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The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation.In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of rRNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.It is now widely accepted that tumor-promoting phorbol esters, which mimic the action of diacylglycerol, can activate a calcium-and phospholipid-dependent protein kinase, protein kinase C (reviewed in references 4, 17, and 20). We have previously observed that protein synthesis can be stimulated within minutes in nondividing Drosophila cells by reagents that act through a pathway involving calcium and the activation of protein kinase C (32). We found that phorbol esters stimulated protein synthesis and that this stimulation was dependent on the presence of calcium, suggesting a common signaling pathway. Since in most systems, the cellular processes of protein and rRNA synthesis are closely coupled (reviewed in reference 21), we have investigated whether in nondividing cells, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) can bring about changes in rRNA synthesis parallel to its affects on protein synthesis.Since TPA is known to affect growth-related events in dividing cells, we also chose to investigate the effects of this reagent on rRNA synthesis in dividing Drosophila cultured cells which were either growing slowly, i.e., under serumreduced conditions, or growing rapidly, in the presence of a high concentration of serum.There are several levels at which stimulation or inhibition of RNA synthesis could potentially be controlled. We have investigated the changes in rDNA transcription which take place in Drosophila cells treated with TPA and serum by using nuclear run-on assays to examine the number of active polymerase I (Pol I) complexes present on the 18S and 28S rRNA genes. MATERIALS AND METHODS Drosophila culture and male accessory gland isolation.Wild-type Drosophila melanogaster (Oregon-R) was grown at 25°C on a standard cornmeal-Karo-yeast agar medium supplemented with live yeast cells. After eclosion, the flies were separated according to sex. Accessory glands from 10 virgin males (7 to 10 days old) were ...

In adipocytes, adenylate cyclase is positively regulated by (3-adrenergic agents and negatively regulated by adenosine. Incubation of adipocytes with adenosine deaminase relieves the inhibition of adenylate cyclase by destroying the adenosine that the cells release into the medium. When adipocytes are incubated with adenosine deaminase and the fl-adrenergic agent isoproterenol, most of their ATP is converted to AMP in 5 min. Either isoproterenol or adenosine deaminase alone has little or no effect. In the additional presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) cAMP accumulates instead of AMP. Under these conditions, cAMP represents 40-50% of the total intracellular adenine nucleotides, and ATP only 5%. N6-(L- MATERIALS AND METHODS(-)-Isoproterenol, 3-isobutyl-1-methylxanthine (iBuMeXan), N6-(L-2-phenylisopropyl)adenosine, adenosine deaminase (type I), and bovine serum albumin (fraction V) were purchased from Sigma. Collagenase was from WorthingtonCooper and 8-(p-sulfophenyl)theophylline was from Research Biochemicals (Wayland, MA). 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) was generously supplied by Hoffmann-La Roche.Adipocytes were isolated from epididymal fat pads of 180-200 g Sprague-Dawley rats by collagenase treatment (3). Cells were resuspended in Krebs-Ringer Hepes buffer (pH 7.4) containing 2% bovine serum albumin and 3 mM glucose and continuously gassed with oxygen. Aliquots containing about 2 x 105 cells per ml were distributed to plastic vials for incubation with the indicated agents. After incubation, cells were separated from the medium by centrifugation (1000 x g for 10 sec), washed with 5 ml of 300 mM sucrose to remove residual medium, and extracted with 5% trichloroacetic acid for 15 min. After centrifugation, the extracts Were filtered through glass wool and ether-extracted. Samples were lyophilized, redissolved in the HPLC buffer, and passed through a 0. RESULTSAn understanding of the pathways and mechanisms of energy metabolism in cells requires information on the levels of Abbreviations: iBuMeXan, 3-isobutyl-1-methylxanthine; Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one. 1614The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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