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Endocrinology Vol. 140, No. 8 3682-3687
Copyright © 1999 by The Endocrine Society

ARTICLES

Role of Protein Kinase C in Facilitation of Luteinizing Hormone (LH)-Releasing Hormone-Induced LH Surges by Neuropeptide Y1

Sarah M. Leupen and Jon E. Levine

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

Address all correspondence and requests for reprints to: Jon E. Levine, Ph.D., Hogan Hall 2–160, 2153 North Campus Drive, Evanston, Illinois 60208. E-mail: jlevine{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In female rats, neuropeptide Y (NPY) facilitates LHRH-induced LH surges without affecting basal LH release. The signal transduction mechanisms mediating this facilitation are unknown. Here, the involvement of PKC in this process was investigated. Anterior pituitaries (APs) were removed from rats at 1400 h proestrus and perifused in vitro with M199 for 5 h. After an equilibration and baseline period, tissue received hourly 5-minute pulses of the PKC inhibitor GF109203X (GFX), 2.5 µM, followed 15 min later by a 5-minute pulse of LHRH (10-8 M), NPY (10-6 M), or phorbol 12-myristate 13-acetate (PMA, 50 nM), or some combination. This regimen was repeated hourly for 3 h. As shown previously, NPY had no effect on basal LH release but greatly facilitated LHRH-induced LH release. Treatment with PMA also facilitated LHRH-induced LH release, to approximately the same degree as NPY. Inhibition of PKC activity with GFX completely prevented NPY’s and PMA’s facilitation of LH release but did not inhibit LH release stimulated by LHRH alone. Because previous work suggested involvement of both NPY and PKC in alterations of LHRH receptor affinity or number, the in vivo effects of NPY on LHRH binding characteristics were also investigated. Although NPY treatment reliably enhanced LHRH-induced LH and FSH surges in proestrous rats, this action was not accompanied by any detectable change in the affinity or concentration of LHRH receptors in anterior pituitary cell membranes. In summary, we have found that NPY’s actions are blocked by PKC inhibition, mimicked by PKC stimulation, and not associated with any overt alterations in LHRH receptor affinity or number. We conclude that PKC activation is required for NPY’s facilitation of LHRH-induced LH surges, and that this mechanism likely involves PKC targets other than those which may alter LHRH receptor number or affinity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROPEPTIDE Y (NPY) is a 36-amino acid brain peptide with a wide tissue distribution and myriad roles in endocrine and cardiovascular physiology. NPY acts under favorable steroid conditions to facilitate LH release, especially the preovulatory LH surge, at both the hypothalamic and pituitary levels. NPY’s action at the pituitary gonadotrope is requisite for normal elaboration of the LH surge; we have shown that blockade of NPY Y1 receptors on the afternoon of proestrus attenuates the LH surge by more than 70% (1). Consistent with a role in surge elaboration, NPY gene expression (2) and release into the portal vasculature (3) are acutely up-regulated just before the LH surge, and peripheral immunoneutralization of NPY results in an attenuation of the surge (4, 5), which is very similar to that produced by blockade of pituitary Y1 receptors at that time. The mechanism of action of NPY’s physiological amplification is unknown; however, because NPY is incapable of releasing LH in the absence of LHRH (3, 6), it is likely that cross-talk occurs between signaling pathways activated by NPY and LHRH.

Protein kinase C (PKC) is a logical candidate for a molecule that could mediate, or at least participate in, this cross-talk. The LHRH-receptor mediated activation of PKC and PKC’s participation in LHRH-induced LH release have been demonstrated in numerous systems both in vivo and in vitro (e.g. 7–9). PKC’s roles in LHRH-induced LH release include amplification of the primary Ca2+ signal (10, 11), MAP kinase activation (12), negative feedback on phosphoinositide hydrolysis pathways (13), and activation of LHß transcription (14); some (15, 16) but not all (17) authors have shown a role for PKC in mediation of LHRH self-priming. However, PKC activation is not sufficient to evoke LH release in the absence of increased [Ca2+]i; the dissociability of PKC activation and LH secretion has been confirmed by a number of laboratories (17, 18, 19). This dissociability is consistent with a role in NPY’s facilitating actions because NPY cannot release LH in the absence of LHRH. NPY has been shown to activate PKC in other preparations, including murine macrophages (20) and Y1-receptor transfected CHO cells (21). Additionally, both PKC and NPY have been implicated in the alteration of LHRH receptor binding characteristics (22, 23, 24, 25). Here, we investigated the ability of a PKC activator to mimic NPY’s action, and a PKC inhibitor to block NPY’s facilitation of LHRH-induced LH secretion in vitro. Our studies used anterior pituitary tissues obtained from female rats on the afternoon of proestrus because previous work demonstrated that tissues obtained at this estrous cycle stage are most responsive to the facilitating actions of NPY. Because both NPY and PKC have both been suggested to evoke changes in LHRH receptor binding characteristics (22, 23, 24, 25), we additionally tested the hypothesis that any PKC-dependent, facilitating effects of NPY may be associated with changes in LHRH receptor number or affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experimental procedures were conducted with the approval of Northwestern University’s Animal Care and Use Committee. Female Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) were housed in groups of four to five rats per cage in a temperature-controlled room, with the lights on from 0500–1900 h. Animals had access to tap water and standard laboratory chow ad libitum. Estrous cycles were monitored through daily examination of vaginal cytology; only animals showing two consecutive 4-day estrous cycles were used in the experiments. For LHRH receptor binding analyses, diestrous rats were anesthetized with methoxyfluorane (Metofane, Pittman-Moore, Inc., Washington Crossing, NJ) and fitted with indwelling atrial catheters (PE-50, Becton Dickinson and Co., Parsippany, NJ). 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 of proestrus.

In vitro perifusion experiments
Animals were killed by decapitation at 1400 h on proestrus; APs were rapidly removed, cut into eighths, and placed in medium (Medium 199 with 25 mM HEPES, Earle’s salts, and L-glutamine (Life Technologies, Inc., Grand Island, NY) with 0.5% BSA (Sigma Chemical Co. Co., St. Louis, MO) and 25 µg/ml gentamicin (Garamycin, Schering-Plough Corp., Kenilworth, NJ) previously oxygenated and warmed to 37 C. The pituitary fragments were then rinsed with fresh medium, and fragments from a single AP were placed into a 200 µl microchamber for perifusion. Six APs were used in each perifusion session. Pituitary fragments were perifused at a rate of 6 ml/h. Ten-minute fractions were collected and frozen at -20 C until subsequent LH RIA.

Parameters of perifusion
After a 1-h equilibration period and a half-hour baseline collection period, the tissue was perifused with warmed, oxygenated medium (described above) containing the PKC inhibitor GF109203X (2.5 µM, Sigma Chemical Co., St. Louis, MO) or vehicle, in a 5-min pulse. Fifteen minutes later, the tissue received a pulse of LHRH (10-8 M) or NPY (10-6 M) or PMA (50 nM), or combinations of these, also in a 5-min pulse. This regimen was repeated every hour for 3 h. At the end of the perifusion, a 10-min challenge with 60 mM K+ was given to test for tissue viability; data from pituitaries not responding to this depolarizing stimulus were not included in statistical analyses. The PKC inhibitor used in these studies, GF109203X (bisindolylmalemide I), has been demonstrated to be both a potent inhibitor and selective for PKC (26).

LHRH receptor binding protocol
In proestrous rats, hourly blood samples were taken from 0900 until the time of decapitation and stored at -20 C until LH and FSH RIA. Animals received PB (sodium pentobarbital, and all other nonpeptides from Sigma Chemical Co., St. Louis, MO) at 1230 h followed by LHRH (10ng/pulse) with or without NPY (10 µg/pulse) half-hourly from 1300 h until 1700 h or time of decapitation (all peptides from Peninsula Laboratories, Inc., Belmont, CA). Rats were decapitated at 1500, 1600, or 1700 h and pituitary glands were quickly removed and snap-frozen on dry ice and stored at -70 C until binding assay. For the saturation binding protocol, a second in vivo protocol was used as above, except that all animals were decapitated at 1500 h.

LHRH receptor binding assay
Single pituitaries were thawed and homogenized in 2 ml ice-cold sucrose buffer (0.25 M sucrose, pH 7.7) and centrifuged at 10,000 x g for 25 min at 4 C. Pellets were resuspended in Tris-HCL buffer (10 mM, pH 7.7) to a final concentration of 40 µg protein/200 µl. Protein concentrations were determined by Bradford assay. The 200 µl membrane protein was added to polypropylene tubes containing 100 µl assay buffer (10 mM Tris-HCl + 1 mM dithiothreitol + 0.5% BSA), 300 pg 125I-LHRHa (D-ala6 Des-Gly10 LHRH) and 600 pg unlabeled agonist in a total volume of 500 µl. Duplicate nonspecific binding tubes contained an additional 100-fold excess (80 ng) of unlabelled agonist. After 80 min incubation on ice, reactions were stopped by addition of 2ml Tris buffer and centrifuged at 27,000 x g for 15 min. Pellets were counted in a {gamma} spectrophotometer. Specific binding was determined by subtraction of nonspecific binding from total binding. Binding capacity was calculated as fmol bound/mg protein. For a saturation binding protocol, 125I-LHRHa was added in concentrations of 50, 100, 150, 200, 400, or 600 pg/tube. The binding protocol in other respects was similar to that described above.

Radioimmunoassays
The LH and FSH levels in plasma samples were determined by RIA, using assay materials generously provided by the NIDDK. The standard used in the LH 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%. For FSH RIA, the FSH RP-2 standard preparation was used, and the intra and interassay coefficients of variation were 5.3% and 4.1%, respectively.

Statistical analysis
Perifusion experiments. For each animal, the three 10-min time periods following the receipt of the pulsatile stimulus were pooled, and the means and standard errors for each experimental group after each of the three pulses were calculated. Two-way ANOVA with repeated measures followed by Neuman-Keuls posthoc test was used to test for differences among each pair of experimental groups. Differences among treatment groups were considered statistically significant if P < 0.05.

Receptor binding experiments. Binding capacity was calculated as fmol bound/mg protein. Means and standard errors of binding capacity values were calculated for each group and a Student’s t test was employed to determine if significant differences existed between the two groups at each time point. In the saturation binding protocol, nonlinear regression analysis was used to determine the number of different sites bound, and standard Scatchard analysis was used to determine the Bmax and Kd for each group. The 95% confidence intervals for each parameter were compared between the groups to determine if significant differences existed. The plasma LH and FSH levels were calculated as the mean level of hormone for each time point, and a Student’s t test was used to assess differences in LH or FSH levels between groups receiving LHRH alone vs. those receiving LHRH with NPY. Results were considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Perifusion experiments
Exposure of tissues to three successive pulses of LHRH significantly stimulated LH release over basal levels (Fig 1Go). Each LHRH pulse stimulated LH secretion to a significant extent compared with pretreatment, basal levels. Furthermore, the magnitudes of the three responses were successively increased (first pulse, 88 ± 26 ng/ml; second pulse, 162 ± 24 ng/ml; third pulse, 183 ± 34 ng/ml) (n = 5), demonstrating a self-priming effect across the perifusion session.



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  		Figure 1. LH responses to stimulation of superfused anterior pituitary glands with each of three pulses of LHRH, NPY, or a combination. *, P < 0.05 vs. basal secretion; **, P < 0.05 greater facilitation than LHRH alone.

 
Addition of NPY strongly facilitated the stimulation produced by LHRH alone for the first and second pulses (175 ± 21; 232 ± 23; 196 ± 31 ng/ml, n = 7, P = 0.03 first pulse/0.049 sec pulse). Responses to the third pulses containing LHRH alone and LHRH plus NPY were not significantly different. Challenges with NPY alone produced no significant effect on LH secretion for any of the three pulse infusion times. When the PKC activator, PMA, was combined with LHRH, it facilitated LHRH-induced LH release (Fig. 2Go). The facilitating effects of PMA closely paralleled those of NPY (195 ± 34; 248 ± 33; 218 ± 27 ng/ml, n = 6, P = 0.05/0.04). The facilitating actions of PMA were completely blocked by concurrent administration of the PKC inhibitor (101 ± 17; 168 ± 22; 188 ± 21 ng/ml, n = 5, data not shown). The effects of PMA and NPY on LHRH-stimulated secretion were not additive (188 ± 18; 240 ± 26; 221 ± 19 ng/ml, n = 6).



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  		Figure 2. LH responses to stimulation of superfused anterior pituitary glands with each of three pulses of LHRH, NPY, PMA, or a combination. *, P < 0.05 greater facilitation than LHRH alone. LHRH and LHRH + NPY data repeated from Fig. 1Go for comparison.

 
Addition of the PKC inhibitor to test solutions containing NPY and LHRH completely blocked NPY’s ability to facilitate LHRH-induced LH release (Fig 3Go; 112 ± 18; 177 ± 19; 177 ± 14 ng/ml, n = 5, P = 0.05/0.03). The PKC inhibitor was found to be without effect, however, on LH responses to LHRH alone (102 ± 25; 143 ± 24; 165 ± 3 ng/ml, n = 6).



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  		Figure 3. LH responses to stimulation of superfused anterior pituitary glands with each of three pulses of LHRH, NPY, GFX, or a combination. *, P < 0.05 greater facilitation than LHRH alone. LHRH and LHRH + NPY data repeated from Fig. 1Go for comparison.

 
Effects of NPY in vivo on LHRH-induced LH and FSH secretion and LHRH receptor binding
As depicted in Fig. 4Go, NPY greatly facilitated the LHRH-induced LH surge in pentobarbital-blocked, proestrous rats. The facilitating effect was highly significant for the 1500 h, 1600 h, and 1700 h time points. In addition, NPY was found to significantly potentiate the LHRH-induced FSH surge at the 1600 h and 1700 h time points (Fig. 4Go). The LHRH receptor binding assay revealed that the LHRH binding capacities of anterior pituitary membranes derived from these same rats were not different across the three time points and were not different between rats receiving LHRH alone vs. those receiving LHRH and NPY (Fig. 5Go). Indeed, the binding capacity under all conditions remained within a narrow range of mean values between 500–550 fmol/mg protein.



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  		Figure 4. LH (upper) and FSH (lower) released during peptide administration and at time the rats were killed in receptor binding studies. Animals received PB at 1230 h, followed by LHRH (10 ng/pulse) with or without NPY (10 µg/pulse) half-hourly from 1300–1700 h or until the time that rats were killed. *, P < 0.05 more LH released than for LHRH alone.

 


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  		Figure 5. Upper, binding capacity of LHRH receptors in pituitaries from proestrous animals treated with LHRH alone or LHRH and NPY. Lower, Scatchard plot of binding characteristics of the LHRH agonist to pituitaries from similarly treated animals.

 
Nonlinear regression analysis of the saturation binding data revealed a best fit describing one-site binding. In subsequent Scatchard analysis (Fig. 5Go), the maximum number of sites, Bmax, and the binding affinity, as reflected by the dissociation constant, Kd, were found to be no different in tissues from rats receiving LHRH alone (Bmax = 355.8 ± 21 fmol/mg protein, Kd = 6.1 ± 0.8 nM) vs. LHRH plus NPY (Bmax = 345.1 ± 19 fmol/mg protein, Kd = 5.4 ± 0.7 nM) at the 1500 h time point.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that PKC is a mediator of NPY’s facilitating effect on LHRH-induced LH release in the pituitary gonadotrope. Activation of PKC in pituitary tissues from proestrous rats was able to mimic NPY’s facilitating effects, and inhibition of PKC blocked NPY’s actions without inhibiting LH release stimulated by LHRH alone. These observations support the idea that NPY’s actions during the preovulatory period are modulatory; while not directly stimulatory, they lead to an up-regulation of some component of LHRH signal transduction that is, in turn, regulated by PKC. It is not known whether NPY acts directly on the gonadotrope or through an intermediate cell type.

Consistent with the idea that PKC mediates NPY’s effects are observations that neither is sufficient to evoke LH release in gonadotropes. Protein kinase C is only able to evoke exocytosis under a sufficiently high intracellular Ca2+ concentration (27), and NPY facilitates only LHRH-stimulated, not basal, LH release (6, 28). Importantly, PKC has been implicated in NPY’s facilitation of norepinephrine-induced vasoconstriction, an effect which, like NPY’s action in the gonadotrope, is mediated by Y1 receptors (21, 29), requires external calcium (30), and is both modulatory and facilitatory in nature. PKC is also involved in Y1-receptor mediated signalling in human syncytiotrophoblast cells (31) and is required for NPY-activated circadian phase shifts in pacemaker neurons of the suprachiasmatic nucleus of the hypothalamus (32) and stimulation of peritoneal macrophage functions in the mouse (20).

A number of PKC isoforms ({alpha}, ßII, {delta}, {epsilon}, and {zeta} (33)) have been found to be expressed in the anterior pituitary, and it is not clear which of these, or what combination, may mediate NPY’s actions. LHRH elevates ß, {delta}, and {epsilon} but not {alpha} mRNA in {alpha}T3–1 cells (34, 35). Divergent roles for different PKCs have been implicated in PKC-PLD signaling; PLD, which activates PKC under conditions of sustained gonadotrope stimulation, may preferentially activate different PKC isoforms than PLC does (36). Specific involvement of any of these isoforms remains to be determined.

There are several possible mechanisms through which PKC activation by NPY could facilitate LHRH-induced LH release. PKC activation could result in the phosphorylation of Ca2+ channels, triggering Ca2+-stimulated exocytosis (11), activation of the MAP kinase pathway, which appears to be involved in {alpha}-subunit transcription and possibly LHRH self-priming (37, 38), or regulation of the transcription or stability of LHß subunit mRNA.

Both PKC and NPY have also been implicated in the regulation of the affinity of LHRH for its receptor or LHRH receptor number (22, 23, 24, 25). Parker et al. (22) demonstrated increased LHRH binding to NPY-treated membrane preparations from chronically OVX rats, an effect that they attributed to increased binding affinity because no changes in receptor number were seen. However, another study demonstrated that NPY increased the number of LHRH receptors available for binding in dispersed anterior pituitary cells from male rats (23). On proestrus, LHRH receptor levels, after a precipitous drop in the early afternoon, rebound to proestrus-morning levels by 1500 h and remain high until about 1800 h (39); we postulated that NPY may be partially or wholly responsible for this rebound. On the basis of these observations with NPY, and since PKC has also been implicated in regulating LHRH binding characteristics (24, 25), we tested the ability of NPY to alter LHRH binding capacity or affinity under physiological conditions in which NPY facilitates LHRH-induced LH surges. In our studies, NPY was found to alter neither LHRH binding affinity nor receptor number, even as it greatly facilitated LHRH-induced LH and FSH surges. It appears likely, therefore, that an alteration in LHRH binding parameters is not necessary for NPY to exert its effects under these conditions. Rather, the ability of NPY to enhance LHRH-induced LH secretion must be mediated by an action exerted at some point in the LHRH signaling pathway downstream of the LHRH receptor. Our findings are somewhat at odds with the previous results (22, 23), showing that NPY is capable of altering one or more characteristics of LHRH binding in pituitary preparations. We consider it unlikely that the discrepancy between the studies is attributable to an inability of our protocols to detect a potentially important alteration in LHRH binding capacity or receptor affinity, as the LHRH binding characteristics that we observed reproduced those obtained in previous studies (40, 41). Moreover, similar receptor binding protocols have been used successfully by others to discern relatively small changes in LHRH binding characteristics (39). There are other possible explanations for this discrepancy, including the fact that the two previous studies were performed in membrane preparations from chronically ovariectomized rats and dispersed pituitary cells from male rats, respectively. Thus, it is possible that NPY may not exert these same effects on LHRH receptors in intact female rats. It is also possible that under some conditions NPY may alter LHRH receptor characteristics but that these actions are independent of the enhancement of LHRH-induced LH secretion. Indeed, NPY’s facilitation of LHRH-induced LH surges only occurs under favorable steroid conditions (28, 42) that were not fulfilled in these prior experiments, suggesting that some other action of NPY which does not result in altered LH release may be mediated by an alteration in LHRH binding.

The ability of NPY to facilitate LHRH-induced LH secretion is demonstrable only under conditions leading to gonadotropin surges, such as on the afternoon of proestrus (28) or in ovariectomized, steroid-primed animals (42). It is not clear how ovarian steroids may up-regulate the responsiveness of pituitary tissue to NPY. Our current observations suggest at least one route through which this pathway may be rendered patent in the gonadotrope; estrogen and progesterone may up-regulate expression of one or more PKC isoforms. Protein kinase C has been shown to be up-regulated by estrogen in the pituitary (43), but no specific estrogen-responsive isoforms have been identified. Alternatively, ovarian steroids may up-regulate one or more downstream targets of PKC, which in turn converge at some unknown locus in a LHRH signal transduction pathway.

In summary, we have demonstrated the involvement of PKC as an obligatory component of the NPY signal transduction pathway mediating enhancement of LHRH-induced LH surges. PKC activation is necessary for NPY’s facilitation of the LHRH-induced LH surge to occur, and sufficient to mimic that action. Signaling events downstream of PKC, and the mechanisms by which ovarian steroids up-regulate this pathway, remain to be characterized.


   Acknowledgments
 
The authors gratefully acknowledge the advice of Dr. Mary Hunzicker-Dunn in the design of these studies. The RIA materials were provided by NIDDK.


   Footnotes
 
1 These studies were supported by NIH Grants R01-HD-20677 and P30-HD-28048. Results were presented in preliminary form at the 80th Annual Meeting of The Endocrine Society, New Orleans, Louisiana, June 1998. Back

Received January 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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