**Autocrine and Paracrine Regulation of Prolactin Secretion by Prolactin Variants and by Hypothalamic Hormones**

Flavio Mena, Nilda Navarro and Alejandra Castilla

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51681

#### **1. Introduction**

96 Prolactin

317–324.

1986; 103: 493–507.

2011c; 31: 921-927.

68:6377–6386.

7089.

Rosnack KJ, Stroh JG, Singleton DH, Guarino BC, Andrews GC. Use of capillary electrophoresis-electrospray ionization mass spectrometry in the analysis of synthetic

Prevot V, Rio C, Cho GJ, Lomniczi A, Heger S, Neville CM, Rosenthal NA, Ojeda SR, Corfas G. Normal female sexual development requires neuregulin-erbB receptor signaling in

Sharif A, Duhem-Tonnelle V, Allet C, Baroncini M, Loyens A, Kerr-Conte J, Collier F, Blond S, Ojeda SR, Junier MP, Prevot V. Differential erbB signaling in astrocytes from the cerebral cortex and the hypothalamus of the human brain. Glia 2009; 57: 362–379. Spuch C, Diz-Chaves Y. Fibroblast growth factor-1 and epidermal growth factor modualate prolactin responses to TRH and dompamine in prmary cultures. Endocrine 2006; 29:

Taylor SB, Taylor AR, Koenig JI. The interaction of disrupted Type II Neuregulin 1 and chronic adolescent stress on adult anxiety and fear related behaviors. Neuroscience

Ueno Y, Sakurai H, Tsunoda S, Choo MK, Matsuo M, Koizumi K, Saiki I. Neuregulininduced activation of ErbB3 by EGFR tyrosine kinase activity promotes tumor growth

Usdin TB, Fischbach GD. Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes. J Cell Biol

Vlotides G, Siegel E, Donangelo I, Gutman S, Ren SG, Melmed S. Rat prolactinoma cell growth regulation by epidermal growth factor receptor ligands. Cancer Res 2008;

Vlotides G, Cooper O, Chen YH, Ren SG, Greenman Y, Melmed S. Neuregulin regulates

Witters L, Scherle P, Friedman S, Fridman J, Caulder E, Newton R, Lipton A. Synergistic inhibition with a dual epidermal growth factor receptor/HER-2/neu tyrosine kinase inhibitor and a disintegrin and metalloprotease inhibitor. Cancer Res 2008; 68:7083–

Yuan G, Qian L, Song L, Shi M, Li D, Yu M, Hu M, Shen B, Guo N. Neuregulin-beta promotes matrix metalloproteinase-7 expression via HER2-mediated AP-1 activation in

Zhao W, Zhao X, Peng S, Pan H, Ma Z, Shen Y. Expression and localization of cell adhesion molecules in the pituitary of C57BL 6 mice. J Shantou Univ Med Coll 2010; 23: 65–67. Zhao W, Ren S. Neuregulin-1 (Nrg1) is mainly expressed in rat pituitary gonadotrope cells and possibly regulates prolactin (PRL) secretion in a juxtacrine manner. Journal of

Zhao W, Shen Y, Ren S. Endogenous expression of Neuregulin-1 (Nrg1) as a potential modulator of prolactin (PRL) secretion in GH3 cells. Cell Tissue Res 2011b; 344:313–320. Zhao W, Ren S. Endogenous Neuregulin-1 expression in the anterior pituitary of female Wistar-Furth rats during the estrous cycle. Journal of Southern Medical University

peptides. J Chromatogr A 1994; 675: 219–225.

hypothalamic astrocytes. J Neurosci 2003; 23: 230–239.

2012; http://dx.doi.org/10.1016/j.neuroscience.2012.09.045

and metastasis in melanoma cells. Int J Cancer 2008; 123: 340–347.

prolactinoma gene expression. Cancer Res 2009; 69: 4209–4216.

MCF-7 cells. Mol Cell Biochem 2008; 318: 73–79.

Neuroendocrinology 2011a; 23:1252-1262.

The synthesis and release of prolactin (PRL) by lactotrophs in the anterior pituitary (AP) are regulated by factors produced in the hypothalamus as well as in the posterior and neurointermediate pituitary lobes, by autocrine and paracrine signals from the anterior pituitary itself (Ben-Jonathan & Hnasko, 2001; Kordon, 1985; Denef, 1988; Denef, 2008; Freeman et al., 2000; Lorenson and Walker, 2001; Schwartz & Cherny, 1992; Schwartz, 2000; Wang & Walker, 1993; Sinha, 1992; Sinha 1995; Moore et al., 2002; Bollengier et al., 1989; Bollengier et al., 1996; Kadowaki et al., 1984; MacLeod et al., 1966; Sgouris & Meites, 1953; Chen et al., 1968; Welsch et al., 1968) and also by gonadal steroids (Maurer & Gorski, 1977; Maurer, 1982). In addition, it has been reported that total PRL and PRL variants (Denef, 2008; Shah & Hymer, 1989) are secreted under different physiological conditions (Denef, 2008; Wang & Walker, 1993; Sinha, 1992; Sinha 1995; Mena et al., 1984; Mena et al., 1992; Boockfor & Frawley, 1987). And, it is known that functional interactions and cytological differences exist among pituitary lactotrophs within the anterior pituitary gland (Denef, 1988; Schwartz & Cherny, 1992; Schwartz, 2000; Boockfor & Frawley, 1987) and that functional variations (Boockfor & Frawley, 1987; Boockfor et al., 1986; Frawley & Boockfor, 1991; Nagy & Frawley 1990), as well as autoregulation (Nagy et al., 1991) and interactions with other pituitary cells (Denef, 2008; Sinha, 1992; Moore et al., 2002; Kadowaki et al., 1984) and with hypothalamic hormones (Ben-Jonathan & Hnasko, 2001; Chen et al., 1968) occur in different circumstances. For instance, lactotrophs from the central AP region of lactating rats, i.e., the region surrounding the neurointermediate pituitary lobe (Boockfor & Frawley, 1987; Frawley & Boockfor, 1991; Papka et al., 1986) are bigger, secrete more PRL than those of the peripheral AP region and after a short period of suckling become more sensitive to the PRL-stimulatory agents, TRH and angiotensin II; moreover, they become unresponsive

© 2013 Mena et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

to dopamine; and interact with lactotrophs in the peripheral region of the gland (Boockfor & Frawley, 1987; Boockfor et al., 1986; Frawley & Boockfor, 1991; Nagy et al., 1991; Nagy & Frawley 1990; Diaz et al., 2002). In these studies, it is possible that the release of PRL variants may have influenced the regulation of PRL release.

Autocrine and Paracrine Regulation of Prolactin Secretion by Prolactin Variants and by Hypothalamic Hormones 99

the *in vitro* release of PRL from pituitary glands of male rats in a dose-dependent manner

Our results suggest that PRL variants are released into the CM from the central and peripheral AP regions of lactating rats, that they interact and selectively and specifically stimulate or inhibit the *in vitro* release of other PRL variants from lactotrophs of lactating rat APs; that hypothalamic hormones selectively regulate and interact with PRL variants released from AP lactotrophs, and finally, whether in response to CM's from lactating rats, changes in electrical activity (EA) occur in male lactotrophs, as well as in astrocytes from the central nervous system, and in intracellular calcium concentration in sympathetic neurons

Animal studies were performed under a protocol similar to the USPHS Guide for the Care and Use of Laboratory Animals and the Official Mexican Guide from Secretary of Agriculture (SAGARPA NOM-062-Z00-1999) published in 2001. Wistar primiparous lactating rats (8-10 pups per litter) were housed individually in a room with a reversed lightdark cycle (14 h light, 10 h darkness) and constant temperature (23-25°C) and were fed *ad libitum* (Purina Chow, Ralston Purina Co., Chicago IL, USA). On postpartum days 10-12 (7 am, local time) groups of mothers had their pups removed, and 6 h later their pups were or were not returned to the mothers and suckled for 15 min. At the end of the suckling or nonsuckling periods, the mothers were killed by decapitation after light ether anesthesia. From all animals employed (see below), the pituitary was removed under a dissecting microscope, the posterior lobe was discarded and, using fine forceps as originally described by Papka et al. 1986, and by Bookfor & Frawley, 1987, the central region around the neurointermediate lobe and the peripheral region i.e., the rest of the AP tissue (Boockfor & Frawley, 1987; Diaz et al., 2002) were dissected independently, and incubated in Earle's medium as described

In individual flasks containing 300 μl of Earle's medium, media were conditioned by incubating tissue fragments corresponding to the central (CR) and peripheral (PR) pituitary regions from lactating rats. The pituitary fragments were incubated immediately after removal to prevent disruption of hormone storage dynamics (Mena et al., 1992; Diaz et al., 2002). Flasks containing the pituitary fragments were gassed with 95% O2, 5% CO2, sealed with rubber stoppers and incubated at 37°C in a water bath shaker (American Optical, Buffalo NY, USA) for 1h. CM from pituitary fragments of each group of rats employed was concentrated and desalted in a Centricon micro-concentrator (Centripep, Millipore, Bredford MA, USA) and stored frozen until assayed, along with the corresponding primary

(Huerta-Ocampo et al., 2007; Mena et al., 2010).

**2.2. Preparation of concentrated conditioned media** 

cultures of pituitary cells or with cultures of sympathetic neurons.

(Mena et al, 2012b).

**2.1 General** 

below.

**2. Materials and methods** 

In previous reports (Huerta-Ocampo et al., 2007; Mena et al., 2010) we showed that conditioned media (CM) and PRL variants i.e., from 7-14 and 70-97 kDa, from lactating rat APs, characterized by Western blotting and eluted from SDS-PAGE, promoted the *in vitro* vesicular release of the hormone from preformed, mature PRL granules of male rat APs, and that such release was independent of PRL synthesis (Huerta-Ocampo et al., 2007). Autocrine and paracrine types of actions have also been shown to occur within the AP (Denef, 2008; Freeman et al., 2000; Lorenson and Walker, 2001; Schwartz & Cherny, 1992; Schwartz, 2000; Welsch et al., 1968; Diaz, et al., 2002; Huerta-Ocampo et al., 2007; Mena et al., 2010), and were demonstrated when the central and peripheral AP regions of lactating rats were incubated *in vitro* with CM from pituitaries of lactating, pregnant and steroid-treated castrated males or females, but not from untreated castrated rats, intact male rats or by a PRL Standard (Huerta-Ocampo et al., 2007; Mena et al., 2010). Also, more potent effects occurred with CM from APs of early- than from mid- or late- lactating rats and from rats non-suckled for 8 or 16 h than from those non-suckled for 32 h (Mena et al., 2010). These results suggest that, under certain conditions, PRL variants released from lactating and nonlactating rat APs may regulate the release of PRL variants from the lactotrophs.

In the present study, CM proteins, i.e., PRL variants, that were released *in vitro* from the AP regions of lactating rats were separated and electroeluted from SDS-PAGE and tested using *in vitro* incubation techniques. We sought to determine first, whether PRL variants, which are known to occur within the AP (Schwartz & Cherny, 1992; Schwartz, 2000; Wang & Walker, 1993; Sinha, 1992; Bollengier et al., 1989; Huerta-Ocampo et al., 2007; Mena et al., 2010; Mena & Grosvenor, 1972; Asawaroengchai et al., 1978; Nicoll et al., 1969; Mansur & Hymer, 1985), and are released *in vitro* (Mena et al, 1984; Mena et al., 1992; Huerta-Ocampo et al., 2007; Mena et al., 2010; Mena & Grosvenor, 1972; Grosvenor et al., 1967; Grosvenor et al., 1979; Mena et al., 1989; Mena et al., 1993) after the suckling-induced PRL transformation i.e., the transfer of the hormone from a pre-releasable to a releasable state (Mena & Grosvenor, 1972; Grosvenor et al., 1967; Mena et al., 1993) would influence the release of PRL variants from lactating rat lactotrophs; and second, whether the effects of dopamine (DA), thyrotropin-releasing hormone (TRH) and oxytocin (OT) upon PRL release would manifest their effects upon PRL secretion by regulating the release of PRL variants from lactating rat lactotrophs (Mena et al., 2011).

Several reports indicate that PRL has some neuro and gliatrophic properties, and that it mediates the development and maturation of dopaminergic neurons in the hypothalamopituitary system (Möderscheim et al., 2007). We showed previously (Morales et al., 2001) that intrathecal injection of PRL in the spinal cord promoted the sympathetic inhibition of milk ejection in lactating rats, and that prolactin variants in CM from the central and peripheral regions of the anterior pituitary from lactating, but not from male rats promoted the *in vitro* release of PRL from pituitary glands of male rats in a dose-dependent manner (Huerta-Ocampo et al., 2007; Mena et al., 2010).

Our results suggest that PRL variants are released into the CM from the central and peripheral AP regions of lactating rats, that they interact and selectively and specifically stimulate or inhibit the *in vitro* release of other PRL variants from lactotrophs of lactating rat APs; that hypothalamic hormones selectively regulate and interact with PRL variants released from AP lactotrophs, and finally, whether in response to CM's from lactating rats, changes in electrical activity (EA) occur in male lactotrophs, as well as in astrocytes from the central nervous system, and in intracellular calcium concentration in sympathetic neurons (Mena et al, 2012b).
