Basic fibroblast growth factor prevents cAMP-induced apoptosis in cultured Schwann cells.

Peripheral nerve axotomy induces apoptosis in Schwann cell precursors; basic fibroblast growth factor (bFGF) protects these cells from axotomy-induced apoptosis (Jessen et al.: Neuron 12:509-527, 1994; Gavrilovic et al.: Eur J Neurosci 7:7-85, 1995). In this study, we investigate the effects of bFGF on apoptosis in neuron-free cultures of neonatal rat Schwann cells. Apoptotic cell death was induced in primary and secondary expanded Schwann cells by treatment with 1 mM concentrations of 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP), a membrane-permeable analogue of cAMP which induces expression of galactocerebroside in the plasma membranes of Schwann cells. Treatment with bFGF reduced the percentage of galactocerebroside-bearing Schwann cells undergoing cAMP-induced DNA fragmentation. These findings suggest that bFGF can enhance the survival of terminally differentiated Schwann cells by preventing apoptosis.

Deoxyadenosine- and cyclic AMP-induced cell cycle arrest and cytotoxicity.

We compared deoxyadenosine (AdR)- and cyclic AMP (cAMP)-induced cell cycle arrest and cytotoxicity in wild type and mutant S49 cells to determine whether they resulted from the same or different mechanisms. Cyclic AMP and deoxyadenosine are synergistic rather than additive in cytotoxicity assays, suggesting different mechanisms of toxicity. Although cyclic AMP causes cell death after 72 h, in concentrations sufficient to result in cell cycle arrest it is reversible with virtually no cytotoxicity for at least 24 h, whereas AdR-induced cell cycle arrest is lethal and irreversible. AdR-induced G1 cell cycle arrest results in diminished ribonucleotide reductase activity but the kinetics of this inhibition differ from cyclic AMP-induced cell cycle arrest. Cyclic AMP arrest and cytotoxicity depend on cyclic AMP-dependent protein kinase (PKA) activity, whereas AdR toxicity does not differ between cell lines with or without PKA activity. Furthermore, deoxycytidine prevents AdR cell cycle arrest and cytotoxicity but has no effect on cyclic AMP G1 arrest. Finally, comparison of cytofluorographic patterns of G1-arrested cells suggests that the AdR block is later in G1 than cyclic AMP-induced cell cycle arrest. In summary, these data show that while the mechanisms of cell cycle arrest and cytotoxicity of cyclic AMP and deoxyadenosine are uncertain, they do appear to involve different pathways.

Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia.

Recent evidence has suggested that cAMP plays a role as a second messenger in the decrease in nociceptive threshold (or hyperalgesia) produced by agents acting on primary afferent terminals. In support of this hypothesis we report that intradermal injection of a direct activator of adenyl cyclase, forskolin, produces a dose-dependent hyperalgesia in the rat. The duration of this hyperalgesia was prolonged by the phosphodiesterase inhibitors, isobutylmethylxanthine and rolipram. Forskolin hyperalgesia was antagonized by the Rp isomer of cyclic adenosine-3'5'-monophosphothioate, an analog of cAMP that prevents the phosphorylation of the cAMP protein kinase. The Rp isomer of cyclic adenosine-3'5'-monophosphothioate also inhibited the hyperalgesia induced by a membrane-permeable analogue of cAMP, 8-bromocyclic adenosine monophosphate, as well as the hyperalgesia induced by agents that are presumed to act directly on primary afferent nociceptors: prostaglandin E2, prostaglandin I2, (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid; and the adenosine A2-agonist 2-phenylaminoadenosine. Although the cAMP second messenger system contributes to primary afferent hyperalgesia, we found no evidence for a contribution of protein kinase C. Thus, hyperalgesia induced by prostaglandin E2, prostacyclin (prostaglandin I2), (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid, the adenosine A2-agonist 2-phenylaminoadenosine, 8-bromocyclic adenosine monophosphate and the direct activator of adenyl cyclase, forskolin, were not significantly attenuated by the selective inhibition of protein kinase C by the 19-31 fragment of protein kinase C. Two other inhibitors of protein kinase C, sphingosine and staurosporine, also failed to attenuate prostaglandin E2-induced hyperalgesia.

Growth arrest of proadipocytes at a distinct predifferentiation G1 state associated with cytotoxic responsiveness to 8-bromo cyclic AMP.

The control of cell proliferation can result from the coupling of growth arrest and differentiation. In this regard, we recently demonstrated that growth arrest which precedes the differentiation of 3T3 T proadipocytes must occur at a distinct state in the G1 phase of the cell cycle (GD). Cells arrested at GD differ in several biological parameters from cells arrested in G1 at other states induced by either serum deprivation (GS) or nutrient deficiency (GN). Specifically, GD-arrested cells can differentiate in the absence of DNA synthesis and GD-arrested cells can be induced to proliferate when stimulated with 1-methyl-3-isobutylxanthine; GS- and GN-arrested cells cannot. In addition, GD-, GS- and GN-arrested cells reside at topographically distinct states in G1. We now report that GD-arrested proadipocytes are also distinct in that they are highly sensitive to a cytotoxic effect of 8-bromocyclic AMP, whereas GS- and GN-arrested cells are not.