Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion

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 Downloaded a UNIVERSIDAD VALLADOLID. BIBLIOTECA on Janua y 19, 2021 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 Downloaded a UNIVERSIDAD VALLADOLID. BIBLIOTECA on Janua y 19, 2021 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) Downloaded a UNIVERSIDAD VALLADOLID. BIBLIOTECA on Janua y 19, 2021 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) Downloaded a UNIVERSIDAD VALLADOLID. BIBLIOTECA on Janua y 19, 2021 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. 1. Ha is, G. W., Reed, M. & Fawce , C. P. (1966) B . 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