Localization of extracellular superoxide dismutase in rat lung: neutrophils and macrophages as carriers of the enzyme.

Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to localize extracellular superoxide dismutase (EC-SOD) and its mRNA in rat lung before and after a lipopolysaccharide (LPS)- and hyperoxia-induced inflammation. In control rats, EC-SOD mRNA was synthesized in macrophages and in cells of the arterial vessel walls and the alveolar septa. The EC-SOD protein was mainly localized in plasma and on the apical side of the epithelial cells located near bronchus-associated lymphoid tissue (BALT). ISH did not reveal major changes in the distribution of EC-SOD mRNA upon induction of inflammation. In contrast, IHC demonstrated a progressive staining of the epithelium of the larger bronchi for the protein. Neutrophils and macrophages invading the lung showed an intensive staining for the EC-SOD protein concomitantly with a decrease of the enzyme in the plasma. Twenty-four hours after LPS stimulation only a spotty positivity remained on neutrophils in and between the alveolar spaces. In the bronchoalveolar lavage fluid (BALF), only macrophages showed a strong positivity for EC-SOD mRNA while the protein was detected in macrophages and neutrophils. Exposure to hyperoxia did not affect the distribution of EC-SOD mRNA and protein. The presented data demonstrated that in lung tissue the EC-SOD enzyme may have a protective function for activated macrophages, neutrophils, and lympoid tissue-associated epithelial cells.

Cyclic AMP-mediated induction of the glial fibrillary acidic protein is independent of protein kinase A activation in rat C6 glioma.

N6-O'2-dibutyryl cAMP (dbcAMP), N6-monobutyryl cAMP (N6-mbcAMP), 8-Chloro cAMP (ClcAMP), and O'2-monobutyryl cAMP (O'2-mbcAMP) were used to study glial fibrillary acidic protein (GFAP) induction in rat C6 glioma. With the exception of O'2-mbcAMP, these cAMP analogs induced GFAP after stimulation of cells with a concentration of 0.5-1 mM. Only dbcAMP and N6-mbcAMP increased the intracellular concentration of cAMP. Protein kinase A (PKA) activation is often proposed to be involved in GFAP expression in astrocytes. Ion-exchange chromatography indicated that protein kinase activity is associated with PKA type II in C6. dbcAMP, N6-mbcAMP, and ClcAMP upregulated the amount of cAMP-binding proteins approximately twofold. RI was upregulated in the cytosol and particulate fraction, whereas RII was not affected after stimulation with dbcAMP. Concomitant, the PKA activity decreased approximately 60% and 40% in the cytosol and particulate fraction, respectively. CREB is constitutively expressed in C6 and is downregulated after stimulation with dbcAMP. The membrane-permeable PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) did not suppress the induction of GFAP-mRNA and its translation into GFAP. On the contrary, depending on the time difference between H89 and dbcAMP addition to C6, GFAP synthesis could even be potentiated more than twofold. Experiments in the presence of cycloheximide showed that protein synthesis is necessary for GFAP transcription. Although all components of the PKA signal transduction pathway are present in C6, GFAP synthesis is not dependent on PKA activation but required the synthesis of an unidentified factor.

Cyclic AMP-induced differentiation increases the synthesis of extracellular superoxide dismutase in rat C6 glioma.

The effect of membrane permeable cAMP analogues on the expression of extracellular superoxide dismutase (EC-SOD) was studied in rat C6 glioma. EC-SOD is constitutively expressed but stimulation with cAMP analogues still increased the EC-SOD transcription and the secreted SOD activity. The potency to enhance EC-SOD expression is correlated with the ability of the cAMP analogue to induce cAMP-dependent differentiation in C6. The increase in EC-SOD mRNA and in secreted activity depended on the concentration of the cAMP analogues and on the cultivation time. Twenty-four hours after addition of 0.5 mM N6, O'2-dibutyryl cAMP (dbcAMP) or N6-monobutyryl cAMP (N6-mbcAMP) EC-SOD mRNA expression increased approximately twofold, while stimulation for 68 h with 0.5 mM N6-mbcAMP or 1 mM 8-Chloro cAMP (ClcAMP) and 1 mM dbcAMP enhanced the mean secreted activity/cell three- and fivefold, respectively. O'2-monobutyryl cAMP (O'2-mbcAMP) did not affect EC-SOD synthesis. The enhancement in EC-SOD activity did not require activation of protein kinase A. ATP, TGF-beta, IFN-gamma, and LPS did not affect EC-SOD synthesis. The presented data point to a cAMP-dependent pathway for the enhanced expression of EC-SOD by glial cells in brain.

Monoclonal antibodies against metallothioneins from the human liver.

Six hybridomas secreting monoclonal antibodies directed against human fetal liver metallothioneins (MTs) have been generated and characterized. One antibody, K5A6, was specific for MT-1, the others recognized all isoMTs present in the human fetal liver. Each of these antibodies showed a unique cross-reactivity pattern when tested with MTs from the livers of different mammals. A double-antibody sandwich enzyme-linked-immunosorbent-assay (ELISA) was developed with the antibodies L2E3 and L5G2. This assay allows the detection of 60 pg human fetal liver MT and exhibits a metal dependent response for Zn-, Cd- and Hg-MT.