- Email: [email protected]
[24] Assessment of in Vivo adrenocorticotropic hormone treatment on adrenal mitochondrial functions in Vitro
[24]
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ration of 3-P-glycerate conversion to glyceraldehyde-3-P (probably resulting from increased reduction of the cytosolic NAD systems).3s Final Comments The method of tissue metabolic analysis has been used most extensively in the study of the control of glycolysis and glycogen metabolism in muscle and of gluconeogenesis in liver. This is mainly because of the ready availability of relevant analytical grade enzymes, the high flux rates, and the relatively high concentrations of the intermediates of these pathways. In addition, the fact that these tissues are relatively homogeneous and that many of the intermediates of these pathways are localized to the cytosol reduces the number of potential problems of interpretation related to cell heterogeneity or compartmentation. Although tissue metabolic analysis has provided much useful information regarding the regulation of other pathways (fatty acid oxidation and ketogenesis, the hexosc monophosphate shunt, triglyceride breakdown and synthesis, Krebs cycle, and protein synthesis) such studies have generally been incomplete or have given equivocal results because of the inability to measure some key intermediates or determine the distribution of metabolites between mitoehondria and cytosol. It is anticipated that assays of improved specificity and sensitivity will be developed to partly overcome these problems and to enable other pathways to be studied. The problem of conIpartmentation seems somewhat intractible at this time, but it should be noted that in no instance has compartmentation been shown to negate interpretations based on metabolic analysis.
[24] Assessment of in Vivo Adrenocorticotropic Hormone Treatment on Adrenal Mitochondrial Functions in V i t r o By ~TANLEY BANIUKIEWICZ,AJAI HAKSAR,and FERNAND PERON Introduction
Mitochondria of steroid-producing tissues are unique in that they contain, in addition to the classic electron transport chain, a cytochrome P4~o chain which is directly involved in the activation of oxygen for several steroid hydroxylating reactions. In the rat adrenal cortex, the
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hydroxylations involved in side chain cleavage of cholesterol as well as hydroxylations at llfl and 18 positions on the steroid nucleus are carried out in the mitochondria. Adrenocorticotropic hormone (ACTH) stimulates the production of corticosterone in this species when given in vivo or when added in vitro to incubations of adrenal sections or cell suspensions but not when added directly to isolated mitochondria. However, mitochondria obtained from adrenals of rats treated with ACTH, in vivo, show differences in some of the functions (including and related to steroidogenesis) when compared with the adrenal mitochondria from untreated control rats. In our laboratory we have compared adrenal mitochondria from ACTH-treated and control rats in terms of their ability to carry out 11fl-hydroxylation of ll-deoxycorticosterone (DOC) in the presence of some Krebs cycle acids. This ability, or extent in which the l lfl-hydroxylation reaction occurs in vitro, depends on the method used to prepare the mitochondria. We have also measured oxygen uptake and ADP-O ratios in our mitochondrial preparations while other laboratories have compared parameters such as side chain cleavage of cholesterol and cytochrome P4~o levels in the adrenal mitochondria from ACTH-treated and untreated animals. 1-3 The purpose of this section is to present the methods used in our laboratory for the preparation of rat adrenal mitochondria to show the importance of such methods for investigating in vivo effect (s) of ACTH action as is reflected in certain parameters of mitochondrial activity in vitro. Animals and Treatment Adult male Sprague-Dawley rats, weighing 150-170 g, from Charles River Company are used in these experiments. The animals are allowed Purina rat chow and water ad libitum and are used in the experiments only after they have spent at least a week in our sound-proofed animal quarters. Every group of rats is divided into two smaller groups: one to serve as the untreated controls and the other as ACTH-treated. The animals in the latter group are given three subcutaneous injections of 0.1 ml of porcine ACTH (Acthar gel, 40 USP units/ml; Armour Co.) in the nape of the neck at 4 P..~I. and 11 P.M. on the day preceding the experiment.and at 8 A.M. on the day of the experiment, respectively. Rats in both groups are sacrificed by decapitation 90 minutes (controls first) 1 A. C. Brownie, E. R. Simpson, C. and H. Beinert, Biochem. Biophys. ZE. R. Simpson, C. R. Jefcoate, A. 28, 442 (1972). 3A. C. Brownie, J. Alfano, C. R. and H. Beinert, in press.
R. Jefcoate, G. S. Boyd, W. It. Orme-Johnson, Res. Commun. 46, 483 (1972). C. Brownie, and G. S. Boyd, Eur. J. Biochem. Jefcoate, E. R. Simpson, W. Orme-Johnson,
[24]
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after the last injection of ACTH to the treatment group. At the time of sacrifice the rats in both groups weigh about 200-220 g.
Preparation of Homogenates The ,~drenals of 20 control or ACTH-treated rats are rapidly excised after sacrifice,, trimmed of all adhering fat, and homogenized in 7 ml of the ice-cold buffers denoted hereafter as mixture A or B. Mixture A: Tris buffer (20 raM), sucrose (250 mM), nicotinamide (20 raM), and EDTA (1 raM), pH 7.4 Mixture B: Tris buffer (20 mM), sucrose (250 raM), and nicotinamide (20 raM), pH 7.4 Homogenization of the whole adrenals is carried out in a glass PotterE1vehjem homogenizer tube fitted with a loose glass pestle which is driven by a variable speed Eberback rotary motor. Generally, the cleaned adrenals from the 20 rats weigh about 800-1000 mg (wet weight) and are completely homogenized in the buffer by 8-10 passes of the pestle in the homogenizer tube. This procedure is easily carried out in the ambient temperature of the laboratory taking care to keep the homogenizer tube submerged in a beaker containing cracked ice.
Preparation of the Mitochondrial Fraction The homogenate prepared above is then centrifuged in a Spinco model L centrifuge at 400 g for 10 minutes. The supernatant is removed carefully to avoid contamination from the loosely packed sediment and recentrifuged at 9000 g to obtain the mitochondrial pellet. After removal of the supernatant with a Pasteur pipette and wiping the tube free of the white lipid layer which floats on top of the supernatant, the mitochondrial pellet is suspended in 3 ml of the buffer using a hollow glass rod sealed at one end by a glass bubble blown previously to exactly fit the bore of the plastic tube containing the pellet. After using as many rapid strokes of the glass rod as are required for complete suspension of the pellet, it is recentrifuged at 9000 g. The suspension and washing procedure is repeated once more, and finally the washed pellet is transfered by means of 2.0 ml of buffer to a clean Potter-Elvehjem homogenizer tube and resuspended in the buffer with the pestle turning at slow speed to yield a homogeneous suspension of mitochondria. The mitochondrial preparation is diluted with buffer to yield 0.10 ml of a suspension of mitochondria derived from 20 mg of original wet weight adrenal tissue. Generally, one adrenal (15-20 mg} yields about 0.5 mg of the mitochondrial protein and the procedure described above results in a mitochondrial
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preparation with minimum contamination from other organelles or components of the tissue. Protein concentrations are determined by the wellknown method of Lowry using bovine serum albumin to standardize the assay. Preparation of the Microsomal Fraction The supernatant obtained after the first 9000 g centrifugation to sediment mitochondria above is recentrifuged at 15,000 g for 15 minutes. The resulting sedimented pellet containing "light mitochondria" and "heavy" microsomes" is discarded, and the supernatant is centrifuged at 105,000 g for 60 minutes. The reddish completely transparent pellet thus obtained contains the microsomes and is suspended in the buffer to give a protein concentration of approximately 5 mg/ml of the suspension. One adrenal generally yields about 0.25 mg of protein. The final supernatant is considered to be made up of the soluble components of the cell and usually contains about 3.2 mg protein per ml. One adrenal yields about 0.5 mg of protein. Incubation Medium The basic buffer for the incubation contains the following: sucrose (141 raM), nicotinamide (53 mM), KC1 (16.4 mM), NaC1 (16.4 mM), KH2PO~ (1.1 raM), bovine serum albumin (BSA, 0.1%), and Tris-HC1 (21 mM), pH 7.4. This mixture is prepared from the following isotonic s~ock aqueous solutions: 10 parts, 1% B SA; 1 part, 0.1 M KH~PO~; 13 parts, 0.154 M Tris in 0.25 M sucrose; 10 parts, 0.5 M nicotinamide; 10 parts, 0.154 M KCI; 10 parts, 0.154 M NaC1; and 40 parts, 0.25 M sucrose. Incubations Incubations of 1.0 ml of incubation medium plus 0.10 ml of homogenate or mitochondrial suspension are carried out in air in 10 ml Erichmeyer flasks or beakers at 37 ° for 10 minutes in a Dubnoff metabolic shaker (108 shakes/minute). Pyruvate or Krebs cycle acids are added as aqueous solutions at the required concentration in a volume of 0.02 ml. The steroid substrate (DOC) dissolved in 0.01 ml of a mixture in ethylene glycol-absolute ethanol (1:1 v/v) is usually added at a concentration (70/~M) known to cause little uncoupling of the respiratory chain of the mitochondria. Thus, the order of preparation for the incubation is as follows: 10 ml beakers on ice in a steel rack to fit the Dubnoff, addition of buffer, addition of pyruvate or Krebs cycle acids, addition
[24]
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of mitoehondria or homogenate, addition of DOC, swirling of the flasks or beakers, and promptly incubating the flasks at 37 ° in the metabolic incubator. The order of addition or preparation for the oxygraph studies is slightly different in that mitoehondria are added to the oxygraph cell before pyruvate or Krebs cycle acids and the whole sequence of addition is carried out when the cell contents are at 37 °. Concentrations of mitoehondria and homogenate, pyruvate and Krebs cycle acids, DOC, and time of incubation (10 minutes) are optimal conditions found in this laboratory. At desired time intervals or usually after 10 minutes, the steroid hydroxylation reactions are stopped hy the addition of 0.1 ml of 1 N H..,SO~ to the incubation vessel. The contents are either extracted immediately or frozen and stored at --5 °. Steroid substrate hydroxylated products are stable for many months in this manner. Extraction and Measurement of Corticosterone
The steroids are extracted by taking appropriate aliquots of the incubation mixture which are added to aqueous ethanol to give a final concentration of 13% ethanol (v/v) in 2 ml of the solution. This ethanolie extract is in turn extracted with 5 ml of petroleum ether by shaking in glass-stoppered centrifuge tubes. After removal of the petroleum ether layer the ethanolie aqueous phase is extracted with 4 ml of diehloromethane in the same tube. At this point the aqueous layer is discarded and the dichloromethane layer can be used for the determination of cortieosterone as well as 18-hydroxylated steroids. Cortieosterone is measured in the above dichlormnethane extract by the sulfuric acid fluorescence method of Silber e t al. 4 To aliquots of the dichloromethane layer is added 3 ml of 80% aqueous H..,SO~ and fluorescence is measured after 50 minutes at room temperature in an AmincoBowman Speetrofluorometer with activating wavelength of 470 nm and fluorescence wavelength of 515 nm. The eortieosterone assays are very accurate, sensitive, and specific when using the rat as the species of animal for the above studies. Cortisol or other steroids which might interfere in the assay are. not produced in the rat from either DOC or endogenous steroid precursors because of the tack of the 17~-hydroxylase enzyme in the rat adrenal. Precautions
1. It is imperative to use purified diehloromethane to carry out the extractions; otherwise, high blank values may be obtained. A convenient R. H. Silber, R. D. Busch, and R. Oslapas, Clin. Chem. 4, 278 (1958).
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method for purification of the dichloromethane is as follows: One-thousand milliliters of dichloromethane are washed with an equal volume of water and dried over a layer of anhydrous Na.oSO4. Five grams of Norite charcoal are added next, the flask swirled, and the dichloromethane filtered into a large Erlenmeyer flask. Sufficient sodium hydroxide pellets are added to cover the bottom of the flask and this is allowed to stand overnight at room temperature before distillation. Only the middle distilled fraction (37.5-38 ° ) is used for extraction purposes in our studies. 2. If female rats are used, the dichloromethane extracts of the incubation medium should be washed with 0.1 N aqueous N a O H before the addition of the 80% H~SO~ to eliminate the possible contamination by estrogens which interfere in the fluorescence assay. I t is imperative to remove all of the sodium hydroxide even at the expense of removing a little dichloromethane extract. If this is not done at the addition stage of the 80% H-_S04, Na_~SO~ will be formed which quenches the fluorescence of the corticosterone measured. 3. For obvious reasons, utmost precautions should be taken to minimize the possibility of any stressful noises or other situations from disturbing the control animals; for example, guinea pigs are not to be placed in the same rooms as rats since the guinea pig squeal is very stressing to the rats. Decapitation of the control rats can be done in 1 or 2 seconds by experienced hands. Stressful effects in terms of increasing plasma corticosterone values in control rats have not been observed using this method of sacrifice when done efficiently and with a minimum level of noise.
Measurement of Oxygen Uptake, Respiratory Control, and A D P - O Ratios Mitochondrial oxygen utilization is measured polarographically with the vibrating platinum electrode of the Gilson, model K M oxygraph. Recordings can be taken at various temperatures by circulating water at the desired temperature through the jacket surrounding the cell in which the respiration studies are carried out. The cell usually contains the buffer mixture outlined under the incubation medium section, an oxidizable substrafe like succinate (5 raM), mitochondria (1 mg protein) as prepared in mixture A or B, and the steroid DOC in a final volume of 1.3 ml. A D P - O ratios are obtained in the absence of DOC after addition of 0.02 ml of an aqueous solution of A D P (200 nmoles), i.e., in state 3 mitochondria, whereas the respiratory control ratios represent values obtained by dividing 02 uptake in state 3 mitochondria by that obtained in state 4. It has been our experience that low respiratory controls are always ob-
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MITOCHONDRIA
TABLE I ADP-O
RATIOS AND RESPIRATORY CONTROL FOR ACTH AND CONTROL MITOCHONDRIA OBTAINFD ~ I T H SUCCINATE
Buffer used in the preparation 1. Mixture A
ADPO I)~Ca
2. Mixture A Mg 2+ added before the experiment 3. Mixture B -k- 1 mM Mg 2+ 4. Mixture B H- 1 mM Mg ~+ 2 mM EDTA added before b the experiment
ADP-O RC
ACTIt mitochondria
Control mitoehondria
2.0 2.07 2.12 2.86 ADP-O ratio could not be obtained since the rate of 02 utilization did not change after the addition of ADP. RC could not be calculated. Same as above 2.08 2.2 1.79 2.25
a Here, RC stands for respiratory control. b EDTA added 5 minutes before addition of 2 mM suecinate. t a i n e d no m a t t e r w h a t o x i d i z a b l e s u b s t r a t e is used ( T a b l e I ) . On t h e o t h e r hand, good A D P - O r a t i o v a l u e s are found i n d i c a t i n g t i g h t coupling of o x i d a t i o n to p h o s p h o r y l a t i o n with K r e b s cycle acids or s u b s t r a t e s (Fig. 1). A D P - O r a t i o s are v e r y low or c a n n o t be o b t a i n e d if the m i t o c h o n d r i a h a v e been p r e p a r e d in the absence of E D T A ( m i x t u r e B ) . I n fact, complete u n c o u p l i n g is o b s e r v e d if m i t o c h o n d r i a arc p r e p a r e d in the presence of 1 m M M g '-'+. I n these p r e p a r a t i o n s a 5 - m i n u t e p r e i n c u b a t i o n of t h e m i t o c h o n d r i a with 2 m M E D T A is a p r e r e q u i s i t e for o b t a i n i n g A D P - O r a t i o s a n d to c o m p l e t e l y reverse t h e u n c o u p l i n g effect of Mg2+. 5,6 I n exp e r i m e n t s where o x y g e n u p t a k e a n d c o r t i c o s t e r o n e p r o d u c t i o n (from D O C ) r a t e s are s t u d i e d as a consequence of o x i d i z a b l e s u b s t r a t e a d d i t i o n , A D P is not a d d e d ( s t a t e 4 m i t o c h o n d r i a ) . T h e a m o u n t of D 0 C which is a d d e d is c a r e f u l l y a d j u s t e d to a c o n c e n t r a t i o n (70 ~ M ) which will m i n i m i z e i n h i b i t i o n of corticosterone f o r m a t i o n b y D O C , which i n h i b i t s at the first p h o s p h o r y l a t i o n site of the r e s p i r a t o r y chain. 7 1l$-Hydroxylation of DOC F o r t h e sake of b r e v i t y we will refer to h o m o g e n a t e s , m i t o e h o n d r i a , a n d s u p e r n a t a n t o b t a i n e d from a d r e n a l s of r a t s t r e a t e d with A C T H simL. A. Sauer and P. J. Mulrow, Arch. Biochem. Biophys. 134, 486 (1969). ~J. L. Purvis, R. G. Battu, and F. G. P~ron, /n "Functions of the Adrenal Cortex" (K. MeKerns, ed.), Vol. II, p. 1007. Appleton, New York, 1968. 7L. Sauer, Arch. Biochem. Biophys. 149, 42 (1972).
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02=100% ~~__
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I0 m l b u f f e r - ~--0..I ml mitocho:ria--J~---/
--0.02 ml ADO(0.01 '\, ~ ~
4 mo,os \
ADp: o o ~.o, ', \
",
,\
\,X
l 48.:5nmoles ADP:O=2.07
I
Fie. 1. ADP-0 ratios for mitochondria prepared in mixture A. Trace A is for ACTH mitoehondria and trace B for control mitochondria. ply as " A C T H homogenate," " A C T H mitochondria," and " A C T H Sup," respectively. Similarly, the tissue fractions from control animals will be referred to as "control homogenate," etc. Table II shows that in the presence of an optimum concentration of pyruvate the A C T H homogenate converts more DOC into eortieosterone than the control homogenate. Similar results are obtained with malate, isoeitrate, ~-ketoglutarate, sueeinate, and fumarate. No differences are observed, however, with lactate and oxalaeetate which support l l B - h y droxylation only very poorly if at all. Table I I I compares the pyruvate-supported l lB-hydroxylation of DOC in A C T H and control mitoehondria. In contrast to the homogenate experiments (Table I I ) , it is clear that the conversion of DOC into cortieosterone in A C T H mitoehondria depends greatly on the medium used for the preparation. When prepared in mixture A (i.e., in the presence of E D T A ) the A C T H mitoehondria produce much less eortieosterone than control mitoehondria. Such an inhibition is still observed, although
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A C T H A C T I O N ON A D R E N A L ~ I I T O C H O N D R I A
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TABLE II CONVERSION OF 1 ) O C TO CORTICOSTERONE :[~Y I)~AT ADRENAL HOMOGENATES IN THE PRESENCE OF PYRUVATE a
Homogenization buffer Mixture A Mixture B
ACTHhomogenate Control homogenate (Corticosterone formed ug/10 minutes/mg protein) 16.8 11.8
8.5 8.3
In these experiments the concentration of ])OC and pyruvate were 300 ~M and 1 raM, respectively. TABLE III CONVERSION OF ] ) O C
Buffer used in the preparation Mixture A Mixture B
TO CORTICOSTERONE I]Y RAT ADRENAL MITOCHONDRIA IN THE PRESENCE OF PYRUVATE a
ACTH mitoehondria Control mitoehondria (Corticosterone formed ~g/10 minutes/rag protein) 2.7 7.5
7.8 9.8
a Each beaker contained 70 ~M DOC and 1 mM pyruvate. to a lesser extent, if the m i t o c h o n d r i a are p r e p a r e d in m i x t u r e B (no EDTA). I f the m i t o c h o n d r i a are prepared in m i x t u r e B a n d p r e i n c u b a t e d with 5.lg '-'+ a n d p y r u v a t e a t 37 ° for 30 m i n u t e s before the a d d i t i o n of D O C , the A C T H m i t o c h o n d r i a produce as much corticosterone as the control m i t o e h o n d r i a ( T a b l e I V ) . Such an effect is n o t observed if i n s t e a d of TABLE IV EFF]:CT OF PREINCUBATION WITH Mg 2+ ON THE SUBSEQUENT CONVERSION OF ] ) O C TO CORTICOSTERONE BY ADRENAL MITOCHONDRI.% IN THE PRESENCE OF PYRUVATE a
Mg 2+ added (uM) 0 100 500 1000 1500 2500
ACTH mitochondria Control mitochondria (Corticosterone formed ug/lO minutes/rag protein) 10.5 11.6 12.7 15.3 14.7 12.2
Each beaker contained 70 gM DOC and 1 mM pyruvate.
12.4 13.8 15.3 14.8 11.6 9.1
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preincubating mitochondria with Mg ~+ this ion is only added immediately before the incubation or Ca .-'+ is replaced for Mg -'+. It appears that Mg 2÷ might be lost fronl the ACTH mitochondria during the preparation, especially in the presence of EDTA in mixture A, but not from the controls. This results in an apparent anomalous effect of ACTH on pyruvate-supported llfl-hydroxylation of DOC.
[25] P r e p a r a t i o n a n d P r o p e r t i e s o f C y t o c h r o m e P from Endocrine Glands By PETER F. HALL
Cytochrome P450 occurs in mitochondrial and microsomal fractions of cells from the steroid-forming organs, namely: adrenal, ovary, testis, and placenta. In the adrenal, P4.~0occurs in the cortex, while in the ovary this cytochrome is found in corpus luteum. It is likely that elsewhere in the ovary and in the testis and placenta, P450 is to be found in those cells responsible for steroid synthesis, although technical problems have so far prevented a direct confirmation of this presumption. It should also be considered that in the placenta, P450 may serve some role in connection with hydroxylation of drugs although this has not been clearly demonstrated. Cytochrome P4~0 oxidizes substrates with the aid of atmospheric oxygen, one atom of which is reduced to water and the other atom becoming the oxygen of a hydroxyl group on the substrate. To reduce oxygen the P450 must in turn be reduced by electrons from TPNH. Mitochondrial P4~o Much more is known about the mitochondrial P450 from adrenal cortex than about that of other endocrine organs. This P450 requires a flavoprotein (TPNH-dehydrogenase) and a nonheme iron protein (adrenodoxin) in order to convey electrons from T P N H to oxygen: 0 2 ~ / ~ Steroid
TPNH
~- Fp ~
NHI
Cytochrome P[:H ~ . ~ Steroid_OH
The steroid substrate is hydroxylated at specific C atoms; namely, ll~and 18- hydroxylation and hydroxylation reactions in connection with the conversion of cholesterol to pregnenolone (so-called side chain cleav-
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ration of 3-P-glycerate conversion to glyceraldehyde-3-P (probably resulting from increased reduction of the cytosolic NAD systems).3s Final Comments The method of tissue metabolic analysis has been used most extensively in the study of the control of glycolysis and glycogen metabolism in muscle and of gluconeogenesis in liver. This is mainly because of the ready availability of relevant analytical grade enzymes, the high flux rates, and the relatively high concentrations of the intermediates of these pathways. In addition, the fact that these tissues are relatively homogeneous and that many of the intermediates of these pathways are localized to the cytosol reduces the number of potential problems of interpretation related to cell heterogeneity or compartmentation. Although tissue metabolic analysis has provided much useful information regarding the regulation of other pathways (fatty acid oxidation and ketogenesis, the hexosc monophosphate shunt, triglyceride breakdown and synthesis, Krebs cycle, and protein synthesis) such studies have generally been incomplete or have given equivocal results because of the inability to measure some key intermediates or determine the distribution of metabolites between mitoehondria and cytosol. It is anticipated that assays of improved specificity and sensitivity will be developed to partly overcome these problems and to enable other pathways to be studied. The problem of conIpartmentation seems somewhat intractible at this time, but it should be noted that in no instance has compartmentation been shown to negate interpretations based on metabolic analysis.
[24] Assessment of in Vivo Adrenocorticotropic Hormone Treatment on Adrenal Mitochondrial Functions in V i t r o By ~TANLEY BANIUKIEWICZ,AJAI HAKSAR,and FERNAND PERON Introduction
Mitochondria of steroid-producing tissues are unique in that they contain, in addition to the classic electron transport chain, a cytochrome P4~o chain which is directly involved in the activation of oxygen for several steroid hydroxylating reactions. In the rat adrenal cortex, the
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hydroxylations involved in side chain cleavage of cholesterol as well as hydroxylations at llfl and 18 positions on the steroid nucleus are carried out in the mitochondria. Adrenocorticotropic hormone (ACTH) stimulates the production of corticosterone in this species when given in vivo or when added in vitro to incubations of adrenal sections or cell suspensions but not when added directly to isolated mitochondria. However, mitochondria obtained from adrenals of rats treated with ACTH, in vivo, show differences in some of the functions (including and related to steroidogenesis) when compared with the adrenal mitochondria from untreated control rats. In our laboratory we have compared adrenal mitochondria from ACTH-treated and control rats in terms of their ability to carry out 11fl-hydroxylation of ll-deoxycorticosterone (DOC) in the presence of some Krebs cycle acids. This ability, or extent in which the l lfl-hydroxylation reaction occurs in vitro, depends on the method used to prepare the mitochondria. We have also measured oxygen uptake and ADP-O ratios in our mitochondrial preparations while other laboratories have compared parameters such as side chain cleavage of cholesterol and cytochrome P4~o levels in the adrenal mitochondria from ACTH-treated and untreated animals. 1-3 The purpose of this section is to present the methods used in our laboratory for the preparation of rat adrenal mitochondria to show the importance of such methods for investigating in vivo effect (s) of ACTH action as is reflected in certain parameters of mitochondrial activity in vitro. Animals and Treatment Adult male Sprague-Dawley rats, weighing 150-170 g, from Charles River Company are used in these experiments. The animals are allowed Purina rat chow and water ad libitum and are used in the experiments only after they have spent at least a week in our sound-proofed animal quarters. Every group of rats is divided into two smaller groups: one to serve as the untreated controls and the other as ACTH-treated. The animals in the latter group are given three subcutaneous injections of 0.1 ml of porcine ACTH (Acthar gel, 40 USP units/ml; Armour Co.) in the nape of the neck at 4 P..~I. and 11 P.M. on the day preceding the experiment.and at 8 A.M. on the day of the experiment, respectively. Rats in both groups are sacrificed by decapitation 90 minutes (controls first) 1 A. C. Brownie, E. R. Simpson, C. and H. Beinert, Biochem. Biophys. ZE. R. Simpson, C. R. Jefcoate, A. 28, 442 (1972). 3A. C. Brownie, J. Alfano, C. R. and H. Beinert, in press.
R. Jefcoate, G. S. Boyd, W. It. Orme-Johnson, Res. Commun. 46, 483 (1972). C. Brownie, and G. S. Boyd, Eur. J. Biochem. Jefcoate, E. R. Simpson, W. Orme-Johnson,
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after the last injection of ACTH to the treatment group. At the time of sacrifice the rats in both groups weigh about 200-220 g.
Preparation of Homogenates The ,~drenals of 20 control or ACTH-treated rats are rapidly excised after sacrifice,, trimmed of all adhering fat, and homogenized in 7 ml of the ice-cold buffers denoted hereafter as mixture A or B. Mixture A: Tris buffer (20 raM), sucrose (250 mM), nicotinamide (20 raM), and EDTA (1 raM), pH 7.4 Mixture B: Tris buffer (20 mM), sucrose (250 raM), and nicotinamide (20 raM), pH 7.4 Homogenization of the whole adrenals is carried out in a glass PotterE1vehjem homogenizer tube fitted with a loose glass pestle which is driven by a variable speed Eberback rotary motor. Generally, the cleaned adrenals from the 20 rats weigh about 800-1000 mg (wet weight) and are completely homogenized in the buffer by 8-10 passes of the pestle in the homogenizer tube. This procedure is easily carried out in the ambient temperature of the laboratory taking care to keep the homogenizer tube submerged in a beaker containing cracked ice.
Preparation of the Mitochondrial Fraction The homogenate prepared above is then centrifuged in a Spinco model L centrifuge at 400 g for 10 minutes. The supernatant is removed carefully to avoid contamination from the loosely packed sediment and recentrifuged at 9000 g to obtain the mitochondrial pellet. After removal of the supernatant with a Pasteur pipette and wiping the tube free of the white lipid layer which floats on top of the supernatant, the mitochondrial pellet is suspended in 3 ml of the buffer using a hollow glass rod sealed at one end by a glass bubble blown previously to exactly fit the bore of the plastic tube containing the pellet. After using as many rapid strokes of the glass rod as are required for complete suspension of the pellet, it is recentrifuged at 9000 g. The suspension and washing procedure is repeated once more, and finally the washed pellet is transfered by means of 2.0 ml of buffer to a clean Potter-Elvehjem homogenizer tube and resuspended in the buffer with the pestle turning at slow speed to yield a homogeneous suspension of mitochondria. The mitochondrial preparation is diluted with buffer to yield 0.10 ml of a suspension of mitochondria derived from 20 mg of original wet weight adrenal tissue. Generally, one adrenal (15-20 mg} yields about 0.5 mg of the mitochondrial protein and the procedure described above results in a mitochondrial
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OF HORMONES
[24]
preparation with minimum contamination from other organelles or components of the tissue. Protein concentrations are determined by the wellknown method of Lowry using bovine serum albumin to standardize the assay. Preparation of the Microsomal Fraction The supernatant obtained after the first 9000 g centrifugation to sediment mitochondria above is recentrifuged at 15,000 g for 15 minutes. The resulting sedimented pellet containing "light mitochondria" and "heavy" microsomes" is discarded, and the supernatant is centrifuged at 105,000 g for 60 minutes. The reddish completely transparent pellet thus obtained contains the microsomes and is suspended in the buffer to give a protein concentration of approximately 5 mg/ml of the suspension. One adrenal generally yields about 0.25 mg of protein. The final supernatant is considered to be made up of the soluble components of the cell and usually contains about 3.2 mg protein per ml. One adrenal yields about 0.5 mg of protein. Incubation Medium The basic buffer for the incubation contains the following: sucrose (141 raM), nicotinamide (53 mM), KC1 (16.4 mM), NaC1 (16.4 mM), KH2PO~ (1.1 raM), bovine serum albumin (BSA, 0.1%), and Tris-HC1 (21 mM), pH 7.4. This mixture is prepared from the following isotonic s~ock aqueous solutions: 10 parts, 1% B SA; 1 part, 0.1 M KH~PO~; 13 parts, 0.154 M Tris in 0.25 M sucrose; 10 parts, 0.5 M nicotinamide; 10 parts, 0.154 M KCI; 10 parts, 0.154 M NaC1; and 40 parts, 0.25 M sucrose. Incubations Incubations of 1.0 ml of incubation medium plus 0.10 ml of homogenate or mitochondrial suspension are carried out in air in 10 ml Erichmeyer flasks or beakers at 37 ° for 10 minutes in a Dubnoff metabolic shaker (108 shakes/minute). Pyruvate or Krebs cycle acids are added as aqueous solutions at the required concentration in a volume of 0.02 ml. The steroid substrate (DOC) dissolved in 0.01 ml of a mixture in ethylene glycol-absolute ethanol (1:1 v/v) is usually added at a concentration (70/~M) known to cause little uncoupling of the respiratory chain of the mitochondria. Thus, the order of preparation for the incubation is as follows: 10 ml beakers on ice in a steel rack to fit the Dubnoff, addition of buffer, addition of pyruvate or Krebs cycle acids, addition
[24]
ACTH ACTION ON ADRENAL MITOCHONDRIA
299
of mitoehondria or homogenate, addition of DOC, swirling of the flasks or beakers, and promptly incubating the flasks at 37 ° in the metabolic incubator. The order of addition or preparation for the oxygraph studies is slightly different in that mitoehondria are added to the oxygraph cell before pyruvate or Krebs cycle acids and the whole sequence of addition is carried out when the cell contents are at 37 °. Concentrations of mitoehondria and homogenate, pyruvate and Krebs cycle acids, DOC, and time of incubation (10 minutes) are optimal conditions found in this laboratory. At desired time intervals or usually after 10 minutes, the steroid hydroxylation reactions are stopped hy the addition of 0.1 ml of 1 N H..,SO~ to the incubation vessel. The contents are either extracted immediately or frozen and stored at --5 °. Steroid substrate hydroxylated products are stable for many months in this manner. Extraction and Measurement of Corticosterone
The steroids are extracted by taking appropriate aliquots of the incubation mixture which are added to aqueous ethanol to give a final concentration of 13% ethanol (v/v) in 2 ml of the solution. This ethanolie extract is in turn extracted with 5 ml of petroleum ether by shaking in glass-stoppered centrifuge tubes. After removal of the petroleum ether layer the ethanolie aqueous phase is extracted with 4 ml of diehloromethane in the same tube. At this point the aqueous layer is discarded and the dichloromethane layer can be used for the determination of cortieosterone as well as 18-hydroxylated steroids. Cortieosterone is measured in the above dichlormnethane extract by the sulfuric acid fluorescence method of Silber e t al. 4 To aliquots of the dichloromethane layer is added 3 ml of 80% aqueous H..,SO~ and fluorescence is measured after 50 minutes at room temperature in an AmincoBowman Speetrofluorometer with activating wavelength of 470 nm and fluorescence wavelength of 515 nm. The eortieosterone assays are very accurate, sensitive, and specific when using the rat as the species of animal for the above studies. Cortisol or other steroids which might interfere in the assay are. not produced in the rat from either DOC or endogenous steroid precursors because of the tack of the 17~-hydroxylase enzyme in the rat adrenal. Precautions
1. It is imperative to use purified diehloromethane to carry out the extractions; otherwise, high blank values may be obtained. A convenient R. H. Silber, R. D. Busch, and R. Oslapas, Clin. Chem. 4, 278 (1958).
300
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[24]
method for purification of the dichloromethane is as follows: One-thousand milliliters of dichloromethane are washed with an equal volume of water and dried over a layer of anhydrous Na.oSO4. Five grams of Norite charcoal are added next, the flask swirled, and the dichloromethane filtered into a large Erlenmeyer flask. Sufficient sodium hydroxide pellets are added to cover the bottom of the flask and this is allowed to stand overnight at room temperature before distillation. Only the middle distilled fraction (37.5-38 ° ) is used for extraction purposes in our studies. 2. If female rats are used, the dichloromethane extracts of the incubation medium should be washed with 0.1 N aqueous N a O H before the addition of the 80% H~SO~ to eliminate the possible contamination by estrogens which interfere in the fluorescence assay. I t is imperative to remove all of the sodium hydroxide even at the expense of removing a little dichloromethane extract. If this is not done at the addition stage of the 80% H-_S04, Na_~SO~ will be formed which quenches the fluorescence of the corticosterone measured. 3. For obvious reasons, utmost precautions should be taken to minimize the possibility of any stressful noises or other situations from disturbing the control animals; for example, guinea pigs are not to be placed in the same rooms as rats since the guinea pig squeal is very stressing to the rats. Decapitation of the control rats can be done in 1 or 2 seconds by experienced hands. Stressful effects in terms of increasing plasma corticosterone values in control rats have not been observed using this method of sacrifice when done efficiently and with a minimum level of noise.
Measurement of Oxygen Uptake, Respiratory Control, and A D P - O Ratios Mitochondrial oxygen utilization is measured polarographically with the vibrating platinum electrode of the Gilson, model K M oxygraph. Recordings can be taken at various temperatures by circulating water at the desired temperature through the jacket surrounding the cell in which the respiration studies are carried out. The cell usually contains the buffer mixture outlined under the incubation medium section, an oxidizable substrafe like succinate (5 raM), mitochondria (1 mg protein) as prepared in mixture A or B, and the steroid DOC in a final volume of 1.3 ml. A D P - O ratios are obtained in the absence of DOC after addition of 0.02 ml of an aqueous solution of A D P (200 nmoles), i.e., in state 3 mitochondria, whereas the respiratory control ratios represent values obtained by dividing 02 uptake in state 3 mitochondria by that obtained in state 4. It has been our experience that low respiratory controls are always ob-
[24]
ACTH ACTION
ON ADRENAL
301
MITOCHONDRIA
TABLE I ADP-O
RATIOS AND RESPIRATORY CONTROL FOR ACTH AND CONTROL MITOCHONDRIA OBTAINFD ~ I T H SUCCINATE
Buffer used in the preparation 1. Mixture A
ADPO I)~Ca
2. Mixture A Mg 2+ added before the experiment 3. Mixture B -k- 1 mM Mg 2+ 4. Mixture B H- 1 mM Mg ~+ 2 mM EDTA added before b the experiment
ADP-O RC
ACTIt mitochondria
Control mitoehondria
2.0 2.07 2.12 2.86 ADP-O ratio could not be obtained since the rate of 02 utilization did not change after the addition of ADP. RC could not be calculated. Same as above 2.08 2.2 1.79 2.25
a Here, RC stands for respiratory control. b EDTA added 5 minutes before addition of 2 mM suecinate. t a i n e d no m a t t e r w h a t o x i d i z a b l e s u b s t r a t e is used ( T a b l e I ) . On t h e o t h e r hand, good A D P - O r a t i o v a l u e s are found i n d i c a t i n g t i g h t coupling of o x i d a t i o n to p h o s p h o r y l a t i o n with K r e b s cycle acids or s u b s t r a t e s (Fig. 1). A D P - O r a t i o s are v e r y low or c a n n o t be o b t a i n e d if the m i t o c h o n d r i a h a v e been p r e p a r e d in the absence of E D T A ( m i x t u r e B ) . I n fact, complete u n c o u p l i n g is o b s e r v e d if m i t o c h o n d r i a arc p r e p a r e d in the presence of 1 m M M g '-'+. I n these p r e p a r a t i o n s a 5 - m i n u t e p r e i n c u b a t i o n of t h e m i t o c h o n d r i a with 2 m M E D T A is a p r e r e q u i s i t e for o b t a i n i n g A D P - O r a t i o s a n d to c o m p l e t e l y reverse t h e u n c o u p l i n g effect of Mg2+. 5,6 I n exp e r i m e n t s where o x y g e n u p t a k e a n d c o r t i c o s t e r o n e p r o d u c t i o n (from D O C ) r a t e s are s t u d i e d as a consequence of o x i d i z a b l e s u b s t r a t e a d d i t i o n , A D P is not a d d e d ( s t a t e 4 m i t o c h o n d r i a ) . T h e a m o u n t of D 0 C which is a d d e d is c a r e f u l l y a d j u s t e d to a c o n c e n t r a t i o n (70 ~ M ) which will m i n i m i z e i n h i b i t i o n of corticosterone f o r m a t i o n b y D O C , which i n h i b i t s at the first p h o s p h o r y l a t i o n site of the r e s p i r a t o r y chain. 7 1l$-Hydroxylation of DOC F o r t h e sake of b r e v i t y we will refer to h o m o g e n a t e s , m i t o e h o n d r i a , a n d s u p e r n a t a n t o b t a i n e d from a d r e n a l s of r a t s t r e a t e d with A C T H simL. A. Sauer and P. J. Mulrow, Arch. Biochem. Biophys. 134, 486 (1969). ~J. L. Purvis, R. G. Battu, and F. G. P~ron, /n "Functions of the Adrenal Cortex" (K. MeKerns, ed.), Vol. II, p. 1007. Appleton, New York, 1968. 7L. Sauer, Arch. Biochem. Biophys. 149, 42 (1972).
302
EVALUATION OF BIOLOGICAL EFFEC TS OF HORMONES
02=100% ~~__
[24]
I0 m l b u f f e r - ~--0..I ml mitocho:ria--J~---/
--0.02 ml ADO(0.01 '\, ~ ~
4 mo,os \
ADp: o o ~.o, ', \
",
,\
\,X
l 48.:5nmoles ADP:O=2.07
I
Fie. 1. ADP-0 ratios for mitochondria prepared in mixture A. Trace A is for ACTH mitoehondria and trace B for control mitochondria. ply as " A C T H homogenate," " A C T H mitochondria," and " A C T H Sup," respectively. Similarly, the tissue fractions from control animals will be referred to as "control homogenate," etc. Table II shows that in the presence of an optimum concentration of pyruvate the A C T H homogenate converts more DOC into eortieosterone than the control homogenate. Similar results are obtained with malate, isoeitrate, ~-ketoglutarate, sueeinate, and fumarate. No differences are observed, however, with lactate and oxalaeetate which support l l B - h y droxylation only very poorly if at all. Table I I I compares the pyruvate-supported l lB-hydroxylation of DOC in A C T H and control mitoehondria. In contrast to the homogenate experiments (Table I I ) , it is clear that the conversion of DOC into cortieosterone in A C T H mitoehondria depends greatly on the medium used for the preparation. When prepared in mixture A (i.e., in the presence of E D T A ) the A C T H mitoehondria produce much less eortieosterone than control mitoehondria. Such an inhibition is still observed, although
[24]
A C T H A C T I O N ON A D R E N A L ~ I I T O C H O N D R I A
303
TABLE II CONVERSION OF 1 ) O C TO CORTICOSTERONE :[~Y I)~AT ADRENAL HOMOGENATES IN THE PRESENCE OF PYRUVATE a
Homogenization buffer Mixture A Mixture B
ACTHhomogenate Control homogenate (Corticosterone formed ug/10 minutes/mg protein) 16.8 11.8
8.5 8.3
In these experiments the concentration of ])OC and pyruvate were 300 ~M and 1 raM, respectively. TABLE III CONVERSION OF ] ) O C
Buffer used in the preparation Mixture A Mixture B
TO CORTICOSTERONE I]Y RAT ADRENAL MITOCHONDRIA IN THE PRESENCE OF PYRUVATE a
ACTH mitoehondria Control mitoehondria (Corticosterone formed ~g/10 minutes/rag protein) 2.7 7.5
7.8 9.8
a Each beaker contained 70 ~M DOC and 1 mM pyruvate. to a lesser extent, if the m i t o c h o n d r i a are p r e p a r e d in m i x t u r e B (no EDTA). I f the m i t o c h o n d r i a are prepared in m i x t u r e B a n d p r e i n c u b a t e d with 5.lg '-'+ a n d p y r u v a t e a t 37 ° for 30 m i n u t e s before the a d d i t i o n of D O C , the A C T H m i t o c h o n d r i a produce as much corticosterone as the control m i t o e h o n d r i a ( T a b l e I V ) . Such an effect is n o t observed if i n s t e a d of TABLE IV EFF]:CT OF PREINCUBATION WITH Mg 2+ ON THE SUBSEQUENT CONVERSION OF ] ) O C TO CORTICOSTERONE BY ADRENAL MITOCHONDRI.% IN THE PRESENCE OF PYRUVATE a
Mg 2+ added (uM) 0 100 500 1000 1500 2500
ACTH mitochondria Control mitochondria (Corticosterone formed ug/lO minutes/rag protein) 10.5 11.6 12.7 15.3 14.7 12.2
Each beaker contained 70 gM DOC and 1 mM pyruvate.
12.4 13.8 15.3 14.8 11.6 9.1
304
EVALUATION OF BIOLOGICAL EFFECTS OF HORMONES
[24]
preincubating mitochondria with Mg ~+ this ion is only added immediately before the incubation or Ca .-'+ is replaced for Mg -'+. It appears that Mg 2÷ might be lost fronl the ACTH mitochondria during the preparation, especially in the presence of EDTA in mixture A, but not from the controls. This results in an apparent anomalous effect of ACTH on pyruvate-supported llfl-hydroxylation of DOC.
[25] P r e p a r a t i o n a n d P r o p e r t i e s o f C y t o c h r o m e P from Endocrine Glands By PETER F. HALL
Cytochrome P450 occurs in mitochondrial and microsomal fractions of cells from the steroid-forming organs, namely: adrenal, ovary, testis, and placenta. In the adrenal, P4.~0occurs in the cortex, while in the ovary this cytochrome is found in corpus luteum. It is likely that elsewhere in the ovary and in the testis and placenta, P450 is to be found in those cells responsible for steroid synthesis, although technical problems have so far prevented a direct confirmation of this presumption. It should also be considered that in the placenta, P450 may serve some role in connection with hydroxylation of drugs although this has not been clearly demonstrated. Cytochrome P4~0 oxidizes substrates with the aid of atmospheric oxygen, one atom of which is reduced to water and the other atom becoming the oxygen of a hydroxyl group on the substrate. To reduce oxygen the P450 must in turn be reduced by electrons from TPNH. Mitochondrial P4~o Much more is known about the mitochondrial P450 from adrenal cortex than about that of other endocrine organs. This P450 requires a flavoprotein (TPNH-dehydrogenase) and a nonheme iron protein (adrenodoxin) in order to convey electrons from T P N H to oxygen: 0 2 ~ / ~ Steroid
TPNH
~- Fp ~
NHI
Cytochrome P[:H ~ . ~ Steroid_OH
The steroid substrate is hydroxylated at specific C atoms; namely, ll~and 18- hydroxylation and hydroxylation reactions in connection with the conversion of cholesterol to pregnenolone (so-called side chain cleav-