Insulin-like growth factor-I induces a rapid increase in calcium currents and spontaneous membrane activity in clonal pituitary cells.

The role of growth factors in the adult brain is largely unknown, although receptors for factors such as insulin-like growth factor-I (IGF-I) have been localized on nondividing mature neurons. Because neurons use the frequency and pattern of action potentials to encode information, we assessed the ability of IGF-I to modulate rapidly the electrical properties of GH4C1 cells, a spontaneously active pituitary line with neuronal L- and T-type calcium currents. Electrical quiescence (the absence of spontaneous activity) was induced by culture in serum-depleted conditions. IGF-I, which is synthesized locally in mammalian brain, induced a rapid increase in electrical activity that was accompanied by increased activation of calcium channel currents. These effects were dose and time dependent. The spontaneous activity of cells exposed to 20 ng/ml IGF-I increased in approximately 10 sec and, after a brief exposure, continued increasing for at least 8 hr. Currents carried by calcium channels doubled within 10 sec. Both the increase in spontaneous activity and the increased activation of calcium channel currents were blocked by tyrosine kinase inhibitors. These results suggest that IGF-I can act as a rapid neuromodulator of calcium currents.

Neurotrophic protein S100 beta stimulates glial cell proliferation.

Nervous system development involves a coordinated series of events, including regulation of cell proliferation and differentiation by specific extracellular factors. S100 beta is a neurotrophic protein that has been implicated in regulation of cellular proliferation, but direct evidence was lacking. In this report, nanomolar concentrations of S100 beta are shown to stimulate proliferation of rat C6 glioma cells and primary astrocytes. An S100 mutant with a single amino acid change was inactive. S100 beta also stimulated increases in the steady-state levels of c-myc and c-fos protooncogene mRNAs and complemented the effects of platelet-derived growth factor. Two neuroblastoma cell lines did not proliferate in response to S100 beta, suggesting that the mitogenic activity of S100 beta is selective for astroglial cells. These results suggest that S100 beta may be involved in the coordinate development and maintenance of the central nervous system by synchronously stimulating the differentiation of neurons and the proliferation of astroglia.

Antisense inhibition of glial S100 beta production results in alterations in cell morphology, cytoskeletal organization, and cell proliferation.

The phenotypic effects of selectively decreasing the levels of S100 beta in cultured glial cells were analyzed. Two separate antisense approaches were utilized for inhibition of S100 beta production: analysis of clonal isolates of rat C6 glioma cells containing an S100 beta antisense gene under the control of a dexamethasone-inducible promoter, and analysis of C6 cells treated with S100 beta antisense oligodeoxynucleotides. Both antisense methods resulted in a decrease in S100 beta levels in the cell, as measured by RIA. The inhibition of S100 beta production correlated with three alterations in cellular phenotype: (a) a flattened cell morphology; (b) a more organized microfilament network; and (c) a decrease in cell growth rate. The studies describe here provide direct evidence for an involvement of S100 beta in glial cell structure and function, and suggest potential in vivo roles for S100 beta in regulation of glial cell morphology, cytoskeletal organization, and cell proliferation.

Rapid isolation of plasmalemma from cultured A431 cells: characterization of epidermal growth factor receptors.

A rapid method for the purification of plasma membrane from a relatively small number of A431 cells is described. The method is a simple, two-step differential centrifugation in the presence of Ca2+ that requires a total centrifugation time of 7 min. The membrane preparations contained a high level of epidermal growth factor (EGF) receptor activity demonstrated by both the quantity of specific ligand binding and the amount of EGF-dependent phosphorylation of the receptor and an exogenous substrate. EGF-dependent autophosphorylation identified the EGF receptor in the purified membranes as an undegraded 170-kDa protein.