Endothelial mechanisms for inactivation of inflammation-induced hyperpermeability.

Microvascular hyperpermeability is a hallmark of inflammation. Many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function. Therefore, we propose that targeted therapeutic approaches focusing on mechanisms that terminate hyperpermeability would avoid the negative effects of prolonged hyperpermeability while retaining its short-term beneficial effects. We tested the hypothesis that inflammatory agonist signaling leads to hyperpermeability and initiates a delayed cascade of cAMP-dependent pathways that causes inactivation of hyperpermeability. We applied platelet-activating factor (PAF) and vascular endothelial growth factor (VEGF) to induce hyperpermeability. We used an Epac1 agonist to selectively stimulate exchange protein activated by cAMP (Epac1) and promote inactivation of hyperpermeability. Stimulation of Epac1 inactivated agonist-induced hyperpermeability in the mouse cremaster muscle and in human microvascular endothelial cells (HMVECs). PAF induced nitric oxide (NO) production and hyperpermeability within 1 min and NO-dependent increased cAMP concentration in about 15-20 min in HMVECs. PAF triggered phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in a NO-dependent manner. Epac1 stimulation promoted cytosol-to-membrane eNOS translocation in HMVECs and in myocardial microvascular endothelial (MyEnd) cells from wild-type mice, but not in MyEnd cells from VASP knockout mice. We demonstrate that PAF and VEGF cause hyperpermeability and stimulate the cAMP/Epac1 pathway to inactivate agonist-induced endothelial/microvascular hyperpermeability. Inactivation involves VASP-assisted translocation of eNOS from the cytosol to the endothelial cell membrane. We demonstrate that hyperpermeability is a self-limiting process, whose timed inactivation is an intrinsic property of the microvascular endothelium that maintains vascular homeostasis in response to inflammatory conditions.NEW & NOTEWORTHY Termination of microvascular hyperpermeability has been so far accepted to be a passive result of the removal of the applied proinflammatory agonists. We provide in vivo and in vitro evidence that 1) inactivation of hyperpermeability is an actively regulated process, 2) proinflammatory agonists (PAF and VEGF) stimulate microvascular hyperpermeability and initiate endothelial mechanisms that terminate hyperpermeability, and 3) eNOS location-translocation is critical in the activation-inactivation cascade of endothelial hyperpermeability.

Microalgae share key features with human erythrocytes and can safely circulate through the vascular system in mice.

As animal cells cannot produce oxygen, erythrocytes are responsible for gas interchange, being able to capture and deliver oxygen upon tissue request. Interestingly, several other cells in nature produce oxygen by photosynthesis, raising the question of whether they could circulate within the vascular networks, acting as an alternative source for oxygen delivery. To address this long-term goal, here some physical and mechanical features of the photosynthetic microalga Chlamydomona reinhardtii were studied and compared with erythrocytes, revealing that both exhibit similar size and rheological properties. Moreover, key biocompatibility aspects of the microalgae were evaluated in vitro and in vivo, showing that C. reinhardtii can be co-cultured with endothelial cells, without affecting each other's morphology and viability. Moreover, short-term systemic perfusion of the microalgae showed a thoroughly intravascular distribution in mice. Finally, the systemic injection of high numbers of microalgae did not trigger deleterious responses in living mice. Altogether, this work provides key scientific insights to support the notion that photosynthetic oxygenation could be achieved by circulating microalgae, representing another important step towards human photosynthesis. KEY POINTS: • C. reinhardtii and endothelial cells are biocompatible in vitro. • C. reinhardtii distribute throughout the entire vasculature after mice perfusion. • C. reinhardtii do not trigger deleterious responses after injection in mice.

TNF-α-activated eNOS signaling increases leukocyte adhesion through the S-nitrosylation pathway.

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Apocynin Treatment Prevents Cardiac Connexin 43 Hemichannels Hyperactivity by Reducing Nitroso-Redox Stress in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a fatal disease that causes cardiomyopathy and is associated with oxidative stress. In the heart, oxidative stress interferes with the location of connexin 43 (Cx43) to the intercalated discs causing its lateralization to the plasma membrane where Cx43 forms hemichannels. We tested the hypothesis that in DMD cardiomyopathy, increased oxidative stress is associated with the formation and activation of Cx43 hemichannels. For this, we used mdx mice as a DMD model and evaluated cardiac function, nitroso-redox changes and Cx43 hemichannels permeability. Mdx hearts presented increased NADPH oxidase-derived oxidative stress and increased Cx43 S-nitrosylation compared to controls. These redox changes were associated with increased Cx43 lateralization, decreased cardiac contractility and increased arrhythmic events. Pharmacological inhibition of NADPH oxidase using apocynin (one month) reduced systemic oxidative stress and reversed the aforementioned changes towards normal, except Cx43 lateralization. Opening of Cx43 hemichannels was blocked by apocynin treatment and by acute hemichannel blockade with carbenoxolone. NADPH oxidase inhibition also prevented the occurrence of apoptosis in mdx hearts and reversed the ventricular remodeling. These results show that NADPH oxidase activity in DMD is associated with S-nitrosylation and opening of Cx43 hemichannels. These changes lead to apoptosis and cardiac dysfunction and were prevented by NADPH oxidase inhibition.

Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly.

The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking ß1-integrin antibodies point to ß1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.

Insulin therapy and its consequences for the mother, foetus, and newborn in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a disease characterised by glucose intolerance and first diagnosed in pregnancy. This condition relates to an anomalous placental environment and aberrant placental vascular function. GDM-associated hyperglycaemia changes the placenta structure leading to abnormal development and functionality of this vital organ. Aiming to avoid the GDM-hyperglycaemia and its deleterious consequences in the mother, the foetus and newborn, women with GDM are firstly treated with a controlled diet therapy; however, some of the women fail to reach the recommended glycaemia values and therefore they are passed to the second line of treatment, i.e., insulin therapy. The several protocols available in the literature regarding insulin therapy are variable and not a clear consensus is yet reached. Insulin therapy restores maternal glycaemia, but this beneficial effect is not reflected in the foetus and newborn metabolism, suggesting that other factors than d-glucose may be involved in the pathophysiology of GDM. Worryingly, insulin therapy may cause alterations in the placenta and umbilical vessels as well as the foetus and newborn additional to those seen in pregnant women with GDM treated with diet. In this review, we summarised the variable information regarding indications and protocols for administration of the insulin therapy and the possible outcomes on the function and structure of the foetoplacental unit and the neonate parameters from women with GDM.

S-nitrosylation of VASP at cysteine 64 mediates the inflammation-stimulated increase in microvascular permeability.

We tested the hypothesis that platelet-activating factor (PAF) induces S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) as a mechanism to reduce microvascular endothelial barrier integrity and stimulate hyperpermeability. PAF elevated S-nitrosylation of VASP above baseline levels in different endothelial cells and caused hyperpermeability. To ascertain the importance of endothelial nitric oxide synthase (eNOS) subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced S-nitrosylation of VASP in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Reconstitution of VASP knockout myocardial endothelial cells with cysteine mutants of VASP demonstrated that S-nitrosylation of cysteine 64 is associated with PAF-induced hyperpermeability. We propose that regulation of VASP contributes to endothelial cell barrier integrity and to the onset of hyperpermeability. S-nitrosylation of VASP inhibits its function in barrier integrity and leads to endothelial monolayer hyperpermeability in response to PAF, a representative proinflammatory agonist.NEW & NOTEWORTHY Here, we demonstrate that S-nitrosylation of vasodilator-stimulated phosphoprotein (VASP) on C64 is a mechanism for the onset of platelet-activating factor-induced hyperpermeability. Our results reveal a dual role of VASP in endothelial permeability. In addition to its well-documented function in barrier integrity, we show that S-nitrosylation of VASP contributes to the onset of endothelial permeability.

S-nitrosylation regulates VE-cadherin phosphorylation and internalization in microvascular permeability.

The adherens junction complex, composed mainly of vascular endothelial (VE)-cadherin, ß-catenin, p120, and γ-catenin, is the main element of the endothelial barrier in postcapillary venules.S-nitrosylation of ß-catenin and p120 is an important step in proinflammatory agents-induced hyperpermeability. We investigated in vitro and in vivo whether or not VE-cadherin isS-nitrosylated using platelet-activating factor (PAF) as agonist. We report that PAF-stimulates S-nitrosylation of VE-cadherin, which disrupts its association with ß-catenin. In addition, based on inhibition of nitric oxide production, our results strongly suggest that S-nitrosylation is required for VE-cadherin phosphorylation on tyrosine and for its internalization. Our results unveil an important mechanism to regulate phosphorylation of junctional proteins in association with S-nitrosylation.

S-Nitrosation of ß-catenin and p120 catenin: a novel regulatory mechanism in endothelial hyperpermeability.

RATIONALE: Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood. OBJECTIVE: We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins. METHODS AND RESULTS: We measured PAF-stimulated SNO of ß-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of ß-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of ß-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin. CONCLUSIONS: Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.

Coordinated endothelial nitric oxide synthase activation by translocation and phosphorylation determines flow-induced nitric oxide production in resistance vessels.

BACKGROUND/AIMS: Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol. METHODS: In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2-10 ml/min) on nitric oxide (NO) production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca(2+). RESULTS: Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3-5-min) that peaked during the first 15 s, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation and dissociation from Cav-1 depended on extracellular Ca(2+), while sustained NO production was abolished by the PI3K-Akt blocker wortmannin. CONCLUSIONS: In intact resistance vessels, changes in flow induce NO production by transient Ca(2+)-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca(2+)-independent PI3K-Akt-mediated phosphorylation.

Development of a Novel Perfusable Solution for ex vivo Preservation: Towards Photosynthetic Oxygenation for Organ Transplantation.

Oxygen is the key molecule for aerobic metabolism, but no animal cells can produce it, creating an extreme dependency on external supply. In contrast, microalgae are photosynthetic microorganisms, therefore, they are able to produce oxygen as plant cells do. As hypoxia is one of the main issues in organ transplantation, especially during preservation, the main goal of this work was to develop the first generation of perfusable photosynthetic solutions, exploring its feasibility for ex vivo organ preservation. Here, the microalgae Chlamydomonas reinhardtii was incorporated in a standard preservation solution, and key aspects such as alterations in cell size, oxygen production and survival were studied. Osmolarity and rheological features of the photosynthetic solution were comparable to human blood. In terms of functionality, the photosynthetic solution proved to be not harmful and to provide sufficient oxygen to support the metabolic requirement of zebrafish larvae and rat kidney slices. Thereafter, isolated porcine kidneys were perfused, and microalgae reached all renal vasculature, without inducing damage. After perfusion and flushing, no signs of tissue damage were detected, and recovered microalgae survived the process. Altogether, this work proposes the use of photosynthetic microorganisms as vascular oxygen factories to generate and deliver oxygen in isolated organs, representing a novel and promising strategy for organ preservation.

Interleukin-8 Secreted by Glioblastoma Cells Induces Microvascular Hyperpermeability Through NO Signaling Involving S-Nitrosylation of VE-Cadherin and p120 in Endothelial Cells.

Glioblastoma is a highly aggressive brain tumor, characterized by the formation of dysfunctional blood vessels and a permeable endothelial barrier. S-nitrosylation, a post-translational modification, has been identified as a regulator of endothelial function. In this work we explored whether S-nitrosylation induced by glioblastoma tumors regulates the endothelial function. As proof of concept, we observed that S-nitrosylation is present in the tumoral microenvironment of glioblastoma in two different animal models. Subsequently, we measured S nitrosylation and microvascular permeability in EAhy296 endothelial cells and in cremaster muscle. In vitro, conditioned medium from the human glioblastoma cell line U87 activates endothelial nitric oxide synthase, causes VE-cadherin- S-nitrosylation and induces hyperpermeability. Blocking Interleukin-8 (IL-8) in the conditioned medium inhibited S-nitrosylation of VE-cadherin and hyperpermeability. Recombinant IL-8 increased endothelial permeability by activating eNOS, S-nitrosylation of VE-cadherin and p120, internalization of VE-cadherin and disassembly of adherens junctions. In vivo, IL-8 induced S-nitrosylation of VE-cadherin and p120 and conditioned medium from U87 cells caused hyperpermeability in the mouse cremaster muscle. We conclude that eNOS signaling induced by glioma cells-secreted IL-8 regulates endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Differential role of S-nitrosylation and the NO-cGMP-PKG pathway in cardiac contractility.

The role of nitric oxide (NO) in cardiac contractility is complex and controversial. Several NO donors have been reported to cause positive or negative inotropism. NO can bind to guanylate cyclase, increasing cGMP production and activating PKG. NO may also directly S-nitrosylate cysteine residues of specific proteins. We used the isolated rat heart preparation to test the hypothesis that the differential inotropic effects depend on the degree of NO production and the signaling recruited. SNAP (S-nitroso-N-acetylpenicillamine), a NO donor, increased contractility at 0.1, 1 and 10 microM. This effect was independent of phospholamban phosphorylation, was not affected by PKA inhibition with H-89 (N-[2((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide), but it was abolished by the radical scavenger Tempol (4-hydroxy-[2,2,4,4]-tetramethyl-piperidine-1-oxyl). However, at 100 microM SNAP reduced contractility, effect reversed to positive inotropism by guanylyl cyclase blockade with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), and abolished by PKG inhibition with KT5823, but not affected by Tempol. SNAP increased tissue cGMP at 100 microM, but not at lower concentrations. Consistently, a cGMP analog also reduced cardiac contractility. Finally, SNAP at 1 microM increased the level of S-nitrosylation of various cardiac proteins, including the ryanodine receptor. This study demonstrates the biphasic role for NO in cardiac contractility in a given preparation; furthermore, the differential effect is clearly ascribed to the signaling pathways involved. We conclude that although NO is highly diffusible, its output determines the fate of the messenger: low NO concentrations activate redox processes (S-nitrosylation), increasing contractility; while the cGMP-PKG pathway is activated at high NO concentrations, reducing contractility.

Gugulipid causes hypercholesterolemia leading to endothelial dysfunction, increased atherosclerosis, and premature death by ischemic heart disease in male mice.

For proper cholesterol metabolism, normal expression and function of scavenger receptor class B type I (SR-BI), a high-density lipoprotein (HDL) receptor, is required. Among the factors that regulate overall cholesterol homeostasis and HDL metabolism, the nuclear farnesoid X receptor plays an important role. Guggulsterone, a bioactive compound present in the natural product gugulipid, is an antagonist of this receptor. This natural product is widely used globally as a natural lipid-lowering agent, although its anti-atherogenic cardiovascular benefit in animal models or humans is unknown. The aim of this study was to determine the effects of gugulipid on cholesterol homeostasis and development of mild and severe atherosclerosis in male mice. For this purpose, we evaluated the impact of gugulipid treatment on liver histology, plasma lipoprotein cholesterol, endothelial function, and development of atherosclerosis and/or ischemic heart disease in wild-type mice; apolipoprotein E knockout mice, a model of atherosclerosis without ischemic complications; and SR-B1 knockout and atherogenic-diet-fed apolipoprotein E hypomorphic (SR-BI KO/ApoER61h/h) mice, a model of lethal ischemic heart disease due to severe atherosclerosis. Gugulipid administration was associated with histological abnormalities in liver, increased alanine aminotransferase levels, lower hepatic SR-BI content, hypercholesterolemia due to increased HDL cholesterol levels, endothelial dysfunction, enhanced atherosclerosis, and accelerated death in animals with severe ischemic heart disease. In conclusion, our data show important adverse effects of gugulipid intake on HDL metabolism and atherosclerosis in male mice, suggesting potential and unknown deleterious effects on cardiovascular health in humans. In addition, these findings reemphasize the need for rigorous preclinical and clinical studies to provide guidance on the consumption of natural products and regulation of their use in the general population.

Nitric Oxide Synthase 1 Modulates Basal and ß-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart.

S-nitrosylation of several Ca2+ regulating proteins in response to ß-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether ß-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (-25-30%) and basal S-nitrosylation of total proteins (-25-60%), RyR2, SERCA2 and LTCC (-60-75%). NOS-1 inhibition reduced (-25-40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (-85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) ß-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.

Stimulated human neutrophils form biologically active kinin peptides from high and low molecular weight kininogens.

Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H- and L-kininogens. Neutrophils isolated from human blood were stimulated with f-Met-Leu-Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time-course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High-performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met-Lys-bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin-immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil-generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time-course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.

Boldine prevents renal alterations in diabetic rats.

Diabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 µmol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects.

The ATP required for potentiation of skeletal muscle contraction is released via pannexin hemichannels.

During repetitive stimulation of skeletal muscle, extracellular ATP levels raise, activating purinergic receptors, increasing Ca2+ influx, and enhancing contractile force, a response called potentiation. We found that ATP appears to be released through pannexin1 hemichannels (Panx1 HCs). Immunocytochemical analyses and function were consistent with pannexin1 localization to T-tubules intercalated with dihydropyridine and ryanodine receptors in slow (soleus) and fast (extensor digitorum longus, EDL) muscles. Isolated myofibers took up ethidium (Etd+) and released small molecules (as ATP) during electrical stimulation. Consistent with two glucose uptake pathways, induced uptake of 2-NBDG, a fluorescent glucose derivative, was decreased by inhibition of HCs or glucose transporter (GLUT4), and blocked by dual blockade. Adult skeletal muscles apparently do not express connexins, making it unlikely that connexin hemichannels contribute to the uptake and release of small molecules. ATP release, Etd+ uptake, and potentiation induced by repetitive electrical stimulation were blocked by HC blockers and did not occur in muscles of pannexin1 knockout mice. MRS2179, a P2Y1R blocker, prevented potentiation in EDL, but not soleus muscles, suggesting that in fast muscles ATP activates P2Y1 but not P2X receptors. Phosphorylation on Ser and Thr residues of pannexin1 was increased during potentiation, possibly mediating HC opening. Opening of Panx1 HCs during repetitive activation allows efflux of ATP, influx of glucose and possibly Ca2+ too, which are required for potentiation of contraction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.