On the Recovery of [3H]Noradrenaline from Different Metabolic Compartments of Rat Brain with Respect to the Role of Catechol-O-Methyltransferase

Abstract: Rats were treated with reserpine, desmethylimipramine, or carrier, either alone or in combination with tropolone. Either 10 min (t₁) or 1 h (t₂) after intraventricular injection of [³H]noradrenaline, they were decapitated. The total ³H activity and the recovery of [³H]noradrenaline were determined in tissue extracts from various brain regions. Maximum total ³H activity was measured at t₁ in all tropolone-treated rats; the mean sum of these results served as an estimate of the initial tissue concentration of [³H]noradrenaline. At t₁, 40–50% of the sum of [³H]noradrenaline and its metabolites was recovered unchanged in normal rats; reserpine and DMI reduced the recovery to 18–27%. In all groups, the decline of [³H]noradrenaline was retarded after t₁. Inhibition of catechol-O-methyltransferase by tropolone caused consistently elevated [³H]noradrenaline levels, but did not affect the metabolic rate after t₁ when compared with similarly pretreated, but tropolone-free rats. Thus, if catechol-O-methyltransferase was inhibited during the injection of [³H]noradrenaline, a higher percentage of the amine had been taken up into spaces with a slow noradrenaline turnover. The maximum increase was seen when the neuronal uptake, was inhibited by desmethylimipramine. This supported the hypothesis that an additional extraneuronal space exists, in addition to the known intraneuronal and extraneuronal compartments, which has a slow noradrenaline turnover. The tropolone effect on the noradrenaline recovery possibly shows that there might be a saturable “methylating system,” similar to that described for the periphery, in which catechol-O-methyltransferase is linked to the extraneuronal uptake₂. By affecting the access of noradrenaline to non-neuronal cells it might influence the rate of noradrenaline elimination from the intercellular space.     

Synthesis of tritiated 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells

Summary The potent and specific inhibitor of anion permeability, 4,4-diisothicyanostilbene-2,2-disulfonic acid (DIDS) was synthesized in tritiated form ([³H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([³H]H₂DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [³H]DIDS was substantially faster than that of [³H]H₂DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [³H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×10⁶ molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.     

Plasma levels of norethisterone measured by radioimmunoassay and gas chromatography-mass fragmentography

Assays designed to quantitate the plasma levels of norethisterone (NET) are compared. Results from radioimmunoassay were compared with those from gas chromatography-mass fragmentography which were established in 5 subjects after a 1-mg oral dose of the steroid. In general, the levels of steroid measured by radioimmunoassay were usually higher than those measured by gas chromatography-mass fragmentography; there was however a correlation coefficient of .96 (P .001). For concentrations of the steroid above 500 pg/ml, the mean overestimate by radioimmunoassay was less than 30%.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

Studies on the metabolism of oestrone and oestradiol-17 beta in the kidneys of minipigs of different ages and sexes (author's transl)]

After incubation of [4-14C]oestrone (E1) with kidney cortex slices of minipigs, [4-14C]oestradiol-17 beta (E2) and small amounts of a polar metabolite were detected in the ether-soluble fraction. E1, E2 and polar metabolites were found in the protein-bound fraction. The water-soluble fraction contained E1-3-glucuronide (80% of total glucuronides), E2-3-glucuronide and trace amounts of the 3-monoglucuronide of oestriol (E3). When E2 was used as substrate, the main product formed was E1; it was detected in both the ether-soluble and protein-bound fractions. E1-3-Glucuronide was the main metabolite in the water-soluble fraction, which also contained some E2-3-Glucuronide. In male minipigs, the mast, the rate of conversion of E1 and E2 as well as the formation of glucuronides were significantly greater in fertile females than in infertile females. Whereas no sex difference was observed in the metabolism of oestrogens in kidneys of infertile minipigs, the rate of oxidoreduction and glucuronidation was more pronounced in fertile female animals than in the corresponding males. The present results suggest that, in the kidneys of minipigs, the ratio of E1 to E2 is shifted towards the former; furthermore, by a comparatively rapid metabolism of the oestrogenic hormone, the renal tissue contributes to the maintenance of hormonal equilibrium.