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Pharmacological characteristics of [3H]taurine release evoked by nitric oxide (NO) were investigated using mouse cerebral cortical neurons in primary culture. NO generators such as S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) dose-dependently increased [3H]taurine release from neurons. Such stimulatory effects of NO generators were completely abolished by hemoglobin, a NO radical scavenger, indicating that these [3H]taurine releases might be due to NO liberated from SNAP and SNP. Sodium withdrawal from incubation buffer significantly inhibited the SNAP- and SNP-induced [3H]taurine releases, whereas the removal of calcium showed no alterations in the [3H]taurine release evoked by NO generators. Beta-Alanine and guanidinoethane sulfonate, inhibitors of carrier-mediated taurine transport system, inhibited the SNAP- and SNP-evoked releases of [3H]taurine in a dose-dependent manner. These results indicate that the NO-evoked [3H]taurine release from cerebral cortical neurons is mediated by the reverse process of sodium-dependent carrier-mediated taurine transport system.

Effect of long-term exposure to ethanol (EtOH) on the phosphatidylinositol 4,5-biphosphate (PIP2)-specific and cytosolic phospholipase C (PLC) activities in neuroblastoma x glioma hybrid (NG 108-15) cells and the brains from EtOH-inhaled mice were investigated. Long-term (2 days) exposure of NG 108-15 cells to EtOH induced significant decrease in PIP2-specific PLC activity dependent on concentration and duration of exposure, although the presence of EtOH in the enzyme assay system induced no alteration in PIP2-specific PLC activity. On the other hand, cytosolic PLC activity in NG 108-15 cells significantly increased by both the long-term exposure of the cells to EtOH and the addition of EtOH into the assay system. These changes in activities of both types of PLC in NG 108-15 cells observed after EtOH exposure recovered rapidly by the removal of EtOH. Moreover, the changes in activities of PIP2-specific and cytosolic PLC in the brain of EtOH-inhaled mice were similar to those found in NG 108-15 cells. These results indicate that EtOH inhibits the activity of PIP2-specific PLC and activates cytosolic PLC in the brain. These changes in cerebral PLC activities are suggested to involve in central action of EtOH and establishment of alcohol dependence.

Capillary electrophoresis (CE) coupled with liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay was used to detect sensitivity both diarrhetic shellfish toxins and hepatotoxic microcystins in marine and freshwater samples. This procedure provided a quantitative bioscreen for the rapid optical resolution of either of these toxin families in complex mixtures such as cultured marine phytoplankton, contaminated shellfish and cyanobacteria (natural assemblages). Following detection, identified toxins were purified by an enzyme bioassay-guided two-step LC protocol. Using the latter approach, at least four microcystins were rapidly isolated from a cyanobacteria bloom (largely Microcystis aeruginosa) collected from a Canadian drinking-water lake, including a novel microcystin termed microcystin-XR, where X is a previously unidentified hydrophobic amino acid of peptide residue molecular mass 193 Da. The unified CE/LC-linked PPase bioscreen described provides a powerful capability to dissect multiple toxin profiles in marine or freshwater samples contaminated with either okadaic acid or microcystin classes of toxin.

Toxins produced by marine phytoplankton represent a severe global health hazard to humans that eat seafood and are also responsible for massive natural fish kills in specialized bloom situations. Tumour-promoting hepatotoxins from the freshwater microcystin/nodularin class were identified in Northeastern Pacific Ocean, Eastern Canadian and European mussels for the first time. These hepatotoxins were detected at biologically active levels up to three-fold higher than accepted quarantine levels for the diarrhetic shellfish toxin okadaic acid (OA), based on their activity (in microcystin-LR equivalent units) in a liquid chromatography (LC)-linked protein phosphatase bioassay. The presence of microcystins/nodularins in oceanic shellfish identifies a potentially novel class of intoxication which is also prevalent in other forms of marine aquatic life, namely sponges and fish. The widespread presence of prokaryotic microcystins and nodularins in the marine environment may be indicative of the importance of signal transduction pathways involving potent inhibition of protein phosphatases in early marine eukaryotes.

Evidence is presented that links microcystins to a severe liver disease that occurs in Atlantic salmon that are netpen-reared in coastal British Columbia. Liquid chromatography-linked protein phosphatase bioassay analysis of extracts of liver tissue taken from Atlantic salmon afflicted with netpen liver disease showed the presence of an inhibitor of protein phosphatase that was chromatographically indistinguishable from microcystin-LR. Analysis of liver tissue from healthy control fish showed a complete absence of microcystin-LR. Intraperitoneal injection of microcystin-LR into healthy Atlantic salmon re-created the pathologic changes of netpen liver disease, including diffuse necrosis and hepatic megalocytosis.

Liquid chromatography (LC)-linked protein phosphatase 1/2A (PP-1/PP2A) bioassay was used to quantitatively identify diarrhetic shellfish toxins in marine phytoplankton (cultured and natural assemblages) and commercially available mussels. Using this approach, multiple protein phosphatase inhibitor profiles of varying composition were found in diarrhetic mussels from Holland and Canada. Based on LC elution positions and relative activity versus PP-1 and PP-2A, at least six inhibitors distinct from known diarrhetic shellfish toxins were identified and termed mussel phosphatase inhibitor (MPI) 19,22,23,25,33 and 42. The levels of these inhibitors, in okadaic acid equivalent units, varied from 100 pg to 3350 ng per g shellfish tissue. The combined levels of PP-1/2A inhibitors in all instances superseded that of okadaic acid/dinophysistoxin-1 and may contribute to the diarrhetic shellfish toxin profile of the contaminated mussels. The efficacy of LC-protein phosphatase bioassay was established for cultured phytoplankton where picogram levels of okadaic acid could be detected from microgram extracts of Prorocentrum lima. Analyses of plankton net tows from estuarine mussel culture sites in Eastern Canada revealed a heterogeneous population of protein phosphatase inhibitors, with dinophysistoxin-1 being most prevalent. This toxin was predominant for at least 2 months in mussel populations in the immediate vicinity of plankton sampling sites. The results are consistent with a hypothetical model in which marine bacteria, cyanobacteria and dinoflagellates combine to produce a variety of protein phosphatase inhibitors effective against signal transduction pathways in higher eukaryotes.