Metabolism and Rate of Secretion

of Aldosterone in the BullfrogSTANLEYULIcK and ERICA FEiNHOLuz

From the Medical Service, Veterans Administration Hospital, and theDepartment of Medicine, Albert Einstein College of Medicine ofYeshiva University, Bronx, NewYork 10468

ABSTRACT A study of the metabolism of al-dosterone in the bullfrog was undertaken to pro-vide a measurable metabolite for the indirect iso-tope dilution technique for measuring secretoryrates. The rate of excretion of labeled aldosteronewas considerably slower in the frog than in manand made necessary the collection of excretoryproducts for 5 days to insure reasonably completerecovery of metabolites. The major identifiablemetabolite was a tetrahydro derivative subse-quently identified as 3,f-hydroxy-5,8-tetrahydroal-dosterone. This metabolite was excreted partly inthe free form and partly as a glucuronic acid con-jugate. The pH 1-hydrolyzable conjugate of al-dosterone was not detected.

For the measurement of secretory rates, aldos-terone-3H was injected into the dorsal lymph spaceand the animal placed in a bath to provide an en-vironment of constant electrolyte composition andintake and a means of collecting excretory prod-ucts. Urine and bath fluid were collected for 5days, tetrahydroaldosterone was isolated, and itsspecific activity determined for the calculation ofthe aldosterone secretory rate. The rate of secre-tion of aldosterone in the bullfrog was increasedby a tap water bath and by bovine adrenocortico-tropic hormone, decreased by a saline bath and bydexamethasone, and unchanged by valine-5-angio-tensin amide.

INTRODUCTIONAldosterone is a quantitatively minor componentof the mammalian adrenal cortex. Interest in its

Received for publication 27 May 1968 and in revisedform 10 July 1968.

secretion and regulation in certain lower verte-brates was stimulated by the finding of Carstensen,Burgers, and Li (1, 2) that aldosterone was a ma-jor corticosteroid in bullfrog interrenal incubates.Subsequent studies of the biosynthetic pattern ofthe tissue, which showed a predominence of al-dosterone and its potential biogenetic precursorsand absent 17a-hydroxylation (3, 4), appeared tosupport the implication of Carstensen and co-workers' findings (2) that the bullfrog interrenalwas functionally homologous with the zona glo-merulosa of the mammalian adrenal cortex.

The regulation of aldosterone secretion in mam-mals is predominantly extrapituitary. Earlier ob-servations demonstrated the role of the pituitaryin supporting adrenocortical function in certainanuran amphibians particularly during periods ofincreased metabolic activity (5). The existence ofextrapituitary regulatory factors as well was sug-gested by the evidence of persistent electrolyte-active hormonal activity in hypophysectomizedfrogs (6, 7). It was of interest therefore to in-vestigate the evolution of the aldosterone regula-tory mechanism in a species in which efficient so-dium conservation was crucial to its adaptationto a semiaquatic fresh water habitat.

The present report describes a method to mea-sure the rate of aldosterone secretion in the bull-frog and the application of the technique to con-trolled studies of the effects of environmental sa-linity and potential regulatory factors. The methodis based on the single injection isotope dilutiontechnique used to measure the secretory rate ofthe hormone in man (8), modified by prolongingthe period of collection of excretory products to5 days because of the bullfrog's slower rate of me-

The Journal of Clinical Investigation Volume 47 1968 2523

tabolism and excretion of the radioactive steroid.The technique utilizes the semiaquatic habitat ofthe bullfrog to provide simultaneously an environ-ment and intake of controlled electrolyte composi-tion and a means of collecting metabolic products.Since the presentation of our preliminary report(9), the results of another technique using post-caval phlebotomy in a pithed preparation of Ranacatesbeiana (10) are available for comparison.

METHODSBullfrogs (Rana catesbeiana) of both sexes, weighingfrom 180 to 550 g, were in the fasting state and werekept in a sink under running tap water when not usedfor experiments. For the experimental periods which re-quired control of external environment and collection ofexcretory products, the animals were kept in 12 inches X6 inches X 5 inches plastic cages containing approxi-mately 200 ml of bath fluid and covered with a wiremesh lid. This procedure allowed partial immersion ofthe frog and contact of the bath with the underside ofits body when the frog was in a normal resting position.At the end of each 24-hr period, bath fluid was replacedand combined with contents of the urinary bladder, emp-tied by means of No. 205 polyethylene tubing insertedinto the cloaca. Two bath fluids were used: tap water and60 meq of NaCl per liter. When hypotonic saline wasused, estimated daily evaporation from the plastic cagewas replaced by adding distilled water to maintain thesodium concentration constant within ± 10%.

Hormones and labeled aldosterone were dissolved insterile distilled water containing 0.9% benzyl alcohol aspreservative, and 0.5 ml was injected into the dorsallymph sac. Aldosterone-1,2-'H (50 mc/,umole) was pre-pared biosynthetically (11).

Radioactivity was measured in a liquid scintillationspectrometer equipped with external standardization.The phosphor solution consisted of 3.0 g of 2,5-diphenyl-oxazole and 6 ml of ethanol per liter of toluene. For thecounting of aqueous solutions, a 0.5-ml aliquot was mixedwith 5 ml of absolute ethanol and 10 ml of toluene phos-phor solution.

Tritium-14C ratios. The previous (12) simplification ofthe equation of Okita, Kabara, Richardson, and Leroy(13)

3H/4C =N1 - N2/b (1)N2 - aN1

where N1 = net counts per minute in tritium channel; N2=net counts per minute in 14C channel; a= N2/N1 forpure tritium and b = N2/N1 for pure "C was based onthe assumption that constant a was negligible comparedto b. With improved liquid scintillation instrumentationbetter discrimination between the two isotopes could beattained. Typical values for the overlap constants werea = 10' and b = 5.0. Under these circumstances con-stant a could be neglected in a further simplification of

the equation,'H/l4C = N1/N2 - 1/b (2)

The N,/N2 ratio was first calculated and then correctedby substracting l/b. When N1/N2 was greater than 5.0,and 1/b was 0.2, the error introduced by neglectingl/b was less than 4%. Under these circumstances, N1/N2was a reasonable approximation of the 'H/"C ratio

3H/P4C = Ni/N2- (3)Preparation of urinary fractions. After the injection

of aldosterone-8H, a 5 day pool of bath fluid and urinewas extracted with ethyl acetate and the extract washedwith dilute sodium hydroxide and water to obtain the freefraction. The combined washings and urine were ad-justed to pH 5 and incubated with approximately 250U/ml of mammalian glucuronidase at 45°C for 2 days.A neutral ethyl acetate extract of the incubate representedthe glucuronidase-hydrolyzable fraction. The combinedwashings and extracted urine from the preceding stepwere adjusted to pH 1 with HCl, allowed to remain atroom temperature for 24 hr, and a neutral ethyl acetateextract, the pH 1-hydrolyzable fraction, was prepared.

Aldosterone secretory rates. When the bath composi-tion was changed, sodium concentration in catheterizedbladder urine was monitored daily and secretory ratesdetermined when equilibration occurred. Measurementswere made during all seasons of the year but were pre-ceded by a control determination to minimize variationdue to seasonal fluctuations (14) in adrenal activity.5 ,uc of aldosterone-8H was injected, and pooled bathfluid and urine were collected for 5 days. An aliquot ofthe pool was counted to verify recovery of at least 65%oof the injected dose. In a few instances when recoverywas less, the experiment was discarded. Tetrahydroal-dosterone was isolated from the pool and its specific ac-tivity measured as described below. The 5 day secretoryrate was calculated by dividing the counts per minute ofaldosterone injected by the specific activity of the metabo-lite, and the average daily secretory rates shown in theFigures and Tables were obtained by dividing by 5.

Isolation of frog tetrahydroaldosterone. The 5 daypool of urine and bath fluid collected after a single in-jection of labeled aldosterone was incubated with mam-malian glucuronidase, extracted with ethyl acetate, andthe extract washed with dilute sodium hydroxide andwater. The extract which represented the combined freeand glucuronidase-hydrolyzable fractions was chromato-graphed on the ethylene dichloride-formamide system for24 hr with cortisol as reference steroid. Teterahydroal-dosterone 1 was located for elution by radioactive scan-ning and by its migration at 0.85 the rate of cortisol.The eluate was dissolved in methylene chloride, washed

1 The chromatographic mobilities of frog tetrahydroal-dosterone differed slightly from those of the major- me-tabolite in human urine, the 3a-hydroxy-5,8 isomer (10).The frog metabolite has subsequently been identified asthe 3,8-hydroxy-5,6 isomer: 3,8,11,6,21-trihydroxy-20-keto-5,8-pregnan-18-al (H. C. Rose and S. Ulick; data inpreparation).

2524 S. Ulick and E. Feinholtz

with water, dried and evaporated, and acetylated withacetic anhydride-"C (0.8 mc/mmole) :toluene (1: 4) inpyridine at 60'C for 18 hr and excess reagents removedby partition (11). The triacetate was chromatographedon the Bush A and methylcyclohexane-formamide sys-tems in which it migrated at approximately 1.1 times therate of deoxycorticosterone acetate. After elution, thedried residue was shaken mechanically with 5 ml of 0.1 NHC1 for 30 min at room temperature in a glass-stopperedtest tube. The aqueous phase was extracted with methyl-ene chloride and the extract washed with 0.1 N NaOHand water, dried, and evaporated. The resulting 3,21-diacetate was isolated after chromatography in methyl-cyclohexane: toluene (9: 1)-formamide in which it mi-grated at 0.55 the rate of deoxycorticosterone. The18-acetoxy group was reintroduced by acetylation withunlabeled acetic anhydride in pyridine in the usual mannerat 80°C in an aluminum test tube heating block and themethylcyclohexane-formamide system was used to re-isolate the triacetate. When this specific procedure ofselective removal and regeneration of the C-18 acetoxygroup was applied, the 'H/4C ratios of diacetate and tri-acetate agreed within 5%. Specific activity was calculatedin the usual manner from the product of the 'H/"C ratioof purified metabolite, the specific activity of the acetylat-ing reagent, and the number of '4C-labeled acetoxygroups (two) in the derivative.

An alternate procedure for purifying tetrahydroaldos-terone triacetate used the previously described chromato-graphic sequence (8) except that an alumina column wassubstituted for Celite in the final step. The triacetate wasapplied to a 10 g column of neutral alumina, prepared asdescribed (11, 15) in ligroin: benzene (4: 1) and thecolumn developed with 25 ml of this solvent. The elutingsolvent was changed to benzene, and 5-ml fractions werecollected automatically. Peak concentrations of the tri-acetate usually appeared in fractions 16-20. Specific ac-tivity was calculated from the 8H/"C ratio of the 14Cpeak fraction to minimize the error of isotope effect,provided the criteria (11) for radiochemical purity weremet. Tetrahydroaldosterone could also be isolated as theetiolactone derivative by direct periodic acid oxidativecleavage (11) of the pool of urine and bath fluid, fol-lowed by saponification and relactonization. Because thedoubly labeled derivative was a monoacetate, the sensi-tivity of the procedure was somewhat less.

RESULTS

Rate of excretion of labeled aldosterone andmetabolites. Fig. 1 shows the rate of appearanceof radioactivity in urine and bath fluid after theinjection of 10 uc of aldosterone-3H in four frogs(shaded area) compared to data obtained in hu-man subjects ( 11 ). The frogs continued to excreteappreciable amounts of radioactivity over thefirst 3 days. Since there was negligible incrementfrom the 4th to the 5th day, a 5 day collection pe-riod was chosen for the subsequent secretory rate

EXCRETION

5DAYS

FIGURE 1 Cumulative rate of excretion of radioactivity(expressed as per cent of dose) into urine and bathfluid in four bullfrogs after the injection of 10 sc ofaldosterone-'H into the dorsal lymph space. Comparativedata in human subjects are from reference 11.

determinations. The cumulative 5 day excretionwas 65-78%o of the dose.

Pattern of radioactive metabolites. 5-day poolsof urine and bath fluid were analyzed for free, glu-curonidase-hydrolyzable, and pH 1-hydrolyzableradioactivity in each of four animals. Table Ishows that a mean of 427% of injected radioactivityremained in the aqueous phase after removal ofthe three fractions. This material could not berendered extractable by solvolysis (16), incuba-tion with sulfatase, or oxidation with periodicacid (11).

The free fraction contained the largest portionof extractable radioactivity. Chromatographic anal-ysis of this fraction showed that a major compo-nent was an isomer of tetrahydroaldosterone. A

TABLE IPattern of Radioactive Metabolites of Aldosterone

in the Bullfrog

Per cent of injected aldosterone-3H (10 /c) excretedin fraction

Aqueous Glucuroni-Bullfrog (nonextract- dase hy- pH 1 Totalweight able) Free drolysis hydrolysis recovered

g

220 43.5 13.3 11.1 1.5 69.4235 39.1 21.5 14.2 2.0 76.8440 35.5 19.5 8.0 1.0 64.0530 49.0 19.4 7.2 0.85 76.5

Mean 41.6 18.4 10.1 1.4 71.5

Metabolism and Rate of Secretion of Aldosterone in the Bullfrog 2525

TAP WATERI SALINE ]

FROG 280gr~~~~ WAK

.'..-........':.'.:.:.-,. ...:::: :::: :::::-:::::::

:-::-::--::--:::::::::: :-:.:;::.-:

tI2 :.:.;::......:.:.::.....

.....::.. .:..:.:--ak :-!-..............0 2 3 4

DAYS

SALINE TAP WATER

FROG 163g:

::::1{s-- q

*-. l.':.--'1

::--:.'13

,: :4...:: .:;:. .*

:- 1.:: :3

-:: :1'-

." "' i.':i

35.3

- * INTA KE

8 100 2 4 6DAYS

FIGURE 2 Effect of a change in bath fluid composition on the sodium concen-

tration of catheterized frog bladder urine.

minor component of the free fraction, generallyless than 10%o, consisted of unmetabolized aldos-terone. Tetrahydroaldosterone was also excretedin conjugated form. It was a major componentof the extractable radioactivity liberated by incu-bation with mammalian glucuronidase. For thesubsequent isolation and analysis of the tetrahydrometabolite the combined free and glucuronide frac-tion was used.

Although the pH 1-hydrolyzable conjugate ofaldosterone, a probable 18-glucuronide (17), ac-

counts for approximately 10%o of the secretedhormone in man (18), this metabolite was a neg-

ligible excretory product in the frog. Hydrolysis atpH 1 yielded a mean of 1.4% of the dose. Pooledextracts of this fraction were analyzed by the ad-dition and reisolation of aldosterone-14C. The3H/14C ratio of purified product indicated thatless than 0.06% of injected aldosterone was in thisform.

Effect of environmental sodium on frog's uri-

FROG 235gSALINE I TAP WATER |

19

nary sodium concentration and aldosterone secre-

tory rate. Fig. 2 shows the effect of a changein bath fluid composition on the sodium concen-

tration of catheterized bladder urine. The changefrom tap water to saline resulted in a prompt in-crease in bladder urinary sodium concentration inthe 280 g frog to values equal to (and in otherexperiments slightly exceeding) that of the ex-

ternal environment. The adaptation from saline totap water shown for a 163 g frog was associatedwith a fall in urinary sodium concentration to a

value of 0.26 meq/liter after 10 days.Fig. 3 correlates the changes in urinary sodium

concentration observed in Fig. 2 with the measure-

ment of aldosterone secretory rate after each ani-mal had come into equilibrium with its environ-ment. The very low urinary sodium concentrationsobserved when the frogs were kept in the tap wa-

ter bath were associated with the secretion of 11and 19 ug of aldosterone per kg per day. Con-versely, the high urinary sodium concentrations

FROG 295 g

TAP WATERI SALINE20T

ALDOSTERONEi 'lSECRETION10g/. FIGURE 3 Correlation of the changes

per DAY 33 l2.2 in urine sodium concentration (Fig.

o | |1ll 1 2) with the measurement of frogaldosterone secretory rate in response

60- r---------INTAKE to changes in bath fluid composition.No 028

Meq/Iiter 4 | ||0.22 027 0.75 Q61

0i a--- INDINTAKE----------Y

DAYS

2526 S. Ulick and E. Feinholtz

60-

URINENo 40-

Meq/Iliter

20-

----O-i

I

TABLE I IEffect of Angiotensin, ACTH, and Dexamethasone

on Aldosterone Secretion in Bullfrogs

Aldosterone secretory rate, ;pg/kg per day

Bath. . NaCl NaCi,60 meq/ 60 meq/ Tap Tap Tap Tap

liter liter water water water water

Dexa-Angio- Angio- meth-

Bullfrog Ri/kg tensin, tensin, ACTH, asone,weight per day 0 40 jug 0 40gg 1.0 U 140 pg

g220 3.6 4.5 22 19 64235 4.0 3.8 16 21 37440 4.6 3.9 20 22 53530 5.9 4.9 19 14 47230 10.9 2.3260 10.4 1.9

ACTH, adrenocorticotropic hormone; Armour bovine ACTH, valine-5-angiotensin amide (Hypertensin-Ciba).* Hormones were administered every 12 hr for 6 days beginning 1 daybefore the determination of the aldosterone secretory rate.

of the frogs kept in hypotonic saline were associ-ated with lowered secretion of 2.2 and 3.3 ,ug ofaldosterone per kg per day. This 5- to 6-folddifference between the sodium-depleted and so-dium-repleted states is comparable to that ob-served in mammals. Thus an environment simu-lating the frog's natural habitat was associatedwith relative hypersecretion of aldosterone. Hypo-tonic saline suppressed the secretory rate to therange of the mean basal rate (2 ,ug/kg per day)in man determined by a similar method (11).

Effect of angiotensin, ACTH, and dexametha-sone on aldosterone secretion. Angiotensin wasadministered to each of four animals both in tapwater and in a hypotonic saline environment.Table II shows that the angiotensin-treated ani-mals did not secrete significantly different amountsof aldosterone from the control values. The animalsin the tap water bath which did not respond toangiotensin were then given ACTH. There was a1.8-3.4-fold increase in aldosterone secretion inthe four animals, indicating that they were notalready maximally stimulated. This evidence forthe role of pituitary factors was extended by theadministration of ACTH-suppressive doses ofdexamethasone. In two experiments, the elevatedaldosterone secretory rates associated with a tapwater environment were decreased to approxi-mately one-fifth of the control value by dexametha-sone.

DISCUSSION

Application of the indirect isotope technique forthe measurement of secretory rates required es-sentially complete excretion of radioactivity as-sociated with the metabolite chosen for measure-ment. Completeness of excretion of the metabolite,3,e,5,8-tetrahydroaldosterone, as judged by therate of excretion of total radioactivity, required5-day collection periods. This relatively slow rateof excretion was probably a reflection of the hy-pometabolism of the poikilothermic state. The5-day collection periods limited the application ofthe technique in the bullfrog to chronic experi-ments, but this limitation was offset by the pre-cision and convenience of the method which al-lowed repeated measurements to be made in theconscious animal serving as its own control andavoided the introduction of uncontrollable vari-ables such as surgery, blood loss, and adrenalblood flow.

Secretion of aldosterone by the bullfrog, sug-gested by the identification of the steroid in inter-renal incubates (2), was established in vivo by theisotope dilution technique used in the presentstudy. The observed reciprocal relationship be-tween sodium intake and urinary sodium concen-tration on the one hand and the rate of secretionof aldosterone on the other agrees with the find-ings in man (8), dog (19), sheep (20), and rat(21). Earlier observations which anticipiated thisrelationship showed that a measure of hormonallystimulated sodium transport, the isolated skinshort circuit current, was increased when frogswere kept in distilled water (22, 23). A suppres-sive effect of a saline environment on plasma al-dosterone has been shown in Bufo marinus (24).A saline infusion failed to decrease plasma aldos-terone in a pithed preparation of Rana cate-sheiana (10). Blood loss and dexamethasonetreatment may account for the conflicting resultin the latter report (10).

Earlier observations demonstrated the role ofthe pituitary in maintaining adrenocortical func-tion in aneuran amphibians, particularly duringperiods of increased metabolic activity (5).Specific effects of ACTHon the secretion of elec-trolyte-active hormone were suggested by the aug-mentation of skin short circuit current in ACTH-treated frogs (6). In vitro stimulation of aldos-terone synthesis in bullfrog interrenal incubates

Metabolism and Rate of Secretion of Aldosterone in the Bullfrog 2527

by the addition of mammalian ACTH and offrog pituitary extract was shown by Carstensen,Burgers, and Li (2). The present data confirmstimulation of aldosterone secretion in vivo bymammalian ACTH. The observed decrease in al-dosterone secretion with ACTH-suppressive dosesof dexamethasone suggest that the frog's endoge-nous ACTH supports aldosterone secretion invivo. Although intramuscular ACTHdid not sig-nificantly increase plasma aldosterone in a bled,pithed preparation of Rana catesbeiana, an intra-venous infusion of the peptide did increase theplasma level in dexamethasone-treated animals(10).

The existence of extrapituitary mechanisms forregulating electrolyte-active hormone in the frogwas suggested by observations after hypophysec-tomy that adrenocortical cell atrophy was incom-plete (25) and that frogs kept in distilled watercontinued to excrete urine of very low sodium con-centration (7) and showed increased skin shortcircuit current after an initial postoperative de-pression (6). Evidence for a role of the renin-angiotensin system in Rana catesbeiana was ob-tained by Johnston, Davis, Wright, and Howards(10) who found that frog renin increased theconcentration of aldosterone in postcaval plasma,although its steroidogenic effect was less than thatof ACTH. The negative results with valine-5-angiotensin amide in the present study suggestthat the homologous angiotensin peptide may berequired. The steroidogenic potency of syntheticangiotensin is variable in different mammalianspecies. Its stimulatory effect is short-lived in thesheep (20) and variable in the rat (21, 26), wheredose (27) and mode of administration (28) maydetermine its effectiveness.

The present study demonstrates secretion ofaldosterone in the bullfrog and reciprocal changesin that secretion in response to changes in the sa-linity of the environment. Aldosterone secretionwas strongly stimulated by mammalian ACTHand suppressed by dexamethasone. The methoddescribed makes the bullfrog a suitable experi-mental subject for controlled physiologic studiesof the regulation of the adrenocortical secretion.Several questions not answered by the availabledata require further investigation. Although bothpituitary and extrapituitary trophic mechanismsexist in the bullfrog, their integration in vivo and

relative participation as mediators of the sodium orvolume depletion stimulus is not known. Anotherquestion is whether anuran interrenal, althoughwithout zonation histologically, exhibits the char-acteristics of function zonation of the mammalianadrenal cortex, namely, the independent regula-tion of the secretion of electrolyte-active and ofcarbohydrate-active steroids. Corticosterone is se-creted by the bullfrog (10), but it is not clearwhether the steroid functions as a glucocorticoidin this species or is released from the interrenalas an aldosterone biogenetic precursor. Frogrenin increased the plasma level of both steroids(10) in Rana catesbeiana, but a saline environ-ment decreased the peripheral plasma level of al-dosterone but not that of corticosterone in Bufomarinus (24).

ACKNOWLEDGMENTS

This work was supported by U. S. Public Health ServiceGrant AM10641-02.

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Metabolism and Rate of Secretion of Aldosterone in the Bullfrog 2529