Rui Pedrosa

Oxidative and non-oxidative mechanisms of neuronal cell death and apoptosis by L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine

The present study was designed to evaluate the nature of intervening agents in L‐DOPA‐ and dopamine‐induced neurotoxicity in Neuro‐2A cells. In the absence of cells and in conditions of light protection, at 37°C, L‐DOPA or dopamine (1 mM) in culture medium degraded spontaneously in a time‐dependent manner, this being prevented by ascorbic acid (200 μM) and other antioxidants, namely glutathione (1 mM), N‐acetyl‐L‐cysteine (1 mM), sodium metabisulphite (200 μM), but not N‐ter‐butyl‐α‐phenylnitrone (1 mM) and deferoxamine (100 μM). The viability of Neuro‐2A cells declined following treatment with L‐DOPA or dopamine in a concentration‐ and time‐dependent manner. The decrease in cell viability by L‐DOPA (10±4% of control) or dopamine (15±4% of control) was markedly attenuated by antioxidants (ascorbic acid, glutathione, N‐acetyl‐L‐cysteine and sodium metabisulphite). Autoxidation of L‐DOPA or dopamine was accompanied by the formation of H2O2 in a time‐dependent manner, this being completely prevented by ascorbic acid at 24 h or markedly reduced at 48 h. Protective effects of 100 U ml−1 catalase (40±1% of control) against L‐DOPA‐induced cell death were lower than those conferred by 200 μM ascorbic acid (70±3% of control). Catalase‐induced protection (59±5% of control) against dopamine‐induced cell death was similar to that conferred by 200 μM ascorbic acid (57±4% of control). L‐DOPA‐induced neuronal cell death was also accompanied by increases in caspase‐3 activity, this being insensitive to ascorbic acid. Dopamine‐induced increase in caspase‐3 activity occurred only when autoxidation of the amine was prevented by ascorbic acid. It is suggested that in addition to generation of H2O2 and quinone formation, L‐DOPA‐ and dopamine‐induced cell death may result from induction of apoptosis, as evidenced by increases in caspase‐3 activity. Dopamine per se induces apoptosis by a mechanism independent of oxidative stress, as evidenced by the fact that increases in caspase‐3 activity occurred only when autoxidation of the amine was prevented.

Dopamine D3 receptor‐mediated inhibition of Na+/H+ exchanger activity in normotensive and spontaneously hypertensive rat proximal tubular epithelial cells

This study evaluated the response of the Na+/H+ exchanger (NHE) to dopamine D1‐ and D2‐like receptor stimulation in immortalized renal proximal tubular epithelial cells and freshly isolated renal proximal tubules from the spontaneously hypertensive rat (SHR) and their normotensive controls (Wistar Kyoto rats; WKY). Stimulation of D1‐like receptors with SKF 38393 attenuated NHE activity in WKY cells (IC50=151 nM), but not in SHR cells. Stimulation of D2‐like receptors with quinerolane (IC50=120 nM) attenuated NHE activity in SHR cells, but not in WKY cells. Forskolin was equipotent in SHR and WKY cells in inhibiting NHE activity. The effect of SKF 38393 was abolished by overnight treatment of WKY cells with cholera toxin (CTX, 500 ng ml−1), but not with pertussis toxin (PTX, 100 ng ml−1). The effect of quinerolane (1 μM) was abolished by overnight treatment of SHR cells with PTX, but not with CTX. The D3 receptor agonist 7‐OH‐DPAT (IC50=0.8 μM) attenuated NHE activity in SHR cells only. This effect was abolished by S‐sulpiride and by overnight treatment with PTX. The D4 receptor agonist RBI 257 did not affect NHE activity. The 7‐OH‐DPAT inhibited NHE activity in freshly isolated renal proximal tubules from 4‐ and 12‐week‐old SHR and 12‐week‐old WKY, but not in freshly isolated renal proximal tubules from 4‐week‐old WKY. It is concluded that D3 receptors coupled to a Gi/o protein play a role in the handling of tubular Na+, namely through inhibition of the NHE activity, this being of particular relevance in the SHR, which fail to respond to D1‐like dopamine receptor stimulation.

Distinct Signalling Cascades Downstream to Gsα Coupled Dopamine D1-like NHE3 Inhibition in Rat and Opossum Renal Epithelial Cells

Dopamine D₁-like receptors are linked via G proteins to multiple cellular signaling pathways, namely adenylyl cyclase (AC) and phospholipase C (PLC). We have previously shown that the D₁-mediated inhibition of Na⁺-K⁺-ATPase activity in OK cells involves the sequential activation of the AC-protein kinase A (AC-PKA) and the PLC-protein kinase C (PLC-PKC) pathways. The present study evaluated signaling cascades involved in dopamine-mediated inhibition of Na⁺/H⁺ exchanger isoform 3 (NHE3) in rat and opossum renal cells. Na⁺/H⁺ exchanger activity was assayed as the initial rate of intracellular pH (pH_i) recovery after an acid load. V_max values (in pH units/s) for Na⁺-dependent pH_i recovery in rat cells (0.0097±0.0007) were greater (P<0.05) those in opossum cells (0.0063±0.0007), with similar K_m values (in mM) for Na⁺ (rat, 35±9; opossum, 24±9). The IC₅₀ values for EIPA and amiloride induced decrease in NHE activity in rat and opossum kidney cells are in agreement with the observation that rat renal proximal tubules and opossum kidney cells express mainly the NHE3 isoform. The D₁-like receptor agonist SKF 38393 inhibited NHE3 activity in a concentration-dependent manner in both rat and opossum cells. The D₁-mediated inhibition of NHE3 was prevented either by the D₁-like receptor antagonist SKF 83566 (1 µM), overnight treatment with cholera toxin (500 ng/ml) and the PKA antagonist H-89 (10 µM) in rat and opossum kidney cells. The effect of SKF 38393 was abolished by the PKC antagonist chelerythrine (1 µM), or the PLC inhibitor U-73,122 (3 µM) in opossum cells, but not in rat cells. In addition, dibutyril cAMP (dB-cAMP; 500 µM) was found to increase PLC activity in OK cells but not in rat cells. The effect of D₁-like dopamine agonist was accompanied by increases in cyclic AMP production in rat and opossum cells. The inhibitory effect of SKF 38393 (1 µM) on NHE3 activity was abolished in rat and opossum cells pre-treated with the anti-G_Sα antibody, but not in cells treated with the anti-G_q/11 α antibody. It is concluded that D₁ agonists decrease NHE3 activity by classical stimulation of AC and PKA via G_Sα proteins in rat kidney cells. By contrast, the D₁-mediated inhibition of NHE3 in renal opossum cells involves a peculiar mechanism with AC-PKA and PLC-PKC pathways.

H2O2 Stimulation of the Cl−/HCO3− Exchanger by Angiotensin II and Angiotensin II Type 1 Receptor Distribution in Membrane Microdomains

The present study tested the hypothesis that angiotensin II (Ang II)–induced oxidative stress and Ang II–stimulated Cl−/HCO3− exchanger are increased and related to the differential membrane Ang II type 1 (AT1) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl−/HCO3− exchanger activity with EC50s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H2O2. The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H2O2 in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT1 receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT1 receptors in both microdomains, mainly the glycosylated form of the AT1 receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H2O2 generation that provokes an enhanced expression of glycosylated and nonglycosylated AT1 receptor forms in lipid rafts.

Cytoprotective effect of seaweeds with high antioxidant activity from the Peniche coast (Portugal)

Screening of antioxidant potential of dichloromethane and methanolic extracts of twenty-seven seaweeds from the Peniche coast was performed by: total phenolic contents (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Seaweeds revealing the highest antioxidant activity were screened for cytoprotective potential in MCF-7 cells, including the mitochondrial membrane potential analysis and the caspase-9 activity. High correlation was found between TPC of seaweed extracts and their scavenging capacity on DPPH and peroxyl radicals. The highest antioxidant activity was displayed by the methanolic fraction of brown seaweeds belonging to Fucales, however Ulva compressa presented the highest cytoprotective effect by blunting the apoptosis process. These results suggest that high antioxidant activity may not be directly related with high cytoprotective potential. Thus, seaweeds reveal to be a promising source of compounds with potential against oxidative stress.

Antitumor and Antimicrobial Potential of Bromoditerpenes Isolated from the Red Alga, Sphaerococcus coronopifolius

Cancer and infectious diseases continue to be a major public health problem, and new drugs are necessary. As marine organisms are well known to provide a wide range of original compounds, the aim of this study was to investigate the bioactivity of the main constituents of the cosmopolitan red alga, Sphaerococcus coronopifolius. The structure of several bromoditerpenes was determined by extensive spectroscopic analysis and comparison with literature data. Five molecules were isolated and characterized which include a new brominated diterpene belonging to the rare dactylomelane family and named sphaerodactylomelol (1), along with four already known sphaerane bromoditerpenes (2–5). Antitumor activity was assessed by cytotoxicity and anti-proliferative assays on an in vitro model of human hepatocellular carcinoma (HepG-2 cells). Antimicrobial activity was evaluated against four pathogenic microorganisms: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans. Compound 4 exhibited the highest antimicrobial activity against S. aureus (IC50 6.35 µM) and compound 5 the highest anti-proliferative activity on HepG-2 cells (IC50 42.9 µM). The new diterpene, sphaerodactylomelol (1), induced inhibition of cell proliferation (IC50 280 µM) and cytotoxicity (IC50 720 µM) on HepG-2 cells and showed antimicrobial activity against S. aureus (IC50 96.3 µM).

Antioxidant and Antimicrobial Potential of the Bifurcaria bifurcata Epiphytic Bacteria

Surface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga, Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 +/- 3.43 nmol p-nitrophenol.mg protein-1.min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). beta-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Activity and Regulation of Na+-HCO3- Cotransporter in Immortalized Spontaneously Hypertensive Rat and Wistar-Kyoto Rat Proximal Tubular Epithelial Cells

The present study evaluates the presence and functional proprieties of the Na+-HCO3− cotransporter (NBC) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive (SHR) and normotensive Wistar–Kyoto (WKY) rats. The expected size and nucleotide sequence of a 1031-bp fragment corresponding to type 1 NBC (NBC1) was identified in both cell lines. The expression of the NBC1 transcript was lower (P<0.05) in SHR than in WKY cells. After intracellular acidification and in the presence of amiloride (1 mmol/L), the addition of sodium (115 mmol/L) in the absence of chloride resulted in rapid intracellular pH recovery that was higher in WKY than in SHR cells. This was an Na+- and HCO3−-dependent process in both cell lines. 4,4′-Diisothiocyanatodihydrostilbene-2,2′-disulphonic acid inhibited NBC activity in both WKY and SHR cells; the inhibitory effect was, however, more pronounced in WKY than in SHR cells. Forskolin (10 &mgr;mol/L) and dibutyryl cAMP (0.5 mmol/L) did not alter NBC activity. Acidosis induced by a 24-hour treatment with NH4+ (20 mmol/L) increased NBC activity to a greater extent in SHR than in WKY cells, without changes in intracellular pH and cell viability. Treatment with acetazolamide (300 &mgr;mol/L) for 24 hours did not change NBC activity in both cell lines. In contrast to NBC, Na+-K+ ATPase activity and expression were higher in SHR than in WKY cells. It is concluded that SHR cells are endowed with lower NBC activity than WKY cells, but the former is more resistant to 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulphonic acid and responds better to acidosis.

Effect of stocking density and different diets on growth of Percula Clownfish, Amphiprion percula (Lacepede, 1802)

The aim of this study was to evaluate the influence of stocking density (0.5, 1, 2 and 3 fishL−1) and commercial marine fish diets (diet A, B, C and D) over four months on specific growth rate, condition factor, percentage without anomalous pigmentation (partial or total lack of white bands -miss-band) and survival of juvenile Amphiprion percula.Results showed that at 0.5 fishL−1 densities induced the best survival (100%) and also the maximum percentage of fish without miss-band (58.33 +/−4.417%). The maximum SGR was obtained for the 0.5 fishL−1 (0.459 ± 0.023% cm/day). However, the best condition factor (2.53 +/− 0.27) was achieved for 2 fishL−1 densities. There were no significant differences in survival (68.9 to 84.5%), fish without miss-bands (18.03 to 26.92%) and condition factor (1.92 to 2.1) among diets during the experimental period. On the other hand, diet C (with 41% crude protein) supported the best SGR (0.485 ± 0.001% cmday−1).The results suggested that stocking density are critical and more relevant when compared with the different diet tested, namely on specific growth rate, condition factor, the miss-band and survival of juvenile percula clownfish.This study has particular significance with regards to anemonefishes husbandry in terms of survival and production efficiency.