An adrenergic control system in Tetrahymena.

The discovery by Janakidevi et al.' that the ciliated protozoan Tetrahymena contains epinephrine and norepinephrine led Blum et al.2 to examine the effect of reserpine on these ciliated protozoa. It was found that reserpine inhibited the growth of these cells and depleted the catecholamine content. The growth inhibition was partially reversed by addition of glucose to the medium.2 These observations suggested that Tetrahymena might contain other components of the catecholamine system found in metazoa and prompted us to examine the effects of a variety of pharmacological agents on the growth of Tetrahymena. We have found that the growth of Tetrahymena is sensitively inhibited by certain a and ,3 adrenergic blocking agents and other drugs which are known to interact with the catecholamine system in mammals. Because one of the major effects of the catecholamines in metazoa is to control the metabolism of glycogen, we also examined the effects of some of these agents on the glycogen content of Tetrahymena and have found effects on glycogen content consistent with the view that an adrenergic metabolic control system is operative in Tetrahymena. Abstracts of part of this work have been published.3' 4 Materials and Methods.-Tetrahymena pyriformis, strain HSM, were grown axenically in Erlenmeyer flasks with Morton closure tops at 250 without shaking. The flasks were filled to less than one fifth their nominal capacity for all growth experiments, and to less than one tenth nominal capacity for all experiments in which glycogen concentration was to be measured. Cells were counted with a Coulter counter (Coulter Co., Hialeah, Fla.). Two media were used. Medium A consisted of 1% proteose peptone and 0.05% liver extract in 0.02 M potassium phosphate at pH 6.5. Medium DK was essentially the synthetic medium described by Dewey and Kidder,5 supplemented with 0.04-0.07% proteose peptone, as specified. Generally, 25 ml of medium was used, and five ml of water, or water containing the reagents to be studied, was added at zero time. The media were sterilized by autoclaving. All other chemicals except triiodothyronine were dissolved in water, the pH adjusted to near neutrality, and sterilized by passage through ultramicro fritted glass filters. Triiodothyronine was suspended in water with vigorous stirring and then boiled for 2 min in a screw-cap test tube. Glycogen was assayed by the phenol-sulfuric acid method as described by Dubois et al.6 Cells were chilled in ice and washed twice by centrifugation for 1 min in a clinical centrifuge, using a buffer consisting of 0.08 M Tris and 0.036 M NaCl, pH 7.5. In this buffer, there was little or no cell lysis.7 After the second wash, the cells were resuspended in ice-cold 0.086 M NaCl, counted, and suitable aliquots were taken in quadruplicate for glycogen assay. The absorbance was measured in a Spectronic 20 colorimeter. Glucose standards were run with each assay, and all results were computed directly from the glucose standard line. In the case of cells grown in the absence of added glucose, virtually all of the color developed in the assay was due to the glucose units of glycogen, but with cells grown in the presence of glucose there may have been large amounts of intracellular glucose.8 For the present purposes, we are concerned only with the total glucose residues of the cell, and have referred to this as glycogen for convenience. Chemicals were obtained from the following sources: reserpine phosphate, Ciba Pharmaceutical Company; dibenzyline, tranylcypromine sulfate, and the sodium salt of 1-triiodothyronine, Smith, Kline, and French; dichloroisoproterenol (DCI), Aldrich Chemical Company; guanethidine, Ciba Pharmaceutical Company; corticosterone, Mann Research Laboratories; Segontin, Hoechst Pharmaceuticals, Inc.; Catron, Lakeside Laboratories, Inc.; desipramine, Geigy Pharmaceuticals; Inderal, Ayerst Laboratories; 3'5'-cyclic adenosine 5'-phosphate, Pabst Labora-