Abstract the ROLE of ESTROGEN in ORPHANIN FQ/NOCICEPTIN INDUCED PROLACTIN RELEASE by Prajakta Dinesh Mangeshkar the Role of Estr

Abstract

THE ROLE OF ESTROGEN IN ORPHANIN FQ/NOCICEPTIN INDUCED PROLACTIN RELEASE

By Prajakta Dinesh Mangeshkar

The role of estrogen in modulating the prolactin secretory response to Orphanin FQ/Nociceptin (OFQ/N) and the involvement of hypothalamic dopaminergic neurons in mediating this response were investigated.

The prolactin secretory response to OFQ/N was significantly attenuated in placebo treated female Sprague-Dawley rats compared to estrogen replaced animals. Also, OFQ/N produced a significant decrease in the phosphorylated tyrosine hydroxylase (pTH) to hydroxylase (TH) ratio only in estrogen treated rats, indicating inhibition of hypothalamic dopaminergic neurons. Estrogen treatment produced a significant decrease in pituitary ERα expression levels, but sensitivity to OFQ/N was still higher than in placebo animals. In the hypothalamus, there were several protein bands that may be isoforms of ERα.

These results indicate that estrogen is necessary for the prolactin secretory response to OFQ/N in female rats and that OFQ/N suppresses hypothalamic dopaminergic activity only in the presence of estrogen.

THE ROLE OF ESTROGEN IN ORPHANIN FQ/NOCICEPTIN INDUCED PROLACTIN RELEASE

A Thesis

Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Zoology by

Prajakta Dinesh Mangeshkar Miami University Oxford, OH

2005

Advisor: ______Dr. James Janik

Co-Advisor: ______Dr. Phyllis Callahan

Reader: ______Dr. Kathy Killian

Reader: ______Dr. Paul Harding Table of Contents

INTRODUCTION Prolactin and its Regulation 1 Tyrosine Hydroxylase 1 Gender Differences 2 Role of Ovarian Steroids 3 Endogenous Opiates 5 Orphanin FQ/Nociceptin 5 Orphanin FQ/Nociceptin and Prolactin Release 6

MATERIALS AND METHODS Animals and Treatment 10 Western Blot Analysis 11 Hormone Assays 12 Statistical Analysis 12

RESULTS Effects of Treatment on plasma 17-β estradiol, wet uterine weights and body weights 13 Effect of Estrogen on OFQ/N induced PRL release 13 Hypothalamic pTH and TH expression levels 13 Effect of estrogen on ERα protein expression in the pituitary and hypothalamus 14

DISCUSSION 30

REFERENCES 34

ii List of Tables

TABLE 1. Effects of estrogen on body weight in ovx/placebo and ovx/estrogen female rats. 18

iii List of Figures

FIGURE 1. Plasma 17-β estradiol levels and uterine weight of ovx/estrogen and ovx/placebo animals. 15

FIGURE 2.The effect of estrogen on OFQ/N induced prolactin secretion in ovx female rats. 19

FIGURE 3. The effect of estrogen replacement on hypothalamic pTH and TH levels and on the pTH: TH ratio. 21

FIGURE 4. The effect of OFQ/N administration on hypothalamic pTH and TH levels and on the pTH:TH ratio. 23

FIGURE 5. The effect of OFQ/N on pTH and TH expression levels on the pTH:TH ratio in ovx/estrogen treated animals. 25

FIGURE 6. The effect of estrogen on ERα in ovx female rats. 27

iv 1. Introduction and Background

Prolactin and its Regulation: Prolactin (PRL) is a polypeptide hormone, composed of 197-199 amino acids and has a molecular weight of 23kDa (Goffin et al, 2002). Prolactin plays a major role in the maintenance and stimulation of the mammary gland during lactation, as well as the regulation of a number of diverse physiological processes including homeostasis, growth, reproduction and metabolism (Neill and Nagy, 1994; Goffin et al, 2002).

Prolactin is primarily secreted by the lactotropic cells of the anterior pituitary gland (Goffin et al, 2002). Prolactin secretion is affected by a number of factors, but is primarily under the tonic inhibitory control of hypothalamic dopamine (Freeman et al, 2000). The dopamine (DA) perikarya, which are located in the periventricular (A14) and arcuate nuclei (A12) of the hypothalamus, provide dopamine to the pituitary (Moore and Lookingland, 1995). These neuronal populations are divided into three systems: the tuberohypophysial dopaminergic (THDA) neurons project from the rostral portion of the arcuate nucleus to the intermediate and neural lobes of the pituitary; the periventricular-hypophysial dopaminergic (PHDA) neurons project from the periventricular nucleus to the intermediate lobe of the pituitary; and the tuberoinfundular dopaminergic (TIDA) neurons project their short axons from the dorsomedial arcuate nucleus to the median eminence of the hypothalamus and release DA into hypophysial portal vessels (Moore and Lookingland, 1995; Freeman et al, 2000). Among these neuronal populations, the TIDA neurons are the major regulators of prolactin secretion (Reymond and Porter, 1985; Moore and Lookingland, 1995; Freeman et al, 2000). Dopamine travels, via the portal system, to the anterior lobe of the pituitary gland, binds to pituitary D2 receptors and, through the action of Gi proteins, leads to inactivation of the voltage-gated calcium channels. This tonically inhibits prolactin release from secretory granules, and it also inhibits adenylyl cyclase which suppresses PRL gene expression (Neill and Nagy, 1994; Ben-Jonathan and Hnasko, 2001).

Tyrosine hydroxylase:

Tyrosine hydroxylase (TH) is the rate –limiting enzyme in the biosynthesis of

1 catecholamines (Ikeda et al, 1965; Kumer and Vrana, 1996). TH has a molecular weight of 60kD and occurs as a tetramer, composed of a regulatory N- terminal and catalytic C- terminal region (Campbell et al, 1986, Grennet et al, 1987, Haycock, 1990). Because TH plays a pivotal role in numerous physiological processes, its synthesis is tightly regulated by a number of different factors. The two primary forms of regulation include long- term regulation, involving changes in gene expression, and short – term regulation, involving phosphorylation by different protein kinases (Kumer and Vrana, 1996). Phosphorylation is a generally accepted form of TH activation in vivo (Kaufman 1995) and sites of phosphorylation have been identified on the TH 40 amino acid N-terminal (Haycock et al, 1990). These include four serine sites: Ser8, Ser19, Ser31 and Ser40. Of these four sites, phopshorylation of Ser40 is known to have a major effect on TH activity (Kumer and Vrana, 1996). Recent evidence suggests that the phosphorylation of Ser40 and Ser19 residues results in an open conformation of the TH molecule and that phosphorylation of Ser19 leads to an increase in the rate of Ser40 phosphorylation (Bevilaqua, et al, 2001). The residues around Ser40 (Arg-Arg-Gln-Ser40-Leu) are known to be commonly recognized by PKA, PKC and Cam-PKII (Kumer and Vrana, 1996).

TH is a non-heme iron protein which exists in two forms, ferric and ferrous (Ramsey et al, 1996). Tetrahydropterins activate TH by reducing the ferric to a ferrous form (Ramsey et al, 1996). Cathecholamines like dopamine and dihydroxyphenylalanine (DOPA) can bind to the ferric form and trap it in an inactive form (Ramsey and Fitzpatrick, 2000). However, it has been suggested that the primary effect of phosphorylation at Ser40 is a decrease in the enzyme’s affinity for catecholamines (Ramsey and Fitzpatrick, 1998, 2000). Thus, phosphorylation activates the enzyme by increasing the rate of dissociation of bound catecholamines allowing reduction to the ferrous form to proceed. Because TH phosphorylation represents the activity of the enzyme, and therefore dopamine synthesis, its regulation by OFQ/N and ovarian steroids is a focus of this study.

Gender Differences: There are fundamental sexual differences in the activity of the TIDA neurons that influence prolactin regulation (Gunnet and Freeman, 1982). Although the concentrations of dopamine

2 and the density of the TIDA neurons are similar in males and females (Arbogast and Voogt, 1990), there are sexual differences in TIDA neuronal activity and responsiveness. Dopamine synthesis and turnover in the median eminence of female rats is 6-8 times higher than in male rats (Gudelsky and Porter, 1981). This sex-related difference is not a function of the suppressive effect of androgens, but rather a stimulatory effect of the ovarian steroids (Gudelsky and Porter, 1981). Females have higher TH mRNA levels in the arcuate nucleus (Arbogast and Voogt, 1990), as well as higher concentrations of TH in the median eminence (Porter, 1986). This greater expression of the TH gene in females could account for the higher TH activity in the TIDA neurons (Arbogast and Voogt, 1990). TH activity in females is decreased by ovariectomy and restored by estrogen, however, in males, the reverse occurs; TIDA neuronal activity is increased by orchidectomy and decreased by testosterone (Ben- Jonathan and Hnasko, 2001). Thus, these differences in the TIDA neuronal activity between male and female rats can be attributed to the steroid environment and the modulation of these neurons by the ovarian steroids.

Role of Ovarian Steroids: Estrogen (E) has direct physiological effects on hypothalamic neurons and pituitary cells. Estrogen has both positive and negative effects on the pituitary (for review see Shupnik et al, 2002). In lactotroph cells, E has been shown to stimulate prolactin synthesis and secretion (Lieberman et al, 1981; Scully et al, 1997). Administration of chronic estradiol, alone or in combination with progesterone, modulates the responsiveness of lactotrophs to dopamine by increasing the proliferation and percentage of PRL secreting lactotrophs (Livingstone et al, 1998). Further, estradiol also causes an increase in PRL-receptor (PRL-R) expression in the dorso-medial arcuate nucleus (Lerant and Freeman, 1998). In the absence of ovarian steroids, PRL secretion is low and stable throughout the day while estrogen stimulates the secretion of PRL in ovariectomized rats (Demaria et al, 2000). Estradiol is also known to exert stimulatory as well as inhibitory effects on TIDA neuronal activity. These effects are dependant on concentration, time of administration and duration of action (Arbogast and Hyde, 2000). Long term exposure to estradiol has an inhibitory effect on dopamine synthesis in TIDA neurons: it suppresses basal, calcium-dependent and cAMP- dependent synthesis of dopamine in TIDA neurons (Arita and Kimura, 1987). Acute estrogen

3 treatment decreases the rate of TH gene transcription while longer estrogen treatments increase its transcription (Blum et al, 1987). This increase in TH transcription is due to PRL negative feedback which is triggered by the stimulatory effect of estrogen on PRL synthesis and secretion. Thus longer estrogen treatments stimulate PRL secretion which, in turn, exerts negative feedback on its own secretion by stimulating dopaminergic neurons and therefore increasing TH levels (Blum et al, 1987).

Estrogen modulates gene expression through estrogen receptors (Shupnik et al, 2000). There exists a plethora of estrogen receptors (ERs) in the brain (for review see Toran – Allerand, 2004) including both plasma membrane bound ERs and intranuclear ERs. Among these, ERα, ERβ and TERP-1 are known to be classic receptors which mediate estrogen action in the pituitary (Shupnik 2000, 2002) with ERα present at much higher levels in the pituitary than ERβ (Toran – Allerand, 2004). ERα and ERβ appear to be complementary but are functionally distinct. Estrogen stimulates ERα with more potency than ERβ and ER mediate the effects of estrogen on pituitary lactotrophs For example, in ERα KO mice, but not in ERβ KO mouse, pituitary lactotrophs were atrophied which strongly suggests that ERα, but not ERβ, plays a role in maintaining these cells at the level of the pituitary (Pelletier al, 2003). The hypothalamus also expresses significant levels of ERα and ERβ (Shupnik, 2002). ERα protein and mRNA levels have been detected mainly in the arcuate and ventromedial nuclei; while ERβ has been found in the paraventricular nucleus and preoptic area (Shupnik, 2002). Among the abundant forms of estrogen receptors, a recently identified receptor in the hypothalamus of rat, mouse and baboon termed ERX, seems to differ from ERα but is recognized by the same antibodies to ERα (Toran- Allerand, 2004). Estrogen modulates hypothalamic ERα expression in young female rats (Voogt et al, 2003). Moreover, TH neurons in the arcuate nucleus also express ERα receptors (Herbison et al, 1999) which are downregulated by estrogen. Thus results of these studies suggest that estrogen, acting through its specific estrogen receptor, plays an important role in the regulation of dopaminergic neurons by first inhibiting activity of these neurons and then stimulating an increase in lactrotroph recruitment as well as PRL synthesis and secretion.

4 Endogenous Opiates: Opiate drugs form the major class of strong analgesics. Endogenously produced opiate peptides and their receptors play an important role in central nervous system function (Taylor and Dickenson, 1998). There are four known endogenous opiate peptide (EOP) families. Among these, three are well characterized and considered the “classic” opiate peptides. These include β-endorphin, the enkephalins, and the dynorphins. Orphanin FQ/Nociceptin (OFQ/N) is a more recently discovered 17 amino acid neuropeptide with high amino acid sequence homology with the classic endogenous opiates, especially the dynorphins (Meunier, et al. 1995; Reinscheid, et al. 1995).

Each opiate peptide has a distinct receptor. The “classic” opiate receptors are δ (OP1)

(Evans, et al. 1992; Kieffer, et al. 1992; Yasuda, et al. 1993), κ (OP2) (Meng, et al. 1993;

Minami, et al. 1993; Yasuda, et al. 1993), and µ (OP3) (Chen, et al. 1993; Thompson, et al.

1993). OFQ/N binds to its own unique ORL1 (OP4) receptor subtype (Mollereau, et al. 1994; Meunier, et al. 1995; Reinscheid, et al. 1995; Mollereau, et al. 1996; Lan, et al. 1997). Classic endogenous opiates are known to regulate PRL secretion in a physiologically relevant manner (Neill and Nagy, 1994). Opiate administration results in an increase in PRL secretion and the increase in females is much more robust than in males (Baumann and Rabii 1990; Janik et al. 1992). Furthermore, the mechanism of action of the opiate peptides involves, at least in part, inhibition of dopaminergic neurons (Baumann and Rabii 1990; Callahan et al. 1996; Arbogast and Voogt 1998; Freeman et al 2000).

Orphanin FQ/Nociceptin: Orphanin FQ/Nociceptin (OFQ/N), a heptadecapeptide was discovered simultaneously in porcine (Reinscheid et al, 1995) and rat brain homogenates (Meunier et al, 1995) and later in the bovine brain (Okuda-Ashitaka et al, 1996). OFQ/N shares a close evolutionary relationship with other endogenous opiates, especially dynorphin (Reinscheid et al, 1995; Meunier et al, 1995; Reinscheid et al, 2000). In spite of its homology with the other endogenous opiates, there is a striking difference in the N-terminus of the peptide. OFQ/N has a phenylalanine in place of the tyrosine residue in other opiate peptides. Like the other opiate peptides, OFQ/N is cleaved from a larger amino acid precursor protein, preproOFQ/N

5 (Darland and Grandy, 1998; Reinscheid, et al 2000). OFQ/N precursor mRNA has been localized in the hypothalamus and striatum but not in the anterior pituitary gland (Nothacker et al, 1996; Neal et al, 1999a). Additionally, the OFQ/N peptide has been co-localized in several hypothalamic nuclei with the precursor protein (Henderson and McKnight, 1997; Neal et al, 1999b). Since the hypothalamus regulates anterior pituitary secretion, this anatomic expression of the precursor mRNA and OFQ/N peptide suggests that OFQ/N likely plays a role in neuroendocrine function.

The OFQ/N receptor (OP4), shares 60% homology with the classic opiate receptor subtypes (Dooley and Houghten, 2000). In spite of the structural and functional similarities to other

opiate receptors, OFQ/N binds exclusively to the OP4 receptor with high affinity (Reinscheid et al, 1995). Like the other opiate receptors, OP4 is a Gi-protein coupled receptor with seven transmembrane domains (Chen et al, 1994). It inhibits adenylyl cyclase activity (Reinscheid et al, 1995) which results in inhibition of voltage -gated calcium channels and activation of + inwardly rectifying K current (Reinscheid et al, 1995; Mogil and Pasternak, 2001). The OP4 receptor is found in the limbic areas, the brain stem and is highly concentrated in the arcuate nucleus of the hypothalamus (Neal et al, 1999; Taylor and Dickenson, 1998). OFQ/N has been implicated in a number of physiological functions including pain perception, spatial learning and locomotor function (Mogil and Pasternak, 2001), however its role in neuroendocrine function remains poorly understood.

OFQ/N and Prolactin Release: Our laboratory first demonstrated the stimulatory effects of OFQ/N on prolactin release (Bryant et al, 1998). OFQ/N stimulates prolactin secretion in both male and female rats in a dose and time-related manner with the response in females being of higher magnitude. This stimulatory response is not mediated by any of the classic opiate receptor subtypes (Bryant et al, 2002). Additional studies from our laboratory indicate that TIDA neuronal inhibition was rapid and transient. These results suggest that OFQ/N inhibits TIDA neurons transiently and this inhibition precedes the prolactin increase (Chesterfield et al, in review 2005). Also, a previous study by Sheih and Pan (2001) followed a different time-course but also reported that OFQ/N decreased DOPAC levels in the hypothalamus. This inhibitory effect of OFQ/N

6 on TIDA neuronal activity corresponds well with its stimulatory effect on plasma prolactin levels. Thus the anatomical localization of OFQ/N and its inhibitory effect on hypothalamic dopaminergic neurons clearly suggest that OFQ/N plays an important role in regulating prolactin secretion. However, the reason(s) and mechanisms responsible for the gender differences in the magnitude of the prolactin secretory response need to be elucidated. Hence the goal of this research was to determine the role of estrogen in mediating the prolactin secretory response to OFQ/N and to investigate inhibition of hypothalamic dopaminergic activity as a possible mechanism of action.

7 1. Introduction: The regulation of PRL secretion is complex, but it is primarily under tonic, inhibitory control by the hypothalamic dopaminergic neurons (Neill and Nagy 1994; Moore and Lookingland, 1995; Ben-Jonathan and Hnasko, 2001, Freeman 2000). Although there are 3 major, hypothalamic, dopaminergic pathways, it is the tuberoinfundibular dopaminergic (TIDA) neurons that are most important in controlling prolactin release (Moore and Lookingland, 1995; Ben-Jonathan et al, 2001, Freeman 2000). Other factors modulate prolactin secretion, including estrogen, which has direct effects on both the hypothalamus and pituitary (Ben- Jonathan and Hnasko, 2001), and the endogenous opioid peptides (EOP), which increase prolactin secretion, at least in part, by inhibiting the TIDA neurons (Moore and Lookingland, 1995; Freeman, et al., 2000).

Estrogen (E) has direct physiological effects on hypothalamic neurons and pituitary cells. In lactotroph cells, E has been proposed to stimulate prolactin synthesis and secretion (Lieberman et al, 1981; Scully et al, 1997). Estrogen also exerts stimulatory or inhibitory effects on TIDA neuronal activity depending on its concentration, time of administration and duration of action (Arbogast and Hyde, 2000). Acute estrogen treatment decreases, while chronic estrogen increases the rate of tyrosine hydroxylase (TH) gene transcription, indicating that estrogen affects the activity of these neurons (Blum et al, 1987; Arita and Kimura, 1987). Estrogen modulates gene expression in the pituitary and hypothalamus typically through two estrogen receptors, ERα and ERβ (Shupnik et al, 2000; Toran – Allerand, 2004). ERα protein and mRNA levels have been detected mainly in the arcuate and ventromedial nuclei (Shupnik, 2002). Moreover, TH neurons in the arcuate nucleus also express ERβ (Herbison et al, 1999). This colocalization provides a possible molecular mechanism through which estrogen may act on dopaminergic neurons to modulate prolactin secretion.

The EOP are also involved in the regulation of PRL secretion, exerting a stimulatory effect due, at least in part, to inhibition of TIDA neuronal activity (Moore and Lookingland, 1995; Freeman et al 2000). Orphanin FQ/Nociceptin (OFQ/N) is a more recently identified neuropeptide (Reinscheid et al 1995; Meunier et al, 1995) that exhibits structural and

8 functional similarities to other opiates (Reinscheid et al, 1995), but that binds exclusively to

the OFQ/N receptor (ORL1 or OP4) with high affinity (Dooley and Houghten, 2000). Although the role of OFQ/N in regulating hormone secretions is poorly characterized, localization studies support a possible role for OFQ/N in mediating neuroendocrine processes. OFQ/N precursor mRNA has been localized in the hypothalamus but not in the anterior pituitary gland (Nothacker et al, 1996; Neal et al, 1999). Also, there is significant overlap of OFQ/N peptide and ERβ receptor in the paraventricular nucleus of the hypothalamus (Isgor, et al, 2003) indicating a potential interaction between estrogen and OFQ/N in the regulation of anterior pituitary hormone secretion at the level of the hypothalamus.

We have previously demonstrated that OFQ/N stimulates prolactin release in male and female rats (Bryant et al, 1998) and this response is not mediated by any of the classic opiate receptor subtypes (Bryant et al, 2002). Because the OFQ/N-induced PRL secretory response was greater in females than in males, the purpose of this study was to determine if estrogen modulates the stimulatory effects of OFQ/N on prolactin secretion. Furthermore, levels of total and phosphorylated tyrosine hydroxylase were determined as an indication of estrogen’s modulatory effect on hypothalamic, dopaminergic neuronal activity.

9 2. Materials and Methods

2.1 Animals and treatment:

Virgin female Sprague-Dawley rats ( 200-250g) (Harlan Labs, Indianapolis, IN, USA) were housed in standard rat cages under conditions of controlled lighting (12 light:12 dark) and temperature (21°C). Food and water were provided ad libitum. All animal procedures were approved by Miami University Institutional Animal Care and Use Committe. Rats were bilaterally ovariectomized (OVX) under isoflurane anesthesia using the dorsal approach and were implanted with placebo pellets or pellets containing 17β-estradiol with an estimated release rate of 0.05 mg/day (Innovative Research of America, FL) at the time of OVX (Brann et al, 1993; Johren et al, 1997). Immediately following ovariectomy, the animals were implanted with chronic i.c.v cannula into the right lateral ventricle following the coordinate system of Pellegrino et, al (1979). The surgery was performed under ketamine/xylazine anesthesia (Ketaset, Phoenix Sc. Inc, St. Joseph, MO; Xyla-Ject, Phoenix Sc. Inc, St. Joseph, MO) (80 mg/kg, 14 mg/kg, intramuscular, respectively). Following 13 days of recovery and one day prior to an experiment, animals were implanted with a jugular catheter under isofluorane anesthesia as previously described (Janik, et al, 1992; Callahan, et al, 1988). All experiments were performed two weeks after ovariectomy.

On the day of the experiment, the animals were allowed to acclimate to the laboratory setting for one hour prior to initiating the experiment. All experiments were performed between 0830-1000 h. At the start of an experiment, an initial blood sample was withdrawn before any drug administration. Saline or OFQ/N (55 pmol or 550 pmol, icv in a 5 µl volume) was injected into the lateral ventricle and additional serial blood samples were withdrawn 1, 3, 10, 20, 30 and 60 minutes later. The blood samples were centrifuged and plasma was removed and stored at -20ºC until assayed for prolactin. At the conclusion of the experiment, 0.1% toluidine blue was injected into the i.c.v. cannula to visually confirm cannula placement. Rats were sacrificed by rapid decapitation and the hypothalamus and anterior pituitary gland were dissected and individually frozen in liquid nitrogen. All tissue was stored at –80°C until used. Wet uterine weights were measured and used as an index of estrogen action.

10 2.2 Western Blot Analysis: 2.2.1 Hypothalamic pTH and TH:

The hypothalamic tissue was sonicated in 100 µl of homogenizing buffer and protein content in a 10 µl aliquot was determined using the BCA protein assay kit (Pierce, Rockford, IL). Homogenized protein (20 and 30 µg) were separated on 8% SDS-polyacrylamide gel for 2 h at 200V. Samples were transferred electrophoretically to Immobilon- P transfer membranes (Millipore Corp, MA) at 0.4A for 4 h. The blots were blocked in 5% BSA/1% Polyvinylpyrrolidone/1% Polyethyleneglycol (Haycock, 1993) overnight and incubated for 48 hrs at 4°C with primary antibody (rabbit anti-tyrosine hydroxylase phosphoSer40, 1: 1,500 dilution, Chemicon, Temecula, CA) diluted in the blocking buffer. After 4 × 10 min washes in Tris buffer containing 0.05% tween (TBST), the blots were incubated for 1. 5 h with horseradish- peroxidase –conjugated secondary antibody in TBST (goat-anti rabbit IgG, 1:5,000 dilution, Chemicon, Temecula, CA) at room temperature. Finally, the blots were washed 5 × 10 min in TBST and the protein was detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and developed on Kodak biomax film (Fisher, Pittsburgh, PA).

After probing for pTH, total TH expression levels were determined in the same blots. The blots were stripped with elution buffer (Restore western blot stripping buffer, Pierce, Rockford, IL) for 1 hour at room temperature and blocked in 8% nonfat milk for 4 hours. They were incubated with primary antibody (anti-TH, 1:10,000 dilution, BD Biosciences/Transduction labs, Hercules, CA) overnight at 4°C. The membranes were rinsed in TBST and incubated with secondary antibody (goat anti-mouse IgG, 1: 10,000 dilution, Chemicon, Temecula, CA) for 1.5 h. The TH protein was visualized as described for pTH.

When probing for pTH and TH, actin was quantified as an internal control using rabbit anti- actin antibody, (1:70, 000 dilution, Sigma-Aldrich, St.Louis, MO) when probing for pTH and mouse anti-actin (1:400,000 dilution, Chemicon, Temecula, CA) when probing for TH. The optical density of the pTH, TH and actin bands were quantified using ImageQuant 5.2 analysis software.

11 2.2.2 Estrogen Receptor α:

Estrogen receptor α (ER α) protein in hypothalamus and pituitary was separated on 8% SDS- polyacrylamide gels. For rat pituitaries, the amount of protein loaded was as follows: Placebo treated animals (5, 10 and 20 µg) and estrogen treated animals (10, 20 and 30 µg), while for rat hypothalami, the protein loaded was 10, 20 and 30 µg for all treatment groups. The samples were run and transferred as described for pTH/TH. The blots were blocked in 8% non-fat milk containing 5% BSA overnight at 4°C. Blots were then incubated overnight at 4°C with primary antibody (anti- rabbit ER α IgG, Santa Cruz, USA) at a dilution of 1:5,000 for pituitaries and 1: 2,500 for hypothalamus. Protein loading was normalized to Actin (anti- rabbit IgG, 1: 70,000 in TBST, Sigma-Aldrich, St. Louis, MO). After 4 × 10 washes in TBST, the blots were incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibody in TBST (1: 5,000 goat-anti- rabbit IgG, Chemicon, Temecula, CA) at room temperature (20ºC). Finally, the blots were washed 4 × 10 min in TBST, detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and developed on Kodak biomax film (Fisher, Pittsburgh, PA).

2.3 Hormone Assays:

Plasma prolactin concentrations were measured in duplicate samples by double antibody radioimmuonoassay using reagents purchased from the National Hormone and Peptide Program (NHPP) and Dr. A.F. Parlow. Plasma 17-β estradiol concentrations were determined using a commercially available kit (ImmuChem Double Antibody, MP Biomedicals, Costa Mesa, CA), according to the manufacturer’s specifications.

2.4 Statistical Analysis:

Prolactin data were analyzed by repeated measures ANOVA with time as the repeated measure. When significant F values were obtained, a post hoc Bonferroni test was performed. Western blots were analyzed using a non-paired student-t-test. The western blots were analyzed by non-paired student t-test with unequal variance. For all western blot analysis, the effects of treatment were expressed as percent change from its respective control (100%).

12 3. Results

3.1 Effects of treatment on plasma 17- β estradiol levels, wet uterine weights and body weights

Circulating concentrations of 17- β estradiol (Fig 1A) and wet uterine weight (Fig 1B) were significantly greater in ovx/estrogen (ovx/E) treated animals compared to placebo controls (p=0.0005 and 0.001, respectively). Body weights were not significantly different between the two groups (Table 1).

3.2 Effect of estrogen on OFQ/N induced PRL release

There were no significant changes in circulating prolactin levels across time in either the ovx/placebo or ovx/E treated animals. However, basal (time 0) prolactin levels in the ovx/E animals were higher than in the ovx/placebo group (Fig. 2). The prolactin response to OFQ/N was much greater in the estrogen replaced rats than in the placebo animals. In fact, OFQ/N administration did not significantly increase prolactin levels in ovx/placebo females except at 10 min after the 55 pmol dose (p= 0.0040) and this was significantly less than the increase in the ovx/E animals at the same time (p=0.0024). In contrast, in the ovx/E treated animals, prolactin levels were significantly greater than basal values at 3 and 10 min following both the 55 pmol (p = 0.02 at 3 min and p = 0.042 at 10 min) and the 550 pmol dose ( p ≤ 0.0001 at 3 and 10 min). Furthermore, the response in estrogen-replaced animals was significantly greater than in placebo controls at both the 55 pmol dose (p = 0.006 at 3 min and p = 0.0024 at 10 min) and the 550 pmol dose (p ≤ 0.0001 at 3 min and p = 0.0016 at 10 min).

3.3 Hypothalamic pTH and TH expression levels

Total TH and phosphorylated TH (pTH) appeared as single bands corresponding to molecular masses of approximately 60kDa (Figs. 3, 4 and 5). Estrogen replacement did not significantly alter the expression levels of either phophorylated TH (p=0.08) or total TH in the hypothalamus (p = 0.38) (Figs.3 A, B and C) and the pTH: TH ratio was also not significantly different (p=0.13).

13 Neither pTH or TH expression was significantly affected by OFQ/N administration to ovx/placebo animals, although there was an overall increase in these levels compared to the ovx/placebo animals that were injected with saline (Fig. 4 A, B and C). In the estrogen- replaced animals, OFQ/N had a significant, inhibitory effect on TH activity (Fig. 5 A, B and C). OFQ/N administration produced a decrease in pTH expression (Fig. 5A). Although this difference was not significant (p=0.063), since TH levels remained unchanged, the pTH:TH ratio indicated that there was significant inhibition of TH activity (p=0.05).

3.4 Effect of estrogen on ERα protein expression in the pituitary and hypothalamus

To assess the effect of estrogen on the sensitivity of the anterior pituitary gland and hypothalamus, ERα protein expression levels were determined in ovx/placebo and ovx/estrogen replaced animals. In the pituitary, there was a clear band of 66kD that corresponds to ERα expression and there was a significant decline in the density of this band in estrogen replaced animals (p=0.008) (Fig. 6A and B). However, in the hypothalamus, in addition to faint bands at 66 kD, which are intact ERα, there were additional bands of lower molecular weight (~62 kD) and another band of ~55 kD which was very highly expressed (Fig. 6 C). These bands probably represent expression of ER isoforms.

14 Figure 1. Plasma 17-β estradiol levels (A) and uterine weight (B) of ovx/estrogen (Ovx/E) and ovx/placebo animals. Values are means ±SEM.

* Significantly different from ovx/placebo animals (p=0.0005 for estradiol and p=0.001 for uterine weight).

15 A.

180 OVX/PLACEBO (n=47) * 160 OVX/ESTROGEN (n=24)

140

120 ) /m l

(pg 100

17 b-estra60 diol

0 TREATMENT

16 B.

0.6 OVX/PLACEBO (n=10) *

OVX/E (n=14)

0.5

0.4

0.3

WEIGHT (gms) WEIGHT

0.2

0.1

TREATMENT

17 Table 1. Effects of estrogen on body weight (grams) in ovx/placebo and ovx/estrogen female rats.

OVX/PLACEBO 255.625± 3.712 (n=47)

OVX/ESTROGEN 253.125± 5.742 (n=24)

Estrogen replacement did not significantly affect body weight. Values are means ±SEM.

18 Figure 2. The effect of estrogen on OFQ/N induced prolactin secretion in ovx female rats. An initial blood sample was withdrawn prior to OFQ/N or saline (control) injections to determine basal levels of prolactin (Time 0). Additional samples were taken at the times indicated on the figure. The prolactin secretory response to both doses of OFQ/N was significantly increased above basal values by 3 minutes (p=0.006 and p<0.0001 for the 55 and 550 pmol doses, respectively) and 10 minutes (p=0.0024 and p=0.0016 for the 55 and 550 pmol doses, respectively). Although prolactin levels were significantly increased at 10 minutes after the 55 pmol dose in ovx/placebo animals (p= 0.0040), these levels were significantly attenuated compared to the ovx/estrogen animals (p=0.0024).

Values are means + SEM. Number of animals in each group are shown in parentheses.

* Significantly different from basal values in the same group.

# Significantly different from levels in the ovx/placebo animals at the same time.

19 900 # PLACEBO/SAL (8) * PLACEBO/55pmol (14) 800 PLACEBO/550pmol (3) E/SAL (4) E/55pmol (9) 700 E/550pmol (6) # * 600

) # 500 /m l * (pg 400

PRL # * 300

200

100 *

0 0 1 min 3 min 10 min 20 min 30 min 60 min TIME

20 Figure 3. Effect of estrogen on hypothalamic pTH and TH levels and on the pTH:TH ratio. Expression levels of each protein, i.e. pTH and TH, as well as the pTH:TH ratio, were expressed as a percent of the protein expression level in the control, Ovx/Placebo/Saline treated group. Control values were set to 100%. Estrogen did not significantly affect expression levels of phosphorylated TH (pTH) or total TH and also did not alter the pTH:TH ratio (A). Representative western blots for pTH (B) and TH (C) are shown. Values are means ±SEM.

21 A.

350 ovx/placebo/Sal (n=3)

300 pTH:Actin (n=3) TH:Actin (n=3) pTH:TH (n=3) 250

200

150 % CONTROL

100

0 TREATMENT

B. Ovx/Placebo/Sal (control) Ovx/E/Sal

pTH (62kD)

Actin (42kD)

C. Ovx/Placebo/Sal (control) Ovx/E/Sal

TH (58kD)

Actin (42kD) 20 µg 30 µg 20 µg 30 µg

22 Fig. 4. The effect of OFQ/N administration on hypothalamic pTH and TH levels and on the pTH:TH ratio. Expression levels of each protein, i.e. pTH and TH, as well as the pTH:TH ratio, were expressed as a percent of the protein expression level in the control, Ovx/Placebo/Saline treated group. Control values were set to 100%. OFQ/N did not produce any significant change in the pTH:TH ratio in the placebo animals (p=0.13) compared to controls (A). Representative western blots for pTH (B) and TH (C) are shown. Values are means ±SEM.

23 A. 250

OVX/PLACEBO/SAL (n=3)

200 pTH:ACTIN (n=3) TH:ACTIN (n=3)

pTH:TH (n=3) 150

% CONTROL 100

0 TREATMENT

B. Ovx/Placebo/Sal (control) Ovx/Placebo/OFQ

pTH (62kD)

Actin

(42kD) C. Ovx/Placebo/Sal (control) Ovx/Placebo/OFQ

(58kD)

Actin

(42kD)

20 µg 30 µg 20 µg 30 µg

24 Fig. 5. Effect of OFQ/N on pTH and TH expression levels and on the pTH:TH ratio in ovx/estrogen treated animals. Expression levels of each protein, i.e. pTH and TH, as well as the pTH:TH ratio, were expressed as a percent of the protein expression level in the control, Ovx/Estrogen/Saline treated group. Control values were set to 100%. Although OFQ/N did not affect the total TH expression, pTH was decreased, but not significantly (p=0.06). However, the pTH:TH ratio was significantly decreased following OFQ/N administration (p=0.05) (A). Representative western blots for pTH (B) and and TH (C) are shown.

* Significantly different from ovx/estrogen/sal animals.

A. 140 OVX/E/Saline (n=3) pTH:Actin (n=3) 120 TH:Actin (n=3) pTH:TH (n=3) 100

80 *

60 % CONTROL

0 TREATMENT

B. Ovx/E/Sal (control) Ovx/E/OFQ/N

pTH (62kD)

Actin (42kD)

20 µg 30 µg 20 µg 30 µg

C. Ovx/E/Sal (control) Ovx/E/OFQ/N

(58kD)

Actin (42kD)

20 µg 30 µg 20 µg 30 µg

Figure 6. Effect of estrogen on ERα in OVX female rats. (A) Representative image of western blot of ERα protein expression in the anterior pituitary of placebo and estrogen replaced ovx animals. (B) Estrogen replaced animals show a significant decrease in the ERα:Actin receptor expression ratio when compared to placebo replaced animals (p=0.008). (C) Representative western blot of ERα protein in the hypothalamus in the two groups of animals show that, in addition to the ERα band seen at 66kD, there were additional bands that are likely isoforms of ERα.

* Significantly different from ovx/E/sal animals. Values are means ±SEM.

Ovx/Placebo/OFQ Ovx/E/OFQ

ERα

(66kD) Actin ( 42kD )

5 µg 10 µg 20 µg 10 µg 20 µg 30 µg

1.8 OVX/PLACEBO (n=5) OVX/E (n=5) 1.6

1.4

1.2 * 1

ER:ACTIN 0.8

0.6

0.4

0.2

0 TREATMENT

Ovx/Placebo Ovx/Estrogen

ER α (66kD)

55kD

Actin

(42kD) 10 µg 20 µg 30 µg 10 µg 20 µg 30 µg

29 4. Discussion

The results of this study indicate that estrogen is necessary for the OFQ/N-induced prolactin secretory response in female rats and this effect occurs, at least in part, due to inhibition of the hypothalamic dopaminergic neurons. Numerous releasing factors regulate prolactin secretion including the opiates and estrogen. Additionally, there are important gender differences in the control of this hormone (Gunnet and Freeman, 1982) that are due to a stimulatory effect of the ovarian steroids (Ben-Jonathan and Hnasko, 2001).

Since we have previously shown that the magnitude of the prolactin secretory response to OFQ/N was much greater in female than in male rats (Bryant, et al., 1998), we investigated the modulatory role of estrogen in mediating this response. Replacing estrogen in ovx females resulted in a significant increase in uterine weight indicating that this dose of estrogen was sufficient to produce a physiological response in this target organ. Furthermore, consistent with estrogen’s stimulatory effect on pituitary lactotrophs (Ben-Jonathan and Hnasko, 2001), estrogen replacement produced a significant increase in basal prolactin levels compared to the placebo treated group. Estrogen replacement also led to an increased sensitivity to OFQ/N. Although OFQ/N did induce a slight increase in prolactin secretion in ovx/placebo females, this response was significantly attenuated compared to the estrogen treated animals. Indeed, the magnitude of the prolactin secretory response to OFQ/N in the estrogen treated group was as great, or greater, than the response in intact, females (Bryant, et al., 1998, 2002).

Prolactin regulation is primarily under the tonic inhibitory control of hypothalamic dopamine and the important role of the TIDA neurons is well known (Freeman et al, 2000; Ben- Jonathan and Hnasko, 2001). It is also well established that other opiates stimulate prolactin secretion, at least in part, by inhibiting hypothalamic dopaminergic neuronal activity, including the TIDA neurons (for review see Freeman, et al., 2000). The results of this study demonstrate that OFQ/N administration produced a significant decrease in the pTH:TH ratio in the hypothalamus only in the estrogen replaced animals possibly due to less phosphorylation. TH is the rate–limiting enzyme in the biosynthesis of dopamine (Ikeda et al, 1965; Kumer and Vrana, 1996) and phosphorylation of TH is an indicator of

30 dopaminergic neuronal activation in vivo (Kaufman 1995). Phosphorylation of the Ser40 residues of TH seems to have a major effect on TH activity (Kumer and Vrana, 1996). Our results indicate that OFQ/N, like the other opiates, also exerts an inhibitory effect on hypothalamic dopaminergic neuronal activity and support previous findings that OFQ/N administration produced a decrease in median eminence DOPAC levels (Shieh and Pan, 2001). However, this only occurred in estrogen treated females, suggesting that estrogen modulates the sensitivity of these dopaminergic neurons to OFQ/N. Our results are consistent with results from several previous studies that demonstrated greater sensitivity and activity of these neurons in females than in males. For example, dopamine synthesis and turnover in the median eminence of female rats is 6-8 times higher than in male rats (Gudelsky and Porter, 1981). Furthermore, females have higher TH mRNA levels in the arcuate nucleus (Arbogast and Voogt, 1990), as well as higher concentrations of TH in the median eminence (Porter, 1986). It is known that this gender difference in TH levels and activity is due, at least in part, to modulation by the ovarian steroids because TH activity in females is decreased by ovariectomy and restored by estrogen (For review see Ben-Jonathan and Hnasko, 2001).

Administration of OFQ/N to placebo animals did not produce any significant changes in pTH or TH expression, however, there appeared to be a trend towards increased activity. This was a surprising result because OFQ/N has been reported to inhibit brain dopaminergic activity (See Schlicker and Morari, 2000 for review). One possible explanation may be that the low levels of estrogen in the placebo animals may have altered hypothalamic responsiveness to OFQ/N. However, OFQ/N inhibits dopamine neurons even in male animals, suggesting that low levels of estrogen do not alter dopaminergic responsiveness. It is necessary to further investigate the mechanisms responsible for the modulatory effects of gonadal steroids on opiate regulation of neuronal activity in order to explain these results.

Finally, although there was a trend toward increased hypothalamic TH activity, results from this study indicate that estrogen replacement did not significantly affect pTH or TH levels. This is in contrast to results reported by Arbogast and Hyde (2000) that mRNA level were increased following estrogen treatment. One possible explanation for the discrepancy between the effects of estrogen on mRNA and protein levels is that all mRNA is not

31 translated. Another possibility is that the differences in the dose and/or duration of estrogen replacement contributed to the seemingly contradictory findings.

Although estrogen did not significantly affect TH activity, our results clearly indicate that estrogen is an important modulator of OFQ/N’s inhibitory effect on hypothalamic, dopaminergic activity and on OFQ/N-induced prolactin secretion. Although there are numerous isoforms of the estrogen receptors (ER) in the brain, ERα is the most important, physiological receptor (Shupnik et al, 2000, 2002; for review see Toran –Allerand, 2004). In agreement with a previous report (Shupnik et al, 2000), pituitary ERα expression levels were decreased in estrogen-replaced animals, even though the prolactin reseponse to OFQ/N was extremely robust. One possible explanation for these results is that ERα expression is upregulated in the pituitary glands of placebo controls in order to increase or maintain sensitivity to very low estrogen levels. On the other hand, pituitary ERα receptor expression was decreased in estrogen-replaced animals because circulating estrogen levels were high. Receptor activation probably still occurred because there were such high levels of estrogen available in the circulation. The hypothalamus also expresses significant levels of ERα (Shupnik, 2002). Moreover, TH neurons in the arcuate nucleus express ERα (Herbinson et al, 1999) and estrogen downregulates ER expression in the arcuate nucleus. In this study, ERα protein corresponding to the 66kD molecular mass was not detected in significant amounts in the hypothalamus, however lower molecular mass bands corresponding to 62kD and especially ~55kD were more prominent. This 62kD band might be the putative isoform of the receptor recently found in the hypothalamus of rat, mice and baboon termed, ERX, which seems to differ from ERα but is recognized by the same antibodies to ERα (Toran- Allerand, 2004). Similarly, the 55 kD band corresponds to a protein which has been identified as β- tubulin, and this protein expression is increased in response to estrogen treatment (Ramirez et al, 2001). Shupnik (2002) suggests that, when full length, ERα expression is reduced, other isoforms are expressed at higher levels (Shupnik et al, 2000, 2002). Thus estrogen, through any of these estrogen receptor isoforms may modulate the sensitivity of dopaminergic neurons. This ability to modify the sensitivity of both the pituitary and hypothalamus may provide an important molecular mechanism through which estrogen exerts its actions which in turn lead to some major physiological changes. Estrogen may exert direct or indirect

32 actions on either hypothalamic neurons to regulate activity, and/or on pituitary cells, to modulate rates of prolactin synthesis and secretion. Clearly, lactotrophs in the the anterior pituitary gland express ER and the sensitivity of these cells to physiological stimuli, including stimulation by the opiates, is modulated by estrogen (Ben-Jonathan and Hnasko, 2000).

In summary, this is the first report to demonstrate that estrogen is essential for mediating the OFQ/N – induced prolactin secretory response and to show that this effect occurs, at least in part, by inhibiting hypothalamic, dopaminergic activity. OFQ/N modulates TH activity by decreasing the levels of phosphorylated TH in estrogen replaced animals only and this alteration in physiological function may be mediated by ERα.

33 References:

L.A. Arbogast, J.L. Voogt, Sex-related alterations in hypothalamic tyrosine hydroxylase after neonatal monosodium glutamate treatment. Neuroendocrin. 5 (1990) 2460-467.

L.A. Arbogast and J. F. Hyde, Estradiol attenuates the forskolin-induced increase in hypothalamic tyrosine hydroxylase activity. Neuroendocrin. 71 (2000) 219-227.

J. Arita and F. Kimura, Direct inhibitory effect of long term estradiol treatment on dopamine synthesis in tuberoinfundibular dopaminergic neurons: In vitro studies using hypothalamis slices. Endocrin. 121(1) (1987) 692-698.

M. Baumann and J. Rabii, µ-selective opioid peptides stimulate prolactin release in lactating rats. J Neuroendo 4 (1990) 701-708.

Ben –Jonathan, R. Hnasko, Dopamine as a prolactin (PRL) inhibitor. Endo Rev. 22 (2001) 724-763.

R.M. Bevilaqua, M.E. Graham, P.R. Dunkley, E. I. Nagy-Felsobuk, P.W. Dickson, Phosphorylation of Ser19 alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser40. J. Biol. Chem. 276 (44) (2001) 40411-40416.

M. Blum, B.S. McEwen, J.L. Roberts, Transcriptional analysis of tyrosine hydroxylase gene expression in the tuberoinfundibular dopaminergic neurons of the rat arcuate nucleus after estrogen treatment. J. Biol. Chem. 262 (1987) 817-821.

D.W. Brann, P.L. Zamorano, L.P. Chorich, V.B. Mahesh, Steroid hormone effects on NMDA receptor binding and NMDA receptor mRNA levels in the hypothalamus and cerebral cortex of the adult rat. Neuroendocrin. 58 (1993) 666-672.

W. Bryant, J. Janik, M Baumann, P. Callahan, Orphanin FQ stimulates prolactin and growth hormone release in male and female rats. Brain Res. 807 (1998) 228-233.

34 1 2 W. Bryant , P. Callahan, J. Janik, E. Murphree, [Phe ψ(CH2-NH)Gly ]NC(1-13)NH2 does not antagonize orphaninFQ/nociceptin-induced prolactin release. Brain Res. 57(5) (2002) 695- 703.

P. Callahan, J. Janik, L. Grandison, J. Rabii, Morphine does not stimulate prolactin release during lactation. Brain Res. 442 (1988) 214-222.

P.Callahan, M. Baumann, J. Rabii, Inhibition of tuberoinfundibular dopaminergic neural activity during suckling: involvement of u and k opiate receptor subtypes. J. Neuroendocrin. 8 (1996) 771-776.

P.Callahan, S. Klosterman, D. Prunty, J. Tompkins, J. Janik, Immunoneutralization of endogenous opioids peptides prevents the suckling-induced prolactin increase and the inhibition of tuberoinfundibular dopaminergic neurons. Neuroendocrin. 71 (2000) 268-276.

D.G. Campbell, D.G. Hardie, P.R. Vuillet, Identification of the four phophorylation sites in the N-terminal region of tyrosine hydroxylase. J. Biol. Chem. 261 (1986) 10489-10492.

Y. Chen, A. Mestek, J. Liu, JA. Hurley, L. Yu, Molecular cloning and functional expression of a u-opioid receptor from rat brain. Mol. Pharm. 44 (1993) 8-12.

Chen Y, Fan Y, Liu J, Mestak A, Tian M, Kozak CA, Yu L, Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family. FEBS Lett. 347 (1994) 279-283.

Chesterfield M, J. Janik, E. Murphree, C. Lynn, E. Schmidt, and P. Callahan, Specificity of orphanin FQ/nociceptin (OFQ/N)-induced prolactin secretion in female rats. Endocrin. In Review.

T. Darland, D.K. Grandy, The orphanin FQ system: an emerging target for the management of pain? Br. J. Anaesth. 81 (1998) 29-37.

35 J.E. Demaria, J.D. Livingstone, M.E. Freeman, Ovarian steroids influence the activity of neuroendocrine dopaminergic neurons. Brain Res. 879 (2000) 139-147.

C.T. Dooley, R.A. Houghten, Orphanin FQ/Nociceptin receptor binding studies. Peptides 21(2000) 949-960.

C. Evans, D. Keith, H. Morrison, K. Magendzo, R.H. Edwards, Cloning of a delta opioid receptor by functional expression. Science 258 (1992) 1952-1955.

M.E. Freeman, B. Kanyicska, A. Lerant, G. Nagy, Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80 (4) (2000) 1523- 1631.

V. Goffin, N. Binart, P. Turaine, P.A. Kelly, Prolactin: The new biology of an old hormone. Ann. Rev. Physiol. 64 (2002) 47-67.

H.E. Grenett, F.D. Ledley, L.L. Reed, S.L.C. Woo, Full-length cDNA for rabbit tryptophan hydroxylase:functional domains and evolution of aromatic amino acid hydroxylases. Proc. Natl. Acad. Sci. USA. 84 (1987) 5530-5534.

G.A.Gudelsky, J.C.Porter, Sex-related difference in the release of dopamine into hypophysial portal blood. Endocrin. 109 (1981) 1394-1398.

J.W. Gunnet, M.E. Freeman, Sexual differences in regulation of prolactin secretion by two hypothalamic areas. Endocrin. 110 (1982) 697-702.

J.W. Haycock, Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40. J Biol. Chem. 265 (1990) 11682-11691.

J.W. Haycock, Polyvinylpyrrolidone as a blocking agent in immunochemical studies. Anal. Biochem 208 (1993) 397-399.

G. Henderson, A.T. McKnight, The orphan opioid receptor and its endogenous ligand- nociceptin/orphanin FQ. Trends Pharm. Sci. 18 (997) 293-300.

36 A.E. Herbison, S.X. Simonian, D.P. Spratt, Identification and characterization of estrogen receptor alpha-containing neurons projecting to the vicinity of the gonadotropin-releasing hormone perikarya in the rostral preoptic area of the rat. J. Comp. Neurol. 411 (1999) 346- 358.

C. Isgor, K.R. Sheih, H. Akil, S.J. Watson, Colocalization of estrogen beta-receptor messenger RNA with orphanin FQ, vasopressin and oxytocin in the rat hypothalamic paraventricular and supraoptic nuclei. Anat Embryol (Berl). 206(6) (2003) 461-9.

J. Janik, P. Callahan, J. Rabii, The role of the µ1 opiate receptor subtype in the regulation of prolactin and growth hormone secretion by B-endorphin in female rats: studies with naloxonazine. J Neuroendo 4 (1992) 701-708.

O. Jöhren, G.L. Sanvitto, G. Egidy, J.M Saavedra, Angiotensin II AT1A receptor mRNA expression is induced by estrogen-progesterone in dopaminergic neurons of the female rat arcuate nucleus. J. Neurosci. 17(21) (1997) 8283-8292.

S. Kaufman, Tyrosine hydroxylase. Advances in Enzymology and Related Areas of Mol. Biol. 70 (1995) 103-220.

B. Kieffer, K. Befort, C. Gaveriauz-Ruf, C.G. Hirth, The delta opioid receptor: Isolation of a cDNA by expression, cloning, and pharmacological characterization. Proc Natl Acad Sci USA 89 (1992) 12048-12052.

S.C. Kumer and K.E. Vrana, Intricate regulation of tyrosine hydroxylase activity and gene expression. J. Neurochem. 67 (1996) 443-462.

W. Lan L. Pu, K. Ling, J. Shao, A. Cheng, L. Ma, G. Pei, Molecular characterization and functional expression of opioid receptor-like 1 receptor. Cell Res. 7 (1997) 69-77.

37 A. Lerant and M.E. Freeman, Ovarian steroids differentially regulate the expression of PRL- R I neuroendocrine dopaminergic neuron populations: a double label confocal microscopic study. Brain Res. 802 (1998) 141-154.

M.E. Lieberman , R.A. Maurer, P. Claude , J. Wiklund , N. Wertz , J. Gorski, Regulation of pituitary growth and prolactin gene expression by estrogen. Adv. Exp. Med. Biol. 138 (1981) 151-63.

M. Ikeda, M. Levitt, S. Udenfriend. Hydroxylation of phenylalanine by purified preparations of adrenal and brain tyrosine hydroxylase. Biochem. Biophys. Res. Commun. Feb 17;18 (1965) 482-8.

J.D. Livingstone, A. Lerant, M.E. Freeman, Ovarian steroids modulate responsiveness to dopamine and expression of G-Proteins in lactotropes. Neuroendocrin. 68 (1998) 172-179.

F. Meng, G. Xie, R.C. Thompson, A. Mansour, A. Goldstein, S.J. Watson, H. Akil . Cloning and pharmacological characterization of a rat kappa opioid receptor, Proc Natl Acad Sci USA 90 (1993) 9954-9958.

J.C. Meunier, C. Mollereau, L. Tool, C. Suaudeau, C. Moisand, P. Alvinerie, J.L, Butour, J.C. Guillemont, P. Ferrara, B. Monsarrat, H. Mazarguil, G. Vassarat, M. Permentier, K. Constentin, Isolation and structure of the endogenous agonist of the opioid receptor-like ORL-1 receptor. Nature 377 (1995) 532-535.

M. Minami, T. Toya, Y. Katao, K. Maekawa, S. Nakamura, T. Onogi, S. Kaneko, M. Satoh, Cloning and expression of a cDNA for the rat kappa opioid receptor. FEBS Lett 329 (1993) 291-295.

J.S. Mogil, G.W. Pasternak, The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol. Rev 53 (2001) 381-415.

38 C. Mollereau, M. Parmentier, P. Mailleux, J.L. Butour, C. Moisand, P. Chalon, D. Caput, G. Vassarat, J.C Meunier, ORL-1, a novel member of the opioid receptor family. FEBS Lett 341 (1994) 33-38.

C. Mollereau, M. J. Simons, P. Soularue, F. Liners, G. Vassart, J.C. Meunier, M. Parmetier, Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene. Proc Natl Acad Sci USA. 93 (1996) 8666-8670.

K. E. Moore, K.J. Loookingland, Dopaminergic neuronal systems in the hypothalamus. In Psychopharmacology: the Fifth Generation of Progress. Bloom F.E, Kupfer D.J (Eds), Raven Press, New York, pp 245-256, 1995.

C.R. Neal, A. Mansour, R. Reinscheid, H.P. Nothacker, O. Civelli, H. Akil, S.J. Watson, Localization of orphanin FQ (Nociceptin) peptide and messenger RNA in the central nervous system of the rat, J. Comp. Neurol. 406 (1999a) 503- 547.

C.R. Neal, A. Mansour, R. Reinscheid, H.P. Nothacker, O. Civelli, H. Akil, S.J. Watson, Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL-1 receptor mRNA expression with 125I-[14Tyr]-orphanin FQ binding, J. Comp. Neurol. 412 (1999) 563-605.

J.D. Neill, G.M. Nagy, Prolactin secretion and its control. In: E. Knobil and J.D. Neill, (Eds) The Physiology of Reproduction, 2nd ed. New York: Raven Press, 1994, 1833-1857.

H.P. Nothacker, R.K Reinscheid, A. Mansour, R. A. Henningsen, A. Ardati, F. J. Jr. Monsama, S.J. Watson, O. Civelli, Primary structure and tissue distribution of the orphanin FQ precursor. Proc. Natl. Acad. Sci. 93 (1996) 8677-8682.

E. Okuda-Ashitaka, S. Tachibana, T. Houtani, T. Minami, Y. Masu, M. Nishi, H. Takeshima, T. Sugimoto, S. Ito, Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus. Mol. Brain Res. 43 (1996) 96-104.

39 L.J. Pellegrino, A.S. Pelligrino, A.J. Cushman, A stereotaxic atlas of the rat brain, 2nd ed, Plenum Press, NY, 1979.

G. Pellitier, S. Li, D. Phaneuf, C. Martel, F.Labrie, Morphological studies of prolactin- secreting cells in estrogen receptor alpha and estrogen receptor beta knockout mice. Neuroendocrin. 77 (5) (2003) 324-33.

J.C. Porter, Relationship of age, sex, and reproductive status to the quantity of tyrosine hydroxylase in the median eminence and superior cervical ganglion of the rat. Endocrin. 118(4) (1986) 1426-1432.

V.D. Ramirez, J.L. Kipp, I. Joe, Estradiol, in the CNS, targets several physiologically relevant membrane-associated proteins. Brain Res Brain Res Rev. 37(1-3) (2001) 141-52.

A.J. Ramsey, P.J. Hillas, P. F. Fritzpatrick, Characterization of the active site iron in tyrosine hydroxylase. J. of Biol. Chem. 271 (1996) 24395-24400.

A.J. Ramsey, P.F. Fitzpatrick, Effects of phosphorylation of serine 40 of tyrosine hydroxylase on binding of catecholamines: Evidence for a novel regulatory mechanism. Biochem. 37 (1998) 8980-8986.

A.J. Ramsey and P. F. Fitzpatrick. Effects of phosphorylation on binding of catecholamines to tyrosine hydroxylase: specificity and thermodynamics. Biochem 39 (2000) 773-778.

R.K. Reinscheid, H.P. Nothacker, A. Courson, A. Ardati, R.A. Henningsen, J.R. Bunzow, D.K. Grandy, H. Langen, F.J. Monsma, O. Civelli, Orphanin FQ: A neuropeptide that activates an opioid-like G-protein coupled receptor. Sc. 270 (1995) 792-794.

R.K Reinscheid, H. P Nothacker, O. Civelli, The Orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice. Peptides 21 (2000) 901-906.

M.J. Reymond and J.C. Porter, Involvement of hypothalamic dopamine in the regulation of prolactin secretion. Hormone Res. 22 (1985) 142-152.

40 M.K. Scully, S.A. Glieberman, J. Lindzey, D.B Lubhan, S.K. Korach, M.G. Rosenfeld, Role of estrogen receptor-α in the anterior pituitary gland. 11(6) (1997) 674-681.

K.R. Sheih, J.T. Pan, Effects of orphanin FQ on central dopaminergic neuronal activities and prolactin secretion. AM J Physiol Regul Integrative Comp Physiol 280 (2001) R705-R712.

M. Shupnik, D.A. Schreihofer, M.H. Stoler, Differential expression and regulation of estrogen receptors (ERs) in the rat pituitary and cell lines: Estrogen decreases ERα protein and estrogen responsiveness. Endocrin. 141(6) (2000) 2174-2183.

M.A. Shupnik, Oestrogen receptors, receptor variants and oestrogen actions in the hypothalamic-pituitary axis. J. Neuroendocrin. 14 (2002) 85-94.

E. Schlicker, M. Morari, Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system. Peptides 21(7) (2000) 1023-9.

F. Taylor, A. Dickenson, Nociceptn/orphanin FQ. A new opioid, a new analgesic. NeuroReport 9(12) (1998) R65-R70.

R. Thompson, A. Mansour, H. Akil, S.J. Watson, Cloning and pharmocological characterization of rat µ-opioid receptor. Neuron 11 (1993) 903-913.

Toran-Allerand Dominique C, Minireview: A plethora of estrogen receptors in the brain: Where will it end? Endocrin. 145(3) (2004) 069-1074.

J.L. Voogt, Y. Hou, S.P. Yang, Changes in estrogen receptor-α expression in hypothalamic dopaminergic neurons during proestrous prolactin surge. Endocrine 20(1/2) (2003) 131-138

K. Yasuda, K. Raynor, H. Kong, C. Breder, J. Takeda, T. Resine and G. Bell, Cloning and functional comparison of kappa and delta opioid receptors from the mouse brain. Proc Natl Acad Sci USA 90 (1993) 6736-6740.