Sessão a modulation of mpp+ uptake by procyanidins in caco-2 cells: involvement of oxidation/reduction reactions



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1. MODULATION OF MPP+ UPTAKE BY PROCYANIDINS IN CACO-2 CELLS: INVOLVEMENT OF OXIDATION/REDUCTION REACTIONS
Ana Faria1,2, Rosário Monteiro1, Nuno Mateus2, Isabel Azevedo1, Conceição Calhau1

1Department of Biochemistry (U38-FCT), Faculty of Medicine of the University of Porto, 4200-319 Porto; 2Chemistry Investigation Centre, Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.
The absorption of certain nutrients and drugs is known to be influenced by concomitant ingestion of other substances. This work aimed to study the modulation of the intestinal uptake of organic cations by diet procyanidins. Five procyanidin fractions with different structural complexity were obtained by fractionating grape seed extract and their effect on 1-methyl-4-phenylpyridinium (MPP+) uptake was evaluated in Caco-2 cells. Apical uptake of 3H-MPP+ was increased by a 60 min exposure to 600 µg/mL of procyanidin fractions, this being positively related with procyanidin complexity. 3H-MPP+ uptake increased with preincubation time, which could be due to procyanidin oxidation during preincubation. Use of oxidizing agents showed that transporter’s redox state could affect its activity. Moreover, Caco-2 cells were maintained in 25 mM glucose through at least 5 passages, aiming to alter their oxidative state. In these cells, 60 min preincubation with procyanidins, increased 3H-MPP+ uptake to values similar to those obtained in normal cells with oxidized procyanidins. Oxidized procyanidins increased 3H-MPP+ uptake even more. A relation between preincubation time and 3H-MPP+ uptake was also found. In conclusion, procyanidins can modulate 3H-MPP+ apical uptake in Caco-2 cells, most probably through oxidation/reduction phenomena. Interactions between these compounds and drugs may affect their bioavailability.

2. Extracellular glucose concentration INFLUENCEs hEMT expression in Caco-2 cells
Ana Faria1,2, Rosário Monteiro1, Isabel Azevedo1,Conceição Calhau1

1Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, 4200-319 Porto; 2Chemistry Investigation Centre, Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.
Chronic exposure to high glucose concentration has been suggested to alter regulation and functional activity of proteins. The aim of this study was to assess the effect of high glucose concentration on organic cation transporter 1 (hOCT1) and extraneuronal monoamine transporter (hEMT) in Caco-2 cells. 3H-MPP+ (1-methyl-4-phenylpyridinium) uptake by control (5.5 mM glucose in media) or high glucose (HGC; 25 mM glucose) Caco-2 cells was evaluated and OCT1 and hEMT expression was assessed by RT-PCR. MPP+ uptake was decreased in HGC relatively to control (0.739±0.09 vs 1.2±0.04 pmol/mg protein, respectively). Additionally, two compounds known to inhibit 3H-MPP+ uptake1, corticosterone and clonidine were used. Corticosterone (300 µM) inhibited uptake both in control and HGC, whereas clonidine (50 µM) only inhibited 3H-MPP+ uptake in control cells. These results suggest that extracellular glucose may alter transporters, possibly due to redox phenomena as previously described2. Kinetic measurements revealed a decrease Km and Vmax in HGC, representing an increase of transport affinity and a decrease in the number of transporter units. Using comparative RT-PCR a decrease in hEMT expression was found in HGC. These results indicate that extracelular glucose concentration, possibly through alterations in cell oxidative state, influences MPP+ uptake in Caco-2 cells.

(1) Martel F et al, 2001, Naunyn-Schmiedeberg’s Arch Pharmacol 363, 40–9.

(2) Faria A et al, 2006, FEBS Letters 580, 155–160.

3. Stimulation of alkaline phosphatase activity from human osteoblasts by polyphenols and polyphenol-rich-beverages
Negrão MR, Azevedo I, Martins MJ

Department of Biochemistry, Faculty of Medicine (U38/FCT), Porto University. 4200-319 Porto, Portugal.


Epidemiologic studies correlate regular consumption of some polyphenol-rich-beverages with a reduction in bone mass loss. Alkaline phosphatase (ALP) regulates bone mineralization as confirmed by hypophosphatasia and ALP “knockout” mice. We studied the effect of chronic exposure (48h) to polyphenols and lyophilized polyphenol-rich-beverages upon ecto-ALP activity and RNA expression, as well as cellular viability, in Saos-2 (osteoblastic cell line). Xanthohumol, quercetin and resveratrol increased enzymatic activity without modifying ALP RNA expression; catechin induced no changes in any of the parameters. Xanthohumol increased, resveratrol reduced and quercetin and catechin had no effect on cellular viability. Lyophilized apple and orange juices increased ecto-ALP activity, without altering its RNA expression. Lyophilized red wine reduced ALP activity while increasing RNA expression. Lyophilized polyphenol-rich-beverages either did not affect Saos-2 cellular viability or slightly decreased it (orange juice). Chronic exposure to xanthohumol, quercetin, resveratrol or lyophilized apple juice increased bone ALP activity, without a negative effect on Saos-2 viability. This may contribute, at least in part, for their osteoporosis protective effect. More studies are needed, namely mineralization assays in the presence of these polyphenols and beverages, in order to correctly infer the physiologic meaning of our results. Support: FCT (POCTI, FEDER, Programa Comunitário de Apoio) and iBeSa.


4. Modulação da Fosfátase Alcalina Hepática e Cardíaca por Ácidos Gordos
Hugo Martins, Maria Conceição Gonçalves, Isabel Azevedo, Maria João Martins

Serviço de Bioquímica (U38/FCT), Faculdade de Medicina, Universidade do Porto, 4200-319 Porto, Portugal.


A fosfátase alcalina (FA) está envolvida na regulação de vários sistemas de transporte transmembranar, incluindo o transporte intestinal de lipídeos. Parece desempenhar também um papel importante na calcificação vascular.

Neste trabalho avaliámos o efeito in vitro de alguns ácidos gordos (0,01 – 2,5 mM) na actividade da FA em homogeneizados de fígado e coração de Rato Wistar. A actividade enzimática foi determinada a pH=10,4 usando p-nitrofenilfosfato (substrato) e Tris (solução tampão).

Os ácidos gordos de cadeia curta (acético, propiónico e butírico) não modificaram a actividade da FA no fígado, tendo apenas o propiónico inibido a FA cardíaca. Também os ácidos cáprico e láurico (cadeia média) inibiram a FA cardíaca, tendo o último inibido também a FA hepática. Os ácidos esteárico, araquídico, palmitoleico e oleico inibiram a actividade enzimática em ambos os tecidos, tendo o palmítico inibido apenas a FA hepática. Os padrões de inibição foram diferentes: as inibições da FA hepática pelos ácidos gordos de cadeia longa saturada foram precedidas duma activação.

A modulação da actividade da FA por ácidos gordos sugere a possibilidade destes interferirem com transportes transmembranares e calcificação vascular, hipóteses que serão interessantes aprofundar no contexto do síndrome metabólico.




5. Vitamin B1 uptake by the BeWo human placental cell line: characterization and nutritional modulation

E Keating, C Lemos, I Azevedo, F Martel

Department of Biochemistry (U38-FCT), Faculty of Medicine of Porto, University of Porto, 4200-319 Porto, Portugal. (keating@med.up.pt)

Our aim was to characterize the placental uptake of vitamin B1 (thiamine), by determining the characteristics of 3H-thiamine uptake by the trophoblast cell line, BeWo, and by studying its modulation by acute or chronic exposure to dietary bioactive substances. We verified that uptake of 3H-thiamine by BeWo cells was: 1) temperature-dependent and energy-independent; 2) pH-dependent (stimulated by acidification of the incubation medium); 3) Na+-dependent and Cl--independent; 4) not affected by the thiamine structural analogs amprolium, oxythiamine and thiamine pyrophosphate; 5) inhibited by the unrelated organic cations guanidine, N-methylnicotinamide, tetraethylammonium, clonidine and cimetidine; 6) inhibited by the organic cation serotonin, and by two selective inhibitors of the serotonin plasmalemmal transporter (SERT), fluoxetine and desipramine.

Moreover, uptake of 3H-thiamine was stimulated by chronic exposure of the cells to caffeine and was inhibited by chronic exposure to xantho-humol and isoxantho-humol. These effects do not seem to be mediated by the modulation of the expression levels of either hThTr-1 or hSERT mRNAs.

In conclusion, 3H-thiamine uptake by BeWo cells seems to occur through a process distinct from ThTr-1 and ThTr-2; rather, it seems to involve SERT. Moreover, uptake of this vitamin seems to be differently modulated by distinct dietary bioactive compounds.

This work was supported by FCT and Programa Ciência, Tecnologia e Inovação do Quadro Comunitário de Apoio (POCTI/SAU-FCF/59382/2004) and IBESA.




6. Acute and chronic effects of ethanol and other dietary bioactive compounds on folate uptake by the BeWo human placental cell line
E Keating, C Lemos, P Gonçalves, I Azevedo, F Martel
Department of Biochemistry (U38-FCT), Faculty of Medicine of Porto, University of Porto, 4200-319 Porto, Portugal. (keating@med.up.pt)

Folate acts as a coenzyme in important cellular reactions being critically important for normal fetal development

The aim of this work was to study interactions between folate and some bioactive substances that may alter folate absorption at the placental level. To do this we tested the acute and chronic effects of some compounds present in alcoholic and non-alcoholic drinks on 3H-folate uptake by BeWo cells and also on the expression of RFC1 and FRalpha mRNA by BeWo cells. Our results demonstrate that the mechanism involved in 3H-folate uptake is differentially modulated by several dietary bioactive substances. Namely, 3H-folate apical uptake by BeWo cells is concentration-dependently reduced by acute exposure to epicatechin, isoxanthohumol or teophyllin, and is increased by chronic exposure to xanthohumol, quercetin or isoxanthohumol. This increase does not seem to result from changes in RFC1 or FRalpha gene expression. Moreover, we also demonstrate that 3H-folate uptake is reduced by chronic exposure of BeWo cells to ethanol and this reduction is accompanied by a reduction in FRalpha expression. Our results suggest that inhibition of placental FRalpha gene expression with the consequent insufficient folate supply to the placenta and the fetus may be involved in the toxicity of ethanol during pregnancy.

This work was supported by FCT and Programa Ciência, Tecnologia e Inovação do Quadro Comunitário de Apoio (POCTI/SAU-FCF/59382/2004) and IBESA.



7. Antioxidant properties of anthocyanins and anthocyanin-derived pigments: structure-activity relationship
Ana Faria, Joana Oliveira, Manuela Ribeiro, Victor de Freitas, Nuno Mateus

Chemistry Investigation Centre, Department of Chemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal.

anafaria@med.up.pt
Pigments from natural sources are safe and have attractive bright colours. There are challenges in food industry to replace synthetic dyes by natural pigments. In previous reports, extracts of anthocyanin-derived pigments (especially Portisins) revealed a higher antioxidant/antiradical capacity than their anthocyanins precursors. In this work, the structure-activity relationship of pure individual anthocyanin (cyanidin and cyanidin-3-glucoside) and anthocyanin-derived pigments (cyanidin-pyruvic acid adducts and vinylpyrano-cianidin-catechin) was assayed. The ability of cyanidin and its cyanidin-derived pigments to inhibit lipid peroxidation, induced by AAPH (2,2’-azobis-2-methyl-propanimidamide, dihydrochloride), in a liposome membrane system was examined. The antioxidant capacities of these compounds were evaluated by monitoring oxygen consumption and formation of conjugated dienes. In addition, antiradical properties and reducing power of these pigments were determined using DPPH and FRAP assays. All the cyanidin pigments provided protection of membranes against peroxyl radicals by increasing the induction time of oxidation. This effect increased with structural complexity of cyanidin pigments probably due to an extended conjugation of π electrons that could easily stabilize the radical scavenged throughout his structure. The results yielded from the DPPH and FRAP assays were in agreement with the ones obtained with the liposome membranes.
8. Efeito da oxiluciferina e do desidroluciferil-adenilato
na luciférase do pirilampo



Diogo Fernandesa, Daniela Maiab, Hugo Fragaa, Rui Fontesc e Joaquim C.G. Esteves da Silvaa

aCentro de Investigação em Química, Departamento de Química, Faculdade de Ciências da Universidade do Porto. bEscola Secundária da Boa Nova - Leça da Palmeira (Ciência Viva).

cLaboratório de Bioquímica (U38-FCT), Faculdade de Medicina da Universidade do Porto.
A luciférase do pirilampo é a enzima responsável pela catálise das reacções químicas que dão origem à produção de luz neste insecto. Contudo, para além da reacção bioluminescente, a luciférase é capaz de catalisar muitas outras reacções frequentemente classificadas como escuras.

Um exemplo destas reacções é a oxidação do intermediário luciferil-adenilato pelo oxigénio em desidroluciferil-adenilato (L-AMP) e a descoberta mais recente da nossa equipa de investigação foi a identificação e quantificação da formação de H2O2 no decurso desta reacção lateral [1]. O L-AMP produzido é um poderoso inibidor da reacção de produção de luz sendo o principal responsável pelo facto de a reacção bioluminescente ser de tipo “flash” [2]. A coenzima A (CoA), quando adicionado ao meio de ensaio, reage com o L-AMP anulando o seu efeito inibidor e estabilizando a velocidade de produção de luz [2].

No entanto, na rápida descida de velocidade de produção de luz, para além da síntese de L-AMP, estão envolvidos outros factores de natureza desconhecida [2].

Usando técnicas de luminometria e cromatográficas testámos a possibilidade de a oxiluciferina, o emissor de luz, poder participar na referida descida de velocidade. Os resultados obtidos mostraram que, quando a velocidade de produção de luz começa a descer, o consumo de substratos é ainda demasiado pequeno para explicar o fenómeno e que a adição de coenzima A ao meio de ensaio não o reverte de forma completa. Os estudos em que se usou oxiluciferina e L-AMP (por nós sintetizados e purificados) mostraram que, embora menos potente que o L-AMP, a oxiluciferina é um inibidor da reacção bioluminescente da luciférase do pirilampo. Também mostraram que, ao contrário do que acontece no caso do L-AMP, o CoA não reverte o efeito inibidor da oxiluciferina.

Assim, confirmando a hipótese testada, concluímos que a oxiluciferina formada no decurso da reacção bioluminescente é, em parte, responsável pela cinética tipo “flash” desta reacção.

1. Fraga, H., Fernandes, D., Novotny, J., Fontes, R., Esteves da Silva, J.C.G. (2006) ChemBioChem. 7, 929-35.

2. Fraga, H., Fernandes, D., Fontes, R., Esteves da Silva, J.C.G. (2005) Febs J. 272, 5206-16.

Agradecimentos

Diogo Fernandes, aluno do 1º ano do curso de Física da Faculdade de Ciências do Porto, agradece à Reitoria da Universidade do Porto e à Caixa Geral de Depósitos (Investigação Científica na Pré-Graduação) e ao Programa Ciência Viva o financiamento do trabalho apresentado neste poster assim como o do trabalho que consta no artigo científico publicado no âmbito deste projecto e referido em [1].



9. Somatostatin Regulates 3H-MPP+ Release from Primary Cultures of Bovine Adrenal Chromaffin Cells via Activation of Distinct Receptor Subtypes
Laura Ribeiro1, Fátima Martel1, Isabel Azevedo1

1Department of Biochemistry, Faculty of Medicine (U38-FCT), 4200-319 Porto, Portugal.
Somatostatin (SS), a neuropeptide widely distributed in the central and peripheral nervous systems, acts through five specific cell surface receptors (SSTR1-5) to elicit different biological functions.

Using reverse transcription followed by PCR amplification (RT-PCR), we have identified in bovine adrenal chromaffin cells the expression of the SS gene.

The purpose of this study was to investigate the effect of SS and SSTR2-5 selective agonists on adrenal catecholamine release, using model substrate 3H-1-methyl-4-phenylpyridinium (3H-MPP+) release from primary cultures of bovine adrenal chromaffin cells.

The effects of SS (0.01-1 M) upon ACH (100-500 M; 10 mM)-evoked 3H-MPP+ release differed between different cultures, allowing us to distinguish two types of cell cultures: type A- cell cultures, in which SS decreased ACH-induced 3H-MPP+ release, and type B- cell cultures, in which SS increased ACH-induced 3H-MPP+ release. The SSTR2, 4 and 5 selective agonists mimicked the inhibitory effects elicited by SS upon ACH-induced 3H-MPP+ release in type A cell cultures, whereas the SSTR2, 3 and 5 selective agonists mimicked the excitatory effect of SS upon ACH-induced 3H-MPP+ release on type B cell cultures.

On the other hand, while SS (0.01-1 M) and the SSTR3 and 5 agonists significantly augmented the release of 3H-MPP+ from type A cell cultures, neither SS nor its SSTR selective agonists did significantly affect this release from type B cell cultures.

Interestingly, type A and B cell cultures were not only heterogeneous in relation to the response to SS and SSTR agonists, but did also differ regarding AD/NA cellular ratio, 3H-MPP+ uptake capacity, basal 3H-MPP+ release, and 3H-MPP+ release response to ACH.



In summary, our data indicate that: (1) adrenal chromaffin cells respond differentially to SS and SSTR agonists as to basal and ACH-stimulated 3H-MPP+ release, and (2) endogenous SS might modulate adrenal catecholamine secretion through its specific receptors.

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