the action of steroid at the cellular · dixon, grayand quincey: steroid hormones and suggested...

POSTGRAD. MED. J. (1964), 40, 448

THE ACTION OF STEROID HORMONESAT THE CELLULAR LEVEL

P. F. DIXON, B.A., M.B., B.Chir., A.R.I.C., C. H. GRAY, M.D., D.Sc., F.R.C.P., F.R.I.C.,R. V. QUINCEY, B.A.

Department of Chemical Pathology, King's College Hospital Medical School, London, S.E.5.

Although the complex series of events whichoccur when a steroid hormone is administeredto a human subject or to an intact animalmight reflect a series of completely unre!latedactivities, they are more likely to be secondaryto a few fundamental actions on the celils ofthe body and it is with this second conceptthat research in this field has been directed.There are two particular features of steroids

which may be important in determining theirmode of action. Firstly, they have oxygensubstituents at certain typical positions whichreadily undergo enzymatic oxido-reduction andcould, therefore, act as coenzymes or pros-thetic groups of enzymes in reactions involvinghydrogen transfer. Secondly, steroids are sur-face active and interact with hydrophobic sur-faces producing energy. If the energy wereto be absorbed iby a receptor molecule it couldmodify the structure and hence the biologicalactivity of that molecule. A less well definedchange is associated with the ability of steroidsto capture electrons; this has recently beenmeasured by Lovelock, Simmonds and Vanden-heuvel (1963) who consider that the highelectron affinities of adrenocortical hormones,a property unusual among organic compounds,might indicate their ability to participate inor control biological oxidative processes.

Laidler and Krupka (1961) compared theassociation between the steroid and a receptorwith the formation of an activated enzyme-substrate Michaelis complex. Entropy andvolume changes during activation of enzymesindicate that structural changes occur in theenzyme molecule and such changes might ex-plain the disturbances of membrane perme-ability in nerve cells associated with structuralchanges in acetylcholinesterase. A similarmechanism might account for changes inducedby steroids in the permeability of cell structures.

In this process parts of the receptor mole-cules having specific binding properties mightbe masked, unmasked, created or destroyed;as well as causing a redistribution of boundsubstrates, an alteration in enzymic propertiesmight result. Kimberg and Yielding (1962)studied the inhibition of pyruvate kinase by

oestrogens and related compounds and showedby viscometry and electrophoresis structuralchanges in the enzyme, without a change inmolecular weight. Yielding and Tomkins(1962) have reported a steroid-hormone inducedloss of activity of crystalline glutamic dehydro-genase due to disaggregation into subunitsfunctioning as alanine dehydrogenase with anuncovering of pyridine-nucleotide binding sites.These authors have been more concerned toshow the possibility of such changes ratherthan to attach great physiological significanceto them. Chemical changes in receptor mole-cules after association could account for thehighly theoretical possibility of the formationof active enzymes from inactive precursors.

That the receptor itsel-f could be an enzymecofactor has been considered by Scott andEngel (1961) who obtained physical evidencefor the interaction between steroid hormonesand purine dinucleotides. This interactionbetween hormone and a coenzyme associatedwith a postulated change in the structureof the coenzyme might abolish its hydrogen-carrying function, but there was no experi-mental evidence for this.Apart from oxido-reductive changes,

metabolism of the steroid molecule is notthought to be of physiological importanceexcept as a mechanism for steroid inactivation.However, competition for active sites onenzymes as opposed to association with themcould be of importance in influencing steroidmetabolism itself.The various theories which have been pro-

posed to explain the action of steroid hormonesat the cellular level will be considered underthe following headings:

1. Effects on membrane permeability andactive transport.

2. Effects on hydrogen transfer.3. Effects on enzyme induction and protein

synthesis.

Effects on Membrane Permeability and onActive Transport.

Roberts and Szego (1953) found that cestro-gens increased the glucose uptake of rat uteri,

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DIXON, GRAY and QUINCEY: Steroid Hormones

and suggested that their primary action mightbe to facilitate the entry of glucose into theuterine cells. Noall, Riggs, Walker and Chris-tensen (1957) showed an increased uptake ofx -amino-iso-butyric acid (AIB) in immaturerat uteri 20 hours after administering aestradioland in rat liver 2 hours after giving hydro-cortisone. Since this synthetic amino acid isnot metabolised, any change in uptake mustbe due to a change in transport. Halkerston,Eichhorn, Feinstein, Scully and Hechter (1960)examined the effects of cestradiol on the uteriof castrate rats using intravenous [14C]labelled AIB as well as [14C] D-xylose; theyfound no change 12 hours after cestrogen injec-tion, although the metabolic effects on glucoseuptake and incorporation of amino acids intoprotein are detectable after one hour. Aftersix hours there was an increase in the watercontent of the tissue and a small increase inAIB and xylose accumulation. They concludedthat there is no primary change in sugar oramino acid transport and interpreted previousresults as secondary effects operating at a latertime. On the other hand Noall and Allen(1961) obtained more than 100 per cent increasein AIB uptake in uteri in vitro removed only30 minutes after intravenous administration ofcestradiol to immature rabbits. They commentthat in vivo experiments were unsuitalble forthe investigation of early changes. No effectwas observed when the excised uteri weretreated wth cestradiol in spite of adequatepenetration of the hormone. Although thissuggested that the increased uptake observedafter in vivo hormone administration may nothave been a primary effect, they postulatedthat cestradiol might be metabolised to a hypo-thetical active form but their evidence wasinconclusive. An increased uptake of AIBby rat levator ani muscles after large doses oftestosterone and of synthetic anabolic steroidswas shown by Metcalf and Gross (1960) butthe effects were not observed until 39 hoursafter the injection of the steroid and ninehours after AIB administration. IncreasedAIB uptake in isolated perfused rat livers wasseen by Bass, Chamibers and Richtarick (1963)two hours after both in vivo and in vitroadministrations of hydrocortisone, suggestingan early direct effect on liver cells.

It is reasonable to conclude that steroidhormones alter transport mechanisms and thusinfluence the availability of substrate to cells,but although early effects have been observedin isolated organs it is not certain that theseare primary events. Since cells are nowregarded not as bags of assorted enzymes but

as highly organised structures, parts of whichare clearly differentiated by membranes andphase boundaries, a possible effect on intra-cellular substrate distribution must also ibeconsidered. Information on this, however, isscanty and indirect. Binding to subceUlularparticles has been studied by Westphal (1961)who demonstrated interaction between livermitochondria and hydrocortisone and cortico-sterone, and by Bellamy (1963) who showed asignificant binding between corticosterone andrat liver ribosomes. There is definite evidencethat the mitochondrial membrane is influencedby steroids. Westphal (1961) found that hydro-cortisone and corticosterone increased theswelling of rat liver mitochondria, indicatingan increased permeability to water. Gallagher(1960) concluded that hydrocortisone inhibitedoxidative metabolism in liver imitochondriaby increasing the membrane permeability caus-ing loss of respiratory cofactors. Blecher(1962) suggested that a similar swelling wasassociated with a release of latent ATPasewhich inhibited metabolism of glucose byreducing the availability of ATP. He citedthis as a mechanism for the ilymphocytolyticaction of steroids.

Structural alterations in the peripheral cellmembrane are generally assumed to accountfor changes in transport (see Tomkins andMaxwell, 1963). Controlled and variablepassive diffusion can be explained on a bio-chemical basis, but explanations of mechanismsof active transport in which energy is used totransfer molecules against concentration gra-dients are only speculative. Hechter and Lester(1960) present and review data suggesting thatpart of the increased glucose uptake of musclecells in response to insulin is the apparentremoval of intracellular barriers to diffusion,enabling the sugar to equilibrate in a largerfraction of the cell water. Extending this theorythe authors outline a model cell based on theresults of their experiments on sodium andpotassium distribution in the mould Neuro-spora Crassa. Potassium was taken up againsta concentration gradient and sodium wasexcluded from the major part of the cell water,but in the presence of desoxycorticosteroneboth ions were distributed in all of the celwater at the same concentration as in the sur-rounding medium. This suggested that thesodium pump operated not at the cell boun-dary but in the cytoplasm. The crystal struc-ture of certain silicate minerals excludes thehighly hydrated sodium ions, whereas the lesshydrated potassium ions are able to diffusefreely through and be adsorbed by the lattice;

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such minerals behaved in a similar mannerto untreated Neurospora. The authors assumedthat in the cytoplasm there are ordered latticeswhich require energy to maintain their struc-tures and which bind potassium ions and watermolecules and exclude sodium ions. In theaibsence of energy or under the influence ofspecific agents the lattices expand, potassiumbinding sites are lost and sodium is no longerexcluded. Other cytoplasmic components mayalso bind sugar and amino acid molecules.The bound substrate equilibrates with sub-strate in the non-bound intracellullar water andat equilibrium the concentration of this equalsthat of the surrounding medium. Active trans-port in this model is the binding of substrateand water to the lattice with equilibration ofthe unbound intracellular phase with the sur-rounding medium through an inert memjbrane.In spite of the authors' assertions to the con-trary, sufficient binding of potassium to accountfor active transport is thermodynamically incon-sistent with the maintainance of an electricalpotential across the membrane. However, theability of this ingenious model to account formany aspects of active transport merits seriousconsideration.

Effects on Hydrogen TransferSeveral steroids inhibit the transfer of

hydrogen from the pyridine nucleotide coen-zymes 'by the mitochondrial cytochrome chain(see Wade and Jones, 1956) reducing respira-tion and oxidative phosphorylation. Yielding,Tomkins, Munday and Cowley, (1960) locatedthe site of action between flavoprotein andcytochrome b; Chance and Hollunger (1963)found that inhiibition of respiration by pro-gesterone was sensitive to reagents such asdinitrophenol which uncouple the oxidativeand phosphorylation processes, and concludedthat the actual site of inhibition was in theenergy-transfer process. They postulated thatthe inhibitor binds a high-energy intermediateand that the uncoupling reagent dissipates thiscomplex with loss of its energy.

Oestrogens stimulate respiration in theirtarget tissues. Hagerman and Villee (1957)studied placental respiration and identified anoestrogen-sensitive isocitric dehydrogenase.This Krebs cycle enzyme requires NADP ascoenzyme, but it was at first thought that theoestrogen-sensitive enzyme utilised NAD; laterthe oestrogen-sensitive step was discovered tobe the transfer of hydrogen from NADPH toNAD (Talalay, Hurlock and Williams-Ashman1958), the ratelimiting factor in the Krebscycle being the availability of NADP:

ISOCITRATE-N NADP NADH

lsocitrk/l hydogeosdehydrogen ,seOXALOSUCCINATE NADPH\'. NAD

Transhydrogenases, which catalyse the trans-fer of hydrogen between pyridine nucleotideccenzymes, have been identified in both mito-chondria and in soluble cell sap of severaltissues and organisms. The transfer of hydrogenfrom NADPH to NAD increases the efficiencyof energy production since NADH transfers itshydrogen to cytochrome c via flavoprotein,cenzyme Q and cytochrome b with a greateryield of ATP than NADPH which transfershydrogen directly to cytochrome c. They mayalso play important regulatory roles in cellularmetabolism, as NADP may be a rate-limitingfactor in dehydrogenations in the pentosephosphate cycle and the availability of NADPHmay influence fatty acid and steroid biosyn-thesis and hydroxylations. In addition to theoestrogen-sensitive transhydrogenase, whichHagerman and Villee (1961) claim can befound in all cestrogen target organs, Hurlockand Talalay (1958) have described a 3-hydroxy-steroid-sensitive transhydrogenase system inliver, but which is probably only of minorimportance (Stein and Kaplan, 1959). Pesch,Piros, and Klatskin (1962), and McGuire andPesch (1962) have identified steroid-sensitivetranshydrogenases in particulate preparationsof liver and anterior pituitary.The mechanism of steroid-sensitive trans-

hydrogenation is the subject of some dispute.Talalay and Williams-Ashman (1958) proposeda dehydrogenase system having dual nucleotidespecificity with the transfer occurring via thesteroid which is alternately reduced andoxidised:-

N% IXC-0 + NADPH+K+H H -C-C01 +tNADP+

H-C-OH+NAD+ sC-00 +NADHH

the nett reaction being:

NADPH + NAD+ NADP+ + NADH

Hagerman and Villee (1959) consider thatthe placental transhydrogenase is stimulated by17 3-OH cestrogens but that they take no directpart in the reaction and using starch-gel electro-

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phoresis they claimed to separate dehydrogen-ase activity from transhydrogenase activity,although in small yield. Williams-Ashman,Jarabak, Adams and Talalay (1962) purifiedthe placental enzyme 2,500-fold with an over-all yield of 29 per cent and could not separatedehydrogenase from transhydrogenase activityat any stage. Moreover the stilbcestrol deriv-tives, having only phenolic oxygen groups,should be inactive if the dehydrogenase theoryis correct. Adams, Jarabak and Talalay (1962)reported that with the purified enzyme, diethyl-stilbeestrol acted as a competitive inhibitor.However Abe, Hagerman and Villee (1964)working with an enzyme isolated from myomet-rium found only slight dehydrogenase activityand that diethylstilboestrol was fully active instimulating transhydrogenation. Whatever themechanism, cestrogen-stimulated transhydro-genation undoubtedly occurs, and Joel, Hager-man and Villee (1961) present data supporting,but not proving, the hypothesis that increasedATP production after stimulation of trans-hydrogenation directly influenced the synthesisof cell constituents.

Lucas, Neufield, Utterback, Martin andStotz (1955) reported that cestrogens stimulateuterine peroxidase activity and Williams-Ashman, Cassman and Calvins (1959) havefound a possible hydrogen transfer by purelyphenolic compounds in model systems utilizingperoxidase. Talalay and Williams-Ashman(1960) have isolated such a system from uteriand shown an cestrogen-mediated increase inoxidation, but its physiological significance isdoubtful.Bush (1962) considers that chemical changes

in steroid molecules are not related to theirfunction and represent metabolism and inacti-vation only. He has examined particularly the11-oxygen function and from a study of hydro-cortisone analogues has concluded that the11(P-hydroxyl group is the only essentialrequirement for glucocorticoid activity; parti-cularly significant is the finding that 9x-fluoro-hydrocortisone does not give rise to 1 1-oxometabolites, and it therefore seems unlikely thatoxidation plays any part in its biologicalactivity since this fluoro compound is manytimes more active than hydrocortisone.

Effects on Enzyme Induction and ProteinSynthesisThe administration of cestrogens to females

causes a rapid increase in uterine weight andadministration of androgens to males causes anincrease in the weights of the secondary sex

organs and of other sensitive tissues. Theincrease in weight is due largely to proteinalthoug,h in the uterus there is also a rapiduptake of water. Glucocorticoids cause a netdecrease in peripheral-tissue protein whilstappearing to enhance protein synthesis in theliver. In addition to the general weight responseof target tissues to cestrogens or androgens,administration of these hormones increases theeffective concentration of many enzymes andglucocorticoids increase the effective concentra-tion of some liver enzymes. The response toandrogens and probably to other steroidhormones is highly dependent on both tissueand species.Induced enzyme synthesis. Tihe effect of steroidhormones on protein synthesis is most easilystudied by determining changes in enzymeactivity because of the ease with which smallnet changes may be measured. An increase inactivity of an enzyme after steroid administra-tion could be due to either enzyme activation oran increase in enzyme synthesis, i.e. enzymeinduction. Induction, however, might not neces-sarily be a primary action of the steroid butcould be secondary to some other action of thesteroid in modifying the intracellular environ-ment. Much experimentation has been designedto establish whether there is activation orinduction, often by immunological studies.The effect of testosterone on the activity of

mouse kidney (-glucuronidase has been studiedby Riotton and Fishman (1953). The activityof this enzyme after treatment of the animalwith testosterone showed a thirtyfold increaseand could be maintained at this level for 60days by continued testosterone administration.The response was later attributed to an increasein enzyme synthesis because the specific activityof f-glucuronidase isolated from the kidneysof mice treated with testosterone and given[14C1-glycine was higher than that from thekidneys of untreated mice or the livers of bothtreated and untreated animals (Pettengill andFishman, 1960). Tryptophan pyrrolase activityin liver is stimulated by the administration ofhydrocortisone to rats but this increase isprevented by simultaneous administration ofthe methionine analogue ethionine (Horton andFranz, 1959); in perfused-liver preparationsthe increase occurs after the hydrocortisone hasdisappeared (Goldstein, Stella and Knox, 1962).The increase in enzyme activity is, therefore, aresult of increased enzyme synthesis.

Hydrocortisone produces a rapid increase intyrosine- cx-ketoglutarate transaminase activityin perfused rat-liver preparations (Goldstein

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and others, 1962). Kenney (1962a) has purifiedthis enzyme and shown that there are bothactive and inactive enzyme components but theproportions of active and inactive componentswere the same in preparations from both controland hydrocortisone-treated animals. He con-cluded that hydrocortisone did not promote theconversion of an inactive enzyme form to theactive form and he also showed (Kenney 1962b)that the rise in enzyme activity was paralleledby a rise in antigenic material suggesting thatincreased enzyme synthesis was primarilyresponsible for the hydrocortisone-inducedincrease in activity. Kenney and Flora (1961)showed that the increase in activity wasrelated to the concentration of steroid in theliver and believed that the steroid was actingdirectly on the protein-synthesising system.

Glutamic-pyruvic transaminase activity in ratliver is increased by hydrocortisone administra-tion and this increase could be inhibited byethionine (Segal, Beattie and Hopper, 1962).Segal, Rosso, Hopper and Weber (1962) haveshown that the rise in enzyme activity isparalleled by an increase in antigenic materialagain suggesting that the hormone inducesenzyme synthesis. However, fasting, diabetes ora high protein diet also increases hepaticglutamic-pyruvic transaminase activity (Rosen,Roberts and Nichol, 1959) which suggests thatthe observed increase may be secondary to aperipheral effect. Similarly, although hydrocor-tisone increases glucose-6-phosphatase activityin the livers of adrenalectomized or normal ratsso does starvation or a high protein diet(Harper and Young, 1959; Harper, 1959) oralloxan diabetes (Frcesch, Ashmore andRenold, 1958).Effects on uptake of amino acids. Severalworkers have studied the uptake of radioactiveamino acids by target tissues after oestrogenor androgen administration. Frieden, Laby,Bates, and Layman (1957) showed that theuptake of [14C]-glycine into mouse kidney wasstimulated by testosterone propionate andButenandt, Gunther and Turba (1960) showedthat the incorporation of [14C]-leucine by semi-nal vesicles of immature rats receiving testo-sterone was increased fourfold whilst Mueller(1961) has shown that the amount of radio-activity in protein was greater in uteri fromcestradiol-treated rats after ["4C]-glycineadministration. Mueller (1961) also showed thatthe incorporation of radioactivity from [14C]-glycine into nucleic acid adenine and of 82p intophospholipid of uteri was increased when ratswere treated with cestradiol. Puromycin in-

hibited these effects of cestradiol. He concludedthat cestradiol-stimulated protein synthesis andthat the effect of puromycin on the incorpora-tion of radioactivity from glycine into nucleicacid adenine and of phosphorus into phospho-lipid was due to an inhibition of the enzymesresponsible for adenine and phospholipidsynthesis. Such experiments do not define thestep in protein synthesis on which steroids act.Wilson (1962a) in an attempt to define this

more closely has studied the rise in specificactivity of free intracellular tyrosine and ofprotein during incubation of slices of ratseminal vesicles with [14C]-tyrosine. Heconcluded that testosterone did not act byenhancing amino acid transport and that theincrease in amino acid incorporation wassecondary to an increased protein synthesis.Effects on amino acid activation. Steroids maynot only act directly on amino acid transport(see above) but have been shown to increasealso the activity of some amino-acid-activatingenzymes, which are essential for the synthesisof protein. Thus, Mueller, Herranen and Jervell(1958) have shown that cestradiol causes anearly increase in the activity of seven of theknown amino-acid-activating enzymes andKochakian, Tanaka and Hill (1961) haveshown that the activity of amino-acid-activatingenzymes in the seminal vesicles of guinea pigswas reduced 40 per cent 40 days after castra-tion. The activity could be restored byandrogens.Effects on ribosomal activity and the formationof ribonucleoprotein. Much recent work onsteroids has been designed to define their exactrole in protein synthesis, the main steps ofwhich are outlined in Figure 1.Amino acids are activated in the cytoplasm by

specific amino-acid-activating enzymes and can thencomplex with specific soluble ribonucleic acidmolecules (s-RNA). The s-RNA-amino-acid complexwhich is forned is transferred to a specific positionon an RNA template situated on the surface of aribosome. Peptide-bonding of the amino acidsattached to the template occurs and is followed byrelease of the newly formed protein from thetemplate. The genetic determination of the structureof a protein is due to the synthesis of that protein ona ribosomal RNA template whose base sequence isdetermined by that of the deoxyribonucleic acid(DNA) of the structural gene. The template ormessenger RNA is synthesised in the nucleus andtransferred to a ribosome; its rate of synthesis isdetermined by an operator-repressor gene system(Jacob and Monod, 1961). The operator is normallyinhibited by the repressor and cytoplasmic induceract by reducing the inhibitory action of the repressoron the operator. Since the ability of ribosomes tosynthesise protein is related to its template RNA

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amino amino ATP activated amino acid GT P formation of ribonucdeoprotein releaseacid j - acid -- . amino acid - * sRNAcomplex- * on ribosomal RNA template -* of protein

Itransport across specific amino acid transfer RNA releasingcIl membrane amino acid specific sRNA enzyme polymerase\ factors

activatingenzyme /

messenger RNA synthesis RNA breakdownin nucleus controlledby a gene complex

FIG. 1.-The Biosynthesis of Protein.

content protein synthesis will be affected. Steroidscould act on protein synthesis by regulating theavailability of template RNA through control of itsrate of synthesis or breakdown, or alternatively bymodifying ribosomal structure as Tissi6res, Schles-singer and Gros (1960) have shown that this isimportant in determining the rate of protein synthesisin bacterial systems.Wilson (1962a) investigated the effect of

testosterone administration on the increase withtime in specific activity of ribosomal proteinand s-RNA-amino-acid complex in slices ofseminal vesicle incubated with ['4C1-tyrosine or['4CJ-leucine. He found that testosteroneaccelerated the rise in specific activity ofribosomal protein but did not affect the rise inspecific activity of the s-RNA-amino-acid. Heconcluded that testosterone acted by enhancingthe formation of ribonucleoprotein on theribosomes and in similar experiments on theeffect of cestradiol on protein synthesis in ratuterine slices, Wilson (1962b) showed thatcestradiol also acted on this step. Using pre-parations of ribosomes and soluble factorsisolated from the oviducts of cestradiol-treatedor control hens, Wilson (1962b) showed thatcestradiol modified ribosomal activity but didnot affect the activity of the soluble factorsrequired in the formation of ribonucleoprotein.The action of glucocorticoids on protein

synthesis in the liver has also been defined atthe ribosomal level by Korner (1960) whoshowed that ribosomes from the livers of ratsadrenalectomized some weeks previously wereless active than those of controls. Treatment ofnormal rats with hydrocortisone increased liverribosome activity. The immediate effect ofadrenalectomy, however, was to increaseribosomal activity and this could be abolishedby treatment with hydrocortisone. The activityof the soluble factors was unchanged duringthese procedures. These paradoxical early andlate effects of adrenalectomy have not yet beenexplained.Effects on synthesis of template (ribosomal)ribonucleic acid. Mueller (1961) has shown that

increased nucleic acid synthesis is an early effectof cestrogen treatment; Kochakian, Hill andAonuma (1963) have found that template RNAfrom mouse kidney, but not soluble RNA, wasincreased by testosterone propionate admimnstra-tion and Cantarow, Pashkiss and Williams(1959) have found that the administration oftestosterone increased ["ICI-uracil incorporationby rat liver and hepatoma cells. More directly,Hancock, Zelis, Shaw and Williams-Ashman(1962) have shown that the incorporation ofradioactive cytosine triphosphate into RNA byDNA-dependent RNA polymerase from thenuclei of rat ventral prostate was decreased inpreparations from castrated rats and theactivity could be restored by treating the ratswith testosterone. These experiments show thatsteroids increase the synthesis of template RNA.Work on the inhibition of induced enzyme

synthesis by 8-azaguanine (which preventsRNA synthesis) and by actinomycin (whichprevents the messenger RNA from attachingto the ribosomes) has shown that the synthesisof template RNA and its attachment to theribosomes are necessary for induced enzymesynthesis. Thus, Kvam and Parkes (1960) haveshown that the simultaneous administration of8-azaguanine and hydrocortisone to adrenal-ectomized rats prevented the increase in glucose-6-phosphatase and fructose 1, 6-diphosphatasewhich normally occurred after hydrocortisoneadministration. Similarly Greengard and Acs(1962) showed that actinomycin prevented therise in tryptophan pyrrolase and tyrosine- a-ketoglutarate transaminase activity after hydro-cortisone administration. However, chloram-phenicol, which inhibits protein synthesis inbacteria and is thought to act by preventingthe messenger RNA from attaching to theribosomes, does not inhibit the incorporation of[14C]-leucine into kidney ribosomes from test-osterone-treated mice (Kochakian and others,1963) nor does it inhibit protein synthesis in theoviduct system of hens treated with cestradiol(Wilson, 1962b). This does not invalidate the

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454 POSTGRADUATE MEDICAL JOURNAL August, 1964

view that steroids act by increasing the synthesisof template RNA, but shows that there is nota high turnover of labile messenger RNAsensitive to chloramphenicol inhibition as inbacteria.

Direct evidence that steroids act on proteinsynthesis by regulating the availability of tem-plate RNA has been obtained by Liao andWilliams-Ashman (1962). They found that thetransfer of radioactivity from s-RNA-valine-[14Cq to trichloracetic acid-insoluble materialby ribonucleoprotein particles from rat ventralprostate was markedly reduced in preparationsfrom animals castrated 2-3 days previously andthe activity could be increased threefold by theaddition of polyuridine-guanidine (UG), a syn-thetic polynucleotide having a high uracil/guanine ratio. The administration of testo-sterone to the animals completely reversed theeffects of castration and the addition of poly-UG to preparations from testosterone-treatedcastrates had no effect. On the other hand,Wilson (1962b) found that the addition ofribosomal RNA, extracted from the oviducts ofhens treated with cestradiol, to the protein-synthesising system from the oviducts of un-treated hens did not increase the rate of proteinsynthesis. However, the RNA might have beendegraded during the extraction procedure.Effects of glucocorticoids on protein synthesis inperipheral tissues. The glucocorticoid-induceddecrease in peripheral-tissue protein seems tobe due to a decreased rate of synthesis ratherthan to an increased rate of breakdown. Man-chester, Randle and Young (1959) showed thatadrenalectomy raised, whilst hydrocortisonedepressed, the rate of incorporation of [14C]-glycine into protein of rat diaphragm andWhite, Hoberman and Szego (1948) showed thatthe rate of incorporation of ['5N]-glycine intomuscle and some other tissues was increasedby adrenalectomy. From a study of the rateof excretion of isotopic nitrogen after theadministration of [15N]-glycine to normal,adrenalectomized and adrenalectomized ratstreated with cortisone, Clarke (1953) concluded

that cortisone acted by inhibiting synthesisrather than by promoting protein breakdown.ConclusionsMany of the physiological effects of steroids

on electrolyte distribution and on the avail-ability of cellular substrates might result froma single action of these hormones on activetransport. Although the exact mechanism ofactive transport is unknown, the theory ofHechter and Lester is interesting. Steroidscould interact with those intracellularstructures which are involved in the transportmechanism, modify their structure and hencethe uptake of substances by the cell. However,the uptake of substrate by cells is probably notrate-limiting in all cases and steroids certainlyaffect the activity of many intracellular enzymesystems so that an apparent action on transportcould be secondary to some other action on anintracellular enzyme system.

Steroid-activated enzyme systems whichcatalyze transhydrogenation occur in manytissues but their presence in all cestrogen targettissues is questionable (Mueller, 1961) whichthrows doubt on their physiological significance.The mechanism proposed by Talalay for trans-hydrogenation is the only example of chemicalchange in the steroid molecule being importantin its mode of action.The action of steroids on protein synthesis,

with the possible exception of the influence ofglucocorticoids on peripheral tissues, isprobably to regulate the synthesis of messengerRNA, perhaps through the operator-repressorgene system. Such a mechanism permits specificenzyme synthesis and a differentiation in tissueresponse because in different tissues enzymeswith similar functions may have differentstructures (Kaplan, Ciotti, Hamolsky andBieber, 1960) and hence be controlled bydifferent gene complexes. However, the increasein synthesis of some enzymes after steroidadministration might not be due to a directaction of the steroid on the protein-synthesisingmechanism but could be secondary to someother change in cellular environment.

REFERENCESABE, T., HAGERMAN, D. D., and VILLEE, C. A. (1964): Estrogen-Dependent Pyridine Nucleotide Transhydro-genase of Human Myometrium, J. biol. Chem., 239, 414.ADAMS, J. A., JMABAK, J., and TALALAY, P. (1962): The Steroid Specificity of the 173-hydroxysteroidDehydrogenase of Human Placenta, J. biol. Chem., 237, 3069.BASS, A. D., CHAMBERS, J. W., and RIcHTARICK, A. A. (1963): The Effect of Hydrocortisone on AIB Uptakeby the Isolated Perfused Rat Liver, Life Sci., 4, 266.BELLAMY, D. (1963): The Adsorption of Cortticosteroids to Particulate Preparations of Rat Liver, Biochem.J., 87, 334.BLECHER, M. (1962): Effect of Deoxycorticosterone on Soluble Beef Heart Mitochondrial ATPase, Proc.Soc. exp. Biol. (N.Y.), 111, 529.

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August, 1964 DIXON, GRAY and QUINCEY: Steroid Hormones 455

BUSH, I. E. (1962): 'Chemical and Biological Factors in the Actions of Steroid Hormones' in the HumanAdrenal Cortex. Eds. A. R. Currie, T. Synington, and J. K. Grant, London: E. & S. Livingstone.

BUTENANDT, A., GUNTHER, H. and TURBA, F. (1960): Primary Metabolic Effects of Testosterone, Z. Physiol.Chem., 322, 28.

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the action of steroid at the cellular · dixon, grayand quincey: steroid hormones and suggested...

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