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Effect of actinomycin D and glucagon on glucose uptake by spheroplasts of E. coli
Life Sciences Vol . 9, Part II, pp . 235-239, 1970. Printed in Great Britain
Pergamon Press
EFFECT OF ACTIIIOMYCIN D AND GLUCAGON ON GLUCOSE UPTAKE BY SPHEROPLA3T3 OF E . COLI J. L. R-Candela and M . Carmen Garcia Instituto G . Maraüon . C.S.I. C . Velazquez 144 . Madrid 6 . Spain (Received 15 September 1969; in final form 30 November 1969) Recent observations have suggested that Actinomycin D (AMD) may produce subtle cellular changes that are not explained by inhibition of RNA synthesis . Cooper (1) observed that AMD markedly inhibited poliovirus growth and that this inhibition was reversed by insulin . Laszlo et al . (2) showed that AMD inhibited respiration and anaerobic glycolisis of human leukemic leukocytes. In view of these results we were interested to know if AMD has any action on the glucose uptake by spheroplasts of E . cola and if either insulin or glucagon had any effect on the passage of this sugar .
Methods E. coli Hfr . 3000 (thiamine amsotroph) was obtained from E . Viïiuela, New-York University, New-York . This bacteria was grown is a synthetic medium (MTPA) containing 0.1 M Tris-HCI . pH 7.5, 0.0085 M NaCI, 0.1 M KCl, 0.02 M NH4C1, 0.34 mM KH2P04, 0.16 mM Na2S04, 2 .5 mM CaC12 , 2.5 mM MgC12, 0.01 M glucose, twenty La.minoacids (each 0.1 mM) and thiamine hydrochloride (10 p.g/ml) . The culture was incubated at 37°- C with shaking until an optical density of 0.48 was obtained (Spectronic w.1. :420 mp,) . Spheroplasts were obtained 235
GLUC03E UPTAKE
2gg
Vol. 9, No. 4
_a as described by Vißuela. et al . (g) by incubation (g7°- C) of E. coli with lysozyme (10 Fig/ml) in the presence of EDTA (0 .8 mM) . The epheroplasts suspension was diluted (2 . 5 ml) in a medium which had the same composition as the growth medium except for the omission of aminoacids and supplemented with glucose (S mg/ml), glucose U-C14 (0 .158 p,Ci-specific activity g20 mCi/mM) and albumin (0 .5 mg/ml) . FJnal concentration of spheroplasts ~ 3 . 5 x 10 9/ml. Aliquots (1 ml) of the spheroplasts dilution were transfered bo different glasses in order to test the effect on the glucose uptake of AMD (0 .5-1 l,~g/ml), glucagon (100 F+.g/ml) and AMD plus glucagon . The fourth glass containing only the spheroplast suspension was used as control. All together were incubated at g7°- C with shaking for an hour, is a atmosphere of C02-02 5~ - 95~. One hundred Eil of each suspension was filtered through Millipore (25 mm diameter : 0.22
w pore size) in triplicate . The radioactivity of the filters
was measured in a glass flow counter . The results are expressed in per cent of inhibition or enhancement of the glucose uptake (} S . E. M. )
Results and Discussion The results are summarised in the figures 1 and 2 . The glucose uptake by spheroplasts was depressed by the Actinomycin D. When concentrations of 0.5 l~, g/ml of this chromopeptide were used, the inhibition was completely reversed by glucagoa (100 F+.g/ml) . When the concentration of AMD was higher (1 F+,g/ml) and the concentration of glucagoa was the same as in the former experiment, there was only a partial rever-
Vol . 9, No. 4
GLUCOSE UPTAKE
237 _g
FIG . 1
Effect of AID (0.5W,g/ml), Glucagon (100Kg/ml) and A~ plus Glucagon (0, 5pg - 100~r.g/ml) on the Glucose Uptake by Spheroplasts of E. Coli (Hfr . 3000) .
... C e E d u
(7) Gluc.
" 20
C O L C d (
J )
~rt . " Gluc. 0 C O "_+ ,Q t c
Act. (7)
w 0 -20
Act , (ANNIV) = 10 per cent of inhibition Gluc (glucagon) = 17 .7 per cwt of enhancem~rt Act (AA+ID) + Gluc (glucagon) = 1,1 per cent of enhancement Comparison of the effects : Glucagon versas AMD : 3. E. M. ± 0. 78 ( P < 0. 01) Glucagon + AMID versus AIVID : S. E. M. ± 1 . 9a ( P < 0. 01) In parenthesis: number of experiments
238
GLUC05E UPTAKE
Vol. 9, No. 4 4
FIG . 2 Eüect of AMD (iN,g/ml), Glucagoa (100N,g/ml) and AMD plus Glucagon (if+,g/ml - 100pg/ml) on the Glucose Uptake by Spheroplasts of E . Coli (Hfr . 3000) . +30
cd E
d
(6 )
Gluc .
+20
c v
t c d
ô
+10
0
-10 c
L C
; ". . .. . . .............. : .. . . . .
.~~i~~~ . ~ii~~~
0
.a
..
-so
Ad .+Gluc. (6 )
iiiïi. "iiiiii iiiiii Act. (6)
-30
Act (AMD) = 20.3 per cent of inhibition Glut (glucagon) = 18.2 per cent of enhancement Act + Gluc (AMD plus Glucagon) = 9.3 per cent of inhibition Comparison of the effects : Glucagon versus AMD : S . E. M. ± 1 . 72 + (P < 0.01) Glucagon + AMID versus AMD : 3. E. M. - 2 .9 (P<0.02) In parenthesis : number of eacperiments.
Vol. 9, No. 4
GLUCOSE UPTAKE
2S9 - 5
sion . Furthermore, in both figures it is shown that glucagon per se produced as increase of the glucose uptake. These preliminary experiments do not elucidate the mechanism how AMD or glucagon produced the observed effects . We may be able to explain these results with experiments now in progress.
Summarq Actinomycin D inhibited glucose uptake by spheroplasts of E. soli (Hfr. 3000) . This effect was completely reversed by glucagon. The passage of glucose into the celle was enhanced by glucagon,
Acknowledgements. - We wish to thank Drs . 3 . Ochoa, M. Salas and E. ViSnlâfor encouragement, advice sad maay fruitful discussions . Thaaks are also due to Drs . D . Vazquez and R. Pastille for their help is our initial experiments . Thaaka to Novo Lab . Danemark (Dr. Arias-Paz, Madrid) for the generous gift of glucagon. The skillful technical assistance of M. Dolores Hermoeo ~ad M. Asunciôn Lôpez Munguira is gratefully acknowledged . This investigation was supported by a great from Lépetit, Milaao-Madrid .
References 1 . - P. D. Cooper, Virology 28, 66S (1988) 2 . - J . I+asZlo, D. 3. Miller, K. S . McCarty sad P. Hochsteia, Science 151, 1007, (1988) 3 . - E. Vifiuela, I. D. Algranaty and 3. Ochoa, European J. Biochem . It 3, (1987)
Pergamon Press
EFFECT OF ACTIIIOMYCIN D AND GLUCAGON ON GLUCOSE UPTAKE BY SPHEROPLA3T3 OF E . COLI J. L. R-Candela and M . Carmen Garcia Instituto G . Maraüon . C.S.I. C . Velazquez 144 . Madrid 6 . Spain (Received 15 September 1969; in final form 30 November 1969) Recent observations have suggested that Actinomycin D (AMD) may produce subtle cellular changes that are not explained by inhibition of RNA synthesis . Cooper (1) observed that AMD markedly inhibited poliovirus growth and that this inhibition was reversed by insulin . Laszlo et al . (2) showed that AMD inhibited respiration and anaerobic glycolisis of human leukemic leukocytes. In view of these results we were interested to know if AMD has any action on the glucose uptake by spheroplasts of E . cola and if either insulin or glucagon had any effect on the passage of this sugar .
Methods E. coli Hfr . 3000 (thiamine amsotroph) was obtained from E . Viïiuela, New-York University, New-York . This bacteria was grown is a synthetic medium (MTPA) containing 0.1 M Tris-HCI . pH 7.5, 0.0085 M NaCI, 0.1 M KCl, 0.02 M NH4C1, 0.34 mM KH2P04, 0.16 mM Na2S04, 2 .5 mM CaC12 , 2.5 mM MgC12, 0.01 M glucose, twenty La.minoacids (each 0.1 mM) and thiamine hydrochloride (10 p.g/ml) . The culture was incubated at 37°- C with shaking until an optical density of 0.48 was obtained (Spectronic w.1. :420 mp,) . Spheroplasts were obtained 235
GLUC03E UPTAKE
2gg
Vol. 9, No. 4
_a as described by Vißuela. et al . (g) by incubation (g7°- C) of E. coli with lysozyme (10 Fig/ml) in the presence of EDTA (0 .8 mM) . The epheroplasts suspension was diluted (2 . 5 ml) in a medium which had the same composition as the growth medium except for the omission of aminoacids and supplemented with glucose (S mg/ml), glucose U-C14 (0 .158 p,Ci-specific activity g20 mCi/mM) and albumin (0 .5 mg/ml) . FJnal concentration of spheroplasts ~ 3 . 5 x 10 9/ml. Aliquots (1 ml) of the spheroplasts dilution were transfered bo different glasses in order to test the effect on the glucose uptake of AMD (0 .5-1 l,~g/ml), glucagon (100 F+.g/ml) and AMD plus glucagon . The fourth glass containing only the spheroplast suspension was used as control. All together were incubated at g7°- C with shaking for an hour, is a atmosphere of C02-02 5~ - 95~. One hundred Eil of each suspension was filtered through Millipore (25 mm diameter : 0.22
w pore size) in triplicate . The radioactivity of the filters
was measured in a glass flow counter . The results are expressed in per cent of inhibition or enhancement of the glucose uptake (} S . E. M. )
Results and Discussion The results are summarised in the figures 1 and 2 . The glucose uptake by spheroplasts was depressed by the Actinomycin D. When concentrations of 0.5 l~, g/ml of this chromopeptide were used, the inhibition was completely reversed by glucagoa (100 F+.g/ml) . When the concentration of AMD was higher (1 F+,g/ml) and the concentration of glucagoa was the same as in the former experiment, there was only a partial rever-
Vol . 9, No. 4
GLUCOSE UPTAKE
237 _g
FIG . 1
Effect of AID (0.5W,g/ml), Glucagon (100Kg/ml) and A~ plus Glucagon (0, 5pg - 100~r.g/ml) on the Glucose Uptake by Spheroplasts of E. Coli (Hfr . 3000) .
... C e E d u
(7) Gluc.
" 20
C O L C d (
J )
~rt . " Gluc. 0 C O "_+ ,Q t c
Act. (7)
w 0 -20
Act , (ANNIV) = 10 per cent of inhibition Gluc (glucagon) = 17 .7 per cwt of enhancem~rt Act (AA+ID) + Gluc (glucagon) = 1,1 per cent of enhancement Comparison of the effects : Glucagon versas AMD : 3. E. M. ± 0. 78 ( P < 0. 01) Glucagon + AMID versus AIVID : S. E. M. ± 1 . 9a ( P < 0. 01) In parenthesis: number of experiments
238
GLUC05E UPTAKE
Vol. 9, No. 4 4
FIG . 2 Eüect of AMD (iN,g/ml), Glucagoa (100N,g/ml) and AMD plus Glucagon (if+,g/ml - 100pg/ml) on the Glucose Uptake by Spheroplasts of E . Coli (Hfr . 3000) . +30
cd E
d
(6 )
Gluc .
+20
c v
t c d
ô
+10
0
-10 c
L C
; ". . .. . . .............. : .. . . . .
.~~i~~~ . ~ii~~~
0
.a
..
-so
Ad .+Gluc. (6 )
iiiïi. "iiiiii iiiiii Act. (6)
-30
Act (AMD) = 20.3 per cent of inhibition Glut (glucagon) = 18.2 per cent of enhancement Act + Gluc (AMD plus Glucagon) = 9.3 per cent of inhibition Comparison of the effects : Glucagon versus AMD : S . E. M. ± 1 . 72 + (P < 0.01) Glucagon + AMID versus AMD : 3. E. M. - 2 .9 (P<0.02) In parenthesis : number of eacperiments.
Vol. 9, No. 4
GLUCOSE UPTAKE
2S9 - 5
sion . Furthermore, in both figures it is shown that glucagon per se produced as increase of the glucose uptake. These preliminary experiments do not elucidate the mechanism how AMD or glucagon produced the observed effects . We may be able to explain these results with experiments now in progress.
Summarq Actinomycin D inhibited glucose uptake by spheroplasts of E. soli (Hfr. 3000) . This effect was completely reversed by glucagon. The passage of glucose into the celle was enhanced by glucagon,
Acknowledgements. - We wish to thank Drs . 3 . Ochoa, M. Salas and E. ViSnlâfor encouragement, advice sad maay fruitful discussions . Thaaks are also due to Drs . D . Vazquez and R. Pastille for their help is our initial experiments . Thaaka to Novo Lab . Danemark (Dr. Arias-Paz, Madrid) for the generous gift of glucagon. The skillful technical assistance of M. Dolores Hermoeo ~ad M. Asunciôn Lôpez Munguira is gratefully acknowledged . This investigation was supported by a great from Lépetit, Milaao-Madrid .
References 1 . - P. D. Cooper, Virology 28, 66S (1988) 2 . - J . I+asZlo, D. 3. Miller, K. S . McCarty sad P. Hochsteia, Science 151, 1007, (1988) 3 . - E. Vifiuela, I. D. Algranaty and 3. Ochoa, European J. Biochem . It 3, (1987)