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). β-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.

Our objective was to characterize the modulation of the activity of Saccharomyces cerevisiae alkaline phosphatases (ALPs) by classic inhibitors of ALP activity, cholesterol and steroid hormones, in order to identify catalytic similarities between yeast and mammalian ALPs. S. cerevisiae expresses two ALPs, coded for by the PHO8 and PHO13 genes. The product of the PHO8 gene is repressible by Pi in the medium. ALP activity from yeast (grown in low or high phosphate medium) homogenates was determined with p-nitrophenylphosphate as substrate, pH 10.4 (lPiALP or hPiALP, respectively). Activation of hPiALP was observed with 5 mM L-amino acids (L-homoarginine _ 186%, L-leucine _ 155% and L-phenylalanine - 168%) and with 1 mM levamisole (122%; percentage values, in comparison to control, of recovered activity). EDTA (5 mM) and vanadate (1 mM) distinctly inhibited hPiALP (2 and 20%, respectively). L-homoarginine (5 mM) had a lower activating effect on lPiALP (166%) and was the strongest hPiALP activator. Corticosterone (5 mM) inhibited hPiALP to 90%, but no effect was observed in low phosphate medium. Cholesterol, ß-estradiol and progesterone also had different effects on lPiALP and hPiALP. A concentration-dependent activation of lPiALP minus hPiALP was evident with all three compounds, most especially with ß-estradiol and cholesterol. These results do not allow us to identify similarities of the behavior of S. cerevisiae ALPs and any of the mammalian ALPs but allow us to raise the hypothesis of differential regulation of S. cerevisiae ALPs by L-homoarginine, ß-estradiol and cholesterol and of using these compounds to discriminate between S. cerevisiae lPiALP and hPiALP.

Dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) are metabolically important in estuaries. Their availability is influenced by organic matter inputs (internal and external) as well as by internal microbial transformations within the system. In this study, spatial and seasonal dynamics of DOC and DON were evaluated in the Douro River estuary (Portugal). Monthly surveys were performed from October 2005 to December 2006 along a salinity gradient at three different depths. DOC and DON were calculated by subtracting the respective inorganic components (dissolved inorganic carbon, DIC, and dissolved inorganic nitrogen, DIN) from total dissolved carbon (TDC) and total dissolved nitrogen (TDN). In the Douro River estuary, DIC exhibits a linear and positive distribution as a function of salinity, indicating an input of DIC into the estuary from coastal waters. This pattern of distribution along the salinity gradient was also observed for total particulate matter, since a decreasing trend upstream was observed. In contrast, the results showed a general nonconservative behaviour of DOC along the estuarine transect during most months. This typical deviation from conservative mixing was mainly associated with sewage DOC sources into the lower and middle estuary. On average, DON concentrations represented 52-88% of the TDN. Along the transect, relationships between DIN and DON with salinity were generally conservative, decreasing towards the mouth, highlighting the fluvial source of such nitrogen forms; however, occasional DON sewage loads were registered. Estimated global Douro River watershed exports of DOC and DON to coastal waters yielded values of 420 kg C m-2 yr-1 and 125 kg N m-2 yr-1, respectively, which are high when compared with previously predicted levels from global models of DOC and DON export.

El carbono orgánico disuelto (COD) y el nitrógeno orgánico disuelto (NOD) son compuestos metabólicamente importantes en los sistemas estuarinos. Su disponibilidad es influenciada por aportes de materia orgánica (internos y externos) y por transformaciones microbianas internas. En este trabajo se evaluaron las dinámicas espaciales y temporales del COD y NOD en el estuario del Río Duero. Los muestreos se realizaron mensualmente entre octubre de 2005 y diciembre de 2006 a lo largo del gradiente salino del estuario, a tres profundidades distintas. El COD y NOD fueron calculados mediante la substracción de los respectivos componentes inorgánicos del CDT (carbono disuelto total) y el NDT (nitrógeno disuelto total). El carbono inorgánico disuelto (CID) en el estuario del Duero mantiene una relación lineal positiva con la salinidad, indicando un aporte de CID al estuario por parte de las aguas costeras. Este patrón de distribución a lo largo del gradiente salino fue también observado para el material particulado total, ya que se observó una tendencia decreciente corriente arriba. En contraste, los resultados mostraron un comportamiento no conservativo del COD durante gran parte de los meses, en su mayoría asociado al aporte de COD de origen antropogénico en las porciones media y inferior del estuario. Las concentraciones de NOD representan 52-88% del NDT. A lo largo del transecto las relaciones entre nitrógeno inorgánico disuelto (NID) y NOD con la salinidad mostraron un comportamiento conservativo, lo que indica una fuerte influencia del Río Duero en las concentraciones de NID y NOD del estuario. No obstante, se han registrado aportes ocasionales de NOD de origen antropogénico. Las exportaciones globales de COD y NOD de la cuenca hidrográfica del Duero a las aguas costeras fueron estimadas en 420 kg C m-2 año-1 y 125 kg N m-2 año-1, respectivamente, las cuales son elevadas al compararse con los resultados de modelos de exportacion global de COD y NOD para la misma cuenca.