Proc. Natl. Acad. Sci. USAVol. 93, pp. 4240-4245, April 1996Pharmacology

Nerve growth factor in the anterior pituitary: Localization inmammotroph cells and cosecretion with prolactin by adopamine-regulated mechanism

(neuroimmune interactions/stress/D2 receptor/gpl4Otrk)

CRISTINA MISSALE*, FLORA BORONI*, SANDRA SIGALA*, ALESSANDRO BURIANIt, MICHELE FABRISt,ALBERTA LEONt, ROBERTO DAL Tosot, AND PIERFRANCO SPANO**Division of Pharmacology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy; and tResearchlife,Castelfranco Veneto, Italy

Communicated by Rita Levi-Montalcini, Institute of Neurobiology, Consiglio Nazionale Ricerche, Rome, Italy, December 22, 1995 (received forreview May 16, 1995)

ABSTRACT Nerve growth factor (NGF) is well charac-terized for its neurotrophic actions on peripheral sensory andsympathetic neurons and on central cholinergic neurons ofthe basal forebrain. Recent evidence, however, has shown highlevels of NGF to be present in a variety of biological fluidsafter inflammatory and autoimmune responses, suggestingthat NGF is a mediator of immune interactions. IncreasedNGF serum levels have been reported in both humans andexperimental animal models of psychological and physicalstress, thus implicating NGF in neuroendocrine interactionsas well. The possible source(s) and the regulatory mechanismsinvolved in the control of serum NGF levels, however, stillremain to be elucidated. We now report the presence of bothNGF gene transcripts and protein in the anterior pituitary.Immunofluorescence analysis indicated that hypophysialNGF is selectively localized in mammotroph cells and storedin secretory granules. NGF is cosecreted with prolactin frommammotroph cells by a neurotransmitter-dependent mecha-nism that can be pharmacologically regulated. Activation ofthe dopamine D2 receptor subtype, which physiologicallycontrols prolactin release, resulted in a complete inhibition ofvasoactive intestinal peptide-stimulated NGF secretion invitro, whereas the specific D2 antagonist (-)-sulpiride stim-ulated NGF secretion in vivo, suggesting that the anteriorpituitary is a possible source of circulating NGF. Given theincreased NGF serum levels in stressful conditions and thenewly recognized immunoregulatory function of this protein,NGF, together with prolactin, may thus be envisaged as animmunological alerting signal under neuronal control.

Nerve growth factor (NGF) (1, 2) belongs to a family ofstructurally related neurotrophic molecules, which includesbrain-derived neurotrophic factor (BDNF) (3), neurotrophin(NT) 3, and NT-4/5 (4-7). NGF acts via transmembranereceptors to regulate many cell functions, including cell sur-vival and differentiation. Two NGF receptors have beencloned and sequenced: the 75-kDa glycoprotein p75NGFR,which mediates low-affinity, high-capacity NGF binding (8),and gp140trk, which mediates high-affinity, low-capacity bind-ing (9-11). Two other members of the trk family of protoon-cogenic tyrosine kinases, gp145trkB (12-14) and gpl40trkC (15),are receptors for BDNF and NT-4/5, and NT-3, respectively.NGF, first identified as a neurotrophic factor for peripheral

sympathetic and sensory neurons (1) and cholinergic neuronalpopulations (16), is now recognized as being involved in amultitude of biological functions. Evidence recently obtainedclearly implicates NGF in tissue inflammatory (17-19) and

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immune (20-23) responses, thus proposing an immunoregu-latory role for this NT. Further, the ability of NGF to activatethe pituitary-adrenocortical axis with a concomitant enhance-ment of serum glucocorticoids concentrations (24, 25), to-gether with increased serum NGF concentrations during stress(26), pregnancy, and lactation (27), supports the concept ofNGF as a modulator of neuroendocrine functions.The possible source(s) of circulating NGF and which regu-

latory mechanisms are involved in the control of serum NGFlevels remains to be identified. In a stressful condition such asaggressive behavior in male mice, it has been suggested thatcirculating NGF originates from the submaxillary salivarygland (26). On the other hand, NGF-like immunoreactivity hasalso been found in the pituitary (28-30). Given the central roleof the hypophysis in hormonal responses to stress, NGF ofhypophysial origin might be secreted into the bloodstreamunder cognitive control during stressful events.The present study was designed to test this hypothesis. Here

we show that in the adult anterior pituitary, NGF is bothselectively expressed by mammotroph and somatomam-motroph cells and stored in secretory granules. In vitro and invivo results indicate that NGF is cosecreted with prolactin(PRL) by neurotransmitter-dependent mechanisms, which canbe regulated pharmacologically, thus proposing that the an-terior pituitary represents a newly recognized source of cir-culating NGF. These results also demonstrate the expressionof gp140trk in PRL-containing cells, thereby suggesting anautocrine role for NGF in the maintenance of the mam-motroph cell differentiated phenotype.

MATERIALS AND METHODSPreparation of Pituitary Cell Cultures. The anterior pitu-

itary lobes from adult male and lactating female rats (CharlesRiver Breeding Laboratories) were minced and placed inEarle's balanced salt solution containing 0.2% bovine serumalbumin (EBSS) previously equilibrated at pH 7.4 with 02/CO2 (95:5); trypsin was added to 2.5%, and the lobes wereincubated at 37°C for 15 min and dispersed by 20 passesthrough a pipette. The undigested tissue was allowed to settleand was again incubated with trypsin as described. The solu-tions containing the dispersed cells were centrifuged at 500 xg for 2 min. Pellets were resuspended in EBSS containingtrypsin inhibitor (0.5 mg/ml), centrifuged, and resuspendedthree additional times. The final suspension was in Dulbecco's

Abbreviations: NGF, nerve growth factor; NT, neurotrophin; PRL,prolactin; FACS, fluorescence-activated cell sorting; GH, growthhormone; ACTH, adrenocorticotropic hormone; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; VIP, vaso-active intestinal peptide; mAb, monoclonal antibody; BDNF, brain-derived neurotrophic factor.

4240

Proc. Natl. Acad. Sci. USA 93 (1996) 4241

modified Eagle's medium containing 10% fetal calf serum, 4mM glutamine, penicillin (100 units/ml), and streptomycin(100 ,ug/ml) and plated in poly(L-lysine)-coated dishes. PRLrelease studies were carried out 4 days after plating. Immu-nofluorescence studies were carried out on cells grown onpoly(L-lysine)-coated slides for 4 days. For fluorescence-activated cell sorting (FACS), cells were fixed immediatelyafter dispersion.Immunofluorescence. Cells were fixed in 4% paraformal-

dehyde/2% (wt/vol) sucrose (15 min at room temperature),permeabilized in 0.5% saponin (10 min at room temperature),and washed for 30 min with phosphate-buffered saline con-taining 0.1 M glycine and 10% bovine serum albumin.

In double-labeling experiments, pituitary cells were double-labeled for 1 h at 37°C with a mouse monoclonal antibody(mAb) to NGF (clone 27/21; 20 ng/ml) and a rabbit anti-PRLantibody (5 ,tg/ml). Other cells were incubated with theanti-NGF antibody (20 ng/ml) and then with the secondaryantibody coupled to fluorescein (1 h at 37°C in the dark). Thesame cells were then labeled with 100 ,tl of culture mediumfrom hybridoma cells producing a mouse antibody (mAb219.6) against secretogranin I (31, 32) and then with thesecondary antibody coupled to rhodamine (1 h at 37°C in thedark).Other cells were incubated with the anti-NGF antibody (20

ng/ml) and 100 tul of culture medium from hybridoma cellsproducing a mouse antibody (mAb 219.6) against secretogra-nin I (31, 32). All double-labeled cells were then incubated for1 h at 37°C in the dark with 10 jug of the appropriate secondaryantibodies per ml coupled to either fluorescein or rhodamineand examined under a fluorescence microscope.For FACS, cells (2 x 105 cells per sample) were fixed in 3%

paraformaldehyde/2% sucrose and permeabilized in 0.1%saponin (10 min at room temperature); cells were then double-labeled for 1 h at room temperature with the followingantibodies: (i) anti-PRL (1:10,000 dilution; 100 ,l per sample)and a monkey antibody to growth hormone (GH) (1:5000dilution; 100 ,tl per sample); (ii) anti-PRL (1:10,000 dilution;100 tul per sample) and anti-NGF (20 ng/ml); (iii) anti-GH(1:5000 dilution; 100 ,l per sample) and anti-NGF (20 ng/ml);(iv) a rabbit antiserum against adrenocorticotropic hormone(ACTH) (100 ,tl per sample) and anti-NGF; (v) a rabbitantiserum to thyroid-stimulating hormone (TSH) (1:40 dilu-tion; 100 pIl per sample) and anti-NGF; and (vi) a mouse mAbto follicle-stimulating hormone (FSH) (1:50 dilution; 100 ,tlper sample) and anti-NGF. Cells were also double-labeled withantibodies against pituitary hormones and a rabbit antiserumto the 14 carboxyl-terminal amino acids of gp140trk (1 ,ug/ml).This latter antibody reacts with gpl40trk and cross-reacts tolesser degrees with gpl45trkB and gpl40trkc. Cells were incu-bated for 1 h at room temperature in the dark with theappropriate secondary antibodies coupled to either fluores-cein or rhodamine and analyzed using multichannel cytometer(FACStar; Becton Dickinson). Total counts from 104 cellswere accumulated for each group. Isotype control with irrel-evant antibodies and omission of the primary antibody wereperformed as internal controls.

Immunoblotting. The anterior and posterior lobes of ratpituitaries were separately homogenized in 10 mM Tris HCl,pH 7.5/5 mM EDTA/1 mM phenylmethylsulfonyl fluoride/leupeptin (10 ,ug/ml)/pepstatin (10 ,ug/ml). After centrifuga-tion at 6500 rpm (Sorvall SS34 rotor), the supernatants werecentrifuged at 35,000 rpm (Beckman T150 rotor) for 30 min at4°C. Cell extracts were resolved on SDS/12% polyacrylamidegels (200 ,Lg of protein per lane). Proteins were transferred tonitrocellulose and immunoblotted for 2 h with anti-NGF (mAb27/21; 20 ng/ml). Immunoreactivity was revealed by a 1-hincubation with secondary antibodies coupled to alkalinephosphatase.

Poly(A)+ RNA Isolation and Hybridization. Poly(A)+ RNAwas isolated according to Badley et al. (33), electrophoresed ona 1% agarose gel (5 ,ug per lane), and transferred to a nylonmembrane in 20x SSC according to Maniatis et al. (34). Blotswere hybridized with a specific 32P-labeled Pst I restrictionfragment of the murine NGF cDNA (35). Hybridization wasperformed in 50% formamide/5x SSC/50 mM phosphatebuffer/0.1% SDS/5X Denhardt's solution/salmon spermDNA (100 ,ug/ml) at 42°C overnight; excess probe was washedfrom membranes once in 1 x SSC/0.1% SDS for 20 min at 55°Cand then twice at higher stringency in 0.1 x SSC/0.1% SDS for15 min at 55°C. Blots were then exposed to Kodak X-Omatfilm.

In Vitro Release ofPRL and NGF. Dissociated pituitary cells(106 cells per well) were incubated at 37°C for 10 min in EBSS,and the supernatants were collected to determine basal re-lease. The cell samples were then incubated for 10 min at 37°Cwith 100 nM quinpirole; 100 nM vasoactive intestinal peptide(VIP) was subsequently added for an additional 10 min, andthe supernatants were collected. After extensive washing, cellswere incubated at 37°C for 10 min with 100 nM VIP, and thesupernatants were collected. The amount of PRL in thesupernatants was determined by a double-antibody RIA.Briefly, aliquots of the supernatants were incubated at roomtemperature in the presence of 125I-labeled PRL and theantibody specific for rat PRL [anti-ratPRL-S-9 (antisera AFP131581570; National Institute of Diabetes and Digestive Kid-ney Diseases)]. Reaction mixtures were then incubated for 1 hat room temperature with the appropriate secondary antibodyin the presence of 8% polyethylenglycol and centrifuged at3000 rpm (1000 x g) for 30 min. The amount of radioactivityin the pellet was then evaluated.A two-site ELISA (kit from Boehringer Mannheim with

mAb 27/21) was used to quantitate NGF in supernatants,using mouse 2.5S NGF as standard (36). Blanks consisted ofsamples added to microwells coated with mouse myeloma IgG(Calbiochem) instead of anti-NGF antibody.

In Vivo Studies. Male Sprague-Dawley rats (250 g; CharlesRiver Breeding Laboratories) were given a single injection of(-)-sulpiride (20 mg/kg) (n = 6) or saline (n = 6). One hourlater, the animals were sacrificed and blood was collected.Serum PRL levels were measured by the RIA previouslydescribed. Serum NGF was determined by using dissociatedcultures of chicken embryonic day 8 sympathetic neurons.Sympathetic neurons were cultured for 48 h without or withvarious amounts (0.3-5%) of rat serum. The percentage ofcells bearing neurites was determined in random fields, andNGF was quantified as described (36, 37). Biospecificity wasevaluated by adding the mouse anti-NGF mAb (27/21; 1Kg/ml).Drugs. VIP was from Sigma; quinpirole was from Research

Biomedicals; (-)-sulpiride was from Ravizza (Milan). 125I-labeled prolactin was obtained from DuPont/New EnglandNuclear. The antibodies against ACTH, FSH, and TSH werefrom BioGenex Laboratories (San Ramon, CA). The antibodyagainst NGF (clone 27/21) and the secondary antibodies werefrom Boehringer Mannheim. The antibody against gpl40trkwas from Santa Cruz Biotechnology. The antibodies to PRL[anti-rat PRL-S-9 (antisera AFP 131581570) and anti-ratPRL-IC-5 (antisera CYTO AFP 425-10-91], rat PRL (PRL-RP-3 AFP4459B), and GH (anti-rGH-S-5) were kindly pro-vided by National Institute of Diabetes and Digestive andKidney Diseases, National Hormone and Pituitary Program,Ogden BioServices Corporation, Rockville, MD. Culture me-dium and fetal calf serum were from Seromed (Milan).

RESULTSNGF Expression in the Pituitary. Poly(A)' RNA was

isolated from either the anterior and the intermediate-

Pharmacology: Missale et al.

4242 Pharmacology: Missale et al.

posterior lobes of the adult pituitary and resolved on an

agarose gel (5 jug per lane). Hybridization with a murine NGFcDNA probe detected the presence of the 1.3-kb NGF mRNAgene transcript in the anterior pituitary; by contrast, little or noNGFmRNA was detectable in the intermediate-posterior lobeof the gland (Fig. 1A). Detection of cyclophilin mRNA was

included in the Northern blot analysis as an internal control.No differences were found in cyclophilin mRNA content in theanterior and posterior pituitary (data not shown). The pres-ence of the translation product ofNGF mRNA in the pituitarywas shown by Western blotting (Fig. 1B). The NGF mAbrecognized a band with an apparent molecular mass of 13 kDaand an additional faint band with a molecular mass of -35 kDain cell extracts from anterior pituitaries of both 4-day-old andadult rats. In contrast, little or no signal was detected inextracts from the intermediate-posterior lobe of the gland.Densitometric quantification of the bands revealed about 300ng/mg of protein. These results were confirmed by the NGFbioassay with sympathetic neurons showing between 200 and300 ng of NGF per mg of protein (data not shown).

Cellular Localization of Hypophysial NGF. Immunofluo-rescence studies were performed to define the localization ofNGF in specific cell populations of the anterior pituitary. Arepresentative FACS analysis of anterior pituitary cells dou-ble-stained with antibodies to PRL and GH is reported in Fig.2, which showed that 37% ± 2% of all pituitary cells were

immunopositive for PRL, whereas 32% ± 2% of the cells werereactive with the GH antibody. Further, 18% ± 2% of the cellswere double-stained for both PRL and GH, suggesting that19% ± 2% of the cellular elements present in the gland are

mammotroph cells containing only PRL, whereas 14% ± 3%are somatotroph cells with GH only, and 18% ± 2% have a

somatomammotroph phenotype with both GH and PRL (Ta-ble 1). A considerable fraction of all pituitary cells were

immunopositive for either NGF (32% ± 4%) or gpl40trk (38%± 0.2%); both figures are very similar to that obtained for thepopulation of PRL-immunoreactive cells (37% ± 2%) (Table1). This last result was reconfirmed by immunologically dou-ble-staining pituitary cells for hypophysial hormones andNGF, using appropriate secondary antibodies coupled toeither fluorescein or rhodamine. Results of this FACS analysis(Table 1) showed, indeed, that the percentage of cells withboth PRL and NGF immunoreactivities to be identical to thatobserved after labeling for only NGF or PRL (39% ± 1%).The percentage of cells double-labeled with NGF and GH

A B

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FIG. 1. Expression of NGF in rat pituitary. (A) Pituitary mRNAwas isolated, separated on a 1% agarose gel (5 tig per lane), andhybridized with a Pst I restriction fragment of the murine NGF cDNA.Lanes: a, 1-day-old pituitary; b, 4-day-old pituitary; c, adult anteriorpituitary; d, intermediate-posterior pituitary. (B) Cell extracts were

resolved by SDS/12% PAGE, and the proteins were transferred ontonitrocellulose and immunoreacted with an mAb to NGF. Lanes: a,standard NGF (50 ng); b, adult anterior pituitary; c, 4-day-oldpituitary; d, intermediate-posterior pituitary.

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FIG2. Cytofluorimetric analysis of pituitary cells stained by PRLand GH antibodies. Anterior pituitary cells were double-labeled withantibodies to GH and PRL and then with the appropriate secondaryantibodies coupled to fluorescein (PRL) or rhodamine (GH) andanalyzed by FACS. (A) Nonspecific labeling. (B) Cells labeled by PRLantibody. (C) Cells labeled by GH antibodies. (D) Cells double-labeledbyGH and PRL antibodies. Total counts of 104 cells were accumulatedfor each group. A representative FACS scan is shown. The percentageof mammotroph and somatotroph cells was calculated as the differ-ence between the total number of cells labeled by PRL or GHantibodies and the number of cells double-labeled by both PRL andGH antibodies.

antibodies (13% ± 1%) was very close to the number of cellspositive for both PRL and GH (18% ± 2%). Some TSH-positive cells also double-stained for NGF, although the resultwas quite variable. No NGF labeling was observed in cellsstained for ACTH and TSH.Analogous results were obtained in double-labeling exper-

iments using pituitary hormone antibodies and an antibodyagainst the 14 carboxyl-terminal amino acids of gp140trk. Thisantibody reacts with the trkA receptor for NGF and cross-reacts to a lesser extent with trkB and trkC receptors for BDNFand NT-3. As shown in Table 1, gp140trk is selectively andspecifically expressed by mammotroph and somatomam-motroph cells. No gp140trk staining was detected in soma-

totroph, corticotroph, thyreotroph, or gonadotroph cells.Intracellular Localization of NGF. The subcellular distri-

bution of NGF was established by fluorescence microscopicexamination of cells double-stained with NGF and PRLantibodies. PRL-like immunoreactivity was seen in punctate

-45 Table 1. Localization of NGF and its receptors in pituitary cells

Hormone + Hormone +Cell type Hormone NGF trk

PRL 19 ±+ 2.0 39 ±+ 1.0 39 +± 0.7GH 14 ±+ 0.7 ND NDGH/PRL 18 ± 2.0 13 ± 1.0 20 ± 3.0TSH 5 ±+ 0.7 ND NDACTH 18 ±+ 2.0 ND NDFSH 12 + 0.5 5+ 3.0 NDNGF 32 + 4.0 - -gp140trk 38 ± 0.2 - -

ND, not detectable. Cells were fixed, permeabilized, double-labeledwith antibodies to pituitary hormones and to NGF or to gpl4o0rk andthen with secondary antibodies coupled to fluorescein or rhodamine,and analyzed by FACS. Total counts of 104 cells were accumulated foreach group. Data are mean + SEM percentages for three independentdeterminations.

Proc. Natl. Acad. Sci. UISA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 4243

structures, sometimes more concentrated in the perinuclearregion, suggesting its proper localization in the secretorygranules (Fig. 3A). NGF immunoreactivity in cells costainedfor PRL displayed a dotted appearance and apparently was inthe same position as the punctate structures labeled by thePRL antibody (Fig. 3B). The intracellular NGF-immunoposi-tive structures were hardly distinguishable from the structuresvisualized by the secretogranin I antibody (Fig. 3 C and D). TheNGF antibody labeled only a portion of these cells, whichstained for secretogranin I, confirming the specific localizationof NGF to a discrete subset of pituitary cells. Staining was notobserved when primary antibodies were omitted (Fig. 3 E andF).NGF Release from the Anterior Pituitary. NGF and PRL

were cosecreted by mammotroph cells in vitro (Table 2). Abasal release of NGF was detectable in pituitary cell culturesfrom male rats. The physiological PRL secretagogue VIP (38),which induced a 19-fold increase in PRL secretion, stimulatedthe NGF secretion 10-fold. Addition to the culture medium ofquinpirole, a highly selective D2 receptor agonist (39), restoredto basal levels the VIP-induced secretion of both PRL andNGF. The basal secretion of PRL and NGF, as well as theabsolute amounts of hormonal peptides secreted in response toVIP, was markedly elevated in cell cultures derived from theanterior pituitary of lactating female rats, as compared to cellsderived from the pituitaries of male rats (Table 2). Thebiological activity of the released NGF was defined in a neuriteoutgrowth assay with sympathetic neurons and PC-12 pheo-chromocytoma cells and confirmed the levels measured byELISA (data not shown).

In vivo experiments were next performed to investigate thepossible increase ofNGF serum levels after a pharmacologicalchallenge with an inducer of PRL secretion. For this purposethe selective D2 receptor antagonist (-)-sulpiride (40) wasused. Administration of (-)-sulpiride at 20 mg/kg to male ratsstrongly increased serum PRL levels, with maximal effects

Table 2. Pharmacological regulation of PRL and NGF releasein vitro

PRL, ng/106 cells NGF, ng/106 cells

Addition Male Female Male Female

None (basal) 6.8 ± 0.7 33 ± 1 38 ± 2.2 58 ± 0.6VIP 132 ± 10* 384 ± 5* 383 ± 15* 3121 ± 25*VIP + quin 4.8 0.3 37± 1 41 7 7±1*

In vitro PRL and NGF release was determined as described. PRLwas measured by a double-antibody RIA and NGF was measured bya two-site ELISA. VIP and quinpirole (quin) were used at 100 nM.Data are means ± SEM for three independent determinations.*P < 0.001 vs. basal.

reached 60 min later. As shown in Fig. 4, quantitation of serumNGF by bioassay (neurite outgrowth in sympathetic neurons)revealed biological activity equivalent to 3 ± 0.4 ng of NGF perml in control animals. Serum NGF rose to 10 ± 1 ng/ml(3.3-fold increase) 1 h after (-)-sulpiride administration, atwhich time the PRL concentration also reached its peak [25.4± 0.4 ng/ml before (-)-sulpiride and 85.3 + 1.4 ng/ml after(-)-sulpiride; 3.4-fold increase]. Antibodies to NGF (mAb27/21) reduced the biological response of sympathetic neuronsto the background levels detectable in the presence of controlserum.

DISCUSSIONThe present study shows that both NGF gene transcripts andNGF protein are present in the adult anterior pituitary. Bycontrast, little or no NGF was detectable in the intermediate-posterior lobe of this gland. Western blot analysis of NGF inthe anterior pituitary revealed the presence of a major bandrepresenting ,3-NGF and an additional species, which mayrepresent a glycosylated form of the NGF precursor protein(41). Densitometric quantification of Western blots and a NGF

FIG. 3. Immunofluorescent staining of pituitary cells with antibodies to NGF, PRL, and secretogranin I. Cells grown on poly(L-lysine)-coatedslides were fixed, permeabilized, and double-labeled with antibodies to either PRL and NGF (A and B) or to NGF and secretogranin I (C andD) as described. (A) Staining for PRL with a secondary antibody coupled to fluorescein. (B) Staining for NGF with a secondary antibody coupledto rhodamine. (C) Staining for NGF with a secondary antibody coupled to fluorescein. (D) Staining for secretogranin I with a secondary antibodycoupled to rhodamine. (E) Rabbit preimmune serum with secondary antibody coupled to fluorescein. (F) Staining of preimmune mouse serumwith secondary antibody coupled to rhodamine. (Bar = 3 gjm.)

Pharmacology: Missale et al.

4244 Pharmacology: Missale et al.

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50-

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NGF

FIG. 4. Stimulation of NGF and PRL secretion into circulation invivo. Male rats were handled for 10 days and then treated with a singlei.p. injection of (-)-sulpiride (20 mg/kg) (hatched bars) or saline(open bars). One hour after injection, rats were sacrificed and bloodwas collected for PRL and NGF. Bars represent the means ± SEM forsix rats in each group. *, P < 0.001 vs. saline-treated rats.

bioassay with sympathetic neurons showed that the levels ofNGF in the anterior pituitary are between 200 and 300 ng/mgof protein, suggesting that the gland is an extraordinarily richsource of NGF. Immunofluorescence clearly demonstratedthat, among the five different pituitary cell phenotypes, NGF-like immunoreactivity was selectively localized to mam-

motroph and somatomammotroph cells and was largely asso-

ciated with intracellular punctate structures that apparentlywere hardly distinguishable from the secretory granules la-beled by PRL antibodies. Further, some of the cellular struc-tures labeled by anti-NGF antibody were apparently immuno-reactive for secretogranin I, a protein belonging to the graninfamily and specifically associated with the matrix of secretorygranules of endocrine cells (31, 32). Whether the structureslabeled by the NGF antibody represent authentic secretorygranules or vesicles of different nature, such as endocytoticvesicles, cannot be definitely addressed by our present data.However, it is worth noting that localization of preformedNGF to granules has been described for cell lines of pituitaryorigin transfected with the NGF gene (42) and, more recently,in mast cells (36). Reported NGF-like immunoreactivity in theanterior pituitary is restricted mainly to the somatomam-motroph lineage (43). However, NGF-like immunoreactivityhas also been shown to be present in small percentages of otherpituitary cell phenotypes (43). The apparent discrepancy be-tween this last study (43) and the present data is most probablydue to differences in experimental conditions used to colo-calize NGF with other hypophysial hormones.There is strong evidence that hypophysial NGF is cosecreted

with PRL by an active, neurotransmitter-regulated mecha-nism. PRL secretion from the anterior pituitary is known to beunder hypothalamic stimulatory and inhibitory controls. VIPis one of the most potent PRL secretagogues (38), whereasPRL secretion is tonically inhibited by dopamine (44) throughactivation of D2 receptors located on pituitary mammotrophs(45). A two-site ELISA detected the presence of abundantf3-NGF protein in the medium of resting pituitary cells. Here,VIP dramatically increased both PRL and NGF release frompituitary cell cultures. On the other hand, stimulation ofdopamine D2 receptors by the selective agonist quinpirole (39)completely prevented the VIP-stimulated increase of bothPRL and NGF in vitro. The observation that blockade ofpituitary D2 receptors with the selective antagonist (-)-sul-piride (40) increased serum PRL and NGF levels is consistent

with the in vitro results and, further, suggests that NGF may bereleased into the bloodstream from the pituitary. Two otherpoints strengthen the concept of NGF and PRL cosecretion:(i) basal and VIP-stimulated secretion of NGF and PRL invitro increase in cell cultures derived from the pituitaries oflactating female rats, a condition of mammotroph cell hyper-plasia (46, 47); (ii) levels of circulating NGF markedly increaseduring human labor and lactation (27), a time when serumPRL levels are also elevated.The present findings propose a neurotransmitter-regulated

release of NGF from PRL-secreting cells of the pituitary andstrongly suggest that the anterior pituitary is a possible sourceof circulating NGF. Increased NGF serum levels followingbehaviorally or psychologically stressful events has been ex-tensively described both in humans and in experimental animalmodels (48). Higher NGF levels have also been found inhuman serum in situations where anxiety may be the mainpsychological feature and prior to activation of the hypo-thalamo-pituitary-adrenal endocrine axis (49).These observations raise an important question concerning

possible targets and, hence, the biological significance ofelevated serum NGF levels. Any cell type expressing high-affinity NGF receptors within range of serum proteins couldpotentially be affected by circulating NGF. Cellular elementsmost likely susceptible to NGF actions include components ofthe immune system such as lymphocytes, monocytes, neutro-phils, mast cell precursors, and basophils. NGF is known tohave proliferating, chemotactic, differentiating, and activatingactions on these cell types (20-23, 49-55). Neuronally regu-lated release of NGF from the pituitary may constitute a newlink between the nervous and immune systems. In this context,NGF may be viewed as a general "alert" signal, which, togetherwith prolactin, may act in concert to prime the immune systemtoward potentially noxious perturbations.Somatomammotroph and mammotroph cells express the

gp140trk protein as detected with an antibody that cross-reactswith the known trk receptors. Two considerations suggest thatstaining could mainly be due to trk: (i) it has been recentlyreported, by using a specific anti-gp140trk antibody, that thismolecular species is present in the somatomammotroph lin-eage (43); (ii) in a previous study, we have shown thatmammotroph and somatomammotroph cells respond to NGF(56), which is well known not to interact with trkB and trkC (2).However, due to the characteristics of the antibody used, thepossibility cannot be excluded that these two molecular speciesare also present in the pituitary. Taken together, these datasuggest that somatomammotroph and mammotroph cells areboth sources and targets of NGF. The anterior pituitary maythus be subject to NGF-mediated autocrine actions. In thisregard, we (56) have recently shown endogenous NGF to be acritical factor in promoting conversion of bipotential progen-itor somatomammotrophs into mature mammotroph cellsduring pituitary development in vitro, thus suggesting itspossible role in pituitary maturation also in vivo. Since bipo-tential somatomammotroph cells may provide plasticity to theadult anterior pituitary, NGF could also be important in theconversion of somatomammotroph cells into mammotrophs inparticular instances of mammotroph hyperplasia such as preg-nancy, lactation, or estrogen administration (46, 47, 57).

In conclusion, the experiments described here show thatpituitary somatomammotroph and mammotroph cells pro-duce, store, and secrete NGF and that NGF is cosecreted withPRL, suggesting that the pituitary gland is a source of circu-lating NGF in physiological conditions and during stress.

We are grateful to Patrizia Rosa, CNR Center of Cytopharmacol-ogy, Department of Pharmacology, University of Milan, for her gift ofanti-secretogranin I antibody.

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[E

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Proc. Natl. Acad. Sci. USA 93 (1996) 4245

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