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Single-cell amperometry is a powerful technique that permits the detection of electrochemically active transmitters, such as catecholamines, histamine, or serotonin, released by exocytosis from secretory cells.Amperometry has two main characteristics that make it ideal for the study of exocytosis at the single-cell level with single-vesicle resolution quantal release. (i) It is noninvasive. The carbon fiber microelectrode can be carefully positioned on plasma membrane of a single cell, allowing the detection of the oxidation current of the secreted molecules. (ii) High temporal resolution and sensitivity. Exocytosis can be monitored with a real-time resolution that allows the determination of the kinetics release with an attomol detection sensitivity, which ensures an accurate calculation of the amount of transmitter released.Here, we compile some recommendations and advices to perform amperometry quantal analysis.

Objectives Lipid profiles appear to be altered in SLE patients due to disease activity and inflammation. Cholesterol efflux capacity (CEC) is the ability of high-density lipoprotein cholesterol to accept cholesterol from macrophages. CEC has been linked to cardiovascular events in the general population and is impaired in SLE patients. The aim of this study was to establish whether CEC is related to subclinical carotid atherosclerosis in SLE patients. Methods The present report is of a cross-sectional study that encompassed 418 individuals: 195 SLE patients and 223 controls. CEC, using an in vitro assay, and lipoprotein serum concentrations were assessed in patients and controls. Carotid intima-media thickness and carotid plaques were evaluated in SLE patients. A multivariable analysis was performed to study the relationship of CEC to SLE-related data, lipid profile and subclinical carotid atherosclerosis. Results CEC was downregulated in SLE patients [8.1 (4.2) % vs 16.9 (10.4) %, P = 0.004). This occurred independently of traditional cardiovascular risk factors, statin use or other variations in the lipid profile related to the disease. Traditional cardiovascular risk factors, both in patients and controls, and SLE-related data such as activity, severity or damage were not associated with CEC. After multivariable regression analysis including lipid profile–related molecules, CEC was inversely and independently associated with the presence of carotid plaques in SLE patients [odds ratio 0.87 (95% CI: 0.78, 0.97), P = 0.014]. Conclusion CEC is impaired in SLE patients independently of other inflammation-related lipid profile modifications that occur during the disease. CEC is associated with carotid plaques in SLE patients.

Cholesterol efflux capacity (CEC) is the ability of high-density lipoprotein (HDL) cholesterol to accept cholesterol from macrophages. CEC is linked to cardiovascular events in the general population, and it has been shown to be disrupted in inflammatory states. The aim of this study was to establish whether CEC is impaired in PsA patients and if this could be explained by disease-related features like disease activity. Case-control study that encompassed 105 individuals: 52 PsA patients and 53 controls. CEC, using an in vitro assay, and lipoprotein serum concentrations were assessed in patients and controls. Disease activity in patients with PsA was measured using the Disease Activity Index for Psoriatic Arthritis (DAPSA). Multivariate analysis was performed to study the differences between CEC in patients and controls, and the relation of CEC with PsA activity-related data and lipid profile. Total cholesterol, apolipoprotein A1, and LDL cholesterol serum levels were downregulated in PsA patients. CEC did not differ between controls and patients (17 ± 10 vs. 18 ± 2%, p = 0.15) after adjusting for traditional cardiovascular risk factors or other variations in the lipid profile related to the disease. Traditional cardiovascular risk factors, both in patients and controls, were not related to CEC. After multivariate regression analysis, the DAPSA score was inversely and independently associated with CEC (beta coefficient − 0.75 [95%CI − 1.39–− 0.11] %, p = 0.023). CEC is inversely associated with disease activity in PSA patients, reinforcing the role of disease activity as a key factor in the development of accelerated atherosclerosis in these patients.

We have tested whether the lack of chromogranins (Cgs) A and B could provoke CNS disorders when combined with an excess of dopamine. We chronically treated (over 6 months) mice lacking both chromogranins A and B (Cgs-KO) with a low oral dosage of L-DOPA/benserazide (10/2.5 mg/kg). Motor performance in the rota-rod test, open field activity, and metabolic cages indicated a progressive impairment in motor coordination in these mice, and an increase in rearing behavior, which was accompanied by an increase in DA within the substantia nigra. We conclude that mild chronic L-DOPA treatment does not produce nigro-striatal toxicity that could be associated with parkinsonism, neither in control nor Cgs-KO mice. Rather, Cgs-KO mice exhibit behaviors compatible with an amphetamine-like effect, probably caused by the excess of catecholamines in the CNS.

Zika virus (ZIKV) infection and its associated congenital and other neurological disorders, particularly microcephaly and other fetal developmental abnormalities, constitute a World Health Organization (WHO) Zika Virus Research Agenda within the WHO’s R&D Blueprint for Action to Prevent Epidemics, and continue to be a Public Health Emergency of International Concern (PHEIC) today. ZIKV pathogenicity is initiated by viral infection and propagation across multiple placental and fetal tissue barriers, and is critically strengthened by subverting host immunity. ZIKV immune evasion involves viral non-structural proteins, genomic and non-coding RNA and microRNA (miRNA) to modulate interferon (IFN) signaling and production, interfering with intracellular signal pathways and autophagy, and promoting cellular environment changes together with secretion of cellular components to escape innate and adaptive immunity and further infect privileged immune organs/tissues such as the placenta and eyes. This review includes a description of recent advances in the understanding of the mechanisms underlying ZIKV immune modulation and evasion that strongly condition viral pathogenesis, which would certainly contribute to the development of anti-ZIKV strategies, drugs, and vaccines.

Objective: Cholesterol efflux capacity (CEC) is the ability of high-density lipoprotein (HDL) cholesterol to accept cholesterol from macrophages. Lipid profiles and CEC appear to be altered in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) due to disease activity and inflammation. CEC has been linked to cardiovascular events in the general population and to subclinical atherosclerosis in SLE and RA patients. The aim of this study was to establish whether CEC varies between patients with SLE and those with RA. Methods: The study encompassed 460 individuals (195 SLE patients and 265 patients with RA). CEC (using an in vitro assay) and concentrations of lipoprotein serum were assessed in both populations. A multivariable regression analysis was performed to study whether CEC differs between SLE patients and RA patients. Results: Comparison of lipid patterns revealed that patients with RA have lower HDL cholesterol and higher apolipoprotein B serum levels than SLE patients. CEC was downregulated in SLE patients compared to patients with RA (β -12 [95% confidence interval -13, -10], P < 0.001). It occurred independently of traditional cardiovascular risk factors, statin use, disease-related data, and other variations in the lipid profile related to the diseases. Conclusion: Patients with RA have a more proatherogenic lipid pattern compared to those with SLE. However, CEC seems to be more damaged in SLE patients than in RA patients. Sin financiación 5.178 JCR (2021) Q2, 12/34 Rheumatology 1.566 SJR (2021) Q1, 7/61 Rheumatology No data IDR 2021 UEC

Objective It is well established that patients with systemic sclerosis (SSc) have a disrupted lipid profile and an increased cardiovascular risk. Cholesterol efflux capacity (CEC), the ability of high-density lipoprotein (HDL)-cholesterol to accept cholesterol from macrophages, has been linked to cardiovascular events. The aim of this study was to establish whether CEC and lipid profile were impaired in SSc patients with respect to controls and whether these changes were associated with disease-related data. Methods Cross-sectional study encompassed 188 individuals: 73 SSc patients and 115 controls. CEC, using an in vitro assay, and lipoprotein serum concentrations were assessed in patients and controls. A multivariable analysis was performed to study the differences in CEC between patients and controls, and if SSc-related data could explain such differences. Results The multivariable analysis adjusted for demographic characteristics, cardiovascular risk factors, and lipid-related molecules showed that total cholesterol (beta coefficient: − 22 [95%CI – 37 to – 7], p = 0.004), triglycerides (beta coefficient: 24 [95%CI 2–47], p = 0.033), lipoprotein A (beta coefficient: 22 [95%CI 2–43], p = 0.033), and CEC (beta coefficient: – 6 [95%CI − 10 to – 2]%,p = 0.002) were significantly different between patients and controls. Skin thickness, as assessed by modified Rodnan skin score, was independently associated with a lower CEC (beta coefficient: – 0.21 [95%CI – 0.37 to – 0.05]%, p = 0.011) after multivariable adjustment. Conclusion SSc patients show an abnormal lipid profile with respect to controls including CEC. Skin thickness is independent and inversely associated with CEC in SSc patients.

Glucagon-like peptide-1 receptor (GLP-1R) agonists’ ability to increase insulin secretion and promote satiety have led to their development for the treatment of type 2 diabetes and obesity; while these beneficial effects have been attributed to receptors in the pancreas and hypothalamus, the function and importance of extra-pancreatic GLP-1Rs in other organ systems is not well understood despite the possibilities for targeting these receptors for other indications. We have analysed the contribution of GLP-1R to the secretory responses of adrenal medulla, a key player in stress responses. Exendin-4 (Ex-4), a synthetic analogue of GLP-1, increased the synthesis and the release of catecholamines (CA) by increasing cAMP production, without the participation of EPAC (Exchange Proteins Activated by cAMP). Ex-4 increased CA synthesis by promoting the activation of tyrosine hydroxylase (TH), and moreover increased the quantum size of exocytotic events by switching exocytosis from partial to full fusion. Our results give a strong support to the role of GLP-1 in the fine control of glycaemia and blood pressure by sympathetic tissues.

Cholesterol efflux capacity (CEC) is the ability of high-density lipoprotein (HDL) cholesterol to accept cholesterol from macrophages. CEC is linked to cardiovascular events in the general population, and it has been shown to be disrupted in inflammatory states. The aim of this study was to establish whether CEC is impaired in PsA patients and if this could be explained by disease-related features like disease activity.Case-control study that encompassed 105 individuals: 52 PsA patients and 53 controls. CEC, using an in vitro assay, and lipoprotein serum concentrations were assessed in patients and controls. Disease activity in patients with PsA was measured using the Disease Activity Index for Psoriatic Arthritis (DAPSA). Multivariate analysis was performed to study the differences between CEC in patients and controls, and the relation of CEC with PsA activity-related data and lipid profile.Total cholesterol, apolipoprotein A1, and LDL cholesterol serum levels were downregulated in PsA patients. CEC did not differ between controls and patients (17 ± 10 vs. 18 ± 2%, p = 0.15) after adjusting for traditional cardiovascular risk factors or other variations in the lipid profile related to the disease. Traditional cardiovascular risk factors, both in patients and controls, were not related to CEC. After multivariate regression analysis, the DAPSA score was inversely and independently associated with CEC (beta coefficient - 0.75 [95%CI - 1.39-- 0.11] %, p = 0.023).CEC is inversely associated with disease activity in PSA patients, reinforcing the role of disease activity as a key factor in the development of accelerated atherosclerosis in these patients.Key Points• Cholesterol efflux capacity is linked to cardiovascular events in the general population.• In patients with psoriatic arthritis, cholesterol efflux capacity is inversely associated with disease activity (beta coefficient - 0.75[95% CI - 1.39-- 0.11] %, p = 0.023).• This finding reinforces the role of disease activity as a key factor in increasing cardiovascular risk in psoriatic arthritis patients.

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (−30%) and Imax (−21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. (Figure presented.). Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

The aim of the work was to examine whether abnormalities in the lipid profile that tocilizumab (TCZ), an anti-IL-6 receptor Ab, exerts in rheumatoid arthritis (RA) patients is related to changes in either proprotein convertase subtilisin/kexin-9 (PCSK9) serum concentrations or in serum cholesterol efflux capacity (CEC). TOCRIVAR is a one-year prospective clinical trial that analyzes the influence of TCZ on cardiovascular risk factors. Twenty-seven RA patients receiving TCZ (8 mg/kg IV/q4w) were assessed at baseline and weeks 12, 24, and 52. Disease activity indexes, adiposity composition, physical activity, serum CEC, PCSK9, and lipoproteins serum concentrations were assessed at every visit. Basal high-sensitivity C-reactive protein (hs-CRP) and disease activity were markedly reduced throughout one-year TCZ treatment. While initially total cholesterol and LDL cholesterol increased their plasma concentration, decreasing to basal afterwards, lipoprotein(a) was significantly lower than basal in all visits of the study. CEC increased after 24 week of treatment proportionally to hs-CRP reduction, and remained significantly higher after week 52 [median % change 32 (3–141), p=0.021]. Interestingly, variations in LDL cholesterol basal concentration along the one year of TCZ treatment correlated directly with changes of PCSK9 serum concentration (r=0.37, p=0.003). Basal abdominal adiposity, BMI, and physical activity remained stable during the study. Long-term TCZ-treated RA patients show an increment in CEC inversely proportional to hs-CRP reduction and changes in LDL cholesterol that might be explained, at least in part, by variations in PCSK9 plasma concentration. Overall, TCZ treatment produces a favorable qualitative net effect in terms of atherogenic implication in RA patients.

En el estudio de los mecanismos de invasión por patógenos (virus, bacterias, parásitos), es importante abordar, a nivel avanzado y molecular, los procesos que gobiernan la fusión de membrana, como uno de los eventos biológicos clave en la infección por microorganismos y en el contexto de las Enfermedades Infecciosas. En este contexto, es importante entender cómo funcionan las proteínas de los patógenos que regulan la invasión de células y tejidos, mediante el control y promoción del proceso de fusión de membrana entre el patógeno y la célula diana. Donde el empleo de la miscroscopía de onda evanescente es importante. Las ilustraciones científicas en este tema suelen ser láminas o esquemas 2D fijos, que ilustran la función de las proteínas implicadas y la fusión de membranas entre patógeno y célula diana, con cadenas secuenciales de imágenes no dinámicas, y difícilmente se ilustra el funcionamiento de este microscopio de última generación. En este capítulo se presenta, a modo de resumen, nuestro proyecto de innovación docente ULL, cuyo objetivo ha sido la creación y generación, por vez primera, de material de Animación 3D para ilustrar de forma pedagógica, y con el mayor reigor ciéntifico, la aplicación de la tecnología de microscopía fluorescente de onda evanescente (TIRFM) en el estudio de células vivas y del mecanismo de su infección por patógenos, aplicándolo a la comprenión del proceso de fusión de membranas y, en particular, al proceso de infección temprana por el virus VIH-1. In the study of the mechanisms of invasion by pathogens (viruses, bacteria, parasites), it is important to address, at an advanced and molecular level, the processes that govern membrane fusion, as one of the key biological events in infection by microorganisms and in the context of Infectious Diseases. In this scenario, it is important to understand how the proteins of the pathogens that regulate the invasion of cells and tissues work, by controlling and promoting the process of membrane fusion between the pathogen and the target cell. In this matter, the use of evanescent wave microscopy (TIRFM) is key and very important. The scientific illustrations in this topic are usually fixed sheets or 2D-diagrams that illustrate the function of the proteins involved and the fusion of membranes between pathogen and target cell, with sequential chains of non-dynamic images, where it is difficult to illustrate how this last generation microscope works. In this chapter we briefly present our ULL teaching innovation project, whose objective has been the creation and generation, for the first time, of a 3D Animation material to illustrate in a pedagogical way, and with the greatest scientific rigor, the application of the evanescent wave fluorescence microscopy technology in the study of living cells and the mechanism of their infection by pathogens, applying it to the understanding of the membrane fusion process and, in particular, to the process of early infection by the HIV-1.

HIV Nef is a central auxiliary protein in HIV infection and pathogenesis. Our results indicate that HDAC6 promotes the aggresome/autophagic degradation of the viral polyprotein Pr55Gag to inhibit HIV-1 production. Nef counteracts this antiviral activity of HDAC6 by inducing its degradation and subsequently stabilizing Pr55Gag and Vif viral proteins. Nef appears to neutralize HDAC6 by an acidic/endosomal-lysosomal processing and does not need the downregulation function, since data obtained with the non-associated cell-surface Nef-G2A mutant – the cytoplasmic location of HDAC6 – together with studies with chemical inhibitors and other Nef mutants, point to this direction. Hence, the polyproline rich region P72xxP75 (69–77 aa) and the di-Leucin motif in the Nef-ExxxLL160-165 sequence of Nef, appear to be responsible for HDAC6 clearance and, therefore, required for this novel Nef proviral function. Nef and Nef-G2A co-immunoprecipitate with HDAC6, whereas the Nef-PPAA mutant showed a reduced interaction with the anti-HIV-1 enzyme. Thus, the P72xxP75 motif appears to be responsible, directly or indirectly, for the interaction of Nef with HDAC6. Remarkably, by neutralizing HDAC6, Nef assures Pr55Gag location and aggregation at plasma membrane, as observed by TIRFM, promotes viral egress, and enhances the infectivity of viral particles. Consequently, our results suggest that HDAC6 acts as an anti-HIV-1 restriction factor, limiting viral production and infection by targeting Pr55Gag and Vif. This function is counteracted by functional HIV-1 Nef, in order to assure viral production and infection capacities. The interplay between HIV-1 Nef and cellular HDAC6 may determine viral infection and pathogenesis, representing both molecules as key targets to battling HIV.

The accumulation of neurotransmitters within secretory vesicles (SVs) far exceeds the theoretical tonic concentrations in the cytosol, a phenomenon that has captivated the attention of scientists for decades. For instance, chromaffin granules can accumulate close to molar concentrations of catecholamines, along with many other products like ATP, calcium, peptides, chromogranins, ascorbate, and other nucleotides. In this short review, we will summarize the interactions that are currently believed to occur between the elements that make up the vesicular cocktail in the acidic environment of SVs, and how they permit the accumulation of such high concentrations of certain components. In addition, we will examine how the vesicular cocktail regulates the exocytosis of neurotransmitters. In this review, we have highlighted the mechanisms that permit the storage of neurotransmitters and hormones inside secretory vesicles. We also have proposed a novel model based in the intravesicular interactions of the main components of this inner cocktail - catecholamines, ATP, and chromogranins - to allow the accumulation of near molar concentrations of transmitters in secretory vesicles. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Background:It is well established that patients with systemic sclerosis (SS) show a disrupted lipid profile and an increased cardiovascular risk. Cholesterol efflux capacity (CEC) is the ability of high-density lipoprotein (HDL)-cholesterol to accept cholesterol from macrophages. CEC has been linked to cardiovascular events in the general population and to subclinical atherosclerosis in patients with rheumatoid arthritis and systemic lupus erythematosus.Objectives:The main purpose of our study was to assess, for the first time, whether CEC is disrupted in patients with SS compared to controls. We also aimed to identify patients’ characteristics that could explain such potential CEC disturbance.Methods:Cross-sectional study that encompassed 188 individuals; 73 SS patients and 115 age- and sex-matched controls. CEC, using anin vitroassay, and lipoprotein serum concentrations were assessed in patients and controls. A multivariable analysis was performed to study the differences in CEC between patients and controls, and if SS-related data could explain CEC differences.Results:CEC was downregulated in SS patients as compared to controls (beta coefficient -6 [95%CI -10- -2] %, p=0.002). This occurred independently of traditional cardiovascular risk factors, statin use or other variations in the lipid profile produced by the disease. Demographics and lipid profile were, in general, not related with CEC in both patients and controls. In this sense, only abdominal circumference showed a positive association with CEC in patients (beta coefficient 0.09 [95%CI 0.03-0.14], p=0.002) but not in controls. Similarly, no traditional cardiovascular risk factors were related with CEC in both populations. Regarding lipid profile, no correlations were identified between the standard lipid profile molecules and CEC. Remarkably, the use of statins was not related to CEC in both patients and controls. Lastly, concerning SS related data, a negative association between mRSS and CEC was identified (beta coef. -1.08 [95%CI -2.03- -0.12] %, p=0.028).Skin thickness through modified Rodnan (mRSS) was positively related to age and the presence of hypertension, but negatively associated with apolipoprotein B, apo B:A1 ratio, and CEC when univariate correlations were assessed (Table 4). When the relation of mRSS to these lipid-related molecules was adjusted for traditional CV risk factors, the statistical significance of mRSS with those molecules was maintained. Moreover, when the relation between mRSS and CEC was additionally adjusted for other lipid-related molecules, its significance was conserved (beta coef. -1.35 [95%CI -2.62- -0.08]) %, p=0.038)Conclusion:CEC is downregulated in SS patients independently of other inflammation-related lipid profile modifications that occur in the disease. Skin thickness is independent and inversely associated with CEC in SS patients.Disclosure of Interests:Iván Ferraz-Amaro Grant/research support from: Pfizer, Abbvie, Speakers bureau: Pfizer, Abbvie, MSD., delgado frias esmeralda Speakers bureau: Pfizer, Abbvie, MSD, Vanessa Hernández-Hernández Speakers bureau: Pfizer, Abbvie, MSD, Hiurma Sánchez-Pérez: None declared, Laura de Armas-Rillo: None declared, Estefania Armas González: None declared, Jose David Machado: None declared, Federico Díaz-González Grant/research support from: Abbvie, Pfizer, MSD, Speakers bureau: Abbvie, Pfizer, MSD

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

Primary Sjögren’s syndrome (pSS) is an autoimmune exocrinopathy in which the role that the immune response plays in reducing exocrine gland function, including the glandular microenvironment of cytokines, has not been fully understood. Epithelial cells from biopsies of human parotid gland (HPG) were used to establish a model of human salivary gland in vitro. In this model, the functional consequences of several proinflammatory soluble factors present in the pSS glandular microenvironment were assessed. Stimulation with isoproterenol and calcium produced a significant increase in the basal activity of amylase in the HPG cell supernatants. Under these conditions, the presence of TNF-α and CXCL12 increased amylase mRNA cellular abundance, but reduced the amylase activity in the cell-free supernatant in a dose-dependent manner. IL-1β and IFN-γ, but not TGF-β, also diminished amylase secretion by HPG cells. These results suggest that the glandular microenvironment of cytokine, by acting post-transcriptionally, may be responsible, at least in part, for the reduced exocrine function observed in pSS patients. These data may help to a better understanding of the pathogenesis of SS, which in turn would facilitate the identification of new therapeutic targets for this disorder.

The study of chromaffin secretory vesicles (SVs) has contributed immensely to our understanding of exocytosis. These organelles, also called chromaffin granules, are a specific type of large dense secretory vesicle found in many endocrine cells and neurons. Traditionally, they have been isolated from bovine adrenal glands due to the large number of SVs that can be obtained from this tissue. However, technical advances now make it possible to obtain very pure preparations of SVs from mice, which is particular interesting for functional studies given the availability of different genetically modified strains of mice. Despite the small size of the mouse adrenal medulla (400–500 μm and less than 2 mg in weight), we have successfully carried out functional studies on SVs isolated from WT and knockout mice. As such, we present here our method to purify crude vesicles and to fractionate mouse chromaffin SVs, along with examples of their functional characterization.

Chromogranins (Cgs) are the most abundant intravesicular proteins of chromaffin granules. Using Cgs knockout mice, we found that the lack of chromogranin A (CgA), chromogranin B (CgB) or both drastically reduce the vesicular content of catecholamines (CA), impair its accumulation in granules and largely affect the kinetics of exocytosis. Conversely, the overexpression of CgA induces the genesis of vesicles, increases their quantal content and even transforms non-secretory in cells capable to secrete substances. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and regulates the exocytotic process.

Adrenergic receptors are expressed on the surface of inflammation-mediating cells, but their potential role in the regulation of the inflammatory response is still poorly understood. The objectives of this work were to study the effects of α2-adrenergic agonists on the inflammatory response in vivo and to determine their mechanism of action. In two mouse models of inflammation, zymosan air pouch and thioglycolate-induced peritonitis models, the i.m. treatment with xylazine or UK14304, two α2-adrenergic agonists, reduced neutrophil migration by 60%. The α2-adrenergic antagonist RX821002 abrogated this effect. In flow cytometry experiments, the basal surface expression of L-selectin and CD11b was modified neither in murine nor in human neutrophils upon α2-agonist treatment. Similar experiments in HUVEC showed that UK14304 prevented the activation-dependent upregulation of ICAM-1. In contrast, UK14304 augmented electrical resistance and reduced macromolecular transport through a confluent HUVEC monolayer. In flow chamber experiments, under postcapillary venule-like flow conditions, the pretreatment of HUVECs, but not neutrophils, with α2-agonists decreased transendothelial migration, without affecting neutrophil rolling. Interestingly, α2-agonists prevented the TNF-α–mediated decrease in expression of the adherens junctional molecules, VE-cadherin, β-catenin, and plakoglobin, and reduced the ICAM-1–mediated phosphorylation of VE-cadherin by immunofluorescence and confocal analysis and Western blot analysis, respectively. These findings indicate that α2-adrenoceptors trigger signals that protect the integrity of endothelial adherens junctions during the inflammatory response, thus pointing at the vascular endothelium as a therapeutic target for the management of inflammatory processes in humans.

Chromogranins (Cgs) are acidic proteins that have been described in the large, dense core vesicles (LDCVs) of adrenal chromaffin cells and that have been shown to promote LDCV formation, even in nonsecretory cells. Catecholamines (CAs) are adsorbed by Cgs in vitro, and the absence of Cgs modifies the storage and exocytosis of CAs in chromaffin cells. In this study, we set out to assess the role of CgA in the accumulation and exocytosis of CAs in cells when the levels of CgA and CA are manipulated. We overexpressed CgA in nonsecretory HEK293 cells and in secretory PC12 cells, to study the formation, movement, and exocytosis of newly formed granules by evanescent wave microscopy. We analyzed the association of Cgs/CA by HPLC and amperometry and their role in the accumulation and exocytosis of amines, both under resting conditions and after l-DOPA overloading. To our knowledge, this is the first demonstration that CgA expression in a nonsecretory cell line facilitates the storage and exocytosis of CA. In addition, CgA overexpression causes a doubling of the accumulation of CA, although it slows down exocytosis in PC12 cells. We propose a model to explain how the CgA/CA complex governs the accumulation and exocytosis of secreted amines.

Background Lipid profiles appear to be altered in rheumatoid arthritis (RA) patients because of disease activity and inflammation. Cholesterol efflux capacity (CEC), which is the ability of high-density lipoprotein cholesterol to accept cholesterol from macrophages, has been linked not only to cardiovascular events in the general population but also to being impaired in patients with RA. The aim of this study was to establish whether CEC is related to subclinical carotid atherosclerosis in patients with RA. Methods We conducted a cross-sectional study that encompassed 401 individuals, including 178 patients with RA and 223 sex-matched control subjects. CEC, using an in vitro assay, lipoprotein serum concentrations, and standard lipid profile, was assessed in patients and control subjects. Carotid intima-media thickness (CIMT) and carotid plaques were assessed in patients with RA. A multivariable analysis was performed to evaluate the relationship of CEC with RA-related data, lipid profile, and subclinical carotid atherosclerosis. Results Mean (SD) CEC was not significantly different between patients with RA (18.9 ± 9.0%) and control subjects (16.9 ± 10.4%) (p = 0.11). Patients with RA with low (β coefficient −5.2 [−10.0 to 0.3]%, p = 0.039) and moderate disease activity (β coefficient −4.6 [−8.5 to 0.7]%, p = 0.020) were associated with lower levels of CEC than patients in remission. Although no association with CIMT was found, higher CEC was independently associated with a lower risk for the presence of carotid plaque in patients with RA (odds ratio 0.94 [95% CI 0.89–0.98], p = 0.015). Conclusions CEC is independently associated with carotid plaque in patients with RA.

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.

As Arf6 is key to coordinating plasma membrane trafficking and regulates cellular invasion by several microorganisms, the authors studied Arf6 function during early HIV-1 infection. The data suggest that HIV-1 requires Arf6-driven plasma membrane dynamics and depends on GTP/GDP activity to efficiently fuse, enter, and infect CD4+ T lymphocytes., As the initial barrier to viral entry, the plasma membrane along with the membrane trafficking machinery and cytoskeleton are of fundamental importance in the viral cycle. However, little is known about the contribution of plasma membrane dynamics during early human immunodeficiency virus type 1 (HIV-1) infection. Considering that ADP ribosylation factor 6 (Arf6) regulates cellular invasion via several microorganisms by coordinating membrane trafficking, our aim was to study the function of Arf6-mediated membrane dynamics on HIV-1 entry and infection of T lymphocytes. We observed that an alteration of the Arf6–guanosine 5′-diphosphate/guanosine 5′-triphosphate (GTP/GDP) cycle, by GDP-bound or GTP-bound inactive mutants or by specific Arf6 silencing, inhibited HIV-1 envelope–induced membrane fusion, entry, and infection of T lymphocytes and permissive cells, regardless of viral tropism. Furthermore, cell-to-cell HIV-1 transmission of primary human CD4+ T lymphocytes was inhibited by Arf6 knockdown. Total internal reflection fluorescence microscopy showed that Arf6 mutants provoked the accumulation of phosphatidylinositol-(4,5)-biphosphate–associated structures on the plasma membrane of permissive cells, without affecting CD4-viral attachment but impeding CD4-dependent HIV-1 entry. Arf6 silencing or its mutants did not affect fusion, entry, and infection of vesicular stomatitis virus G–pseudotyped viruses or ligand-induced CXCR4 or CCR5 endocytosis, both clathrin-dependent processes. Therefore we propose that efficient early HIV-1 infection of CD4+ T lymphocytes requires Arf6-coordinated plasma membrane dynamics that promote viral fusion and entry.

Several, if not all adrenergic β-blockers (β-Bs), accumulate progressively inside secretory vesicles in a time- and concentration-dependent manner, and could be considered to be false neurotransmitters. This transmitter effect is most likely unrelated to their ability to block adrenergic receptors, but it could explain the delay in lowering arterial pressure in hypertensive patients. We have developed a new drug to monitor the accumulation of β-Bs inside living cells, RCTM-3, which fluoresces in the visible spectrum. Here we describe the procedure to synthesize this new compound, as well as its fluorescent properties, pharmacological profile and its accumulation inside the secretory vesicles of PC12 cells., JDM and BB hold a CONSOLIDER contract (CSD2008-00005 to RB) from the Spanish Ministry of Science and Innovation (MICINN). This work was supported by the Spanish Ministry of Science and Innovation (BFU2007-64963, CTQ2008-03334/BQU and CTQ2008-06806-C02-01/BQU) and CIARIIS/FEDER, PI2007/017 (JDM). The authors have no conflicts of interest to declare.

Chromogranins (Cgs) are acidic proteins that have been implicated in several physiological processes such as vesicle sorting, the production of bioactive peptides and the accumulation of soluble species inside large dense core vesicles (LDCV). They constitute the main protein component in the vesicular matrix of LDCV. This latter characteristic of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). It is likely that Cgs are behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion, due to their low affinity and high capacity to bind solutes present inside LDCV. The recent availability of mouse strains lacking Cgs, combined with the arrival of several techniques for the direct monitoring of exocytosis, have helped to expand our knowledge about the mechanisms used by granins to concentrate catecholamines and Ca(2+) in LDCV, and how they affect the kinetics of exocytosis. We will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

J. Neurochem. (2010) 114, 335–343. Abstract Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca2+. Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca2+ in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

Clathrin-coated vesicles are responsible for the trafficking of several internalized biological cargos. We have observed that the endogenous F-actin-linker moesin co-distributes with constitutive components of clathrin-coated structures. Total internal reflection fluorescence microscopy studies have shown that short interference RNA of moesin enhances the lateral movement of clathrin-coated structures and provokes their abnormal clustering. The aggregation of clathrin-coated structures has also been observed in cells overexpressing N-moesin, a dominant-negative construct unable to bind to F-actin. Only overexpressed moesin constructs with an intact phosphatidylinositol 4,5-bisphosphate-binding domain co-distribute with clathrin-coated structures. Hence, this N-terminal domain is mostly responsible for moesin/clathrin-coated structure association. Biochemical endosome fractioning together with total internal reflection fluorescence microscopy comparative studies, between intact cells and plasma-membrane sheets, indicate that moesin knockdown provokes the accumulation of endocytic rab5-clathrin-coated vesicles carrying the transferrin receptor. The altered trafficking of these endocytic rab5-clathrin-coated vesicles accounts for a transferrin receptor recycling defect that reduces cell-surface expression of the transferrin receptor and increases the amount of sequestered transferrin ligand. Therefore, we propose that moesin is a clathrin-coated vesicle linker that drives cargo trafficking and acts on nascent rab5-clathrin-coated vesicles by simultaneously binding to clathrin-coated vesicle-associated phosphatidylinositol 4,5-bisphosphate and actin cytoskeleton. Hence, functional alterations of moesin may be involved in pathological disorders associated with clathrin-mediated internalization or receptor recycling.

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

Exocytosis occurs via fusion of secretory granules with the cell membrane, whereupon the granule content is at least partially released and the granule membrane is temporarily added to the plasma membrane. Exocytosis is balanced by compensatory endocytosis to achieve net equilibrium of the cell surface area and to recycle and redistribute components of the exocytosis machinery. The underlying molecular mechanisms remain a matter of debate. In this review, we summarize and discuss recent progress in the understanding of compensatory endocytosis, with the focus on chromaffin cells as a useful model for studying mechanisms of regulated secretion.

The effects of the antihypertensive agent hydralazine (1 to 100 nmol/L) on the exocytotic process of single adrenal chromaffin cells have been studied using amperometry. Hydralazine does not reduce the frequency of exocytotic spikes but rapidly slows the rate of catecholamine release from individual exocytotic events by reducing the quantal size of catecholamine exocytosis. Confocal and standard epifluorescence microscopy studies show that hydralazine rapidly accumulates within secretory vesicles. The blockade of the vesicular H + pump with bafilomycin A 1 inhibits hydralazine uptake. Experiments with permeabilized cells show that hydralazine displaces catecholamines from secretory vesicles. The drug also displaces vesicular Ca 2+ , as shown by fura-2 microfluorimetry. These data suggest that hydralazine acts, at least partially, by interfering with the storage of catecholamines. These effects of hydralazine occurred within seconds, and at the tissue concentrations presumably reached in antihypertensive therapy; these concentrations are a thousand times lower than those described for relaxing vascular tissues in vitro. We proposed that these novel effects could explain many of the therapeutic and side effects of this drug that are likely exerted in sympathetic nerve terminals.

The role of nongenomic action of estrogens on elicited catecholamine secretion and exocytosis kinetics was studied in perfused rat adrenals and in cultured bovine chromaffin cells. 17beta-Estradiol as well as the estrogen receptor modulators raloxifene and LY117018, but not 17alpha-estradiol, inhibited at the micromolar range the catecholamine output elicited by acetylcholine or high potassium. However, these agents failed to modify the secretion elicited by high Ca(2+) in glands treated with the ionophore A-23187 (calcimycin), suggesting that estrogens did not directly act on the secretory machinery. At the single cell level, estrogens modified the kinetics of exocytosis at nanomolar range. All of the drugs tested except 17alpha-estradiol produced a profound slowing down of the exocytosis as measured by amperometry. LY117018 also reduced the granule content of catecholamines. 17beta-Estradiol reduced the intracellular free Ca(2+) but only at micromolar concentrations, whereas nanomolar concentrations increased the cAMP levels. These effects were reproduced with the nonpermeable drug 17beta-estradiol-horseradish peroxidase and antagonized with nanomolar concentrations of the antiestrogen ICI 182,780 (fulvestrant). Our data suggest the presence of membrane sites that regulate both the exocytotic phenomenon and the total catecholamine release with high and low affinity, respectively.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba(2+)-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO, S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N(omega)-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba(2+). NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

Chromogranins (Cgs) are acidic proteins implicated in several physiological processes, including the biogenesis and sorting of secretory vesicles, the generation of bioactive peptides, and the accumulation of soluble species inside large dense core vesicles (LDCV). Indeed, Cgs are the main protein component of the vesicular matrix in LDCV, and they are involved in the concentration of soluble species like neurotransmitters and calcium. Experiments using electrochemical techniques such amperometry, patch amperometry, and intracellular electrochemistry have clarified the functional roles of Cgs in the accumulation and release of catecholamines. We have focused this review at a single event of exocytosis of chromaffin cells from three mouse strains lacking Cgs. Accordingly, in this brief review, we will focus on the role of Cgs in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from studies on adrenal chromaffin cells.

Background HIV-1 entry into target lymphocytes requires the activity of actin adaptors that stabilize and reorganize cortical F-actin, like moesin and filamin-A. These alterations are necessary for the redistribution of CD4-CXCR4/CCR5 to one pole of the cell, a process that increases the probability of HIV-1 Envelope (Env)-CD4/co-receptor interactions and that generates the tension at the plasma membrane necessary to potentiate fusion pore formation, thereby favouring early HIV-1 infection. However, it remains unclear whether the dynamic processing of F-actin and the amount of cortical actin available during the initial virus-cell contact are required to such events. Results Here we show that gelsolin restructures cortical F-actin during HIV-1 Env-gp120-mediated signalling, without affecting cell-surface expression of receptors or viral co-receptor signalling. Remarkably, efficient HIV-1 Env-mediated membrane fusion and infection of permissive lymphocytes were impaired when gelsolin was either overexpressed or silenced, which led to a loss or gain of cortical actin, respectively. Indeed, HIV-1 Env-gp120-induced F-actin reorganization and viral receptor capping were impaired under these experimental conditions. Moreover, gelsolin knockdown promoted HIV-1 Env-gp120-mediated aberrant pseudopodia formation. These perturbed-actin events are responsible for the inhibition of early HIV-1 infection. Conclusions For the first time we provide evidence that through its severing of cortical actin, and by controlling the amount of actin available for reorganization during HIV-1 Env-mediated viral fusion, entry and infection, gelsolin can constitute a barrier that restricts HIV-1 infection of CD4+ lymphocytes in a pre-fusion step. These findings provide important insights into the complex molecular and actin-associated dynamics events that underlie early viral infection. Thus, we propose that gelsolin is a new factor that can limit HIV-1 infection acting at a pre-fusion step, and accordingly, cell-signals that regulate gelsolin expression and/or its actin-severing activity may be crucial to combat HIV-1 infection.

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with l-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.

Chromogranins/secretogranins (Cgs) are the major soluble proteins of large dense-core secretory vesicles (LDCVs). We have recently reported that the absence of chromogranin A (CgA) caused important changes in the accumulation and in the exocytosis of catecholamines (CAs) using a CgA-knock-out (CgA-KO) mouse. Here, we have analyzed a CgB-KO mouse strain that can be maintained in homozygosis. These mice have 36% less adrenomedullary epinephrine when compared toChgb+/+[wild type (WT)], whereas the norepinephrine content was similar. The total evoked release of CA was 33% lower than WT mice. This decrease was not due to a lower frequency of exocytotic events but to less secretion per quantum (∼30%) measured by amperometry; amperometric spikes exhibited a slower ascending but a normal decaying phase. Cell incubation withl-DOPA increased the vesicle CA content of WT but not of the CgB-KO cells. Intracellular electrochemistry, using patch amperometry, showed thatl-DOPA overload produced a significantly larger increase in cytosolic CAs in cells from the KO animals than chromaffin cells from the WT. These data indicate that the mechanisms for vesicular accumulation of CAs in the CgB-KO cells were saturated, while there was ample capacity for further accumulation in WT cells. Protein analysis of LDCVs showed the overexpression of CgA as well as other proteins apparently unrelated to the secretory process. We conclude that CgB, like CgA, is a highly efficient system directly involved in monoamine accumulation and in the kinetics of exocytosis from LDCVs.

The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with beta-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle.

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca(2+), but the free intragranular [Ca(2+)], the mechanisms for Ca(2+) uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate.We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca(2+) from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca(2+) from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells overexpressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca(2+) released from the vesicles to the cytosol in the presence of bafilomycin, dramatically increased the granule motion of chromaffin- or PC12-derived granules, and triggered exocytosis (measured by amperometry).We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of the local cytosolic Ca(2+) around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1.