Vascular effects of the 21-aminosteroid tirilazad mesylate: protection against oxidant stress.

The vascular effects of tirilazad mesylate (U-74006F), a 21-aminosteroid antioxidant under development for the treatment of acute CNS trauma and ischemia, have been investigated using isolated rings of rabbit aorta. Endothelium-dependent relaxation was measured in rings contracted with 0.25 microM phenylephrine. Acetylcholine produced a dose-dependent relaxation that was slightly attenuated by the addition of U-74006F (0.1-10.0 microM). At a concentration of 10.0 microM, U-74006F shifted the EC50 for acetylcholine relaxation from 60 +/- 10 to 150 +/- 20 nM and reduced the maximum response by 20%. U-74006F (0.1-10.0 microM), did not reduce relaxation to the endothelium-independent vasodilator nitroglycerin nor did it affect the tone of either resting or phenylephrine contracted rings. ACh relaxation was abolished by a 40 min treatment with the superoxide generating system xanthine oxidase (XO; 0.1 U/ml) plus xanthine (0.4 mM). Relaxation to nitroglycerin was not impaired by XO, nor were the phenylephrine-induced contractions. U-74006F, at doses of 0.05-10 microM, protected against XO mediated damage to endothelium-dependent relaxation. These results demonstrate that tirilazad mesylate can protect endothelial function from damage by reactive oxygen species. Preservation of endothelial function might represent an important component of the activity of tirilazad mesylate in vivo.

Two novel antioxidants, U74006F and U78517F, inhibit oxidant-stimulated calcium influx.

Incubation of UC11MG human astrocytoma cells with 250 microM hydrogen peroxide (H2O2) plus 50 microM ferrous ammonium sulfate resulted in two temporally distinct increases in intracellular calcium. The first peak, which occurred within 1 min, was due to release from intracellular stores, whereas the second increase was the result of influx of extracellular calcium. Both U74006F and U78517F, two novel antioxidants, specifically inhibited the second, but not the first peak of calcium. The calcium increase was inhibited 70-80% with incubation with either 0.1 microM U78517F or 10 microM U74006F. Treatment with the drugs also protected against oxidant-induced loss in cell viability. In experiments using the membrane potential dye DiBAC4(3), hydrogen peroxide treatment of cells resulted in membrane depolarization. However, at concentrations up to 100 microM, neither of the drugs had any effect on the H2O2-induced depolarization. The drugs also did not affect membrane depolarization resulting from treatment with tetrapropyl ammonium bromide, a potassium channel blocker. These results indicate that U74006F and U78517F act to inhibit injury-induced calcium fluxes, which may contribute to their in vivo efficacy.

Tissue distribution and cellular localization of hsp56, an FK506-binding protein. Characterization using a highly specific polyclonal antibody.

Heat shock protein 56 (hsp56) has been shown to be involved in two cellular pathways, as an immunophilin for FK506 and as a component of steroid receptor complexes. To help define its role in these cellular pathways, we have developed UPJ56, a polyclonal antibody raised against hsp56 purified from Jurkat cells. In Western blot experiments, hsp56 was highly expressed in rat thymus, liver, and spleen, with low levels in lung and muscle. In immunofluorescence experiments using untreated LLC-PK1 cells, fibrillar staining was seen in the cytoplasm, suggesting a cytoskeletal localization of hsp56. The nuclei were brightly stained, except for the nucleoli. Confocal microscopy demonstrated that the staining was present in all planes of the nucleus. These results suggest that hsp56 is expressed in tissues enriched in steroid receptors and is highly expressed in tissues involved in T cell function. Furthermore, the localization of hsp56 with the cytoskeleton and throughout the nucleus is consistent with its association with steroid receptor complexes.

Research note: effects of age and immunization interval on the anamnestic response to T-cell-dependent and T-cell-independent antigens in chickens.

Measurement of a humoral anamnestic response is a common procedure used to evaluate immunological competence. The purposes of the present study were to determine the optimum time period between primary and secondary immunizations needed to obtain the maximum antibody titer and to determine whether this interval differs with the age of the chicken or with the type of inducing antigen. The anamnestic responses of birds of two ages to a T-cell-dependent antigen (SRBC) and to a T-cell-independent antigen (Brucella abortus, BA) were evaluated. Birds of 4 wk or 6 mo of age were injected i.v. with either BA or SRBC, and a secondary injection was given 2, 4, 6, or 8 wk later. Agglutination titers for total and mercaptoethanol (ME)-resistant antibodies were determined from serum samples collected at 3, 6, and 9 days postsecondary immunization. Generally, titers were highest at 6 days postsecondary immunization. For both antigens, the chick total antibody levels at Day 6 postsecondary immunization were higher at 4-, 6-, and 8-wk immunization intervals than at 2-wk intervals; adult titers were independent of immunization interval. There was an interaction between age of birds and type of antigen.