Hypophysiotropic and neuromodulatory regulation of adrenocorticotropin in the human fetal pituitary gland

(1)

Hypophysiotropic and neuromodulatory

regulation of adrenocorticotropin in the human

fetal pituitary gland.

Z Blumenfeld, R B Jaffe

J Clin Invest.

1986;

78(1)

:288-294.

https://doi.org/10.1172/JCI112563

.

Synthetic human corticotropin-releasing factor (hCRF) stimulated ACTH secretion by

human fetal pituitaries in superfusion and dispersed human fetal pituitary cells cultured on

an extracellular matrix in static incubation from 14 to 23 wk gestational age. The action of

hCRF in vitro was potentiated by arginine vasopressin (AVP) at all ages studied.

8-Br-cAMP induced a response similar to hCRF. The AVP effect on ACTH was synergistic with

both CRF and 8-Br-cAMP. hCRF-mediated secretion of ACTH was noncompetitively

inhibited by 24-h pretreatment, or by 3-h concomitant treatment, with dexamethasone.

Neither oxytocin, catecholamines, prostaglandins, nor indomethacin exerted significant

effects on ACTH secretion, either alone or in combination with hCRF or AVP during the

gestational ages studied. These results support a physiologic role for CRF in the regulation

of secretion by corticotropic cells as early as 14 wk gestation, by which time corticotropes

and ability to secrete ACTH have been demonstrated.

Research Article

Find the latest version:

http://jci.me/112563/pdf

(2)

Hypophysiotropic and Neuromodulatory Regulation

of Adrenocorticotropin in

the

Human Fetal Pituitary Gland

Zeev Blumenfeld and Robert B. Jaffe

ReproductiveEndocrinologyCenter, Departmentof Obstetrics,

Gynecology

and Reproductive Sciences, Universityof California,San

Francisco, California 94143

Abstract

Synthetic human corticotropin-releasing factor (hCRF) stimu-lated ACTH secretionby humanfetalpituitaries in superfusion and dispersed human fetal pituitary cells culturedon an

extra-cellular matrix in static incubation from 14to23 wk gestational

age. The action ofhCRF in vitro was potentiated byarginine

vasopressin (AVP)at allages studied. 8-Br-cAMP induced a responsesimilartohCRF. The AVP effectonACTHwas

syn-ergistic with both CRF and 8-Br-cAMP. hCRF-mediated

secre-tionof ACT7Hwasnoncompetitively inhibited by 24-h pretreat-ment, or by 3-h concomitant treatment, withdexamethasone.

Neither oxytocin, catecholamines, prostaglandins, nor

indo-methacin exerted significant effectsonACTH secretion, either

aloneorincombination with hCRForAVPduring the gestational

agesstudied.

These resultssupportaphysiologic role for CRF in the

reg-ulation ofsecretion by corticotropic cellsasearlyas14 wk

ges-tation, by which time corticotropes and abilitytosecreteAC'TH havebeen demonstrated.

Introduction

Adenohypophyseal secretion ofACTHcanbeelicited by intrinsic

andextrinsic stimuli.Many studies have been performed

con-cerning the physiologic, anatomic, andpharmacologicaspects

ofthe ACTHregulatorysystem(1-6). These studiessupportthe

conceptofacomplex systemsubservedbymultiple factors of hypothalamic, neurohypophyseal, and peripheral origins (1). The constellation of putative regulatory factors presently associated with the control network includescorticotropin-releasing factor (CRF)' (7), arginine vasopressin (AVP) (8), epinephrine (9),

and,

possibly, oxytocin (10). Thepresenceof each of these factors in hypophyseal portal plasma of therathas beenreported (1, 1

1-17), and high-affinity receptors for CRF (18), AVP (19), and

This workwaspresented inpart atthe AnnualMeeting oftheSociety

forGynecologic Investigation, Phoenix, AZ, 1985;andatthe 67th Annual

Meeting of the Endocrine Society, Baltimore,MD, 1985.

Dr.Blumenfeld'spresentaddress isDepartmentof Obstetrics and

Gynecology, Rambam Medical Center, Haifa, Israel. Address

corre-spondencetoDr.Jaffe.

Receivedforpublication 18 November 1985.

1.Abbreviations usedinthispaper:AVP,arginine vasopressin; CMF,

Hanks' calcium andmagnesium-free buffer, CRF,corticotropin-releasing factor; ECM, extracellular matrix; M199/EBSS, medium 199/Earle's

balancedsaltsolution; PGs, prostaglandins; PGE2, prostaglandin E2.

epinephrine (20) have been identified in adenohypophyseal tis-sue. A variety of in vitro and in vivo studies suggest that the action of CRF may be modulated by glucocorticoids (21-28), catecholamines (23, 29), neurohypophyseal peptides (21, 23, 30-32), prostaglandins (33), and other substances.

Therefore, we assessed the ability of human fetal pituitary cells torespond to a variety of substances which have been re-ported to regulate ACTH secretion in adult animals. Others have suggested that the human fetal pituitarycannotrespondtoCRF until after 20 wkgestation (34, 35). However,wehaveidentified human fetal corticotropic cells by the 12th wk of pregnancy, using immunocytochemical techniques(36); others have dem-onstrated theability of human fetalpituitaries to secrete ACTH by the end of the first trimester (37). Therefore we sought to ascertain whether the human fetal

pituitary

could respond to CRF and AVPbefore 20 wkgestation. Todo this, we performed bothsuperfusion and static incubation studies.

Methods

33 humanfetuses at 14-23 wk ofgestationwereobtained within 2 h of

therapeutictermination of pregnancybyevacuation after laminaria-in-duced dilatation.

Superfusion.Inthefirsteightcases, the fetalpituitarywasremoved andplacedinice-cold medium199/Earle'sbalanced salt solution(M199/

EBSS) and transportedtothelaboratory.Eachpituitarywasdivided into sixfragmentstoincreasesurface area,placedinatissue chamber

con-tainingsuperfusion medium,andsubmergedina37°Cwaterbath. The

superfusion systemwas modified from that describedby Kraicer and Chow(38).Thesuperfusionchamberwascomposedofan0-ring Pyrex joint (G9235-1/11,Corning 6780, AmericanScientificProducts,McGaw Park, IL) withaninternal diameterof5mm.The

pituitary

fragments

wereplacedon a0.5-mm thickscinteredglassdisc filter fitted witha

rubbergasket and held between the Pyrexjoints byametalclamp.The

superfusionmediumwas

M199/EBSS

(prepared

by

the cell culturefacility

of theUniversity ofCalifornia, San Francisco [UCSF]) consistingof 0.1% bovineserumalbumin(BSA,fractionV,98-99%albumin; Sigma

ChemicalCo.,St.Louis, MO),20X10- M

bacitracin,

10-'

Mascorbate,

25 mM Hepesbuffer, and saturatedwith 95%

02

and 5%

CO2.

The mediumwaspumpedfroma37°Creservoirbya

peristaltic

pump

(Man-ostatCorp.,NewYork,NY)at a rateof 0.3

ml/min

through

a

four-way

valve(SRV-4; Pharmacia FineChemicals,Piscataway, NJ).This system

permittedinstantchangingof the medium inflow into the chamber with minimal pressurechanges.AllconnectingtubingwasPE

160,

i.d.,

0.045 in. (Clay Adams,Parsippany, NJ)and theconnectors werestainless steel

(SmallPartsInc.,Miami, FL).Theeffluent from the tissue chamberwas

collectedonice insequential10-minfractions,

using

afraction collector (model 328; Isco, Inc.,Lincoln, NE).Theeffluentwasdivided into three

aliquotsandstoredat-70°Cuntil assay.

SynthetichumanCRFwaspurchased from Peninsula Laboratories (Belmont,CA), dissolved in 10-3NHCIinnormalsaline,andslowly

injectedinto thesuperfusionsystem(firstthreesuperfusions).Inthenext

fivesuperfusions, the CRFwasdiluted inthesuperfusion medium within

5minandequilibrated with

O2/CO2

in areservoiridenticaltothe medium reservoir. It wasthensuperfusedas a 10-min square-wavethroughthe

peristalticpump,usingthefour-wayvalvetominimize pressurechanges.

J.Clin. Invest.

(3)

AVP(Bachem, Torrance, CA) wasdissolved in normal saline (pH 4.5)and thendiluted in medium. KCI was dissolved in superfusion me-diumataconcentrationof60 mM and administered as a 10-min square-wave atthe endof eachsuperfusion.

Thepurity of CRF and AVP wasconfirmed by high performance liquid chromatography. CRF was chromatographed on a C- 18 column using aflow rate of 1 ml/min and a linear gradient over 40 min from 100% 0.05 M NaH2PO4 to 40% acetonitrile in the phosphate buffer. AVP was analyzed on a C- 18 column using the method of O'Hare and Nice (39).

8-Br-cAMP, used inbothsuperfusions and incubations, was obtained fromSigma Chemical Co.

Preparationofdispersedcellsand staticincubations.Dexamethasone, norepinephrine, isoproterenol, prostaglandin E, indomethacin, and oxy-tocin wereobtained from Sigma Chemical Co. Dulbecco's Modified Ea-gle'sMedium H-16; M199/EBSS; Hanks' calcium (0.9%) magnesium-free (CMF) buffer; fetal bovine serum; and saline with 0.1% trypsin (0.05%) and versine(0.02%); weresupplied by the cell culture facility at UCSF. All media contained 100

gg/ml

penicillin, 100 Mg/ml strepto-mycin, 2 MMglutamine, and 25mMHepesbuffer. Fibroblast growth factor waspurifiedfrom bovine brains accordingtothemethodof Gos-podarowiczetal.(40). Tissue culture disheswerepurchasedfrom Falcon Labware, Becton, Dickinson & Co., Oxnard, CA. All cultures were maintainedat37°C inaCH/Pincubator (Forma Scientific, Div. of

Mal-linckrodt,Marietta, OH) with 95%02-5%

CO2-ExtracellularmatriLx (ECM).The use ofan ECM derived from bovine

corneal endothelial cells for maintenance of human pituitarycells in culture has beenpreviouslydescribed (41,42).TheECM produced by bovine cornealendothelium hasnowbeen usedextensivelyfortheculture ofvarious normal andneoplastic cells (43). Recently, theuseof this matrixprovidedasuccessfulmeansfor culturing human pituitary

ade-nomacells, which adherepoorlytoplastic(41, 42). Theuseof ECMcan

alsocircumvent the need for various attachment factors inserum (43-45). Cultureof bovine corneal endothelial cells and the production of ECM wereperformedaccordingtothe methodofGospodarowiczet al.

(46).Inbrief,for matrixproduction,stock culturesofcorneal endothelium weresplitwithsaline with0.1% trypsin and versine, and the corneal endothelial cells (3X

I04'/ml)

wereplatedin 24-well plates with Dulbecco's modified Eagle's mediumcontaining 15%fetal bovine serum, 5% (wt/ vol) dextran T-40, and antibioticsasdescribed above. Fibroblast growth factor (100 ng/ml)wasadded threetimes during the first week. After reaching confluence (within 6-7 d), the cultureswereincubated for

7-10additional days,during which time the matrixwas deposited. The cellswereremovedwitharapidwash in 20mMammoniumhydroxide.

Thematrixwasrinsed twice with phosphate-buffered saline pH 7.4,and

the platescontaining phosphate-bufferedsaline and antibioticswerestored

at40C.

Pituitarydispersion. Eachof the 25 human fetalpituitariesusedfor static incubations(14-23wkgestation)wasremovedwithin2 hof preg-nancytermination and placed in Hanks' CMF bufferat room

temper-ature.Fourtermrhesusfetal pituitaries, utilized for comparison,were

obtained after hysterotomy. The tissuewasminced in freshHanks'CMF,

and thefragmentsweretransferredto a15-mlconical tube andcentrifuged

at300 g for 1 min. Thebufferwasaspirated and5 mlofHanks'CMF

containing0.5%collagenase andatotalof 50

Mg

DNAasewereadded. Thiswasincubatedat 37°C for 1 hina Dubnoff metabolicshaking

incubator (Precision Scientific 66722; American Scientific Products, McGaw Park, IL),afterwhichanadditional50

Mg

DNAase wereadded and thetissuewasmechanically dispersedinSml Hanks' CMFby trit-uration witha5 mlpipette. Any remaining fibrous tissuefragmentswere

allowedtosettle andwerethenremoved. The cellsuspensionwas

cen-trifugedat150 g for5min,and thecellpelletwasresuspended inM199/ EBSScontaining 10% FBS and platedat adensity of7.5-10X 104cells

per ECM-coatedwell.

Incubation. After 3-4d inculture, thecells, bythis timefirmly

at-tached to theECM,werewashed andpreincubatedwith serum-free

me-diumcontaining0.1%BSA.Aftera90-minpreincubation period, the cells werewashedagainand secretagogueswereadded in M199/EBSS

containing 0.1% BSA,penicillin/streptomycin, Hepes buffer, and glu-tamine; 1 ml per well. Eachtreatment wasperformed in quadruplicate

orquintuplicate wells. Aftera3-hincubation with either medium

(con-trol), 10 nMCRF, 10or100 nMAVP, or acombination of these

se-cretagogues, the medium wascollected in coldpolystyrene Eppendorf tubes,aliquoted,andkeptat-70°C until assay.

After eachincubation,serum-containing mediumwasadded tothe cells, whichwereincubated for 24-48 h. Afterwards the cellswerewashed andpreincubated with serum-free medium,asdescribed above. After preincubation andafter 24-48hincubation, the mediawerecollected and assayedfor ACTHtoverifythatthebaseline concentrationwasthe

samein allof the wells andthatthe numberofACTH-secretingcells in

each wellwascomparable. Therewas nodifference between the increase of ACTH secretionafter exposuretoCRF,AVP,orbothon day3-5 (firstexperiment)orin subsequentexperiments with thesamecells.

After theexperiments performed with each fetalpituitary,thecells were detachedusing STV solution andcounted,usingaCoultercounter

(Coulter ElectronicsInc.,Hialeah, FL).

ACTHradioimmunoassay.The ACTHradioimmunoassay (47)

em-ployed rabbit anti-ACTH(1-24) serum,generouslyprovided byDr.S. Hane,Metabolic ResearchUnit,UCSF. ACTH(I1-39)(Calbiochem,La

Jolla,CA)wasusedfor the standardcurve. The ACTHtracesupplied

byDr. Hane wasiodinatedusingamodification ofthe method of Hunter and Greenwood(48).

Aftera48-h incubationof samples with antiserum and trace, sepa-ration of bound from free hormonewasaccomplished by addition of goat anti-rabbit y-globulin.Thecross-reactivitywithalpha melanocyte-stimulatinghormonewas<0.1%, and theintraassayandinterassay vari-ationswere<7.5 and<10%,respectively.Theassaysensitivitywas1 pg ACTH/tube. The sample volumewas20-100Ml/tube.Thefinal antiserum concentrationwas 1:75,000.

Statisticalanalysis. Comparisonsweremadeusingone-wayanalysis

of variance (Newman-Keuls test)(49)for eachexperiment. Similar

ex-perimentswereanalyzedusinglinearregressionandleast squaresmeans

analysis of variance with Bonferroni correction formultiple

compari-sons(50).

Results

Theaddition of CRFtofetal anteriorpituitary cells in culture

or fragments in

superfusion

significantly

increased ACTH

se-cretioninto the medium

(Figs.

1 and

2).

Superfusion

ofthe

eight

14-23-wk-old human fetal

pituitaries

resulted in a

significant increase

in ACTH in the

effluent

after CRFadministrationatall ages studied (four less

than,

and four greaterthan, 20wks) (Fig. 2AandB). Theability of the tissue

to secrete ACTHwasconfirmed

by

administration of 60 mM

KCIattheend

of

each

superfusion.

AVP atconcentrations of

l0-'

and 10-8 M also increased

CL

1,000-7E

CONTROL CRF AVP CRF+AVP 10 nM 100nM

Figure 1. MeanACTH secretion bydispersedhumanfetalpituitary

cellsfrom 12fetuses after3-h exposuretoCRF(10nM),AVP(100

nM),or acombination ofthese secretagogues. Least square

(4)

CRF

10 nM

n

CRF+AVP KCI

60 mM

n

200

C,I)

'E

I

a 150

100

50

0 & - --4rl

I

0.01 0.1 1.0 10 100

log [CRF] (nM)

Figure3.ACTH response tovarious concentrations of CRF in static

incubationofdispersedhumanfetal pituitary cells from a 22-wk-old

fetus.Mean±SD.

-_

(Fig. 4). The combination of 3 mM 8-Br-cAMP with AVP

brought aboutasynergistic effect onACTH secretion (Fig. 4).

Nosucheffectwasdetectedwhendispersed pituitary cellswere

60 120 180 exposed to a combination of CRF and 8-Br-cAMP.

M I N U T E S No correlation was found between gestational age and the extentof increase in ACTH secretion by the human fetal pituitary

Vehicle CRF CRF CRF 8-Br-cAMP KCI cells (between 15 and 23 wk) after exposure to CRF alone or

InM 10 nM 10 nM 3 mM 60 mM CRF + AVP. Further, inexperiments with fetalpituitarycells

Fl n n n n from four term rhesus monkeys treated similarly, there was no

significant difference between the

increase

in ACTH inresponse toCRF aloneorCRF+ AVPand thatobserved in the human fetal pituitarycells(datanotshown).

Oxytocin didnotelicitany

significant

changein ACTH se-cretion at concentrations

ranging

from 0.2 to20nM (range of physiologic concentration insystemicand portal circulation in

baseline and stressconditions) (1).Neither did the combination ofoxytocin and CRF or AVP elicit any significant change in

ACTHsecretion from that

achieved

by CRFor AVPalone.

Dexamethasone inhibition ofACTH secretion. Pretreatment of the

_pituitary

cells for 24 h with dexamethasone

(20 nM)

sig-60 120 180

MINUTE S

240 300

800

Figure2.(A) ACTHresponsetoCRF, AVP,acombination of these

secretagogues, andKCIinsuperfusionof 14-wkoldhuman fetal

pitui-tary. (B) ACTHresponsetoCRF, 8-Br-cAMP, and KCIin

superfu-sionofpituitary froman 18-wk-old human fetus.

ACTHsecretionby humanfetalpituitarycells, butwasless

po-tentthanCRF. However, the combination of CRF and 10-7or

10-8 M AVP was synergistic and induced significantly higher

ACTH secretion than that induced by eithersecretagoguealone (Fig. 1). The 50% effective concentration of CRF for ACTH secretionwas0.5-1 nM in five different experimentsatfetalages

rangingfrom 17 to23 wk. A representative example is shown in Fig. 3. The intrinsic activity (secretory response inducedat

maximally effective concentrations) of CRFwassimilartothat

ofthe cAMP derivative, 8-Br-cAMP. Stimulation with 3 mM 8-Br-cAMP in static incubations of dispersed cells (Fig. 4)orin

superfusion (Fig. 2) brought aboutasignificant increase in ACTH

secretion. However, 8-Br-cAMP was an effective secretagogue

forACTHsecretion onlyinconcentrations higherthan l0-3M

cL.

I)

600

400

200

0

CONTROL AVP 8BrcAMP AVP+ 100 nM 3 mM 8BrcAMP

CRF 10nM

Figure4.Mean ACTHsecretionby dispersedhumanfetalpituitary

after 3-hexposuretoAVP, CRF,and 8-Br-cAMP administeredsingly

orincombination(n= _5)._Least_squaremeans±SEM.***P <0.001, **P<0.01, *P<0.05 from control.

A

2,000

AVP

0.1

_gM

n

1,500

1,000

C

500

B

2,400 1

2,000

1600

1,200

V)

i

UL

Soo

(5)

nificantly reduced the amount of ACTH secreted by the cells exposed to medium alone. Subsequent 3-h exposure of the cells to

CRF (10 nM)

or

8-Br-cAMP (3

mM) elicited significantly less

ACTH secretion

in the dexamethasone-pretreated wells than the wellswhich were not pretreated with dexamethasone but were exposed to CRF or 8-Br-cAMP (n=5)(Fig. 5). ACTH secretion

in

the control wells exposed to medium alone wasnot signifi-cantly

different

from secretion in the wells exposed to M 199 for 3 h that had been

previously

exposed to 24-h dexamethasone

(n

=

5)

(Fig. 5).

Inanother series of experiments, incubation of fetal pituitary

cells

with dexamethasone for 3 h didnotinduce significantly less

secretion of

ACTH than in control incubations. However, 3-h

incubation of

the cells

with

dexamethasone and CRF induced

significantly

lower ACTH secretion than that induced by 3-h exposureto

CRF alone (n

=

5)

(Fig. 6).

Theeffects of exposure

to

dexamethasone with CRF,

AVP, or other secretagogueswere

completely reversed

24 h

after removal of

the

steroid

by

multiple

medium

changes.

Interaction between CRF and catecholamines. The

a-

or

,B-adrenergic agonists, norepinephrine,

or

isoproterenol

(

10-6

M),

did

notresult in

significant

change

in ACTH secretion

(n =5). There was no change in ACTH

secretion after

a 3-h exposure

to

catecholamines

alone or toacombination of

catecholamines

andCRF or AVP,

compared

with CRForAVPalone

(n

=

5).

Interaction between CRF andprostaglandin

or

indomethacin.

Exposure

of fetal pituitary

cells to

prostaglandin

E1 (10-6 M; n

=

4)

or to

prostaglandin E2

(PGE2)

(10-6

M;

n =

3)

did not

result in

any

significant change

in ACTH secretion

compared

with

that

of control cells, either

aloneorin combination

with

CRF

orAVP.

Similarly,

3-h

exposuretothe

prostaglandin

syn-thetase

inhibitor, indomethacin (14

,M),

didnotelicit any

sig-nificant change in ACTH secreted by

the

pituitary

cells

exposed

to

indomethacin

alone orin

combination

with CRFor

AVP,

compared with control

cells or

cells

exposed

for 3

h toCRF

orAVP.

Discussion

Synthetic

hCRF

canstimulate ACTH secretion

by

human fetal

pituitary cells, at least as early as 14 wk of fetal life. This is

360-270

90

CONTROL 8BrcAMP CRF 3 mM 10nM

Figure 5.MeanACTHsecretion by dispersed human fetal pituitary

cellsexposedtoCRF, 8-Br-cAMP, and control after24-hincubation inthepresence or absence of 20 nM dexamethasone (n=6). o,

M199;

.,

dexamethasone. Leastsquaremeans±SEM. ***P <0.001.**P <0.01.

m

If)

41C

EP

gL

300-

200-100 A

CONTROL AVP 100 nM CRF 10 nM

Figure 6. Mean ACTH secretion by dispersed human fetal pituitary cells

after

3-h exposure to CRV, AVP, or medium in the presence and absence of 20 nM dexamethasone (n=4). o,M199;m, dexametha-sone.Leastsquare means±SEM. ***P <0.001.

consonant

with

our

previous immunohistochemical

demon-stration of corticotropes in

the human

fetal

pituitary

by this

stage

of

gestation (36).

Thereason

for the

inability

of others

to

demonstrate

fetal

pituitary

ACTH

responsiveness until after 20

wk(34, 35) is not immediately apparent.

In human

fetal pituitaries,

as

in

rat adult

pituitaries (27),

AVP

exhibits

markedly lower potency than

CRF in stimulating

ACTH

secretion;

synergism

was observed between CRF and

physiologic concentrations of

AVP.

Our study demonstrated

that

glucocorticoids

can act

directly

on human

fetal

pituitary

cellsto

blunt the ACTH

responseto

CRF.

Maximal

inhibition of CRF-mediated ACTH secretion

wasobserved

in

rat

pituitaries after

severl

hours

of

pretreatment with dexamethasone (27), as

is typically

seen

with

other

tran-scriptionally mediated steroid effects. However,

theacute

inhi-bition of

ACTH

release, detected in

human

fetal

pituitary

cells

as

early

as3 h

after

incubation with dexamethasone

and

CRF,

suggests that

glucocorticoids

mayexert atleast part

of their

neg-ative feedback

effects by affecting

release

of stored ACTH

aswell

as

transcription.

The

similar

effects of CRF

and cAMP

derivatives

and the

ACTH-releasing actions of

agentsknown to elevate

intracellular

cAMP, suchas

phosphodiesterase inhibitors, cholera toxin,

and

forskolin,

have

suggested

an

involvement of

the

adenylate cyclase

system in

ACTH secretion (23, 27, 51, 52).

CRFwas

found

to

rapidly elevate intracellular

cAMP

levels in

rat

pituitary

cell

cultures, and

glucocorticoid

pretreatment

markedly inhibited

CRF-mediated increases

in

intracellular

cAMP

concentrations

(27, 28).

This

was thought to

indicate

that the

glucocorticoid

effect might

be

mediated by

altered cAMP levels. Our

observation

that

glucocorticoids

can

inhibit

ACTH

secretion

brought about

by

exogenous

8-Br-cAMP in

human

fetal

pituitary

cells suggests

that

glucocorticoids

also act, in part,

beyond

the

cAMP-gener-ating

step in

their

inhibition of CRF action.

The

synergistic effects of

AVPand CRF and ofAVPand 8-Br-cAMP,but not

of

CRF and 8-Br-cAMP, on ACTH secretion

by

human

fetal

pituitary

cellsareconsonant

with

the

hypothesis

that thereare

different

second messengers

involved

in the

cellular

actions

of

CRF and AVP

(53).

Whereas

CRF action

onACTH release is mediatedby the adenylate

cyclase-protein

kinase

path-way, AVPaction on ACTH release

is

modulatedbya cAMP-independent

mechanism

that appearsto be initiatedbya rise in

cytosolic

calcium

concentration (53).

(6)

synergistically with CRF in rat pituitary cells inonestudy (27). However, others havedescribed an antagonistic action of oxy-tocin onACTH secretion whenadministered with AVP alone

orwith AVP andCRF in adult women (54). Plotsky (1) has foundoxytocin concentrationsinthe range of

10-12

M in sys-temic blood and 10-9 Min theportalblood of adult rats. How-ever, none of the concentrationsofoxytocinused in our

exper-iments (0.2-20 nM) resulted in significant changeinACTH

se-cretion by human fetal pituitary cells, either alone or in

combination with CRF, AVP,orCRF +AVP. Thus,whether

oxytocin has any physiologic roleinACTHregulationremains

tobe

elucidated.

Itdoes not seemtoplayamajor role in human fetal life, eitheras asecretagogue or as an inhibitor ofACTH

secretion at the ages studied.

In contrast to their

effects

on adultanimal pituitary cells (27), neither a-nor

13-adrenergic

agonistic catecholamines sig-nificantly modified ACTH secretion by human fetal pituitary cells under the study

conditions

employed.

Prostaglandins (PGs) are known to exist in the anterior

pi-tuitary

gland, as are

specific PGE2 binding sites

(33). PGs are known tostimulate cAMP accumulation in anterior pituitary tissue (53, 55).

Buckingham

and Hodges

(56)

found an increase in the release of ACTH

from

adenohypophyseal segments in vitro in response to PGE2, and others (57) reported a slight

ele-vation

of

plasma

corticosterone

levels

after injection

of prosta-glandin

El

orprostaglandin

Fl,

into the anterior pituitary of

anesthetized, dexamethasone-pretreated

rats.Incontrast, no ef-fects of

prostaglandin

El

or

PGE2

onbasal ACTH release from rat

anterior pituitary

halves (58) or on basal ACTH and

,B-en-dorphin

release

from

rat

anterior pituitary

cell cultures(59) were observed.

PGE2

even

inhibited ACTH production

in anterior pituitary cell cultures after stimulation by a hypothalamic extract

with CRF-like

activity (59). More recently (33), PGE2 was found

tobe

formed locally, after

binding of

the

neurohormones,

and

to act as a

negative feedback-modulator of vasopressin

and CRF

activity

in the

pituitary

gland. Our

findings

that PGsor

indomethacin

alone or in

combination with

CRF or AVP do not

stimulate

or

inhibit

ACTH

secretion

are

similar

to

studies

inrats(58).Although PGs may havea

physiologic

role in

modulating

ACTH

secretion

in the adult,

this ability

may not

develop until

late in

intrauterine,

orearly in

extrauterine,

life. Wedid not find a

significant

correlation

between

fetal

age and theACTH responsetoCRFor toCRF+AVPbetween 14

and 23 wkof human

gestation,

norbetween the response of thesesecond

trimester

human

pituitary

cells and the response

of four

rhesusterm

fetal pituitary

cells.A

correlation

with age,

however,

hasbeen

found in sheep fetuses (59, 60)

at a

gestational

age

comparable

to28-34 wk in

humans,

astagenotamenable

to

testing

in the human

fetus.

Theontogenesis

of

the

corticotrophs

andtheir responseto

CRF,

AVP, and

glucocorticoids

occurs

before

the 14th wk of fetal life. The response to

catecholamines

and

prostaglandins

maydevelop later in

intra-

or

extrauterine

life.Inany case, the human

fetal

pituitary

hasthe

capacity

torespondto stressstimuli inuteroby the

beginning of

the second

trimester

of pregnancy.

As

previously

reported (1,2, 8, 15, 16,

21-26, 32),

the responses

to stressstimuli

consist mainly of

regulation

of ACTH secretion

by

CRF andAVPand

feedback by

adrenal

glucocorticoids.

Our

studies

in the human

fetus

demonstrate that the

capacity

forall

of these responses existsat orbefore 14wkof intrauterine life.

A drawback ofstatic incubation is the

inability

to assess

precisely the timecourseofchanges in hormonal secretion after

exposure to areleasing factororother modulator of secretion. The combination of superfusion and static incubationmaybe complimentary in understanding the complex interactions of variousmodulators of fetal pituitary hormone synthesis and

se-cretion.

Acknowledgments

Wearegratefulto Dr.RobertKuhnfor his assistance withhigh

perfor-manceliquidchromatographyand Dr. Mary C. Martin for her invaluable help inobtainingspecimens.

This workwassupported inpartby grantHD08478 from the National Institute of Child Health andHumanDevelopment.

Dr.Blumenfeld isaPostdoctoralFellow in Reproductive Endocri-nology andarecipient ofanAmericanPhysician Fellowship.

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