New Insights Into Permeation of Large Cations Through ATP-Gated P2X Receptors

The permeability of large cations through the P2X pore has remained arguably the most controversial and complicated topic in P2X-related research, with the emergence of conflicting studies on the existence, mechanism and physiological relevance of a so-called “dilated” state. Due to the important role of several “dilating” P2X subtypes in numerous diseases, a clear and detailed understanding of this phenomenon represents a research priority. Recent advances, however, have challenged the existence of a progressive, ATP-induced pore dilation, by demonstrating that this phenomenon is an artifact of the method employed. Here, we discuss briefly the history of this controversial and enigmatic dilated state, from its initial discovery to its recent reconsideration. We will discuss the literature in which mechanistic pathways to a large cation-permeable state are proposed, as well as important advances in the methodology employed to study this elusive state. Considering recent literature, we will also open the discussion as to whether an intrinsically dilating P2X pore exists, as well as the physiological relevance of such a large cation-permeable pore and its potential use as therapeutic pathway

ATP-gated P2X receptors in health and disease

Extracellular ATP is currently recognized as one of the most widely distributed neurotransmitters and neuromodulators in the peripheral and central nervous system. ATP-gated P2X receptors are expressed by neurons, glial and many other non-neuronal cells and represent an attractive target for therapeutic interventions. Diverse molecular and cellular mechanisms have been identified for P2X receptor functioning, including the ability to enlarge the size of the ion pore associated with the release of several key immune molecules. A major recent breakthrough was the determination of the X-ray crystal structures of zebrafish P2X4 receptor in ATP-bound and ATP-free states. The P2X receptor research field is rapidly growing, as evidenced by the almost 2000 papers published in the last 5 years. However, despite the fundamental signalling function of extracellular ATP in the nervous system, the widespread roles of P2X receptors have not been widely elucidated and presented in textbooks. In this volume of papers we aim to gather a collection of high quality papers, detailing the latest insights from the most accomplished international P2X receptor researchers. Importantly, basic research into P2X receptors has a strong translational impact and our collection of articles could be a valuable guide for the development of new pharmacological and biotechnological tools addressing the function of P2X receptors. Within this collection we plan to cover receptor structure-function relationships, receptors trafficking, to highlight the special properties of P2X receptors and their pharmacological profiles, and to describe the translational aspects of cellular ATP signaling in pain and in other neurological and vascular diseases

Molecular mechanisms of the cardiovascular protective effects of polyphenols

Epidemiological studies have reported a greater reduction in cardiovascular risk and metabolic disorders associated with diets rich in polyphenols. The antioxidant effects of polyphenols are attributed to the regulation of redox enzymes by reducing reactive oxygen species production from mitochondria, NADPH oxidases and uncoupled endothelial NO synthase in addition to also up-regulating multiple antioxidant enzymes. Although data supporting the effects of polyphenols in reducing oxidative stress are promising, several studies have suggested additional mechanisms in the health benefits of polyphenols. Polyphenols from red wine increase endothelial NO production leading to endothelium-dependent relaxation in conditions such as hypertension, stroke or the metabolic syndrome. Numerous molecules contained in fruits and vegetables can activate sirtuins to increase lifespan and silence metabolic and physiological disturbances associated with endothelial NO dysfunction. Although intracellular pathways involved in the endothelial effects of polyphenols are partially described, the molecular targets of these polyphenols are not completely elucidated. We review the novel aspects of polyphenols on several targets that could trigger the health benefits of polyphenols in conditions such as metabolic and cardiovascular disturbances

ENDOTHELIUM-INDEPENDENT AND ENDOTHELIUM-DEPENDENT VASORELAXATION BY A DICHLOROMETHANE FRACTION FROM ANOGEISSUS LEIOCARPUS (DC) GUILL. ET PERR. (COMBRETACEAE): POSSIBLE INVOLVEMENT OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE INHIBITION.

Many traditional medicinal herbs from Burkina Faso are used to treat arterial hypertension (HTA). Among them, Anogeissus leiocarpus (A. Leiocarpus) which is well known and widely used in Burkina traditional medicine. Herein we assess the effects of dichloromethane fraction from A. leiocarpus stem bark (ALF), selected as the most active on cyclic nucleotide phosphodiesterases (PDEs) and characterized its specificity towards purified vascular PDE1 to PDE5 isoenzymes and study its effects on a vascular model. ALF potently and preferentially inhibits (IC50=1.6 ± 0.6 µg/mL) the calmodulin-dependent phosphodiesterase PDE1, being mainly present in vascular smooth muscle and preferentially hydrolyses cGMP. In the same range (IC50 =2.8 ±0.2 µg/ml) ALF inhibits PDE2, a cGMP-activated enzyme that is only present in endothelial cells and hydrolyses both cAMP and cGMP. PDE5, which specifically hydrolyses cGMP and which mainly contributes to cGMP hydrolysis is also potently inhibited by ALF (IC50=7.6±3.5 µg/ml). The potencies of ALF on cAMP hydrolyzing isoenzymes was lesser, being more effective on PDE4 (IC50= 17.6±3.5 µg/ml) than on PDE3 (60.9 ± 1.8 µg/ml). Since the major effect of ALF were against cGMP hydrolysis and since cGMP is implicated in endothelium-dependent relaxation, the endothelium-dependent vasorelaxation was studied on isolated porcine coronary arteries rings pre-contracted with U46619. The endothelium-dependent vasorelaxation is significantly inhibited by Nω-nitro-L-arginine (LNA 300 µmol/L, an inhibitor of endothelial NO synthase), but not affected by charybdotoxin (CTX, 100nM) plus apamin (APA, 100nM) (two inhibitors of EDHF-mediated responses). The combination of 4-aminopyridine (4-AP, 1 mmol/L, inhibitor of voltage-dependent potassium channels, Kv) plus baryum (Ba2+, 30 µmol/L, inhibitor of the potassium channels with entering correction, Kir) plus ouabain (3 µmol/L, inhibitor of ATPase Na+/K+ channels) partially inhibits endothelium-independent vasorelaxant effect. This endothelium-independent relaxant effect was also sensitive to combination of 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one (ODQ, 10 µM, soluble guanylyl cyclase inhibitor) and N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 100 nM, Protein Kinase A inhibitor). Taken together, these results indicate that ALF is a powerful vasodilator modulated by the formation of NO from endothelium, but also act by directly relaxing the vascular smooth muscle cells, by inhibiting cGMP hydrolyzing PDEs (PDE1, PDE2 and PDE5) and to a lesser extend on cAMP degradation (PDE3 and PDE4), cAMP and cGMP being second messengers involved in vascular relaxation

P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH.

P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells

Molecular mechanisms of the cardiovascular protective effects of polyphenols

Epidemiological studies have reported a greater reduction in cardiovascular risk and metabolic disorders associated with diets rich in polyphenols. The antioxidant effects of polyphenols are attributed to the regulation of redox enzymes by reducing reactive oxygen species production from mitochondria, NADPH oxidases and uncoupled endothelial NO synthase in addition to also up-regulating multiple antioxidant enzymes. Although data supporting the effects of polyphenols in reducing oxidative stress are promising, several studies have suggested additional mechanisms in the health benefits of polyphenols. Polyphenols from red wine increase endothelial NO production leading to endothelium-dependent relaxation in conditions such as hypertension, stroke or the metabolic syndrome. Numerous molecules contained in fruits and vegetables can activate sirtuins to increase lifespan and silence metabolic and physiological disturbances associated with endothelial NO dysfunction. Although intracellular pathways involved in the endothelial effects of polyphenols are partially described, the molecular targets of these polyphenols are not completely elucidated. We review the novel aspects of polyphenols on several targets that could trigger the health benefits of polyphenols in conditions such as metabolic and cardiovascular disturbances

Differential Signal Transduction of Progesterone and Medroxyprogesterone Acetate in Human Endothelial Cells

AbstractThe conjugated equine estrogens-only arm of the Women's Health Initiative trial, showing a trend toward protection from heart disease as opposed to women receiving also medroxyprogesterone acetate (MPA), strengthens the debate on the cardiovascular effects of progestins. We compared the effects of progesterone (P) or MPA on the synthesis of nitric oxide and on the expression of leukocyte adhesion molecules, characterizing the signaling events recruited by these compounds. Although P significantly increases nitric oxide synthesis via transcriptional and nontranscriptional mechanisms, MPA is devoid of such effects. Moreover, when used together with physiological estradiol (E2) concentrations, P potentiates E2 effects, whereas MPA impairs E2 signaling. These findings are observed both in isolated human endothelial cells as well as in vivo, in ovariectomized rat aortas. A marked difference in the recruitment of MAPK and phosphatidylinositol-3 kinase explains the divergent effects of the two gestagens. In addition, both P and MPA decrease the adhesiveness of endothelial cells for leukocytes when given alone or with estrogen. MPA is more potent than P in inhibiting the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. However, when administered together with physiological amounts of glucocorticoids, MPA (which also binds glucocorticoid receptor) markedly interferes with the hydrocortisone-dependent stabilization of the transcription factor nuclear factor κB and with the expression of adhesion molecules, acting as a partial glucocorticoid receptor antagonist. Our findings show significant differences in the signal transduction pathways recruited by P and MPA in endothelial cells, which may have relevant clinical implications

Grape-Derived Polyphenols Improve Aging-Related Endothelial Dysfunction in Rat Mesenteric Artery: Role of Oxidative Stress and the Angiotensin System

Aging is characterized by the development of an endothelial dysfunction, which affects both the nitric oxide (NO)- and the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations, associated with vascular oxidative stress and the activation of the angiotensin system. This study investigated whether red wine polyphenols (RWPs), antioxidants and potent stimulators of NO- and EDHF-mediated relaxations improve aging-related endothelial dysfunction, and, if so, examined the underlying mechanism. Mesenteric artery reactivity was determined in organ chambers, vascular oxidative stress by dihydroethidine and MitoSOX staining, and expression of target proteins by immunohistochemical staining. Control young rats (16 weeks) received solvent (ethanol, 3% v/v), and middle-aged rats (46 weeks) either solvent or RWPs (100 mg/kg/day) in the drinking water. The acetylcholine-induced endothelium-dependent NO component was slightly reduced whereas the EDHF component was markedly blunted in rings of middle-aged rats compared to young rats. The endothelial dysfunction was associated with oxidative stress, an upregulation of angiotensin II and AT1 receptors and a down-regulation of SKCa, IKCa, and angiotensin converting enzyme. Intake of RWPs for either one or two weeks improved the NO and the EDHF components of the relaxation, and normalized oxidative stress, the expression of SKCa, IKCa and the components of the angiotensin system. The protective effect of the 2-week RWPs treatment persisted for one and two weeks following stopping intake of RWPs. Thus, intake of RWPs caused a persistent improvement of the endothelial function, particularly the EDHF component, in middle-aged rats and this effect seems to involve the normalization of the expression of SKCa, IKCa and the angiotensin system