Journal

of Molecular

andCellular

Accelerated Isolated

Cardiology

(1978)

10,827-845

RNA and Protein Synthesis in Mitochondria From Unperfused Myocardium. Possible Involvement of Cyclic AMP

BERNHARD KLEITKE AND ALBERT WOLLENBERGER Division of Cellular and Molecular Cardiology, Central Institute of Heart and Circulatory Regulation Research, Academy of Sciences of the DDR, 1115 Berlin-Buch, German Democratic Republic (Received

16 August

1977,

accepted in revisedform

15 November

1977)

B. KLEITKE AND A. WOLLENBERGER. Accelerated RNA and Protein Synthesis in Mitochondria Isolated From Unperfused Myocardium. Possible Involvement of Cyclic AMP. 3ournal of Molecular and Cellular Cardiology (1978) 10, 827-845. Mitochondria were isolated under aseptic conditions from the ventricles of excised rat hearts that were kept totally ischemic (unperfused) at 37°C. The rates of incorporation of [14C]-labelled precursors of DNA, RNA, and protein were measured up to 23 h following excision of the hearts. Incorporation of [‘4C]thymidine into DNA remained unaltered during thii period. Incorporation of [i%]leucine into protein was accelerated by 40% after 20 min of ischemic storage. [i%]Uridine incorporation into RNA was markedly accelerated, the maximum effect (1200/c stimulation) being noted 20 min after excision of the heart. An even greater but more slowly occurring increase in the rate of [iJC]uridine incorporation was observed in mitochondria isolated from unperfused livers. Increased permeability of mitochondrial membranes to uridine and reduction of the mitochondrial uridine pool were excluded as causes of the increased incorporation of [i%]uridine into cardiac mitochondrial RNA. The rate of incorporation of [i%]UMP using [14C]UTP as substrate was increased by about 70% at 20 min of ischemic storage of the hearts. These results indicate that the observed acceleration of uridine incorporation was due in a large measure to stimulation of the transcription process in the isolated cardiac mitochondria. Cardiac cyclic AMP levels were elevated about Z-fold for at least 20 min following excision of the heart. Incorporation of [l*C]uridine into mitochondria isolated from fresh hearts was modestly stimulated by 10-r M cyclic AMP. Treatment of a group of rats with theophylline, which produced a protracted elevation of cardiac cyclic AMP levels, caused a significant increase in the rate of [%I uri ‘d’ me incorporation into the isolated cardiac mitochondria of these rats. The latter set of data is suggestive of a possible role of cyclic AMP in the observed ischemia-induced stimulation of mitochondrial RNA synthesis.

KEY WORDS : Myocardial RNA

synthesis;

Protein

ischemia; Liver ischemia; Mitochondria; synthesis; Cyclic AMP; Theophylline.

DNA

synthesis;

1. Introduction In 1973 we reported that mitochondria isolated from totally ischemic (unperfused) rat hearts, incorporated [14C]uridine at a higher rate than did mitochondria from fresh control hearts [18, 441, the rate of RNA degradation being apparently unchanged [44]. Since isolated cardiac mitochondria have been shown to be capable 0022-2828/78/090827+

19 $02.00/0

0

1978 Academic

Press Inc.

(London)

Limited

828

B. KLEITKE

AND

A.

WOLLENBERGER

of incorporating this nucleoside into their RNA [9], it seemed quite possible that the effect observed by us was due to stimulation in these organelles of the synthesis of RNA from its immediate precursors, the ribonucleoside triphosphates. However, in such a complex system as the mitochondrion, stimulation of the incorporation of [ W] uridine might conceivably be due to changes preceding stimulation of the final step in RNA synthesis, such as an increase in the permeability of the mitochondrial membrane to uridine, a decrease in the mitochondrial uridine pool, or an accelerated conversion of [Wluridine into [W]UTP. The present study was undertaken to test these possibilities and as an attempt to uncover possible factors linking &hernia to the acceleration of cardiac mitochondrial [Wluridine incorporation. The rate of [Wluridine incorporation into mitochondria isolated from unperfused livers was determined for the purpose of comparison. 2. Materials

and Methods

Biochemicals Actinomycin D, ethidium bromide, RNA-free sucrose, N-tris(hydroxymethy1) methyl-2-aminomethane sulfonic acid (TES), marker RNA from wheat seedlings (18s and 25S), and Dowex 1 x 8, 200 to 400 mesh, were products of Serva, Heidelberg; tris(hydroxymethyl)aminomethane (Tris), mercaptoethanol, and rifampicin were obtained from Ferrak, Berlin; chloramphenicol was obtained from VEB Berlin-Chemie; pyruvate kinase, RNase, DNase, RNA from yeast, nucleoside triphosphates, and uridine were purchased from Boehringer Mannheim GmbH; L-adrenaline bitartrate was obtained from Schuchardt, Miinchen. Other nonradioactive chemicals used were of highest purity available. [U-W]UTP, 471 mCi/mmol, [U-WI]uridine, 477 mCi/mmol, [U-Wlthymidine, 495 mCi/mmol, and L-[U-W]leucine, 311 mCi/mmol, were obtained from the Radiochemical Centre, Amersham. Ischemic storage Male Wistar rats, 4 to 5 months old and weighing about 250 g, were used. They were lightly anesthetized with ether, and chest and abdomen were shaved and rinsed with 70% ethyl alcohol. The hearts and livers were rapidly excised and placed for approximately 30 s in a 0.25 M sucrose-20 mM Tris solution of pH 7.4 and 37”C, in which the hearts continued to beat. The organs were then transferred to a moist stoppered tube, in which they were kept unperfiused at 37°C for different times [see ref. a. They were then transferred into ice-cold sucrose-Tris solution. i’Xeophylline treatmznt One group of rats was given intraperitoneal

injections of theophylline

ethylene-

ACCELERATED

RNA

AND

PROTEIN

diamine (aminophylline, 75 mg/kg body weight, water) 30 min prior to killing.

SYNTHESIS

829

1 g dissolved in 5 ml of distilled

Preparation of mitochondria The handling of the tissuesand the preparation of the mitochondria were done under aseptic conditions [38] and, unless otherwise stated, at about 0°C. The 10 000 g pellet obtained was suspended in 0.1 M potassium phosphate solution of pH 7.4 and kept in this solution at 30°C for 30 min in order to make the mitochondrial membrane freely permeable to substrates [37]. About 10 vols of ice-cold 0.25 M sucrose-20 mM Tris solution of pH 7.4 were added to the suspensionand mitochondria were recovered by a 10 min centrifugation at 10 000 gaV. They were washed twice with the samesucrosesolution and finally suspendedin the sucrose solution in a dilution of about 20 mg of mitochondrial protein per ml. The mitochondrial suspensionwas frozen and stored at -20°C for incorporation experiments on the next day (see below). Preliminary experiments had shown that heart mitochondria can be stored frozen under these conditions for several days without loss in their incorporation activity. Rat liver mitochondria were isolated by the same method employed for the preparation of cardiac mitochondria except that a teflon-glass homogenizer was used. Contamination of the mitochondrial preparations by bacteria was negligible, as indicated by electron microscopic inspection, kindly performed by Dr W. Schulze. Incorporationexperiments Synthesis of mitochondrial DNA and RNA was estimated at 37°C by following the rates of incorporation of [Wlthymidine and of [Wluridine or [W]UTP, respectively, into cold trichloroacetic acid (TCA)-insoluble material of the isolated organelles. Mitochondrial protein synthesis was estimated by measuring the incorporation of [Wlleucine at 30°C into hot (90°C) TCA-insoluble mitochondrial material. Incorporation experiments were carried out in a shaking water bath in 5 ml glassvials which contained 0.5 ml of one of the following reaction mixtures : (a) For [WYJthymi diru incorporation,performed with slight modifications according to Parsonsand Simpson [31] : 50 ITM TES, 15 nm potassium phosphate, 4 nm KCl, 7 rnM MgCls, 2 rnM ATP, 15 pi each of deoxyATP, deoxyGTP, and deoxyCTP, 1 pCi of [Wlthymidine, 1 mg mitochondrial protein. The pH was 7.4, the reaction period 30 min. (b) For [W]uridiru incorporation, performed with slight modifications according to Gamble and McCluer [9] : 50 mu TES, 15 mM potassium phosphate, 4 mbn. MgCls, 40 rnM KCl, 10 nm mercaptoethanol, 0.5 nm ethylene diaminotetraacetate (EDTA), 0.5 t&i of [Wluridine, 1 mg mitochondrial protein. The pH was 7.4, the reaction period 20 min.

830

B. KLEITKE

AND

A. WOLLENBERGER

(c) For [l*C] UMP incorporation with [W’] UTP as substrate, performed according to Saccone et al. [3q: 40 mM Tris-HCI, 4 mM MgCls, 66 mu KCl, 0.4 mu each of ATP, GTP, and CTP, 2.5 nm phosphoenol pyruvate, 0.1 yCi of [14C] UTP, 10 pg pyruvate kinase, 1 mg mitochondrial protein. The pH was 7.4, the reaction period 10 min. (d) For [W]leucine incorporation, performed with slight modifications according to Wheeldon and Lehninger [40] : 50 mM TES, 15 mM potassium phosphate, 15 mM MgCls, 100 mM KCl, 100 mM sucrose, 10 mM succinate, 2 mM ATP, 0.5 pCi of [14C]leucine, 1 mg mitochondrial protein. The pH was 7.2 ,the reaction period 20 min. The addition of a mixture of 19 unlabelled amino acids (excepting leucine) did not increaseincorporation rates. Incorporation rates were linear with time under all conditions during the reaction periods indicated above. Each reaction was started by addition of the mitochondrial suspension.At the start and at the end of each run, two 100 ~1 aliquots were taken from the reaction mixture and were quickly placed each on a Whatman 3MM filter paper disc, 22 mm in diameter, which had been prewashed in distilled water. The discswere then transferred into ice-cold 5% TCA solution for termination of the incorporation. The subsequenttreatment was done essentially as outlined by Mans and Novelli [2.5], except that the extraction with hot TCA was omitted when DNA and RNA syntheseswere measured. Becauseof uncontrollable day-to-day variations in the incorporation rates, each set of experiments was carried out with experimental and control rats that were killed at approximately the same time. For the most part the results are expressedin per cent of the incorporation rates of the controls. Determination

of radioactivity

The radioactivity on the filter paper discs was measured in a Nuclear Chicago Mark I liquid scintillation spectrometer using vials filled with 10 ml of a scintillation solution of the following composition: 5 g 2,5-diphenyloxazole and 0.2 g 1,4-bis 2(5-phenyloxazyl) benzene in 1000 ml of toluene. The values of the counts listed in the tables refer to the count numbers per min. Preparation

and sucrose a2nsity gradient centrifugation [W’]-labelled mitochondrial RNA

of

Mitochondria were incubated in the presence of [i%]uridine for 30 min under conditions described for incorporation experiments. The reaction was terminated by the addition of 20 vols of ice-cold 0.25 M sucrose-20 mM Tris solution of pH 7.4 and the mitochondria were recovered by centrifugation at 10 000 gaV. for 10 min. They were washed once with sucrosesolution and then suspendedin 0.5% sodium dodecyl sulfate solution. RNA was extracted and precipitated according to Peacock and Dingman [33], except that carrier RNA was added to the aqueous phaseof the

ACCELERATED

RNA

AND

PROTEIN

SYNTHESIS

831

phenol extract (about 1 mg of yeast RNA to the extract of 20 to 40 mg of mitochondrial protein) and that the precipitated and dissolved RNA was not dialyzed. The precipitated RNA was dissolved in 2 ml of 0.1 M NaCl-1 mu EDTA solution of pH 6.2. Prior to density gradient centrifugation one portion of this RNA solution was treated with 40 p.g RNase/ml in the presence of 5 mM MgClz at 37°C for 30 min. RNase-treated and untreated mitochondrial RNA as well as the 18s and 25s marker RNA (dissolved in the same solution as the mitochondrial RNA) were layered onto a linear sucrose gradient (4 to 40%, w/w, RNase-free sucrose in 0.1 M NaCl-1 mM EDTA solution of pH 6.2). Gradients were run at 40 000 rev/min in a SW 41 rotor of a Beckman L2-65B ultracentrifuge at 0°C for 16 h. Fractions of three drops each were collected and radioactivity was counted according to Hall and Cocking [12]. The absorbancy of each fraction was measured at 260 nm in a Unicam SP 800 spectrophotometer. Determination

of uridine content

(a) Uridine extraction Cardiac mitochondria, prepared as described before, were suspended in ice-cold distilled water to give a protein concentration of 10.0 mg/ml. This suspensionwas frozen and ground to a fine powder in a mortar in the presenceof liquid nitrogen. A volume of 10 N perchloric acid sufficient to make the thawed suspension0.5 N in HC104 was added. The thawed suspensionwas stirred for several minutes and centrifuged at 10 000 gaV and 0°C for 10 min. The supernatant was carefully decanted and neutralized at 0°C by addition of 10 N KOH. Precipitated KC104 was removed at 0°C by centrifugation. Aliquots of the supernatant, 3 ml each, were brought to a pH of 8 to 9 by the addition of 0.02 N NHIOH, mixed with 1.5 g Dowex 1 x 8 ion exchanger in the formate form [14], and stirred at room temperature for 15 min. The resin was then separated from the solution by filtration through a Schott-Jena G2 glassfilter funnel and washed several times with 0.02 N NH40H solution. The washed Dowex was suspendedin 1.5 ml of 0.02 M acetic acid and stirred at room temperature for 15 min in order to selectively elute the bound uridine [3]. The mixture was then centrifuged and the decanted and neutralized supernatant was stored up to the time of the determination of its uridine content. In order to determine the efficiency of this method an aliquot of each mitochondrial extract was mixed with a small quantity of [l%]uridine and submitted to the extraction procedure outlined above. Recovery in the Dowex eluates of added radioactivity amounted to 60 to 80%. For blank correction distilled water was treated in the sameway aswere the extracts. (b) Uridinedetermination None of the available methods for the assay of uridine proved to be sufficiently

832

B. KLEITKE

AND

A.

WOLLENBEROBR

sensitive for the determination of the minute amounts of uridine present in mitochondrial preparations from one or even several rat hearts. Therefore, the content of uridine in the eluates was determined indirectly by following the inhibitory action of the eluates on the incorporation of [l‘X]uridine into RNA of isolated rat liver motochondria, an inhibition due to the dilution of the added [WZ]uridine by the [W+rridine in the extracts. The uridine content of the eluate fraction added to the reaction mixture (see reaction mixture for cardiac mitochondria) was read from a calibration curve comprising the range of 3 to 19 nmol of [W] uridine/ml of reaction mixture. Each reading was corrected for the corresponding blank and recovery values. Determination of cyclic AMP The ventricles of rat hearts were almost instantly frozen [46J (a) in situ prior to and 60 s after cutting the aorta in thoracotomized untreated and theophylline-treated animals given chloralose (50 mg/kg body weight intraperitoneally) - ether anaesthesia and artificial respiration and (b) after storage in the moist chamber at 37°C for 10 and 20 min. Cyclic AMP was extracted [IO], purified by chromatography on aluminum oxide and Dowex 1 x 2 columns [LX], and determined according to Gilman [IO], using a partly purified protein kinase from bovine adrenal cortex (kindly supplied by Dr E.-G. Krause) as binding protein. Determination of fiotetn Mitochondrial protein was estimated bumin as standard.

according

to Lowry

et al. [.?3], using

oval-

Expression of results Unless otherwise

stated results are presented as means f standard errors

(S-E.).

3. Results Efict of inhibitors, nucleases, and hot trichloroacetic acid compounds used into mitoEvidence that the incorporation of the W-labelled chondrial material probably represented synthesis of DNA, RNA, and protein in the isolated organelles, was obtained by following the incorporation in the presence of specific inhibitors of these syntheses and by analyzing some of the labelled reaction products with respect to their susceptibility to degradation by DNase, RNase, and hot TCA (Table 1). The results in Table 1 indicate :

Rifampicin, 1.6 x 10-4~

Chloramphenicol, IO-3 iu

[‘4C]UTP

[14C]Leucine

80-90

60-80

60-80

30-50

30-80

Incorporation reaction 0/0 inhibition by

Cycloheximide, 6 x 10-4~

RNase 20 kcdml

20 f&ml

RNase,

20 w/ml

DNase,

not inhibited

Hot 5% TCA

-

DNase, 40 &ml

RNase, 4.0 w/ml

-

RNase, 40 t.rfiW Hot 5% TCA

DNase, 40 w/ml Hot 5% TCA

[r4C]-labelled incorporation product sensitive to insensitive to

For measurement of breakdown of the labelled products of incorporation, loaded filter paper discs (see Methods) were placed either for 30 min at 37% in a solution of 15 mM potassium phosphate-50 mM TES-5 mu MgCla of pH 7.2, containing the corresponding nucleases, or for 15 min at 90°C in 5% TCA solution. Radioactivity on the discs was measured before and after incubation.

Rifampicin, 1.6 x 10-4~

by

[14C]Uridine

inhibited

Actinomycin D, 4 x 1o-sM Ethidium bromide, 10-4 M

Precursor

1. Effect of inhibitors of the synthesis of mitochondrial DNA, RNA, and protein on the incorporation of [14C]-labelled precursors into acid-insoluble material of isolated cardiac mitochondria. Breakdown by DNase, RNase, and hot trichloroacetic acid

[14C]Thymidine

TABLE

a34

B. KLEITKE

AND

A. WOLLENBERGER

(1) The incorporation of [I’%] thymidine was markedly reduced by actinomycin D and ethidium bromide, two inhibitors of the synthesis of DNA in mitochondria [27, 411. The labelled reaction product was sensitive to DNase and hot TCA, but not to RNase. These results suggest that the incorporation of [l%]thymidine measured was an index for the synthesis of mitochondrial DNA. (2) Rifampicin, an inhibitor of mitochondrial RNA synthesis [9, 211, significantly reduced the incorporation of [Wluridine and [W]UTP. The labelled product of the incorporation of [Wluridine was found to be sensitive to RNase and hot TCA, but not to DNase. These findings suggest that we were measuring the synthesis of RNA in the isolated mitochondria. Possible contamination of the mitochondrial preparations by nuclear material could be excluded as a source of error, since preliminary experiments had shown that isolated heart nuclei did not incorporate [14C]uridine under present incubation conditions. (3) The incorporation of [r%]leucine was strongly inhibited by chloramphenicol, but not by cycloheximide. Chloramphenicol has been found to be a specific inhibitor of mitochondrial protein synthesis in eukaryotic cells [20, 401, whereas cycloheximide mainly inhibits the extramitochondrial protein synthesis in these cells [22]. The observed rates of incorporation of [W]leucine may therefore be taken as a measure of the rates of mitochondrial protein synthesis. (4) The incorporation of [r%]thymidine remained insensitive to DNase and that of [Wluridine insensitive to RNase. This finding is not surprising in view of reports [28, 291 that the mitochondrial membrane is impermeable to these enzymes. Synthesis of DNA,

RNA,

and protein in mitochottdria from unperfwedhearts

Figure 1 indicates that RNA synthesis in the isolated cardiac mitochondria was markedly accelerated following storage of the heart at 37”C, the maximum effect (120% acceleration) being seen after 20 min of storage. At this time the rate of protein synthesis in the mitochondria was enhanced by about 35%. The rate ofsynthesis of DNA remained unchanged in the organelles during the entire period of observation. Subsequent experiments with cardiac mitochondria were concerned solely with the synthesis of RNA. An additional set of experiments was performed in which control and ischemic mitochondria were each isolated from separated parts of the sameheart. The results (Table 2) show that after 20 min of ischemic storage of the myocardium, mitochondria were isolated which incorporated [%]uridine nearly twice as fast as did mitochondria from fresh myocardium, thus confirming the finding shown in Figure 1. Evidence that RNA

wus the reaction product of th incorporation

of [Wluridine

In order to obtain more direct evidence that it was cardiac mitochondrial RNA

ACCELERATED

r

RNA

AND

PROTEIN

835

SYNTHESIS

T

Time

of storoge

bin)

FIGURE 1. Rates of incorporation of [W2]thymidine (O-O), [W]uridine (w), and [r%]leucine (A-A) into mitochondria isolated after various periods of storage of the hearts. Incorporation rates in mitochondria from fresh hearts (0 min) are taken as 100%. The points marked by an asterisk differ significantly (P < 0.001) from the values at zero minute.

TABLE

2. Incorporation myocardium

of [WZ] u&line into RNA of mitochondria prepared and from myocardium kept unperfused for 20 min

Heart no.

1 2 3 4 5 6

Rate (ct

from fresh

of incorporation

mg mitochondrial protein-l Fresh tissue Storage 1 2 x

x

min-1)

20

153 219

363

393

1.8

305 101 74 96

597 157

2.0 1.6 1.7 2.1

129 200

Mean f S.E.

2.4

1.9 * 0.1

The ventricles of each heart were divided into two portions, each consisting of half of the left and half of the right ventricle. One portion was kept in the moist tube at 37”C, the other served as control.

which was labelled by [Wluridine in our product was isolated and characterized by after treatment with RNase. The results of in Figure 2. The gradient profile resembles berger and Tuppy in yeast mitochondria

cell-free system, its sedimentation a representative very closely that [42]. It is seen

the labelled reaction properties before and experiment are shown obtained by Wintersthat RNA species of

836

B. KLRITKJ?,

AND

A.

W0LLRNBERCX.R

different size were labelled and that they all were sensitive to R&se, expected if one was dealing with RNA.

Lineor

FIGURE 2. Sedimentation after treatment with RNase.

of [~*C&ridin~labcllcd

sucrose

as is to be

gradient

cardiac

mitochondrial

RNA

prior

to and

Evkience that RJVA synth-esis was stimulated In such a complex system as the mitochondrion, stimulation of the incorporation of [14C]uridine need not necessarily be equivalent to stimulation of synthesis of RNA. In this system, an acceleration of the incorporation of [Wluridine might likewise be caused by (a) an increase in the permeability of the mitochondrial membrane to uridine, (b) a rise in the rate at which uridine is transformed to UTP, or (c) a decrease in the mitochondrial uridine pool. These possibilities were tested in the following sets of experiments. (a) Permeability of tlu mitochondtial membrane to t&dim? Treatment of isolated mitochondria with 0.1 M potassium phosphate solution has been found to render the membranes of the organelles more permeable to substrates [37j. Isolated cardiac mitochondria that were submitted to such a treatment exhibited a 3- to 4-fold increase in the incorporation of [14C]uridine (Table 3). Nevertheless, the stimulatory effect of myocardial &hernia on this incorporation remained the same, regardless of whether or not the organelles were treated with

ACCELERATED

RNA

AND

PROTEIN

a37

SYNTHESIS

phosphate (Table 3). If this ischemia-induced effect had been due to a phosphate treatment-like alteration of the mitochondrial membrane, one would have expected the effect to be reduced or abolished after treatment of the ischemic mitochondria with 0.1 Mpotassiumphosphate solution.

TABLE

3. Effect of phosphate treatment on the incorporation of [r~C]uridine chondria prepared from fresh and from stored myocardium

Experiment no.

Rate of incorporation ct x mg mitochondrial protein-l x min-1 Phosphate-treated Mitochondria not treated Fold stimulation with phosphate mitochondria 2/l 413 20 min of Fresh tissue 20 min of Fresh tissue

storage

storage

1 1 2 3

47 48 72

2

3

4

108 89 229

153 219 305

363 393 597

Mean f 8.n. Frsh

and stored

into mito-

cardiac

tissues were obtained

from

the same heart

2.0 1.8 3.2

2.4 1.8 2.0

2.3 f 0.4

2.1 & 0.1

(see Table

2).

(b) UridittGcontentof mitochondria The data in Table 4 show that the isolated ischemic cardiac mitochondria contained at least as much uridine as did the corresponding control organelles, although the former incorporated [W]uridine at a considerably higher rate than did the latter. These data speak against the possibility that a reduced uridine pool in the ischemic cardiac mitochondria was responsible for their accelerated [‘*Cl uridine incorporation.

TABLE

4. Content of uridine and rate of [W]uridine isolated from fresh and from stored myocardium

Uridine content (nmol/mg mitochondrial protein) Fresh tissue 20 min of storage 0.83 & 0.39 Fresh and stored

ct

tissues were obtained

in mitochondria

Rate of incorporation mitochondrial protein-1 X min-1 Fresh tissue 20 min of storage

X

1.26 f 0.26 cardiac

incorporation

mg

70 f2 from

the same heart,

117 &Q as described

in Table

2.

838

B. KLEITKE

(c) Incoqoration of[W]

AND

UMP with [W]

A. WOLLENBERUER

UTP as substrate

Since nucleoside triphosphates, but not nucleosides, serve as substrates for the synthesis of RNA, a reaction mixture containing [14C]UTP was used in order to see whether ischemic mitochondria would incorporate [14C]UMP into their RNA at an increased rate. Table 5 shows that this was the case, even though the stimulation of the incorporation was less pronounced than that observed with [14C]uridine. These findings indicate that at least part of the stimulation of incorporation of [14C]uridine due to ischemic storage of the hearts was caused by an increase in the rate of polymerization of the mitochondrial ribonucleoside phosphates to form RNA. TABLE 5. Incorporation of [14C]uridine and of [lQC]UMP+ into RNA ofmitochondria prepared from fresh myocardium and from myocardium kept unperfused for 20 min Experiment no.

Incorporation of [14C]uridine, ct x mg mitochondrial protein-l

x min-1

Freshtissue Storage

1 2 3 4

Incorporation of [14C]UMP*, ct x mg mitochondrial

1

2

130 102 139 118

377 310 297 303

Mean f S.E.

protein-l

211 2.9 3.0 2.1 2.8

X

min-1

Freshtissue Storage 3

4

68 48 40 60

87 67 124 80

2.7 & 0.2

413

1.3 1.4 2.8 1.3 1.7 f 0.3

* The radioactive substrate was[14C]UTP.

Freshandstoredcardiactissues wereobtainedfromthesameheart,asdescribed in Table2.

RNA synthsisin mitochondria from unperfusedrat liver Since it seemedof interest to find out whether total ischemia would cause a stimulation of mitochondrial RNA synthesisin tissuesother than the heart, mitochondria were prepared from excised livers which had been stored in the moist tube at 37°C. They were incubated in the same [14C]uridine incorporation medium that was used for the heart mitochondria. Figure 3 shows that mitochondria from ischemic livers incorporated [14C]uridine into RNA at a higher rate than did mitochondria from fresh livers. A longer period of ischemic storage was required to produce a significant stimulation of the incorporation than was the casein the heart experiments. After 80 min of storage of the livers the rate of incorporation of [14C]uridine into mitochondrial RNA was increased by 95%, after 120min it was increased by almost 150%.

ACCELERATED

2

280

-

260

-

240

-

220

-

RNA

AND

PROTEIN

839

SYNTHESIS

.-s ‘j :2cx3B .E % 2 e

180-

.: z Tl a

160 -

140 -

120

-

L

0

I

I

40 Time

FIGURE 3. Relative rates of incorporation excised unperfused rat livers. The points marked the value at zero minute.

Cyclic AMP

,

I

I

80 of storage

I

120 (mid

of [14C]uridine by an asterisk

into mitochondria differ significantly

isolated (P < 0.02)

from from

levels in unperjksed myocardium and e$ect of added cyclic AMP

Table 6 shows, in confirmation and extension of previous observations on myocardial tissue made in this and other laboratories [7, 35, 431, that the level of cyclic AMP rose quickly in the rat heart after cessationof the coronary flow and remained elevated for at least 20 min. Since there is evidence for a participation of cyclic AMP in the regulation of bacterial [32] and of nuclear [S, 15, 391 RNA synthesis, the question arose whether this nucleotide might have a triggering function in the stimulation of RNA synthesis that was observed in ischemic mitochondria. Exposure of five different preparations of mitochondria from fresh rat hearts to 10-7 M cyclic AMP in [14C] urr‘d’me incorporation medium resulted in increasesin the rate of [W]uridine incorporation between 10 and 30%, the average increase being 22%. The reaction, both in the absence and presence of 10-7 M

840 TABLE

B. KLEITKE

AND

6. Cyclic AMP levels in unperfused of theophylline-treated rats Unperfirsed ventricles Time after cessation Cyclic AMP, of coronary flow pmol/mg heart Cm4 0 1 10 20

0.42 1.23 0.91 1.19

& f * f

A.

WOLLENBEROER

rat heart ventricles

and in heart ventricles

Ventricles oftheophylline-treated rats Time of killing Cyclic AMP after administration pmol/mg heart of theophylline (min)

0.08 0.36 0.21 0.05

control* 10 20 30

0.69 0.84 1.21 0.94

f 0.05 f 0.08 ho.08 f 0.06

* Rats not given theophyhine. The experiments with the unperfused hearts and those with the theophylline-treated animals were performed 3 years apart on different rat populations.

cyclic AMP, was not affected by addition of 10-s M theophylline or of a partly purified cyclic AMP-dependent protein kinase from bovine adrenal cortex (8 to 40 pgglml). R.NA synthesisin isolatedcardiacmitochondriaof t&ophyllin&reated rats Table 6 shows furthermore that the level of cardiac cyclic AMP was elevated almost Z-fold in rats 20 min after an intraperitoneal injection of 75 mg of theophylline ethylenediamine per kg. It was of interest, therefore, to see whether treatment of rats with theophylline might imitate the effect of myocardial ischemia on the synthesis of RNA in isolated cardiac mitochondria. Mitochondria were isolated from the hearts of theophylline-treated rats and were indeed found to incorporate [i*C]uridine at a higher rate than did the mitochondria from control animals. At a dose of theophylline ethylenediamine of 75 mg/kg body weight, corresponding to 3.3 x lo-* mol/kg, given 30 min before excision of the heart, the increase in the rate of [r*C]uridine into mitochondrial RNA amounted, on the average, to 38 f 6%. Higher dosesof theophylline ethylenediamine and intervals other than 30 min were not tried.

4. Discussion

The present experiments have provided evidence that the previously noted [18, 441 increase in the rate of incorporation of [r*C]uridine into cardiac mitochondria isolated from excised rat hearts following 20 min of ischemic storage is due neither to increased permeability of the mitochondrial membranes to uridine nor to a reduction of the mitochondria1 uridine pool, but that it represents in a large measure an accelerated synthesisof mitochondrial RNA from its immediate precursors, the

ACCELERATED

RNA

AND

PROTEIN

SYNTHESIS

841

ribonucleoside triphosphates. Evidence was also obtained for an acceleration of protein synthesis in these mitochondria. On the other hand, synthesis of mitochondrial DNA was not accelerated. As pointed out in the Results section, it is highly unlikely that bacteria and non-mitochondrial structures and components from the source tissue could have contributed to any significant degree to the RNA and protein synthesis activities in the mitochondrial preparations. The observed stimulation of RNA and protein synthetic activities in mitochondria prepared from unperfused myocardium, provided that it is representative of events in these organelles in situ, may be an in vitro manifestation of repair processesthat occur in vivo in ischemia-damaged mitochondria during recovery of heart muscle from an ischemic insult. According to Rabinowitz and co-workers [ll stimulation of the synthesis of cardiac mitochondrial protein is an early event following temporary hypoxia of the rat heart. Their finding corresponds well to ours, since an acceleration of the synthesis of mitochondrial protein requires an acceleration of both intra- and extramitochondrial macromolecular syntheses [4, 341. The finding that RNA synthesis was also increased in mitochondria isolated from unperfused rat livers suggests that stimulation of mitochondrial transcription by ischemia may be a phenomenon of more widespread occurrence among mammalian tissues. In mitochondria isolated from fresh hearts, RNA synthesis was not stimulated by anaerobic incubation of the organelles [441, suggestingthat factors present in intact ischemic cardiac muscle may be acting as stimulants of mitochondrial genetic transcription. The question may be raised, whether cyclic AMP could be one of thesefactors. Injection of this nucleotide into rats has been reported to mimic the stimulatory effect of ACTH on the synthesis of RNA in the mitochondria of adrenocortical cells [30]. In heart muscle the level of cyclic AMP rises sharply following arrest of blood flow [451 and remains elevated for at least 20 min (Table 6). This elevation is believed to be due initially to stimulation of adenylate cyclase causedby endogenouscardiac catecholamine liberation [451 and to be maintained as a result of a decrease in the activity of cyclic AMP phosphodiesterase[43]. Administration of catecholamines capable of raising cellular cyclic AMP levels have been reported to increasein heart muscle the incorporation of labelled amino acids into protein [16, 17, 24 and of labelled ribose into ribosomal RNA [5l. In the present experiment a sustained elevation of myocardial cyclic AMP was produced by a single intraperitoneal injection of the cyclic AMP phosphodiesterase inhibitor, theophylline [13], and this treatment caused a significant increase in the incorporation of [Wluridine into isolated cardiac mitochondria. In bacteria, which resemble the mitochondria with respect to some features of their macromolecular syntheses [2], cyclic AMP is an essential component of controlled transcription [32]. The nucleotide has also been reported to be a regulator of nuclear RNA synthesis [S, 15, 391. In our experiments incorporation of [WZ] uridine into isolated cardiac mitochondria appeared to be subject to stimulation

842

B. KLEITKEAND

A. WOLLENBERGER

by 10-T M cyclic AMP. Since mitochondria isolated from mammalian tissues, including heart muscle, have been found to contain a cyclic AMP-dependent protein kinase as well as substrate for this enzyme [ref. 19 and unpublished data), the system required for the expression of the biological action of cyclic AMP in animal cells [II] seemsto be present in theseorganelles. It must be noted, however, that the observed stimulator-y effect of cyclic AMP on uridine incorporation into heart mitochondria was rather weak. It is possible that present experimental conditions were not favourable for revealing a stimulation of mitochondrial RNA synthesis by added cyclic AMP. Moreover, in addition or alternatively to cyclic AMP, other effecters or factors may be responsiblefor the increased rate of RNA synthesis

seen in heart

and also liver

mitochondria

isolated

from

the unperfused

source organs. Acknowledgetnent We thank Mrs Hanna Sydow for excellent technical assistance in all phases of the present study and Miss Christel Kemsies for performing the [W]thymidine incorporation measurements.

REFERENCES 1. ASXENBRENNER, V., ZAK, R., CUTILETTA, A.F. & RABINO~I~Z, M.Effectofhypoxia on degradation of mitochondrial component in rat cardiac muscle. American 3ournal of Physiolopy221, 1418-1425 (1971). 2. ASHWELL, M. & WORK, T. S. The biogenesis of mitochondria. Annual Review of Biochemistry39,251-290 (1970). 3. COHN, W. E. Methods of isolation and characterization of mono- and polynucleotides by ion exchange chromatography. In Methods in Enzymology, Vol. III, pp. 724-743. S. P. Colowick and N. D. Kaplan, Eds. New York: Academic Press Inc. (1957). COOTE, J. & WORK, T. S. Proteins coded by mitochondrial DNA in mammalian cells. EuropeanJournal of Biochemistry 23,564-574 (1971). CORTI, A., CASTI, A., RJX.ALI, N. & CALDARERA, C. M. Effect of norepinephrine on soluble Mn++-stimulated poly (A) synthetase activity and ribonucleic acid of perfused rabbit heart. Biochemical [email protected] Research Communications 71, 1125-l 130 (1976). DEUTICKE, B., GERLACH, E. & DIERKESMANN, R. Abbau freier Nukleotide in Hem, Skeletmuskel, Gehirn und Leber der Ratte bei Sauerstoffmangel. Pjiigers Archiu ftir diegesamte Physiologie 292,239-254 (1966). 7. DOBSON, J. G. & MAYER, S. E. Mechanism of activation of cardiac glycogen phosphorylase in ischemia and anoxia. Circulation Research 33,412-420 (1973). L. J. Increased RNA synthesis in nuclei 8. DOICAS, M., BOTNEY, D. & KLEINSMITH, isolated from rat liver slices incubated with cyclic adenosine-3’,5’-monophosphate or glucagon. Archives of Biochemistry and Bio&hysics 159, 7 12-72 1 ( 1973). 9. GAMBLE, J. G. & MCCLIJER, R. H. In vitro studies with rifampicine on the stability of heart mitochondrial RNA. 3ournal of Molecular Biology 53,557-560 ( 1970). 10. GILMAN, A. G. A protein binding assay for adenosine-3’,5’-cyclic monophosphate. Proceedings of the National Acaa!emyof Sciences67,305-3 12 ( 1970). 11. GREENCARD, P. & Kuo, J. F. On the mechanism of action of cyclic AMP. In Role of

ACCELERATED RNA AND PROTEIN SYNTHESIS

12. 13. 14. 15.

16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27.

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Cyclic AMP in Cell Function, pp. 287-306. P. Greengard and E. Costa, Eds. Advances in Biochemical Psychoopharmacology,Vol. 2. New York : Raven Press ( 1970). UL, T. C. & COOKING, E. C. High-efficiency liquid scintillation counting of l*Clabelled material in aqueous solution and determination of specific activity of labelled protein. BiochemicalJournal 96,626-633 (1965). HE-, M., MEINERTZ, T., SCHMELZLE, B. & SCHOLZ, H. Effects of theophylline on force of contraction and cyclic AMP in isolated guinea pig auricles. JvaunynSchmiedebergs Archivfir Pharmakologie 293 Suppl. R, 24 (1976). HURLBERT, R. B. Preparation of nucleoside diphosphates and triphosphates. Methoa!s in Enzymology, Vol. II, pp. 785-805. S. P. Colowick and N. 0. Kaiilan, Eds. New York : Academic Press Inc. ( 1957). JOST, J. P. & SMIB, M. K. Role of cyclic adenosine-3’,5’-monophosphate in the induction of hepatic enzymes. II Effect of Ns, Os-dibutyryl cyclic adenosine-3’, 5’-monophosphate on the kinetics of ribonucleic acid synthesis in purified rat liver nuclei.3ournal of BioLogical Chemistry 2441623-1629 (1972). KALLFELT, B. J., HJALMARSON A. C. & ISAKSSON,0. G. In vitro effects of catecholamines on protein synthesis in perfused rat heart. 3ourndof Molecular and Cellular Cardiology 8, 787-802 (1976). KALLFELT, B.J., WALDENSTR~M, P. & HJALMARSON, A. C. Effects of adrenaline in vivo on protein synthesis and sensitivity to ischemia of the perfused rat heart. 3ound of Molecukr and Cellular Cardiology 9,383-398 (1977). KLEITKE, B., HINTERBERGER, U., ONNEN, K., R.umzsm,G. & WOLLENEZRGER, A. Der EinfluP von totaler Ischlmie auf die Ribonukleinsgureund Eiweiasynthese im Herzmuskel der Ratte, untersucht an Schnitten und zellfreien Systemen. A&z biologica et medica germanica 30,33-55 (1973). KLEITKE, B., Snow, H. & WOLLENBERGER, A. Evidence for cyclic AMP-dependent protein kinase activity in isolated guinea pig and rat liver mitochondria. Acta biologica et medica germanica 35, Kg-K1 7 ( 1976). KROON, A. M. Protein synthesis in mitochondria. III. On the effects of inhibitors on the incorporation of amino acids into protein by intact mitochondria and digitonin fractions. Biochimica et biobhysica acta 108,275-284 (1965). K~~NTZEL, H. & SC-R, K. P. Mitochondrial RNA polymerase from Neurospora crassa. Nature, New Biology, London231,265-269 (1971). LAMB, A. J., CLARK-WALKER, G. D. & LINNANE, A. W. The biogenesis ofmitochondria. 4. The differentiation of mitochondrial and cytoplasmic protein synthesizing systems in vitro by antibiotics. Biochimica et biojhysica acta 161,415-427 (1968). LOWRY, 0. H., ROSEBROUGH, N.J., FARR, A.L. & RANDALL, R.J. Protein measurement with the Folin phenol reagent.Journal of Biological Chemistry 193,265-275 (1951). &LOV, S. Effect of sympathomimetic amines and monoamino-oxidase inhibitors on protein synthesis in rat heart. Biochemical Pharmacology25, 1645-1651 (1976). w, R. J. & NOVELLI, G. D. Measurement of incorporation of radioactive amino acids into protein by a filter-paper disc method. Archives of Biochemistry and Biophysics 94,48-53 (1961). h/z40, C. C. & GUIDOTTI, A. Simultaneous isolation of adenosine-3’,5’-cyclic monophosphate (CAMP) and guanosine-3’,5’-cyclic monophosphate (cGMP) in small tissue samples. Analytical Biochemistry 59,63-68 (1974). h&yER, R. R. & Snmso~, M. V. DNA biosynthesis in mitochondria. Differential inhibition of mitochondrial and nuclear DNA polymerases by the mutagenic dyes ethidium bromide and acriflavin. Biochemical and Biophysical Research Communications 34, 238-244 (1969).

a44 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

44.

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WOLLENBEROER

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ACCELERATED

RNA

AND

Note Added

PROTEIN

SYNTIiESIS

845

in Proof

As this paper goes to press, we learn of the work of L. V. Puchkova, “Detection of [sH] cyclic AMP in the transcription complex of rat liver mitochondria”, Molecular and Cellular Biochmistry 14, 115-l 19 (1977), which indicates that cyclic AMP binds to the transcription complex of rat liver mitochondria and may regulate RNA synthesis in these organelles.

Accelerated RNA and protein synthesis in mitochondria isolated from unperfused myocardium. Possible involvement of cyclic AMP.

Journal of Molecular andCellular Accelerated Isolated Cardiology (1978) 10,827-845 RNA and Protein Synthesis in Mitochondria From Unperfused My...
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