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Neuropeptide Y Y1-Receptor Stimulation Is Required for Physiological Amplification of Preovulatory Luteinizing Hormone Surges¹

Department of Neurobiology & Physiology, Northwestern University, Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Jon E. Levine, Ph.D., Department of Neurobiology and Physiology, 2153 North Campus Drive, Northwestern University, Evanston, Illinois 60208. E-mail: jlevine{at}nwu.edu

	Abstract

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Neuropeptide Y (NPY) has been shown to potentiate the actions of LHRH during the generation of preovulatory LH surges. It is not yet known, however, if activation of a specific subtype of NPY receptors in the anterior pituitary gland is an obligatory event in the stimulation of spontaneous LH surges. A battery of NPY receptor agonists, as well as the specific NPY Y1 receptor antagonist BIBP3226, were used to assess the role of Y1 receptors in the amplification of LH surges. In Exp 1, the potencies of a number of NPY agonists in facilitating LHRH-induced LH surges were assessed in pentobarbital (PB)-blocked, proestrous rats. The rank-ordered potencies of these compounds were determined to be PYY = [Leu³¹Pro³⁴]NPY > NPY >> hPP = rPP = NPY(13–36), which most closely reproduces the known rank-ordered affinties of these compounds for the Y1 receptor. In Exp 2, a Y1 subtype- specific antagonist, BIBP3226, was administered to unanesthetized, proestrous rats to assess the involvement of the Y1 receptor in the stimulation of spontaneous LH surges. The BIBP3226 compound strongly attenuated the endogenous proestrous LH surge, reducing the integrated value of LH secretion during the proestrous surge by more than 70%. In Exp 3, we assessed the ability of the Y1 receptor antagonist to block exogenous NPY effects on LHRH-induced LH surges. Treatment with BIBP3226 was found to completely prevent NPY amplification of LHRH-induced LH surges in pentobarbital-blocked, proestrous rats, thus confirming a pituitary locus of action of the drug. Taken together, these data clearly demonstrate that activation of neuropeptide Y receptors of the Y1 subtype is required for the physiological amplification of the spontaneous preovulatory LH surge in rats.

	Introduction

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NEUROPEPTIDE Y (NPY) is a 36-amino acid neuropeptide that is expressed throughout the arcuate nucleus of the hypothalamus and elsewhere in brain and functions as a neurochemical mediator in a variety of physiological systems. In the hypothalamic-pituitary-gonadal axis, its actions appear to be critical for the physiological elaboration of preovulatory LH surges. The peptide has been demonstrated to act in the hypothalamus to augment LHRH release (1, 2), as well as in the anterior pituitary gland to facilitate LHRH-induced release of LH surges (3, 4, 5). NPY gene expression (6) and release into the hypophysial portal vessels (7) are acutely augmented in association with LH surges, and peripheral immunoneutralization of NPY results in an attenuation of steroid-induced LH surges (8, 9). These observations have supported the idea that a preovulatory NPY surge functions as a physiological amplification signal in the generation of preovulatory gonadotropin surges. It has yet to be determined, however, which NPY receptor mediates this physiological amplification process, and to what extent the activation of this receptor is obligatory for the elaboration of preovulatory LH surges.

Six NPY receptor subtypes have been described (Y1-Y6), based on both their rank-order affinities for NPY receptor agonists (Table 1) and, more recently, on the cloning and characterization of five of the receptor subtypes (10, 11, 12, 13, 14, 15, 16, 17, 18). Previous pharmacological evidence has suggested that the receptor subtype that mediates LHRH release is either the Y1 or a Y1 -like receptor (19, 20); however, the receptor subtype mediating NPY’s effects at the pituitary is completely unknown, its elucidation delayed by the unavailability, until recently, of subtype-specific antagonists.

	Materials and Methods

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Animals and surgical protocol
All animal and surgical experimental procedures were conducted in accordance with the policies of Northwestern University’s Animal Care and Use Committee. Female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed in groups of four or five rats per cage in a temperature-controlled room, with lights on from 0500–1900 h. Animals had free access to tap water and standard laboratory rat chow before and during experiments. Estrous cycles were monitored daily through examination of vaginal cytology. After exhibiting at least two normal 4-day estrous cycles, rats were anesthetized with methoxyfluorane (Metofane, Pitman-Moore, Inc., Washington Crossing, NJ) and fitted with indwelling atrial catheters (PE-50) on the afternoon of diestrus. Catheters were inserted through the jugular vein, exteriorized at the nape, and secured with a plastic cuff. A stainless steel plug was used to occlude the catheter until experiments were conducted on the following day.

Exp 1
From 0900–2000 h on the day of proestrus, hourly 0.25 ml blood samples were taken from the animals. Samples were centrifuged and the plasma stored at -20 C for subsequent RIA. At 1230 h, animals received pentobarbital (40 mg/kg BW) ip to block endogenous peptide release from the hypothalamus. At half-hour intervals from 1300–1700 h, animals received iv pulses of LHRH (15 ng/pulse) and either saline or one of six NPY agonists (2.2 nmol/pulse): NPY, Peptide YY (PYY), rat pancreatic polypeptide (rPP), human pancreatic polypeptide (hPP), [Leu³¹Pro³⁴] NPY or NPY (13–36) (all peptides from Peninsula Laboratories, Belmont, CA). It does not seem to be the case that significant differences in clearance of NPY receptor agonists occur, given their similar metabolic fate via processing by the membrane peptidases dipeptidyl peptidase IV, aminopeptidase P, and endopeptidase 24.11 (25). Both LHRH and NPY were administered in a pulsatile manner to roughly mimic the endogenous secretory profiles of the peptides (26, 27). Estrous vaginal cytology was confirmed on the day after the experiment.

Exp 2
On the day of proestrus, hourly 0.25 ml blood samples were taken from 1300–2100 h and centrifuged; plasma was stored at -20 C until LH RIA. Every 15 min from 1400–1800 h, animals received iv pulses of BIBP3226 (Peninsula Laboratories), 50 µg/pulse or saline vehicle. The time period of drug delivery was chosen so as to correspond as closely as possible with the priming of the pituitary to the actions of LHRH, and thus to the presumed window of activity of NPY’s actions at the pituitary gonadotrope. The frequency of the drug delivery was chosen due to the short half-life of BIBP3226 in vivo (15–45 min). The dose of BIBP3226 used in this experiment is based on effective doses used in the original in vivo characterization of the antagonist (24, 28). Estrous vaginal cytology was confirmed on the day after the experiment.

Exp 3
From 0900–2000 h on proestrus, hourly 0.25 ml blood samples were taken through the catheter and centrifuged. The plasma was stored at -20 C until LH RIA. Animals received 40 mg/kg BW pentobarbital ip at 1230 h to block hypothalamic peptide secretion. BIBP3226 (50 µg) or vehicle was administered iv at 15-min intervals from 1300–1700 h; during the same time period, LHRH (15 ng/pulse) either alone, or concomitantly with NPY (10 µg/pulse), was administered at half-hour intervals. On the following day, estrous vaginal cytology was confirmed.

RIAs
LH levels in plasma samples were determined by RIA, using assay materials generously provided by the NIDDK. The standard used in the assay was LH RP-3. The sensitivity of the LH RIA was 20 pg/tube, and the intrassay coefficient of variation was less than 6%. The interassay coefficient of variation at 0.18 ng/tube was 9%.

Statistical analysis
Plasma LH levels for each experimental group were calculated in all experiments as the mean amount of LH released for each hourly time point as well as for the average amount of LH released from 1500–2000 h. Standard errors were calculated for each experimental group and either a Student’s t test or a one-way ANOVA followed by a Neuman-Keuls post-hoc analysis was used to determine differences between LH surge levels in experimental groups. In all studies, results were considered significant if P 0.05.

	Results

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Exp 1
Effects of pentobarbital on LH release. Administration of pentobarbital at 1230h to rats treated with saline from 1300–1700 h effectively (n = 5) blocked the endogenous LH surge, as reported previously (3, 21, 22).

Effects of pulsatile administration of LHRH and NPY agonists in pentobarbital-treated rats. Rats in all treatment groups showed LH surges. LHRH alone (n = 6) elevated basal plasma LH levels to a mean peak value of 7.7 ng/ml; total LH released during and after LHRH administration was 27.6 ng/ml. NPY (P < 0.05; n = 5), PYY (P < 0.001; n = 5), and [Leu³¹Pro³⁴]NPY (P < 0.001; n = 6) all significantly potentiated the LHRH-induced LH surge vs. animals treated with LHRH alone, both in terms of peak surge levels (Fig. 1) and total LH released during and after peptide administration (Fig. 2). In addition, [Leu³¹Pro³⁴]NPY (P < 0.01) and PYY (P < 0.01) showed significantly greater facilitation than NPY. NPY (13–36) (n = 5), rPP (n = 6) and hPP (n = 5) did not significantly augment total LH release in comparison with the control. Thus, the rank order potency of the peptides used in this experiment was found to be: PYY = [Leu³¹Pro³⁴]NPY > NPY >> hPP = rPP = NPY(13–36).

View larger version (20K): [in this window] [in a new window]   Figure 1. LH release during and after peptide administration in animals treated with PB (40 mg/kg) followed by half-hourly pulses of LHRH (15 ng) and either saline or one of six NPY agonists (2.2 nmol) from 1300–1700 h (arrows). Y1=[Leu31Pro34]NPY. Y2 = NPY(13–36). Values expressed as means ± SEM.

View larger version (45K): [in this window] [in a new window]   Figure 2. Sum over time of LH released during and after peptide administration in animals treated with PB (40 mg/kg) followed by pulses of LHRH (15 ng) and either saline or one of six NPY agonists (2.2 nmol) half-hourly from 1300–1700 h. Y1=[Leu31Pro34]NPY. Y2 = NPY(13–36). Values expressed as means ± SEM. *= P < 0.05.

View larger version (26K): [in this window] [in a new window]   Figure 3. Effect of administered BIBP3226 delivered at 15-min intervals from 1400–1800 h at 50 µg/pulse on the proestrous LH surge of normally cycling females. Values expressed as means ± SEM. *, P < 0.05 vs. saline-treated controls.

View larger version (27K): [in this window] [in a new window]   Figure 4. LH release on proestrus of PB-blocked animals treated with BIBP3226 (50 µg) at 15-min intervals from 1300–1700 h or saline vehicle and replaced with pulses of LHRH (15 ng) and either saline or NPY (10 µg) in half-hourly pulses during this time period. Values expressed as means ± SEM. *, P < 0.05 vs. animals treated with LHRH and NPY.

	Discussion

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The NPY Y1 receptor belongs to a family of G protein-coupled receptors that have distinctive ligand-binding characteristics. Y1 (10), Y2 (11, 12), Y4 (13, 14), Y5 (15), and most recently Y6 (16, 18) receptors have been cloned and expressed in mammalian cells, and their signature NPY-agonist binding properties have been determined. In agreement with the results using the specific Y1 receptor antagonist, BIBP3226, we determined that the pharmacological profile of the pituitary receptor that mediates NPY’s actions on LHRH-induced LH secretion matches most closely with that of the Y1 receptor (Table 1). A Y2 receptor can be ruled out based on two factors: the C-terminal fragment [NPY(13–36)] did not significantly augment LHRH-induced LH release, and the substituted peptide, [Leu³¹Pro³⁴]NPY, has no activity at Y2 receptors but was a strong agonist in this experiment. The pharmacologically defined Y3 receptor does not bind PYY, while PYY was a strong agonist in this study. The profile bears no resemblance to a Y4 receptor, in which both rPP and hPP are strong agonists and PYY is not an agonist. The profile of the newly discovered Y5 receptor is in many respects similar to that of a Y1, the primary difference being that hPP is a very weak agonist at Y1 receptors but a good agonist at Y5 receptors (15). Because in this experiment LH release in hPP-treated animals did not differ significantly from that in animals receiving no agonist, we conclude that the likely candidate for mediation of NPY’s effects on LHRH-induced LH release is a Y1 receptor. The newly discovered Y6 receptor is not found in rats (16, 17).

The Y1 receptor is also implicated in the second experiment, in which administration of the highly potent and selective NPY Y1 antagonist, BIBP3226, severely attenuated the endogenous LH surge on the afternoon of proestrus. This compound has no antagonistic activity at any other known receptor subtype (15, 24, 29). In addition to attenuating the surge, BIBP3226 produced a delay of approximately 2 h in the onset of the surge (Fig. 3); indeed, onset occurred in treated animals at approximately the time of termination of treatment. These and other experiments (data not shown) in which administration of BIBP3226 after surge onset had no effect on surge development or peak LH values provide clues to the timing of NPY’s involvement in the priming of gonadotropes to LHRH action. We cannot say from these experiments that NPY Y1 receptor stimulation must occur during the 2 h preceding the surge; nevertheless, NPY actions are without effect in the absence of LHRH stimulation, and the effects of NPY are thought to be mediated in part via up-regulated receptor binding (30, 31), possibly via an unmasking of a cryptic pool of receptors (30). Because these events must occur before LHRH receptor stimulation, it would seem likely that the neurosecretion of NPY and activation of Y1 receptors occurs during the hours or minutes leading up to the initial ascent of LH on the afternoon of proestrus.

In previous work, the involvement of NPY Y1 receptors in the stimulation of LHRH release was suggested on the basis of in vivo administration of several peptide agonists (20). These tests predated the further characterization of NPY receptor subtypes and their respective pharmacologies, and the involvement of Y1 receptors remains to be confirmed by blockade of the hypothalamic actions of NPY with BIBP3226. Nevertheless, the possible actions of BIBP3226 on LHRH release, as opposed to LHRH-induced LH secretion, were in need of consideration. Due to the calculated mol wt of BIBP3226 (MW 491.3), it is not expected that the systematically administered antagonist could cross the blood-brain barrier and reach central structures other than circumventricular organs such as the organum vasculosum of the lamina terminalis or the median eminence. Therefore, in our second experiment, BIBP3226 may have blocked receptors located in the median eminence as well as the anterior pituitary, both known sites of NPY modulation or stimulation of LHRH effects. Our third experiment was conducted to isolate the effect of the drug to the pituitary level; because hypothalamic peptide release is blocked in the pentobarbital-treated rat, any BIBP3226 binding to median eminence NPY receptors would be without effect on LH levels. The fact that LH levels in BIBP3226-treated rats from Exp 2 and 3 are very similar suggests that the effect seen in Exp 2 is largely, if not entirely, due to occupation of pituitary Y1 receptors. The belief that these Y1 receptors are located within the gonadotrope itself is suggested by the recent finding that Y1 receptor mRNA is expressed predominantly within this pituitary cell type (32).

This data corresponds well with recent work indicating that Y1 receptor gene expression in the anterior pituitary is subject to dynamic regulation during the estrous cycle, with acutely increased expression following the proestrous LH surge (33). This pattern is highly consistent with the involvement of the Y1 receptor in mediation of proestrous pituitary events. The possibility of a heterogeneity of receptors being involved in NPY’s facilitation cannot be ruled out; the involvement of another subtype could be masked in this study. However, given that the Y1-specific antagonist, BIBP3226, attenuates endogenous LH surges to the same extent that the nonsubtype specific antagonist, PYX2, does (34), it is probable that it is the Y1 receptor that is primarily or wholly responsible for the facilitation of the LHRH-induced LH surge. Th similarity of LH levels in animals from Exp 3 treated with BIBP3226 with those in animals treated with LHRH alone confirms this idea.

NPY is without effect on LH release in the absence of LHRH (3, 34); its action is to amplify the LH-releasing activity of LHRH. These data confirm that NPY strongly facilitates the action of LHRH. Furthermore, the failure of BIBP3226 to completely block the LH surge confirms that subphysiological surges do occur in the absence of NPY. It is possible that a higher dose of antagonist would have resulted in complete blockade of the LH surge; however, this is unlikely, especially given the data from this and previous (3) PB-block experiments in which blocked rats treated with LHRH alone show a very similar, subphysiological, LH surge. Immunoneutralization of NPY (4) results in a similar LH profile. These data, taken together, confirm NPY’s role as an amplifier and not releasing factor.

In summary, we have shown that the binding of NPY to Y1-receptors at the pituitary gonadotrope is a necessary event for the full elaboration of the proestrous LH surge. Current work in the lab is focused on signaling pathways through which NPY’s facilitation may be carried out. Given that NPY’s pituitary-level facilitation only occurs under steroidal conditions in which LH surges are generated (23), we are also investigating possible mechanisms by which steroids regulate the patency of NPY’s signaling pathways to produce amplified LH surges.

	Acknowledgments

 
The authors gratefully acknowledge the technical assistance of Mr. David Kim and Mr. Jeffrey Norgle.

	Footnotes

 
¹ This work was supported in part by NIH Grants R01-HD-20677, P01-HD-21921, and P30-HD-28408.

Received January 17, 1997.

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				S. M. Leupen and J. E. Levine Role of Protein Kinase C in Facilitation of Luteinizing Hormone (LH)-Releasing Hormone-Induced LH Surges by Neuropeptide Y Endocrinology, August 1, 1999; 140(8): 3682 - 3687. [Abstract] [Full Text]

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