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Purpose: Visual impairment poses significant challenges in daily life, especially when navigating unfamiliar environments, resulting in inequalities and reduced quality of life. This study aimed to gain an in-depth understanding of the needs and perspectives of visually impaired people in sports-related contexts through surveys and focus groups, and to understand whether their needs are being met by current technological solutions., Materials and methods: To accomplish this, opinions gathered from focus groups and interviews were compared to the technological solutions found in the literature. Since many unmet needs were identified, participants from associations and organizations were asked to identify key characteristics for the development of a robot guide. The results underscored the paramount importance of an easy-to-use guide that offers accurate and personalized assistance. Participants expressed a strong desire for advanced features such as object recognition and navigation in complex environments, as well as adaptability to the user's speed while providing the necessary safety features to ensure a high level of autonomy., Results: This research serves as a bridge between technological advances and the needs of the visually impaired, contributing to a more accessible and inclusive society. By addressing the unique challenges faced by the visually impaired individuals and tailoring technology to meet their needs, this study takes a significant step toward reducing disparities and improving the independence and quality of life for this community., Conclusions: As technology continues to advance, it has the potential to be a powerful tool in breaking down barriers and fostering a world where everyone, regardless of their visual ability, can navigate the world with confidence and ease.

Background: The escalating prevalence of diabetes, with its multifaceted complications, poses a pressing challenge for healthcare systems globally. In response, the advent of continuous glucose monitoring (CGM) systems, offering technological solutions for daily diabetes management, presents significant opportunities. However, the widespread adoption faces several barriers, linked both to the technological configuration of the devices and to the psychological dimension of patients. Therefore, this study aims to apply and test a theoretical model that investigates the antecedents of the intention to use Continuous Glucose Monitoring systems., Methods: The research model was built to unveil the impacts of psychological factors, functional components and rational constructs derived from the Technology Acceptance Model (TAM) on CGM systems sustained adoption. To ensure the comparability of results, we have collected data from people who had used Dexcom ONE Dexcom (San Diego, CA) for the first time for at least one month. Employing Structural Equation Modelling (SEM) techniques, the hypothesized relationships among constructs were assessed., Results: The analyses confirmed the positive correlation of rational factors to the Intention to Use. Subjective Norm, intended as the physicians' influence, is positively correlated with the Perceived Usefulness. Trend Arrows, albeit being negatively correlated with Perceived Usefulness, have a positive correlation on Perceived Ease Of Use, reinforcing its mediating effect towards Perceived Usefulness. Among psychological factors, Trust in the CGM technology positively correlates with Intention to Use. Health Literacy is negatively correlated to the Intention to Use., Conclusions: These findings contribute to theoretical and managerial understanding, providing recommendations to enhance the adoption of CGM systems like Dexcom ONE., (© 2024. The Author(s).)

Background: The rising incidence of chronic diseases among the population, further exacerbated by the phenomenon of aging, is a primary concern and a serious challenge for the healthcare systems worldwide. Among the wide realm of health digital technologies, the rise of Digital Therapeutics (DTx), which are medical devices able to deliver evidence-based treatments to manage and treat diseases, opens new opportunities. However, their diffusion and usage are still fragmented among countries. As the diffusion results from the adoption of technology from a social system and individual acceptance, this study aims to design and test a theoretical model that investigates the intention to use DTx, with a particular focus on the treatment of obesity, as a widespread and burdensome chronic condition., Methods: This research is built on 336 answers coming from a survey to test the proposed model, which consists of a combination of organizational mechanisms, derived from Institutional Theory, and rational factors, derived from the Technology Acceptance Model (TAM). The survey has been delivered to patients and former patients of Istituto Auxologico Italiano, a hospital with several locations in northern Italy, recognized as a center of excellence for the treatment of obesity., Results: The analyses of the answers, performed through the Structural Equation Modelling (SEM) technique, confirmed the influence of the Perceived Usefulness on Intention To Use, and of the Perceived Ease Of Use on the Perceived Usefulness, confirming the validity of the assumptions derived from the TAM. On the other hand, institutional factors were introduced as antecedents of the Perceived Usefulness, and the Perceived Ease Of Use. Results show that the Regulative Pillar influences both the TAM constructs, the Normative Pillar (peer influence) has a positive effect only on the Perceived Usefulness, and finally, the Cultural Pillar impacts the Perceived Ease Of Use., Conclusion: This study allows filling the knowledge gap regarding the usage of the Institutional as a means to predict individuals' intentions. Moreover, managerial contributions are available as the results have been operationalized into practical advice to managers and healthcare professionals to foster the adoption, and thus the diffusion, of Digital Therapeutics., (© 2023. The Author(s).)

Background and Aims: The most recent guidelines suggest treating patients whose FRAX 10-year fracture risk scores are ≥ 20%. However, this method of evaluation does not take into account parameters that are nonetheless relevant to the therapeutic choice. Our aim was to compare the therapeutic choices for treatment based on a wider assessment (real-world practice) with those based on FRAX scores, taking 20% as the cut-off score., Methods: We obtained the medical history, bone mineral density (BMD) values, and the presence of major fragility fractures in a sample of 856 postmenopausal women. The 10-year FRAX risk of major osteoporotic fracture was calculated, and patients were grouped into risk classes ("FRAX < 20%" = low, "FRAX ≥ 20%" = high); we then compared the treated and untreated patients in each class. After an average interval of 2.5 years, changes in lumbar and femoral BMD and appearances of new fragility fractures were recorded., Results: 83% of high-risk patients and 57% of low-risk patients were treated. The therapeutic decision was based mainly on densitometric values and the presence of vertebral fractures. At the 2.5 year follow-up, lumbar spine and femur BMD had decreased in the untreated group; 9.9% of the treated patients developed new vertebral fragility fractures, compared with 5.3% of the untreated patients., Discussion and Conclusions: Our wider assessment designated as at high fracture risk a group of patients who had not been identified by the FRAX assessment. FRAX could underestimate the risk of fracture in older people, for which the therapeutic choice should consider a broader approach, also based on individual patient's needs., (© 2022. The Author(s).)

Objectives: Aging is associated with low-grade chronic inflammation contributing to a decline in lung performance. The Dietary Inflammatory Index (DII) has been introduced to evaluate the inflammatory potential of different diets, which may further affect individuals' respiratory function. This study investigates the association between DII and lung performance in older adults., Methods: This cross-sectional study included 155 adults aged ≥65 y recruited at public gyms in Padua, Italy. Participants were assessed on medical history, biochemical parameters, body composition (through dual energy x-ray absorptiometry), anthropometry, and lung function (by spirometry). Based on individuals' dietary habits, we computed their DII and categorized participants in the lower DII (comprising those in the lowest DII tertile) or the higher DII (comprising those in the highest DII tertiles) groups. The association of DII with forced expiratory volume in the first second (FEV1) and forced vital capacity (FVC) was tested through multivariable linear regression analyses in the total sample and stratified by body mass index (<25 kg/m 2 versus ≥25 kg/m 2 )., Results: The mean age of the sample was 71.2 y and 80% were women. Individuals in the higher DII group had FEV1 and FVC values reduced by 0.15 L (95% CI, -0.29 to -0.01 L) and 0.25 L (95% CI, -0.43 to -0.07 L), respectively, as compared with those in the lower DII group. Results seemed to be more marked among participants with excess weight conditions., Conclusions: Pro-inflammatory diets may affect lung function in fit older people, and this effect may be exacerbated in overweight or obese individuals., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background & Aims: In oncology, the dosage of anti-neoplastic drugs is generally adapted to the patient's body surface area (BSA). We investigated the potential differences between BSA and body weight (BW) in estimating the variability in body composition among individuals, especially older adults., Materials and Methods: The study population included 322 community-dwelling individuals with different age and sex: 45 adult men (AM, age 18-65 years), 86 older men (OM, age >65 years), 54 adult women (AW, age 18-65 years), and 137 older women (OW, age >65 years). For each participant, we estimated the body composition with dual-energy X-ray absorptiometry, and we calculated the BSA using the DuBois and DuBois formula. The strength of relationships between fat free mass (FFM) and fat mass (FM) with BSA, BW, and BMI were expressed as correlation (r) and determination coefficients (R 2 )., Results: Most of the included sample was normal weight (45.7%) or overweight (41.9%). FFM demonstrated a stronger association with BSA than with BW or BMI in all age/sex groups, with r ranging from 0.831 to 0.924 (p < 0.001 for all) and R2 from 0.691 to 0.853. Conversely, BW and BMI were more strongly related to FM than BSA, especially in women. For such relationship, BW, in particular, showed r ranging from 0.793 to 0.924 (p < 0.001 for all)., Conclusions: This study suggests that BSA may be more appropriately used to estimate FFM, compared with BW. Instead, alternative parameters should be considered to estimate FM in patients at risk for adverse effects of lipophilic drugs, especially in older age., Competing Interests: Declaration of competing interest None., (Copyright © 2021 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Background: Smoking is recognized among the risk factors for osteoporosis, but only few studies have comprehensively explored its influence on bone metabolism and strength. We aimed to evaluate smoking effects on calcium-phosphate metabolism, bone mineral density (BMD) and fracture risk in postmenopausal women., Methods: Our sample included 1067 postmenopausal women who arrived to our osteoporosis outpatient clinic. Anamnestic data, smoking habits (categorized as never, former, and current; and by smoking intensity and duration), biochemical parameters, lumbar/femoral BMD, and presence of vertebral fractures were recorded. In a subsample of 357 women, the changes in BMD after a 2-yr follow-up period were also assessed., Results: Current smokers had shorter reproductive age, lower body mass index, and higher prevalence of heavy alcohol consumption than former/never smokers. They also had lower PTH values and weaker linear association between serum vitamin D and parathyroid hormone (current β = -0.11[SE = 0.004]; former β = -0.14[SE = 0.01]; never β = -0.20[SE = 0.003]; p < 0.01 for all). Baseline BMD did not reflect differences based on smoking habits, duration or intensity. However, after 2 years, only current smokers significantly worsened in femural BMD. After adjustment for confounders, the chance of having sustained vertebral fractures at the first evaluation increased by 74% (95% confidence interval:1.07-2.83) in current compared with never smokers, especially among heavy smokers., Conclusions: Smoking may negatively affect bone by inhibiting vitamin D-parathyroid hormone axis, reducing estrogen exposure, promoting risky health behaviors, and accelerating bone loss, especially at the femur. No significant differences were observed in these outcomes among former smokers, suggesting that quitting smoking has beneficial effects on bone health., (Copyright © 2019 The International Society for Clinical Densitometry. Published by Elsevier Inc. All rights reserved.)

Objectives: The aim of this prospective study was to investigate whether two cellular and metabolic health indices, phase angle (PhA) and metabolic equivalents (METs), can predict changes in frailty states in fit community-dwelling older people., Methods: A sample of 118 individuals aged ≥65 years who attended a twice-weekly mild fitness program of aerobic and/or resistance exercises was enrolled in the study. At baseline and after three years, individuals underwent a clinical examination, biochemical determinations, bioelectrical impedance analysis, body composition assessment with dual energy X-ray absorptiometry, physical performance tests, and frailty and sarcopenia assessment. In 78 participants was executed indirect calorimetry, too. Based on frailty transitions during the follow-up between non-frailty, pre-frailty and frailty, participants were categorized as improved, stable (non-frail or pre-frail), and worsened or remaining frail. The chances to experience different frailty changes by baseline PhA and METs were explored through multinomial regression analysis and expressed as odds ratios (OR) and 95% Confidence intervals (95%CI)., Results: During the follow-up, 8 participants improved in frailty status, 84 were stable and 26 worsened or remained frail. For each one-unit increase in PhA, the odds of improving in frailty increased by 4.53 times (95%CI:1.18-17.46); while for each one-unit increase in METs, the odds of worsening in frailty decreased by 65% (95%CI:0.16-0.79)., Conclusions: PhA and METs may be indirect measures of functional reserve, with lower values being potential biomarkers of evolving frailty., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In brain mitochondria succinate activates H(2)O(2) release, concentration dependently (starting at 15 μM), and in the presence of NAD dependent substrates (glutamate, pyruvate, β-hydroxybutyrate). We report that TCA cycle metabolites (citrate, isocitrate, α-ketoglutarate, fumarate, malate) individually and quickly inhibit H(2)O(2) release. When they are present together at physiological concentration (0.2, 0.01, 0.15, 0.12, 0.2 mM respectively) they decrease H(2)O(2) production by over 60% at 0.1-0.2 mM succinate. The degree of inhibition depends on the concentration of each metabolite. Acetoacetate is a strong inhibitor of H(2)O(2) release, starting at 10 μM and acting quickly. It potentiates the inhibition induced by TCA cycle metabolites. The action of acetoacetate is partially removed by β-hydroxybutyrate. Removal is minimal at 0.1 mM acetoacetate, and is higher at 0.5 mM acetoacetate. We conclude that several inhibitors of H(2)O(2) release act jointly and concentration dependently to rapidly set the required level of H(2)O(2) generation at each succinate concentration.

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H(2)O(2) release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ((.)NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its (.)NO release) induces an inhibition of the succinate dependent H(2)O(2) production consistent with a (.)NO dependent covalent modification. However maximal inhibition of the succinate dependent H(2)O(2) release is obtained in the presence of low GSNO (20-100 μM), but not with SNP. This inhibition appears independent of (.)NO release since μM GSNO does not affect mitochondrial respiration, or the H(2)O(2) detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O (2) (.-) since GSNO chemically competes with NBT and cytochrome C in O (2) (.-) detection.

Mitochondrial production of H(2)O(2) is low with NAD substrates (glutamate/pyruvate, 3 and 2 mM) (G/P) and increases over ten times upon further addition of succinate, with the formation of a sigmoidal curve (semimaximal value at 290 microM, maximal H(2)O(2) production at 600 microM succinate). Malate counteracts rapidly the succinate induced increased H(2)O(2) release and moves the succinate dependent H(2)O(2) production curve to the right. Nitric oxide (NO) and carbon monoxide (CO) are cytochrome c oxidase inhibitors which increase mitochondrial ROS production. Cyanide (CN(-)) was used to mimic NO and CO. In the presence of G/P and succinate (300 microM), CN(-) progressively increased the H(2)O(2) release rate, starting at 1.5 microM. The succinate dependent H(2)O(2) production curve was moved to the left by 30 microM CN(-). The V(max) was little modified. We conclude that succinate is the controller of mitochondrial H(2)O(2) production, modulated by malate and CN(-). We propose that succinate promotes an interaction between Complex II and Complex I, which activates O(2)(-) production.

Complex I is the main O(2)(-) producer of the mitochondrial respiratory chain. O(2)(-) release is low with NAD-linked substrates and increases strongly during succinate oxidation, which increases the QH(2)/Q ratio and is rotenone sensitive. We show that the succinate dependent O(2)(-) production (measured as H(2)O(2) release) is inhibited by propargylamine containing compounds (clorgyline, CGP 3466B, rasagiline and TVP-1012). The inhibition does not affect membrane potential and is unaffected by DeltapH modifications. Mitochondrial respiration is similarly unaffected. The propargylamines inhibition of O(2)(-)/H(2)O(2) production is monitored also in the presence of the Parkinson's disease toxin dopaminochrome which stimulates O(2)(-) release. Propargylamine-containing compounds are the first pharmacological inhibitors described for O(2)(-) release at Complex I.

Complex I is responsible for most of the mitochondrial H(2)O(2) release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H(2)O(2) production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125-129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H(2)O(2) release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on DeltapH or Deltap and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.

Complex I (NADH:ubiquinone oxidoreductase) is responsible for most of the mitochondrial H2O2 release, both during the oxidation of NAD-linked substrates and during succinate oxidation. The much faster succinate-dependent H2O2 production is ascribed to Complex I, being rotenone-sensitive. In the present paper, we report high-affinity succinate-supported H2O2 generation in the absence as well as in the presence of GM (glutamate/malate) (1 or 2 mM of each). In brain mitochondria, their only effect was to increase from 0.35 to 0.5 or to 0.65 mM the succinate concentration evoking the semi-maximal H2O2 release. GM are still oxidized in the presence of succinate, as indicated by the oxygen-consumption rates, which are intermediate between those of GM and of succinate alone when all substrates are present together. This effect is removed by rotenone, showing that it is not due to inhibition of succinate influx. Moreover, alpha-oxoglutarate production from GM, a measure of the activity of Complex I, is decreased, but not stopped, by succinate. It is concluded that succinate-induced H2O2 production occurs under conditions of regular downward electron flow in Complex I. Succinate concentration appears to modulate the rate of H2O2 release, probably by controlling the hydroquinone/quinone ratio.

Inhibitors of Complex I of the mitochondrial respiratory chain, such as rotenone, promote Parkinson disease-like symptoms and signs of oxidative stress. Dopamine (DA) oxidation products may be implicated in such a process. We show here that the o-quinone dopaminochrome (DACHR), a relatively stable DA oxidation product, promotes concentration (0.1-0.2 mum)- and respiration-dependent generation of H(2)O(2) at Complex I in brain mitochondria, with further stimulation by low concentrations of rotenone (5-30 nm). The rotenone effect required that contaminating Ca(2+) (8-10 mum) was not removed. DACHR apparently extracts an electron from the constitutively autoxidizable site in Complex I, producing a semiquinone, which then transfers an electron to O(2), generating O(2)(.) and then H(2)O(2). Mitochondrial removal of H(2)O(2) monoamine, formed by either oxidase activity or DACHR, was performed largely by glutathione peroxidase and glutathione reductase, which were negatively regulated by low intramitochondrial Ca(2+) levels. Thus, the H(2)O(2) formed accumulated in the medium if contaminating Ca(2+) was present; in the absence of Ca(2+), H(2)O(2) was completely removed if it originated from monoamine oxidase, but was less completely removed if it originated from DACHR. We propose that the primary action of rotenone is to promote extracellular O(2)(.) release via activation of NADPH oxidase in the microglia. In turn, O(2)(.) oxidizes DA to DACHR extracellularly. (The reaction is favored by the lack of GSH, which would otherwise preferably produce GSH adducts of dopaminoquinone.) Once formed, DACHR (which is resistant to GSH) enters neurons to activate the rotenone-stimulated redox cycle described.

In brain mitochondria, state 4 respiration supported by the NAD-linked substrates glutamate/malate in the presence of EGTA promotes a high rate of exogenous H2O2 removal. Omitting EGTA decreases the H2O2 removal rate by almost 80%. The decrease depends on the influx of contaminating Ca2+, being prevented by the Ca2+ uniporter inhibitor ruthenium red. Arsenite is also an inhibitor (maximal effect approximately 40%, IC50, 12 microm). The H2O2 removal rate (EGTA present) is decreased by 20% during state 3 respiration and by 60-70% in fully uncoupled conditions. H2O2 removal in mitochondria is largely dependent on glutathione peroxidase and glutathione reductase. Both enzyme activities, as studied in disrupted mitochondria, are inhibited by Ca2+. Glutathione reductase is decreased by 70% with an IC50 of about 0.9 microm, and glutathione peroxidase is decreased by 38% with a similar IC50. The highest Ca2+ effect with glutathione reductase is observed in the presence of low concentrations of H2O2. With succinate as substrate, the removal is 50% less than with glutamate/malate. This appears to depend on succinate-supported production of H2O2 by reverse electron flow at NADH dehydrogenase competing with exogenous H2O2 for removal. Succinate-dependent H2O2 is inhibited by rotenone, decreased DeltaPsi, as described previously, and by ruthenium red and glutamate/malate. These agents also increase the measured rate of exogenous H2O2 removal with succinate. Succinate-dependent H2O2 generation is also inhibited by contaminating Ca2+. Therefore, Ca2+ acts as an inhibitor of both H2O2 removal and the succinate-supported H2O2 production. It is concluded that mitochondria function as intracellular Ca2+-modulated peroxide sinks.

The treatment of cerebral cortex synaptosomes with the membrane impermeable thiol reagent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) induces a long-lasting partial inhibition (about 40%) of the KCl-stimulated Ca2+-dependent exocytosis of glutamate. Synaptosomes are not damaged by the treatment. The increase of cytoplasmic free Ca2+ concentration ([Ca2+]i) upon depolarization is not affected by DTNB. The inhibition is observed also if exocytosis is induced with the Ca2+-ionophore ionomycin. In all cases the inhibition is reversed by the impermeable reductant glutathione (GSH). Similarly the inhibition of exocytosis by H2O2 (Zoccarato, F., Valente, M. and Alexandre, A., Hydrogen peroxide induces a long-lasting inhibition of the Ca2+-dependent glutamate release in cerebrocortical synaptosomes without interfering with cytosolic Ca2+. J. Neurochem., 64 (1995) 2552-2558.) is reversed by GSH. It is concluded that redox changes (possibly thiol-disulfide transitions) of superficial groups modulate the exocytotic apparatus directly. In an attempt to identify the protein(s) involved in this novel type of control, we evidenced DTNB (H2O2) reactive bands at 35 and at 85-150 kDa which can be labeled with a monobromotrimethylammoniobimane bromide (qBBr) derivatization.

We introduce the use of the pH-sensitive dye acridine orange (AO) to monitor exo/endocytosis of acidic neurotransmitter-containing vesicles in synaptosomes. AO is accumulated exclusively in acidic v-ATPase-dependent bafilomycin (Baf)-sensitive compartments. A fraction of the accumulated AO is rapidly released (fluorescence increase) upon depolarization with KCl in the presence of Ca2+. The release (completed in 5-6 s) is followed by reuptake to values below the predepolarization baseline. The reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In a similar protocol, Baf does not affect the initial fast phase of glutamate release measured enzymatically, but it abolishes the subsequent slow phase. Thus, the fast AO release corresponds to the rapid phase of glutamate release and the slow phase depends on vesicle cycling. AO reuptake depends in part on the progressive accumulation of acid-loaded vesicles during cycling. Stopping exocytosis at selected times after KCl by Ca2+ removal with EGTA evidences endocytosis: Its T(1/2) was 12 +/- 0.6 s. The K(A)+, channel inhibitors 4-aminopyridine (100 microM) and alpha-dendrotoxin (10-100 nM) are known to induce glutamate release by inducing the firing of Na+ channels; their action is potentiated by the activation of protein kinase C. Also these agents promote a Ca2+-dependent AO release, which is prevented by the Na+ channel inhibitor tetrodotoxin and potentiated by 4beta-phorbol 12-myristate 13-acetate (PMA). With alpha-dendrotoxin, endocytosis was monitored by stopping exocytosis at selected times with EGTA or alternatively with Cd2+ or tetrodotoxin. The T(1/2) of endocytosis, which was unaffected by PMA, was 12 +/- 0.4 s with EGTA and Cd2+ and 9.5 +/- 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.

The treatment of cerebrocortical synaptosomes with low concentrations of H2O2 induces a long-lasting inhibition of the Ca2+ -dependent release of glutamate induced by KCl or ionomycin, without interfering with the cytosolic calcium and without damaging the synaptosomes (Zoccarato, F., Valente, M., and Alexandre, A. (1995) J. Neurochem. 64, 2552-2558). We report now that the inhibition exerted by H2O2 decreases (from 50 +/- 9% to 25 +/- 11%) if exocytosis is triggered by high (80 mM) rather than by low (30 mM) KCl. Similarly the inhibition decreases when glutamate release is triggered by high rather than by low ionomycin. The decreased inhibition by H2O2 on increasing KCl is accompanied by an increase of [Ca2+]i. We conclude that the treatment with H2O2 decreases the CA2+ sensitivity of the synaptosomal exocytotic apparatus.

We studied the action of H2O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2O2 (50-150 microM) for a few minutes results in a long-lasting depression of the Ca(2+)-dependent exocytosis of glutamate, induced by KCl or by the K(+)-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2O2, although a transient decrease was observed after the treatment. H2O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H2O2 did not modify significantly the KCl-induced increase of [Ca2+]i. H2O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+]i with the Ca2+ ionophore ionomycin. The effects of H2O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2O2, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.

The addition of microM Ca2+ to synaptosomes incubated in the presence of EGTA and NADH activates a slow production of H2O2, which is promptly inhibited by excess EGTA and reactivated by Ca2+. The H2O2 output is inhibited by the Fe chelator deferoxamine. Higher Ca(2+)-dependent H2O2 productions are induced in the presence of added FeCl3 in the absence of lipid peroxidation. The apparent Km for Fe is 28 microM. NADH is oxidized parallel to the production of H2O2 (NADH/H2O2 = 0.82 +/- 0.1). If NADH is omitted, some H2O2 is still generated, with electrons from ferrocytochrome c; in these conditions the inhibition by excess EGTA develops slowly being completed only after some minutes. The semimaximal activation of the H2O2 production is obtained at 1-1.2 microM free Ca2+. Millimolar Ca2+ is inhibitory. After treatment with digitonin, the H2O2 production increases by 40-50%. This 'internal' H2O2 probably corresponds to the previously described Ca(2+)-ionophore-induced H2O2 generation which is observable after glutathione depletion. In no case could a production of O2- be monitored. All the synaptosomal Ca(2+)-Fe oxidase activity is recovered in the plasma membrane fractions. NADH provides most of the reducing equivalents in the heavier fraction, which is the richest in postsynaptic components. A significant proportion of the H2O2 production utilizes electrons from cytochrome c in the lighter plasma membrane fractions.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.

We studied the action of the glutathione transferase substrate, 1-chloro-2,4-dinitrobenzene (CDNB) on the synaptosomal production of H2O2. We found that CDNB (30-40 microM) readily depletes the cytosolic glutathione but is almost without effect on the mitochondrial fraction. The depletion of the cytosolic glutathione induced by CDNB affords the detection in the extracellular space of H2O2 produced intrasynaptosomally upon increasing the cytosolic Ca2+ concentration that is otherwise destroyed by glutathione peroxidase. Higher concentrations of CDNB induce a H2O2 production which is not related to the glutathione content. This H2O2 is of mitochondrial origin and requires that NAD be reduced. The primary product of the mitochondrial CD-NB-dependent oxygen reduction is at least in part the superoxide anion.

We previously reported on the release of hydrogen peroxide from guinea pig cerebral cortex synaptosomes (13). An important finding was that in glutathione depleted synaptosomes a linear release of hydrogen peroxide is rapidly induced on addition of the Ca++ -ionophore ionomycin (in the presence of Ca++) or upon depolarization of the plasma membrane. We report here that the ionomycin induced hydrogen peroxide is reversed following the addition of bovine serum albumin which strongly binds the ionophore, to be reactivated by further addition of excess ionomycin, or of the depolarizing agent KC1. Similarly, the effect of ionomycin is removed on decreasing the concentration of free Ca++. Bovine serum albumin, which counteracts the effect of ionomycin on the release of H2O2, also counteracts the effect of the ionophore on the movements of Ca++ and the release of gamma-aminobutyrate. These findings support the idea that the synaptosomal production of H2O2 is a carefully controlled important physiological event.

The rate of spontaneous efflux of Ca from liver mitochondria incubated in the absence of ATP and Mg increases with time and is associated with a synchronous collapse of membrane potential and with Pi efflux. In the presence of Mg and ATP the ruthenium-red-induced Ca efflux does not change with time. The activity of the Ca efflux pathway in Pi-depleted mitochondria is 15-fold greater than in mitochondria equilibrated with 3.3 mM Pi. 50% inhibition is caused by 0.3 mM Pi. The membrane potential is not affected by changes in Pi concentration, although the steady-state extra-mitochondrial free Ca concentration reflects the alterations in efflux rate. In the presence of Pi, the ruthenium-red-induced efflux rate is independent of the total matrix Ca content; however in Pi-depleted mitochondria, with acetate substituting as permeant anion, the efflux rate increases with total matrix Ca content. The lowered efflux rate in the presence of Pi is not due to a limitation in the rate of dissociation of the matrix Ca-phosphate complex. The efflux pathway is activated by a lowered membrane potential, but the relative effect of Pi is retained. Under the present conditions Na slightly inhibits the efflux rate. The lack of an effect of total matrix Ca content on the efflux rate in the presence of Pi is used as the basis of a highly accurate determination of the activity of the Ca uniporter as a function of external free Ca concentration.