Golgi complex disassembly caused by light-activated calphostin C involves MAPK and PKA.

We examined the participation of MAPK and PKA in the Golgi complex disassembly caused by light-activated Calphostin C in HT-29 cells. When these cells were incubated with Calphostin C, fragmentation and dispersal of the Golgi complex was observed as assessed by immunofluorescence microscopy. Electron microscopy analysis showed that clusters of vesicles and large tubule-vesicular membrane structures, resembling the Golgi remnants present in mitotic cells, substituted the Golgi stacks. In addition, Calphostin C treatment caused inhibition of the endocytic route. We confirmed that the Golgi disassembly was not due to PKC inhibition, and suggested, based on the use of specific inhibitors, that other kinases are involved. It was shown that pretreatment with PD98059 and H-89, both inhibitors of MAPK and PKA, respectively, prior to incubation with Calphostin C, caused blockade of the Golgi disassembly, as well as the inhibition of the endocytic pathway caused by this drug. This finding supports the existence of a novel mechanism by which MAPK and PKA may regulate the Golgi breakdown caused by Calphostin C in HT-29 cells.

Bioavailability of albendazole sulphoxide after netobimin administration in sheep: effects of fenbendazole coadministration.

After oral co-administration of two dosages of netobimin (7.5 and 20 mg kg-1 with fenbendazole (1.1 mg kg-1) to Merino sheep, the AUC0-infinity of albendazole sulphoxide at the lower dosage of netobimin, was significantly increased (75.5 per cent) from control value (34.43 +/- 7.91 versus 60.33 +/- 11.93 microg h ml-1). The pharmacokinetic parameters MRT and T1/2 were also increased: 18.96 +/- 2.54 vs 26.44 +/- 4.69 h and 10.31 +/- 1.72 vs 22.28 +/- 6.75 h respectively. No data corresponding to the higher dosage of netobimin (20 mg kg-1) were statistically different from control values. It is concluded that fenbendazole increases the bioavailability of albendazole sulphoxide in sheep at the 7.5 mg kg-1 dosage, and this may produce a potentiated anthelmintic action.

Physiological response to experimentally induced anemia in rats: a comparative study.

To induce anemia experimentally in rats to provide a hematocrit of 30 to 35% and provide data about the physiologic response to anemia during induction and after it is established, two methods were selected: the application of repetitive doses of phenylhydrazine and bleeding. Blood sample collection was carried out at various times, and hematologic profiles and osmotic resistance were evaluated. The morphologic features of cells and distinct organs also were examined. Results indicated similar decrease of hematocrit and hemoglobin concentration for the two experimental groups, although the reticulocyte response was higher in rats treated with phenylhydrazine, where the presence of young erythrocyte populations was linked to increases in osmotic resistance and glucose utilization. 2,3-Diphosphoglycerate was induced only during the recovery phase of the study and corresponded to a gradual response to hypoxia. Histologically, marked erythroid hyperplasia was found in the bone marrow, and extramedullary hematopoiesis was seen in the spleen and liver.

Influence of surfactants on oral bioavailability of albendazole based on the formation of the sulphoxide metabolites in rats.

The influence of two surfactants, sodium taurocholate (STC) and polysorbate 80 (P80), on the bioavailability of albendazole (ABZ), orally administered to rats, has been studied. To assess the effect of the surfactants, they were administered at critical micellar and supramicellar concentrations, 5 and 10 mM for STC and 0.0022 and 0.22% for P80, along with a subclinical dose of 5 mg kg-1 of ABZ. Doses of 5 and 10.6 mg kg-1 ABZ were also administered as controls. The results show an increase in the sulphoxide AUC values: 55% in the STC 5 mM and 88% in the STC 10 mM and P80 0.0022% treatments when compared to the control ABZ 5 mg kg-1 dose. MRT values also show longer-lasting plasma albendazole sulphoxide concentration following these three treatments, particularly for the P80 0.0022% treatment. While there was no change in the apparent rate of formation of ABZSO, the amount of ABZSO formed was increased significantly by the addition of surfactants (STC 10 mM and P80 0.0022%).

Presystemic metabolism of albendazole: experimental evidence of an efflux process of albendazole sulfoxide to intestinal lumen.

Albendazole (ABZ) presystemic clearance was studied in rat by perfusion of a 25 microM ABZ solution in isolated intestinal loops. Significant secretion of the active metabolite, ABZSO, into the lumen was observed. The metabolite was also present in mesenteric blood. After 30 min of intestinal perfusion, 64% of the ABZ dose had disappeared from lumen. The total amount of ABZSO measured was 0.341 +/- 0.04 nmol/cm with 0.176 +/- 0.03 nmol/cm in mesenteric blood. The metabolite secretion to intestinal lumen was 0.165 +/- 0.05 nmol/cm. Intestinal sulfoxidation was induced by repeated administration of ABZ and ABZ coadministered with surfactants, especially polysorbate 80. The enantioselectivity of the in vitro intestinal sulfoxidation of ABZ showed that the relative contribution of P-450 and flavin-containing monooxygenase was quite similar, but after the induction by ABZ coadministered with polysorbate 80, the cytochrome P-450 system contribution was significantly increased. The appearance of ABZSO in mesenteric blood clearance was also increased under these conditions.

Assembly and functional analysis of tight junction in a colon adenocarcinoma cell line: effect of glucose depletion.

We describe morphologic and biochemical changes in the colonic epithelial HCT-116 cell line following depletion of glucose from the culture medium. Cultured cells under permissive differentiation conditions (inosine-supplemented glucose-free medium) exhibited, after confluence, an enterocytic differentiation, in contrast to cells grown under standard culture conditions, where they remain in an undifferentiated state. The differentiated phenotype was characterized by the presence of a monolayer of polarized cells displaying an apical tight junction, and by the presence of alkaline phosphatase, a well known brush border marker. We demonstrated that the formed tight junctions were functional using the following criteria: a) labeling of the junctions with antibodies recognizing the tight juntion proteins occludin and ZO-1, as observed by immunofluorescence and immunoblotting analysis; b) characteristic organization of the tight junction strands, as observed in freeze-fracture replicas; c) increase ofthe transepithelial resistance across the monolayer; d) not permeation of the ruthenium red stain across the tight junction, and e) presence of the hyperphosphorylated form of occludin.