Role of synthesis of nucleic acids and protein in adaptation to the external environment.

PHY610LOGICAL

Vol.

$5, No. Printed

REVIEW6

1, January in U.S.A.

1075

Role of Synthesis of Nucleic Acids and Protein in Adaptation to the External Environment F. Z. MEERSON Laboratory

oJ Experimental USSR

I. II. III.

IV.

VI. VII. VIII.

I.

Institute

of Medical

of Normal

and Pathological

Sciences, A4oscow

...................................................... Introduction. Role of Activation of Synthesis of Nucleic Acids chondria Formation in Adaptation to Long-Term

Physiology,

USSR 79

and

Proteins and of MitoWork Loads. .........

Role

of Synthesis of Nucleic Acids and Proteins and of Rlitochondria tion in Adaptation to Altitude Hypoxia. ............................. A. Heart .......................................................... B. Brain. ..........................................................

Forma-

Role

Forma-

tion

V.

Cardiology,

Academy

of Synthesis in Adaptation

of Nucleic Acids and Proteins and to Cold. ........................................ ..................................... Nervous System.

A. Sympathetic B. Thyroid. ....................................................... Hypothesis of Common Link in Mechanisms Loads, Adaptation Structural Conclusion

of hlitochondria

81 91 93 98 102 103 104

of Adaptation .................................

Altitude Hypoxia, and Cold. and Prophylaxis. ......................................... Price of Adaptation ........................................ .........................................................

to Intensive

Work 107 114 115 116

INTRODUCTION

Life of an organism becomes possible by virtue of the broad spectrum of evolutionally determined adaptative reactions arising in response to the action of factors of the external cnvironmcnt. Despite their diversity these reactions quite obviously fall in to two in terconnectcd classes, namely, short-term adaptive reactions (“on the spot”) and gradually forming long-term adaptation reactions. The short-terrn adaptive reactions are those for which the adult organism always has ready-made mcchanisrns and include the jerking back of a limb or in response flight of an animal in response to pain, an increase in heat production to cold, an increase in heat emission in rcsponsc to heat, an incrcasc in pulmonary ventilation and cardiac output in response to an oxygen deficiency, etc. These reactions, as a rule, are realized immediately after a stimulus begins to act, but of themselves can ensure perfect adaptation only for a short period. Long-term adaptation embraces the reactions for which the organism does not have ready-made mechanisms, but has only genetically determined prerequisites that ensure gradual formation of the components of such mechanisms under repeated or suficiently long action of factors of the external environment. Such adaptation ensures performance by the organism of formerly unattainable intensive physical work, the organism’s resistance to considerable altitude hypoxia, which was formerly incompatible with life, resistance to cold and heat, and ad79

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justment to changed food. Also included in these reactions are the qualitatively more complex adaptation that manifests itself in formation of conditioned reflexes and a system of reactions of growing complexity in the making of the human personality. Hypertrophy of the skeletal muscles developing in response to work loads is the simplest example of structural changes in a physiological system that play the principal role in achieving a given adaptive reaction. This hypertrophy is combined with an increase in the number of mitochondria and, correspondingly, in the capacity for aerobic resynthesis of ATP (43). This structural change is apparently an essential component of the system of adaptive changes ensuring the best adjustment of the organism to the future “encounters” with greater work loads. Another example of a long-term adaptive reaction is observed in the formation of skills required for coordination of movements and maintenance of the equilibrium of the body. Thus a successful development of the skill of tightrope walking in animals is accompanied by an increase in the function and then the rnass and power of Deiters’ nuclei (73), and this structural change plays an important role in the future encounters of the organism with situations requiring maintenance of body balance under complex conditions. Quite analogously the formation of certain structural changes in the cells of lymph nodes underlies such long-term adaptive reactions as immunogenesis (8) and modification of cells of digestive glands to ensure a change in the spectrum of digestive enzymes being synthesized. The latter change may play a role in the adjustment of the organism to changed food (212). Structural changes underlying the adaptation to long-term factors of the environment usually form simultaneously in several organs that compose a single functional system. For example, the kidneys respond to a deficiency of sodium chloride in the food with an increase in their juxtaglomerular apparatus and renin production. Acting on the alpha globulin of the blood plasma, renin stimulates the formation of an increased amount of angiotensin. Angiotensin, in its turn, causes hyperfunction of the cells of the glomerular zone of the adrenals (2 10). These cells hypertrophy and secrete into the blood increased amounts of aldosterone, which stimulates sodium reabsorption in the distal tubule. Thus the development of a structural change in the adrenals increases the capacity of the renin-aldosterone mechanism, the reabsorption of sodium, and the ability of the organism to deal with future encounters with a deficiency of salt (166). For the more complex forms of adaptation connected with the activity of the brain and behavior of the organism, the idea of a decisive role of the transition from functional to structural changes has been traditional. This transition is conceived to be the cornerstone of the formation of temporary connections (7, 28, 62, 72, 75, 165). Most investigators agree that formation of a temporary connection is achieved through sufficiently intensive excitation of certain neurons involved in the conditioned and unconditioned stimuli. Such a physiological increase in activity in some manner or other always leads to structural changes that in the end ensure


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formation or consolidation of synaptic connections between the excited nerve centers. This structural change that remains after cessation of the excitation ensures the maintenance of the temporary connection that is essentially what is now called a “memory trace” or “memory engram.” It is precisely this kind of multineuronal, structurally ensured memory engram or temporary connection that underlies more complex phenomenon such as the conditioned reflex that constitutes an important mechanism for the organism’s active adaptation to its environment. In all the above examples the structural changes underlying adaptation develop as a result of more or less protracted physiologic activity. Such stable changes, which are not injuries, but a result of physiological activity, may be designated as changes due to use; the shortest of modern definitions of memory is precisely such a change. This definition may be given in a longer form as follows: individual memory of living systems is a change resulting from physiologic activity caused by environmental factors; such changes stably persist after cessation of the activity itself and to a great extent determine the future reactions of the organism to the external environment. These considerations led us to the conclusion that there is a permanent connection between the phenomena of memory and adaptation and that memory in the form of retention of a stable structural change is an indispensable element of the basic long-term adaptive reactions of the organism (118). All the structures of an organism form as a result of the synthesis of nucleic acids and proteins. It is apparently for this reason that activation of the synthesis of nucleic acids and proteins must be regarded a priori as a decisive factor. In this review the role of the synthesis of nucleic acids and proteins in the mechanism of adaptation to intensive work loads, hypoxia, and cold is examined. Attention is dcvotcd mainly to two questions. First, what mechanism causes the activation of the synthesis of nucleic acids and proteins; second, what structural changes occur as a result of activation of the synthesis of these macromolecules.

II.

ROLE

OF

MITOCHONDRIA

ACTIVATION

OF

FORMATION

SYNTHESIS IN

OF

ADAPTATION

NUCLEIC

ACIDS TO

AND

LONG-TERM

PROTEINS WORK

AND LOADS

The intensive activity of physiological systems of a healthy organism-for example, of the cerebral nerve centers or the motor, respiratory, and circulatory apparatus --usually arises in response to signals addressed to definite receptors of the external environment. This activity is not indefinitely protracted and intensive but is limited. In the studies of factors that limit the intensity of physiological functions, which arc still far from having been identified, it was shown that when the work loads on the physiological systems of an unadapted organism are sharply increased the aerobic resynthesis of ATP in the mitochondria may lag behind ATP utilization. As a result the concentration of phosphocreatine and ATP decreases and that of the products, ADP and inorganic phosphate, increases. The (ADP P i)/ATP ratio, designated as the phosphorylation potential, also increases (88). As is well known, the increase in the phosphorylation potential activates the l


osici;~tiv~~ rq-nthc’sis of‘ ATE’ in the rnitochondria. 1Cjirrl,,ltan~~~~lsl?., glymlysis is xtka td and lacta tc ac~cumulatcs due to the ~v~ll-k~ww~~ rrwchanisrn of isostui( mci &st(~ric rc%AbtioIl (1 70). ‘1-11~~ciwr~as~~ in ATP mnccntration, incrcxasv in the phosphoryla tion INtul t i al, and tlw x-t ka t ion of glycolysis during in tensiw ac>tki ty of physiological wstm1s in an intact organism have been dcrnonstrated by nurncrous studies. Thus ILrlw*~ and Saltin (83) cmploycd the rncthod of biopsy and showed that in tlw OqinniIq of‘ physical work in the human cpadriccps fctrnoris the concentration of XI-I’ cI~‘(‘I‘~as(‘s 115( ; and l~hosphocrmtinc 70 “;, glycolysis is actkatcd, and the (,(_,Ilc.t.I1tratioIl of lactate in the ~nuscles and the blood plasma increasw An analuqorls cw~qhx of changcx has bwn discovered in heart rnusclc during corrqxnsatory 11\.I”~~‘1’rll~tiori carwd tx aortic: stenosis (35, 36, 1 12) or by tcrnporary ligation of‘ tk mrta (128) and prikr to that by intensive stimulation of mm-e cm&s (2 14) ii nd wnpat Iwtic g-anglion (68). III 211 swh caws tlic incrcasc in the pliosl~liorylation potential is, on tlic 01x hi1nc1, ;1 rmniksta t ion of inwfkknt capacity for aerobic synthmis of ATP and, on th other, a signal that urgcmtly activates this Ixoccrss that is indispcnsa.bk to Ixm~iciin~ 0’ mcrgy , for the incrcascd function. In our t lic~ory, it is csscntial that in protracted hyperfunction the incrcasv in t 1~ I)lI(~‘il~l’or)-lation potential is always follouwl by activation of the synthesis of 111i&k acids and proteins. 1 loreo\w-, the first to lx activated is the synthesis 01 I1lit(,(~11oI1(1~ial lxotc‘ins leading to biogcncsis of rnitochondria. Such changes haw tx~m ol)suwd wlm~ the contractile function of sklctal muscl~~s increases during training (2 I I ), lcadin g to formation of new rnitochondrial structures (98, 99, 160). 1ntwsiw stimulation of ncrw ccntctrs is accornpankd by, accclcratcd syntlwsis of‘ I< SA and putcin and incrc~ascd activity of Initochondrial mzy~nw (55, 77 ), (13, 51, 73). An in2s u-(91 215 2111 incrcasc in tlr size and wciqht < of the nwrom UKN in the f’urwt ion of‘ mdwrinc glands, such as t hc thyroid and the adrenals, ih ;ho ~lc\c~ollll)~~.r~ic~ti by activation of the‘ synthesis of nuckic acids and protcim iI> t lair cTlls (29, 30, 34, I 79). 1 I orcwvr, Fiala and Glinsman (34) 1lai.c sl~ow~ 1hat format ion 01‘ nlitochondria is an initial csc‘nt in thv adrmal ~11s. Such stud& work loads on organs 21-r (pi tc IHII~~~TOIIS and indicatc~ that in casts of incrcascd ;mci wwmis thv incrcasC in the‘ I~liosI~horylation potmtial is followed by actkation 01’ t lw s\mthc5is of rmclck acids and proteins with an initial incrcasc in thv t)ioqcwc% 01‘ mi tochonctria. ,‘\u im t ion of t lw t)iosvn is apparcn tl\. an indislxnsaI t lwsis of ~llacrornolccul~s MC link of’ adaptation kcausc the inhibitors of tlw wntksis of nk-kic acids and I protch hinckr the dwckpnmt of adaptation to work loads (I ,N), elaboration of ccmdi tioncd rc+kx~s ( 139), and the other adaptkc reactions of the organism (148’). ( >ri t lr dwr hand, factors f‘awring actkalion of tlw syntlivsis of nucleic acids a combination of erotic arid, folk acid, and \itaInin ;\I)( 1 prcwins, f‘or- narq~lc, f‘c,qvI* an acwlvratcd clc~~&q~rncnt of thcsc adaptkc reactions (132, 150). h2, ‘1‘1~ process of’ compensatory hylxrfunction and hylxrtrophy of vitally iInport;ln t o~*gans is ;a good mod4 for s tudyin g the control of Iiurlcic acid and protviI1 .c;vIit lit+. SW+ nwdcls inrludc the conqxmsatory hyp~rfunction of the heart


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in stable aortic stenosis (48, 113, 119) and of the single kidney (105, 142, 15 1) and lung (180, 181) after removal of one of the pair of organs. All these cases are accompanied by adaptation of the organ to an abrupt and irreversible increase in the work load. Immediately after such an increase in the work load, the amount of function per unit of the organ’s mass (intensity of functioning of structures) sharply increases. This is followed by an increase in the phosphorylation potential and by activation of the synthesis of DNA, R,njA, and protein. In the course of 6-l 2 days, these changes increase the mass of the organ by 50-100 % As a result of distribution of the function over the increased mass of the organ, the intensit Y of returns to normal. At the same time the functioning of the structures ‘gradually synthesis of nucleic acids and proteins decreases, the organ ceases to grow, and its hypertrophy ends. On the basis of these data, an idea has formed that the intensity of functioning of the structures in some manner regulates the actiGty of the apparatus for synthesis of macromolecules (116-l 18). Activation of the synthesis arising under the influence of an increase in the “intensity of functioning of structures” (IFS) in hyperfunction of the heart, kidncys, and other organs can be completely prevented by administration of actinomycin D (134, 151), which inhibits the transcription of RnTA on the chromosomal genes of the DNA. This was interpreted as an indication that the activating influence of IFS through the particular links of intracellular regulation is addressed to the genetic apparatus of the cell and that the mechanism of formation of structural changes in adaptation to long-term work loads may be understood on the basis of studying the interrelations between the physiologic function and genetic apparatus of the cell (114). It is important to identify the connections between these events. The direct connection apparently involves activation of transcription of genes located in the chromosomes of the cell nucleus and the small mitochondrial genomes and results in formation of RNA and subsequently of proteins. A feedback relationship exists between the intensity of functioning and the activity of the genetic apparatus. This mechanism works so that the intensity of functioning of the structures is simultaneously the determinant of the activity of the genetic apparatus and at the same time a physiologic constant maintained at an invariable level by timely changes in the activity of this apparatus, i.e., hypertrophy and atrophy of cells (116, 120). Subsequently this idea was substantiated in the greatest detail by studies of’ compensatory hypcrfunction and hypertrophy of the heart caused by aortic stenosis. It was found that the RNA-polymerasc activity in the nuclei of the myocardium increased immediately after an increase in the contractile function of the heart (159). In the next 12-48 h, with a total absence of DNA replication in the nuclei of myocardial cells (115, 125), activation of transcription develops in these cells so that the amount of messenger, transfer, and ribosomal RKA increases. The concentration of RNA in the myocardium and the number of ribosomes per unit of its mass increase considerably (156, 2 18). The extent of activation of transcription was estimated on the basis of the obvious proposition that the rate of R,KA synthesis equals the sum of the rate of increase in RNA content and the rate of RNA degradation.


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DAYS FIG.

I. Dynamics

cardiac hyperfunction time from beginning

of RNA concentration and content in myocardium (CCH). Vertical axis indicates percent of control; of hyperfunction.

FIG. 2. Dynamics of RNA degradation in myocardium during CCH. Continuous line indicates total RNA radioactivity in control; dotted line denotes total RNA radioactivity during CCH. Radioactivity is plotted vertitally and time from beginning of hyperfunction horizontally.

during horizontal

compensatory

axis

denotes

5 k 0”

5

2

The curves in Figure 1 reflect the dynamics of RNA concentration and content in the myocardium observed in our experiments in the process of compensatory cardiac hyperfunction (CCH) caused by coarctation of the aorta in rats (113, 158). They show that within 24 h the RNA concentration increased 11 %, in 2-3 days by 25-27 %, but in the course of the successive week changed little. After 11-12 days, RSA concentration tended to decrease. The total RNA content in the heart increased much faster because of the hypertrophy of the organ. It increased 50 % by the 3rd day, and then reached a plateau amounting to 75 %. During the period


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I3 15 I9

DAYS FIG.

curve) verticallv.

3. Dynamics du ring CCH. Time from

of

RNA

Relative beginning

synthesis

(upper

curve)

rate of RNA synthesis and of hyperfunction is plotted

and

phosphorylation

phosphorylation horizontally.

potential pote :ntial are

(lower plot ted

of decrease in R,li’A concentration, content remained elevated to the same extent as the total mass of the heart. We estimated the rate of RNA degradation in the heart by following the decrease in total radioactivity after labeling with [14C]orotic acid. Figure 2 enables us to compare the rate of decrease in total radioactivity of RNA in control (u/$w line) and in compensatory hyperfunction of the heart (lower line). The radioactivity values are given on a logarithmic scale. One can see that in the hearts of control animals the total RKA radioactivity decreased 60 % in 11 days, while in cases of hyperfunction it dccrcascd 74 %. During; the same period, the RNA content of the hypertrophying heart increased 65%. cIt must be pointed out, however, that the estimates of RNA degradation indicated by thcsc lines arc too low due to reincorporation of the radioactive degradation products back into RNA. The data do suggest that during the short period after imposition of hypcrfunction nearly 50 %, more RNA was synthesized in the genetic apparatus of the myocardial cells than was initially present. Thus the rate of RNA synthesis in compensatory cardiac hyperfunction is very high. The dynamics of this process were calculated on the basis that the rate of RNA synthesis at each moment of time equals the sum of the rate of increase in RX4 content and the rate of RNA degradation. The rate of RNA synthesis was compared with the phosphorylation potential (Fig. 3). The comparison of these parameters shows that the rise of phosphorylation potential, the hypothetical signal, corresponds to t‘he increase in RI?;A synthesis. Xot only does the RNA concentration increase in hypcrtrophying hearts as a result of accelerated t,ranscription, but the number of polysomes is raised. To obtain ribosomes from heart muscle, we employed the method of Heywood et al. (65), which makes it possible to isolate more than 50 % of all the cell RXA, i.e., 3-4 times as many ribosomes as were isolated from the myocardium by other


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FIG. 4. ScdiIncntation profile Inyocardial ribosomes of intact rats. control; 2, treated with RNAase;

of I, 3,

treated plotted

is

horizon

with EDTA. vertically and tally.

Radioactivity fraction number

2.

MEERSON

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methods (156, 2 19). Tl re ribosomcs obtained in our experiments were contaminated with protein, apparently myosin, and the presence of protein hindered the analysis of RNA by ultraviolet absorption. To study the relationship between the number of polysomcs, monomers, and ribosomal subunits, the animals were given the labeled RNA precursor [14C]orotic acid, which after 48 h of exposure was concentrated in ribosomal and transfer RKA (Fig. 4). The profile of radioactivity has a characteristic appearance with a sharp peak in the region of the 80s monomer and a less pronounced peak in the region of dimcrs. Treatment with ribonuclease leads to disappearance of radioactivity in the heavy zone of the gradient and a shift to the region of monomers. Treatment with cthylenediaminetetraacetic acid (EDTA), which causes dissociation of ribosomes into subunits, leads to a shift of the radioactivity peak to this region. These characteristic effects of RrC’Ase and EDTA on the distribution of radioactivity in the sucrose-density gradients indicate that the sedimentation profile reflects the relative proportions of the various ribosomal structures. In Figure 5 the ribosomal profile of normal heart is compared with the heart 48 h after the beginning of hypcrfunction. It can be seen that 48 h after the beginning c of hypcrfunction the radioactivity of RNA in the fractions of the gradient increases and the rclativc content of polyribosomes in the total ribosome population is somewhat higher in the hypertrophying heart. In the normal heart about 50% of the radioactivity is in polyribosomes; in cardiac hyperfunction, the polyribosomes constitute about 60% of the total. Subsequent experiments showed that the relative increase in polyribosomes observed in the initial stage of CCH no longer existed after 8 days, although the synthesis of ribosomal RNA was still faster and the content of ribosomes was increased. As a result of the increase in the number of ribosomes and the proportion of polysomes, the capacity of the myocardial cells for protein synthesis increased. The incorporation of labeled amino acids into total protein of the myocardium increased 60-100 % (48, 112, 170), and the average increase in the quantity of proteins synthetized per gram of myocardium reached 1 mg/h (171). As a result the mass of the heart rapidly grows; for example, in rabbits the weight


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right,

profile is plotted

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1200

FRACTION 5. Sedimentation CCH. Radioactivity

FIG.

AND

of myocardial vertically

NO.

ribosomes and number

of rats during CCH. of fractions horizontally.

I,fft,

control;

of the heart may increase 80 % during the first 5 days after constricting the aorta by 75 ‘.G (112). The synthesis of mitochondrial protein is the first to be activated, and for this reason the area occupied by mitochondria on electron-microscopic sections is increased 30 ‘7/o in the early stage of hypertrophy (152). The activity of the main mitochondrial enzymes per unit of myocardial mass is increased 4060’,;; (171, 173). In order to make a special study of the dyanmics of the synthesis of nucleic acids and proteins of the mitochondria we employed methods that provided partially purified samples of DNA, RNA, and protein from mitochondria and determined the rate of their biosynthesis by addition of corresponding precursors (123, 149). The curves in Figure 6 characterize the changes in mitochondrial DNA synthesis and the concentration of DKA per milligram of mitochondrial protein after constriction of the abdominal aorta. It can be seen that 24 h after the beginning of hyperfunction the incorporation of [3H] thymidine into mitochondrial DNA increases fivefold; in 2 days it increased 8.5-fold, in 4 days 12-fold, and in 7 days 17-fold. After completion of this emergency stage of the hypertrophy (21 days), the rate of thymidine incorporation into mitochondrial DrCTA is increased only twofold and 2 mo later does not differ from that of the control. The concentration of DKA increases somewhat slower and to a lcsscr extent than IX\A synthesis. In earlier studies it was demonstrated by autoradiography that DNA is not synthesized in the nuclei of myocardial cells of either normal or hypertrophying hearts (125, 180). At the same time the bulk of mitochondria were from muscle cells. It follows that adaptive hyperfunction of the heart causes a selective activation of the replication of mitochondrial genomes in muscle cells, while there is no biosynthesis of DNA in the nuclei of these cells. The lower curve in Figure 6 shows that the accelerated biosynthesis of mitochondrial DKA at the height of the process


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leads to the accumulation of 6 times as much DNA per unit of mitochondrial protein as normally. Regardless of how the increased amount of DNA is distributed in the mitochondria, this increase in DNA synthesis correlates with accelerated formation of mitochondrial RnTA and protein and the increase in mitochondrial mass. A comparison of changes in RNA synthesis with DXA concentration in mitochondria is shown in Figure 7 and reveals coincident changes in these pa-

-e-

DNA -concentration DNA -synthesis

-

FIG.

centration increase

6. Dynamics of mitochondrial in rate is plotted

of

incorporation DNA4 vertically

of in

myocardium and tilne

-a-

chondrial

I;IG.

7. Changes RNA of left

and

horizontally.

time

of DNA ventricle

concentration of rats during

[3H]thymidine of left horizontally.

into ventricle

mitochondrial of rats

DNA during

CCH.

and

con-

Relative

RNA - synhsis

and CCH.

incorporation Percent

of of control

[14C]orotic values

acid is plotted

into mitovertically


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FIG. 8. Dynamics of incorporation of [‘“C]orotic acid into nuclear and mitochondrial RNA of left ventricle of rats during CCH. Incorporation (counts/rnin per 100 pg RNA) of [14C]orotic acid is plotted vertically and time after beginning of CCH horizontally.

rameters. This coincidence could indicate that the rate of mitochondrial RNA synthesis is controlled by the availability of mitochondrial DNA, a situation that does not appear to be true in lower organisms such as yeast. Alternatively, the mitochondrial RNA may be contaminated with nuclear and cytoplasmic RNA and as a result the coincidence may be an artifact. The curves shown in Figure 8 make it possible to compare the rate of RNA synthesis in the nuclei and mitochondria of the heart muscle by measuring the incorporation of labeled erotic acid. Changes in the rate of RNA synthesis in the nuclei are very similar to those in the mitochondria. Moreover, the rate of nuclear RNA synthesis in the hypertrophying hearts increases sooner and to a much greater extent than that of the mitochondrial RNA synthesis. This phenomenon may represent synthesis of ribonuclear protein particles, ribosomes, hctcrogencous high-molecular-weight RNA, and messenger RNA. Formation of functionally competent mitochondria depends on synthesis of enzymes of the respiratory chain in the cytoplasm on messenger RNA of nuclear origin (172, 185). The curves in Figure 9 present changes in the rate of synthesis of mitochondrial and nuclear proteins in the process of CCH. It can be seen that the rate of protein synthesis as estimated by incorporation of a mixture of labeled amino acids increases to 260-300 % of the control in the nuclei and to 340-370 ‘% in the mitochondria. These findings suggest that the extent of activation of mitochondrial protein synthesis is 2.3 times greater than for incorporation into total myocardial protein. It is well known that later stages of compensatory cardiac hyperfunction are associated with marked activation of the biosynthesis of myofibrillar and other proteins of the muscle cells. As a result the initial activation of mitochondrial biosynthesis leveling off the content of mitochondrial proteins in the hypertrophied heart does not differ from normal (113), whereas in a late stage of myocardial


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200 100

FIG.

methionine) of control

9. Dynamics of incorporation of a mixture into mitochondrial and nuclear protein values is plotted vertically and time from

of 34C-labeled amino acids (lysine, of left ventricle of rats during CCH. beginning of CCH horizontally.

alanine, Percent

wear it may turn out to be decreased (113, 153). In connection with this it should be remembered that continuous compensatory hyperfunction of the heart caused by aortic stenosis in animals, as well as hypertension or heart disease in man, is not an optimal variant of the organism’s adaptation to a long-term work load. In cases of a less substantial and especially an intermittent work load, as in training for athletics, one can conceive of a situation where initial activation of mitochondrial biogenesis is not followed by a general activation of the synthesis of all proteins. In such a case the increase in the capacity of the mitochondrial system per unit of the muscle mass must be maintained throughout the period of adaptation with a lower degree of hypertrophy of the muscle as a whole. Indeed, Holloszy (67) f ound in animals trained by intensive running for long periods of time that activity of the mitochondrial enzymes, protein content in the mitochondria, and consumption of oxygen by the mitochondria per unit of skeletal muscle mass increased about 100 % In agreement with this, Gollnick and King (43) sThowed, by electron microscopy, that the number of mitochondria per sarcomcre in the muscle fibers of these animals was increased to about the same extent. Finally, Arcos et al. (1) reported that in the hearts of animals trained by swimming the concentration of protein in the mitochondria increased more than 50 %. These experimental data support the proposition that training increases the so-called aerobic capacity for work (31). Since mitochondria are the main structures where oxygen is utilized, this observation indicates an increased capacity of the mitochondrial system in trained organisms. It must be noted, however, that skeletal muscle uses anaerobic metabolism to a greater extent than heart, which is almost exclusively aerobic. In heart there may be no significant change on exercise. In evaluating these data, it should be remembered that an increase in the phosphorylation potential and mobilization of cglycolysis arise when considerable work loads are imposed on the physiologic systems of an unadapted organism. This is often accompanied by fatigue whose extent correlates with lactate accumulation in the bload and tissues (109). Subsequently, as adaptation occurs,


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the deficit of energy-rich phosphates, the phosphorylation potential, and lactate accumulation diminish under the same work loads and the resistance to fatigue increases correspondingly. These changes indicate that the genetic apparatus of the cells becomes activated primarily to synthesize mitochondria along with general growth of the cell. This sequence of events suggests that the enhanced function activates the genetic apparatus either primarily by increasing the phosphorylation potential or secondarily by changes caused by this initial signal. If this assumption is close to being correct, an increase in the phosphorylation potential produced without any increase in function-for example, by oxygen insufliciency or cold- must also cause an activation of the genetic apparatus with a primary increase in the biogenesis of mitochondria. From this point of view it is desirable to examine the data on the role played by the synthesis of nucleic acids and proteins in adaptation to altitude hypoxia and cold.

III.

ROLE

OF

FORMATION

SYNTHESIS IS

OF

KUCLEIC

ADAPTATIO,W

ACIDS TO

ALTITUDE

AND

PROTEINS

AND

OF

MITOCHONDRIA

HYPOXIA

Hvpoxia is not a stimulus of any external sense organ, but invades the internal environment of the organism and causes hypoxemia. Hypoxemia acts as a stimulator of the chemoceptors of the aortic-carotid zone, of the centers that regulate respiration and blood circulation, and of other organs and causes an adaptive hyperfunction of the systems responsible for the transport of oxygen and its distribution. Eaten with the mobilization of these transport systems, hypoxemia is not eliminated even in altitude hypoxia that is compatible with a long life and leads to a decrease in oxygen tension in the tissues. Thus it was shown by the polarographic method that in heart muscle oxygen tension at an altitude of 4 km dccreases by 2 1 % and at an altitude of 6 km by 41 % (91). In other work it was established by the same method that oxygen tension in the brain decreases by 13-15 5 at an altitude of 2 km and by 21 o/c,at an altitude of 4 km (204). When a gas mixture containing 10 ‘5, oxygen was inhaled, hypoxemia was manifested by a 30 % decrease in oxygen tension in the arterial blood and a 3 1 % decrease in the cerebral cortex (92) By virtue of the high affinity of cytochrome oxidase for oxygen, it is thought that these decreases in oxygen tension cannot limit the transport of electrons in the respiratory chain. However, actual data show that, in an intact organism, tissue hypoxia resulting from hypoxemia restricts rcspiration. This is supported by at least three groups of facts. First, in hypoxia of the brain, heart muscle, and other organs, marked activation of glycolysis is observed; this activation is manifested by an increase in the concentration of lactate, increase in the lactate-pyruvate ratio, and a decrease in the concentration of glycogen (38, 40, 190). The most probable mechanism of this activation involves a decrease in the concentration of ATP and an increase in the concentration of the products of its hydrolysis, ADP and Pi, arising from a restriction of respiration. These changes lead to activation of the key enzymes of glycolysis.


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Volume 55

Second, in intensive hypoxia a substantial decrease in phosphocreatine concentration and a slight diminution in ATP in the brain (26) and skeletal muscles (38) were observed. Third, it was demonstrated that in cases of hypoxia compatible with life in even so “privileged” an organ, in the sense of blood supply, as the brain, the decrease in concentration of energy-rich phosphorus compounds is accompanied by a considerable increase in the relation between the reduced and oxidized components of the respiratory chain (46). In general, the situation developing in the cells of an unadapted organism in acute hypoxia may be pictured as follows. The utilization of ATP in the functioning organelles is unchanged or decreased, the concentration of phosphocreatine and ATP is diminished, the concentration of the substrates of oxidative phosphorylation is increased, the intensity of glycolysis and the concentration of its products-pyruvate and lactateare increased, the concentration of NADH and succinate is increased, and the rate of respiration is decreased. The factor restricting the rate of respiration and ATP formation and thereby the function of the organs is deficiency of oxygen. The gradually developing perfect adaptation to hypoxia somehow “expands” this restricting link and ensures utilization by the cells of an amount of oxygen sufficient not merely for preservation of function and survival at an altitude, but for vigorous physical work. The studies conducted by Haldane and Priestly (66), Barcroft (6), Krebs (93, 95), Sirotinin (196, 197), Barbashova (3), Hurtado (71), and in recent decades by Reynafarje (176), 0 u and Tenney (164), and Harris et al. (59) support the conelusion that this result is achieved by two principal factors: I) by increasing the capacity and especially the functional effectiveness of the oxygen transport systems, i.e., the systems of external respiration, blood circulation, and the erythrocytes ; 2) by increasing the capacity for the oxygen utilization and ATP synthesis in the cells, i.e., by increasing the number of mitochondria and their total capacity per unit of cell mass. The idea that these coordinated factors determined the development of

adaptatian constituted ar\. impartant

advance in its titx~

Hewer,

it left tk

major yucstion open, namely, how the organism ensures a stable increase in the capacity of oxygen utilization and transport that forms the very basis of adaptation. In the last few years we have provided evidence that activation of the synthesis of nucleic acids and proteins regularly develops during adaptation to hypoxia (120, 146, 148). E x p eriments have shown that such activation always takes place in the oxygen transport systems, i.e., the blood system (10, 157, 177), the heart (144, 149), and the lungs (127), as well as in systems not directly involved in oxygen transport, in particular the brain (137, 138, 145). An essential role for activation of the synthesis of nucleic acids in adaptation to hypoxia is suggested by experiments in which actinomycin, an inhibitor of RNA synthesis, was administered. The drug disturbed the adaptive response, suppressed 0 2 consumption, and proved fatal to most of the animals (148). Administration of this drug over a period of 5 days, between the 20th and 25th day


January

1975

SYNTHESIS

OF

NUCLEIC

ACIDS

AND

93

PROTEIN

TABLE 1. Efect of actinomycin 2703 on oxygen consumption in intact and altitude-hypoxia-adapted animals -~ - -____ - _--_ __----.. Oxygen

Experiment ____-

_~

Control

(20)

Control

+

- - ---~

actinomycin

Adaptation to hypoxia days (12) Adaptation to hypoxia days (12) Adaptation to hypoxia days + actinomycin Values

are means

Consumption,

Sea level

(12)

ml/kg

7000 m

Difference,

2506

=t 101

1688 zt

101

2290

A

1438 zk

130

186

over

a period

of 10

2285

xk 87

1848 zk

180

over

a period

of 25

2275

zt 56

2043

54

over (12)

a period

of 25

1741 z!z 147

zt SE; numbers

in parentheses

indicate

zt

per h y0

-32.6 P < 0.01 -37.2 P < 0.01 -18.9 P < 0.01 -10.2 P < 0.02 -26.6 P < 0.05

1278 zk

161

number

of animals.

of adaptation, in a dose of 10 mg/kg, which does not kill the control animals, resulted in the death of 65 % of the adapted rats. In the surviving rats, oxygen consumption was reduced. As shown in Table 1, the oxygen consumption of unadapted animals on ,heir first exposure to an altitude of 7000 m decreased about 30 %. In the process of adaptation this defect in oxygen consumption gradually decreased and by the 25th day the animals consumed nearly as much oxygen as they did at sea level. This phenomenon, discovered by Krebs et al. (94), is a clear manifestation of the organism’s adaptation to oxygen deficiency. After 5 days of actinomycin administration the defect in oxygen consumption again rose to 26 %; moreover, under the influence of actinomycin the oxygen consumption of the adapted animals decreased at sea level. Thus the defect in oxygen consumption after inhibition of RNA synthesis was observed without an increased load on the respiratory and circulatory systems. In this connection, it should be remembered that suppression of RNA synthesis by actinomycin is more marked where RNA is synthesized most rapidly. Thus actinomycin suppresses biogenesis of mitochondria stimulated by thyroxine (208). On this basis it was assumed that during adaptation to altitude hypoxia the synthesis of nucleic acids and proteins involved primarily mitochondria. This assumption was an attempt to postulate activation of mitochondrial biogenesis as an important factor of adaptation to hypoxia. On the basis of these ideas, a detailed study was made of the rate of synthesis of nucleic acids and proteins in the heart and brain during adaptation to hypoxia. In most studies the rats were adapted to altitude hypoxia by being placed daily in a pressure chamber for 5-6 h with hypoxia corresponding to that at an altitule of 5000-7000 m; adaptation required 40-45 hypoxic exposures. ,-I. Heart During adaptation to altitude hypoxia the heart oxygen-impoverished blood, but is also hyperfunctioning

is not only supplied with to ensure the necessary


94

F. %. MEEKSON

70

Right

heart

ventride

Volume

%I

left

heart

55

venttidb

180 170 160 150

140 130 120 110 100 90 80 a

IO 20

f

,

$0 50

,

80 days

I

_

.

10 20

80 days

40 50

At t et cessaClbn

of FIG. 10. Dynanks adaptation to altitude ginning of adaptation

hg~oxip

of synthesis of protein (I) and weight (II) o f ventricles Percent of control value is plotted vertically hypoxia. horizontally.

of rat heart during and time from be-

increase in minute volume and to overcome the increased resistance in the pulrnonary circulation (103). Since the minute volume is increased equally for both ventricles and the resistance to the ejection of blood is increased only for the right is higher for the right ventricle, it is obvious that the degree of hyperfunction the activation of the synthesis of ventricle than for the left. Correspondingly, nucleic acids and proteins in all the subsequent experiments is higher in the right ventricle than in the left. During adaptation of animals to hypoxia by this method, the synthesis of proteins, as estimated in the right ventricle by incorporation of labeled methionine into protein, was increased 80 % and the weight of the ventricle by 28 % on the 10th day. Subsequently synthesis decreased somewhat, and the weight of the ventricle stabilized (Fig. 10). Cessation of adaptation is followed by a considerable decrease in the rate of the synthesis and an involution of the hypertrophy. The RSA content of the heart increased 90 % in the right ventricle and 60 % in the left. After cessation of the adaptation, RNA content sharply decreased to normal (Fig. 11). Thus a marked activation of the synthesis of nucleic acids and proteins was observed in the hyperfunctioning heart during adaptation to hypoxia (144). In regard to an increase in the capacity of the mitochondrial systems during to assume that the most rapid rate of synadaptation to hypoxia, it was natural thesis would be found in mitochondria. To check up on this possibility, a series of methods were employed to isolate mitochondrial DNA, RNA, and protein and to determine their rate of synthesis by the same method that was used in the studies of compensatory cardiac hyperfunction ( 149). The curves in Figure 12 reflect the dynamics of the synthesis and concentra-


January

SYNTHESIS

1975

70 200 -

OF

NUCLEIC

ACIDS

,4ND

95

PROTEIN

A /Mm

190

l

0’

I

180 170,

l

,i

/

160* 150

I

/

I I I

i

l

I

1 I

FIG. 11. Dynamics of concentration (I) and content (11) of RNA in ventricles of rat heart during adaptation to altitude hypoxia. Data for right ventricle are shown in A and for left ventricle in B. Percent of control value is plotted vertically and time from beginning of adaptation horizontally.

Right

vcn

tricee

Left

vcfff~ide

B

?I /o

-

DNA -concentration

---

DNA-synthesis

500

too

rb

2‘0

jo

+?i days

lb

2b

30

] thymidine into mitochondrial FIG. 1 2. Dynamics of incorporation of centration of mitochondrial DNA4 in myocardium of right and left ventricles adaptation to altitude hypoxia. Percent of control values is plotted vertically ginning of adaptation horizontally.

40 dags DNA and conof rat heart during and time from be-

tion of D&A in the mitochondria of the right and left ventricles of the heart. It may be asserted that as measured by incorporation of [3H]thymidine, the rate of synthesis of mitochondrial DNA increased ninefold for the right ventricle and fourfold for the left by the 10th day. As a result of this accelerated rate, the concentration of DNA in the mitochondria of the right and left ventricles increased 150 % and 90 %, respectively. During the final period of adaptation, the concentration of DNA was maintained at this level.


96

F.

0

/ 0

I

13.

Dynamics

RNA of right values is plotted

.

1

.

30

20

10

FIG.

Volume

MEERSON

55

RNA -synthesis DNA - concentration

B--w

1-

chondrial control

2.

and

incorporation

10

days

of DNA

concentration

ventricle vertically

of rat heart during adaptation to altitude and time from beginning of adaptation

t

of [14C]orotic

acid

hypoxia. horizontally.

into Percent

mitoof

In evaluating these data it should be remembered that in the nuclei of the myocardial cells during adaptation to hypoxia, as also in cardiac hyperfunction, no DNA synthesis takes place (124). At the same time the mitochondria isolated from the myocardium were largely from muscle cells. It follows that in the mitochondria of heart muscle cells during adaptation to altitude hypoxia there is a selective activation of DNA biosynthesis and a considerable increase in the number of particles of mitochondrial DNA. This increase in DNA concentration in the mitochondria may have various structural manifestations. It may occur either by ‘division of mitochondria (49, 106) or by formation of several DNA molecules in one mitochondrion (172). Whatever the distribution of the increased amount of D&VA in the mitochondria, it signifies an increase in the total capacity of the with an increase in mitochondrial mitochondrial genomes and, in association RSA, it may play an important role in activating the synthesis of proteins of the mitochondria and in increasing their oxidative capacity. The curves in Figure 13 show that in the right ventricle there is more than a twofold increase in incorporation of labeled orotate into mitochondrial RNA. The changes in the biosynthesis of mitochondrial RNA coincide with the dynamics of DNA concentration. This coincidence could indicate that the rate of transcription in mitochondria during adaptation to hypoxia is, as in CCH, determined by the number of genetic matrices. During adaptation to hypoxia a more marked activation of RNA synthesis is observed in myocardial nuclei than in mitochondria. The curves in Figure 14 compare the dynamics of activation of RNA synthesis, i.e., transcription in the nuclear and mitochondrial genomes of the myocardial cells or the right ventricle. From this comparison it follows that the rate of RNA renewal in the nuclei of control hearts is at least twice as high as in the mitochondria. Twenty days after the beginning of adaptation the activation of transcription in the nuclei and mitochondria reaches its maximum, increasing 3.2-fold in nuclei and 2.3-fold in mitochondria. At later periods, the activation of RNA synthesis is less pronounced. This more rapid rate of RNA synthesis in nuclei may be due to synthesis of mRNA coding for enzymes of the respiratory chain (185, 195), but these changes in incorporation of erotic acid into nuclear RNA are difficult to interpret since the


January

197.5

SYNTHESIS

OF

NUCLEIC

ACIDS

AND

97

PROTEIN

1500

FIG. 14. Dynamics of incorporation of [14C]orotic acid in nuclear and mitochondrial RNA of myocardium of right ventricle of heart of rats during adaptation to altitude hypoxia. Incorporation of [14C]orotic acid is plotted vertically and time from beginning of adaptation horizontally.

n

u z a

IO00 --/ f’ \ 0 t

2 b a 0

500

r

/

mitochondria

/

? /

/ 1’

I-

200

nucee/

0

/0 15. Dynamics of rate of incorporation of a mixture of 14C-labeled amino acids (lysine, alanine, methionine) into rnitochondrial and nuclear protein of right ventricle of rats during adaptation to altitude hypoxia. Percent of control values is plotted vertically and time after adaptation horizontally. FIG.

. ,9- --

300 /

200

// /

/ rooJ

--+\

mifochondria Y

/v, S. I’. h? itslov. A< oscow : Ak;ld. N211lk SSR 1962, p. 55-76. K. M. ‘I’imc st1ldy of ac11te cold-ind11ced 89. KNIGGE, acceleration of thvroidal 1131 release in the hamster. Proc. J’oc. iixptl. Hi;/. Mrd. 104 : 368-37 1, 1960. IT. A. F1lnktsionalnoyc sostoyaniye en90. KOLI’SNIK, dokrinnykh zhelyoz u zhivotnykh pri sochetanil kholodovogo i t),omnoogo razdrazhitelel. (Functional state of endocrine glands in animals 11nder combined cold and dark stimuli). In : ‘I’eorctzcheJkiye problemy deistviya nizkikh .flctlon

91.

tem/j(Jratur o/ i.ow

na cyqanizm

L. K. Mejstr;lkh. 1969, p. 235. KOROLKOV, V. /jri

gipoksii

(l‘hroretical

Problems

of

071 thr Organism), edited by 1,eningr;ld : Akad. Na1lk SSSR,

Yf’rm/7rraturr.r

(0 xygrn

I\;.

kCislorodnoye

Supply

of

thr

snabzheniye Heart

in

serdtsa

Hypoxia).

dissertatsii (Synopsis of Cand. Avtorefcrat ki1nd. Thesis). MOSCOW : Ministry of l lealth of the IJSSR, 1966. E. -4. Iz7nrneniya naprya,$leniya 92. KOVAI.I

Molecular mechanisms of adaptation emerging from the physics and evolution of nucleic acids and proteins.

Synthesis, characterization, and evaluation of radiometal-containing peptide nucleic acids.

Temperature adaptation in yeasts: the role of fatty acids.

Adaptation of transferrin protein and glycan synthesis.

Nucleic acids. Adding to the genetic alphabet.

The metabolism of nucleic acids in mice.

The binding of an avian myeloblastosis virus basic 12,000 dalton protein to nucleic acids.

Visualization and characterization of tobacco mosaic virus movement protein binding to single-stranded nucleic acids.

In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

Physical studies of the interaction between the Escherichia coli DNA binding protein and nucleic acids.

Nuclear penetration and stimulation of nucleic acids synthesis by poly(A)-poly(U) in mammalian cells.

Iodination of herpesvirus nucleic acids.

Incorporation of 5-substituted uracil derivatives into nucleic acids. Part IV. The synthesis of 5-ethynyluracil.

Nucleic acids--chemistry and applications.

The ecology of adaptation to a new environment.

Trinitrophenylation of nucleic acids and their constituents.

Symmetry in nucleic acid structure and its role in protein-nucleic acid interactions.

Photoinduced processes in nucleic acids.

Vaccines, cells and nucleic acids.

[Vaccines, cells and nucleic acids].

Circuit topology of proteins and nucleic acids.

Synthesis of peptide nucleic acids containing a crosslinking agent and evaluation of their reactivities.

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.

Adaptation to environment--the Pasteur anthrax vaccine in Australia.