br Acknowledgments The research leading to these
Acknowledgments The research leading to these results was funded by European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 222719 – LIFECYCLE and by the Foundation for Science and Technology of Portugal (FCT), through projects PTDC/MAR-BIO/3890/2012 and PEst-C/MAR/LA0015/2011 and fellowships to PISP (SFRH/BPD/25247/2005) and RSTM (SFRH/BPD/66742/2009). The authors acknowledge Natalia Moncaut and Elsa Couto for practical help.
Introduction Galanin is a neuropeptide isolated from the porcine intestine and is built up of 29 PRIMA-1 (Tatemoto et al., 1983) versus 30 in humans (Bersani et al., 1991, Evans and Shine, 1991, Schmidt et al., 1991). It is broadly expressed in the brain and spinal cord (Melander et al., 1986, Skofitsch and Jacobowitz, 1985), and also in the peripheral nervous system and in endocrine glands including the pituitary (Polak et al., 1991). The most pronounced effect of galanin on the hypophysis in human is to stimulate growth hormone (GH) secretion (Bauer et al., 1986), but galanin has also been associated with other neuroendocrine axes (Merchenthaler, 2010). Interestingly, both in rat (Meister et al., 1989) and monkey (Meister et al., 1990) galanin is co-localized with growth hormone-releasing hormone (GHRH) in nerve endings in the external layer of the median eminence, thus probably being co-secreted with this releasing hormone. Galanin is present in all normal corticotrophs in human post-mortem pituitaries (hpmPs) and also in human pituitary tumors, with the highest levels in adreno-corticotropin hormone (ACTH)-producing adenomas (Polak et al., 1991, Bennet et al., 1991, Hsu et al., 1991, Hulting et al., 1989, Invitti et al., 1999, Sano et al., 1991, Vrontakis et al., 1990, Grenbäck et al., 2004). In fact, galanin has been related to somato- and lacto-troph hyperplasia and adenoma formation in rat and mouse pituitaries (Hulting et al., 1989, Moore et al., 1994, Wynick et al., 1993). Nevertheless, Cushing’s disease patients with galanin-IR positive tumors have better surgical outcome, suggesting that the expression of galanin is related to a less aggressive tumor phenotype (Leung et al., 2002). However, galanin has also been associated with several endocrine and other types of tumors and in some cases correlated to tumor stage, influencing cell proliferation and survival (Kanazawa et al., 2010, Rauch and Kofler, 2010). The effects of galanin are exerted through at least three G-protein-coupled receptors, GalR1–3 (Branchek et al., 2000). In rat they are widely distributed, the GalR1 and –R2 especially in the central nervous system, whereas GalR3 has a more limited distribution and is mainly found in peripheral organs (Waters and Krause, 2000, O’Donnell et al., 2003). In the rat anterior pituitary, as in many other peripheral tissues in this species, only GalR2 and -R3 could be detected (Waters and Krause, 2000). Lack of good antibodies against the human (and rodent) galanin receptors is a limiting factor for the study of protein expression in general, which appears to be the case, also for galanin antibodies (Michel et al., 2009, Lu and Bartfai, 2009). Thus, for this study on tumors we have chosen to use quantitative real time PCR (qPCR) to investigate the transcript levels of galanin and its three receptors in adenomatous tissue from thirteen human pituitary tumors after surgery. As controls we used tissues from twelve normal human pituitaries. Some of these results were presented at meetings (Barde S et al., 2011, Tofighi et al., 2008a).
Results The twelve human post-mortem pituitaries used as controls expressed transcripts for galanin, GalR1 and R2 but not GalR3. All tumors expressed galanin at low levels, except for the two ACTH-producing tumors and one patient with acromegaly who all showed high levels. The thirteen pituitary adenomas showed individual patterns for the three receptor mRNAs. Data on the patients are given in Table 1, Table 2 and Fig. 1. Both sexes and different types of tumors are represented among the thirteen adenomas.