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Introduction: To access the common bile duct in endoscopic retrograde cholangiopancreatography (ERCP), needle-knife fistulotomy (NKF) can be associated with a shorter sphincterotomy compared to standard cannulation. We aimed to compare the success and safety of NKF versus standard cannulation in the treatment of choledocholithiasis. Methods: A cohort of 379 naïve patients with choledocholithiasis who underwent ERCP between 2005 and 2022 was retrospectively analyzed. The patients were divided into two groups: group A (179 consecutive patients) underwent NKF, while group B (180 patients) received standard biliary access and were matched for stone characteristics and ERCP year. Results: Stone removal success rate for group A was significantly lower than that for group B in the initial ERCP (82.0% vs. 92.1%, p = 0.003). In group A, success rates for stone removal were 90.2%, 80%, and 29.4% for stone sizes 15 mm, respectively (p < 0.001). In contrast, group B showed success rates of 99.2%, 81.5%, and 71.4% for the same stone size categories (p < 0.001). Pancreatitis occurred in 3.7% of group A and 5.8% of group B patients (p = 0.340). Regression analysis revealed that NKF cannulation, stone size (>10 mm), and having 4 or more stones were associated with lower stone removal success compared to standard cannulation in the initial ERCP (OR 0.34, p = 0.015; stone size 10–15 mm: OR 0.20, p < 0.001; stone size >15 mm: OR 0.05, p < 0.001; 4 or more stones: OR 0.4, p = 0.040). Conclusions: The removal of common bile duct stones after NKF access, although safe and effective, is less successful than after a standard cannulation, especially at the baseline ERCP.

Introduction and Objectives: Center-based cardiac rehabilitation (CR) programs have been forced to close due to COVID-19. Alternative delivery models to maintain access to CR programs and to avoid physical inactivity should be considered. The aim of this study was to assess physical activity (PA) levels after completing a home-based digital CR program. Methods: A total of 116 cardiovascular disease (CVD) patients (62.6±8.9 years, 95 male) who had been attending a face-to-face CR program were recruited and assessed (baseline and at three months) on the following parameters: PA, sedentary behavior, adherence, cardiovascular and non-cardiovascular symptoms, feelings toward the pandemic, dietary habits, risk factor control, safety and adverse events. The intervention consisted of a multidisciplinary digital CR program, including regular patient assessment, and exercise, educational and psychological group sessions. Results: Ninety-eight CVD patients successfully completed all the online assessments (15.5% drop-out rate). A favorable main effect of time was an increase in moderate to vigorous PA and a decrease in sedentary time at three months. Almost half of the participants completed at least one online exercise training session per week and attended at least one of the online educational sessions. No major adverse events were reported and only one minor event occurred. Conclusion: During the pandemic, levels of moderate to vigorous PA improved after three months of home-based CR in CVD patients with previous experience in a face-to-face CR model. Diversified CR programs with a greater variety of content tailored to individual preferences are needed to meet the motivational and clinical requirements of CVD patients. Resumo: Introdução e objetivos: Os programas convencionais de reabilitação cardíaca (RC) foram forçados a encerrar devido à Covid-19. Modelos alternativos para que os doentes tenham acesso a um programa de RC evitando a inatividade física devem ser considerados. O objetivo deste estudo foi avaliar os níveis de atividade física (AF) de um programa digital de RC em casa. Métodos: Foram recrutados e avaliados (inicialmente e aos três meses) 116 doentes cardiovasculares (CV) (62,6±8,9 anos, 95 homens) que frequentavam um programa presencial de RC, nos seguintes parâmetros: AF, comportamento sedentário, adesão, sintomas CV e não CV, sentimentos face à pandemia, hábitos alimentares, fatores de risco, segurança e eventos adversos. A intervenção consistiu num programa digital multidisciplinar de RC, inclusive acompanhamento regular, sessões de exercício, de ensino e de psicologia em grupo. Resultados: Completaram com sucesso todas as avaliações online (15,5% drop-out) 98 pessoas com doença CV. Houve um efeito favorável no aumento da AF moderada a vigorosa e diminuição do tempo sedentário aos três meses. Quase metade da amostra fez, pelo menos, mais de uma sessão de exercício físico online por semana e assistiu a pelo menos uma das sessões educacionais online. Não se verificaram eventos major e registou-se apenas um minor. Conclusão: Em tempo de pandemia, os níveis de AF moderada a vigorosa melhoraram após três meses em doentes CV que frequentavam previamente um modelo presencial de RC. São necessários mais programas de RC com maior variedade de conteúdos adaptados à preferência individual para dar resposta às necessidades motivacionais e clínicas dos doentes CV.

[GAR+] prion-like elements partially relieve carbon catabolite repression in Saccharomyces cerevisiae. They have been hypothesized to contribute to wine yeast survival and alcohol level reduction, as well as communication with bacteria and stuck fermentation. In this work, we selected [GAR+] derivatives from several genetic backgrounds. They were characterized for phenotypic penetrance, heritability and confirmed as prion-like through curing by desiccation. In terms of fermentation kinetics, the impact of the prion on anaerobic wine fermentation (natural grape juice) was either neutral or negative, depending on the genetic background. Likewise, residual sugars were higher or similar for [GAR+] as compared to the cognate [gar-] strains. The prions had little or no impact on glycerol and ethanol yields; while acetic acid yields experienced the highest variations between [GAR+] and [gar-] strains. Strains analyzed under aerobic conditions followed the same pattern, with either little or no impact on fermentation kinetics, ethanol or glycerol yield; and a clearer influence on volatile acidity. Although no clear winemaking advantages were found for [GAR+] strains in this work, they might eventually show interest for some combinations of genetic background or winemaking conditions, e.g., for reducing acetic acid yield under aerated fermentation.

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This work describes the synthesis of nitrogen-doped carbon nanodots (CNDs) synthesized from ethylenediaminetetraacetic acid (EDTA) as a precursor and their application as luminescent agents with a dual-mode theranostic role as near-infrared (NIR) triggered imaging and photodynamic therapy agents. Interestingly, these fluorescent CNDs are more rapidly and selectively internalized by tumor cells and exhibit very limited cytotoxicity until remotely activated with a NIR illumination source. These CNDs are excellent candidates for phototheranostic purposes, for example, simultaneous imaging and therapy can be carried out on cancer cells by using their luminescent properties and the in situ generation of reactive oxidative species (ROS) upon excitation in the NIR range. In the presence of CNDs, NIR remote activation induces the in vitro killing of U251MG cells. Through the use of flow imaging cytometry, we have been able to successfully map and quantify the different types of cell deaths induced by the presence of intracellular superoxide anions ( . O 2 - ) and hydrogen peroxide (H 2 O 2 ) ROS generated in situ upon NIR irradiation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: The oestrogen receptor β (ERβ) selective agonist diarylpropionitrile (DPN) relaxes endothelium-denuded rat aorta, but the signalling mechanism is unknown. The aim of this study was to assess whether protein kinase A (PKA) signalling is involved in DPN action., Methods: cAMP was measured by radioimmunoassay, HSP20 phosphorylation by 2D gel electrophoresis with immunoblotting, and membrane potential and free cytosolic calcium by flow cytometry., Key Findings: DPN increased cAMP content and hyperpolarised cell membranes over the same range of concentrations as it relaxed phenylephrine-precontracted aortic rings (10-300 µM). DPN-induced vasorelaxation was largely reduced by the PKA inhibitors Rp-8-Br-cAMPS (8-bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer) and H-89 (N-(2-bromocynnamyl(amino)ethyl)-5-isoquinoline sulfonamide HCl) (-73%) and by the adenylate cyclase inhibitor MDL12330A (cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine)) (-65.5%). Conversely, the PKG inhibitor Rp-8-Br-cGMP was inactive against DPN vasorelaxation. In aortic smooth muscle segments, DPN increased PKA-dependent HSP20 phosphorylation, an effect reversed by H-89. Relaxant responses to DPN were modestly antagonised (-23 to -48% reduction; n=12 per compound) by the potassium channel inhibitors iberiotoxin, PNU-37883A, 4-aminopyridine, or BaCl(2) . All four potassium channel inhibitors together reduced DPN relaxation by 86±9% (n=12) and fully blocked DPN hyperpolarisation., Conclusions: ERβ-dependent relaxation of rat aortic smooth muscle evokes an adenylate cyclase/cAMP/PKA signalling pathway, likely activating the cystic fibrosis transmembrane conductance regulator chloride channel and at least four potassium channels., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Objectives: This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor (ERalpha) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings., Methods: Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel., Key Findings: PPT vasorelaxation was largely reduced by the selective ERalpha antagonist methyl-piperidinopyrazole (MPP; -91.6+/-2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (-78.6+/-4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; -85.3+/-5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (-59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; -50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (-40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (-80.8%)., Conclusions: ERalpha receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.

Chloride ions play a key role in smooth muscle contraction, but little is known concerning their role in smooth muscle relaxation. Here we investigated the effect of chloride transport inhibitors on the vasorelaxant responses to nitroprusside in isolated and endothelium-denuded rat aorta, precontracted with phenylephrine 1 muM. Incubation of aortic rings in NO(3)(-) media strongly potentiated the vasorelaxant responses to nitroprusside. Bumetanide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and acetazolamide strongly potentiated the vasorelaxant responses to nitroprusside (by 70-100%). EC(50) were 2.3+/-0.5 microM for bumetanide, 26+/-15 microM for DIDS and 510+/-118 microM for acetazolamide (n=6 for condition). Niflumic acid, a selective inhibitor of ClCa (calcium-activated chloride channels), potentiated nitroprusside relaxation to a similar extent as chloride transport inhibitors, in a non-additive manner. Zinc and nickel ions, both modestly potentiated nitroprusside vasorelaxation (by 20-30%). Cobaltum had negligible effect on nitroprusside vasorelaxation. CPA (p-chlorophenoxy-acetic acid), an inhibitor of volume-sensitive chloride channels (ClC), slightly potentiated nitroprusside vasorelaxation (by 15%), and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhibitors CFTR(inh)172 (5-[(4-Carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone), DPC (diphenylamine-2,2'-dicarboxylic acid) and glibenclamide were without significant effect. In conclusion, inhibition of chloride transport proteins strongly potentiates the vasorelaxant responses to nitroprusside in isolated rat aorta. This effect seems mediated by chloride depletion and inhibition of a chloride channel activated by both, calcium and cyclic GMP (cGMP).

The soy phytoestrogen genistein is a potent vasorelaxant, but its mechanism of action is poorly understood. Here, we used endothelium-denuded rat aorta to investigate the role of the cyclic AMP(cAMP)-activated, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, and its associated Na-K-Cl cotransporter NKCC1. Isolated, endothelium-denuded rat aorta was contracted with phenylephrine 1 microM, and the vasorelaxant responses to genistein were investigated under conditions where CFTR was inhibited by DPC (diphenylamine-2-carboxylic acid) or glibenclamide (n=6 for compound). Both compounds fully antagonized the vasorelaxant responses to genistein, with IC50=57+/-18 microM and 42+/-11 microM for DPC and glibenclamide respectively. H-89, a selective protein kinase A (PKA) inhibitor, blocked the vasorelaxant responses to genistein. Finally, the NKCC1 inhibitor, bumetanide fully antagonized the vasorelaxant responses to genistein against phenylephrine- or KCl-induced contractions, with IC50=2.0+/-0.2 microM and 1.6+/-0.5 microM, respectively (n=6 for condition). These results strongly suggest that CFTR opening is involved in the vasorelaxant action of genistein, and that cAMP-dependent CFTR phosphorylation and chloride entry via the NKCC1 cotransporter are required for genistein action.

In mammalian erythrocytes, outward fluxes by the Na-K-Cl cotransporter NKCC have been clearly characterized, but NKCC fluxes are small and their physiological role, if any, is poorly understood. Avian erythrocytes are nucleated cells, in which a physiologically relevant NKCC acts as a cell volume regulator. Therefore, we further investigated outward cotransport and its relation to cell volume by using quail erythrocytes. Unlike human or rat erythrocytes, quail erythrocytes exhibit outward cotransport fluxes: (1) of high magnitude [maximal rate of bumetanide-sensitive Li+ efflux=12.3+/-1.1 mmol (l cells x h)(-1), mean +/-SEM, n=23] and (2) strongly stimulated by hyperosmotic media (by 100-200% in 500 mosmol/l media). Na+- or Li+-loaded quail erythrocytes exhibited rapid cell shrinkage when incubated in K+-free media. Thus, cell volume remained stationary up to 5-10 min and then started to shrink. Shrinkage was first slow, but progressively accelerated, finally reaching a new stationary state where cell volume had decreased by about 20%. Such rapid cell shrinkage was fully inhibited by bumetanide and was associated with outward cotransport stimulation (self-stimulated or an auto-catalytic process, i.e. a reaction stimulated by its product). External K+ reduced all these phenomena, but significant cell shrinkage was still observed at an external K+ concentration of 2.8 mM. K+ removal failed to stimulate outward cotransport in hypotonic media (250 mosmol/l). Finally, reincubation of shrunken erythrocytes in physiological saline revealed that inward cotransport was stimulated more than outward cotransport. In conclusion, isoosmotic hypokalaemia drives a rapid shrinkage of quail erythrocytes, due to auto-catalytic net outward cotransport stimulation. Whether this is an experimental curiosity or indicates that outward cotransport can have some physiological role deserves further investigation.

Soy-based diets reduce blood pressure in spontaneously hypertensive rats, but apparently not in hypertensive humans. In the present study, the antihypertensive potential of soy milk (500 mL twice daily) compared with cow's milk was investigated in a 3-mo double-blind randomized study of 40 men and women with mild-to-moderate hypertension. Before initiation of the study, urinary isoflavonoids (measured by HPLC) were undetectable in most cases (for genistein, they were always <100 micromol/L). After 3 mo of soy milk consumption, systolic blood pressure decreased by 18.4 +/- 10.7 mmHg compared with 1.4 +/- 7.2 mmHg in the cow's milk group (P < 0.0001), diastolic blood pressure decreased by 15.9 +/- 9.8 mmHg vs. 3.7 +/- 5.0 mmHg in the cow's milk group (P < 0.0001) and mean blood pressure decreased by 16.7 +/- 9.0 mmHg compared with 3.0 +/- 4.6 mmHg in the cow's milk group (P < 0.0001). Urinary genistein was strongly (r = -0.588) and significantly (P = 0.002) correlated with the decrease in blood pressure, particularly for diastolic values. In conclusion, chronic soy milk consumption had modest, but significant hypotensive action in essential hypertensive subjects. This hypotensive action was correlated with the urinary excretion of the isoflavonoid genistein.

Antioxidant effects of isoflavonoids have recently been described. To learn whether the isoflavonoids genistein and equol have actions on the intracellular free radicals, human neutrophils and J774 monocyte-macrophage cell line were used to measure the intracellular production of O(2) (superoxide anion) and H(2)O(2) (hydrogen peroxide) by flow cytometry. The results shown significatives decrease in O(2) and H(2)O(2) production after 1 hour of incubation with equol and genistein. The phagcytic oxidant production decreased owing to the effects of both isoflavonoids in a concentration-dependent manner.

A furosemide-sensitive Na-K-Cl cotransporter (NKCC2 isoform) accounts for almost all luminal NaCl reabsorption in the thick ascending limb of Henle's loop (TALH). The activity of this transport protein is regulated by humoral factors known as cotransport inhibitory factors. One family of these compounds is represented by the urinary phytoestrogens equol and genistein, which inhibit cotransport fluxes at concentrations similar to furosemide. Moreover, they possess salidiuretic potency similar to furosemide in the isolated perfused rat kidney, but are less potent than furosemide (in vivo). Thus, dietary phytoestrogens can be responsible, at least in part, for the low blood pressure of vegetarians.

Intake of soyfoods containing isoflavonoids is related to positive effects on heart and kidney diseases. Urinary equol, a potent inhibitor of Na+-K+-2Cl- cotransport, originates from the metabolism of daidzein by intestinal bacteria. Loop diuretics (eg, furosemide), acting through inhibition of Na+-K+-2Cl- cotransport, are used to maintain adequate blood volume. In the present work, we compare isoflavonoids' inhibition of cotransport and effects on the function and hemodynamics of isolated perfused rat kidneys with those of furosemide. Equol [IC50 (half-maximal inhibitory concentration): 23.6 +/- 3.6 micromol/L], genistein (IC50: 34.8 +/- 2.6 micromol/L), and daidzein (IC50: 140.0 +/- 24 micromol/L) inhibited bumetanide-sensitive rubidium uptake in LLC-PK1 cells. The IC50 of equol and genistein was close to that of furosemide (IC50: 10.3 +/- 2.7 micromol/L). Furosemide, equol, and genistein stimulated water, sodium, and potassium excretion by isolated rat kidneys in the same temporal pattern. None of the isoflavonoids significantly increased the glomerular filtration rate, but genistein induced significant vasorelaxation. We conclude that isoflavonoids exhibit biological activities of furosemide in vitro, at concentrations similar to those reported for other in vitro effects. More research is needed to evaluate the participation of cotransport inhibition by isoflavonoids in the healthful effects claimed for soy intake.

A furosemide-sensitive Na-K-Cl cotransporter (NKCC2 isoform) accounts for almost all luminal NaCl reabsorption in the thick ascending limb of Henle's loop (TALH). The activity of this transport protein is regulated by humoral factors (CIF: cotransport inhibitory factors). One family of CIF compounds is represented by the urinary phytoestrogens equol and genistein, which inhibit cotransport fluxes at similar concentrations as furosemide. Moreover, they possess similar salidiuretic potency as furosemide in the isolated perfused rat kidney, but are less potent than furosemide in vivo. Thus, dietary phytoestrogens can be responsible, at least in part, for the low blood pressure of vegetarians. A second type of CIF is represented by a circulating and urinary factor which is evoked by salt-loading. This, which is not a "ouabain-like" factor, appears to be a new retropituitary natriuretic compound. Endogenous CIF is increased in hypertensive Dahl salt-sensitive rats, probably as a compensatory mechanism against the enhanced NaCl reabsorption in the TALH, which characterizes this model of hypertension. Finally, chronic excess of circulating CIF inhibits and induces up-regulation of erythrocyte Na-K-Cl cotransporter NKCC1.

The urinary isoflavonoid genistein inhibits membrane Na-K-Cl cotransporters at similar concentrations as furosemide, but the significance of this action is unknown. Genistein was therefore investigated in rats for its potential salidiuretic actions. In the isolated, perfused rat kidney, genistein induced a maximal salidiuretic action similar to that of furosemide but was 3 to 5 times less potent than furosemide in terms of active doses (natriuresis EC50, 237+/-92 versus 56+/-20 micromol/L for genistein and furosemide, respectively). Genistein and furosemide had no additive salidiuretic actions. Genistein had no significant effect on glomerular filtration rate but was able to significantly reduce renal vascular resistance with respect to vehicle isolated perfused kidney. Indomethacin (10 micromol/L), a blocker of prostaglandin biosynthesis, reduced salidiuresis and renal vasorelaxation by genistein. Subcutaneous genistein (15 mg/kg) induced a statistically significant increase in diuresis and natriuresis with respect to vehicle during the first 6 hours of administration in rats. In conclusion, genistein compares well with furosemide in vitro for its salidiuretic profile and potency in the isolated perfused rat kidney and is also natriuretic by the subcutaneous route in the rat. Further studies are required to investigate potential natriuretic and perhaps hypotensive actions of dietary genistein.

Dahl salt-sensitive (DS) rats seem characterized by a ubiquitous increase in Na-K-Cl cotransport activity. Here, an endogenous inhibitor of the Na-K-Cl cotransport system (cotransport inhibitory factor, CIF) was investigated in inbred Dahl salt-sensitive (DS) and salt-resistant (DR) rats. The animals were orally loaded for 10 days with 2% NaCl. Plasma from salt-loaded DS rats inhibited cotransport with a 50% inhibition concentration value (IC50) of 6.4 +/- 0.6% (% plasma concentration, vol/vol) vs. 24.2 +/- 2.2% in DR rats (P < 0.0001). In urine, IC50 for cotransport inhibition was constantly lower in DS before and all during the whole salt-loading period (after 10 days of salt loading, IC50 was 2.59 +/- 0.11% and 6.00 +/- 0.24% in DS and DR rats, respectively; P < 0.0001). After 3 days of salt loading, higher salt appetite in DS rats magnified the differences in urinary CIF excretion. In erythrocytes from DS rats, increased cotransport activity was strongly correlated with urinary CIF excretion (r = 0.967). In conclusion, DS rats present increased plasmatic and urinary CIF levels. This can be a compensatory phenomenon to reduce cotransport hyperactivity and increased NaCl reabsorption at the thick ascending limb of Henle's loop.

Background: Reactive oxygen metabolites have been associated with gastrointestinal injury., Objective: To investigate whether mucosal reactive oxygen metabolites are involved in acid and pepsin induced oesophagitis, and if so, which specific metabolites., Methods: The effects of free radical scavengers and the anti-inflammatory drug ketotifen on rabbit oesophagitis induced by acidified pepsin were studied. Isolated oesophageal cells were obtained before and after oesophageal injury and the generation of superoxide anion and hydrogen peroxide was analysed by flow cytometry. The presence of inflammatory cells was determined by indirect immunofluorescence with a mouse antirabbit CD11b antibody., Results: Of the free radical scavengers tested, superoxide dismutase, which reacts with the superoxide anion, significantly reduced oesophagitis, whereas catalase, which reacts with hydrogen peroxide, had only a mild effect and dimethylsulphoxide had no effect. Ketotifen significantly reduced the inflammation and also prevented the induction of oesophagitis. Isolated cells obtained from the oesophageal mucosa after acidified pepsin exposure generated increased amounts of superoxide anions, which were mainly produced by CD11b positive cells., Conclusions: Reactive oxygen metabolites, especially superoxide anion, produced by inflammatory cells play a significant part in the genesis of oesophagitis induced by acid and pepsin in rabbits and might be a target for future medical therapy.

Flow cytometry was used to investigate the participation of reactive oxygen species, other than singlet oxygen, in the cytotoxic effect of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in vitro in A-431 squamous cell carcinoma (SCC) cells and human skin fibroblasts (HSF). We used propidium iodide to determine cellular cytotoxicity, hydroethidine to measure intracellular superoxide anion (O2-) and dihydrorhodamine 123 to assess intracellular hydrogen peroxide (H2O2) content. Our data support the importance of the incubation time with ALA in the selectivity of PDT with ALA against SCC cells, inducing minimum damage on normal HSF. Photoradiation mortality curves of the response of these cell lines to ALA-induced PpIX photosensitization correlated with the extent of photosensitizer accumulation. Intracellular O2- production correlated with cell death, increasing both in a light dose-dependent fashion in ALA treated cells. This correlation was not observed with H2O2-intracellular production. These results suggest the effectiveness of PDT with ALA in vitro in SCC, the significant participation of O2- in its phototoxic mechanism, and the usefulness of flow cytometry in the study of the cytotoxic effect of ALA-induced PpIX PDT.

A potent inhibitor of the Na-K-Cl cotransport system was purified from urines of salt-loaded rats. Mass spectroscopy revealed a molecular mass of 242 Da. Nuclear magnetic resonance showed a spectrum identical to that of 3,4-dihydro-3-(4-hydroxyphenyl)-2H-1-benzopyran-7-ol (an "estrogen-like" isoflavonoid: equol). This compound inhibited cotransport fluxes at similar concentrations (IC50=16-24 microM) as furosemide (IC50 approximately 10 microM). Cotransport inhibitory activity of urines from rats drinking tap water was fully explained by urinary equol concentrations (approximately 27 microM, measured by high-performance liquid chromatography). Slat-loading increased urinary equol excretion, but not sufficiently high to fully explain the very important increase in cotransport inhibitory potency. We conclude that: (i) under basal conditions urinary equol can regulate Na-K-Cl cotransport activity in the kidney and (ii) salt-loading should evoke the appearance of other cotransport inhibitors.

Magnesium skeletal (myocardium and gastrocnemius) content has been studied in rats supplemented with 30 mM dissolutions of Mg2+, Na+, Ca2+ and K+ in drinking water, for 7 or 30 days. In both muscles, the ingestion of Mg2+ for 30 days increased Mg2+ content, while Ca2+ and K+ supplementation caused a significant drop. The increase in Na+ ingestion reduced Mg2+ content in gastrocnemius. There were no significant differences between control and animals supplemented for 7 days. These results suggest that, in the case of supplementation situations, the control mechanisms of the Mg2+ tissular content have a lower gain than those of Na+ and K+ of one order of magnitude.

We investigated the site of origin of a potent inhibitor of the Na-K-Cl cotransport system (CIF), which has been previously identified in urines from salt-loaded rats. Rats were given a 2% NaCl solution to drink for 15 and 60 days and the plasma was obtained and tested for cotransport inhibitory activity (on bumetanide-sensitive Li+ efflux in Li-loaded human erythrocytes) in dose-response curves. The IC50 for cotransport inhibition (vol:vol dilution reducing cotransport activity by 50%) was found to be 26.5 +/- 7.2 (mean +/- SEM, n = 5) and 6.9 +/- 0.7% (n = 10) on days 15 and 60, respectively (control samples, day 0, only inhibited about 20% cotransport activity at a 30% plasma vol:vol dilution, n = 7). Organ extracts from salt-loaded rats were prepared and tested for cotransport inhibitory activity. A statistically significant cotransport inhibitory activity was only found in pituitary extracts (-36 +/- 3 vs. -5 +/- 4% in control rats, n = 4, p < 0.01). Dissection of a large number of pituitary glands from salt-loaded rats revealed CIF activity only in the neurohypophysis (cotransport inhibition -46.5 +/- 5.2 vs. -0.5 +/- 16.4% in the anterior lobe, n = 4, p < 0.05). In conclusion, CIF is a new circulating endogenous factor, probably secreted by the neurohypophysis.

We previously found a potent inhibitor of the Na-K-Cl cotransport system in urines from salt-loaded rats (C.I.F. = cotransport inhibitory factor, ref. 1). Here we extracted an urinary fraction (approximately 1% urine dry weight), free from immunoreactive A.N.P. and digoxin activity, which: (i) potently inhibited cotransport fluxes in MDCK (Madin and Darby canine kidney] cells and in human erythrocytes, (ii) inhibited Na(+)-dependent chloride/bicarbonate exchange with 2-3 times less potency than cotransport and (iii) strongly increased natriuresis and diuresis after i.v. infusion in rats with no significant change in kaliuresis (salidiuretic action reduced by probenecid). Therefore, C.I.F. seems to be a new natriuretic factor with part, but not all the biological profile of loop diuretic drugs.

Human red blood cells were incubated in the presence of Na2MoO4 and the initial rate of molybdenum uptake was measured by using inductively coupled plasma emission spectroscopy. About 99% of molybdenum uptake was inhibited by DIDS or by SITS. DIDS-sensitive molybdenum uptake was inhibited by external chloride, bicarbonate, sulphate and phosphate in the range of concentrations previously described for anion carrier fluxes. Trace elements, previously described to be translocated by the anion carrier, i.e. copper, zinc and cadmium, slightly inhibited molybdenum uptake. Molybdenum uptake was strongly stimulated by acidification, suggesting that the monovalent HMoO4- anion species, which is formed in acidic media (pK approximately 4.1), can be more rapidly translocated than the divalent anion complex MoO4(2-), which is the predominant form at physiological pH. In conclusion, the anion carrier can catalyse rapid molybdenum movements across red cells membranes. This supports previous reports of an enterohepatic circulation of molybdenum, with red blood cells acting as molybdenum carrier between the intestine and the liver.

1. The initial rate of Cd2+ uptake in human red cells was measured by atomic absorption spectrophotometry. 2. About 96% of Cd2+ uptake was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) with IC50 (concentration giving 50% of maximal inhibition) of 0.3 microM and by furosemide with IC50 of 500 microM and was resistant to ouabain and amiloride. This indicates the implication of the [Cl(-)-HCO3-] anion exchanger in Cd2+ uptake. 3. DIDS-sensitive Cd2+ uptake required the presence of external HCO3-. HCO3- ions had a biphasic effect on Cd2+ uptake. Low bicarbonate concentrations were stimulatory, suggesting formation of translocating bicarbonate-cadmium complexes. Higher bicarbonate concentrations were inhibitory, suggesting further bicarbonate complexation with formation of non-translocating species. Depending on the presence or absence of external Cl-, a maximal Cd2+ uptake of 1.7 or 0.37 mmol (l cells)-1 h-1 was observed at bicarbonate concentrations of 15.6 or 11 mM respectively. 4. In the presence of bicarbonate, external Cl- ions strongly stimulated Cd2+ uptake, with linear increase between 70 and 125 mM. This suggests that one translocating species may have chloride as ligand. 5. DIDS-sensitive Cd2+ uptake was modestly inhibited by physiological concentrations of external phosphate and was resistant to external K+, Mg2+ and Ca2+. 6. In conclusion, the anion exchanger is the major transport mechanism for red cell cadmium uptake. Translocating species appear to be monovalent anion complexes of cadmium with HCO3- such as [Cd(OH)(HCO3)2]- and [Cd(OH)(HCO3)Cl]-.

The initial rate of Cu2+ uptake in human red blood cells was measured by atomic absorption. About 80% of Cu2+ uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) concentrations greater than 5-10 microM. DIDS-sensitive Cu2+ uptake required the presence of external HCO3- or external Cl-. Cl- strongly stimulated Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (KCl) of 72 +/- 9.4 (SD) mM (n = 6 experiments). HCO3- stimulated DIDS-sensitive Cu2+ uptake following a Michaelis-like function, with apparent dissociation constant (Kbic) of 10 +/- 1.9 (SD) mM (n = 4 experiments). Maximal rates (of Cl(-)- or HCO3(-)-stimulated Cu2+ uptake) were nonadditive. DIDS-sensitive Cu2+ uptake was not modified by physiological concentrations of phosphate or sulfate. Conversely, it was strongly inhibited by physiological concentrations of L-histidine and cysteine (at a Cu2+ concentration of 100 microM, these physiological ligands exhibited KHis and KCys of 50 and 80 microM, respectively). By using a copper-selective electrode, we found that at pH 7-7.4 copper is associated with OH-, particularly in the form of Cu(OH)2 complexes. In conclusion, the anion exchanger is the major transport mechanism for red blood cell Cu2+ uptake. The translocating species can be the monovalent anion complexes of copper with OH-, Cl-, and/or HCO3-.

The association constant and the maximal binding capacity for calcium, magnesium and strontium to human serum proteins were determined using a wide concentration range by a dialysis technique. The association curves are similar for these elements. Each curve may be subdivided in three parts: the first would correspond to the subfraction tightly bound to protein, the second one shows a linear design implying the association of the element to different binding points which have the same association constant, and the third part shows the saturation of proteins, which is achieved by alkaline-earth concentration about 9 times higher than the physiological ones.