Validation of hair type diversification for recruiting shampoo panelists without losing data relevance.

A sensory descriptive method is used by the industries to characterize their various products under development. The sensory panelists are recruited through some general criteria suggested in International Standard Sensory Analysis - General guidelines for the selection, training and monitoring of selected assessors and expert sensory assessors [5] but for hair product evaluation, some hair criteria should also be considered, as a major challenge lied in the difficulty to recruit panelists. Such an issue led us to find practical solutions to make this recruitment easier. Among others, one possible solution was to restrict some requirements in the characteristics of hair. This study aimed at checking if a hair type diversification on either sensitization or curliness could influence the sensory perception of shampoos, and, if so, to which extent. This study demonstrates that, for a shampoo trained panel, the evaluation is impacted by the hair curliness but is not impacted by the hair sensitization level.

Une méthode de description des sensations est utilisée par les industries pour caractériser leurs différents produits en cours de développement. Des membres sont recrutés pour un panel en se basant sur certains critères généraux suggérés dans la Norme Internationale d'Analyse sensorielle - Directives générales pour la sélection, la formation et la supervision des évaluateurs sélectionnés et des évaluateurs sensoriels experts [5], mais pour l'évaluation des cheveux, certains critères doivent également être pris en compte, car une des principales difficultés consiste à recruter des panélistes. Ce problème nous a obligé de trouver des solutions pratiques pour faciliter ce recrutement. Entre autres, une solution possible était de limiter certaines exigences au niveau des caractéristiques des cheveux. Cette étude vise à vérifier si la diversité au niveau de la sensibilité ou de la frisure de la chevelure pourrait influencer la perception sensorielle des shampooings, et, si c'est le cas, dans quelle mesure. Cette étude démontre que, pour une palette de 6 shampooings développés, l'évaluation est impactée par la frisure de la chevelure7mais n'est pas impactée par le niveau de sensibilité.

RAW 264.7 co-cultured with ultra-high molecular weight polyethylene particles spontaneously differentiate into osteoclasts: an in vitro model of periprosthetic osteolysis.

Wear-particle osteolysis affects prosthesis survival leading to implant loosening up to 70% of revisions. Therapeutic strategies are increasing, however alternative testing methods to experimentally evaluate such treatments are lacking. The aim of this study was to reproduce an in vitro osteolysis model recapitulating the events that, starting from the exposure of macrophages to polyethylene, lead to the establishment of osteoclastogenesis and inflammation. Responses to polyethylene, at 3 and 7 days, in a macrophage cell line, RAW 264.7, were determined by DNA quantification, immunofluorescence, pit assay, gene expression, cytokine production and NF-kB activation. Results showed that 3 days exposure to particles could induce a significant production of Tumor Necrosis Factor alpha (p < 0.0005) and Prostaglandin E2 (p < 0.005) compared to controls. Particles also induced macrophages to spontaneously differentiate into mature and active osteoclasts, in terms of identification of multinucleated cells by Phalloidin staining and by the analysis of osteoclast-specific gene markers. In particular, at 3 days polyethylene induced a significant up-regulation of Nuclear Factor of Activated T-cells, cytoplasmic 1, Receptor Activator of Nuclear factor Kappa-B and Receptor Activator of Nuclear Factor Kappa-B Ligand genes (p < 0.0005) compared to controls. At protein level, the particles induced a significant increase of Receptor Activator of Nuclear Factor Kappa-B Ligand at day 7 over controls (p < 0.0005). Osteoclasts were capable to resorb bone even in absence of differentiating factors. The possible mechanism, beside spontaneous osteoclastogenesis mediated by wear debris, was identified in an autocrine up-regulation of Receptor activator of nuclear factor kappa-B ligand gene expression and protein synthesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 510-520, 2017.

Novel halogenated derivates of JWH-018: Behavioral and binding studies in mice.

JWH-018 is a synthetic CB1 and CB2 agonist illegally marketed as products named "Spice" or "herbal blend" for its psychoactive effects which are much higher than those produced by cannabis. In the last year, the European Monitoring Centre for Drugs and Drug Addiction reported to the Italian National Early Warning System the seizure of plant material containing new halogenated derivatives of JWH-018 (JWH-018 Cl and JWH-018 Br). The present study aimed to investigate the in vitro and in vivo activity of these two new synthetic cannabinoids in mice. In vitro competition binding experiments performed on mouse and human CB1 receptors revealed a high affinity and potency of the halogenated compounds. Synthetic cannabinoids (0.01-6 mg/kg i.p.) impaired motor activity and induced catalepsy in mice and their effects were more severe with respect to those evoked by Δ(9)-THC. Moreover, they increased the mechanical and thermal pain threshold and induced a marked hypothermia. It is interesting to note that whereas high doses of JWH-018 cause seizures, myoclonia and hyperreflexia, the halogenated compounds, in particular JWH-018Br, were less effective. Behavioral and neurological changes were prevented by the selective CB1 receptor antagonist AM 251. These data demonstrate for the first time that JWH-018 Cl and JWH-018 Br act similarly to JWH-018 while inducing less convulsive episodes and myoclonias. These data support the hypothesis that the halogenated compounds may have been introduced onto market to produce similar intoxicating effects as JWH-018 while causing less side effects.

A(2A) adenosine receptors and Parkinson's disease severity.

OBJECTIVES: In the last decade, increasing evidence suggests a key role of adenosine in Parkinson's disease (PD) and A2A adenosine receptors (A2A ARs) as an important pharmacological target in PD. An overexpression of A2A ARs has been found in putamen and in peripheral blood cells of PD patients. The primary aim of this study was to verify whether the alterations in A2A ARs in lymphocytes of PD subjects correlate with disease severity. MATERIAL AND METHODS: A consecutive sample of PD patients was enrolled. A clinical examination and a face-to-face interview were carried out. A2A ARs were investigated to verify the affinity and receptor density in lymphocyte membranes. The data were compared with those found in healthy controls. Moreover, the correlation between A2A AR density and affinity and clinical variables was evaluated in PD patients. RESULTS: In human lymphocyte membranes from PD patients, an increase in A2A AR density and a decrease in A2A AR affinity were found if compared with healthy subjects. A statistically significant correlation between the A2A AR density or affinity and specific clinical parameters as motor and cognitive impairment was detected. Patients with higher A2A AR density and lower affinity were more likely to exhibit motor complications. CONCLUSIONS: Parkinson's disease patients show an A2A AR upregulation in lymphocyte membranes if compared with healthy subjects. The correlation found between A2A AR density or affinity and clinical parameters highlights the central role of A2A AR modulation in the pharmacological treatment for PD and could suggest the putative role of A2A AR as a candidate biomarker of PD severity.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin-1ß (IL-1ß), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. EMF exposure induced a selective increase in A(2A) and A(3) ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active also in EMF-exposed cells. Functional data obtained in the presence of selective A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), while stimulating the release of interleukin-10 (IL-10), an antinflammatory cytokine. These effects seem to be mediated by the EMF-induced upregulation of A(2A) and A(3) ARs. No effects of EMFs or ARs have been observed on matrix degrading enzyme production. In conclusion, this study shows that EMFs display anti-inflammatory effects in human OASFs, and that these EMF-induced effects are in part mediated by the adenosine pathway, specifically by the A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes.

BACKGROUND AND PURPOSE: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A(1), A(2A), A(2B) and A(3)) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. EXPERIMENTAL APPROACH: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kappaB activation, tumour necrosis factor alpha (TNF-alpha) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. KEY RESULTS: mRNA and protein for A(1), A(2A), A(2B) and A(3) adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A(2A) and A(3) adenosine receptors inhibited p38 MAPK and NF-kappaB pathways, an effect abolished by selective adenosine antagonists. A(2A) and A(3) receptor agonists decreased TNF-alpha and IL-8 production. The phosphoinositide 3-kinase or G(s) pathways were involved in the functional responses of A(3) or A(2A) adenosine receptors. Synoviocyte A(1) and A(2B) adenosine receptors were not implicated in the inflammatory process whereas stimulation of A(2A) and A(3) adenosine receptors was closely associated with a down-regulation of the inflammatory status. CONCLUSIONS AND IMPLICATIONS: These results indicate that A(2A) and A(3) adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation.

Adenosine analogs and electromagnetic fields inhibit prostaglandin E2 release in bovine synovial fibroblasts.

OBJECTIVE: To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E(2) (PGE(2)) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). METHODS: SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N6-cyclohexyladenosine (CHA, A(1)), 2-[p-(2-carboxyethyl)-phenetyl-amino]-5'-N-ethylcarboxamidoadenosine (CGS 21680, A(2A)), 5'-N-ethylcarboxamidoadenosine (NECA, non-selective), N6-(3-iodobenzyl)2-chloroadenosine-5'-N-methyluronamide (Cl-IB-MECA, A(3)). SFs were treated with TNF-alpha (10 ng/ml) and lipopolysaccharide (LPS) (1 microg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-alpha- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75 Hz, 1.5 mT) during the period in culture (24h). PGE(2) release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. RESULTS: TNF-alpha and LPS stimulated PGE(2) release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE(2) production. EMFs inhibited PGE(2) production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE(2) release induced by CHA, CGS and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE(2) levels were associated to modification of COX-2 expression. CONCLUSIONS: This study supports anti-inflammatory activities of A(1) and A(2A) adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A(2A) receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes.

OBJECTIVE: The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE(2)) release. METHODS: Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X(1) and P2X(3) purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE(2) release) the potency of purinergic agonists and antagonists was evaluated. RESULTS: Bovine chondrocytes expressed P2X(1) and P2X(3) purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5'-triphosphate (ATP) and alpha,beta-methyleneATP were able to increase NO and PGE(2) release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. CONCLUSIONS: These data demonstrate for the first time the presence of functional P2X(1) and P2X(3) purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE(2) release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields.

OBJECTIVE: The present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied. METHODS: Saturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug-receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated. RESULTS: Bovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types. CONCLUSIONS: PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug-receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases.

Caffeine intake induces an alteration in human neutrophil A2A adenosine receptors.

Caffeine is the most widely used drug in the world and acts mainly through antagonism of the effects mediated by the adenosine receptor subtypes A1, A2A, A2B and A3. We determined whether repeated caffeine administration at different doses and for different periods of time (400 or 600 mg/day for 1 week and 400 mg/day for 2 weeks) alters human neutrophil A2A adenosine receptor density and function. Saturation binding assays showed an increase in affinity (K(D)) and density (B(max)) of A2A adenosine receptors after caffeine intake. These changes were accompanied by increases in cAMP accumulation and decreases in superoxide anion production after stimulation of the A2A receptor subtype using the agonist 5'-N-ethylcarboxamidoadenosine (NECA). Binding and functional changes of A2A receptors returned to baseline after 48 h of caffeine withdrawal. The findings are consistent with a potential anti-inflammatory effect of caffeine mediated by neutrophil A2A receptors.

Stobadine: bellwether of a broader view of drug actions.

Stobadine was recognized early in its development as having antioxidant properties. A number of laboratories found associations between the antioxidant properties of stobadine and its potential beneficial effects. We found that stobadine acted as an antioxidant in a modification of an oxygen radical absorbance capacity (ORAC) assay. Similar results were observed with other drugs, including tirilazad and pramipexole. We suggest that stobadine and certain other drugs exhibit antioxidant properties in both hydrophilic and hydrophobic environments. Other drugs have been developed for their antioxidant properties and some currently marketed drugs have antioxidant properties. Although they may not have been explicitly sought during development, at least some of the beneficial effects may be related to antioxidant properties and/or scavenging of free radicals. Because stobadine was one of the first drugs for which useful properties were associated with its antioxidant actions, stobadine may be seen as a bellwether of a broader view of pharmacological actions--a view that encompasses antioxidant properties as a useful basis of therapeutic effects.

Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6,7-dimethyl-2,4-DI-1- pyrrolidinyl-7H-pyrrolo[2,3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger.

Preincubation of red blood cell (RBC) membranes with a model system known to generate reactive oxygen species (ROS) and free radicals (200 microM ferrous sulfate and 200 microM EDTA, Fe2+/EDTA) resulted inhibition of the Na+/K+ -pump ATPases was also associated with membrane protein crosslinking and lipid peroxidation, the latter as monitored by the formation of thiobarbituric acid reactive substances (TBARS). Inhibition of the ion transport ATPases, protein cross-linking and formation of TBARS were prevented by U-89843D in a concentration-dependent manner, with half-maximal protection seen at 0.3 microM. U-89843D was more potent than the classical antioxidant butylated hydroxytoluene. Neither U-89843D nor the solvent DMSO had any effect on the assay of TBARS. U-89843D exerted only minimal inhibitory activity on ATPase activities. Thus, U-89843D was potent in vitro in preventing a variety of membrane-damaging reactions mediated by ROS. It is suggested that protection of membranes from ROS-mediated damage is of potential usefulness in the prevention and treatment of certain disease processes.

Inhibition by activated neutrophils of the Ca2+ pump ATPase of intact red blood cells.

Human neutrophils, activated by phorbol myristate acetate in the presence of intact red blood cells (RBCs), caused inhibition of the Ca2+ pump ATPase of the RBCs and fragmentation of the enzyme as well as other membrane proteins. Inhibition of the Ca2+ pump ATPase of intact RBCs was directly related to the neutrophil concentration and the time of incubation. Ca2+ pump ATPase activity was partially protected by the addition of exogenous glutathione-glutathione peroxidase, but not by superoxide dismutase. The addition of sodium azide, a potent inhibitor of endogenous RBC catalase, enhanced inhibition of the Ca2+ pump ATPase of intact RBCs. Examination by SDS-polyacrylamide gel electrophoresis of membrane proteins isolated from RBCs preincubated with activated neutrophils showed gross changes in banding patterns as compared to controls. Thus, a significant amount of methemoglobin appeared to be associated with the membrane proteins, and, in general, protein bands appeared to be more diffuse and less defined than proteins in control lanes. In addition, there was an increase in the low molecular weight protein bands. Using a monoclonal antibody to the Ca2+ pump ATPase, it was shown that the 140 kDa band representing the Ca2+ pump ATPase decreased, with concomitant appearance of two low molecular weight bands running at 8.2 and 6.8 kDa in the membrane proteins from RBCs preincubated with activated neutrophils. The data are interpreted to suggest that inhibition of the Ca2+ pump ATPase in intact RBCs under these conditions occurred as a result of: neutrophil-derived superoxide, dismutation of superoxide, to H2O2, diffusion of H2O2 into RBCs, a Fenton type reaction between oxyhemoglobin, and H2O2 producing hydroxyl radical and/or a ferryl radical capable of promoting protein fragmentation of RBC membrane proteins, including the plasma membrane Ca2+ pump ATPase.

Inhibition of the Ca pump of intact red blood cells by t-butyl hydroperoxide: importance of glutathione peroxidase.

Incubation of human red blood cells (RBCs) with t-butyl hydroperoxide (tBHP) resulted in inhibition of the Ca-pump ATPase. This was demonstrated using an assay of the Ca-pump ATPase activity in intact RBCs. In this assay, activity of the Ca-pump ATPase is expressed as the rate constant of the initial loss of ATP in RBCs exposed to Ca and A23187. Pseudo-first-order rate constants (Ca-pump ATPase rate constants) were lower in the presence of tBHP versus controls. Incubation of RBCs with tBHP resulted in both a time- and concentration-dependent inhibition of the Ca-pump ATPase (IC50 approximately 1 mM). Incubation of RBCs with tBHP also resulted in decreased oxyhemoglobin, increased methemoglobin and increased thiobarbituric acid reactive substances (TBARS). GSH levels were significantly lower in the presence of tBHP. GSH fell from a control value of 2.2 mmol/l RBC to 0.46 mmol/l RBC after incubation with 0.25 mM tBHP for 15 min. Both butylated hydroxytoluene and stobadine prevented the formation of TBARS and were partially effective in protecting the Ca-pump ATPase from tBHP-induced inhibition. Dithiothreitol was completely effective in preventing the tBHP-induced formation of TBARS as well as inhibition of the Ca-pump ATPase. However, when added after exposure to tBHP, dithiothreitol was unable to restore Ca-pump ATPase activity completely. An activity of dithiothreitol independent of enzymic thiol group reduction was apparent. In the presence of mercaptosuccinate, a potent inhibitor of glutathione peroxidase, the ability of dithiothreitol to protect the Ca-pump ATPase from tBHP-induced inhibition was abolished. Therefore, protection by dithiothreitol may be afforded by its ability to replenish GSH from oxidized glutathione, thus allowing glutathione peroxidase to metabolize tBHP. These results may be interpreted to suggest that inhibition of the Ca-pump ATPase in intact RBCs occurs as a result of tBHP-induced oxidant stress and subsequent lipid peroxidation which can be prevented by certain antioxidants including butylated hydroxytoluene, stobadine, and thiol-containing compounds such as dithiothreitol. These findings provide further insight into the mode of action of hydroperoxides and certain reactive oxygen species that have been implicated in oxidative stress associated with various pathological conditions. The importance of the GSH/glutathione peroxidase system in metabolizing organic hydroperoxides is also demonstrated.

Simultaneous measurement of multiple membrane ATPases in microtiter plates.

An assay for activities of erythrocyte membrane-bound ATPases adapted from the autoanalyzer method of Raess and Vincenzi (1980a) is described in detail. Mg2+ ATPase, Na+/K(+)-ATPase, Ca2+ ATPase, and calmodulin- (CaM) activated Ca2+ ATPases were determined in microtiter plates. Total volume was 100 microL. Ten microliters of 0.75 mg/mL of red blood cells membranes were added to appropriate buffer in microtiter plates. Plates were preincubated at 37 degrees C for 10 min, reactions were started by the addition of ATP, and plates were incubated for an additional 60 min at 37 degrees C. Reactions were stopped by sodium dodecyl sulfate (SDS). Inorganic phosphate (P(i)) was measured by modifications of the method by Fiske and Subbarow, (Fiske and Subbarow, 1925) in the same plate using a plate reader. The P(i) assay range was between 0 and 250 nm/mL. Results obtained for intraassay precision, (n = 7) are as follows: Mg2+ ATPase = 4.39 +/- 0.25 (5.7% CV); Na+/K(+)-ATPase = 7.33 +/- 0.40 (5.4% CV); Ca2+ ATPase = 15.86 +/- 0.76 (4.8% CV); and CaM-activated Ca2+ ATPase = 74.12 +/- 2.34 (3.2% CV) (nmole P(i)/mg protein/min.). This is a rapid, simple, and nonisotopic method for the determination of membrane-bound ATPases activities. All steps are performed in the same microtiter plate, thus reducing handling and associated errors.

Increased cytosolic free calcium in red blood cells is associated with essential hypertension in humans.

Intracellular free calcium (Cai2+) was measured in human red blood cells (RBCs) using the Ca(2+)-sensitive fluorescent probe, fluo-3. Fresh RBCs were loaded with the acetoxymethyl ester of fluo-3 (fluo-3-AM) in vitro. Following incubation at 37 degrees C for 60 min, cells were separated from nonincorporated fluo-3-AM and fluo-3. Fluorescence was quantified with excitation and emission wavelengths of 506 nm and 526 nm, respectively. The relatively long wavelength characteristics of fluo-3 avoid much of the interference from hemoglobin that makes agents such as quin-2 and fura-2 of limited value in RBCs. A protocol with corrections for quenching and for extracellular fluo-3 was employed for each sample. Cai2+ in red blood cells from 15 normotensive volunteers averaged 134 +/- 12 nmol/L. In RBCs from 30 hypertensive patients who were not treated pharmacologically, Cai2+ was 317 +/- 32 nmol/L (P < .05). In a group of 27 hypertensives who were treated with various drugs, RBC Cai2+ was 221 +/- 27 nmol/L. Measurements were also performed in RBCs from 16 patients before and at least 2 weeks following treatment with one or more antihypertensive agents. Reduction of RBC Cai2+ from 342 +/- 47 to 252 +/- 39 (P < .001) was observed in 16 patients whose blood pressure fell with treatment. Overall, there was a correlation between RBC Cai2+ and diastolic blood pressure (r = 0.413, P < .01, d.f. = 71). Thus, the present results confirm an association between elevated RBC Ca2+ and essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion transport ATPases as targets for free radical damage. Protection by an aminosteroid of the Ca2+ pump ATPase and Na+/K+ pump ATPase of human red blood cell membranes.

Preincubation of red blood cell membranes in the presence of ferrous sulfate and EDTA resulted in both a concentration- and time-dependent inhibition of the Na+/K+ pump ATPase, basal Ca2+ pump ATPase, and the calmodulin- (CaM) activated Ca2+ pump ATPase. The IC50 for all three ATPases was approximately 2.5 x 10(-5) M iron. The addition to membranes of ferrous iron and EDTA in an approximately 1:1 ratio resulted in conversion to the ferric iron form in several minutes. However, inhibition of the ion pump ATPases and cross-linking of membrane proteins occurred over the course of several hours. The time course of formation of thiobarbituric acid-reactive substances (TBARS) closely paralleled inhibition of the ion pump ATPases. Inhibition of the ion pump ATPases was prevented by the addition of deferoxamine or superoxide dismutase but not by mannitol, or catalase. Both butylated hydroxytoluene and tirilazad mesylate (U74006F) prevented the formation of TBARS, limited the inhibition of the ion pump ATPases, and reduced cross-linking of membrane proteins. These data may be interpreted to suggest that inhibition of ion pump ATPases in plasma membranes may occur as a result of iron-promoted formation of superoxide and subsequent lipid peroxidation, which can be prevented by free-radical scavengers including butylated hydroxytoluene and U74006F.