Life Sciences Vol. 11, Part I, pp . 545-555,1972 .

Pergamon PressPrinted in Great Britain

EFFECT OF ADRENERGIC REACTIVE DRUGS ON

TETRAHYMENA PYRIFORMIS IN A CHEMICALLY DEFINED MEDIUM

Linda Lowry and Robert S . Gordee

The Lilly Research LaboratoriesEli Lilly and Company

Indianapolis, Indiana 46206(Received 25 February 1972 ; in final form 19 April 1972)

Glucose, reserpine, dichloroisoproterenol (DCI)or propranolol affected glycogen synthesis of Tetrahymenapyriformis strain W with only slight effects on growthin a defined medium . The presence of glucose increasedglycogen synthesis during stationary phase while reser-pine, DCI or propranolol in defined medium with orwithout glucose reduced glycogen synthesis . Incorpora-tion studies with [14C ]tyrosine and [ 14 C]dihydroxyphenyl-alanine did not substantiate the presence of a. functionalcatecholamine system in our strain of Tetrahymenapyriformis .

The presence

Summary

Introduction

of norepinephrine and epinephrine in Tetra-

hymens grown in a defined medium has been reported (1) . Blum

showed that when the organisms were

reserpine and propranolol inhibited cell growth and reserpine,

propranolol and DCI inhibited glycogen synthesis

reported that the inhibitory effect of reserpine

partially reversed by the addition of glucose

Drug-induced reductions in glycogen synthesis

to increased glycogen phosphorylase

citrate lyase activity (4,5) .

such as those found in Tetrahymena , are known

synthetase and phosphorylase .

Tetrahymena may contain a functional

system .

(2) . He also

on growth was

to the medium (3) .

have been attributed

activity and decreased iso-

In mammalian cells, catecholamines,

to control glycogen

These findings indicate that

adrenergic metabolic control

545

grown in an undefined medium,

546

Growth of Tetrahymena pyriformie

Vol . 11, No. il

The conclusion that Tetrahymena contains a functional

adrenergic metabolic control system has been drawn primarily

from results obtained in an undefined medium . We felt that this

conclusion needed to be substantiated in a defined medium and

proceeded to study the effects of adrenergic blocking agents on

Tetrahymena and to investigate the presence of catecholamines in

our strain .

Materials and Methods

Tetrahymena pyriformis , strain W, was grown axenically in

250 ml Falcon tissue culture flasks at 25°C without shaking .

The medium used was the same basal medium described previously

(6) except that 0 .08, (w/v) MgC12 was substituted for MgCO 3 ,

0 .033 (w/v) CaC12 (anhyd) was substituted for CaCO3 ; 0 .03% (w/v)

L-proline, 0 .01% (w/v) L-serine, 0 .5% (w/v) dextrine and 0 .2%

(w/v) Na t glycerophosphate were also added . When 25 mM D-

glucose was added to the medium, dextrine

phate were deleted . The pH of the medium

glucose was 6 .0 . All chemicals were dissolved in deionized

water . The medium and all chemical solutions were sterilized

by passage through a 0 .45 u Millipore filter . Medium containing

dextrine was prefiltered through a #1 Whatman filter paper . The

inoculum was prepared from cultures 72 hrs old . Each test flask

contained a total of 25 ml ; consisting of 23 ml medium, 1 ml

chemical addition or deionized water (control) and 1 ml inoculum .

Growth was measured by counting an aliquot of cells sus-

pended in 0 .996 (w/v) particle-free saline with a model A Coulter

Counter (280 u orifice, 2 ml draw, threshold 10, gain trim 3,

aperture current 80) . Approximately 2 x 10 4 cells removed from

a flask were ruptured by centrifugation at 850 g for 7 min ; the

resulting pellet was washed twice with 0 .996 saline, and then

and Nat glycerophos-

with and without

Vol . 11, No. 11

Growth of Tetrahymena pyriformis

547

immediately frozen in 1 ml deionized water and stored at 0°C for

glycogen determination . The pellet was treated with 30% KOH in

boiling water for 3 min . Two drops of 10% ZnS04 and 2 .5 ml 95%

ethanol were added . After 5 min, samples were centrifuged for

10 min at 350 g and then placed at 0°C for 20 min . The samples

were then centrifuged at 350 g for 10 min . The supernatant was

discarded, and the precipitate was dissolved in 2 ml of 1N H2SO4

and then hydrolyzed in a boiling water bath for 2 hr . The pH was

adjusted to 7.0 with 1.25 N NaOH after samples were cooled . The

volume of each sample was adjusted to 6 ml and then centrifuged

at 350 g for 10 min . Glycogen was measured as glucose (7) . Each

experimental value represents the average of at least two repli-

cates .

Cells grown in 1000 ml Erlenmeyer flasks containing 100 ml

of defined growth medium were used for the labeled catecholamine

precursor studies . Five 1ACi DL[2-14 C]3-(3,4-dihydroxyphenyl)

alanine, hereafter referred to as [ 14C]Dope or 5,uCi [U-1'C1

tyrosine, were added at the time of inoculation . The medium

utilized for the tyrosine incorporation studies contained 0 .005%

(w/v) "carrier" phenylalanine and 0 .00063% (w/v) "carrier" tyro-

sine . Fifty ml of medium containing cells were centrifuged at

850 g for 7 min at 4°C, and the cells were washed twice with cold

medium . The cell pellet was homogenized in an all glass homoge-

nizer containing 4 ml 0 .4 N HC10 4 and centrifuged at 7000 g for

5 min at 4°C . Supernatant was removed and saved and the pellet

was washed with 3 ml 0 .4 N HC10 4 . Five ml 2% disodium salt EDTA

(w/v) and 30 mg sodium metabisulphite were added to the combined

supernatants . After adjusting the pH to 8 .6 with 2 N NaOH, the

sample was placed on an alumina column ; dopamine and epinephrine

were separated on a Dowex column (8) .

548

Growth of Tetrahymena pyriformis

Vol. 11, No . 11

Chemicals were obtained from the following sources : reser-

pine phosphate, Ciba Pharmaceutical Company; propranolol-HC1,

Ayerst Laboratories ; dichloroisoproterenol, Eli Lilly and Company ;

[14C ]Dope, Nuclear Chicago and [ 14C ]tyrosine,

New England Nuclear .

All other chemicals were reagent grade .

Results

Effect of glucose on growth and glycogen content . Changes in

the glucose concentration of the medium had little effect on cell

replication at 46 hr . At 69 hr, 5 and 10 mM glucose caused little

increase in cell number compared to the controls while 15, 20 and

25 mM glucose caused a slight decrease (Table 1) . The glycogen

synthesis of the cells exposed to 5 or 10 mM glucose was reduced

below that of the control at 46 and 69 hrs ; 15 mM glucose caused

little reduction at 46 hr but showed a marked increase at 69 hr .

Twenty and 25 mM glucose increased glycogen synthesis consider-

ably in a dose related manner at both 46 and 69 hr .

TABLE 1

Effect of Glucose on the Growth and Glycogen Content

of Tetrahymena

Percent Change Compared to Controls

46 hr

6 hr

Glucose Concentration

Growth

Glycogen

Growth

Glycogen

5 MM

-2

-47

+13

-91

10 mm

-0 .2

-97

+11

-46

15 mM

-2

-5

-7

+155

20 mM

-4

+141

-10

+251

25 MM

-3

+151

-11

+315

Control cells were grown in defined medium containing Nat glycero-

phosphate and dextrine . When glucose was added to the medium, the

Nat glycerophosphate and dextrine were deleted .

Vol . 11, No. 11

Growth of Tetrahymena pyriformis

549

Effect-of rese rpine on-growth and glycogen content_ . In both

media, 3 x 10 -s M reserpine phosphate had little effect on cell

growth, but inhibited glycogen synthesis (Table 2) .

TABLE 2

Effect of 3 x 10 -5 M Reserpine on the Growth and

Glycogen Content of Tetrahymena after 44 hr Growth

Percent Change Compared

to Controls'

Growth Glycogen

Defined Medium

-9

-62

Defined medium without dextrine and

Nat glycerophosphate, but supple-

mented with 25 mM glucose

+0 .05

-52

'Tetrahymena cells were exposed to reserpine throughout the entire

44 hr period .

Effect of R-adrenergic blocking agents on growth and glycogen

content . In defined medium containing Nat glycerophosphate and

dextrine or defined medium without the dextrine and Nat glycero

phosphate, but supplemented with 25 mM glucose, 0 .1 mM

propranolol-HC1 had little effect on growth, but showed a striking

inhibitory effect on the glycogen synthesis of cells in stationary

phase after 50 hrs growth (Fig . 1) . Glycogen synthesis was

reduced in the complete medium without glucose 42% at 49 hr and

339 at 67 hr . The presence of 25 mM glucose not only increased

glycogen synthesis by 140% at 49 hr and 156% at 67 hr, but also

increased the inhibitory effect of propranolol by 55% at 49 hr and

44% at 67 hr . One mM propranolol was toxic .

550 Growth of Tetrahgmena pyriformis

g 5 .2

2.0b5.0 ~-

/Mi

É

1.8i

4.8f- 1/7

g, 1 .24.8L !N

e 0.8~4 .4

É

0

.4 L-

~'

4.2

. .

i

i i i i i

I

i i i I i

i i

0 10 20 30

40

50 60

70 80

0

10

20 30

40

50 60

70 80

Hours

Murs

FIGURE

1

Vol . 11, No. 11

Effect

of 0

.1

mM propranolol on the growth and glycogen

content

of

Tetrahymena grown

in a defined medium a--c3,

and

defined medium without

dextrine and

Na

t

glycero-

phosphate,

but supplemented with 25 mM glucoseo- o

.Control

cells 0--m and

9-o .

One

mM dichloroisoproterenol (DCI) in defined medium with

or

without 25 mM glucose exhibited the same inhibitory effect on

the

glycogen synthesis of cells in stationary phase after

40 hr

growth

with little effect on growth (Fig

.

2)

.

Glycogen synthesis

was

reduced in the defined medium

38% at 49 hr

and 62% at

67 hr .

The

presence of 25 mM glucose again increased glycogen synthesis

by 138% at 49 hr

and 162% at

6Z

hr and enhanced the inhibitory

effect

of DCI 56% at 49 hr and 71% at 67 hr

.

Vol . 11, No. 11

Growth of Tetrahymena PYriformis

551

4.0m0 3.600 3.2»

2.8

2.4

2.0

É 1.6

ô 1.20i 0.8m

é 0.4

10 20 30 40 50 80 70 80

0 10 20 30 40 50 80 70 80Hours

Hours

FIGURE 2

Effect of 1 .0 mM dichloroisoproterenol on the growthand glycogen content of Tetrahymena grown in definedmedium p--o, and defined medium without dextrine andNat glycerophosphate, but supplemented with 25 mMglucose o--o .

Control cells m--m and a-e .

Incorporation of labeled precursors into catecholamines .

Incorporation of [ 14 C]tyrosine into the catecholamines of Tetra-

hymens after 64 hr incubation in a defined medium was minimal and

separation of the catecholamines was not considered (Table 3) .

The growth of Tetrahymena obtained with defined medium containing

0 .005% phenylalanine was equivalent to that obtained in complete

medium containing 0 .01% phenylalanine . The cells incubated with

[ 14 C]Dope showed only a trace incorporation into dopamine and

norepinephrine ; the majority of counts were identified as

[ 14 C]Dope in the medium (Table 4) . There was no evidence of the[ 14 C]Dope metabolite dihydroxyphenylpyruvic acid or 3-0-methyl-

dope .

552

Growth of Tetrahymena pyriformis

Vol. 11, No. 11

TABLE 3

Incorporation of [ 14 C]Tyrosine into the Catecholamines of

Tetrahymena Grown in Defined Medium for 64 Hours

'Specific activity expressed as dpm/Umole [ 14 C]tyrosine

added .

TABLE 4

Incorporation of [l 4 C]Dope into the Catecholamines of

Tetrehymena Grown in Defined Medium for 4 Hr or 65 Hr

'Specific activity expressed as dpm/)ymole [ 14C]Dopa added .

2 After 65 hr incubation, 5 14Ci [ 14 C]Dopa was added and the

cultures reincubated an additional 4 hr for a total of 69

hr incubation .

Discussion

The addition of glucose as the sole carbohydrate source in

the defined medium greatly increased glycogen synthesis during

stationary phase . This stimulation of glycogen synthesis by

glucose has been noted in undefined media (2,9 and 10) and has

Total (DPM)_ Catecholamines

Cell Number

Incubation

Time Substrate' Dope Norepi Dopamine

23 .4 x 10e 4 13 .5 x 10' 69,580 2,579 4021

23 .3 x 108 4 13 .3 x 10' 111,328 11,769 5510

22 .4 x 108 65 15 .8 x 10 8 447 342 172

22 .9 x Joe 65 12 .4 x 10 8 65 253 171

23 .3 x 10 8 652 13 .2 x loll 1,992 256 301

Cell Number Substrate' Total (DPM) Catecholamines

9 .75 x 10 e 4 .4 x lo 5 246 (0 .0696)

11 .4 x 10 8 4 .8 x log 44 (0 .009%)

9 .63 x 10 8 4 .5 x log 461 (o .lo~)

Vol. 11, No . 11

Growth of Tetrahymena pyriformis

553

been attributed to the increased activity of glycogen synthetase

(11,10 .

In undefined medium without glucose, 3 x 10 _5M reserpine and

0 .1 mM propranolol inhibited growth (2) . When glucose was added

to the undefined medium, the inhibition of growth by 3 x 10 -SM

reserpine was partially reversed (3) . DCI had no effect on

growth in the undefined medium with and without glucose (2) . In

the defined medium with and without glucose, reserpine, proprenol-

of or DCI had little effect on growth . Growth inhibition was not

necessarily correlated with the metabolic effects in a defined

medium as they were in an undefined medium (5) . One metabolic

effect of the drugs tested was similar in both the defined and

undefined media : all drugs tested reduced glycogen synthesis .

The extent of reduction was dependent on the medium and presence

or absence of glucose (2) . In undefined medium with glucose,

reduced glycogen synthesis after treatment with reserpine and DCI

has been attributed to increased glycogen phosphorylase activity

(4) and decreased isocitrate lyase activity (5) . This reduction

in isocitrate lyase activity cannot be attributed to glucose

repression of the enzyme, since there was no reduction in malate

dehydrogenase activity (5) . When glucose was deleted from the

medium, the isocitrate lyase activity was decreased in the pres-

ence of reserpine, DCI or propranolol (5) . In the mammalian

system, however, reserpine, DCI and propranolol, by different

modes of action, decreased the phosphorylase activity of certain

tissues (12,13) . Isocitrate lyase is not known to be operational

in the mammalian system (14) .

Previous radioactive and fluorometric studies showed the

presence of norepinephrine and epinephrine in Tetrahymena and the

pathway necessary for their synthesis from any of the following

554

Growth of Tetrahymena. pyriformis

Vol. 11, No. 11

three precursors : phenylelanine, tyrosine, or dihydroxyphenyl-

alanine (Dope) (1,3) . In our strain of Tetrahymena grown in a

defined medium, we were unable to detect the presence of norepine-

phrine through radioactive studies using labeled tyrosine or Dopa

precursors . Tyrosine incorporation was negligible and [ 14C]Dope

was not utilized by the cells . These results indicate that our

strain of Tetrahymena does not contain a catecholamine system

similar to that described by others (1,3) .

Acknowledgment

The stimulating interest and co-operation of Dr . R . W . Fuller

of The Lilly Research Laboratories was greatly appreciated during

the course of this work .

References

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(l967)

3 . J . J . Blum, N . Kirshner and J . Utley, Mol . Pharmacol . 2,

4 . J . J . Blum, Arch . Biochem . Biophys . 137, 65 -74 (l970) .

5 " J . J . Blum, Mol . Pharmacol . 4, 247-257 (1968) .

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6o6-6o8 (1966) .

6 . D . Cox, 0 . Frank, S . H . Hutner and H . Baker, J . Protozool .

713-716 (1968) .

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8 . R . J . Wurtman, C . Chou and C . Rose, J . Pharmacol . Exp . Ther .

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M . R . Levy and A . E . Hunt, J . Cell . Biol . 34, 911-915 (1967)

J . F . Hogg and C . Wagner, Fed . Proc . 15, 275 -276 (1956) .

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Growth of Tetrahymena pyriformis

555

11 . D . E . Cook, R . 2 . Narayan, N . Best and D . R . Wilken, Arch .

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