Adrenergic mechanism in Tetrahymena. II. Effect of adrenaline on cell proliferation.

Growth of Tetrahymena in the exponential phase was slightly accelerated by transfer of cells to a medium containing 10-3-10-5 M adrenaline or noradrenaline, while that of stationary phase cells was inhibited by 10-3-10-7 M catecholamine. In synchronized cultures of Tetrahymena, adrenaline inhibited RNA and protein syntheses in both the late G1 and the G2 phases. These inhibitory effects in the late G1 and G2 phases result in an inhibition of the introduction of the S phase and of cytokinesis, respectively. These results indicate that an adrenergic mechanism in Tetrahymena plays a role in regulating the cell cycle. Earlier reports on the role of catecholamines on cell proliferation showed that chronic administration of isoproterenol induced hyperplasia of the salivary glands in rats (1) and mice (2). Barker (3) and Beserga (4) reported that a single injection of isoproterenol re sulted in increased DNA synthesis in rodent salivary gland after a lag period of about 20 hr. On the other hand, adrenaline is known to depress mitotic activity in a variety of tissues (5) and to inhibit DNA synthesis in regenerating rat liver (6). As reported by the authors (7) and others (8), Tetrahymena pyriformis, like mammals, has pathways for both biosynthesis and degradation of catecholamines. Recently, we observed (9) that monoamine oxidase activity decreased in the exponential growth phase and increased in the stationary phase. Furthermore, in synchronized cultures, the enzyme activity was shown to decrease in the M and S phases. The present study was on the role of the adrenergic mechanism in the growth of Tetra hyinena. MATERIALS AND METHODS Methods for culturing and synchronization in Tetrahymena pyrijbrinis W were the same as reported previously (9). For experiments on the effect of adrenaline and noradrenaline on growth, cells in the exponential and the stationary growth phase were obtained by culture for 24 and 96 hr respectively. These cells were then transferred to a fresh medium containing a test drug, and after incubation for 24 hr the cell number was determined electronically using * Present adress: Pharmaceutical Research Laboratory, Sakai Chemical Industry Co., Ltd., Matsugaoka-nakamachi, Kawachinagano, Osaka, Japan. a Coulter Counter, Model B. The growth rate is expressed as a percentage of the con trol cell count. To determine the protein and nucleic acid contents, synchronized cultures of Tetra Iryinena were incubated with or without adrenaline (5 x 10 M) at 26°C. Samples were removed by pipette every 15 min. Nucleic acid was extracted by the procedure of Sch neider (10). RNA was measured by the method of Mejbaum (11) and DNA by that of Ceriotti (12). Protein was determined by the method of Lowry et a!. (13). To study labeling of protein and nucleic acid by the radioactive precursor, synchronized cells were incubated at 26°C with the precursor (50 mpCi/ml) and various test drugs (5 x l0' M) affecting the adrenergic mechanism. Drugs were added just after, or 55 min after the end of heat treatment (EHT). Incorporation of radioactive compounds was mea sured by the rapid radioassay technique of Byfield et al. (14), and radioactivity was deter mined using an Aloka Gas Flow Counter. Tested were the effects of the following drugs; 1-adrenaline (E. Merck, Darmstadt), Catron (Chugai Pharmaceutical Co., Ltd., Tokyo), dl-noradrenaline and DL-dihydroxy phenylalanine (DOPA, Nakarai Chemicals Ltd., Kyoto). The radioactive precursors used for studies on protein, RNA and DNA synthesis, respectively, were uniformly labeled L-leucine-'1C (212 mCi/mmol), uracil-2-14C (21 mCi/mmol) and thymidine-2-14C (51 mCi/ mmol). These compounds were obtained from Daiichi pure Chemicals Co., Ltd., Tokyo.