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Endocrinology Vol. 140, No. 11 5171-5177
Copyright © 1999 by The Endocrine Society

ARTICLES

Evidence that Stimulation of Two Modalities of Pituitary Luteinizing Hormone Release in Ovarian Steroid-Primed Ovariectomized Rats May Involve Neuropeptide Y Y1 and Y4 Receptors1

Mukul R. Jain, Shuye Pu, Pushpa S. Kalra and Satya P. Kalra

Departments of Physiology (M.R.J., P.S.K.) and Neuroscience (S.P., S.P.K.), University of Florida College of Medicine and University of Florida Brain Institute, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Professor Satya P. Kalra, Department of Neuroscience, University of Florida Brain Institute, University of Florida College of Medicine, P.O. Box 100244, Gainesville, Florida 32610. E-mail: skalra{at}ufbi.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A large body of evidence indicates that neuropeptide Y (NPY) is involved in stimulation of basal and cyclic release of hypothalamic LHRH and pituitary LH. To identify the NPY receptor subtypes that mediate the excitatory effects of NPY in these two modalities of LH release, we studied the effects of 1229U91, a selective Y1 receptor antagonist and Y4 receptor agonist, in two experimental paradigms that reproduce the two modalities of LH secretion in steroid-primed ovariectomized (OVX) rats. Rats were ovariectomized and implanted with a permanent cannula into the lateral cerebroventricle. In the first experiment, rats received estradiol benzoate (EB, 30 µg/rat) on day 5, followed 2 days later with progesterone (2 mg/rat) at 1000 h to induce an afternoon LH surge. 1229U91 (30 µg/3 µl) or vehicle (control) was injected intracerebroventricularly into these rats either once at 1300 h or twice (15 µg/injection) at 1100 and 1200 h. Blood samples were collected before progesterone injection at 1000 h and at hourly intervals from 1300–1800 h via an intrajugular cannula implanted on the previous day. In control rats, serum LH levels rose significantly at 1400 h, and these high levels were maintained until 1700 h. After two injections of 1229U91, LH levels displayed a tendency to rise at 1300–1400 h, as in controls, but thereafter, decreased rapidly below the control range. In the second experiment, the acute effect of 1229U91 on LH release was evaluated in OVX rats pretreated with EB alone. Saline alone or saline containing 1, 3, 10, or 30 µg 1229U91 was injected intracerebroventricularly at 1000 h, and the effects on LH release were analyzed at 10, 20, 30, and 60 min. 1229U91 elicited a dose-dependent stimulation of LH release, with maximal response (950% of basal levels) occurring at 10 min after the 30-µg dose; elevated levels were maintained for 1 h. Because 1229U91 is a potent Y4 agonist with some affinity for Y5 receptors, these results raised the possibility that activation of Y4/Y5 receptors by 1229U91 may augment LH release. Therefore, we examined the effects of icv administration of rat pancreatic polypeptide, a Y4-selective agonist, and [D-Trp32]-NPY, a Y5 agonist on LH release in EB-primed rats. Rat pancreatic polypeptide (0.5–2 µg/rat) stimulated LH release in a dose-related manner, and peak levels (280% of basal levels) were seen at 10–20 min; the response evoked by a higher dose (10 µg) was smaller than that induced by 0.5 or 2 µg. [D-Trp32]-NPY was relatively less effective, because only the highest (10-µg) dose elicited a modest stimulation (244% of basal levels). These results demonstrate that 1229U91, a Y1 antagonist and Y4 agonist, evokes two types of responses; it suppresses the protracted ovarian steroid-induced LH surge, and acutely, it also stimulates LH. These results imply that normally two different types of NPY receptors may mediate the stimulation of LH release. Because 1229U91 is a Y1 receptor antagonist, inhibition of the steroid-induced LH surge by 1229U91 suggests that Y1 receptors may mediate the cyclic release of LH. On the other hand, acute stimulation of LH by 1229U91 implies that the Y4 agonist-like activity of 1229U91 may mediate the basal release of LH and that either NPY or a yet-to-be-identified endogenous Y4 receptor agonist may activate Y4 receptors in the hypothalamus to stimulate LH release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY) is one of the most abundant neuropeptides in the rat central nervous system. The neurons synthesizing NPY that participate in the control of reproduction are located in the arcuate nucleus of the hypothalamus (1, 2, 3). A considerable body of evidence shows that NPY is involved in stimulation of both the basal episodic and preovulatory surge release of LH in the rat, rabbit, and monkey (1, 4, 5, 6, 7). Intraventricular administration of NPY stimulated LH release in ovariectomized (OVX) rats primed with ovarian steroids (4). These excitatory effects on LH release have been shown to be a result of LHRH stimulation by NPY (5, 8). Furthermore, blockade of NPY action by either passive immunoneutralization or administration of antisense oligodeoxynucleotides blocked the LH surge in steroid-primed OVX rats and attenuated episodic LH secretion in OVX rats (9, 10, 11, 12, 13, 14).

Although at least six NPY receptor subtypes have been characterized (15, 16), the receptor subtypes mediating the excitatory effects of NPY on LH release are not fully characterized. NPY and its synthetic analogs, including [Leu31,Pro34]-NPY, a putative Y1 receptor agonist, stimulated LH release in ovarian steroid-primed OVX rats (17). From these findings, it was inferred that stimulation of LH release by NPY might involve activation of Y1 receptors in the hypothalamus (17). However, because of the nonavailability of specific receptor antagonists and the fact that [Leu31,Pro34]-NPY was later reported to be a mixed Y1 and Y4 agonist (15, 16, 18), the identity of the receptor subtype in the brain mediating the excitatory effects of NPY on pituitary LH secretion was conjectural. In recent years, a number of Y1 receptor antagonists have been synthesized (15, 19, 20). Among these, 1229U91, a nonapeptide dimer, was first shown to be a potent NPY Y1 receptor antagonist (19). However, considerable agonistic activity at Y4 receptors in several in vitro and in vivo studies was reported later (21, 22, 23, 24, 25). Because there exists little evidence in support of Y4 involvement in the hypothalamic control of LH secretion (1), we initially employed 1229U91 to evaluate the role of Y1 receptors in the ovarian steroid-induced LH surge and in basal LH release in ovarian steroid-treated OVX rats. Quite unexpectedly, during the course of these studies, 1229U91 was found to stimulate LH release, thereby raising the possibility of involvement of Y4 receptors in stimulation of LH release. Therefore, to further characterize a possible role for Y4 receptors, we studied the effects on LH release of other endogenous or synthetic peptides (Table 1Go) with relatively selective agonistic activity for Y4 (rat pancreatic peptide, r-PP) and Y5 receptors (D-Trp-32) (15, 16, 26, 27).


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  		Table 1. Amino acid sequence and receptor activity profile of rat-NPY (r-NPY), 1229U91, r-PP, and [D-Trp32]-NPY

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult female Sprague Dawley rats from Zivic-Miller Laboratories, Inc. (Pittsburgh, PA) weighing 200–225 g, were housed in individual cages in air-conditioned rooms (22 C) with lights on from 0500–1900 h. Rat chow (Ralston Purina Co.) and tap water were available ad libitum. Under pentobarbital anesthesia (50 mg/kg, ip), a permanent stainless steel guide cannula (22 gauge) was placed into the lateral ventricle. The guide cannula was secured in place with acrylic dental cement and anchored to the skull with small machine screws, as described earlier (4, 9). Rats were bilaterally OVX immediately after the brain surgery and allowed to recover for 5 days. On the day of the experiment, rats showing efflux of cerebrospinal fluid from the icv cannula were selected for the experiment. The protocol was approved by the University of Florida Institutional Animal Care and Use Committee, which follows the NIH Guide for the Care and Use of Laboratory Animals.

Effects of 1229U91 on the progesterone (P)-induced LH surge in estradiol benzoate (EB)-primed OVX rats
Five days after ovariectomy and lateral ventricle cannulation, rats were injected with EB (30 µg/rat, sc) at 1000 h; this was designated as day 0. On day 1, rats were implanted with an intrajugular cannula under ether anesthesia. On day 2, these rats received P (2 mg/rat, sc) at 1000 h, followed by either one 30-µg icv injection of 1229U91 (a gift from Dr. A. J. Daniels, Glaxo-Wellcome, Research Triangle Park, NC) at 1300 h or two injections (15 µg each, icv) at 1100 and 1200 h. Control animals received either one injection at 1300 h or two injections at 1100 and 1200 h of vehicle (3 µl sterile saline containing 0.05% BSA, icv). Previous studies demonstrated that progesterone injection at 1000 h to EB-primed OVX rats results in increased LH release after 1300 h (28, 29, 30). Therefore, blood samples (0.2–0.3 ml) were withdrawn via the intrajugular cannula before P injection at 1000 h and at hourly intervals between 1300–1800 h. Each blood sample was replaced with an equal volume of saline. Plasma was stored at -20 C until assayed for LH, by RIA.

Effects of 1229U91 on LH secretion in EB-primed OVX rats
Five days after ovariectomy and implantation of the lateral ventricle cannula, rats were injected with EB (30 µg/rat, sc) at 1000 h; this was designated as day 0. On day 1, rats were implanted with an intrajugular cannula under ether anesthesia. On day 2, the rats received an icv injection of either 1229U91 (1, 3, 10, or 30 µg in 3 µl saline containing 0.05% BSA) or vehicle at 1000 h. Blood samples (0.3 ml) were withdrawn immediately before and at 10, 20, 30, and 60 min after the injection. Each blood sample was replaced with an equal volume of saline. Plasma was stored at -20 C until assayed for LH, by RIA.

Effects of a selective NPY Y4 receptor agonist (r-PP) and a Y5 agonist ([D-Trp32]-NPY), on LH secretion in EB-primed OVX rats
Although 1229U91 is a selective Y1 receptor antagonist, it has a high affinity for Y4 receptors, where it acts as an agonist (16, 21, 22, 23, 24, 25). It also has a weak affinity for Y5 receptors. Therefore, in this experiment, we tested the effects of a selective Y4 receptor agonist r-PP and a selective Y5 agonist [D-Trp32]-NPY (Table 1Go) on LH release in EB-primed OVX rats. Five days after ovariectomy and implantation of lateral ventricle cannula, rats were injected with EB (30 µg/rat, sc) at 1000 h. On the following day, an intrajugular cannula was implanted under ether anesthesia. Forty-eight hours after EB, the rats received an icv injection of 0.5, 2, or 10 µg of either r-PP or [D-Trp32]-NPY or 3 µl of vehicle (sterile saline containing 0.05% BSA) at 1000 h. Blood samples (0.5 ml each) were withdrawn immediately before and at 10, 20, 30, and 60 min after injection. Each blood sample was replaced with an equal volume of saline. Plasma was stored at -20 C until assayed for LH, by RIA.

Hormone estimation
Plasma LH was determined by the double-antibody RIA method with the aid of reagents supplied by Dr. A. F. Parlow and the National Hormone and Pituitary Program, NIDDK; and the levels were expressed in terms of r-LH-RP-2. All samples from an experiment were analyzed in a single assay.

Statistical analysis
The data were analyzed by one-way repeated-measures ANOVA using GraphPad Software, Inc. Prizm software (GraphPad Software, Inc., San Diego, CA), followed by Dunnett’s test, to compare all levels with the initial basal value within a treatment group. Comparisons between groups at a single time point were made using one-way ANOVA, followed by Student Neuman Keul’s test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of 1229U91 on the P-induced LH surge in EB-primed OVX rats
The effects of various doses of 1229U91 on LH levels are shown in Fig. 1Go. Because saline injection, either at 1300 or at 1100 and 1200 h, produced similar LH responses, the results were combined. As expected, in control rats, P treatment elicited a characteristic LH surge in the afternoon. Plasma LH levels were significantly higher at 1400 h than at 1000 h, peaked at 1500 h, and then gradually declined. In rats injected with 30 µg 1229U91 at 1300 h, LH levels were significantly elevated at 1400 h vs. 1000 h values (P < 0.05), as in the control rats. However, LH release was suppressed thereafter; plasma LH levels were significantly lower than their respective control values at 1500, 1600, and 1700 h (P < 0.05); and at the latter two time points, levels were not different from the 1000 h values. Two icv doses of 1229U91 at 1100 and 1200h (15 µg/dose) also inhibited the LH surge. LH levels rose initially at 1300 h (P < 0.05 vs. 1000 h) but rapidly returned to the basal range through 1800 h; levels were significantly lower than in control rats at 1500, 1600, and 1700 h (P < 0.05). Also, this initial rise at 1300 h vs. 1000 h levels was not significant, compared with LH levels at 1300 h in control rats.



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  		Figure 1. Effect of the Y1 antagonist 1229U91 on the LH surge induced by progesterone in EB-primed OVX rats. A, Mean LH levels after icv administration of saline or 30 µg 1229U91 at 1300 h (arrowhead); B, mean LH levels after icv administration of saline or two doses of 1229U91 (15 µg each at 1100 and 1200 h, arrowheads). *, P < 0.05 vs. initial basal levels at 1000 h; a, P < 0.05 vs. levels in saline-injected controls at the same time point.

 
Effects of 1229U91 on LH secretion in EB-primed OVX rats
The effect of icv 1229U91 on LH secretion in EB-primed OVX rats is shown in Fig. 2Go. 1229U91 stimulated LH secretion in these rats; the excitatory response was dose- and time-dependent. Administration of 1 µg 1229U91 produced a small increase in LH (68% over initial basal values), lasting for only 10 min. Higher doses of 1229U91 produced a sustained elevation in plasma LH concentration; the 3-, 10-, and 30-µg doses of 1229U91 induced robust stimulation of LH secretion that lasted from 20 min (3 µg) to at least 60 min (10 and 30 µg). The excitatory effect on LH release was greatest after the 30-µg dose. At 10–30 min post injection, levels were 850% higher than basal.



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  		Figure 2. Acute effect of 1229U91 on LH secretion in EB-primed OVX rats. At 1000 h, 1, 3, 10, or 30 µg 1229U91 or saline was injected icv (arrow). Values are mean ± SEM. *, P < 0.05 vs. initial basal values and levels in controls at the same time; a, P < 0.05 vs. control and the 1- or 3-µg 1229U91-treated animals at the same time point; b, P < 0.05 vs. all other groups at the same time point.

 
Effects of r-PP and [D-Trp32]-NPY on LH secretion in EB-primed OVX rats
Because 1229U91 has a high affinity for Y4 and some affinity for Y5 receptors, we investigated the effects of Y4 and Y5 agonist peptides. r-PP, a selective Y4 agonist, also stimulated LH secretion (Table 1Go and Fig. 3Go). The increments at 10 and 20 min were dose-related (0.5–2 µg) and lasted for 30–60 min. Interestingly, the 10-µg dose was less effective than 2 µg, in terms of the magnitude of the response. In comparison, only the highest 10-µg dose of [D-Trp32]-NPY, a selective Y5 receptor agonist, evoked LH release, lasting for only 20 min (Fig. 4Go).



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  		Figure 3. Acute effect of r-PP on LH secretion in EB-primed OVX rats. At 1000 h, 0.5, 2, or 10 µg r-PP or saline was injected (arrow). Values are mean ± SEM. *, P < 0.05 vs. initial levels at 1000 h; a, P < 0.05 vs. levels in saline-injected controls at the same time point.

 


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  		Figure 4. Acute effect of [D-Trp32]-NPY on LH secretion in EB-primed OVX rats. At 1000 h, 0.5, 2, or 10 µg [D-Trp32]-NPY or saline was injected (arrow). Values are mean ± SEM. *, P < 0.05 vs. initial levels at 1000 h; a, P < 0.05 vs. levels in saline-injected controls at the same time point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In rats displaying regular estrous cycles, two types of LH secretion pattern have been identified (1, 30, 31, 32). In general, LH secretion is basal through various stages of the estrous cycle. This basal pattern of LH secretion is interrupted by the preovulatory LH surges lasting several hours in the afternoon of proestrus. In the absence of feedback action of ovarian steroids, estrogen and P, as in OVX rats, LH secretion is markedly elevated, and the phasic LH surge is lost. However, there is compelling evidence to show that estrogen replacement, achieved either through continuous release of estradiol 17ß through sc implants or by multiple injections of either estradiol 17ß or single injection of the long-acting EB, restores the basal pattern of LH secretion (1, 29, 30). Also, sequential treatment of ovarian steroids, i.e. EB treatment followed by P injection 48 h later in OVX rats, reinstates the preovulatory-type LH surge in the afternoon (1, 28, 29). Consequently, these two paradigms have been extensively employed to investigate the role of neurotransmitters/neuromodulators and their receptors in the basal and surge modes of LH secretion (reviews, Refs. 1, 30).

The hypothalamic NPY system is closely associated with the cyclic discharge of LH, as shown by the observation that NPY levels in the median eminence and NPY synthesis (as reflected by NPY messenger RNA (mRNA) in the ARC) increased before and during the LH surge occurring spontaneously on proestrus, or that induced by P in EB-primed OVX rats (5, 33, 34, 35). Blockade of NPY synthesis by antisense oligodeoxynucleotides inhibited NPY accumulation in the median eminence and blocked the P-induced LH surge in OVX rats (9). Similarly, blockade of the effects of NPY by passive immunoneutralization inhibited the LH surge induced by P and that occurring on proestrus (14, 36). In the present study, we report that icv administration of 1229U91 blocked the P-induced LH surge in OVX rats. Because 1229U91 is reported to be a highly selective antagonist of the Y1 subtype (19, 22, 23, 24, 25), these results argue for Y1 receptor involvement in stimulation of the LH surge by NPY. In support of this notion are the studies showing presence of Y1 receptors in the medial preoptic area and arcuate nucleus, the sites implicated in induction of LH surge (37).

Because NPY nerve terminals synapse on LHRH perikarya and dendrites in the medial preoptic area (38), the neural site intimately associated with induction of LH surge (1, 30), and because NPY can stimulate LHRH release from LHRH terminals in the median-eminence (8), it is highly likely that blockage of Y1 receptors on LHRH neurons by 1229U91 results in suppression of LH surge. Because there was a small increase in LH release at 1300 h in one experiment, which was followed by suppression of the release, it is possible that Y1 receptors may engage in the protracted elevation of LH release and not in the neuroendocrine events that initiate LH surge. Further investigation is warranted, to characterize the NPY receptor subtype involved in initiating LH hypersecretion.

Previous studies demonstrated that NPY exerts its excitatory influence on LH secretion at two sites: one at the level of the hypothalamus, where it stimulates the release of LHRH; and the other at the level of pituitary gonadotrophs, where it amplifies the LHRH-induced release of LH (1, 5, 8, 39, 40, 41). In a recent study, Leupen et al. (42) reported that peripheral administration of the selective Y1 antagonist BIBP3226 attenuated the LH hypersecretion induced by LHRH and NPY analogs in proestrus rats. Because BIBP3226 does not cross the blood-brain barrier (43), these findings suggested that Y1 receptors on pituitary gonadotrophs may mediate the effects of exogenous NPY. BIBP3226 is reported to be neurotoxic because it produces undesirable motor effects, such as barrel rolling upon icv administration (43). Furthermore, there is evidence to suggest that the action of BIBP3226 in brain may not be specific for NPY receptors (44). Therefore, we employed 1229U91, a highly specific Y1 receptor antagonist (21, 22, 23, 24, 25, 45). The results of the present study, as discussed above, support the possibility that central Y1 receptors may mediate the LH surge induced by ovarian steroids. Because NPY stimulates LHRH release from the hypothalamus of steroid-primed rats (1, 8) and its secretion is closely linked with induction of the LH surge, the results of the current study imply that increased NPY release, under these conditions, may activate Y1 receptors to stimulate the protracted LH surge in the afternoon.

There is an alternate possibility, involving Y4 receptors, that currently cannot be ruled out. Previous studies have shown that diminution in the tonic restraint, exerted by endogenous opioid peptides, plays an important role in the induction of LH surge on proestrus and in the P-induced LH surge in EB-primed OVX rats (1, 46, 47). Morphological evidence showed that NPY neurons terminate on dendrites and soma of the opioid, ß-endorphin (ß-END), producing neurons located in the arcuate nucleus (48). ß-END-containing neurons are also in contact with LHRH neurons in the medial preoptic area (49, 50). Because 1229U91 is a Y4 receptor agonist (23, 24, 25), it raises the possibility that activation of Y4 receptors may augment the opioid restraint to result in suppression of LH surge. Indeed, injection of NPY has been shown to stimulate ß-END release in the hypothalamus of castrated, but not in intact, male rats (51); but these ß-END-producing neurons possess Y1 receptors (52, 53). Whether the Y4 agonist component of 1229U91 activates inhibitory opioid tone, through distinct Y4 receptors in the opioid neurons, for several hours after P injection, remains to be ascertained. Because Y4 peptide agonists, human pancreatic polypeptide (hPP) and rPP (like other neuropeptides) are short acting, it would require a different paradigm than the current one, in which either continuous icv infusion of these peptides or a pure Y4 agonist with no Y1 receptor antagonistic activity is employed, to affirm the possibility that activation of inhibitory opioid tone by Y4 receptors inhibited the P-induced LH surge.

Additionally, our results showed that 1229U91 rapidly increased LH secretion in EB-primed OVX rats. This excitatory LH response was sustained for at least 1 h. Interestingly, the stimulatory effects of 1229U91 are reminiscent of the similar effects of NPY and the NPY agonist [Leu31,Pro34]-NPY in steroid-primed OVX rats (4, 17). NPY and [Leu31,Pro34]-NPY also inhibited LH release in unprimed OVX rats (4, 17). However, we have observed that 1229U91, in doses that stimulated LH release in EB-primed rats, failed to alter LH secretion in unprimed OVX rats (unpublished data), indicating that the stimulatory effects of 1229U91 are dependent on the presence of ovarian steroids. Alternatively, it is possible that ovariectomized rats secrete LH at maximal rate, a response that could not be further augmented by 1229U91. Because NPY is reported to play an excitatory role in basal LH secretion in OVX and ovarian steroid-primed OVX and ovary-intact rats (1, 5, 8, 9, 10, 11, 12, 13), these findings are suggestive of an involvement of a NPY receptor subtype other than the Y1 receptor in basal LH secretion.

In this context, a reappraisal of previous studies indicates a role of the Y4 subtype in the excitatory effects on LH release in OVX steroid-primed rats. Administration [intracerebro-ventricularly (icv)] of hPP, a ligand for Y4 receptors, stimulated LH secretion (54). Interestingly, [Leu31,Pro34]-NPY, the NPY analog believed previously to act selectively at Y1 receptors (55), also stimulated LH release (17). This analog was later reported to have a high affinity for rat Y4 receptors (15, 18, 26), suggesting a role for Y4 receptors. Our current results, showing that 1229U91 produced robust LH stimulation, support the participation of Y4 receptors in stimulation of LH release. On the other hand, hPP has strong affinity for rat Y5 receptors, in addition to Y4 receptors (16, 26). In this context, r-PP, a highly selective Y4 receptor agonist with poor affinity for Y5, Y1, or Y2 receptors (16, 26), also showed a stimulatory effect on LH secretion. A comparative analysis of the LH response, after 1229U91 and r-PP, clearly demonstrated that 1229U91 was more effective than r-PP. It is possible that 1229U91 may be a relatively more potent and stable agonist for Y4 receptor than r-PP. These observations clearly invoke activation of Y4 receptors by 1229U91 in elicitation of LH release. This inference is in line with the recently reported distribution of r-PP binding sites in a number of central nervous system sites, including the preoptic area and arcuate nucleus of the hypothalamus (56, 57, 58), sites implicated in stimulation of LH secretion (1, 5). Using RT-PCR, Bard et al. (59) also reported the existence of Y4 receptor mRNA in the hypothalamus. Soon after presentation of our results (60), Raposinho, P. D., P. Broqua, A. Hayward, R. Galyean, C. Schteingart, J.-L. Junien, and M. L. Aubert (manuscript submitted) communicated to us that 1229U91 stimulated LH release in male rats, possibly by activating Y4 receptors. Cumulatively, it is apparent that Y4 receptors in the hypothalamus play a role in stimulation of pituitary gonadotropin release. However, the endogenous ligand for Y4 receptors, probably related to r-PP or h-PP, has not yet been characterized in the hypothalamus or other regions in the rat brain. PP-like immunoreactivity or PP mRNA was reported in the central nervous system (62, 63), but Miyazaki and Funakoshi (64) were unable to detect PP-like peptide in the brain.

Finally, high affinity of h-PP and some affinity of 1229U91 for Y5 receptors (16, 24, 65) invokes a role for Y5 receptors in stimulation of LH release. However, [D-Trp32]-NPY, a highly selective Y5 receptor agonist (26, 27), produced only a modest stimulation of LH, at a high dose, in our investigation. Because [D-Trp32]-NPY has only weak affinity for this receptor (26), the role of Y5 receptors in stimulation of LH release remains to be clarified. Use of selective Y5 antagonists, when available, may clarify the role of Y5 receptors in stimulation of LH release.

In summary, the results of these experiments clearly demonstrate that the stimulatory effects of hypothalamic NPY on the LH surge and basal LH secretion in steroid-primed OVX rats may be mediated by disparate receptor subtypes. Because 1229U91, a Y1 receptor antagonist, blocked the steroid-induced LH surge in OVX rats, our results suggest that Y1 receptors may mediate the NPY-induced stimulation of preovulatory LH surge in the afternoon of proestrus. The alternative, that augmentation of inhibitory opioid tone by 1229U91 may lead to suppression of P-induced hypersecretion, cannot be ruled out. On the other hand, because the Y4 agonists 1229U91 and r-PP acutely stimulated LH, it is likely that central Y4 receptors may play a role in stimulating basal LH release in ovary-intact and steroid-primed OVX rats. This evidence of the involvement of two distinct NPY receptor subtypes in the two modalities of LH secretion is novel and warrants further investigation.


   Acknowledgments
 
We are grateful to Dr. A. J. Daniels (Glaxo-Wellcome) for the gift of 1229U91.


   Footnotes
 
1 Presented at the 5th International Neuropeptide Y Meeting, Grand Cayman, British West Indies, April 17–22, 1999 (p. 30). This study was supported by NIH Grant HD-08634. Back

Received April 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kalra SP 1993 Mandatory neuropeptide-steroid signaling for the preovulatory luteinizing hormone-releasing hormone discharge. Endocr Rev 14:507–538[Abstract]
  2. Chronwall BM, DiMaggio DA, Massari VJ, Pickel VM, Ruggiero DA, O’Donohue TL 1985 The anatomy of neuropeptide-Y-containing neurons in rat brain. Neuroscience 15:1159–1181[CrossRef][Medline]
  3. Everitt BJ, Hokfelt T 1989 The coexistence of neuropeptide Y with other peptides and amines in the central nervous system. In: Mutt V, Hokfelt T, Fuxe K, Lundberg JM (eds) Neuropeptide Y. Raven Press, New York, pp 61–71
  4. Kalra SP, Crowley WR 1984 Norepinephrine-like effects of neuropeptide Y on LH release in the rat. Life Sci 35:1173–1176[CrossRef][Medline]
  5. Kalra SP, Crowley WR 1992 Neuropeptide Y: a novel neuroendocrine peptide in the control of pituitary hormone secretion, and its relation to luteinizing hormone. Front Neuroendocrinol 13:1–46[Medline]
  6. Khorram O, Pau KY, Spies HG 1987 Bimodal effects of neuropeptide Y on hypothalamic release of gonadotropin-releasing hormone in conscious rabbits. Neuroendocrinology 45:290–297[Medline]
  7. Woller MJ, Terasawa E 1992 Estradiol enhances the action of neuropeptide Y on in vivo luteinizing hormone-releasing hormone release in the ovariectomized rhesus monkey. Neuroendocrinology 56:921–925[Medline]
  8. Crowley WR, Kalra SP 1987 Neuropeptide Y stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamus in vitro: modulation by ovarian hormones. Neuroendocrinology 46:97–103[Medline]
  9. Kalra PS, Bonavera JJ, Kalra SP 1995 Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release. Regul Pept 59:215–220[CrossRef][Medline]
  10. Xu B, Pu S, Kalra PS, Hyde JF, Crowley WR, Kalra SP 1996 An interactive physiological role of neuropeptide Y and galanin in pulsatile pituitary luteinizing hormone secretion. Endocrinology 137:5297–5302[Abstract]
  11. Bonavera JJ, Kalra PS, Kalra SP 1996 L-arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: involvement of neuropeptide Y. Endocrinology 137:1956–1962[Abstract]
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