Production of thrombin and antithrombin III by brain and astroglial cell cultures.

There is increasing evidence that some proteases and protease inhibitors are produced within the central nervous system. It has been proposed that the balance between these two classes of proteins may be an important modulator of brain cell growth and differentiation. Here we report that antithrombin III (ATIII) is produced in brain and primary astroglial cultures. In addition, we show that human astroglial cultures contain prothrombin mRNA, and secrete a thrombin-like protein that makes complexes with antithrombin III.

Experimental basis for increasing the therapeutic index of cis-diamminedicarboxylatocyclobutaneplatinum(II) in brain tumor therapy by a high-zinc diet.

Metallothionein (MT), a ubiquitous intracellular protein, confers resistance to the toxic effects of platinum compounds. Since a high-zinc diet has been shown to induce MT synthesis in extracerebral tissues but not in brain, we investigated whether it could provide an experimental basis for decreasing the hematotoxicity of carboplatin without impairing its activity against brain tumors. After 2 weeks on either a high-zinc diet or a control diet (zinc content, 180 vs 10 ppm), mice and rats received various doses of carboplatin or Hanks' balanced salt solution by i.p. injection. The hematotoxicity of carboplatin was evaluated with an assay of colony-forming units of granulocytes and mononuclear cells in mice. The high-zinc diet enabled a 50% increase in the carboplatin dose without increasing hematotoxicity. The antitumor activity was evaluated with an assay of the colony-forming efficiency of gliosarcoma cells from 9L brain tumors in rats. The high-zinc diet did not alter the efficacy of carboplatin against this brain tumor. Northern blot analysis confirmed that the high-zinc diet induced MT mRNA in the kidney but not in the brain of mice and rats; it also showed MT mRNA induction in bone marrow cells of mice but not in rat 9L brain tumors. These results suggest that increasing the dietary intake of zinc might increase the therapeutic index of carboplatin in the treatment of brain tumors.

Characterization of "plasma proteins" secreted by cultured rat macroglial cells.

The brain is isolated behind a blood-tissue barrier that restricts the access of circulating proteins to neural cells. There is evidence that some of these proteins are synthesized within the central nervous system. The present study examines the synthesis and secretion of such proteins by cultured macroglial cells. Primary glial cultures were derived from cortical and subcortical regions of neonatal rat brains, and subsequent secondary cultures were enriched in type-1 astrocytes, type-2 astrocytes, or oligodendrocytes. Newly synthesized proteins were immunoprecipitated from the culture media using antisera directed against whole rat serum. All three types of glial cells secreted a range of plasma proteins. In general, type-1 astrocytes secreted more of these proteins than did type-2 astrocytes or oligodendrocytes, although the one-dimensional polyacrylamide gel electrophoresis (PAGE) profiles were specific for each cell type. Anti-sera directed against specific plasma proteins identified three of the most abundant proteins secreted by type-1 astrocytes as transferrin, alpha-2-macroglobulin, and ceruloplasmin. Northern blot analysis of cellular RNA confirmed that type-1 astrocytes contained transferrin mRNA, and that it was more abundant in cultures derived from subcortical regions than from cortical regions. In situ hybridization studies revealed that virtually all type-1 and type-2 astrocytes contained transferrin mRNA. Since the proteins identified in this study have been proposed to have a variety of neurotrophic roles in the central nervous system, these data further extend the range of possible functions that glial cells may serve in the CNS.

Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes.

Interest in obtaining cell lines for use in studies on the development and biochemistry of the central nervous system has motivated efforts to establish cells from primary brain cultures by the use of oncogene-transfer techniques. In previous reports, cell lines derived from astrocytes in this way have had immature or abnormal phenotypes. We have explored the possibility of specifically "targeting" expression of exogenous oncogenes to differentiated astrocytes by using the promoter of the gene encoding glial fibrillary acidic protein, which is expressed almost exclusively in such cells. We report here that cell lines displaying the phenotypic characteristics of type 1 astrocytes can be established reproducibly in this manner. Given the heterogeneity of primary cultures, the availability of clonal cell lines displaying characteristics of type 1 astrocytes should greatly facilitate our understanding of the biology of these cells.

A dual thrombin receptor system for platelet activation.

Platelet-dependent arterial thrombosis triggers most heart attacks and strokes. Because the coagulation protease thrombin is the most potent activator of platelets, identification of the platelet receptors for thrombin is critical for understanding thrombosis and haemostasis. Protease-activated receptor-1 (PAR1) is important for activation of human platelets by thrombin, but plays no apparent role in mouse platelet activation. PAR3 is a thrombin receptor that is expressed in mouse megakaryocytes. Here we report that thrombin responses in platelets from PAR3-deficient mice were markedly delayed and diminished but not absent. We have also identified PAR4, a new thrombin-activated receptor. PAR4 messenger RNA was detected in mouse megakaryocytes and a PAR4-activating peptide caused secretion and aggregation of PAR3-deficient mouse platelets. Thus PAR3 is necessary for normal thrombin responses in mouse platelets, but a second PAR4-mediated mechanism for thrombin signalling exists. Studies with PAR-activating peptides suggest that PAR4 also functions in human platelets, which implies that an analogous dual-receptor system also operates in humans. The identification of a two-receptor system for platelet activation by thrombin has important implications for the development of antithrombotic therapies.