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The action of testosterone at the cell level. Testosterone stimulation of the respiration of Euglena gracilis
ARCHIVES
OF
BIOCHEMISTRY
AND
The Action Testosterone
BIOPHYSICS
94,
358-363 (1961)
of Testosterone
Stimulation
at the Cell
of the Respiration
D. E. BUETOW
Level.
of Euglena
gracilid
AND B. H. LEVEDAHL
From the Gerontology Branch, National Heart Institute, National Institutes of Health, Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland; and the Baltimore City Hospitals, Baltimore, Maryland; and the Department of of California, Los Angeles, California Zoology, University Received
March
27,196l
The respiration rate of Euglena in the presence of 2.0 pg. or 0.1 pg. testosterone/ml. medium is about 20 and 14% higher, respectively, than that of controls lacking the steroid. This result obtains whether or not the cells were previously grown in the presence of testosterone. Further, the increased respiration rate of Euglena in the presence of testosterone is dependent upon the presence of a nitrogen source. The testosterone “effect” is reduced when nitrogen is absent from the respiration medium and is restored when nitrogen is reintroduced into the medium. The data suggest that an early effect of this steroid on Euglena is on the permeability of these cells to nitrogen-containing compounds with a resultant effect on protein metabolism.
gation of any possible effects of the steroids on the respiration of these cells. Early experiments (5) showed that Euglena had a higher respiration rate (measured as ~1. OS/ lo6 cells/hr.) in the presence of various concentrations of testosterone, estrone, and hydrocortisone than in the absence of these steroids. The present experiments are an extension of this observation that testosterone affects the respiration rate of intact’ Euglena cells. In addition, since Euglena can grow and respire on a simple, defined medium with inorganic nitrogen as the sole nitrogen source, its respiration in the presence of testosterone with or without nitrogen could also be tested.
INTRODUCTION
The addition of low concentrations (O.l0.001 pg./ml.) of steroid hormones to the medium of the streptomycingrowth bleached free-living flagellate protozoan, Euglena gracilis var. bacillaris, results in an accelerated growth rate for this organism (i-3). Also, the total protein content of Euglena cells grown in the presence of 6.9 x 10-6 or 3.5 X lo-’ M testosterone is higher than that found in cells grown in the absence of testosterone (4). The processes of growth as defined by the synthesis of new material require energy which is supplied to the cells by the oxidation of exogenous substrate. Thus, a consideration of the effects of testosterone and other steroids on the growth and protein content of Euglena prompted an investi-
MATERIALS
AND
METHODS
GROWTH
‘A portion of these data are taken from a dissertation presented (by D.B.) in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of California, Los Angeles, 1959. Work completed at this time was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles (NR-120-336).
The single cell system used throughout these studies was a streptomycin-bleached strain (SMLl) of Euglena gracilis var. bacillaris. Experimental and stock cultures of Euglena were grown axenically on the defined Cramer-Myers medium (6) with the following modifications: (a) vitamin B1 content was increased from 0.01 t,o 0.02 mg./l. and vitamin B,, content was increased from 0.0005 358
ACTION
OF TESTOSTERONE
to 0.01 mg./l.; (b) CoClz was used routinely in place of Co(NO&. Experimental cultures were grown in 300 ml. of the medium contained in l-1. Erlenmeyer flasks kept throughout the growth period in the dark in an incubator maintained at 25°C. In experiment,s in which the cells were grown in the presence of test,osterone, the steroid was dissolved in absolute ethanol (5 mg./lO ml.) and then sterilely pipctted in the desired concentrat,ions into antorlaved culture medium. Corresponding amounts of ethanol were added to t,he control cultures (see Tables). Cultures were inoculated to begin growth with an initial density of 2-10 X lo3 cells/ml.
TABLE
Acetd' OXK".
RESPIRATIONS
Ethano,b
No.
pl. 02/106 cells/hr.
of experiments
_____. N
(a) Washed in basal CramerMyers medium with nitrogen --___-~ 0.061 0.061 0.061
13 0.068 M 0.0034 M
10.1 * 0.4c 10.4 f 0.9 10.5 f 0.5
4 11 __
(b) Washed in basal CramerMyers medium minus nitrogend
RESPIRATION The cells were harvest,ed during the logarithmic phase of growth, concentrated by centrifugation at 2000 r.p.m. for 10 min., and washed twice in either of the following: (a) basal Cramer-Myers medium (minus acetate and vitamins B, and B13) or (b) basal medium wit,h Na,HPOa substituted for (NH&HPO, Cells were then suspended in a few milliliters of the wash medium. Method (b) removed external nitrogen sources from the cells and placed them in a nitrogen-free environment. Final cell concentration range was 1.2-13 X lo6 cells/ml. However, most experiments were run with cell populations of 2.7-4.8 X 10” cells/ml. Unless otherwise not,ed, testosterone in absolute et,hanol (5 mg./lO ml.) was added to CramerMyers medium to give a final steroid conccntration of either 6.9 x lo-“ or 3.5 X 10.’ M. Corresponding amounts of absolute ethanol were added to t,he control flasks. Generally, an additional control was run with each experiment, namely, a flask containing basal medium only (i.e., the “endogenous cont.rol”). In order to examine any effect of testosterone on the endogenous respiration of Euglena. the steroid (6.9 X 10m61M) was added directly to the basal medium. The solution was autoclaved and, upon cooling to room temperature, was used for the respiration experiments. This concentration of t,estosterone is wit,hin the water solubility range of the steroid (7). When 3.5 X 10.’ dl test,osterone was needed, the above solution was diluted with basal medium. d fresh solution was prepared for each experiment. In all experiments, respiration rates were determined in air by the direct method of Warburg (8) at 26.5”C. The cells were equilibrated for lO20 min. After the substrate was tipped in, readings were taken at IO-min. intervals. Only 30 min. was required to est.ablish a linear rate of oxygen upt,ake in all cases. All other conditions were as previously reported (9).
I
OF EUOLEJA CELLS GROWN WITH ETHANOL AND/OR ACETATE AS CARBON SOURCES
ENDOGENOUS
O.OGl
-
12
10.2 f
0.5
a (ias phase, air; temp. 2G.5’C.; pH 6.8. With the exception of a few experiments, the endogenous rate in all cases remained linear for only 3040 min. Values are calculated on the basis of the rates during the first 30 min. of the experiments. b Molarities used during growth. c Mean f S.E. n Nitrogen (i.e., (NH,)zHPOJ reintroduced at start of respiration experiments. TABLE
II
ENINGENOVS RESPIRATIONS OF EUCLEA-A CELLS (DROWNWITH ACETATE AND ETHANOL AS CARBOX S~IJRCES AND ADDED TESTOSTERONE I Acetat@ CClnC”
IIEthanol” UXX”.
Testosterone’ concn.
Numbe r of experiments
Ml. O*/lO” cells/hr.
-I-
M 0.061
10.5 f
l.lr
0.061
10.2 zt 0.G
ClGas phase, air; temp. 26.5’C.; pH 6.8. B Molarities used during growth. c Mean f S.E. RESULTS ENDOGENOUH
RESPIRATION
OF Euglena
The endogenous respiration of Euglena cells grown under various conditions is given in Tables I and II. It was the same in these experiments whether or not the cells had been grown in the presence of testoster-
360
BUETOW TABLE
AND
III). The oxygen uptake values for Euglena cells respiring in the presence of the steroid were significantly different from the control values at about the 0.025 level. The respiration rate of the acetate-ethanol control was the same as that previously reported for the respiration rate of cells in the presence of acetate alone (9, 10).
III
RESPIRATION” OF EUGLENA CELLS GROWN WITH ACETATE AS CARBON SOURCE
Acetate
CiXlC”.
Testosterone” concn.
N”$ber
*“e52ag’ I
ww;;-
uptake
Per cent of controlC
&i$jG =Y
-I&f ! 0.061 0.061 6.9 X 1O-6 M (2.0 pg./ml.)
7 7
22.9 27.5
7% 100 I119.8 f 2.9d
ACETATE-ETHANOL-GROWN
one. In addition, the temporary removal of all external nitrogen sources from these cells (i.e., washing in basal medium minus nitrogen) and the introduction of nitrogen again at the time of respiration had no effect on the endogenous respiration (Table I). The mean endogenous rate under all conditions was 10.3 ~1.02/106 cells/hr.
TESTOSTERONE-GROWN
CELLS
TABLE
Acetateb concn. M
A. 0.061 0.061
B. 0.061 0.061
IV
CELLS GROWN WITH ACETATE AND ETHANOL
Ethanolh concn.
0.0034 0.0034
AS CARBON
SOURCES
Number of experiments
Average 01 uptake in pl. Ox/106 cells/hr.
Per cent of control
3 3
21.7 26.2
100 119.6 f 3.6d
3 3
22.7 25.7
100 113.7 Et 1.3
M
0.068 0.068
CELLS
Euglena were grown on medium with 2.0 pg./ml. or 0.1 pg./ml. testosterone. The amounts of testosterone added to the respiration vessels were the same as those used during growth. Cells respiring in the presence of 2.0 pg. or 0.1 pg. testosterone/ml. showed an average respiration rate of 119 and 115% of the control rate, respectively (Table V). Control values were like those for cells grown in the absence of the steroid
The mean respiration rate of cells in the presence of 6.9 x 10m6 M testosterone was 120% of that of the control cells (Table
RESPIRATIONS OF EUCLENA
CELLS
Table IV gives the results of these experimcnts. The respiration rate of cells in the presence of 2.0 pg. testosterone/ml. was 120% of the acetate-ethanol control cells, an increase like that noted for the corresponding experiment on acetate-grown cells (Table III). One-tenth microgram of testosterone/ml. medium also stimulated the respiration of acetate-ethanol-grown cells, but less so than testosterone in a concentration of 2.0 pg./ml. (i.e., 114% vs. 120%).
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Dissolved in absolute ethanol and added at time of respiration experiments. Controls contained appropriate amounts of ethanol. c Control standardized at 100 in each experiment. d Mean & S.E.
ACETATE-GROWN
LEVEDAHL
O’u
6.9 X 10-O M (2.0 pg./ml.)
3.5 x lo-‘n/r (0.1 pg./ml.)
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Added to basal medium at time of respiration growth. c Added at time of respiration experiments. dMean f S.E.
experiments;
molarities
were the same as used during
ACTIOS
OF TESTOSTEROSE TABLE
R~SI~IRATION~~
V
OF Ec~cms,t CELLS GROWN WITH ACETATE ASD &HANOL S~LTRCES ASD ADDED TESTOSTERONE Number of experiments
Testosterone” concn:
A. Grown
R. Grown 0.0034 0.0034
AS
Average 0~ uptake injdl. O?,‘W cells/hr.
CARBON
Per cent of control
in 6.9 X 1OM6M testosteroner 3 3
6.9 x 10-G u (2.0 pg./ml.)
O.Oiil 0. Oiil
361
21.5 25.5
in 3.7 X 10-j M testosterone” ‘4 4
3.5 x 10-T 112 (0.1 /Jg./‘ml.)
20.7 23.7
100 114.7 Xt 3.5
4 Gas phase, air; temp., 26.5’C.; pH ti.8. * Added to basal medium at time of respiration experiments; molarities were the same as used during growth. c For respiration experiments, cells were washed in testosterone-free medium and divided into t,wo groups: testosterone was added to one and omitted from the other (i.e., the controls). d Mean f S.E.
(Table IV). ils with the endogcnous experiments, there appeared to be no “carryover” effect of the steroid from growth to the respiration measurements. EXDOGENOCS
TABLE
Testosterod concn.
RESPIRATION IN PRESEKCE OF TESTOSTERONE
These experiments were designed to check any effect of testosterone on the respiration of cells in the absence of a carbon source. Acetat’c-grown cells were washed with basal medium tither containing or lacking nitrogen (i.e., (NHJ2HP04). Nitrogen with or without testosterone was then introduced at the start of the respiration experiment. When 3.5 X lop7 M testosterone was present! the respiration rate of cells washed in medium containing nitrogen was 110% of the control while the respiration rate of cells washed in medium lacking nitrogen was 123% of the control (Table VI). mhen 6.9 X lo-” M testosterone was present, corresponding values were 114 and 122% of the controls. Testosterone increased the cndogenous respiration of Euglena, but the increase m-as less in cells continually exposed to a nitrogen source than in cells prepared in nitrogen-free medium and then exposed again to nitrogen at the same time as the steroid. Further, in the latter case, both
VI
ENDOGENOGS RESPIR~TI~N~~ OF BC’CZESA~~ IN THE PRESENCE OF TESTOSTEROXE Plumber of experiments
Jl
Per cent of control 0:
B. Washed in basal medium with nitrogen G.9 x IO-6 3.5 x 10-i
3 2
114.1 f 0.7” 109.7 zt --E
B. Washed in basal medium minus nitrogen/ 8.9 x IO-6 3.5 x 10-T
3 3
122.3 f 123.2 f
2.6 5.0
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Grown with sodium acetate as carbon source. c Added at time of respiration experiments. diMean f S.E.; control in each experiment was standardized at 100yl; for control values, see Table I. c Mean of two experiments. f Nitrogen (i.e., (NH,)*HPO,) reinkoduced at start of respiration experiments.
concentrations of testosterone showed about) the same per cent increase over the conkols.
BUETOW
X2
ASI)
IXVEL)AHI,
merit with NI-I,Cl as a nitrogen source gave tlir saniv results.’
1Number Mediumminus Testosteroned! of nitrogenCc” cxpcriconcn. c”“trol)~i merits .v 0.9 x IO--* 3.5 x 10-7
.\lediumplus nitropzn c’b control) I
1 3
104.3 f 1.0 105.1 f 3.0
117.2 f 111.6 f
I.0 2.6
(1(hs phase, air; kmp., 2li.5’C.; pH 6.8. * (;rown with sodium acetate as wrtwn source. c (SH,),Hl’O4 d IXssolved in absolute ethanol and added at time of respiraf,ion expcrimente. Conl.rols contained appropriate amounts of ethanol. 0 Controls were standardized at loO~/~ in each cnse. See text for control wlues.
~ITROCXS DEPESL)ENCE OF TESTOSTEROSE EFFECT These experiu1cnt.s were designed to test whet.her or not the effect of testosterone on the respiration of Euglena was dependent upon the presence of nitrogen. Acetategrown cells were washed in nitrogen-free basal medium. The respiration rate of the controls (i.e., on medium containing sodium acetate as carbon source but lacking a nitrogon source) was 95.27’, of t.hc rwl)iration rate of Ezcglenn on medium containing both carbon and nitrogen sources. All values in Table VII arc relat.ed t.o the nit.rogen-free cont,rols. \\‘hcn the cells wcrc resljiring in the :tbwnce of nitrogen but with aclticd tcstostclrone, little differctnco was noted owr the cont.rolS, i.e., 104 and 105% of t.ht: control wlucs for the two concentrations of testost.crOIW used. However, when a nitrogen source? i.e., (XH., )JIPOq , was added at the s:~tn(’ tiniv its the tcstostcronc, the values were inrrcascd to 117 and 112% of t.he controls, rwlwtively. Thcee 1ntt.w increases in the rwpiration rate of E~glcnn wew like those noted for Euglena respiring in the lw3ence of total medium and tclstorteronc (Tablts 111-Y). The effect. of tcatoswrone on the respiration of Euglenn was rcduccd in the :~bscnce of nitrogen and was restowd when nitrogen was ad&v1 again. A single oxperi-
The respiration rate of Euglenu in the presence of testosterone is higher t.lian that obscrvcd in t,hc absence of this steroid hormone. Approximately the sanw quantitative inrrcaw in respiration rate in t.lie presence of a giwn conccnt.ration of tcstoswone results whether or not the cells had heen grown in the presence of the steroid. Also, the respiration rate of cells grown in the prcsencc of testosterone hut respiring in its abscncc is the same as cells which wcrc never e.cposed to the steroid. The same is true of the cndogcnous respiration rutc of Btcglencr. Such observations argue against any “carry-over” effect of the steroid from tlw growth to that respiration expcrimrnts. It is further noted that the effect of tcetostvronc on tlic respiration of Buqlew is like t.liat olwrvcd with similmr concentrations of other stwoids on luminous bacteria I ll)! yeast (12)) and mammalian fibroblast cells maintained in culture (13). Tlic influence of tcstostcrone on the respiration of EwgZe,zn also parallels iLs a&on in intact mammalian target organ svstems ( 14). Increased respiratory activity Is genclrallv noted in the mammal when t.cstostcrone is added in viva. In vitro addition of steroids:, tiowcwr, has resulted in conflicting reports concerning the influence of stwoids on rcqiration. This problem does not ap~W:II* to IX encountered rvith t.lw intact kuglena single wll qstcm. The same me(liuiil (i.e., “(lnVir(~rliilerlt” I is uwd for both growth :ui(l rcsl)iration, and steroids can be aclclc~l to thv nwtlium either during growth or during respiration or during both. Thv relationship of tvstost~crone to protwin ant1 amino acid metabolism and tot.aI nitrogcri contt>nt in various systems has rcwived con>idvr:tblc attt,nt.ion ( 14-19 I. It 11:~ also bwn notctl that Euglentr grown in t.lw prewnce of t&ostcronc contain a higlirr total protAn content, than .E:‘lcglerrcc grown in t,hc :ib+encc of this stwoid (4). Furtlwr, tlw prcwnt c~xpcrimt~nt!: show that
ACTION
OF TESTOSTEROSE
the effect of testosterone on the respiration of Euglena gracilis is dependent upon the presence of a nitrogen source (Table VII). In addition, the effect of testosterone on Euglena cells continually exposed to nitrogen is less than when the cells are denied nit,rogen for a period of time prior to the respiration measurements (Table VI). Similar studies with other steroid hormones would be of use in determining urhether or not the present results are testosteronespecific. However, t,he present experiments, along with the observation that the effect of testosterone occurs as soon as the cells are exposed to the steroid (i.e., O2 uptake was linear throughout the time of measurement), suggest that the effect of testost,erone is on the permeability of the cells to nitrogen-containing compounds or, perhaps, on some enzyme system involved in the synthesis of proteins from simple nitrogencontaining compounds. ACKNOWLEDGMENT The authors express their gratitude to Mrs. M. Robinson for technical assistance during these experiments. REFERENCES 1. B~ETOW, D. E., .~ND LEVEDAHL, B. H., Arch. Biochem. Biophys. 73,273 (1958). 2. LEVEDAHL, B. H., BUETOW, D. E., AND COLLINS, J. D., Federation Proc. 17, 95 (1958).
x:3
3. BUETOW, D. E., ASD LEVED~HL, B. H., Arch. Rio&em. Biophys. 86,34 (1960). 4. B~ETOW, D. E., AND LEVEDAHL, B. H., Fcderation Proc. 20,197 (1961). 5. BUETOW, D. E., Ph.D. Thesis, Univ. of California, Los Angeles, 1959. 6. CRAMER, M., ASD ME*ERS, J., Arch. Xikrobiol. 17,384 (1952). 7. EIK-YES, B., SCHELL~LIAN, J. A., LWIRY, R., AND SAMUELS, I,. T., J. Biol. Chem. 206, 411 (1954). 8. KX~REIT, W., BURRIS, R., AND STAUFFER, J., “Manometric Techniques.” Burgess Publ. Co., Minneapolis, 1959. 9. WILSOX, B. R., B~ETOW, D. E., JAHN, T. L., AND LEVEDAHL, B. H., Exptl. Cell Research
18,454 (1959). 16. D.4NFORTH, Jv. F., AND &-ILSON, B. w., J. Protozool. 4,52 (1957). 11. TAYLOR, G. W., J. Cellular Comp. Physiol. 1, 297 (1932). 12. SALMONY, D., Biochem. J. 62, 411 (1956). 13. GROSSFELD,H., Endocrinology 65, 777 (1959). 14. ROBERTS, S., AND f&co, C. M., Physiol. Reus. 33,593 (1953). 15. MARVIN, H. PI’., AND AWAP.4R.4, J., Proc. sot. Exptl. Biol. ,Ved. 72, 93 (1949). 16. GASSSER, F. X., AND HOPWOOD, M. L., Proc. Sot. Exptl. Biol. Med. 81, 37 (1952). 17. ~OALL, M. u'., RIGGS. T. R.. R.4LKER. L. M.. AND CHRISTESSEN, H. N., Science li6, 1002 (1957). 18. METCALF, W:., AND GROSS, E., Science 132, 41 (1960). 19. PORTER, J. C., AND MELA~Y, R. M., Endooinology 51,412 (1952).
OF
BIOCHEMISTRY
AND
The Action Testosterone
BIOPHYSICS
94,
358-363 (1961)
of Testosterone
Stimulation
at the Cell
of the Respiration
D. E. BUETOW
Level.
of Euglena
gracilid
AND B. H. LEVEDAHL
From the Gerontology Branch, National Heart Institute, National Institutes of Health, Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland; and the Baltimore City Hospitals, Baltimore, Maryland; and the Department of of California, Los Angeles, California Zoology, University Received
March
27,196l
The respiration rate of Euglena in the presence of 2.0 pg. or 0.1 pg. testosterone/ml. medium is about 20 and 14% higher, respectively, than that of controls lacking the steroid. This result obtains whether or not the cells were previously grown in the presence of testosterone. Further, the increased respiration rate of Euglena in the presence of testosterone is dependent upon the presence of a nitrogen source. The testosterone “effect” is reduced when nitrogen is absent from the respiration medium and is restored when nitrogen is reintroduced into the medium. The data suggest that an early effect of this steroid on Euglena is on the permeability of these cells to nitrogen-containing compounds with a resultant effect on protein metabolism.
gation of any possible effects of the steroids on the respiration of these cells. Early experiments (5) showed that Euglena had a higher respiration rate (measured as ~1. OS/ lo6 cells/hr.) in the presence of various concentrations of testosterone, estrone, and hydrocortisone than in the absence of these steroids. The present experiments are an extension of this observation that testosterone affects the respiration rate of intact’ Euglena cells. In addition, since Euglena can grow and respire on a simple, defined medium with inorganic nitrogen as the sole nitrogen source, its respiration in the presence of testosterone with or without nitrogen could also be tested.
INTRODUCTION
The addition of low concentrations (O.l0.001 pg./ml.) of steroid hormones to the medium of the streptomycingrowth bleached free-living flagellate protozoan, Euglena gracilis var. bacillaris, results in an accelerated growth rate for this organism (i-3). Also, the total protein content of Euglena cells grown in the presence of 6.9 x 10-6 or 3.5 X lo-’ M testosterone is higher than that found in cells grown in the absence of testosterone (4). The processes of growth as defined by the synthesis of new material require energy which is supplied to the cells by the oxidation of exogenous substrate. Thus, a consideration of the effects of testosterone and other steroids on the growth and protein content of Euglena prompted an investi-
MATERIALS
AND
METHODS
GROWTH
‘A portion of these data are taken from a dissertation presented (by D.B.) in partial fulfillment of the requirements for the degree of Doctor of Philosophy, University of California, Los Angeles, 1959. Work completed at this time was aided by a contract between the Office of Naval Research, Department of the Navy, and the University of California, Los Angeles (NR-120-336).
The single cell system used throughout these studies was a streptomycin-bleached strain (SMLl) of Euglena gracilis var. bacillaris. Experimental and stock cultures of Euglena were grown axenically on the defined Cramer-Myers medium (6) with the following modifications: (a) vitamin B1 content was increased from 0.01 t,o 0.02 mg./l. and vitamin B,, content was increased from 0.0005 358
ACTION
OF TESTOSTERONE
to 0.01 mg./l.; (b) CoClz was used routinely in place of Co(NO&. Experimental cultures were grown in 300 ml. of the medium contained in l-1. Erlenmeyer flasks kept throughout the growth period in the dark in an incubator maintained at 25°C. In experiment,s in which the cells were grown in the presence of test,osterone, the steroid was dissolved in absolute ethanol (5 mg./lO ml.) and then sterilely pipctted in the desired concentrat,ions into antorlaved culture medium. Corresponding amounts of ethanol were added to t,he control cultures (see Tables). Cultures were inoculated to begin growth with an initial density of 2-10 X lo3 cells/ml.
TABLE
Acetd' OXK".
RESPIRATIONS
Ethano,b
No.
pl. 02/106 cells/hr.
of experiments
_____. N
(a) Washed in basal CramerMyers medium with nitrogen --___-~ 0.061 0.061 0.061
13 0.068 M 0.0034 M
10.1 * 0.4c 10.4 f 0.9 10.5 f 0.5
4 11 __
(b) Washed in basal CramerMyers medium minus nitrogend
RESPIRATION The cells were harvest,ed during the logarithmic phase of growth, concentrated by centrifugation at 2000 r.p.m. for 10 min., and washed twice in either of the following: (a) basal Cramer-Myers medium (minus acetate and vitamins B, and B13) or (b) basal medium wit,h Na,HPOa substituted for (NH&HPO, Cells were then suspended in a few milliliters of the wash medium. Method (b) removed external nitrogen sources from the cells and placed them in a nitrogen-free environment. Final cell concentration range was 1.2-13 X lo6 cells/ml. However, most experiments were run with cell populations of 2.7-4.8 X 10” cells/ml. Unless otherwise not,ed, testosterone in absolute et,hanol (5 mg./lO ml.) was added to CramerMyers medium to give a final steroid conccntration of either 6.9 x lo-“ or 3.5 X 10.’ M. Corresponding amounts of absolute ethanol were added to t,he control flasks. Generally, an additional control was run with each experiment, namely, a flask containing basal medium only (i.e., the “endogenous cont.rol”). In order to examine any effect of testosterone on the endogenous respiration of Euglena. the steroid (6.9 X 10m61M) was added directly to the basal medium. The solution was autoclaved and, upon cooling to room temperature, was used for the respiration experiments. This concentration of t,estosterone is wit,hin the water solubility range of the steroid (7). When 3.5 X 10.’ dl test,osterone was needed, the above solution was diluted with basal medium. d fresh solution was prepared for each experiment. In all experiments, respiration rates were determined in air by the direct method of Warburg (8) at 26.5”C. The cells were equilibrated for lO20 min. After the substrate was tipped in, readings were taken at IO-min. intervals. Only 30 min. was required to est.ablish a linear rate of oxygen upt,ake in all cases. All other conditions were as previously reported (9).
I
OF EUOLEJA CELLS GROWN WITH ETHANOL AND/OR ACETATE AS CARBON SOURCES
ENDOGENOUS
O.OGl
-
12
10.2 f
0.5
a (ias phase, air; temp. 2G.5’C.; pH 6.8. With the exception of a few experiments, the endogenous rate in all cases remained linear for only 3040 min. Values are calculated on the basis of the rates during the first 30 min. of the experiments. b Molarities used during growth. c Mean f S.E. n Nitrogen (i.e., (NH,)zHPOJ reintroduced at start of respiration experiments. TABLE
II
ENINGENOVS RESPIRATIONS OF EUCLEA-A CELLS (DROWNWITH ACETATE AND ETHANOL AS CARBOX S~IJRCES AND ADDED TESTOSTERONE I Acetat@ CClnC”
IIEthanol” UXX”.
Testosterone’ concn.
Numbe r of experiments
Ml. O*/lO” cells/hr.
-I-
M 0.061
10.5 f
l.lr
0.061
10.2 zt 0.G
ClGas phase, air; temp. 26.5’C.; pH 6.8. B Molarities used during growth. c Mean f S.E. RESULTS ENDOGENOUH
RESPIRATION
OF Euglena
The endogenous respiration of Euglena cells grown under various conditions is given in Tables I and II. It was the same in these experiments whether or not the cells had been grown in the presence of testoster-
360
BUETOW TABLE
AND
III). The oxygen uptake values for Euglena cells respiring in the presence of the steroid were significantly different from the control values at about the 0.025 level. The respiration rate of the acetate-ethanol control was the same as that previously reported for the respiration rate of cells in the presence of acetate alone (9, 10).
III
RESPIRATION” OF EUGLENA CELLS GROWN WITH ACETATE AS CARBON SOURCE
Acetate
CiXlC”.
Testosterone” concn.
N”$ber
*“e52ag’ I
ww;;-
uptake
Per cent of controlC
&i$jG =Y
-I&f ! 0.061 0.061 6.9 X 1O-6 M (2.0 pg./ml.)
7 7
22.9 27.5
7% 100 I119.8 f 2.9d
ACETATE-ETHANOL-GROWN
one. In addition, the temporary removal of all external nitrogen sources from these cells (i.e., washing in basal medium minus nitrogen) and the introduction of nitrogen again at the time of respiration had no effect on the endogenous respiration (Table I). The mean endogenous rate under all conditions was 10.3 ~1.02/106 cells/hr.
TESTOSTERONE-GROWN
CELLS
TABLE
Acetateb concn. M
A. 0.061 0.061
B. 0.061 0.061
IV
CELLS GROWN WITH ACETATE AND ETHANOL
Ethanolh concn.
0.0034 0.0034
AS CARBON
SOURCES
Number of experiments
Average 01 uptake in pl. Ox/106 cells/hr.
Per cent of control
3 3
21.7 26.2
100 119.6 f 3.6d
3 3
22.7 25.7
100 113.7 Et 1.3
M
0.068 0.068
CELLS
Euglena were grown on medium with 2.0 pg./ml. or 0.1 pg./ml. testosterone. The amounts of testosterone added to the respiration vessels were the same as those used during growth. Cells respiring in the presence of 2.0 pg. or 0.1 pg. testosterone/ml. showed an average respiration rate of 119 and 115% of the control rate, respectively (Table V). Control values were like those for cells grown in the absence of the steroid
The mean respiration rate of cells in the presence of 6.9 x 10m6 M testosterone was 120% of that of the control cells (Table
RESPIRATIONS OF EUCLENA
CELLS
Table IV gives the results of these experimcnts. The respiration rate of cells in the presence of 2.0 pg. testosterone/ml. was 120% of the acetate-ethanol control cells, an increase like that noted for the corresponding experiment on acetate-grown cells (Table III). One-tenth microgram of testosterone/ml. medium also stimulated the respiration of acetate-ethanol-grown cells, but less so than testosterone in a concentration of 2.0 pg./ml. (i.e., 114% vs. 120%).
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Dissolved in absolute ethanol and added at time of respiration experiments. Controls contained appropriate amounts of ethanol. c Control standardized at 100 in each experiment. d Mean & S.E.
ACETATE-GROWN
LEVEDAHL
O’u
6.9 X 10-O M (2.0 pg./ml.)
3.5 x lo-‘n/r (0.1 pg./ml.)
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Added to basal medium at time of respiration growth. c Added at time of respiration experiments. dMean f S.E.
experiments;
molarities
were the same as used during
ACTIOS
OF TESTOSTEROSE TABLE
R~SI~IRATION~~
V
OF Ec~cms,t CELLS GROWN WITH ACETATE ASD &HANOL S~LTRCES ASD ADDED TESTOSTERONE Number of experiments
Testosterone” concn:
A. Grown
R. Grown 0.0034 0.0034
AS
Average 0~ uptake injdl. O?,‘W cells/hr.
CARBON
Per cent of control
in 6.9 X 1OM6M testosteroner 3 3
6.9 x 10-G u (2.0 pg./ml.)
O.Oiil 0. Oiil
361
21.5 25.5
in 3.7 X 10-j M testosterone” ‘4 4
3.5 x 10-T 112 (0.1 /Jg./‘ml.)
20.7 23.7
100 114.7 Xt 3.5
4 Gas phase, air; temp., 26.5’C.; pH ti.8. * Added to basal medium at time of respiration experiments; molarities were the same as used during growth. c For respiration experiments, cells were washed in testosterone-free medium and divided into t,wo groups: testosterone was added to one and omitted from the other (i.e., the controls). d Mean f S.E.
(Table IV). ils with the endogcnous experiments, there appeared to be no “carryover” effect of the steroid from growth to the respiration measurements. EXDOGENOCS
TABLE
Testosterod concn.
RESPIRATION IN PRESEKCE OF TESTOSTERONE
These experiments were designed to check any effect of testosterone on the respiration of cells in the absence of a carbon source. Acetat’c-grown cells were washed with basal medium tither containing or lacking nitrogen (i.e., (NHJ2HP04). Nitrogen with or without testosterone was then introduced at the start of the respiration experiment. When 3.5 X lop7 M testosterone was present! the respiration rate of cells washed in medium containing nitrogen was 110% of the control while the respiration rate of cells washed in medium lacking nitrogen was 123% of the control (Table VI). mhen 6.9 X lo-” M testosterone was present, corresponding values were 114 and 122% of the controls. Testosterone increased the cndogenous respiration of Euglena, but the increase m-as less in cells continually exposed to a nitrogen source than in cells prepared in nitrogen-free medium and then exposed again to nitrogen at the same time as the steroid. Further, in the latter case, both
VI
ENDOGENOGS RESPIR~TI~N~~ OF BC’CZESA~~ IN THE PRESENCE OF TESTOSTEROXE Plumber of experiments
Jl
Per cent of control 0:
B. Washed in basal medium with nitrogen G.9 x IO-6 3.5 x 10-i
3 2
114.1 f 0.7” 109.7 zt --E
B. Washed in basal medium minus nitrogen/ 8.9 x IO-6 3.5 x 10-T
3 3
122.3 f 123.2 f
2.6 5.0
a Gas phase, air; temp., 26.5’C.; pH 6.8. b Grown with sodium acetate as carbon source. c Added at time of respiration experiments. diMean f S.E.; control in each experiment was standardized at 100yl; for control values, see Table I. c Mean of two experiments. f Nitrogen (i.e., (NH,)*HPO,) reinkoduced at start of respiration experiments.
concentrations of testosterone showed about) the same per cent increase over the conkols.
BUETOW
X2
ASI)
IXVEL)AHI,
merit with NI-I,Cl as a nitrogen source gave tlir saniv results.’
1Number Mediumminus Testosteroned! of nitrogenCc” cxpcriconcn. c”“trol)~i merits .v 0.9 x IO--* 3.5 x 10-7
.\lediumplus nitropzn c’b control) I
1 3
104.3 f 1.0 105.1 f 3.0
117.2 f 111.6 f
I.0 2.6
(1(hs phase, air; kmp., 2li.5’C.; pH 6.8. * (;rown with sodium acetate as wrtwn source. c (SH,),Hl’O4 d IXssolved in absolute ethanol and added at time of respiraf,ion expcrimente. Conl.rols contained appropriate amounts of ethanol. 0 Controls were standardized at loO~/~ in each cnse. See text for control wlues.
~ITROCXS DEPESL)ENCE OF TESTOSTEROSE EFFECT These experiu1cnt.s were designed to test whet.her or not the effect of testosterone on the respiration of Euglena was dependent upon the presence of nitrogen. Acetategrown cells were washed in nitrogen-free basal medium. The respiration rate of the controls (i.e., on medium containing sodium acetate as carbon source but lacking a nitrogon source) was 95.27’, of t.hc rwl)iration rate of Ezcglenn on medium containing both carbon and nitrogen sources. All values in Table VII arc relat.ed t.o the nit.rogen-free cont,rols. \\‘hcn the cells wcrc resljiring in the :tbwnce of nitrogen but with aclticd tcstostclrone, little differctnco was noted owr the cont.rolS, i.e., 104 and 105% of t.ht: control wlucs for the two concentrations of testost.crOIW used. However, when a nitrogen source? i.e., (XH., )JIPOq , was added at the s:~tn(’ tiniv its the tcstostcronc, the values were inrrcascd to 117 and 112% of t.he controls, rwlwtively. Thcee 1ntt.w increases in the rwpiration rate of E~glcnn wew like those noted for Euglena respiring in the lw3ence of total medium and tclstorteronc (Tablts 111-Y). The effect. of tcatoswrone on the respiration of Euglenn was rcduccd in the :~bscnce of nitrogen and was restowd when nitrogen was ad&v1 again. A single oxperi-
The respiration rate of Euglenu in the presence of testosterone is higher t.lian that obscrvcd in t,hc absence of this steroid hormone. Approximately the sanw quantitative inrrcaw in respiration rate in t.lie presence of a giwn conccnt.ration of tcstoswone results whether or not the cells had heen grown in the presence of the steroid. Also, the respiration rate of cells grown in the prcsencc of testosterone hut respiring in its abscncc is the same as cells which wcrc never e.cposed to the steroid. The same is true of the cndogcnous respiration rutc of Btcglencr. Such observations argue against any “carry-over” effect of the steroid from tlw growth to that respiration expcrimrnts. It is further noted that the effect of tcetostvronc on tlic respiration of Buqlew is like t.liat olwrvcd with similmr concentrations of other stwoids on luminous bacteria I ll)! yeast (12)) and mammalian fibroblast cells maintained in culture (13). Tlic influence of tcstostcrone on the respiration of EwgZe,zn also parallels iLs a&on in intact mammalian target organ svstems ( 14). Increased respiratory activity Is genclrallv noted in the mammal when t.cstostcrone is added in viva. In vitro addition of steroids:, tiowcwr, has resulted in conflicting reports concerning the influence of stwoids on rcqiration. This problem does not ap~W:II* to IX encountered rvith t.lw intact kuglena single wll qstcm. The same me(liuiil (i.e., “(lnVir(~rliilerlt” I is uwd for both growth :ui(l rcsl)iration, and steroids can be aclclc~l to thv nwtlium either during growth or during respiration or during both. Thv relationship of tvstost~crone to protwin ant1 amino acid metabolism and tot.aI nitrogcri contt>nt in various systems has rcwived con>idvr:tblc attt,nt.ion ( 14-19 I. It 11:~ also bwn notctl that Euglentr grown in t.lw prewnce of t&ostcronc contain a higlirr total protAn content, than .E:‘lcglerrcc grown in t,hc :ib+encc of this stwoid (4). Furtlwr, tlw prcwnt c~xpcrimt~nt!: show that
ACTION
OF TESTOSTEROSE
the effect of testosterone on the respiration of Euglena gracilis is dependent upon the presence of a nitrogen source (Table VII). In addition, the effect of testosterone on Euglena cells continually exposed to nitrogen is less than when the cells are denied nit,rogen for a period of time prior to the respiration measurements (Table VI). Similar studies with other steroid hormones would be of use in determining urhether or not the present results are testosteronespecific. However, t,he present experiments, along with the observation that the effect of testosterone occurs as soon as the cells are exposed to the steroid (i.e., O2 uptake was linear throughout the time of measurement), suggest that the effect of testost,erone is on the permeability of the cells to nitrogen-containing compounds or, perhaps, on some enzyme system involved in the synthesis of proteins from simple nitrogencontaining compounds. ACKNOWLEDGMENT The authors express their gratitude to Mrs. M. Robinson for technical assistance during these experiments. REFERENCES 1. B~ETOW, D. E., .~ND LEVEDAHL, B. H., Arch. Biochem. Biophys. 73,273 (1958). 2. LEVEDAHL, B. H., BUETOW, D. E., AND COLLINS, J. D., Federation Proc. 17, 95 (1958).
x:3
3. BUETOW, D. E., ASD LEVED~HL, B. H., Arch. Rio&em. Biophys. 86,34 (1960). 4. B~ETOW, D. E., AND LEVEDAHL, B. H., Fcderation Proc. 20,197 (1961). 5. BUETOW, D. E., Ph.D. Thesis, Univ. of California, Los Angeles, 1959. 6. CRAMER, M., ASD ME*ERS, J., Arch. Xikrobiol. 17,384 (1952). 7. EIK-YES, B., SCHELL~LIAN, J. A., LWIRY, R., AND SAMUELS, I,. T., J. Biol. Chem. 206, 411 (1954). 8. KX~REIT, W., BURRIS, R., AND STAUFFER, J., “Manometric Techniques.” Burgess Publ. Co., Minneapolis, 1959. 9. WILSOX, B. R., B~ETOW, D. E., JAHN, T. L., AND LEVEDAHL, B. H., Exptl. Cell Research
18,454 (1959). 16. D.4NFORTH, Jv. F., AND &-ILSON, B. w., J. Protozool. 4,52 (1957). 11. TAYLOR, G. W., J. Cellular Comp. Physiol. 1, 297 (1932). 12. SALMONY, D., Biochem. J. 62, 411 (1956). 13. GROSSFELD,H., Endocrinology 65, 777 (1959). 14. ROBERTS, S., AND f&co, C. M., Physiol. Reus. 33,593 (1953). 15. MARVIN, H. PI’., AND AWAP.4R.4, J., Proc. sot. Exptl. Biol. ,Ved. 72, 93 (1949). 16. GASSSER, F. X., AND HOPWOOD, M. L., Proc. Sot. Exptl. Biol. Med. 81, 37 (1952). 17. ~OALL, M. u'., RIGGS. T. R.. R.4LKER. L. M.. AND CHRISTESSEN, H. N., Science li6, 1002 (1957). 18. METCALF, W:., AND GROSS, E., Science 132, 41 (1960). 19. PORTER, J. C., AND MELA~Y, R. M., Endooinology 51,412 (1952).