Mitochondrial protein synthesis and the stimulation of steroidogenesis by cyclic adenosine 3′,5′-monophosphate in isolated rat adrenal cells

86

Biochimica et Biophysica Acta, 383 (1975) 86--92

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98223 M I T O C H O N D R I A L PR O T E I N SYNTHESIS AND T H E STIMULATION OF S T E R O I D O G E N E S I S BY CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE IN ISOLATED R A T A D R E N A L CELLS

SEYMOUR B. KORITZ and RAKOMA WIESNER Department of Biochemistry, Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029 (U.S.A.)

(Received August 5th, 1974) Summary The stimulation by cyclic AMP of steroidogenesis in rat adrenal cells isolated by trypsin t r e a t m e n t was inhibited by D-threo-chloramphenicol and by its L - t h r e o - i s o m e r . The f o r m e r is an inhibitor of mitochondrial protein synthesis while the latter is not. Both substances, at concentrations which inhibit steroidogenesis, inhibit amino acid i ncor po rat i on into the proteins of microsomes. Inhibition in ot her subcellular fractions also occurs depending on the isomer and its conc e nt r at i on. In no case was there a preferential inhibition of amino acid in co r por a t i on into mitochondrial proteins. Carbomycin, a n o t h e r inhibitor of mitochondrial protein synthesis, gave similar results. In addition, subfractionation of m i t o c h o n d r i a in these experiments revealed no preferential inhibition of amino acid incorporation into the proteins of either the soluble or m e m b r a n e fractions of this organelle. The above results were obtained at several concentrations of the inhibitors when only partial inhibition of steroidogenesis was present. Both isomers of chloramphenicol inhibited steroidogenesis in a cell-free system to an e x t e n t equal to t ha t f o u n d with cyclic AMP-stimulated steroidogenesis in intact cells. It is concluded t hat these inhibitors of mitochondrial protein synthesis have multiple metabolic effects in adrenal cells.

Introduction Ferguson [1] has shown t hat the acute stimulation by ACTH of steroidogenesis in the adrenal can be abolished by p u r o m y c i n , an inhibitor of protein synthesis. Extension of this t y p e of exper i ment by Garren et al. [2] revealed that cycloheximide administration during maximal stimulation of steroidogenesis by ACTH resulted in a rapid decrease to basal levels. Since b o t h the onset

87 and the maintenance of the ACTH effect are sensitive to inhibitors of protein synthesis the involvement of a protein, or proteins, with a rapid turnover rate is indicated. Little information is available, however, about the nature, the role, or the site of synthesis of this rapidly turning-over protein. It has been f o u n d by Davis and Garren [3] that the inhibition of steroidogenesis by cycloheximide is located in the conversion of cholesterol to pregnenolone, the same site where ACTH stimulation of steroidogenesis occurs [4,5]. These reactions take place in the mitochondria [6] and it has been found that only these reactions of steroidogenesis are increased in mitochondria isolated from the adrenals of rats which had been given ACTH [7]. The observation that cycloheximide, which affects only extra-mitochondrial protein synthesis [8,9] is an inhibitor of ACTH action suggests that the rapidly turning-over protein of Garren et al. [2] is synthesized outside the mitochondria and may then be incorporated into the mitochondria. This is the situation which prevails for perhaps 90--95% of mitochondrial proteins [10--12]. However, it has been reported that D~chloramphenicol, but n o t the L-(+)-threo-isomer, inhibited both protein synthesis and ACTH~stimulated steroidogenesis in rat adrenal quarters [13]. Since D-chloramphenicol inhibits protein synthesis in mitochondria [9,14--16] but not protein synthesis which is extra-mitochondrial [16,17] the possibility is presented that rapidly turning-over proteins of both mitochondriai and extra-mitochondrial origins are involved in ACTH action. The data presented in this report do not support a role for protein synthesis in mitochondria in the stimulation of steroidogenesis by cyclic AMP in adrenal cells. Reduction of steroidogenesis by inhibitors of mitochondrial protein synthesis was accompanied by inhibition of protein synthesis in subcellular fractions other than the mitochondria. In addition, D-chloramphenicol and L-chloramphenicol inhibited corticosterone synthesis in short-term incubations with cell-free systems. Materials and Methods Adrenal cells were prepared by the m e t h o d of Sayers et al. [18] and were suspended in Krebs-Ringer bicarbonate medium which contained 0.2% glucose, 0.5% bovine serum albumin and 0.1% lima bean trypsin inhibitor. In experiments which involved subsequent fractionation of the ceils 5 - 1 0 s - - 6 • l 0 s cells per ml of incubation medium were present, otherwise 3 • 10 s cells per ml were used. The incubation medium also contained 5 mM cyclic AMP, and, when present, 0.5 pCi of L-[U-14C]leucine with a specific activity of 342 Ci/mol. The incubations were carried out at 37°C in silicone-coated beakers in a D u b n o f f incubator in an atmosphere of 95% 02 and 5% CO2. At the end of the incubation aliquots were extracted with dichloromethane and corticosterone determined as described previously [7]. In those experiments where protein synthesis in the various subcellular fractions was determined the cells were collected, at the end of the incubation, by centrifugation at 3000 × g for 3 min and washed twice with 0.15 M NaC1 which contained 2 mg of L-leucine per ml. The cells were then suspended in

88 0.25 M sucrose/5 mM Tris, pH 7.5. To get adequate cell breakage, as determined by microscopic examination, up to 20 strokes with a Teflon homogenizer were required. The hom ogenat e was centrifuged at 750 X g for 10 min t o obtain the crude nuclear debris and the supernatant from this centrifuged at 7700 X g for 10 min. The mitochondrial fraction thus obtained was washed twice by resuspending it in the sucrose/Tris and centrifuging at 5900 X g for 10 min. The supernatant from the initial mitochondrial fraction was centrifuged at 105 000 X g for 60 min to obtain the microsomes and the cytosol. Separation of the m i t o c h o n d r i a into KCl-soluble and -insoluble fractions was carried out by the p r o ced u r e described by Beattie et al. [12] except that 0.6 M KC1 was used and th e mitochondrial suspension was frozen t w o times. The incorporation of the [U -14C]leucine into the proteins of the various fractions was determined by the procedures described by Beattie et al. [ 1 9 ] . Protein was determined by the m e t h o d of L o w r y et al. [ 2 0 ] . Corticosterone synthesis in a cell-free system was carried out as described elsewhere [7]. Carbomycin was a gift f r om Pfizer Pharmaceutical Com pany and L-threochloramphenicol was a gift f r om Parke-Davis Pharmaceutical Company. Dthreo-chloramphenicol was obtained f r om t he Sigma Chemical Company. The L-chloramphenicol sample used in this study had been f o u n d to have no effect on in vitro mitochondrial protein synthesis while the D-isomer at the same concentrations caused an 88% inhibition [ 3 1 ] . Results The stimulation of steroidogenesis in the experiments o f this r e p o r t was carried o u t with cyclic AMP rather than ACTH to obviate any effect the inhibitors may have on the binding of ACTH to receptors or on t he adenylcyclase system. In the absence of cyclic AMP (or ACTH) the rate of steroidogenesis in the isolated adrenal cells was essentially zero. It is seen f r om the data in Table I that b o t h the D- and L-threo-isomers of chloramphenicol inhibit cyclic AMP-stimulated steroidogenesis at the concentrations used. Lower concentrations resulted in negligible, if any, inhibition. At the lower concentrations the D-isomer was a b o u t 30% more effective than t he L-isomer. TABLE

I

THE EFFECT The incubation CAP ( M X 1 0 4)

0 3 6 10 20

OF CHLORAMPHENICOL

ON STEROIDOGENESIS

IN RAT ADRENAL

CELLS

time was 60 rain.

Corticosterone synthesis (nmol/ml

incubation

medium)

D-CAP

% inhibition

L-CAP

% inhibition

8.3 5.6 3.8 2.2 1.1

33 54 74 87

8.3 6.1 5.0 3.6 1.7

26 40 57 79

89 T A B L E II THE EFFECT OF CHLORAMPHENICOL RAT ADRENAL CELLS

ON PROTEIN

SYNTHESIS

AND STEROIDOGENESIS

IN

T h e c o n c e n t r a t i o n o f c h l o r a m p h e n i c o l in E x p t I w a s 5 . 1 0 - 4 M a n d in E x p t II 1 ' 1 0 - 3 M. T h e i n c u b a t i o n t i m e w a s 60 rain.

Expt

Cell f r a c t i o n

Protein synthesis (cpm/~g protein)

% decrease

Corticosterone % decrease synthesis (nmol/ml incubation medium)

Control

DCAP

LCAP

DCAP

LCAP

Control

DCAP

LCAP

DCAP

LCAP

14.5

9.4

10.6

35

27

Homogenate Crude nuclear debris Mitochondria Microsomes Supernatant

27.4 28.6 11.4 41.4 44.7

22.7 24.0 9.4 30.6 35.6

26.4 27.6 11.0 35.7 44.6

17 16 18 26 20

0 0 0 14 0 16.7

5.8

9.2

65

45

Homogenate Crude nuclear debris Mitochondria Microsomes Supernatant

20.8 19.9 10.3 38.8 30.7

12.7 10.9 5.3 19.9 23.1

18.8 15.3 9.1 30.8 31.9

39 45 48 49 25

10 23 12 21 0

I

To determine w het her the inhibition of steroidogenesis was uniquely involved with protein synthesis in the mitochondria, t he effect of D-chloramphenicol and L-chloramphenicol on t he i ncorporat i on of [U -14C] leucine into the proteins o f the subcellular fractions of the adrenal cells was determined. Experiments carried o u t at t w o concentrations of bot h isomers are given in Table II. It is seen t hat at the lower c o n c e n t r a t i o n , D ~ h l o r a m p h e n i c o l inhibited amino acid i ncor por at i on into the proteins of all fractions with the greatest effect on the microsomes. The presence of L-chloramphenicol at this c o n cen tr atio n resulted in an inhibition only in the microsomes. Both isomers inhibited steroidogenesis. At t he higher c on cent rat i on, b o t h isomers inhibited amino acid in co r p or a t i on into the proteins o f all fractions except for the supernatant fraction in the presence of L-chloramphenicol. In b o t h experiments t he effects on protein synthesis and on steroidogenesis, was greater with D-chloramphenicol than with L-chloramphenicol, b ut in no case was there a preferential inhibition of amino acid i nc or por a t i on into t he mitochondrial proteins. The macrolides are a group o f antibiotics which inhibit protein synthesis in isolated m i t o c h o n d r i a [ 2 1 ] . On the basis of its ability to penetrate at least liver mitochondria, one of these, carbomycin, was chosen to investigate its effect on steroidogenesis and protein synthesis in t he adrenal cells. In these experiments the m i t o c h o n d r i a were subfractionated into 0.6 M KCl-soluble and -insouble fractions, a procedure which was n o t possible in t h e experiments with chloramphenicol since t he use of bot h t he D- and L-isomers in an experi m ent limited the a m o u n t o f m i t o c h o n d r i a obtained. The data obtained with carbomycin are given in Table III. Concentrations lower than those given resulted in no, or negligible, inhibition of steroidogenesis. At the concentrations used,

90 TABLE III THE EFFECT OF CARBOMYCIN ADRENAL CELLS

ON PROTEIN

SYNTHESIS

AND STEROIDOGENESIS

IN RAT

T h e c o n c e n t r a t i o n s o f c a r b o m y c i n w e r e 9 ~tM in E x p t I, 18 # M in E x p t II a n d 1 1 9 pM in E x p t I n . T h e i n c u b a t i o n t i m e w a s 6 0 rain.

Expt

Cell f r a c t i o n

Protein synthesis (cpm/pg protein) Control

% decrease

Corticosterone synthesis ( n m o l / m l incubation medium)

Carbomycin

I Homogenate Crude nuclear debris Mitochondria (insoluble fraction) M i t o c h o n d r i a (soluble fraction) Microsomes Supernatant

44.9 43.6 20.0 18.2 78.4 80.2

43.9 43.5 19.1 16.0 69.4 80.5

0 0 5 12 12 0

Homogenate Crude nuclear debris Mitochondria (insoluble fraction) M i t o c h o n d r i a (soluble f r a c t i o n ) Microsomes Supernatant

39.7 39.4 12.5 18.3 87.6 64.2

38.6 38.4 11.2 15.2 74.7 65.9

0 0 10 17 15 0

Homogenate 45.6 Crude nuclear debris 40.0 Mitochondria (insoluble fraction) 29.1 M i t o c h o n d r i a (soluble fraction) 22.4 Microsomes 113.0 Supernatant 80.7

32.3 29.5 15.3 10.8 53.0 43.8

29 26 47 52 53 46

II

III

% decrease

Control

Carbomycin

10.0

6.4

36

14.7

7.2

51

10.9

0.9

92

inhibition o f steroidogenesis increased with t h e increase in c o n c e n t r a t i o n . At the t w o lower c o n c e n t r a t i o n s inhibition of a m i n o acid i n c o r p o r a t i o n into proteins was seen only in the m i t o c h o n d r i a l fractions and the m i c r o s o m e s , b u t with no preferential inhibition in the m i t o c h o n d r i a . At the highest concentraTABLE IV THE EFFECT OF CHLORAMPHENICOL SYSTEM FROM RAT ADRENALS

ON CORTICOSTERONE

SYNTHESIS

The incubation was carried out for 3 min. Addition

-

Concentration (mM)

-

D-CAP L-CAP

0.67 0.67

Corticosterone synthesis (nmol/ml incubation medium) 0.92 0.30 0.37

% inhibition

67 60

IN A CELL-FREE

91 tion inhibition occurred in all fractions and to about equal extents in the mitochondria, microsomes and soluble fractions. The effect of D-chloramphenicol and L-chloramphenicol on corticosterone synthesis from endogenous precursors in a succinate-dependent cell-free system is given in Table IV. It is to be noted that at the concentrations used these substances inhibited steroidogenesis in the cell-free system to an extent equal to or greater than t h a t f o u n d with cyclic AMP-stimulated intact cells. The incubation time in these experiments was only 3 min. Discussion The data presented indicate that at concentrations at which t h e y inhibit cyclic AMP-stimulated steroidogenesis in adrenal cells, the inhibitors of mitochondrial protein synthesis, D-chloramphenicol and carbomycin, have other metabolic effects also. This is reflected in the intact cell experiments (Tables II and III) by the inhibition of amino acid incorporation into the proteins of subcellular fractions other than the mitochondria, particularly in the microsomes. In no instance could a preferential effect on mitochondrial proteins be demonstrated with either substance. To the contrary, at the lower concentration of D-chloramphenicol (Table II) a greater effect on the amino acid incorporation into the proteins of the microsomal fraction was seen. Carbomycin also inhibited amino acid incorporation into the proteins of the microsomes. In the mitochondria this substance had a preferential effect on the soluble proteins (Table III). These are the proteins which have an extra-mitochondrial origin [10--12]. It is to be noted that the above effects on protein synthesis were obtained when only partial inhibition of steroidogenesis was seen. It is n o t known what metabolic effects, other than on mitochondrial protein synthesis, carbomycin has on eukaryotic cells. As inhibitors of bacterial and mitochondrial protein synthesis the macrolides act by binding to the 50 S ribosomes [22]. No effects of carbomycin on respiration or oxidative phosphorylation have been observed [21,23]. The substance also has no effect on the intracellular concentration of K ÷ or Mg2÷ in HeLa cells [23]. On the other hand, both the D- and L-threo-isomers of chloramphenicol have been shown to inhibit the mitochondrial respiratory chain [24--29] with a major effect apparently on NADH oxidation [26--28]. The relative effects of the two isomers on the components of the respiratory chain vary with the species [27] and even within a given organ the effects of the isomers depends on the mitochondrial energy producing c o m p o n e n t measured [27--29]. This variation precludes any prediction as to which isomer may be more inhibitory of the energy producing system in a specific case. However, it is clear that with isolated mitochondria the D-isomer inhibits protein synthesis [9,14--16] and the L-isomer does not [30]. In addition, D-chloramphenicol has no effect on microsomal protein synthesis [16,17]. An interpretation of the results obtained by Farese [13] involving mitochondrial protein synthesis in the stimulation of steroidogenesis by ACTH was based on the inhibition of both protein synthesis (total tissue protein) and steroidogenesis by D-chloramphenicol and with no effect on either process by L-chloramphenicol. The discrepancy between these data and those presented in this report is n o t apparent especially

92 since inhibition of steroidogenesis by L-chloramphenicol at a third the concentration used by Farese was observed. A n o t unreasonable explanation for the difference is th at the permeability of the adrenal cells may be different for the t w o isomers. The adrenal cells used in the present study were isolated by a process which involved digestion with trypsin. This may have altered the cell m e m b r an e so th at b o t h isomers could enter, while with the adrenal slices used by Farese the L-isomer may have been relatively excluded. The difficulty in ascribing the effects of D-chloramphenicol on steroidogenesis to a specific inhibition of mitochondrial protein synthesis is emphasized by the data on succinate-dependent corticosterone synthesis in a cell-free system using a very short incubation t i m e (Table IV). It is seen t hat at equivalent concentrations inhibitions equal to or greater than those obtained for cyclic AMP-stimulated steroidogenesis in intact cells were observed.

Acknowledgement This work has been supported by Grant AM-13361 from the United States Public Health Service.

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