Effect of 17β-estradiol and testosterone on guanosine 3′, 5′-cyclic monophosphate in the rat adrenal cortex

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Pages 1727-1734

June 30, 1981

EZ?PECIOF 17B-ESTRADIOLANDTES'IOSTERD~ON 1<

GrJAN3SDJE

3’,

MONOPHOSPHATF IN THE

5’-CYCLJC

RAT FUXENALCOl-tI'EX Josette GUIILEWNI'~

Serge CXTILLENU'T

Faculte de Midecine Pitie-Salp&riere 75634

Received

April

PARIS

CEDEX

13

- FRANCE

1981

21,

The levels of guanosine 3', 5'-cyclic monophosphate (CGMP)were measured in the rat adrenal cortex after administration of a single dose of either 176-estradiol or testosterone. Young immature rats received 10 ug 17B-e&radio1 (females) or 100 ug testosterone (males). After testosterone administration, cCW levels progressively rose to about 150 per cent of the control values after 4-6 hrs, and remained elevated until at least 9 hr. Administration of 176-e&radio1 resulted in a similar increase in cZ%?, which began at 2 hr and persisted until 9 hr, reaching levels of about 180 per cent of the controls. Our data are further evidence of general effect of steroid horrmnes on &%? in their target tissues.

There is nm increasing evidence that hormonal steroids affect c%lP levels in their

respective

target

tissues. At first,

were shown to be able to induce a transitory

estrogens

rise in cCM? in the

rat uterus (1) as well as in the chicken oviduct

(2). Then, two other

steroid hormnes were found to orcduce a similar

effect

target

tissues : 1,25-dihydroxyvitamin

(3), and dexamethasone, a synthetic adrenal cortex

the steroid-induced

parallelled

D3 in the chick intestinal glucocorticosteroid,

mucosa

in the rat

(4). The mechanismof the &@ response to estrogens in

the uterus has been the mst

nuclear receptor

in tm distinct

thoroughly

studied.

increase in cc'rlp level

It has been shown that

is dependent on the cytosol-

system since (a) the time course of changes in c<;Mplevels

that of the steroid nuclear occupancy, and (b) the effect

on

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&NPlevelswas tested

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propxtionaltotheestrogenicpotencyofeach

(5). Moreover, the parallelism

knmn estrogenic effects

between the @

such as water Mition

ampound response and other

of the uterus suggest a

hormonal action. Since the existence of receptors for andmgens (6, 7) and for estrogens (8) hadbeendemxstrated toverifywhetherbothkinds increase in adrenccortical

in the ratadrenalgland,wedecided

of steroidhomneswere

able to induce an

c.QQ levels as we had previously

with dexamethasone in the sametissue

shown to occm

(4).

MATERIALsANDME!rHoDs Inmature Wistar rats weighing 55 g (104 males and 120 females) ore supplied by IFFA CREDO,Saint-C&main-sur-l'A.rbresle, France. They were housed in the laboratory for 3 days before being used. Meanwhile, they were fed dry rat pellets (UAR, Villeaoisson, France) and tap water ad libitum and kept at their adrenals quickly removed 20 + 2' C. They were killed by decapitation, and the fasciculata-reticularis zones isolated as previously described (4). Female rat uterine hornswere collected, freed frcm fatandkept on dry ice until lyotiilisation. The lyophylized tissues were homogenized by sonication and the cyclic nucleotides measured by a radioimmunological method (9, 10). Protein was detemined by the mthod of Imry et al. (11) with E%SA as standard. Homeadministration : Testostemne andl7B-estradiolwere dissolved in absolute ethanol and adninistered by im injection in the thigh (50 M/rat). Controlanirnalswere injectedwithethauolonlyandkilledatthe sane time as the corresponding home-injected groups. RESULTS Effect

of testcetercme Twodistinct

experimentswere

presented in Table 1. In the first

carriedout,

the results ofwhich

(short-tern)

experiment, oZ%' and cAW

were measmed before and 2, 4 and 6 hrs after (100 pg/rat).

A significant

administration

(P < 0.05 and P < 0.01) rise in cc;Mpwas observed

at 4 and 6 hrs, shming that testosterone A

testosterone

are

action lasted for a longer time.

second, long-term experiment was designed to account for the circadian

variations

incyclicn~leotidelevelswhichhavebeen~trated

rat adrenal cortex

(12). cCNPand cAMPwere measured at different

1728

inthe times after

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TABLE1 : CYCLIC NXLEIXILlELEVELSINTHE ADRBGGCORTEXOFYouNGIMWlVREM?GEPATS. EFF'EYXOFTES~ ADlINI~TICN EXP-1

Tim after 'injection

n

CxaP CAMP % ofunt.reatedcontrols mean -+ SE-I IEarl -+ SEM

hOWS

Untreated controls Testosterone

2 4 6

6

loo+10

loO+

6 6 6

103 + 10 141 T lsa 190 T- 24b

loo+11 loo + 17 95 z 5

ExPERIMTSr2

CXWP

8

CAMP

%of vehicle-injected oxkrols lTlxin+sm mean-+ SEX Testosterone

2 4 6 9 24

8 8 8 8 8

111 117 149 149 113

+ T T T +

11 13 lib 12b 10

92 124 132 62 88

+ 7 + T -T

11 16 9 gb 10

Testosterone (100 ug/rat) in 50 1.1ethanol was injected intra-muscularly et zero t&x. Experiment

1. &mrone-treated

(Student's t-test).

animals

were ampared

with

untreated

controls

E5qerimnt 2.At each tims point, each hormne-injected group was cxqared with a vehicle-injected one (Student"s t-test). a RO.05 b RO.01

the injection

of either

testosterone

or the vehicle

alone. The pattern of

changes in dXP is presented in Figure 1. It shows a progressive rise waking at 6 hr and returning Effect

to ccntrol

levels at 24 hr.

of 17&estradiol The results

of two distinct

Short-term eqerinrmt

experimants are sumarized

1 shows the effect

tissues, the adrmalcortexandthe before and 1, 2, 3 and 4 hrs after

of 17B-estradiol

uterus.

administration.

was different

1729

on two different

c@P andcAMPweremasured

17&estradiol

course of changes in cGlP concentration

in Table 2.

The tims

between the adrenals

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.v

2

4

6

9

hours

24

Figure 1. T~courseofchangesincCMPintheratadrenalcortexaftertheadministration of a single dose (100 pg/rat) of testosterone to young imnatumrnale rats. Ateach tim-pointkmmne-treated animalsware canparedwith vehicle-injected controls (Student's t-test). Significant increases (P cO.01) were observedat and 9 hr.5 after testos~ aclministration . Eachvaluerepresentsthemean-sEMof8subjects.

andthe uterus. at2

In the uterus the rise in cG4Pwa.s very t.ransient,peaking

hrandretmningtobasallevels

at4

hr,whereas

persisted up to 4 hr. Experiment 2 was therefore tern effects

of 17B-estradiol

on adrenocortical

in the adrenals it

designed to test the longc%BP. As explained above

(Table 1, experiment 2), each hormone-injected group was roved vehicle-injected variations

with a

one at the samehours in order to account for the circadian

and the effects

of the vehicle,

if any. The tima comse of changes

in cQ4P is presented in Figure 2. As it could be expected from the results of experimant 1, the effects

of 178-estradiol

on c@lP were more prolonged in the

adrenals than in the uterus.

DISCUSSION The pssibility effects

nucleotides

to mediate part or all of the

of steroid honmnes was hypothesized scme years ago and &VP was

the first brief

for cyclic

to be investigated.

rise in uterine

After

administration

CAMPwas described, similar

1730

of estrogens, an early to that observed in other

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: CYCLIC NKLKYTJDELEVFLSINTHEADRDlALCDRTEX ANDTHEUl'EFUJSOFYCUNGlMATUPE

FTimTAE RATS.

EFFECT OF 17B-ESTVADIOL ADXINISTRATI@l EXPE~PIMDJT 1

Adrenal

Tine after injection hours

n

c??e CM@ %ofuntreatedomtrols m3Il+sl31 Iwan + SE31

cortex

8

Untreated controls

100 + 9

Estradiol

83 132 145 189

+ i 5 -

9 3d 17b 22c

75 109 81 100

+ 7 : ;

8 9 5 9

uterus Untreated controls

8

100 + 10

Estradiol

83 170 123 98

EXPEPJl?ENT2

4 6 9 24

17Bestradiol

(10 pg/rat)

8 8 8 8

tissues

1. Hormne-treated t-test).

animals

was injected

ware cmpared

+ T 7 -

gc 6d lSc 10

124 119 82 106

intra-muscularly with

untreated

+ + 7 7

14 23 6 10

at zero tine. controls

each hormone-treated grow was ccqxred with t-test). The 2 hr tima Fint (see figure 2) vehicle-injected was available for -ison.

P
under

the

influence

experiments

shmed

to suppress

the estrogen-induced

cal

136 171 178 112

in 50 pl ethanol

Experiamt 2. At each time pint, a vehicle-injected one (Student's was anitted since no corresponding a b c d

21 23a 19 12

CQ-fP CAMP % of vehicle-injected controls aWl+SEX Il-E?Xl+tTEV

Estradiol

Experiment (Student's

+ + -f +

responses

(14,

that

15).

of peptide

propanolol,

Nevertheless,

homnes a B-adrenergic

CAMD respmse scma other

1731

(13).

Hmever,

blocking without

altering

mdifications

subsequent agent,

was able

other in the

physiologi@$W level

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Estrsdiol

0

Vehicle

.*

I 246

9

houra

24

F@LIE 2. Tlme~seofchangesincGMPintheratadrenalcortexaftertheadministration of a single dose (10 pg/rat) of 178-estradiol to young im mture fenalerats. Ateachtim-pointbxnme -trea~animalS~ m with vehicle-injected controls (Student's t-test). Significant increases vere observed at 4 hr (PC0.011, at 6 hr (P
have been described. A modest but significant

decrease was shcxm to occur in the

uterus between 2 and12 hrs afterestradioladministration hasbeenshcxm tooccurwithdexmethasone decreasewas neither

adrenals (4). Hmever, that

found in the chick intestinalmucosa

of 1 cx, 25-dihydxoxycholecalciferol generalized.

inthe

(1) : the samething

after

administration

(3) nor in the present work, and cannot be

Ontheotherhand,administrationofestrogen

to immtureor

ovariectcenized female rats hasbeen found to increase cQlp concentrations the uterus (1). That&@

responsewas characterizedbya

delayed (about1 hr)

progressive and prolonged rise.

Lateron,

response on the cytosol-nuclear

receptor system was established

other tissues inwhicha&NP

the dependency of the uterine cQIP

response tosteroidhomes

are kncxm to contain the corresponding hormone receptors of the uterine cG@ response have been shcmn to follm involved

in the transfer

in

of the cytosol-receptor

1732

has beendemmstrat~~3 (16, 17). The kinetics by about an hour those

amplex to the nucleus and

then to persist roughly as long as the steroid-receptor In the present study the estradiol-induced

(5), and all

nuclear occupancy (5).

cXXP rise was mre prolonged in the

Vol. 100. No. 4,1981

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adrenals than in the uterus. That phenamnon might therefore reflecting

be interpreted

a longer steroid nuclear cxxupancy. In addition,

obsemedafter

testosterone

intracellular

administration

nkatabolization

elucidated.

Recent findings

in the transitory

shming a direct

by steroids favor the first

and ventral

the consequenceofits

accumulation of &ZJP, either

prostate

hypothesis. Z-to 5-fold

activation

the stimulation

in rat liver,

of guanylate cyclase oxurred

(19). It

within

trating

that testosterone

adrenal function,

That discrepancy was determined in the thus

mechanisms.

series, one could expect dexamthasone, estradiol adrenal effects.

should be noted that

ha-rcgenization of the tissue,

If we consider the cSW respmse as the first

similar

muscle

on helxatic and uterine

steroid adninistration.

37,000 X g supernatant obtained after

skeletal

15 rain, whereas -in vivo

may be explained by the fact that the enzymatic activity

bypassing cmplex intracellular

of guanylate cyclase

kidney,

(18). E&radio1 had the sameeffect

&NP did not rise before 1 hr after

remains to be

Testosterone was found to enhance

guanylate cylase of female rats and guinea-pigs

biochemical event in a

and testosterone

to induce

Although many experimants have been performed demnsand estradiol

they have failed

confusing results are not surprising where these steroids are knm

and dexamethasone were able to influence

to give uneguivocal results

(20). These

with regard to the extra-adrenal

sites

to act.

The discovery of an adrenal cGQ response after and e&radio1

the delayed response

of synthesis or by suppression of degradation,

guanylate cyclase activity

as

into dihydrotestosterone.

The mechanisminvolved by stimulation

couldbe

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administration

of testosterone

should suggest a new approach to the role of that cyclic

tide in the adrenal and is new evidence that the alterations may be considered as a general cellular

in cc;Mp levels

response to steroid hormones.

1733

nucleo-

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1. Kuehl, F.A., Jr., Ham, E.A., Zanetti, M.E., Sanford, C.H., Nicol, S.E., and Goldberg, N.D. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1866-1870. 2. Schumacher, M., Seidel, I., and Strgtling, W.H. (1977) Biochem. Biophys. Rfs. coMnun. 74, 614-620. 3. Guillmant, J., and Guillemnt, S. (1980) Biochem. Bioohys. Res. Ccmmn, 93, 906-911. 4. Guillemnt, J., and Guillemnt, S. (1979) Biochem. Biophys. Res. Ccamun, 88, 163-169. 5. Flandroy, L., and Galand, P. (1979) I%l. Cell. Endccrinol. 13, 281-290. 6. Calandra, R.S., Purvis, K., Naess, O., Attramdal, A., Djoseland, O., and Hanson, V. (1978) J. Steroid Biochem. 9, 1009-1015. 7. Rifka, S.M., Cutler, G.B., Jr., Sauer, M.A., and lmiaux, D.L. (1978) Endocrinology 103, 1103-1110. 8. Cutler, G.B., Jr., Barnes, K.M., Sauer, M.A., and Loriaux, D.L. (1978) Endocrinolcqv, 102, 252-257. 9. Cailla, H-L., Racine-Weisbuch, MS., and Delaage, M.A. -(1973) Anal. Bicchem. 56, 394-407 10. Cailla, H.L., Vannier, C.J., and Delaage, M.A. (1976) Anal. Biochem. 70, 195-202. 11. Lc%rrlr, D.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chm. 193, 265-275. 12. Guillemnt, J., Reinberg, A. and Guillemnt, S. (1980) Acta Endocrinol. (KM) 95, 382-387. 1.3. szego, C.M., and Davis, J.S. (1967) Pi-cc. Natl. Acad. Sci. USA 58, 17111718. 14. Rosenfeld, MC,., and OWalley, B.M. (1970) Science 168, 253-255 15. Dupont-Mairesse, N., Van Sande, J., Rooryck, J., Fastrez-Boute, A., and Galand, P. (1974) J. Steroid Biochem. 5, 173-178. 16. Tsai, H.C., and Norman, A.W. (1973) J. Biol. Chem. 248, 5967-5975. 17. Loose, D.S., Do, Y.S., Chen, T.L., and Felcbxan, D. (1980) Endocrinology, 137-146. 18. Vesely, D.L. (1979) Proc. Natl. Acad. Sci. USA 76, 3491-3494. 19. Vesely, D.L., and Hill, D.E. (1980) Endocrinology 107, 2104-2109. 20. Kitay, J-1. (1968) in Functions of the Adrenal Cortex, p 775-811, North Holland, Amsterdam.

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