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. 897 898 Hsu et al AJP April 1991, Vol. 138, No. 4 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) intra- venous 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 adrenocortico- tropic 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 pitu- itary 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