The importance of the rapid vascular effects of aldosterone is increasingly appreciated. Through these rapid pathways, aldosterone has been shown to regulate vascular contractility, cell growth, and ...apoptosis. In our most recent studies, we demonstrated the effects of aldosterone on cell growth and contractility in vascular smooth muscle cells. We showed that these effects could occur via activation of the classic mineralocorticoid receptor, as well the recently characterized G protein-coupled estrogen receptor (GPER), initially characterized as an estrogen-specific receptor. However, the mechanisms underlying aldosterone's endothelium-dependent actions are unknown. Furthermore, the ERK regulatory and proapoptotic effects of aldosterone mediated by GPER activation in cultured vascular smooth muscle cells were only apparent when GPER was reintroduced into these cells by gene transfer. Whether GPER activation via aldosterone might be an important regulator in native vascular cells has been questioned. Therefore, to determine the role of GPER in mediating aldosterone's effects on cell growth and vascular reactivity in native cells, we examined rat aortic vascular endothelial cells, a model characterized by persistent robust expression of GPER, but without detectable mineralocorticoid receptor expression. In these endothelial cells, the GPER agonist G1 mediates a rapid increase in ERK phosphorylation that is wholly GPER-dependent, paralleling the actions of aldosterone. The effects of G1 and aldosterone to stimulate ERK phosphorylation paralleled their proapoptotic and antiproliferative effects. In previous studies, we reported that aldosterone mediates a rapid endothelium-dependent vasodilatory effect, antagonistic to its direct vasoconstrictor effect in endothelium-denuded preparations. Using a rat aortic ring/organ bath preparation to determine the GPER dependence of aldosterone's endothelium-dependent vasodilator effects, we demonstrate that aldosterone inhibits phenylephrine-mediated contraction. This vasodilator effect parallels the actions of the GPER agonist G1. Furthermore, the effects of aldosterone were completely ablated by the GPER antagonist G15. These data support an important role of GPER activation in aldosterone-mediated regulation of endothelial cell growth, as well as in aldosterone's endothelium-mediated regulation of vasoreactivity.
It has been increasingly appreciated that steroids elicit acute vascular effects through rapid, so-called nongenomic signaling pathways. Though aldosterone, for example, has been demonstrated to ...mediate rapid vascular effects via both mineralocorticoid receptor-dependent and -independent pathways, the mechanism(s) of this mineralocorticoid receptor-independent effect of aldosterone is yet to be determined. For estrogen, its rapid effects have been reported to be, at least in part, mediated via the 7-transmembrane-spanning, G protein-coupled receptor GPR30. Previous studies have demonstrated common response outcomes in response to both aldosterone and estrogen on GPR30 expression, ie, activation of phosphatidylinositol 3-kinase-dependent contraction and extracellular signal-regulated kinase activation in vascular smooth muscle cells. The present studies were undertaken to test the hypothesis that the rapid response to aldosterone in smooth muscle is dependent on the availability of a GPR30-dependent signaling pathway. These findings not only reconcile differences in the literature for aldosterone response in freshly isolated versus cultured aortic smooth muscle cells but also suggest alternative therapeutic strategies for modulating aldosterone actions on the vasculature in vivo.
Abstract
The arterial myogenic response to intraluminal pressure elicits constriction to maintain tissue perfusion. Smooth muscle Ca
2+
is a key determinant of constriction, tied to L-type (Ca
V
...1.2) Ca
2+
channels. While important, other Ca
2+
channels, particularly T-type could contribute to pressure regulation within defined voltage ranges. This study examined the role of one T-type Ca
2+
channel (Ca
V
3.1) using C57BL/6 wild type and Ca
V
3.1
−/−
mice. Patch-clamp electrophysiology, pressure myography, blood pressure and Ca
2+
imaging defined the Ca
V
3.1
−/−
phenotype relative to C57BL/6. Ca
V
3.1
−/−
mice had absent Ca
V
3.1 expression and whole-cell current, coinciding with lower blood pressure and reduced mesenteric artery myogenic tone, particularly at lower pressures (20–60 mmHg) where membrane potential is hyperpolarized. This reduction coincided with diminished Ca
2+
wave generation, asynchronous events of Ca
2+
release from the sarcoplasmic reticulum, insensitive to L-type Ca
2+
channel blockade (Nifedipine, 0.3 µM). Proximity ligation assay (PLA) confirmed IP
3
R1/Ca
V
3.1 close physical association. IP
3
R blockade (2-APB, 50 µM or xestospongin C, 3 µM) in nifedipine-treated C57BL/6 arteries rendered a Ca
V
3.1
−/−
contractile phenotype. Findings indicate that Ca
2+
influx through Ca
V
3.1 contributes to myogenic tone at hyperpolarized voltages through Ca
2+
-induced Ca
2+
release tied to the sarcoplasmic reticulum. This study helps establish Ca
V
3.1 as a potential therapeutic target to control blood pressure.
Acetylcholine, the first chemical to be identified as a neurotransmitter, is packed in synaptic vesicles by the activity of VAChT (vesicular acetylcholine transporter). A decrease in VAChT expression ...has been reported in a number of diseases, and this has consequences for the amount of acetylcholine loaded in synaptic vesicles as well as for neurotransmitter release. Several genetically modified mice targeting the VAChT gene have been generated, providing novel models to understand how changes in VAChT affect transmitter release. A surprising finding is that most cholinergic neurons in the brain also can express a second type of vesicular neurotransmitter transporter that allows these neurons to secrete two distinct neurotransmitters. Thus a given neuron can use two neurotransmitters to regulate different physiological functions. In addition, recent data indicate that non-neuronal cells can also express the machinery used to synthesize and release acetylcholine. Some of these cells rely on VAChT to secrete acetylcholine with potential physiological consequences in the periphery. Hence novel functions for the oldest neurotransmitter known are emerging with the potential to provide new targets for the treatment of several pathological conditions.
Obesity-associated chronic inflammation contributes to metabolic dysfunction and propagates atherosclerosis. Recent evidence suggests that increased dietary cholesterol exacerbates inflammation in ...adipose tissue and liver, contributing to the proatherogenic milieu. The ability of the citrus flavonoid naringenin to prevent these cholesterol-induced perturbations is unknown. To assess the ability of naringenin to prevent the amplified inflammatory response and atherosclerosis induced by dietary cholesterol, male Ldlr−/− mice were fed either a cholesterol-enriched high-fat or low-fat diet supplemented with 3% naringenin for 12 weeks. Naringenin, through induction of hepatic fatty acid (FA) oxidation and attenuation of FA synthesis, prevented hepatic steatosis, hepatic VLDL overproduction, and hyperlipidemia induced by both cholesterol-rich diets. Naringenin attenuated hepatic macrophage infiltration and inflammation stimulated by dietary cholesterol. Insulin resistance, adipose tissue expansion, and inflammation were alleviated by naringenin. Naringenin attenuated the cholesterol-induced formation of both foam cells and expression of inflammatory markers in peritoneal macrophages. Naringenin significantly decreased atherosclerosis and inhibited the formation of complex lesions, which was associated with normalized aortic lipids and a reversal of aortic inflammation. We demonstrate that in mice fed cholesterol-enriched diets, naringenin attenuates peripheral and systemic inflammation, leading to protection from atherosclerosis. These studies offer a therapeutically relevant alternative for the prevention of cholesterol-induced metabolic dysregulation.
The thoracic aortic wall can degenerate over time with catastrophic consequences. Vascular smooth muscle cells (SMCs) can resist and repair artery damage, but their capacities decline with age and ...stress. Recently, cellular production of nicotinamide adenine dinucleotide (NAD
) via nicotinamide phosphoribosyltransferase (Nampt) has emerged as a mediator of cell vitality. However, a role for Nampt in aortic SMCs in vivo is unknown.
To determine whether a Nampt-NAD
control system exists within the aortic media and is required for aortic health.
Ascending aortas from patients with dilated aortopathy were immunostained for NAMPT, revealing an inverse relationship between SMC NAMPT content and aortic diameter. To determine whether a Nampt-NAD
control system in SMCs impacts aortic integrity, mice with
-deficient SMCs were generated. SMC-
knockout mice were viable but with mildly dilated aortas that had a 43% reduction in NAD
in the media. Infusion of angiotensin II led to aortic medial hemorrhage and dissection. SMCs were not apoptotic but displayed senescence associated-ß-galactosidase activity and upregulated p16, indicating premature senescence. Furthermore, there was evidence for oxidized DNA lesions, double-strand DNA strand breaks, and pronounced susceptibility to single-strand breakage. This was linked to suppressed poly(ADP-ribose) polymerase-1 activity and was reversible on resupplying NAD
with nicotinamide riboside. Remarkably, we discovered unrepaired DNA strand breaks in SMCs within the human ascending aorta, which were specifically enriched in SMCs with low NAMPT.
promoter analysis revealed CpG hypermethylation within the dilated human thoracic aorta and in SMCs cultured from these tissues, which inversely correlated with
expression.
The aortic media depends on an intrinsic NAD
fueling system to protect against DNA damage and premature SMC senescence, with relevance to human thoracic aortopathy.
The autonomic nervous system is an important modulator of cardiac signaling in both health and disease. In fact, the significance of altered parasympathetic tone in cardiac disease has recently come ...to the forefront. Both neuronal and nonneuronal cholinergic signaling likely play a physiological role, since modulating acetylcholine (ACh) signaling from neurons or cardiomyocytes appears to have significant consequences in both health and disease. Notably, many of these effects are solely due to changes in cholinergic signaling, without altered sympathetic drive, which is known to have significant adverse effects in disease states. As such, it is likely that enhanced ACh-mediated signaling not only has direct positive effects on cardiomyocytes, but it also offsets the negative effects of hyperadrenergic tone. In this review, we discuss recent studies that implicate ACh as a major regulator of cardiac remodeling and provide support for the notion that enhancing cholinergic signaling in human patients with cardiac disease can reduce morbidity and mortality. These recent results support the idea of developing large clinical trials of strategies to increase cholinergic tone, either by stimulating the vagus or by increased availability of Ach, in heart failure.
Heart activity and long‐term function are regulated by the sympathetic and parasympathetic branches of the nervous system. Parasympathetic neurons have received increased attention recently because ...acetylcholine (ACh) has been shown to play protective roles in heart disease. However, parasympathetic innervation is sparse in the heart, raising the question of how cholinergic signaling regulates cardiomyocytes. We hypothesized that non‐neuronal secretion of ACh from cardiomyocytes plays a role in cholinergic regulation of cardiac activity. To test this possibility, we eliminated secretion of ACh exclusively from cardiomyocytes by targeting the vesicular acetylcholine transporter (VAChT). We find that lack of cardiomyocyte‐secreted ACh disturbs the regulation of cardiac activity and causes cardiomyocyte remodeling. Mutant mice present normal hemodynamic parameters under nonstressful conditions; however, following exercise, their heart rate response is increased. Moreover, hearts from mutant mice present increased oxidative stress, altered calcium signaling, remodeling, and hypertrophy. Hence, without cardiomyocyte‐derived ACh secretion, hearts from mutant mice show signs of imbalanced autonomic activity consistent with decreased cholinergic drive. These unexpected results suggest that cardiomyocyte‐derived ACh is required for maintenance of cardiac homeostasis and regulates critical signaling pathways necessary to maintain normal heart activity. We propose that this non‐neuronal source of ACh boosts parasympathetic cholinergic signaling to counterbalance sympathetic activity regulating multiple aspects of heart physiology.—Roy, A., Fields, W. C., Rocha‐Resende, C., Resende, R. R., Guatimosim, S., Prado, V. F., Gros, R., Prado, M. A. M., Cardiomyocyte‐secreted acetylcholine is required for maintenance of homeostasis in the heart. FASEB J. 27, 5072–5082 (2013). www.fasebj.org
Aldosterone, oestrogens and other vasoactive steroids are important physiological and pathophysiological regulators of cardiovascular and metabolic function. The traditional view of the ...cardiovascular actions of these vasoactive steroids has focused on their roles as regulators of transcription via activation of their ‘classical’ receptors mineralocorticoid receptors (MR) and oestrogen receptors (ER). However, based on a series of observations going back more than half a century, scientists have speculated that a range of steroids, including oestrogen and aldosterone, might have effects on regulation of smooth muscle contractility, cell growth and differentiation that are too rapid to be accounted for by transcriptional regulation. Recent studies performed in our laboratories (and those of others) have begun to elucidate the mechanism of rapid steroid‐mediated cardiometabolic regulation. GPR30, now designated as GPER‐1 (http://www.iuphar‐db.org/DATABASE/FamilyIntroductionForward?familyId=22), a newly characterized ‘orphan receptor’, has been implicated in mediating the rapid effects of estradiol and most recently those of aldosterone. Studies to date have taught us that to understand the rapid vascular mechanisms of steroids, one must (i) know which vascular ‘compartment’ the steroid is acting; (ii) know which receptor the steroid hormone is activating; and (iii) not assume the receptor specificity of a steroid receptor ligand based solely on its selectivity for its traditional ‘transcriptional’ steroid receptor. Our newfound appreciation of the rapid effects of steroids such as aldosterone and oestrogens opens up a new vista for advancing our understanding of the biology and pathobiology of vascular regulation.
LINKED ARTICLES This article is one of a set of reviews submitted to BJP in connection with talks given at the September 2010 meeting of the International Society of Hypertension in Vancouver, Canada. To view the other articles in this collection visit http://dx.doi.org/10.1111/j.1476‐5381.2010.01167.x, http://dx.doi.org/10.1111/j.1476‐5381.2011.01235.x and http://dx.doi.org/10.1111/j.1476‐5381.2011.01260.x
The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved ...remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.
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