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Effects of hormones on the RNA-synthesis of Tetrahymena pyriformis
C()rnp, Biochem Ph.)'sio! Vol. 68C. pp. 251 to 253
0306=4.492/81,/030251-03502,00/0
O Pergamon Press Ltd 1981. Printed in Greal Britain
EFFECTS OF HORMONES ON THE RNA-SYNTHESIS OF TETRAH YMENA PYRIFORMIS* G. CSAnA and L. UBORNY,~K Department of Biology, Semmelweis University of Medicine, Budapest, Hungary
(Received 15 July 1980) Abstract--I. Prednisolone, Ta, serotonin and epinephrine were found to enhance the RNA synthesis of Tetrahymena pyriformis considerably, while thyroxine (T4) and histamine did so to a lesser degree. 2. The curve (rate of synthesis) for hormonal action showed 2 peaks, a minor at 5 rain and a major one at 30 min. 3. The effects of triiodothyronine (T3) and T4 differed considerably, the latter is much less active. 4. The highest stimulatory effect was developed by serotonin: the cells exposed to it still showed an cightfold increase of newly synthesized RNA over the control after 2 hr.
INTRODUCTION
prednisolone (Di-adreson-F-aquosum, Organon, Oss). Each hormone was applied at the concentration of 10-to M, for periods of 5, 15, 30, 60 and 120 rain. Cells washed or incubated for 24 hr in Losina-Losinsky solution were treated with 5pCi/ml 08.5 x l04Bq) [aH]uridin¢ (Amersham, U.K.). The specific activity of the radioisotope was ! pCi/ml (3.7 x l04 Bq). The RNA was extracted by the following procedure (Andersen, 1971 ; Smillie & Krotkov, 1960): the synchronized cells were suspended in Losina solution, and were centrifuged at 3000g. The supernatant was decanted, the cell sediment was homogenized in 100% methanol at 0--4"C,and was centrifuged again at 3000 g. The precipitate was suspended in 0.2% CH3COOH, centrifuged at 3000 g, the sediment was suspended in 5% HCIO4, centrifuged again at 3000g, and dehydrated in 3 changes of ethanol, ethanol-ether and ether. The dehydrated material was incubated in 0.3 M KOH for 15-20 hr at 37°C, cooled, and centrifuged at 3000 g. The supernatant was, alter addition of 1 m170% HCIO,~, centrifuged at 3000 9, and the RNA was recovered from the fluid phase. The T. pyriformis cells were exposed simultaneously to the hormone and the radioisotope, and were fixed in 4% formaline after expiration of the appropriate exposure time. After extraction of the RNA, radioactivity was determined with a Packard tricarb scintillation counter. An appropriate control group, exposed to [3H]uridine only, was set up with each (time-hormone) test. In each series of the experiment 20 measurements were made, and the mean percentage of cpm was calculated for l06 cells relative to the control as 100.
Testrahymena pyriformis, although it contains some hormone-like materials (Blum, 1967; lwata & Kariya, 1973; Janakivedi et al., 1966; Kariya et al., 1974) does not itself possess hormonal regulation, but responds to various hormones including those of vertebrates (Csaba et al., 1977) on artifical exposure. The response is usually specific, as insulin (Csaba & Lantos, 1975) and epinephrine (Csaba & Lantos, 1976) were found to stimulate the glucose metabolism of the protozoon, histamine and serotonin its phagocytic activity (Csaba & Lantos, 1973, 1975, 1977)and some members of the thyroxin series its mitotic activity (Csaba & N6meth, 1979; Romanov et al., 1974). In the present studies we examined the influence of various hormones on the RNA synthesis of T. pyriformis for the following reasons: (l) to utilize the RNA synthesis of T. pyriformis as a non-specific index for activity assay of various hotmones including those not known to evoke a specific response in Tetrahymena, and (2) to clarify through follow-up of the RNA synthesis whether the hormonal action expires after alteration of the cAMP level or proceeds at the genetic level. A genetic effect has been postulated on the grounds that certain hormones, for which T. pyriformis displays membrane receptors, have been shown to enter into the cell body and to assume a heterochromatic nuclear localization (Csaba & Sudfir, 1979; Korneliussen, 1976; Sudfir & Csaba,.1979).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Tetrahyraena pyriforrais GL cells maintained in 1~ bacto-Trypton medium (Difco, MI) containing 0.1~ yeast extract were used in the experiment after synchronization by thermal shock (Zeuthen, 1971). Appropriate batches of cells were treated with the folIowing hormones:triiodothyronine(Ta, Serva, Heidelberg), thyroxine (T4, Bender-Hobein, Ziirich), epinephrine (Richter, Budapest), histamine (Reanal, Budapest), serftonin (serotonin creatinin sulphate, Reanal, Budapest), * Supported by the Scientific Research Council, Ministry of Health, Hungary, 1-01-0302-02-1/Cs. 251
The first conclusion drawn from the experiment was that the hormones affected the RNA synthesis of T. pyriformis differently, although the tendency of the effect was similar with some of them (Fig. I & Table 1). In every case, 2 peaks appeared after 5 and 30 min, respectively, but while under the influence of histamine RNA synthesis showed a decrease relative to the control after 5min, the other hormones accounted for a moderate, but significant increase at 5 mitt, and a powerful one at 30rain. Epinephrine and, especially, serotonin, Ta and prednisolone characteristically showed a decrease of activity at 15 rain, followed by an enormous increase thereof by 30min. By 60rain, the activities of all hormones
252
G. CSAI~Aand L. UBOR.,;YAK
7o0~[
~i~ j ~ i ~ i~ ~ ,0oo.
','~77-----~'~ "~
~
", ~ ', ~ "~ ,,~ '8 ~ / ~ i ~.z . . , . ~ . ~ " :~':. . . . . . . . . . . ... . . . . . . . . . . .-II ,oo I [ ~17 / ' _ / ,,,\ ...... " " ',,, .... ~ _ i
~..
m ~ *--*~'-~,,~* ,,.--At, r~. ON,°,N,,. ~ - - , h,..,~, m---I~
5o ", ~ ",~
t
.'s
~o
go
6o .,~.
Fig. I. Incorporation of [3H]uridine by RNA in Tetrahymena pyriformis cells exposed to different hormones. except serotonin dropped to practically the same level approximating the control, and by 120 min those of all except serotonin and T,, fell below the control level, The hormones used in the experiment represented 4 different types; firstly molecules gaining access to the nucleus of vertebrate cells and acting at the genetic level; secondly molecules capable of entering into the nucleus of T. pyriformis; thirdly, hormones representing members of a series in which one hormone is considerably more active in vertebrates than the other; fourthly, molecules with known effect on T. pyr!formis. The forthcoming discussion follows this classification, The hormones capable of entering the cellular nu-
cleus in the vertebrate organism developed the greatest stimulatory action on RNA synthesis of T. pyriformis. Prednisolone--like all steroids--is bound by a cytosol receptor and develops action at the nuclear level (Cake & Litwack, 1975); the same applies to T3 (Samuels et al., 1974: Sterling et al., 1974). Evidence has also been presented of the intranuclear access of serotonin (Korneliussen, 1976; Sud:ir & Csaba, 1979). In view of this, one might be tempted to conclude that the hormones responsible for stimulation of RNA synthesis in vertebrates develop the greatest stimulatory action also at the unicellular level. This interpretation is, however, recognized as false if it is taken into consideration that although T4 also gains access to the cellular nucleus in vertebrates, its effect on T pyriformis was negligible compared to Ta. while epinephrine, having an intranuclear access only in the unicellular (Csaba et al., 1980) had a considerable, although not maximal, influence on the latter. At the same time, we showed in earlier experiments (Csaba et al., 1978) that Tetrahymena does not possess a cytosol receptor for steroids (at least not in the form therefore, have no access to its nucleus, while histamine has (Csaba & Sud~ir, 1979), but it developed nonetheless little (positive) activity under the given conditions of experiment. It appears, in view of the foregoing considerations, that the presence of a hormone in the nucleus it not in itself an indispensable prerequisite of the hormone's nuclear action. Nevertheless, hormonal effects developed at the genetic level do show a parallelism in vertebrates and T. pyriformis. The response of T. pyriformis to T3 and T,L presents a special problem. Both hormones are bound by cytosol receptors and gain access into the nucleus, but T3 is several times as active as T, in the vertebrate organism (Astwood, 1971). This activity difference was still more pronounced under the conditions of experiment, for while vertebrates show a T3-T 4 binding ratio of 4:1. the T. pyriformis cells bound the 2 hormones at the ratio of 10:1. This seems logical as it is known that of the iodine hormones those with a lower iodine content, e.g. diiodothyrosine, stimulate mitosis to a greater degree than those containing more iodine (Csaba & N6meth, 1979). Serotonin differed in action from all other hormones included in the study. It stimulated the synthesis of RNA markedly at 30min, and while the RNA synthesis of the cells exposed to the other hor-
Table 1. Incorporation of [-~H]uridine by RNA in Tetrah.vmena p.vriformis cells exposed to different hormones. (Calculated for 106 cells relative to the control as 100) Control
Epinephrine
Prednisolone
T3
100 100 100 100 100
130.05" 87.04* 393.92 202.61 56.13
287.93 62.95 3623.11 245.97 39.44
188.76 63.54 1667.4 159.78 26.51
Significance of difference from the control: *P < 0.05. t P < 0.02. NS = not significant. Not otherwise indicated = P < 0.01.
Histamine 80.39 43.57 183.4 135.72"162.19
Serotonin 203.04 46.62 1198.56 990.25 824.96
T4 163.44"f" 95.48 "~s 158.51 164.52 126.983'
Time of exposure (rain) 5 15 30 60 120
RNA synthesis in Tetrahymena m o n e s was a p p r o x i m a t i n g the control level by 60 min, and fell below it by 120 min, the serotonin-exposed cells still showed a more than 8-fold increase of RNA over the control after 2 hr. This can be attributed either to a long-term action of serotonin, or to a long lifetime of the R N A stimulated by serotonin. T h e latter h o r m o n e stimulated also the growth of T. pyriformis cells to an extraordinary degree (Csaba & N6meth, 1979), but the information emerging from the present study permits no conclusion on the interrelationship of this p h e n o m e n o n with the increased synthesis of RNA. The 2 peaks of h o r m o n a l action, which were invariably found in each experimental series, suggest that the first peak signifies the first Imembrane-level) encounter o f t h e h o r m o n e with the cell via the c A M P system (Langan, 1970), probably through activation of histone kinase, while the second---superior--peak might be the issue of the development of h o r m o n a l action at the genetic level,
REFERENCES ANDERSEN R. S. (1971) Ribosomal synthesis in greening primary leaves of bean seedling IPhaseolus vulgaris) BiDchem. Physiol. Pflanzen 162, 245-264. ASTWOOD E. B. (1971) Thyroid and antithyroid drugs. In The Pharmacological Basis of Therapeutics, (Edited by GOODMAN L. S. & GILMAN A.), pp. 1466 1500. MacMillan, London. BLt;M J. J. (1967) An adrenergic control system in Tetrahymena. Proc. natn. Acad. Sci. (USA)51, 81-88. CAKE M. H. & LITWACK G. (1975) The glucocorticoid receptor. In Biochemical Action of Hormones (Edited by LI'rWACK G.). pp. 317-390. Academic Press, New York. CSABA G. & LANTOST. (1973) Effect of hormones on protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indoleacetic acid in Tetrahymena pyriformis. Cytobiologie 7, 361-365. CSAaA G. & LA.~TOS T. (1975) Specificity of hormone receptors in Tetrahymena. Experiments with serotonin and histamine antagonists. Cytobiologie ! i. 44--49. CSABA G. & LA.~IOS T. (1975) Effect of insulin on the glucose uptake of protozoa. Experientia 31, 1097-1098. CSAaA G. & LANTOS T. (1976) Effect of epinephrine on glucose metabolism in Tetrahyme~la. Endokrinolo¢lie 68, 239 240.
253
CSABAG. & LANrOS T. (1977) An attempt to differentiate selection and amplification in hormone receptor development. The unicellular model. Differentiation $, 57-59. CSAB^ G., SUDAR F., NAG': S. U. & Domgzv O. (1977) Localization of hormone receptors in Tetrahymena. Protoplasma 91, 179- 189. CSABAG. LANTOST., N^CV S. U., AR/,NVl P. & N,~RAY A. (1978) Effects of steroids on Tetrahymena. Acta biol. reed. germ. 37, 1377-1380. CSABA G. & SL'D,~R F. (1979) Localization of [3H]histaming in the nucleus of Tetrahymena. Aeta morph. Acad. Sci. hung, 27, 89-94. CSAB^ G. & NEM~rH G. (19801 Effect of hormones and their precursors an unicellulars. The selective responsiveness of Tetrahymena. Comp. Biochem. Physiol. 65B, 387-390. CSAaAG., SUDAR F. & PADOS R. 11980) Binding and internalization of [aH]epinephrine in Tetrahymena. Endokrinolo qie. In press. lW^TA H. & KARIYA K. (1973) Adrenergic mechanism in Tetrahymena I. Changes in monoamine oxidase activity during growth. Experientia 29, 265-266. JANAKIVEDI D., DEWEY J. C. & KIDDER G. W. (1966) The biosynthesis of catecholamines in two genera of protozoa. J. biol. Chem. 24, 2576-2578. KARIYA K., SAITO R. & IWATA H. (19741 Adrenergic mechanism in Tetrahymena Ill. cAMP and cell proliferation Jap. J. Pharmae. 24, 129-134. KORNELIUSSEN H. (1976) 5-hydroxytryptamine: autoradiographic evidence for uptake into fibroblast cell nuclei Experientia 32,443-445. LANGANT. A. 11970) Phosphorylation of histones in vivo under the control of cyclic AMP and hormones. Acta Biochem. Psychopharmac. 3, 307-310. ROMANOVYU. A., SAVCHENKO T. V. & ZARENKOVA V. P. (1974) Effect of thyroxin on cell division and the mitotic cycle of Tetrahymena pyr(formis. Biol. Zh. Arm. 27, 9--15. SAMt:EI,S A. H., TSAI J. G. & CASANOVA J. (1974) Thyroid hormone action: in vitro demonstration of putative receptors in isolated nuclei and soluble nuclear extracts. Science 184, 1188 1190. SMILLItN. M. & KROTKOV G. (1960) The estimation of nucleic acid in some algae and higher plants. Can. J. Bot. 38, 31 49. STERLINGK., SALDANHAV. F., BRENXER N. A. & MILCH P. O. (1974~ Cytosol binding protein of thyroxine and triodothyronine hormone in kidney tissue. Nature 250, 661-663. Suo,~R F. & CSABA G. 11979) Localization of [3H]serotonin in the adrenal medulla cells of newborn rats. ,4cta morph. Acta. Sci. hung. 27, 83 87. ZEUTHEN E. (1971) Synchrony in Tetrahymena by heat shocks spread a normal cell generation apart. E xpl cell. Res. 68, 49-52.
0306=4.492/81,/030251-03502,00/0
O Pergamon Press Ltd 1981. Printed in Greal Britain
EFFECTS OF HORMONES ON THE RNA-SYNTHESIS OF TETRAH YMENA PYRIFORMIS* G. CSAnA and L. UBORNY,~K Department of Biology, Semmelweis University of Medicine, Budapest, Hungary
(Received 15 July 1980) Abstract--I. Prednisolone, Ta, serotonin and epinephrine were found to enhance the RNA synthesis of Tetrahymena pyriformis considerably, while thyroxine (T4) and histamine did so to a lesser degree. 2. The curve (rate of synthesis) for hormonal action showed 2 peaks, a minor at 5 rain and a major one at 30 min. 3. The effects of triiodothyronine (T3) and T4 differed considerably, the latter is much less active. 4. The highest stimulatory effect was developed by serotonin: the cells exposed to it still showed an cightfold increase of newly synthesized RNA over the control after 2 hr.
INTRODUCTION
prednisolone (Di-adreson-F-aquosum, Organon, Oss). Each hormone was applied at the concentration of 10-to M, for periods of 5, 15, 30, 60 and 120 rain. Cells washed or incubated for 24 hr in Losina-Losinsky solution were treated with 5pCi/ml 08.5 x l04Bq) [aH]uridin¢ (Amersham, U.K.). The specific activity of the radioisotope was ! pCi/ml (3.7 x l04 Bq). The RNA was extracted by the following procedure (Andersen, 1971 ; Smillie & Krotkov, 1960): the synchronized cells were suspended in Losina solution, and were centrifuged at 3000g. The supernatant was decanted, the cell sediment was homogenized in 100% methanol at 0--4"C,and was centrifuged again at 3000 g. The precipitate was suspended in 0.2% CH3COOH, centrifuged at 3000 g, the sediment was suspended in 5% HCIO4, centrifuged again at 3000g, and dehydrated in 3 changes of ethanol, ethanol-ether and ether. The dehydrated material was incubated in 0.3 M KOH for 15-20 hr at 37°C, cooled, and centrifuged at 3000 g. The supernatant was, alter addition of 1 m170% HCIO,~, centrifuged at 3000 9, and the RNA was recovered from the fluid phase. The T. pyriformis cells were exposed simultaneously to the hormone and the radioisotope, and were fixed in 4% formaline after expiration of the appropriate exposure time. After extraction of the RNA, radioactivity was determined with a Packard tricarb scintillation counter. An appropriate control group, exposed to [3H]uridine only, was set up with each (time-hormone) test. In each series of the experiment 20 measurements were made, and the mean percentage of cpm was calculated for l06 cells relative to the control as 100.
Testrahymena pyriformis, although it contains some hormone-like materials (Blum, 1967; lwata & Kariya, 1973; Janakivedi et al., 1966; Kariya et al., 1974) does not itself possess hormonal regulation, but responds to various hormones including those of vertebrates (Csaba et al., 1977) on artifical exposure. The response is usually specific, as insulin (Csaba & Lantos, 1975) and epinephrine (Csaba & Lantos, 1976) were found to stimulate the glucose metabolism of the protozoon, histamine and serotonin its phagocytic activity (Csaba & Lantos, 1973, 1975, 1977)and some members of the thyroxin series its mitotic activity (Csaba & N6meth, 1979; Romanov et al., 1974). In the present studies we examined the influence of various hormones on the RNA synthesis of T. pyriformis for the following reasons: (l) to utilize the RNA synthesis of T. pyriformis as a non-specific index for activity assay of various hotmones including those not known to evoke a specific response in Tetrahymena, and (2) to clarify through follow-up of the RNA synthesis whether the hormonal action expires after alteration of the cAMP level or proceeds at the genetic level. A genetic effect has been postulated on the grounds that certain hormones, for which T. pyriformis displays membrane receptors, have been shown to enter into the cell body and to assume a heterochromatic nuclear localization (Csaba & Sudfir, 1979; Korneliussen, 1976; Sudfir & Csaba,.1979).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
Tetrahyraena pyriforrais GL cells maintained in 1~ bacto-Trypton medium (Difco, MI) containing 0.1~ yeast extract were used in the experiment after synchronization by thermal shock (Zeuthen, 1971). Appropriate batches of cells were treated with the folIowing hormones:triiodothyronine(Ta, Serva, Heidelberg), thyroxine (T4, Bender-Hobein, Ziirich), epinephrine (Richter, Budapest), histamine (Reanal, Budapest), serftonin (serotonin creatinin sulphate, Reanal, Budapest), * Supported by the Scientific Research Council, Ministry of Health, Hungary, 1-01-0302-02-1/Cs. 251
The first conclusion drawn from the experiment was that the hormones affected the RNA synthesis of T. pyriformis differently, although the tendency of the effect was similar with some of them (Fig. I & Table 1). In every case, 2 peaks appeared after 5 and 30 min, respectively, but while under the influence of histamine RNA synthesis showed a decrease relative to the control after 5min, the other hormones accounted for a moderate, but significant increase at 5 mitt, and a powerful one at 30rain. Epinephrine and, especially, serotonin, Ta and prednisolone characteristically showed a decrease of activity at 15 rain, followed by an enormous increase thereof by 30min. By 60rain, the activities of all hormones
252
G. CSAI~Aand L. UBOR.,;YAK
7o0~[
~i~ j ~ i ~ i~ ~ ,0oo.
','~77-----~'~ "~
~
", ~ ', ~ "~ ,,~ '8 ~ / ~ i ~.z . . , . ~ . ~ " :~':. . . . . . . . . . . ... . . . . . . . . . . .-II ,oo I [ ~17 / ' _ / ,,,\ ...... " " ',,, .... ~ _ i
~..
m ~ *--*~'-~,,~* ,,.--At, r~. ON,°,N,,. ~ - - , h,..,~, m---I~
5o ", ~ ",~
t
.'s
~o
go
6o .,~.
Fig. I. Incorporation of [3H]uridine by RNA in Tetrahymena pyriformis cells exposed to different hormones. except serotonin dropped to practically the same level approximating the control, and by 120 min those of all except serotonin and T,, fell below the control level, The hormones used in the experiment represented 4 different types; firstly molecules gaining access to the nucleus of vertebrate cells and acting at the genetic level; secondly molecules capable of entering into the nucleus of T. pyriformis; thirdly, hormones representing members of a series in which one hormone is considerably more active in vertebrates than the other; fourthly, molecules with known effect on T. pyr!formis. The forthcoming discussion follows this classification, The hormones capable of entering the cellular nu-
cleus in the vertebrate organism developed the greatest stimulatory action on RNA synthesis of T. pyriformis. Prednisolone--like all steroids--is bound by a cytosol receptor and develops action at the nuclear level (Cake & Litwack, 1975); the same applies to T3 (Samuels et al., 1974: Sterling et al., 1974). Evidence has also been presented of the intranuclear access of serotonin (Korneliussen, 1976; Sud:ir & Csaba, 1979). In view of this, one might be tempted to conclude that the hormones responsible for stimulation of RNA synthesis in vertebrates develop the greatest stimulatory action also at the unicellular level. This interpretation is, however, recognized as false if it is taken into consideration that although T4 also gains access to the cellular nucleus in vertebrates, its effect on T pyriformis was negligible compared to Ta. while epinephrine, having an intranuclear access only in the unicellular (Csaba et al., 1980) had a considerable, although not maximal, influence on the latter. At the same time, we showed in earlier experiments (Csaba et al., 1978) that Tetrahymena does not possess a cytosol receptor for steroids (at least not in the form therefore, have no access to its nucleus, while histamine has (Csaba & Sud~ir, 1979), but it developed nonetheless little (positive) activity under the given conditions of experiment. It appears, in view of the foregoing considerations, that the presence of a hormone in the nucleus it not in itself an indispensable prerequisite of the hormone's nuclear action. Nevertheless, hormonal effects developed at the genetic level do show a parallelism in vertebrates and T. pyriformis. The response of T. pyriformis to T3 and T,L presents a special problem. Both hormones are bound by cytosol receptors and gain access into the nucleus, but T3 is several times as active as T, in the vertebrate organism (Astwood, 1971). This activity difference was still more pronounced under the conditions of experiment, for while vertebrates show a T3-T 4 binding ratio of 4:1. the T. pyriformis cells bound the 2 hormones at the ratio of 10:1. This seems logical as it is known that of the iodine hormones those with a lower iodine content, e.g. diiodothyrosine, stimulate mitosis to a greater degree than those containing more iodine (Csaba & N6meth, 1979). Serotonin differed in action from all other hormones included in the study. It stimulated the synthesis of RNA markedly at 30min, and while the RNA synthesis of the cells exposed to the other hor-
Table 1. Incorporation of [-~H]uridine by RNA in Tetrah.vmena p.vriformis cells exposed to different hormones. (Calculated for 106 cells relative to the control as 100) Control
Epinephrine
Prednisolone
T3
100 100 100 100 100
130.05" 87.04* 393.92 202.61 56.13
287.93 62.95 3623.11 245.97 39.44
188.76 63.54 1667.4 159.78 26.51
Significance of difference from the control: *P < 0.05. t P < 0.02. NS = not significant. Not otherwise indicated = P < 0.01.
Histamine 80.39 43.57 183.4 135.72"162.19
Serotonin 203.04 46.62 1198.56 990.25 824.96
T4 163.44"f" 95.48 "~s 158.51 164.52 126.983'
Time of exposure (rain) 5 15 30 60 120
RNA synthesis in Tetrahymena m o n e s was a p p r o x i m a t i n g the control level by 60 min, and fell below it by 120 min, the serotonin-exposed cells still showed a more than 8-fold increase of RNA over the control after 2 hr. This can be attributed either to a long-term action of serotonin, or to a long lifetime of the R N A stimulated by serotonin. T h e latter h o r m o n e stimulated also the growth of T. pyriformis cells to an extraordinary degree (Csaba & N6meth, 1979), but the information emerging from the present study permits no conclusion on the interrelationship of this p h e n o m e n o n with the increased synthesis of RNA. The 2 peaks of h o r m o n a l action, which were invariably found in each experimental series, suggest that the first peak signifies the first Imembrane-level) encounter o f t h e h o r m o n e with the cell via the c A M P system (Langan, 1970), probably through activation of histone kinase, while the second---superior--peak might be the issue of the development of h o r m o n a l action at the genetic level,
REFERENCES ANDERSEN R. S. (1971) Ribosomal synthesis in greening primary leaves of bean seedling IPhaseolus vulgaris) BiDchem. Physiol. Pflanzen 162, 245-264. ASTWOOD E. B. (1971) Thyroid and antithyroid drugs. In The Pharmacological Basis of Therapeutics, (Edited by GOODMAN L. S. & GILMAN A.), pp. 1466 1500. MacMillan, London. BLt;M J. J. (1967) An adrenergic control system in Tetrahymena. Proc. natn. Acad. Sci. (USA)51, 81-88. CAKE M. H. & LITWACK G. (1975) The glucocorticoid receptor. In Biochemical Action of Hormones (Edited by LI'rWACK G.). pp. 317-390. Academic Press, New York. CSABA G. & LANTOST. (1973) Effect of hormones on protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indoleacetic acid in Tetrahymena pyriformis. Cytobiologie 7, 361-365. CSAaA G. & LA.~TOS T. (1975) Specificity of hormone receptors in Tetrahymena. Experiments with serotonin and histamine antagonists. Cytobiologie ! i. 44--49. CSABA G. & LA.~IOS T. (1975) Effect of insulin on the glucose uptake of protozoa. Experientia 31, 1097-1098. CSAaA G. & LANTOS T. (1976) Effect of epinephrine on glucose metabolism in Tetrahyme~la. Endokrinolo¢lie 68, 239 240.
253
CSABAG. & LANrOS T. (1977) An attempt to differentiate selection and amplification in hormone receptor development. The unicellular model. Differentiation $, 57-59. CSAB^ G., SUDAR F., NAG': S. U. & Domgzv O. (1977) Localization of hormone receptors in Tetrahymena. Protoplasma 91, 179- 189. CSABAG. LANTOST., N^CV S. U., AR/,NVl P. & N,~RAY A. (1978) Effects of steroids on Tetrahymena. Acta biol. reed. germ. 37, 1377-1380. CSABA G. & SL'D,~R F. (1979) Localization of [3H]histaming in the nucleus of Tetrahymena. Aeta morph. Acad. Sci. hung, 27, 89-94. CSAB^ G. & NEM~rH G. (19801 Effect of hormones and their precursors an unicellulars. The selective responsiveness of Tetrahymena. Comp. Biochem. Physiol. 65B, 387-390. CSAaAG., SUDAR F. & PADOS R. 11980) Binding and internalization of [aH]epinephrine in Tetrahymena. Endokrinolo qie. In press. lW^TA H. & KARIYA K. (1973) Adrenergic mechanism in Tetrahymena I. Changes in monoamine oxidase activity during growth. Experientia 29, 265-266. JANAKIVEDI D., DEWEY J. C. & KIDDER G. W. (1966) The biosynthesis of catecholamines in two genera of protozoa. J. biol. Chem. 24, 2576-2578. KARIYA K., SAITO R. & IWATA H. (19741 Adrenergic mechanism in Tetrahymena Ill. cAMP and cell proliferation Jap. J. Pharmae. 24, 129-134. KORNELIUSSEN H. (1976) 5-hydroxytryptamine: autoradiographic evidence for uptake into fibroblast cell nuclei Experientia 32,443-445. LANGANT. A. 11970) Phosphorylation of histones in vivo under the control of cyclic AMP and hormones. Acta Biochem. Psychopharmac. 3, 307-310. ROMANOVYU. A., SAVCHENKO T. V. & ZARENKOVA V. P. (1974) Effect of thyroxin on cell division and the mitotic cycle of Tetrahymena pyr(formis. Biol. Zh. Arm. 27, 9--15. SAMt:EI,S A. H., TSAI J. G. & CASANOVA J. (1974) Thyroid hormone action: in vitro demonstration of putative receptors in isolated nuclei and soluble nuclear extracts. Science 184, 1188 1190. SMILLItN. M. & KROTKOV G. (1960) The estimation of nucleic acid in some algae and higher plants. Can. J. Bot. 38, 31 49. STERLINGK., SALDANHAV. F., BRENXER N. A. & MILCH P. O. (1974~ Cytosol binding protein of thyroxine and triodothyronine hormone in kidney tissue. Nature 250, 661-663. Suo,~R F. & CSABA G. 11979) Localization of [3H]serotonin in the adrenal medulla cells of newborn rats. ,4cta morph. Acta. Sci. hung. 27, 83 87. ZEUTHEN E. (1971) Synchrony in Tetrahymena by heat shocks spread a normal cell generation apart. E xpl cell. Res. 68, 49-52.