in F rats. In younger MA, beneficial and protective effects of estrogen are evident (decreased vasoconstriction, increased dilator prostanoid function). Conversely, in older RS, detrimental effects of estrogen begin to be manifested (enhanced vasoconstriction and CP function). These findings may lead to age-specific estrogen replacement therapies that maximize beneficial and minimize detrimental effects of this hormone on small cerebral arteries that regulate blood flow.
Assuntos
Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Artéria Cerebral Média/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Fatores Etários , Animais , Relação Dose-Resposta a Droga , Epoprostenol/metabolismo , Estradiol/toxicidade , Terapia de Reposição de Estrogênios/efeitos adversos , Artéria Cerebral Média/metabolismo , Ovariectomia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tromboxano A2/metabolismo , Vasoconstritores/farmacologia , Vasopressinas/farmacologiaRESUMO
Testosterone (TES) and other androgens exert a direct vasorelaxing action on the vasculature in vitro that is structurally specific and independent of cytosolic androgen receptor (AR). The effects of intravenous androgen infusions on mean arterial blood pressure (BP) and heart rate (HR) were determined in conscious, unrestrained, chronically catheterized, ganglionically blocked (hexamethonium, HEX; 30 mg/kg ip) male Sprague-Dawley (SD) and testicular-feminized male (Tfm; AR-deficient) rats, 16-20 wk of age. BP and HR were recorded at baseline and with increasing doses of androgens (0.375-6.00 µmol·kg(-1)·min(-1) iv; 10 min/dose). Data are expressed as means ± SE (n = 5-8 rats/group). In SD rats, baseline BP and HR averaged 103 ± 4 mmHg and 353 ± 12 beats/min (bpm). TES produced a dose-dependent reduction in BP to a low of 87 ± 4 mmHg (Δ16%), while HR was unchanged (354 ± 14 bpm). Neither BP (109 ± 3 mmHg) nor HR (395 ± 13 bpm) were altered by vehicle (10% EtOH in 0.9% saline; 0.15 ml·kg(-1)·min(-1), iv). In Tfm, TES produced a similar reduction in BP (99 ± 3 to 86 ± 3 mmHg, Δ13%); HR was unchanged (369 ± 18 bpm). In SD, 5ß-dihydrotestosterone (genomically inactive metabolite) produced a greater reduction in BP than TES (102 ± 2 to 79 ± 2 mmHg, Δ23%); HR was unchanged (361 ± 9). A 20-µg iv bolus of sodium nitroprusside in both SD and Tfm rats reduced BP 30-40 mmHg, while HR was unchanged, confirming blockade by HEX. Pretreatment of SD rats with neuronal nitric oxide synthase (nNOS) inhibitor (S-methyl-thiocitrulline, SMTC; 20 µg·kg(-1)·min(-1) × 30 min) abolished the hypotensive effects of TES infusion on BP (104 ± 2 vs. 101 ± 2 mmHg) and HR (326 ± 11 vs. 324 ± 8 bpm). These data suggest the systemic hypotensive effect of TES and other androgens involves a direct vasodilatory action on the peripheral vasculature which, like the effect observed in isolated arteries, is structurally specific and AR-independent, and involves activation of nNOS.
Assuntos
Androgênios/administração & dosagem , Pressão Arterial/efeitos dos fármacos , Artérias/efeitos dos fármacos , Hipotensão/induzido quimicamente , Óxido Nítrico Sintase Tipo I/metabolismo , Testosterona/administração & dosagem , Síndrome de Resistência a Andrógenos/genética , Síndrome de Resistência a Andrógenos/metabolismo , Síndrome de Resistência a Andrógenos/fisiopatologia , Androgênios/química , Animais , Artérias/enzimologia , Artérias/fisiopatologia , Di-Hidrotestosterona/administração & dosagem , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipotensão/enzimologia , Hipotensão/fisiopatologia , Infusões Intravenosas , Masculino , Estrutura Molecular , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Relação Estrutura-Atividade , Testosterona/análogos & derivados , Testosterona/química , Fatores de Tempo , Vasodilatação/efeitos dos fármacosRESUMO
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.
Assuntos
Vasos Coronários/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Vasodilatação , Animais , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Transdução de Sinais/efeitos dos fármacos , Suínos , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND: Although the mechanisms underlying the beneficial effects of estrogen on cerebrovascular function are well known, the age-dependent deleterious effects of estrogen are largely unstudied. It was hypothesized that age and sex interact in modulating cerebrovascular reactivity to vasopressin (VP) by altering the role of prostanoids in vascular function. METHODS: Female (F) Sprague-Dawley rats approximating key stages of "hormonal aging" in humans were studied: premenopausal (mature multigravid, MA, cyclic, 5-6 months) and postmenopausal (reproductively senescent, RS, acyclic, 10-12 months). Age-matched male (M) rats were also studied. Reactivity to VP (10(-12)-10(-7) M) was measured in pressurized middle cerebral artery segments in the absence or presence of selective inhibitors of COX-1 (SC560, SC, 1 µM) or COX-2 (NS398, NS, 10 µM). VP-stimulated release of PGI2 and TXA2 were measured using radioimmunoassay of 6-keto-PGF1α and TXB2 (stable metabolites, pg/mg dry wt/45 min). RESULTS: In M, there were no changes in VP-induced vasoconstriction with age. Further, there were no significant differences in basal or in low- or high-VP-stimulated PGI2 or TXA2 production in younger or older M. In contrast, there were marked differences in cerebrovascular reactivity and prostanoid release with advancing age in F. Older RS F exhibited reduced maximal constrictor responses to VP, which can be attributed to enhanced COX-1 derived dilator prostanoids. VP-induced vasoconstriction in younger MA F utilized both COX-1 and COX-2 derived constrictor prostanoids. Further, VP-stimulated PGI2 and TXA2 production was enhanced by endogenous estrogen and decreased with advancing age in F, but not in M rats. CONCLUSIONS: This is the first study to examine the effects of age and sex on the mechanisms underlying cerebrovascular reactivity to VP. Interestingly, VP-mediated constriction was reduced by age in F, but was unchanged in M rats. Additionally, it was observed that selective blockade of COX-1 or COX-2 produced age-dependent changes in cerebrovascular reactivity to VP and that VP-stimulated PGI2 and TXA2 production were enhanced by endogenous estrogen in younger F. A better understanding of the mechanisms by which estrogen exerts its effects may lead to new age- and sex-specific therapeutic agents for the prevention and/or treatment of cerebrovascular diseases.
RESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Vasos Coronários/citologia , Ciclopentanos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/fisiologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Receptores de Estrogênio , Suínos , Regulação para Cima/efeitos dos fármacosRESUMO
Our knowledge of how androgens influence the cardiovascular system is far from complete, and this lack of understanding is especially true of how androgens affect resistance vessels. Our aim was to identify the signaling mechanisms stimulated by testosterone (TES) in microvascular arteries and to understand how these mechanisms mediate TES-induced vasodilation. Mesenteric microvessels were isolated from male Sprague-Dawley rats. Tension studies demonstrated a rapid, concentration-dependent, vasodilatory response to TES that did not involve protein synthesis or aromatization to 17ß-estradiol. Dichlorofluorescein fluorescence and nitrotyrosine immunoblot experiments indicated that TES stimulated peroxynitrite formation in microvessels, and functional studies demonstrated that TES-induced vasodilation was inhibited by scavenging peroxynitrite. As predicted, TES enhanced the production of both peroxynitrite precursors (i.e., superoxide and nitic oxide), and xanthine oxidase was identified as the likely source of TES-stimulated superoxide production. Functional and biochemical studies indicated that TES signaling involved activity of the phosphoinositide 3 (PI3) kinase-protein kinase B (Akt) cascade initiated by activation of the androgen receptor and culminated in enhanced production of cGMP and microvascular vasodilation. These findings, derived from a variety of analytical and functional approaches, provide evidence for a novel nongenomic signaling mechanism for androgen action in the microvasculature: TES-stimulated vasodilation mediated primarily by peroxynitrite formed from xanthine oxidase-generated superoxide and NO. This response was associated with activation of the PI3 kinase-Akt signaling cascade initiated by activation of the androgen receptor. We propose this mechanism could account for TES-stimulated cGMP production in microvessels and, ultimately, vasodilation.
Assuntos
Androgênios/farmacologia , Microvasos/efeitos dos fármacos , Ácido Peroxinitroso/biossíntese , Testosterona/farmacologia , Resistência Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Western Blotting , GMP Cíclico/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Técnicas In Vitro , Masculino , Microvasos/metabolismo , Microvasos/fisiopatologia , Óxido Nítrico/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Xantina Oxidase/metabolismoRESUMO
Although the vasorelaxing effects of testosterone (T) and various androgen metabolites have been observed in a variety of blood vessels and species, previous studies have not systematically compared the vasorelaxing effects of androgen metabolites in different vascular beds within the same species. Therefore, we studied the vasorelaxing effects of T and its 5-reduced metabolites (5α- and 5ß-DHT) on KCl-induced contractions of the canine left coronary artery, femoral artery and saphenous vein, using standard isometric recordings. KCl contractions were inhibited by each androgen in a concentration-dependent manner from 1.8 to 310µM. Vascular sensitivity and efficacy were expressed as inhibitory concentration 50 (IC50) and maximal relaxation (R(max)), respectively. The coronary artery was significantly more sensitive to androgen-induced vasorelaxation than the saphenous vein or femoral artery. These vasorelaxing responses were unaffected by an antiandrogen (Flutamide) or the sulfhydryl reagent, N-ethylmaleimide, suggesting a nongenomic mechanism independent of signaling mediated by the androgen receptor or G proteins. Concentration-response curves were unchanged in endothelium-denuded preparations; thus, the endothelium appears to have no role in androgen-induced vasorelaxation. 5ß-DHT was the most potent androgen in both coronary and femoral artery, but all three androgens were equipotent in the saphenous vein. It is concluded that: 1) significant regional differences exist in vasorelaxing effects of androgen metabolites in the canine vasculature; 2) structural differences in these androgens determine their vasorelaxing efficacy; and 3) regional differences in androgen-induced vasorelaxation may account for some of the conflicting findings reported on the vasorelaxing effects of the androgens.
Assuntos
Androgênios/metabolismo , Vasos Sanguíneos/metabolismo , Di-Hidrotestosterona/metabolismo , Testosterona/metabolismo , Vasodilatação , Vasodilatadores/metabolismo , Antagonistas de Androgênios/farmacologia , Androgênios/química , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Di-Hidrotestosterona/antagonistas & inibidores , Di-Hidrotestosterona/química , Cães , Etilmaleimida/farmacologia , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/metabolismo , Flutamida/farmacologia , Técnicas In Vitro , Masculino , Concentração Osmolar , Reprodutibilidade dos Testes , Veia Safena/efeitos dos fármacos , Veia Safena/metabolismo , Estereoisomerismo , Reagentes de Sulfidrila/farmacologia , Testosterona/antagonistas & inibidores , Vasodilatação/efeitos dos fármacos , Vasodilatadores/antagonistas & inibidores , Vasodilatadores/químicaRESUMO
Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BK(Ca)) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BK(Ca) channel activity by â¼100-fold and that inhibition of nitric oxide synthase (NOS) activity by N(G)-monomethyl-L-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BK(Ca) channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BK(Ca) channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (â¼100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BK(Ca) channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/enzimologia , Testosterona/metabolismo , Vasodilatação , Animais , Vasos Coronários/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Potenciais da Membrana , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio/farmacologia , Transdução de Sinais , Suínos , Fatores de Tempo , Vasodilatação/efeitos dos fármacosRESUMO
Despite an abundance of evidence to the contrary from animal studies, large clinical trials on humans have shown that estrogen administered to postmenopausal women increases the risk of cardiovascular disease. However, timing may be everything, as estrogen is often administered immediately after ovariectomy (Ovx) in animal studies, while estrogen administration in human studies occurred many years postmenopause. This study investigates the discrepancy by administering 17ß-estradiol (E2) in a slow-release capsule to Norway Brown rats both immediately following Ovx and 9 wk post-Ovx (Late), and studying differences in gene expression between these two groups compared with age-matched Ovx and sham-operated animals. Two different types of microarray were used to analyze the left ventricles from these groups: an Affymetrix array (n = 3/group) and an inflammatory cytokines and receptors PCR array (n = 4/group). Key genes were analyzed by Western blotting. Ovx without replacement led to an increase in caspase 3, caspase 9, calpain 2, matrix metalloproteinase (MMP)9, and TNF-α. Caspase 6, STAT3, and CD11b increased in the Late group, while tissue inhibitor of metalloproteinase 2, MMP14, and collagen I α1 were decreased. MADD and fibronectin were increased in both Ovx and Late. TNF-α and inducible nitric oxide synthase (iNOS) protein levels increased with Late replacement. Many of these changes were prevented by early E2 replacement. These findings suggest that increased expression of inflammatory genes, such as TNF-α and iNOS, may be involved in some of the deleterious effects of delayed E2 administration seen in human studies.
Assuntos
Envelhecimento/sangue , Terapia de Reposição de Estrogênios , Estrogênios/sangue , Estrogênios/uso terapêutico , Regulação da Expressão Gênica , Inflamação/tratamento farmacológico , Inflamação/genética , Miocárdio/metabolismo , Animais , Apoptose/genética , Western Blotting , Matriz Extracelular/genética , Feminino , Humanos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos BN , Transdução de Sinais/genéticaRESUMO
Estrogens can either relax or contract arteries via rapid, nongenomic mechanisms involving classic estrogen receptors (ER). In addition to ERα and ERß, 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.
Assuntos
Vasos Coronários/efeitos dos fármacos , Estradiol/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Animais , Cálcio/metabolismo , Células Cultivadas , Vasos Coronários/citologia , Vasos Coronários/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Relaxamento Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Receptores de Estrogênio , Receptores Acoplados a Proteínas G/metabolismo , Suínos , Regulação para Cima/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
The marked sexual dimorphism that exists in human cardiovascular diseases has led to the dogmatic concept that testosterone (Tes) has deleterious effects and exacerbates the development of cardiovascular disease in males. While some animal studies suggest that Tes does exert deleterious effects by enhancing vascular tone through acute or chronic mechanisms, accumulating evidence suggests that Tes and other androgens exert beneficial effects by inducing rapid vasorelaxation of vascular smooth muscle through nongenomic mechanisms. While this effect frequently has been observed in large arteries at micromolar concentrations, more recent studies have reported vasorelaxation of smaller resistance arteries at nanomolar (physiological) concentrations. The key mechanism underlying Tes-induced vasorelaxation appears to be the modulation of vascular smooth muscle ion channel function, particularly the inactivation of L-type voltage-operated Ca(2+) channels and/or the activation of voltage-operated and Ca(2+)-activated K(+) channels. Studies employing Tes analogs and metabolites reveal that androgen-induced vasodilation is a structurally specific nongenomic effect that is fundamentally different than the genomic effects on reproductive targets. For example, 5alpha-dihydrotestosterone exhibits potent genomic-androgenic effects but only moderate vasorelaxing activity, whereas its isomer 5beta-dihydrotestosterone is devoid of androgenic effects but is a highly efficacious vasodilator. These findings suggest that the dihydro-metabolites of Tes or other androgen analogs devoid of androgenic or estrogenic effects could have useful therapeutic roles in hypertension, erectile dysfunction, prostatic ischemia, or other vascular dysfunctions.