In the present study we have examined the ability of 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP; the membrane permeant analog of cAMP which can activate protein kinase A) to mimic hormone action and stimulate glucose transport and glucose transporter (GLUT-1) gene expression as well as the expression of several growth-related protooncogenes in quiescent 3T3-L1 fibroblasts. 8-Bromo-cAMP induced a rapid and prolonged increase in the rate of hexose transport. Early activation of hexose transport (within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters, which corresponded to a doubling in the number of D-glucose-displaceable, plasma membrane cytochalasin B binding sites. The time course for 8-bromo-cAMP-induced hexose transport preceded the accumulation of GLUT-1 mRNA, which peaked between 4 and 8 h after exposure to the agent, and subsequently declined to approach basal (control) levels. Expression of the immediate-early genes c-fos and jun-B was induced by 8-bromo-cAMP on a rapid, but sustained time course, whereas induction of c-jun expression was delayed. Alterations in specific mRNAs following exposure to 8-bromo-cAMP were due to increased gene transcription (as judged by nuclear transcription run-on assays), although with respect to GLUT-1, an increase in mRNA stability was also observed. Treatment of the cells with forskolin resulted in the induction of GLUT-1 expression as well as expression of the immediate early genes. Exposure of quiescent 3T3-L1 fibroblasts to 8-bromo-cAMP resulted in a substantial increase in rates of total protein and RNA synthesis, but had little effect on DNA synthesis. The results demonstrate that 8-bromo-cAMP initiated a G0/G1 transition, but did not permit progression into S-phase. The results further suggest that increased cytosolic cAMP results in the stimulation of glucose transport by three distinct mechanisms to include translocation of pre-existing transporters, increased transcription of the GLUT-1 gene and increased stability of GLUT-1 mRNA.

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The regulation of ornithine decarboxylase (ODC) was examined in an intestinal epithelial crypt cell line (IEC-6). Addition of fetal bovine serum or growth factors to quiescent preconfluent cells resulted in a 20- to 30-fold increase in the specific activity of ODC, which was maximal at approximately 4 h. In contrast, ODC mRNA levels either did not change or increased only twofold over the time period examined. The increased enzymatic activity was blocked by cycloheximide, putrescine, and the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-napthalinesulfonamide (W-7). Cycloheximide alone increased mRNA levels and potentiated the induction in response to serum, suggesting that protein synthesis is not required for the increase in mRNA accumulation. In contrast to its effect on ODC activity, W-7 was without effect on the serum-stimulated increase in ODC or c-fos mRNA levels. Putrescine decreased ODC activity, but not mRNA content, in a dose-dependent manner with an IC50 between 0.1 and 1.0 microM. Also, serum stimulation resulted in a threefold increase in the stability of the enzyme in the presence of cycloheximide; this effect was blocked by pretreatment with W-7. Enzymatic activity was paralleled by ODC protein content as determined by [3H] difluoromethylornithine binding. Finally, the induction of enzyme activity was due entirely to an increase in Vmax as no detectable change in Km for ornithine was detected. These results suggest that ODC is regulated at multiple levels by independent signaling pathways in cultured intestinal epithelial cells. Increased levels of active ODC protein and enzymatic activity are sensitive to W-7 and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

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