Adrenocorticotropic Hormone in Human Pituitary and Pituitary Adenomas

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American Journal ofPathology, Vol. 138, No. 4, April 1991 Copyright © American Association ofPathologist Coexpression of Galanin and Adrenocorticotropic Hormone in Human Pituitary and Pituitary Adenomas

Dora W. Hsu, Shing C. Hooi, E. Tessa corticotroph lineage. Studies oftumors confirmed the Hedley-Whyte, Robert M. Strauss, and observed coexpression of galanin and adrenocorti- Lee M. Kaplan cotropic hormone. The divergent cell type specificity From the C. S. Kubik Laboratoryfor Neuropathology and the ofgalanin production in human and ratpituitaries Gastrointestinal Unit, Massachusetts General Hospital, and reflects different patterns ofgene activation in these the Departments ofPathology and Medicine, Harvard two species. In addition these results suggest that Medical School, Boston, Massachusetts galanin in the human pituitary may participate lo- cally in the regulation of the hypothalamic- pituitary-adrenal axis. (AmJPathol 1991, 138:897- Galanin is a neuropeptide that regulates the secre- 909) tion ofseveralpituitary hormones includingprolac- tin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in Hormone secretion by mammalian anterior pituitary cells the rat anteriorpituitary is cell lineage specific, with is modulated by a number of well-characterized hypotha- predominant expression in lactotrophs and soma- lamic releasing and inhibitory factors.1' 2 More recently, totrophs. The authors examined the cellular distribu- several additional peptides that regulate the release of tion of human Gal-IR in seven normal postmortem anterior pituitary hormones have been found in the hypo- pituitaries and 62 pituitary tumors by immunoper- thalamus. Many of these regulatory peptides also are oxidase staining. In contrast to the rat, Gal-IR in present in cells of the anterior pituitary, suggesting a local human anteriorpituitaries waspresent in corticotro- (paracrine or autocrine) regulatory function. Such pep- phs scattered throughout the gland but not in lac- tides include cholecystokinin (CCK),3 vasoactive intesti- totrophs somatotrophs thyrotrophs; or gonadotro- nal peptide (VIP),4 and galanin.57 phbs Distinct Gal-IR also was present in hyperplastic Galanin is a small neuropeptide initially isolated from and neoplastic corticotrophs in 19 of 22 patients porcine intestine.e Immunoreactive galanin has been with Cushing's disease. In noncorticotroph cell tu- shown to be widely distributed throughout the central ner- mors, unequivocal Gal-IR waspresent in 5 of 11 GH- vous system and the peripheral nerves, with its highest secreting tumors associated with clinical acromega- concentration in regions of the hypothalamus, including ly, 9 of 18 nonfunctioning pituitary adenomas, and the paraventricular (PVN), supraoptic, and arcuate 2 of 14 prolactinomas. Of these galanin-positive tu- nuclei.911 Presence of a high-density galaninlike immu- mors, four of thefive GH-secreting adenomas, six of noreactive (Gal-IR) fiber network and specific galanin- the nine nonfunctioning adenomas, and both of the binding sites in the hypothalamus and the median emi- prolactinomas also contained adrenocorticotropic nence (ME)9'12-14 suggest a neuroendocrne role for this hormone immunoreactivity (ACTH-IR). Immuno- peptide. staining and in situ hybridization on adjacent sec- Involvement of galanin in the regulation of pituitary tions using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant A portion of this work was presented at the 19th Annual Meeting of the galanin expression in normal corticotroph& Immu- Society for Neuroscience, Phoenix, Arizona, November 1989. noelectron microscopy confirmed the presence of Supported in part by grant number DK42189 from the National Insti- Gal-IR in pituitary cells characteristic of corticotro- tutes of Health. Lee M. Kaplan is a Stuart/American Gastroenterological phs in both normal and neoplasticpituitaries. Thus Association Research Scholar. as in the rat, galanin gene expression in the human Accepted for publication December 3, 1990. Address reprint requests to Dora W. Hsu, Neuropathology, Warren 3, pituitary is cell-type specific. Unlike the rat, however, Department of Pathology, Massachusetts General Hospital, Boston, MA human galanin gene expression is restricted to the 02114.

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function is suggested by the observations that 1) admin- ing only hyperplastic nodules without clearly defined ad- istration of porcine galanin into the third ventricle of rats enomas, and two ACTH-producing lung carcinoid causes a dose-dependent increase in plasma prolactin tumors from separate patients. (PRL) and growth hormone (GH) levels1`19 and 2) intravenous injection of galanin increases plasma GH levels in humans20,21 and rats.22 Recent studies in rats show that Immunocytochemistry galanin may also regulate the secretion of adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone Six-micron paraffin sections of the tumors and normal pi- (TSH).16,23 tuitaries were immunostained using the avidin- We and others have previously demonstrated that biotin-peroxidase complex (ABC) method (Vectastain galanin is synthesized and secreted by rat anterior pitu- ABC Elite kit for Gal-IR, and Vectastain ABC kits for the itary cells,5'24'25 and that pituitary expression of the rat anterior pituitary hormones-lR).' Staining was performed galanin gene is strongly dependent on circulating levels in accordance with the protocols suggested by the man- of estrogen5'24 and progestins (Hooi and Kaplan, unpub- ufacturer (Vector Laboratories, Burlingame, CA). Sec- lished observations). In rat pituitaries, Gal-IR is located tions were incubated overnight at room temperature with predominantly in lactotrophs (PRL cells) and somatotro- rabbit antiserum directed against synthetic porcine gala- phs (GH cells), and to a lesser extent in thyrotrophs6'25 nin (Peninsula Labs, Belmont, CA), diluted 1:1500 in (Hsu and Kaplan, unpublished observations). Estrogens phosphate-buffered saline (PBS) containing 0.3% Triton appear to increase pituitary galanin concentrations by X-1 00. This antiserum does not cross-react with the fol- stimulating lactotrophs and somatotrophs specifically lowing regulatory peptides: secretin, PHM-27, VIP, sub- rather than by recruiting significant numbers of additional stance P, L-enkephalin, thyrotropin-releasing hormone, cell types to produce this peptide5,6 (Hsu and Kaplan, arginine vasopressin, pancreatic polypeptide, glucagon, unpublished observations). This combination of cell type growth hormone-releasing hormone, neuropeptide Y, or specificity and hormone responsiveness provides addi- somatostatin, and has been shown to bind human tional support for the involvement of local pituitary galanin galanin.27 To assess the presence and distribution of an- in the regulation of reproduction and lactation, at least in terior pituitary hormones in tumors and normal glands, the rat. the following antisera and dilutions were used: antisera to In the present studies, we sought to determine the human PRL (1:1500), GH (1:2000), B-subunits of TSH levels of galanin expression in human pituitary and pitu- (1:1000), follicle-stimulating hormone (FSH, 1:500), and itary adenomas, and whether similar cell type specificity luteinizing hormone (LH, 1:600), and the a-subunit is observed. We used light and electron microscopic im- (1:2500) of the glycoprotein hormones (all supplied by munocytochemistry as well as blot and in situ hybridiza- Dr. S. Raiti of the National Hormone and Pituitary Pro- tion to examine galanin expression in normal and neo- gram, Baltimore, MD). Rabbit antiserum to ACTH was plastic human pituitary tissues. These studies demon- generated by injection with porcine ACTH139 (INCSTAR, strate that galanin is coexpressed with ACTH in normal Stillwater, MN) and was used at a 1:400 dilution. This human pituitaries and pituitary adenomas. antiserum does not cross-react with other anterior pituitary hormones. The numbers of tumors immunostained for each of the various hormones are indicated in Table 1. Materials and Methods Sections were incubated with these primary antibodies for 45 minutes at room temperature. After PBS washes, Tissues all sections were incubated with biotinylated secondary antibody and avidin-biotin-peroxidase complex. The Pituitary glands from seven individuals (three men aged antigen-antibody binding was visualized with 31 to 60 years and four women aged 22 to 65 years) 3,3'-diaminobenzidine/H202 (DAB; Polysciences, War- without known endocrine disorders were obtained within rington, PA) to produce a brown reaction product. After 24 hours postmortem, fixed in 10% phosphate-buffered washes with water, sections were counterstained with he- formalin, dehydrated, and embedded in paraffin. Pituitary matoxylin, dehydrated, and mounted in Permount (Fisher tumor tissues, obtained through transsphenoidal surgery, Scientific, New York, NY). were fixed and processed in a similar fashion. These in- The specificity of galanin staining was confirmed on cluded 14 prolactinomas, 11 tumors from patients pre- adjacent sections by preabsorbing the anti-galanin an- senting with acromegaly, 19 adenomas associated with tiserum with 1 ,umol/l (micromolar) purified synthetic por- Cushing's disease, and 18 clinically nonfunctioning pitu- cine galanin (Peninsula Labs) for 48 hours at 40C before itary adenomas. In addition, we examined pituitary biop- incubation with tissue sections. sies from three patients with Cushing's disease contain- The extent and intensity of Gal-IR in tumors were Galanin in Human Pituitary 899 AJP April 1991, Vol. 138, No. 4

Table 1. Galanin and Pituitaty Hormone Immunoreactivity in Human PituitaryAdenomas Hormone-IR (1-4+) Gal-IR % Gal-IR n (2-4+) 1 + - (2-4+) PRL GH ITSH ,BFSH ,LH a ACTH Corticotrophic 19 16 1 2 84 0 nd nd nd nd nd 19 Prolactinomas 14 2 2 10 14 14 1 nd nd nd nd 4 GH cell adenomas 11 5 2 4 45 11 11 nd nd nd 8 5 Nonfunctioning 18 9 5 4 50 4 2 9 13 11 13 8 nd: not done. graded on a scale from 0 (no staining) to 4 + (heavy cRNA, immersed in Kodak NTB-2 photographic emul- staining), with 1 + representing weak or equivocal immu- sion, exposed for 5 days, developed, and counterstained noreactivity, or occasional cells stained, 2 + representing with methyl green, as previously described.5 5S-labeled diffuse staining and with focal areas unambiguously pos- cRNA probes were prepared from the human galanin itive, 3 + representing widespread immunoreactivity of cRNA subcloned into pBLUESCRIPT-KS (Stratagene, La moderate intensity, and 4 + representing an overall Jolla, CA) and transcribed with T7 RNA polymerase, as strong immunoreactivity. previously described.29 Control sections were hybridized After preliminary observations suggested localization with 35S-labeled cRNA probes derived from plasmid of Gal-IR to normal corticotrophs, sections of each of the pGEM-3Z (Promega, Madison, WI). adenomas were stained with antisera to ACTH. To confirm that cells containing galanin mRNA were corticotrophs, we immunostained adjacent sections with an antiserum to ACTH, as described above. Double Immunoenzyme Labeling Paraffin sections of normal pituitaries were incubated with Immunoelectron Microscopy antiserum to galanin, applying the ABC-peroxidase protocol as described above and using DAB as substrate to Immunostaining for galanin, using ABC-peroxidase and label the galanin-immunoreactive cells brown. The same DAB as described above, was performed before embed- sections were subsequently incubated with an antiserum ding on 80-ix vibratomed sections of two normal human against one of the anterior pituitary hormones listed pituitary glands and three pituitary tumors from patients above, followed by the ABC-glucose oxidase protocol with Cushing's disease. The tissues were fixed with 4% (Vector Laboratories), using nitro-blue tetrazolium (NTB) paraformaldehyde/0.1% glutaraldehyde in phosphate as substrate to produce a purple/blue reaction product. buffer for 1 to 2 days and then stored in PBS before vi- bratome sectioning. Areas with galanin immunoreactivity apparent by light microscopy were selected, exposed RNA Preparation and Blot Hybridization sequentially to 1% osmium tetroxide for 1 hour, and 1% uranyl acetate in 70% ethanol for 10 minutes, dehydrated Total RNA was isolated from pituitary tissue obtained 6 in graded ethanol solutions, and embedded in an Epon- hours after death using standard methods.5 RNA was araldite mixture (Ladd, Burlington, VT). Sixty-nanometer separated by formaldehyde/agarose gel electrophoresis, sections were examined in a Phillips 301 electron micro- transferred to Nytran membranes (Schleicher and scope without further contrast enhancement. Schuell), and hybridized with complementary DNA (cDNA) probes at 500C, as previously described.5 Radi- olabeled cDNA probes were prepared from a human Results galanin cDNA (Strauss and Kaplan, unpublished data) by the random primer method.28 Galanin Immunoreactivity in Normal Human Pituitary

In Situ Hybridization In the seven normal postmortem pituitaries, Gal-IR was observed in many scattered cells throughout the anterior Whole pituitaries were fixed in 10% formalin, processed, lobe. These cells had a morphology (large ovoid to an- and embedded in paraffin, as described above. Six- gular cells) and distribution similar to ACTH- micron sections were affixed to poly-L-lysine-coated immunoreactive (ACTH-IR) cells identified on adjacent glass slides, hybridized to 35S-labeled human galanin sections (Figure 1 A, B). Galanin-immunoreactivity ap- 900 Hsu et al AJP April 1991, Vol. 138, No. 4 Galanin in Human Pituitary 901 AJP Apil 1991, Vol. 138, No. 4

Figure 1. Immunoperoxidase staining ofadjacent sections ofa normal humanpituitary. The distrbution ofgalanin-immunoreactive cells in the anterior lobe (A) corresponds to that ofACTH-immunoreactive cells (B). Note that ACTH-IR cells outnumber Gal-IR cells, suggesting that galanin is expressed in a subset of corticotrophs (hematoxylin counterstain, x200). Figure 2. Dual staining for galanin and other pituitary hormones using sequential double-immunoenzyme labeling. Galanin-IR is stained brownfrom theperoxidase/DAB reaction, and the anterior pituitary hormone-IR is colored purple/blue by the glucose oxidaselATB reaction. An overlapping of brown and puwple coloration indicates colocalization. Colocalization ofGal-IR and ACTH-IR is apparent (A) (arrows). In contrast, larger brown Gal-JR cells are clearly separatefrom the smaller bluish GH-IR cells (B), orPA#L-IR cells (C), respectively. Open arrowspoint to cells containing only Gal-IR, and arrowheads point to cells containing only GH-IR (B), or PRL-IR (C), respectively (no counterstain, X510). peared as diffuse cytoplasmic staining, similar to the as described in Methods. Hybridization to normal human ACTH-IR. Adrenocorticotropic hormone-positive baso- pituitary disclosed distinct clusters of cells containing phil invasion cells in the posterior pituitary were also gala- galanin mRNA (Figure 4A, C). These labeled cells corre- nin immunoreactive (not shown), and Gal-IR was present sponded to groups of cells intensely stained for ACTH by in nerve fibers within the posterior pituitary. No differ- immunocytochemistry (Figure 4B), confirming that gala- ences in either the distribution or the intensity of Gal-IR nin is expressed specifically in ACTH-producing pituitary were discernible between samples from men and cells. There was no hybridization to 355-labeled cRNA women. derived from plasmid pGEM-3Z in control sections (not shown). No galanin mRNA was detected in the posterior pituitary, confirming that Gal-IR was limited to nerve fibers Colocalization of Galanin and Other (rather than cell in this not Hormones in Normal Pituitary bodies) region (data shown). Sequential double-enzyme labeling was used to delin- eate the pituitary hormone type(s) that colocalized with Immunoreactivity of the Pituitary Tumors galanin. The galanin-peroxidase/DAB reaction produced a brown color, whereas the hormone-glucose oxidase/ As shown in Table 1, 19 tumors from patients with Cush- NTB reaction rendered the cells purple/blue. Colocaliza- ing's disease all exhibited diffuse staining for ACTH tion was indicated by an overlap of the two colors, giving (graded as 2 + to 4+). The 14 prolactinomas were all rise to near-black pigmentation within cells. This pigmen- strongly positive for PRL (all graded as 4+), with one tation was observed in a subset of corticotrophs in the tumor also containing 4+ immunoreactivity for GH. Of normal anterior pituitary, as shown by staining for Gal-IR these prolactinomas, two contained distinct ACTH-IR and and ACTH-IR (Figure 2A). In contrast, there was no ap- two were focally positive for ACTH (Tables 1 and 2). All of parent colocalization of galanin with the other anterior pi- the 11 tumors from the acromegalic patients were immu- tuitary hormones, including PRL, TSH, LH, and GH. As noreactive for GH (graded as 3 + or 4+) and PRL examples, Gal-IR cells (brown) were of a distinctly different cell size and shape from the smaller cells (purple), which were immunoreactive for GH (Figure 2B) or PRL 124o (Figure 2C).

Northern Blot Analysis Total pituitary RNA isolated from postmortem specimens was separated by agarose gel electrophoresis, blotted onto a nylon membrane, and hybridized to a 32P-labeled galanin cDNA probe. This probe contains the full coding 850I: sequence for human galanin (Strauss and Kaplan, unpublished data). As shown in Figure 3, there was a single 850-nucleotide band representing galanin mRNA in human pituitary. Galanin mRNA was present at similar levels in pituitaries from men and women (Figure 3), and was considerably more abundant than galanin mRNA in the pituitary of normal rats of either sex (not shown). Figure 3. RNA blot hybridization analysis. Total RNA was isolated from humanpituitary tissue, separated by gel electrophoresis, blotted, andhybridized to a 32P-klbeled cDNAprobe encoding human In Situ Hybridization galanin as described in Methods. Fifteen micrograms oftotal RNA was loaded in each lane. The concentration of galanin mRNA A 35S-labeled antisense cRNA probe was prepared from from malepituitary (lane 1) is approximately equal to that ofthe female (lane 2). 7Te arrow sbows the single hybridizing band, a human cDNA clone and hybridized to tissue sections, migrating as an approximately 850 base RNVA species. 902 Hsu et al AJP April 1991, Vol. 138, No. 4 Galanin in Human Pituitary 903 AJP Aptil 1991, Vol. 138, No. 4

Figure 4. In situ hybridization of normnal human pituitary tissue with a 35S-labeled cDNA probe encoding human galanin (A,C) and immunoperoxidase stainingfor ACTH in an adjacentparaffin section (B). Hybridization ofgalanin is seen over clusters ofcells (A,C) in the same focal areas as the ACTH-positive cells (B). Arrows point to corresponding groups of cells containing both galanin mRNA and ACTH-IR. Asterisks mark corresponding cells in each section (methyl green counterstain in A and C, hematoxylin counterstain in B, A,B: x130; C: x320). Figure 5. Immunoperoxidase staining ofan ACTH-secretingpituitary adenoma in a patient with Cushing's disease. Adjacent paraffin sections were stainedfor galanin (A), and ACTH (B). 7be majority of tumor cells contain both Gal-IR and ACTH-IR (bematoxylin counterstain, x320).

(graded as 1 + to 4 +). Seven of the eleven tumors also of the five galanin-positive, GH-cell tumors were also showed strong immunoreactivity for the a-subunit of the ACTH-positive (Table 2, Figure 6). The heterogenous glycoprotein hormones (TSH, FSH, and LH), and one group of nonfunctioning tumors showed less strict corre- was weakly positive for a. Of these 11 tumors, five were spondence (six of nine) for the two peptides. immunoreactive for ACTH (graded as 2+ to 4+) (Ta- Conversely there was no significant association be- ble 2). tween the presence of galanin and the other anterior pi- The nonfunctioning pituitary adenomas were immuno- tuitary hormones in these tumors. As noted, only 14% of reactive for a variety of hormones (Table 1), most com- the PRL-secreting tumors and 45% of the GH-secreting monly the a- and 13-subunits of the glycoproteins. Eleven tumors were galanin positive. No apparent correlation of eighteen contained cells immunoreactive for at least was detected between the presence of Gal-IR and any of two of the p-subunits as well as the a-subunit. Two con- the glycoprotein hormones in the nonfunctioning tumors. tained predominantly a-subunit-IR, and another con- Furthermore, although many of the tumors from the non- tained only ,B-subunits and PRL. In addition, one 'nonfunctioning and the GH-secreting groups contained functioning' tumor was strongly positive for PRL, suggest- a-subunit, these a-immunoreactive tumors were divided ing that it was a clinically silent prolactinoma. When these evenly between the galanin-positive and galanin- nonfunctioning tumors were examined for ACTH-IR, 8 of negative tumors. 18 stained positively (graded as 1 + to 3 +). Gal-IR in Crooke's Hyalinized Cells, Hyperplastic Corticotrophs, and Lung Galanin Immunoreactivity in Pituitary Adenomas Carcinoid Tumors Crooke's hyalinization is a specialized feature of nonad- As summarized in Table 1, unequivocal Gal-IR (graded enomatous corticotrophs found in the pituitary tissue of as 2+ to 4+) was observed in 16 of 19 (84%) corti- Cushing's disease. Crooke's hyalinized cells are charac- cotroph cell tumors associated with Cushing's disease. In terized by the large accumulation of hyaline materials in most of these tumors, a majority of cells were positive for the cytoplasm, displacing secretory granules to the pe- both galanin and ACTH (Figure 5A, B). Gal-IR was also riphery of cells. Gal-IR was observed in the periphery of present in 5 of 1 1 (45%) GH-secreting tumors and 9 of 18 these ACTH-immunoreactive cells (not shown), further (50%) of the nonfunctioning tumors. Only 2 of 14 (14%) confirming the association between galanin and ACTH. prolactinomas were found to contain unequivocal Gal-IR. In addition, in pituitary tissue from three patients with Of interest, however, ACTH-IR was detected in discrete Cushing's disease in whom no tumor was identified, subsets of cells in both of these tumors. The correspon- strong Gal-IR was present in the hyperplastic ACTH cells. dence between Gal-IR and ACTH-IR was further demon- Two lung carcinoid tumors contained many ACTH-IR strated in the tumors from patients with acromegaly. Four and Gal-IR cells. Staining of adjacent sections (Figure 7A,

Table 2. ACTH Immunoreactivity in Galanin-containing Human Pituitary Tumors Gal-IR % of Gal-IR (2-4+) that n (2-4+) 1 + - are also ACTH-IR Corticotrophic 19 16*/1 6t 1 */1 t 2t/2§ 100 Prolactinomas 13 2/2 2/2 0/9 100 GH cell adenomas 11 4/5 1/2 0/4 80 Nonfunctioning 18 6/9 1/5 1/4 66 * ACTH positive and galanin positive. t Galanin positive. t ACTH positive but galanin negative. § Galanin negative. 904 Hsu et al AJP Apil 1991, Vol. 138, No. 4

:'-:7 %-- I - 9 * A

Figure 6. Immunoperoxidase staining of a GH-secreting pituitary adenoma from a patient with clinical acromegaly. Adjacent paraffin sections were stainedfor:galanin (A), GH (B), ACTH (C), andPRL (D). Areas ofthis GH-containing tumor that show diffuse Gal-IR (A) also stainfor ACTH (C). Only occasional tumor cells arepositivefor PRL (D) (hematoylin counterstain, x200). Figure 7. Immunoperoxidase staining ofa lung carcinoid tumor. Adjacentparaffin sections were stainedforgalanin (A), andACTH (B), revealing Gal-IR andACTH-IR in the same clusters of tumor cells (hematoxylin counterstain, X320). B) showed that the two peptides were located in the absorption of galanin antiserum with purified synthetic same clusters of cells. porcine galanin completely abolished Gal-IR in sections In each of the studies of Gal-IR described above, pre- tested. Galanin in Human Pituitary 905 AJP Aptil 1991, Vol. 138, No. 4

Subcellular Localization of Gal-IR the Cushing's disease-associated pituitary tumors, cells were uniformly characteristic of corticotrophs. Gal-IR was We used immunoelectron microscopy to determine the observed in many such cells, and was largely associated subcellular localization of Gal-IR within pituitary cells. In with secretory granules measuring 200 to 250 nm in di- normal pituitaries, Gal-IR was observed in cells with the ameter (Figure 9A, B). ultrastructural characteristics of corticotrophs: round nuclei and prominent nucleoli, secretory granules measuring 250 to 500 nm, with the majority at 400 nm, large Discussion lysosomal bodies ('enigmatic bodies'30), and bundles of microfilaments measuring 7 nm in width. High-density Our results demonstrate that galanin and galanin mRNA material corresponding to Gal-IR was most often associ- are abundant in normal human pituitary. Furthermore pi- ated with polyribosomes and dilated rough endoplasmic tuitary galanin expression is lineage specific and limited reticulum, with less immunoreactivity in association with to a subset of corticotrophic cells. Similar cell-specific secretory granules (Figure 8A). In control sections incu- expression is observed in human pituitary tumors, where bated with PBS in place of the galanin antibody, the ob- Gal-IR is most prevalent in corticotroph cell adenomas served high-density material was absent (Figure 8B). In associated with Cushing's disease.

Figure 8. Immunoelectron microscopy of nonral human pituitary to detect immunoreactive galanin. A: A corticotroph cell with characteristic bundles of type 1 microfilaments and secretory granules ranging from 250 to 400 nm in diameter in the cytoplasm. 7The electron-dense galanin reaction product is seen associated with polyribosomes (arrows) in the cytoplasm, rendering these structures very conspicuous (X21,300). B: A corticotroph cell in a control section, incubated with PBS in place of the primary antibody, shows no immunoreactivity. In the absence of the immuno-reaction product, polyribosomes are lessprominent than in A (X21,340). Bars represent 1 ,u 906 Hsu et al AJP April 1991, Vol. 138, No. 4 Galanin in Human Pituitary 907 AJP Apil 1991, Vol. 138, No. 4

Figure 9. Immunoelectron microscopy oftwo corticotroph cell adenomas (A,B) to detect immunoreactivegalanin. Cells are characterized by the presence ofprominent microfilaments. 7Te electron-dense reaction product is seen associated with small secretory granules (200 to 250 nm in diameter), as well as scattered in the cytoplasm amidst various organelles. Arrowspoint to the margin of immunoreactive cells. (A:x 14,250; B:x17,750). Bars represent 1 F.

These observations are largely consistent with the re- galanin in the ME decreased by 30%, whereas the pitu- cent findings of Vrontakis and coworkers,7 who demon- itary galanin concentration doubled. Both effects were strated the presence of galanin immunoreactivity in hu- reversed by glucocorticoid administration. These obser- man pituitary corticotrophs and corticotroph cell adeno- vations provide further support for the involvement of mas. In contrast to their results, however, we also galanin in the regulation of hypothalamic-pituitary-adrenal observed Gal-IR in several of the noncorticotrophic tu- function. The exact nature of that involvement remains mors, where the presence of galanin correlated closely unclear, however, and the observed differences in func- with the presence of ACTH-IR. Additional correlations be- tion and regulation of galanin located within the hypothal- tween galanin and ACTH were observed in normal ba- amus and pituitary suggest that its regulatory role is com- sophil invasion cells in the posterior pituitary, Crooke's plex. hyalinized cells, and two lung carcinoid tumors. Thus In contrast to the situation in rats, where galanin is galanin and ACTH are coexpressed in a variety of normal found primarily in estrogen-stimulated lactotroph cells and neoplastic tissues. and its expression is strongly responsive to estrogen,56 The presence of galanin mRNA in corticotrophs sug- we have shown that human galanin is located in a differ- gests that the observed Gal-IR is produced by these cells ent subset of pituitary cells, the ACTH-producing cells. rather than taken up from the extracellular space. The We speculated earlier on the physiologic significance of similar distribution of mRNA and immunoreactivity indi- this species-dependent variation. This different pattern of cates that the cell type specificity is mediated at the level cell type specificity may also disclose important informa- of gene expression. As well, this colocalization of Gal-IR tion about the regulation of human pituitary hormone and galanin-specific mRNA provides additional confir- gene expression and development. In the rat and mouse, mation of the specificity of the galanin antiserum. Cross- pituitary hormone gene expression is largely regulated at reactivity of the galanin antiserum with ACTH is also un- the level of RNA transcription. A cell-specific transcription likely because only a subset of the normal ACTH- factor (pit-1; GHF-1) has been isolated that regulates the containing cells contained Gal-IR. Furthermore there is no expression of GH and PRL,32,33 and another has been obvious similarity between the structures of these two suggested to activate TSH-,B gene expression.34 Al- peptides, and the antiserum used was raised against though these factors cannot fully account for cell type synthetic porcine galanin, all but eliminating the possibil- specificity (eg, a factor besides pit-1 is required to dist- ity of contamination with other peptides. ingish PRL from GH cells), they do provide a basis for The observed coexpression of galanin and ACTH in understanding the development of differentiated cell pituitary cells (and at least two examples of other neuro- types within the pituitary.35 In the rat pituitary, there is an endocrine cells) suggests that the genes encoding these ordered pattern of cellular differentiation, with the sequen- two peptides may be similarly regulated. Galanin expres- tial appearance of specific differentiated phenotypes.36 sion may be subject to regulation by many of the stimuli The cell type specificity of pituitary galanin expression in that control ACTH synthesis and release, including corti- the rat is similar to that of pit-1 protein, suggesting that the costeroids, corticotropin-releasing hormone, stress, and rat galanin gene may be transcriptionally regulated by dehydration. Such coregulation would implicate galanin this factor. The variant cell type specificity of human gala- in the control of the human hypothalamic-pitui- nin may arise from differences in the regulatory regions of tary-adrenal axis. It is not possible to examine prospec- the human and rat galanin genes. Alternatively the genes tively the regulation of galanin expression, or the local may be structurally similar but regulated differently in the actions of galanin, within the human pituitary. Studies in two species because of variations in the expression or rats demonstrated that galanin inhibits stress-induced activity of cell-specific transcription factors. Altered spec- ACTH secretion, however, and that intravenous injection ificity of transcription factors in different species could of anti-galanin antisera increases plasma ACTH change the patterns of cellular development as well as concentrations.23 Although these effects appear to be the anatomic and functional relationships of pituitary cell modulated primarily at the level of the hypothalamus, a subtypes. We have recently isolated cDNA and genomic local pituitary effect of galanin has not been excluded. clones encoding rat and human galanin. Further charac- Involvement of galanin in the rat hypothalamic- terization of these clones will help to determine whether pituitary-adrenal axis was also suggested by our studies the different pituitary cell type specificity of human and rat of adrenalectomized animals.31 In response to bilateral galanin expression results from variations in the two adrenalectomy, we observed that the concentration of genes or altered cell phenotypes. In either case, detailed 908 Hsu et al AJP April 1991, Vol. 138, No. 4 characterization of the regulation of the two galanin 3. Rehfeld JF, Lindholm J, Andersen BN, Bardram L, Cantor P, genes will provide important insight into the control of cell Fenger M, LOdecke DK: Pituitary tumors containing chole- differentiation within the anterior pituitary. cystokinin. N Engl J Med 1987, 316:1244-1247 Cholecystokinin, another gastrointestinal peptide with 4. Hsu DW, Riskind PN, Hedley-Whyte ET: Vasoactive intesti- a neuroanatomic distribution similar to galanin, has been nal peptide in human pituitary gland and adenomas: An detected in normal human anterior pituitary and pituitary immunocytochemical study. Am J Pathol 1989, 135:329- tumors.3 Elevated tissue concentrations of CCK were 338 with Nelson's 5. Kaplan LM, Gabriel SM, Koenig JI, Sunday ME, Spindel ER, found in pituitary tumors from patients syn- Chin WW: Galanin is an estrogen-inducible secretory prod- drome, Cushing's disease, and acromegaly. All of these uct of the rat anterior pituitary. Proc Natl Acad Sci USA 1988, adenomas contained ACTH, suggesting a similar pattern 85:7408-7412 of CCK and galanin expression in the human pituitary. In 6. Hsu DW, El-Azouzi M, Black PMcL, Chin WW, Hedley- guinea pigs, CCK-IR is found in a large subset of the Whyte ET, Kaplan LM: Estrogen increases galanin immuno- corticomelanotrophs in the anterior pituitary, as well as in reactivity in hyperplastic prolactin-secreting cells in Fischer cell bodies in the arcuate nucleus and nerve endings in 344 rats. Endocrinology 1990, 126:3159-3167 the external layer of the ME.37 In rats, CCK colocalizes 7. Vrontakis ME, Sano T, Kovacs K, Friesen HG: Presence of with CRF and vasopressin in the PVN,38 and has been galanin-like immunoreactivity in nontumorous corticotrophs shown to stimulate ACTH release from cultured rat ante- and corticotroph adenomas of the human pituitary. J Clin rior pituitary cells.-8 39 The similar patterns of pituitary and Endocrinol Metab 1990, 70:747-751 hypothalamic expression of CCK and galanin make it 8. Tatemoto K, Rokaeus A, Jornvall H, McDonald TJ, Mutt V: tempting to speculate that these two peptides may jointly Galanin-A novel biologically active peptide from porcine participate in the regulation of ACTH release. intestine. FEBS Lett 1983, 164:124-128 Cell lineage specificity is not an invariant characteris- 9. Skofitsch G, Jacobowitz DM: Immunohistochemical map- tic of pituitary peptides. In contrast to galanin and CCK, ping of galanin-like neurons in the rat central nervous sys- VIP shows no preferential colocalization with any of the tem. Peptides 1985, 6:509-546 human pituitary tumor subtypes. VIP-IR has been identi- 10. Melander T, Hokfelt T, Rokaeus A, Fahrenkrug J, Tatemoto K, Mutt V: Distribution of galanin-like immunoreactivity in the fied in all types of pituitary tumors and colocalizes with gastro-intestinal tract of several mammalian species. Cell their various hormone products. In normal human pitu- Tissue Res 1985, 239:253-270 itaries, VIP is located preferentially but not exclusively in 11. Melander T, Hokfelt T, Rokaeus A, Cuello AC, Oertel WH, lactotrophs.4 Verhofstad A, Goldstein M: Coexistence of galanin-like im- In conclusion, the population of pituitary cells express- munoreactivity with catecholamines, 5-hydroxytryptamine, ing galanin is dependent on the species examined. Al- GABA and neuropeptides in the rat CNS. J Neurosci 1986, though the full implications of this species and cell lin- 6:3640-3654 eage specificity are currently unknown, the observed 12. Ch'ng JLC, Christofides ND, Anand P, Gibson SJ, Allen YS, variation suggests that further examination of the physio- Su HC, Tatemoto K, Morrison JFB, Polak JM, Bloom SR: logic factors and molecular processes that regulate the Distribution of galanin immunoreactivity in the central ner- human and rat galanin genes will provide important in- vous system and the response of galanin-containing neuro- sight into the functions of this peptide and the control of nal pathway to injury. Neuroscience 1985, 16:343-354 pituitary cell differentiation. 13. Palkovits M, Rokaeus A, Antoni FA, Kiss A: Galanin in the hypothalamo-hypophyseal system. Neuroendocrinology 1987, 46:417-423 Acknowledgments 14. Melander T, HokfeltT, Rokaeus A: Distribution of galanin-like immunoreactivity in the rat central nervous system. J Comp The authors thank Dr. N. T. Zervas for his encouragement and Neurol 1986, 248:475-517 support and Dr. Salvatore Raiti of the National Hormone and 15. 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