Anti-NPPB monoclonal antibody, clone 3H10

Effects of ATP on enzymatically isolated rat ventricular fibroblasts maintained in short-term (36-72 h) cell culture were examined. Immunocytochemical staining of these cells revealed that a fibroblast, as opposed to a myofibroblast, phenotype was predominant. ATP, ADP or uridine 5-triphosphate (UTP) all produced large increases in [Ca2+](i). Voltage-clamp studies (amphotericin-perforated patch) showed that ATP (1-100 m) activated an outwardly rectifying current, with a reversal potential very close to the Nernst potential for Cl-. In contrast, ADP was much less effective, and UTP produced no detectable current. The non-selective Cl- channel blockers niflumic acid, DIDS and NPPB (each at 100 m), blocked the responses to 100 m ATP. An agonist for P2Y purinoceptors, 2-MTATP, activated a very similar outwardly rectifying C1(-) current. The P2Y receptor antagonists, suramin and PPADS (100 m each), significantly inhibited the Cl- current produced by 100 m ATP. ATP was able to activate this Cl- current when [Ca2+](o) was removed, but not when [Ca2+](i) was buffered with BAPTA-AM. In the presence of the phospholipaseC inhibitor U73122, this Cl- current could not be activated. PCR analysis revealed strong signals for a number of P2Y purinoceptors and for the Ca2+-activated Cl- channel, TMEM16F (also denoted ANO6). In summary, these results demonstrate that activation of P2Y receptors by ATP causes a phospholipase C-dependent increase in [Ca2+](i), followed by activation of a Ca2+-dependent Cl- current in rat ventricular fibroblasts.

Currently, there are no fast in vitro broad spectrum screening bioassays for the detection of marine toxins. The aim of this study was to develop such an assay. In gene expression profiling experiments 17 marker genes were provisionally selected that were differentially regulated in human intestinal Caco-2 cells upon exposure to the lipophilic shellfish poisons azaspiracid-1 (AZA1) or dinophysis toxin-1 (DTX1). These 17 genes together with two control genes were the basis for the design of a tailored microarray platform for the detection of these marine toxins and potentially others. Five out of the 17 selected marker genes on this dedicated DNA microarray gave dear signals, whereby the resulting fingerprints could be used to detect these toxins. CEACAM1, DDIT4, and TUBB3 were up-regulated by both AZA1 and DTX1, TRIB3 was up-regulated by AZA1 only, and OSR2 by DTX1 only. Analysis by singleplex qRT-PCR revealed the up- and down-regulation of the selected RGS16 and NPPB marker genes by DTX1, that were not envisioned by the new developed dedicated array. The qRT-PCR targeting the DDIT4, RSG16 and NPPB genes thus already resulted in a specific pattern for AZA1 and DTX1 indicating that for this specific case qRT-PCR might a be more suitable approach than a dedicated array.