P oc. Na l. Acad. Sci. USA
Vol. 94, pp. 14132–14137, Decembe 1997
Physiology
Mul i- esponsi eness o single an e io pi ui a y cells o
hypo halamic- eleasing ho mones: A cellula basis o
pa adoxical sec e ion
(calcium
y
hy o opin- eleasing ho mone
y
g ow h ho mone
y
p olac in)
CARLOS VILLALOBOS*†,LUCI
´ANU
´N
˜EZ*†,L.STEPHEN FRAWLEY†,JAVIER GARCI
´A-SANCHO*‡,AND ANA SA
´NCHEZ*
*Ins i u o de Biologı´a y Gene´ ica Molecula (IBGM), Uni e sidad de Valladolid y Consejo Supe io de In es igaciones Cien ı´ icas, Depa amen o de Fisiologı´a y
Bioquı´mica, Facul ad de Medicina, 47005-Valladolid, Spain, and †Depa men o Cell Biology and Ana omy, Medical Uni e si y o Sou h Ca olina, 171 Ashley
A enue, Cha les on, SC 29425
Edi ed by S. M. McCann, Penning on Biomedical Resea ch Cen e , Ba on Rouge, LA, and app o ed Oc obe 14, 1997 ( ecei ed o e iew
Augus 1, 1997)
ABSTRACT The classic iew o hypo halamic egula ion
o an e io pi ui a y (AP) ho mone sec e ion holds ha
elease o each AP ho mone is con olled speci ically by a
co esponding hypo halamic- eleasing ho mone (HRH). In
his scena io, binding o a gi en HRH ( hy o opin-, g ow h
ho mone-, co ico opin-, and lu einizing ho mone- eleasing
ho mones) o speci ic ecep o s in i s a ge cell inc eases he
concen a ion o cy osolic Ca
21
([Ca
21
]
i
), he eby selec i ely
s imula ing he elease o he app op ia e ho mone. Howe e ,
‘‘pa adoxical’’ esponses o AP cells o he ou well-
es ablished HRHs ha e been obse ed epea edly wi h bo h in
i o and in i o sys ems, aising he possibili y o unc ional
o e lap be ween he di e en AP cell ypes. To explo e his
possibili y, we e alua ed he e ec s o HRHs on [Ca
21
]
i
in
single AP cells iden i ied immunocy ochemically by he ho -
mone hey s o ed. We ound ha each o he i e majo AP cell
ypes con ained disc e e subpopula ions ha we e able o
espond o se e al HRHs. The ela i e abundance o hese
mul i- esponsi e cells was 59% o lac o opes, 33% o hy-
o opes, and in he ange o 47–55% o gonado opes,
co ico opes, and soma o opes. Analysis o p olac in elease
om single li ing cells e ealed ha each o he ou HRHs
es ed we e able o induce ho mone elease om a disc e e
lac o ope subpopula ion, he size o which co esponded
closely o ha in which [Ca
21
]
i
changes we e induced by he
same sec e agogues. When iewed as a whole, ou di e se
unc ional measu emen s o mul i- esponsi eness sugges ha
hypo halamic con ol o pi ui a y unc ion is mo e compli-
ca ed han p e iously en isioned. Mo eo e , hey p o ide a
cellula basis o he so-called ‘‘pa adoxical’’ beha io o
pi ui a y cells o hypo halamic hypophysio opic agen s.
The pi ui a y gland is a majo neu oendoc ine modula o ha
egula es many pe iphe al glands and issues h ough he
sec e ion o an e io pi ui a y (AP) ho mones. These ho -
mones include p olac in (PRL), g ow h ho mone (GH), hy-
o opin (TSH), ad enoco ico opin, and he gonado opins
[ ollicle-s imula ing ho mone (FSH) and lu einizing ho mone
(LH)]. The sec e o y ac i i y o he gland is con olled, in u n,
by signals de i ed om he hypo halamus in he o m o
hypo halamic- eleasing ho mones (HRHs) such as hy o-
opin- eleasing ho mone (TRH), g ow h ho mone- eleasing
ho mone (GHRH), co ico opin- eleasing ho mone (CRH),
and gonado opin- eleasing ho mone (LHRH). The classic
iew o hypo halamic con ol o AP ho mone sec e ion holds
ha each majo HRH modula es he sec e ion o a single
pi ui a y ho mone (1), bu he e a e some no able excep ions
o his ule. Fo example, TRH is acknowledged o be a
physiological modula o o bo h TSH and PRL sec e ion, jus
as LHRH con ols he elease o bo h LH and FSH. In addi ion
o hese acknowledged excep ions, he e ha e been spo adic
epo s abou ‘‘pa adoxical’’ esponses o a ious HRHs. Such
obse a ions o AP ho mone elease elici ed by a nonco e-
sponding HRH de i e om a b oad spec um o s udies
in ol ing bo h no mal and pa hological AP issue e alua ed in
i o o in i o (2, 3). The easons unde lying hese e ec s a e
no known, al hough hey equen ly a e a ibu ed o he lack
o p ope con ol mechanisms in es ablished umo cell lines
o in cells de i ed om pa hological specimens.
Qui e ecen ly wo g oups (including ou own) ha e ob-
ained e idence o sugges ha ‘‘pa adoxical’’ esponses o
HRHs may be as much he ule as he excep ion. Mo e
speci ically, when a emp s we e made o cha ac e ize AP cell
pheno ypes on he basis o hei cy osolic calcium concen a-
ion ([Ca
21
]
i
) esponses o HRH s imula ion, many cells we e
ound o espond o wo o mo e o hese agen s (4, 5). These
indings no only cas doub s on he u ili y o [Ca
21
]
i
esponses
as an index o AP cell pheno ype, bu also aised a couple o
in e es ing ques ions. Is mul i- esponsi eness es ic ed o
ho mone-p oducing cells o a gi en ype? Do such pleio opic
[Ca
21
]
i
esponses ex end o ho mone elease by li ing AP
cells? In he p esen s udy we a emp ed o add ess hese
in iguing ques ions by combining calcium imaging and immu-
nocy ochemis y wi h he aim o co ela ing mul i- esponsi e-
ness o cellula pheno ype o by quan i ying HRH-induced
ho mone elease om single li ing cells h ough he use o
e e se hemoly ic plaque assay.
MATERIALS AND METHODS
AP cells we e ob ained om 8- o 10-week-old male Wis a
a s and a ached o poly-L-lysine-coa ed glass co e slips as
desc ibed p e iously (4). The cells hen we e ei he used
di ec ly o [Ca
21
]
i
measu emen s o cul u ed in RPMI 1640
medium (Sigma) con aining 10% e al cal se um (GIBCO)
and an ibio ics o 2–3 days. Measu emen s o [Ca
21
]
i
we e
pe o med in cells loaded wi h u a-2 (6) by digi al-imaging
The publica ion cos s o his a icle we e de ayed in pa by page cha ge
paymen . This a icle mus he e o e be he eby ma ked ‘‘ad e isemen ’’ in
acco dance wi h 18 U.S.C. §1734 solely o indica e his ac .
© 1997 by The Na ional Academy o Sciences 0027-8424y97y9414132-6$2.00y0
PNAS is a ailable online a h p:yywww.pnas.o g.
This pape was submi ed di ec ly (T ack II) o he P oceedings o ice.
Abb e ia ions: AP, an e io pi ui a y; [Ca
21
]
i
, cy osolic calcium con-
cen a ion; HRH, hypo halamic- eleasing ho mone; PRL, p olac in;
GH, g ow h ho mone; TSH, hy o opin; FSH, ollicle-s imula ing
ho mone; LH, lu einizing ho mone; TRH, TSH- eleasing ho mone;
GHRH, GH- eleasing ho mone; CRH, co ico opin- eleasing ho -
mone; LHRH, lu einizing ho mone- eleasing ho mone o gonado-
opin- eleasing ho mone.
‡To whom ep in eques s should be add essed. e-mail: jgsancho@
cpd.u a.es.
14132
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luo escence mic oscopy. All expe imen s we e conduc ed a
37°C. The cells we e unde con inuous pe usion wi h ei he
con ol o es solu ions a 2–3 mlymin. This a e o pe usion
allowed .95% exchange o he medium wi hin 5–10 s. The
s anda d con ol solu ion had he ollowing composi ion: 145
mM NaCl, 5 mM KCl, 1 mM CaCl
2
, 1 mM MgCl
2
,10mM
glucose, 10 mM Na
1
-Hepes, pH 7.4. Tes solu ions con ained
in addi ion ei he GHRH, LHRH, o CRH a 4 nM o TRH
a 100 nM. The cells we e epi-illumina ed al e na ely a 340
and 380 nm, and ligh emi ed abo e 520 nm was eco ded by
using a Magical Image P ocesso (Applied Imaging, Newcas le,
U.K.). Pixel-by-pixel a ios o consecu i e ames ob ained a
340 and 380 nm exci a ion we e p oduced, and [Ca
21
]
i
was
es ima ed om hese a ios by compa ison wi h u a-2 s an-
da ds. Addi ional de ails can be ound elsewhe e (4, 7, 8).
A he end o he [Ca
21
]
i
measu emen s, AP cells in he
mic oscopic ield we e yped by immuno luo escence by using
an ibodies aised agains one o he pi ui a y ho mones, ei he
as desc ibed p e iously (9) o in acco dance wi h he abb e-
ia ed p o ocol ha ollows. Cells we e ixed wi h 4% pa a-
o maldehyde in PBS o 10 min, pe meabilized wi h 0.3%
T i on X-100 in he abo e solu ion o 3 min, and washed wi h
PBS o 5 min. Then 10% goa se um in PBS was added. A e
5 min he an ibody agains one o he AP ho mones labeled
wi h O egon g een 488 (see below) was added and he
incuba ion con inued o 15–30 min. A e washing, a luo es-
cence image was cap u ed (exci a ion, 490 nm; emission, .510
nm) wi h he image p ocesso . Occasionally, his p ocess was
epea ed o a second luo escen an ibody agains ano he AP
ho mone. Then nuclei we e s ained wi h Hoechs 33258 (0.5
m
gyml, 10 min), and ano he luo escence image was acqui ed
(exci a ion, 340 nm; emission, .420 nm). The image om he
luo escen ly s ained nuclei acili a ed de ini ion o cellula
bounda ies in cells ha we e physically close.
Single-cell measu emen s o PRL elease we e pe o med
by e e se hemoly ic plaque assay in cells p epa ed om male
a s (250 g, Ha lan Sp ague–Dawley, Hol zman; Madison, WI)
as desc ibed p e iously (10). B ie ly, AP cells cul u ed o 2
days on plas ic Pe i dishes (5 310
5
cellsyml) in DMEM
(GIBCO) supplemen ed wi h 0.1% BSA, an ibio ics, and 10%
e al bo ine se um we e esuspended by a b ie ypsiniza ion,
insed wi h DMEM-0.1% BSA, mixed wi h p o ein-A-coa ed
o ine e y h ocy es, and in used in o Cunningham chambe s a
an AP cell densi y o 30 cellsy
m
l. A e a 1-h a achmen
pe iod (37°C; 95% ai -5% CO
2
), chambe s we e insed wi h
assay medium (DMEM-0.1% BSA), looded wi h 150
m
lo
assay medium con aining PRL an ise um (1:80) alone o in
combina ion wi h di e en ea men s ( h ee chambe s each),
and incuba ed o he speci ied imes (1 o 2 h ). Guinea pig
complemen (1:80, 50 min) was added o de elop he plaques.
A e ixa ion o e nigh wi h 2% glu a aldehyde and s aining
wi h oluidine blue, he pe cen age o all pi ui a y cells ha
o med PRL plaques was de e mined a e a 1-h an ibody
incuba ion conduc ed in he absence (con ol) o p esence o
100 nM o each HRH. A leas 300 AP cells we e coun ed in
each chambe . Fo measu emen s o plaque a eas, cells we e
incuba ed wi h an ibody o 2 h unde he same condi ions as
be o e. A his ime he pe cen age o cells o ming PRL
plaques was maximum as de e mined in p elimina y expe i-
men s. The measu emen s o plaque a eas we e made by use
o a calib a ed ocula e icule. The a eas o a leas 50
plaquesyslide we e quan i ied o de i e he mean plaque a ea
and equency dis ibu ions o plaque sizes. The da a om
plaque assays a e he esul o i e (pe cen age o PRL
sec e o s) and ou (plaque a eas) independen expe imen s in
which he e we e h ee eplica es o each ea men . A
wo-way ANOVA was used o assess s a is ical signi icance,
and ea men means we e compa ed by use o he Bonn e -
oni’s Mul iple Compa isons Tes . Di e ences we e consid-
e ed o be signi ican a P,0.05.
An ise a agains a PRL ( abbi , AFP425–10
291
),
b
-TSH
( abbi , AFP1274789), GH (monkey, AFP4115),
b
-FSH (guin-
ea pig, AFP85GP9691BFSHB), and an i-human ad enoco i-
co opin ( abbi , AFP39013082) we e gene ous gi s om he
Na ional Ho mone and Pi ui a y P og am, he Na ional In-
s i u e o Diabe es and Diges i e and Kidney Diseases, he
Na ional Ins i u e o Child Heal h and Human De elopmen ,
and he U.S. Depa men o Ag icul u e, Rock ille, MD.
Fluo escein-labeled an i- abbi , an i-guinea pig, o an i-
monkey IgGs, he hypo halamic- eleasing ho mones (GHRH,
TRH, LHRH, and CRH) and he LHRH an agonis [D-
Phe2,6,P o3]-LHRH all we e ob ained om Sigma. Fu a-
2yAM and O egon g een-iso hiocyana e we e pu chased om
Molecula P obes.
RESULTS
Many AP Cells Respond o Mo e Than One HRH. In he
expe imen illus a ed by ep esen a i e example in Fig. 1, AP
cells we e consecu i ely s imula ed wi h each o he ou
HRHs. Cells A and B esponded wi h an inc ease in [Ca
21
]i o
only one o he eleasing ho mones (TRH and GHRH, e-
spec i ely). In con as , cell C esponded o h ee o he HRHs
and cell D o all ou . The cumula i e esul s ob ained in 25
simila expe imen s wi h AP cells cul u ed o 2–3 days (2,133
cells analyzed in o al) a e summa ized in he las column o
FIG. 1. E ec s o HRHs on [Ca
21
]
i
in ou ep esen a i e single
cells. Cells we e s imula ed wi h solu ions con aining 4 nM CRH,
LHRH, o GHRH, and 100 nM TRH, as shown.
Physiology: Villalobos e al. P oc. Na l. Acad. Sci. USA 94 (1997) 14133
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Table 1. Shown he e a e he anges o di e en combina ions
o induced esponses ob ained in h ee se s o 6–10 expe i-
men s ca ied ou o e a 2-yea pe iod. Consis en wi h ou
p e ious obse a ions (4), hese cells (sec e ing ho mone
pheno ypes no de e mined) dis ibu ed in o h ee subpopu-
la ions o oughly equal size. One subg oup was comp ised o
cells ha did no espond o any o he HRHs (non esponsi e
cells in Table 1) and accoun ed o 19–31% o all AP cells.
In e es ingly, mos o he cells in his subg oup (85–95%)
exhibi ed a [Ca
21
]i ise a e depola iza ion wi h high K
1
(50
mM) solu ions (da a no shown). A second subg oup (36–50%
o all cells) esponded selec i ely o jus one o he HRHs
(mono- esponsi e cells). The inal g oup (29–31%) was made
up o cells ha esponded o mo e han one HRH (mul i-
esponsi e cells) and included some (3–7%) ha we e espon-
si e o all ou . The equencies ound o each o he possible
combina ions o HRH- esponsi e cells also a e de ailed in
Table 1. As es ablished in ou ea lie s udy (4), cells esponding
o mo e han one HRH almos always esponded o TRH.
O e all, mos AP cells we e ound o be esponsi e o TRH
(53 62%), ollowed by GHRH (36 61%), CRH (15 62%),
and LHRH (14 61%) (mean 6SE o he h ee se s o
expe imen s e e ed o abo e).
Mul i-Responsi eness Exis s wi hin All Ho mone-
Con aining Cell Types. The e a e a leas wo plausible
explana ions o mul i- esponsi eness o HRHs. In one sce-
na io, such appa en indisc imina ion migh be es ic ed o
ho mone-sec e ing cells o a gi en ype (e.g., lac o opes).
Al e na i ely, se e al AP cell ypes migh con ibu e o his
phenomenon. In an a emp o dis inguish be ween hese
possibili ies, we immunocy ochemically iden i ied AP cell
ypes a he end o he [Ca
21
]
i
measu emen s, and Fig. 2
illus a es he way in which his was achie ed. Fi s , he cells
we e s imula ed sequen ially wi h he ou HRHs as be o e
(Fig. 1). Fig. 2Ashows h ee images o [Ca
21
]
i
, coded in g ay
le els, aken ei he a es (CONT.) o du ing s imula ion wi h
GHRH o TRH. A he end o he [Ca
21
]
i
measu emen s, he
cells we e ixed and s ained wi h a luo escen an ibody agains
one o he AP ho mones. In ou ine expe imen s, only one
an ibody was used, bu in some ins ances wo we e used
successi ely (Fig. 2B). As shown by ep esen a i e example in
Fig. 2, one o he cells con ained GH and esponded only o
GHRH, whe eas he cell con aining PRL esponded jus o
TRH.
FIG. 2. P o ocol o combining calcium imaging wi h immunocy-
ochemical iden i ica ion o he cell s udied. A single mic oscopic ield
(30 330
m
m) con aining h ee single AP cells is shown. (A) Images
we e cap u ed du ing he measu emen o [Ca
21
]
i
, ei he be o e
s imula ion (REST) o du ing s imula ion wi h ei he 4 nM GHRH o
100 nM TRH. Highe [Ca
21
]
i
appea s b igh e in he g ay image. (B)
Images we e aken du ing he immunocy ochemical iden i ica ion
p o ocol, a e s aining wi h an i-GH-O egon g een (aGH) o a e
s aining wi h an i-PRL-O egon g een (aPRL). NUCLEI, nuclea
localiza ion a e s aining wi h Hoesch 33258. See Ma e ials and
Me hods o de ails.
Table 1. Responses o ho mone-con aining cell ypes o HRHs
Cell ypes % PRL cells % GH cells % ACTH cells % FSH cells % TSH cells % All* cells
Non esponsi e cells 13 [3] 13 [9] 14 19 9 19–31 [22]
SMono- esponsi e cells 28 [66] 31 [61] 33 33 57 36–50 [51]
CRH 0 [0] 0 [1] 17 3 0 3–7 [3]
LHRH 3 [0] 3 [4] 5 19 9 3 [4]
GHRH 0 [1] 11 [54] 0 0 0 9–14 [16]
TRH 25 [65] 17 [2] 11 11 48 17–30 [28]
SMul i esponsi e cells 59 [30] 55 [30] 54 47 33 29–31 [27]
CRH-LHRH 0 [0] 0 [0] 2 0 0 ,1 [0]
CRH-GHRH 2 [0] 0 [3] 6 0 0 0–2 [1]
CRH-TRH 2 [8] 1 [0] 14 0 6 2–3 [3]
LHRH-GHRH 0 [0] 3 [7] 0 3 0 0–1 [2]
LHRH-TRH 6 [7] 9 [0] 0 19 18 1–5 [5]
GHRH-TRH 14 [7] 23 [13] 6 0 0 12–16 [8]
CRH-LHRH-GHRH 0 [0] 0 [3] 0 0 0 ,1 [1]
CRH-LHRH-TRH 5 [2] ,1 [0] 5 0 3 0–3 [2]
CRH-GHRH-TRH 2 [3] 0 [,1] 11 0 0 2–3 [1]
LHRH-GHRH-TRH 7 [1] 6 [2] 2 8 3 1–3 [2]
All ou 23 [2] 8 [1] 8 17 3 3–7 [2]
P opo ional abundance o [Ca
21
]
i
esponses o HRHs in di e en AP cell ypes, iden i ied pos ac o by he ho mone hey s o ed. The di e en
HRH esponsi eness in e ed om he [Ca
21
]
i
a e shown in he i s column. Non esponsi e cells indica es no esponse o any o he HRHs.
Numbe s co espond o he pe cen ages wi hin each cell ype ob ained in se s o 3–10 di e en expe imen s in AP cells cul u ed o 2–3 days o
cells s o ing PRL (P, n5128), GH (n579), ad enoco ico opin (ACTH cells, n564), FSH (n542), o TSH (n533). The alues in he
las column (All cells) co espond o he anges ob ained in h ee se s o 6–10 expe imen s, each one (n5591, 682 and 960 cells) pe o med a
di e en imes du ing a 2-yea pe iod. Cell ype was no iden i ied in hese expe imen s. The numbe s in b acke s co espond o esul s ob ained
in 5–7 expe imen s using eshly p epa ed AP cells o PRL cells (n5236), GH cells (n5200) o all cells wi hou iden i ica ion (n5761).
*Ho mone-sec e ing pheno ype no iden i ied.
14134 Physiology: Villalobos e al. P oc. Na l. Acad. Sci. USA 94 (1997)
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Fig. 3 compa es he pe cen ages o pheno ypically iden i ied
AP cells ha esponded o each o he ou HRHs. On he
whole, each cell ype esponded be e o i s classical, co e-
sponding HRH han i did o he o he sec e agogues, wi h he
excep ion o soma o opes whe e a highe ac ion esponded
o TRH han o GHRH. Su p isingly, mul i- esponsi eness o
HRHs was exhibi ed by all AP cell ypes, and TRH along wi h
LHRH was he mos likely o induce [Ca
21
]
i
esponses ega d-
less o cell pheno ype. Among he AP cell ypes, hy o opes
exhibi ed he mos s ingen deg ee o selec i i y o HRHs in
ha good esponses we e ob ained only wi h TRH and LHRH.
The i s i e columns in Table 1 p o ide addi ional de ails
ega ding he combina ions o esponses exhibi ed by he
di e en cell ypes. Cells esponding o mo e han one HRH
(mul i- esponsi e cells in Table 1) accoun ed o oughly hal
(47–59%) o all lac o opes, soma o opes, co ico opes, and
gonado opes and a smalle p opo ion o hy o opes (33%).
The pe cen age o cells esponding o all ou HRHs was
pa icula ly high o lac o opes (23%), negligible o hy o-
opes (3%), and in e media e o he o he cell ypes (8–
17%). The associa ion o GHRH and TRH esponsi eness
(pa icula ly he a ian s GHRH-TRH and LHRH-GHRH-
TRH) was qui e common in soma o opes and lac o opes.
Thy o opes, on he o he hand, we e he only cells in which
he dominan esponse was es ic ed o a single HRH (TRH,
48% o he TSH cells).
Mul i-Responsi eness Also Exis s wi h Respec o Ho mone
Release. Up o his poin , we used changes o [Ca
21
]
i
as a
biological end-poin wi h which o assess he e ec s o HRHs.
Un o una ely, such changes may no necessa ily culmina e in
a ele an physiologic esponse. In pu sui o a mo e dis al
biological end-poin , we decided o e alua e he e ec s o
HRHs on ho mone elease om single, li ing cells by using he
e e se hemoly ic plaque assay (10). To his end, we ocused
on a single, ep esen a i e cell ype, he lac o ope, because i
exhibi ed he highes deg ee o HRH mul i- esponsi eness in
e ms o [Ca
21
]
i
changes. Acco dingly, we subjec ed AP cells
om male a s o a plaque assay o PRL in he absence o
p esence o a ious HRHs, and hen measu ed wo ele an
pa ame e s. The i s o hese was he pe cen age o all AP
cells ha o med a plaque a e a 1-h incuba ion wi h PRL
an ibody. The a ionale he e is ha a h eshold amoun o PRL
mus be eleased o induce plaque o ma ion. In he case o
lac o opes, a 2-h an ibody incuba ion is equi ed o all cells
ha elease PRL o induce plaque o ma ion. Thus, only a
ac ion o PRL plaques can be de ec ed a e 1 h unde basal
condi ions, bu he addi ion o HRHs, which accele a e he
a e o PRL elease, also can accele a e he a e o plaque
de elopmen . In sho , his s a egy can be used as a e y
sensi i e assay o iden i ying egula o y sec e agogues, pa -
icula ly i such agen s a ec only a subpopula ion o cells. The
o he pa ame e measu ed was ha o plaque a ea (a e a 2-h
an ibody incuba ion), which p o ides a eliable index o he
cumula i e amoun o ho mone eleased (10).
The e ec s o a ious HRHs on he a e o PRL plaque
de elopmen is illus a ed in Fig. 4A. Clea ly, all he HRHs had
a quan i iable e ec on PRL elease, bu as an icipa ed om
he [Ca
21
]
i
esul s, TRH e oked he g ea es esponse. To
con i m ha he ‘‘pa adoxical’’ e ec o a leas one HRH was
media ed by i s co esponding ecep o , we a emp ed o block
he ac ion o LHRH (100 nM) by concu en exposu e o he
LHRH an agonis [D-Phe2,6,P o3]-LHRH (300 nM). As
shown in Fig. 4B, he an agonis diminished he a e o
LHRH-induced PRL elease by 60%, a alue ha compa es
a o ably wi h he 50% educ ion o LH elease obse ed (11)
when he an agonis was used a an iden ical mola a io (3:1).
Nei he he LHRH an agonis no soma os a in (a nonspeci ic
pep ide con ol) had any e ec on he basal sec e ion o PRL
when ea ed alone (Fig. 4B). Finally, he pe cen o plaque-
o ming cells signi ican ly was inc eased by all he ou HRHs
and e ec o he di e en HRHs did no change on a ying he
FIG. 3. Responses o he AP cell ypes o each o he HRHs es ed.
Cells we e conside ed esponsi e o a gi en HRH when an inc emen
o [Ca
21
]
i
exceeding 50 nM was ob ained in expe imen s simila o
hose illus a ed in Fig. 1. The numbe o cells analyzed (3–10
expe imen s o each cell ype) was: PRL, 128; GH, 79; ad enoco i-
co opin (ACTH), 64; FSH, 42; TRH, 33.
FIG. 4. E ec s o HRHs on PRL sec e ion as quan i ied by e e se
hemoly ic plaque assay. Cells we e incuba ed concu en ly wi h PRL
an ibody and he s a ed HRHs (100 nM) o 60 min. (see Ma e ials and
Me hods). (A) E ec s o HRHs on he pe cen age o plaque- o ming
cells. Da a a e he mean 6SEM o i e independen expe imen s. (B)
Inhibi ion o he LHRH e ec by he an agonis [D-Phe2,6,P o3]-
LHRH (1A) a 300 nM; 1SOM, e ec s o soma os a in a 100 nM.
Da a a e ep esen a i e o wo simila expe imen s. Di e ences we e
conside ed signi ican (p)a P,0.05.
FIG. 5. E ec s o cell dilu ion on he sec e ion o PRL induced by
di e en HRHs. AP cells we e in used in o he Cunningham chambe s
a ou di e en dilu ions (7.5–60 cellsy
m
l, as shown). A e ages o
h ee eplica es wi hin a single expe imen a e shown. All he alues
ob ained wi h he ou di e en HRHs di e ed signi ican ly (P,0.05)
om he con ol alues. P o iles a e ep esen a i e o esul s ob ained
in wo simila expe imen s. O he de ails a e as in Fig. 4.
Physiology: Villalobos e al. P oc. Na l. Acad. Sci. USA 94 (1997) 14135
Downloaded a UNIVERSIDAD VALLADOLID. BIBLIOTECA on Janua y 19, 2021
dilu ion o AP cells (Fig. 5). These esul s sugges ha he PRL
sec e ion induced by HRHs was no because o pa ac ine
e ec s o subs ances eleased by cells o he han lac o opes,
which would be expec ed o dec ease wi h cell dilu ion, bu o
di ec ac ion o HRHs on lac o opes. Fig. 6 shows he e ec s
o he ou HRHs on he equency dis ibu ion o plaque
a eas. Each o he ou HRHs e oked an inc ease in he
ela i e amoun o PRL elease by single cells, as e idenced by
he shi o he igh o plaque a eas in he plo s. In all cases,
excep o TRH, he equency dis ibu ion e ealed a e
s imula ion was bimodal, indica ing ha only a subpopula ion
o lac o opes was s imula ed o elease PRL by HRHs o he
han TRH.
Mul i-Responsi e Cells A e No An A i ac o Cul u e. I
could be a gued ha AP cells main ained in p ima y cul u e
o 2–3 days may unde go changes ha ende hem poo ly
ep esen a i e o hei physiologic condi ion in he in ac
animal. In conside a ion o his possibili y, we epea ed se-
lec ed expe imen s wi h eshly dispe sed cells, which we e
allowed o a ach o poly-L-lysine-coa ed co e slips o only 1
h be o e use. P opo ional abundance alues o mono- and
mul i- esponsi e AP cells om esh dispe sions a e p esen ed
wi hin b acke s in Table 1. No e ha he pe cen ages ound in
he whole popula ion (Table 1, ex eme igh column) we e
e y simila o hose abo e o cells cul u ed o 2–3 days. In
lac o opes and soma o opes, howe e , he subpopula ions o
cells esponding o only one HRH was g ea e in he eshly
p epa ed cells han in hei coun e pa s. This inc ease in
mono- esponsi e cells was balanced by a dec eases in bo h cells
non esponsi e o HRHs and he mul i- esponsi e cells, no jus
one o he o he .
DISCUSSION
Acco ding o he classic iew o hypo halamic con ol o AP
ho mone sec e ion, each HRH modula es he elease o a
single AP ho mone. I ollows logically, he e o e, ha each
AP cell ype should be esponsi e o jus one HRH. Howe e ,
ou indings indica e clea ly ha his is no he case. In ac ,
when HRH s imula ion was assessed by [Ca
21
]
i
dynamics, only
one- hi d o he AP cells we e ac ually esponsi e o jus one
HRH, which was no necessa ily hei classical, co esponding
HRH. Ano he hi d o he cells did no espond o any HRH,
e en hough s imula ion wi h high-K
1
solu ions e ealed ha
hey we e exci able cells, and immunocy ochemis y demon-
s a ed ha many o hem con ained AP ho mones. Mo e
su p isingly, he emaining hi d o AP cells esponded o mo e
han one o all HRHs. Immunocy ochemical iden i ica ion o
he s o ed ho mone e ealed ha mul i- esponsi e cells we e
no es ic ed o any pa icula cell ype. Ins ead, a signi ican
popula ion o mul i- esponsi e cells we e p esen wi hin each
o he i e AP pheno ypes. I could be a gued ha HRHs migh
c oss eac nonspeci ically wi h se e al HRH ecep o s. How-
e e , he di e en sizes and chemical s uc u es o he ou
HRHs, as well as he low HRH concen a ions used in his
s udy, a e no consis en wi h his possibili y. I likewise could
be a gued ha mul i- esponsi eness andyo pa adoxical e-
sponses o HRHs de elop as an a i ac o p ima y cul u e, and
he e o e a e no e lec i e o he no mal, physiological si u-
a ion. We ha e shown, howe e , ha mul i- esponsi e cells
we e also p esen in eshly dispe sed AP p epa a ions, al-
hough hei equencies we e somewha lowe han in hose in
cul u es main ained o 2–3 days. The possibili y ha such
seemingly ‘‘inapp op ia e’’ esponses ac ually de i e om ac-
i a ion o ‘‘app op ia e’’ ecep o s is suppo ed by ecen
epo s ha oughly hal o he AP mRNA o he TRH
ecep o is localized o soma o opes (12) and ha he mRNA
encoding he ecep o o soma os a in ( he classic hypo ha-
lamic GH inhibi ing ac o ) is dis ibu ed among all i e majo
cell ypes o he AP (13). In addi ion, i has been epo ed
ecen ly ha 38% pe cen o soma o opes bind bio inyla ed
GnRH, demons a ing ha a ep esen a i e subpopula ion o
GH cells bea s GnRH ecep o s (14). When iewed as a whole,
ou esul s s ongly suppo he iew ha mul i- esponsi eness
o hypo halamic ho mones is a genuine p ope y o no mal AP
cells, and no jus an anomaly es ic ed o pa hophysiological
condi ions.
Ou p esen da a demons a e ha a a ie y o HRHs can
inc ease [Ca
21
]
i
wi hin a gi en ho mone-sec e ing cell ype.
Inasmuch as [Ca
21
]
i
is belie ed o media e he sec e o y
ac ions o HRHs (15–17), i seemed easonable o p opose ha
mul iple HRHs should con e ge on a single AP cell o
modula e he elease o each ho mone. We decided o es his
line o easoning on lac o opes, he AP cell ype ha exhibi ed
he la ges ac ion o mul i- esponsi e cells. By using he
e e se hemoly ic plaque assay, we ound ha all ou HRHs
inc eased bo h he a e and he cumula i e amoun o PRL
eleased om single, li ing lac o opes. Analysis o equency
dis ibu ions o plaque a eas e ealed ha TRH s imula ed
mos o he lac o opes o elease PRL, whe eas he o he
h ee HRHs induced PRL expo om jus selec subpopula-
ions o cells. Thus he e ec s o he ‘‘app op ia e’’ HRHs we e
mo e uni e sal han hose o he ‘‘inapp op ia e’’ coun e -
pa s, al hough he la e we e clea ly unequi ocal. As was he
case o [Ca
21
]
i
mobiliza ion, he e ec s o HRHs on PRL
sec e ion appea ed o be because o speci ic s imula ion o
co esponding HRH ecep o s on lac o opes, because hey
we e no ep oduced by pep ides o compa able sizes and
s uc u es and he e ec s o LHRH we e inhibi ed by a speci ic
LHRH an agonis (Fig. 4). I could be a gued ha pa ac ine
e ec s could con ibu e o s imula ion by nonco esponding
HRHs. Al hough his possibili y canno be absolu ely uled
ou , he esul s om he cell dilu ion expe imen s (Fig. 5)
sugges ha his mechanism does no play a majo ole. The
inding ha mul i- esponsi e cells we e no es ic ed o a gi en
ho mone-sec e ing cell ype sugges s ha sec e ion o AP
FIG. 6. E ec s o HRHs on equency dis ibu ion o plaque a eas.
The sizes o plaques we e quan i ied a e a 2-h an ibody incuba ion
wi h he di e en HRHs (100 nM). The equency dis ibu ions o
con ol, uns imula ed cells (open ci cles and do ed line) and cells
challenged wi h one o he ou HRHs (closed ci cles and con inuous
line) a e compa ed. A e ages o h ee eplica es wi hin a single
expe imen a e shown. P o iles a e ep esen a i e o esul s ob ained
in ou simila expe imen s.
14136 Physiology: Villalobos e al. P oc. Na l. Acad. Sci. USA 94 (1997)
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ho mones o he han PRL also may be con olled in a mul i-
ac o ial manne .
The esul s o he p esen s udy aise an in e es ing and
p o oca i e ques ion: I mul i- esponsi eness o AP cells is as
much he ule as he excep ion, hen why ha e o he s no
epo ed i p e iously? In ac , he e ha e been nume ous
epo s en i ely consis en wi h his possibili y, bu in e p e-
a ion o hose indings appea s o ha e been in luenced by he
con en ional wisdom o he ime. Fo example, i has been
epo ed ha GHRH is capable o elici ing sec e ion o LH
and FSH om a an e io pi ui a ies in i o (18), and ha
TRH is almos as po en as LHRH in e oking LH elease (19).
In addi ion, TRH has been p oposed o be a GH- eleasing
ac o (2) on he basis o he abili y o induce GH elease by
pi ui a y cells om a s and o he mammals (20). The e also
ha e been epo s ha LHRH can s imula e sec e ion o GH
and TSH om AP cell cul u es (21) as well as PRL elease
om agg ega e AP cells (22). Repo s abou so-called ‘‘pa -
adoxical’’ esponses o HRHs a e likewise qui e common o
heal hy and in i m human subjec s (2), bu such obse a ions
a e no ully in e p e able, owing o he po en ial o indi ec
e ec s o HRHs adminis e ed in i o. In summa y, ou
indings ha sec e ion o a gi en AP ho mone can be egula ed
di ec ly by se e al HRHs a e no isola ed; he esul s o he
nume ous s udies by o he s suppo he same conclusion.
The mul i- esponsi e na u e o AP cells in i es specula ion
ha HRHs subse e egula o y oles ha anscend ho monal
syn hesis, elease, and mi o ic a es wi hin hei adi ional
a ge cells. One possibili y is ha in addi ion o egula ing he
acu e elease o i s co esponding ho mone, a gi en HRH
could exe a ch onic in luence on he p opo ion o cells ha
sec e e a di e en ho mone. In his manne , changes in
physiologic equi emen s o he p oduc o a pa icula cell
ype could be me wi hou a ne inc ease in he size o he AP
gland. An example o such pheno ypic plas ici y is he unc-
ional in e con e sion (i.e., ansdi e en ia ion) o mammo-
opes and soma o opes ha is induced by LHRH (see e . 23
o a e iew). A a ia ion on his heme is ha cells wi h
‘‘inapp op ia e’’ ecep o s migh comp ise a ese e popula-
ion, which, when ac i a ed by he ‘‘inapp op ia e’’ HRH, can
be ec ui ed o sec e e he co esponding ho mone. This
possibili y is p edica ed on he no ion ha es ablished AP cell
ypes con ain ace amoun s o ‘‘inapp op ia e’’ ho mones, and
epo s abou immunocy ochemically iden i ied gonado hy o-
opes and soma ogonado opes (14) along wi h co ico opes
ha co-s ain o LH, FSH, TSH, o PRL (24) a e ce ainly
consis en wi h his idea. A inal possibili y wo hy o consid-
e a ion is ha combina ions o wo o mo e HRHs migh
comp ise codes ha a e deciphe ed by a ge cells o e ec a
speci ic esponse. Such a modula ed esponse migh ake he
o m, o example, o he induc ion o mono-ho monal sec e-
ion by a bi-ho monal cell. The speci ic biological endpoin
no wi hs anding, i is clea ha hypo halamic con ol o AP
unc ion is conside ably mo e complica ed han p e iously
en isioned.
Financial suppo om he Spanish Di eccio´n Gene al de In es i-
gacio´n Cien ı´ ica y Te´cnica (DGICYT, G an PB92-0268) and om
he Na ional Ins i u es o Heal h (G an DK38251) is g a e ully
acknowledged.
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