The role of signal transduction mechanisms in the production of the characteristic orthogonal arrays of particle assemblies in the astroglial plasma membrane was investigated in vitro by freeze-fracture electron microscopy. Agents which raise cellular cAMP levels and subsequently activate protein kinase A, such as forskolin (50 μm), isoproterenol (10 μm) and 8-bromo-cAMP (1 mm), increased the density, the number of assemblies per unit area of cleaved cell membrane, and the frequency of astrocytes with assemblies. Agents that lead to the activation of protein kinase C, such as phorbol 12,13-myristate acetate (at 50 nm) and choline-dependent phospholipase C (at 0.01-0.1 U ml-1), did not affect the assembly concentration. Thus, protein kinase A but not protein kinase C appears to be involved in the production of assemblies or their insertion into the astroglial plasma membrane. Although choline-dependent phospholipase C did not affect the astroglial assemblies, it caused the non-assembly, background particles to aggregate. A choline-dependent phospholipase C from a different source (B. cereus) was also active though at a higher concentration. Phospholipases of different specificities, such as phospholipase A2, phospholipase D or inositol-dependent phospholipase C were inactive over a wide range of concentrations. Two other astroglia derived cells, Müller cells and cells of the C6 glioma cell line, were also similarly affected by choline-dependent phospholipase C, while six other cells types including neurons, endothelial cells and fibroblasts were unaffected. It appears that phosphatidylcholine plays a significant role in determining the membrane structure of astrocytes. In a search for a means of isolating the assemblies, the binding of three lectins: ConA, WGA and PNA, conjugated to gold, was tested by label-fracture to ascertain whether the assemblies have an external oligosaccharide component. None of the lectins bound specifically to assemblies.
ASJC Scopus subject areas
- Cell Biology