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Adenosine 3′,5′-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone
ELSEVIER
Molecular
and Cellular Endocrinology
107 (1995) l-7
Adenosine 3 ’ ,S-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone George Abdilnour, Gregory A. Bourne* Department
of Physiology
Vniversity
of Saskatchewan,
Saskutoon, Saskatchewan,
Received 6 May 1994; 6 October
S7N OWO, Canada
1994
Abstract An in vitro perifusion system was used to ascertain the role of CAMP in the genesis of the self-priming effect of gonadotrophinreleasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 pmol/l cycloheximide. Forskolin (1 PmoYl) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial IO-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskohn + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in CAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on CAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that CAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon. Keywords:
Cycloheximide; Flufenamate; Forskolin; Luteinizing hormone; Perifusion system; Pituitary (rat)
1. Introduction Studies on the secretory actions of gonadotrophinreleasing hormone (GnRH) have established that repeated pulsatile administration of equal amounts of GnRH result in the secretion of progressively greater amounts of luteinizing hormone (LH) (Aiyer et al., 1974; Edwardson and Gilbert, 1976; Waring and Turgeon, 1980, Evans et al., 1984; Buckingham and Cover, 1986). This phenomenon, known as the self-priming effect of GnRH, is dependent on estradiol, progesterone (Aiyer et al., 1974; Beck et al., 1978; Cover and Buckingham, 1989; Turgeon 1990, 1991) and protein synthesis and Waring, (Edwardson and Gilbert, 1975; Pickering and Fink, 1976a, 1979; Turgeon and Waring, 1991) and requires a
*
Corresponding
author, Department
of Livestock
Science, Faculty of
Agriculture, University of the West Indies, St Augustine, Trinidad. Tel.: (809) 6622002;
Fax: (809) 6639686.
0 1995 Elsevier Science Ireland Ltd. All rights reserved 0303-7207/95/$09.50 SSDl 0303-7207(94)03418-8
minimum of 30-60 min to develop (Aiyer et al., 1974; Waring and Turgeon, 1983). Continuous infusions of GnRH result in a biphasic secretion of LH (Fink et al., 1976; Bourne and Baldwin, 1980; Waring and Turgeon, 1983; Das et al., 1989). The response is characterized by an initial acute release of the hormone (phase I), followed approximately 30-60 min later by a greatly augmented secretion rate (phase II). The initial release occurs independently of de novo protein synthesis, while the secondary phase is dependent on protein synthesis (Bourne and Baldwin, 1980; Das et al., 1989) and estradiol (Baldwin et al., 1983). The similarities in the time lag required for the manifestation of the biphasic response and the self-priming effect, coupled with the mutual dependency on de novo protein synthesis and estradiol, strongly suggest that these two events result from common underlying cellular mechanisms. Studies designed to elucidate potential second messengers of the biphasic secretion of LH have demonstrated that adenosine 3’S’-cyclic monophosphate (CAMP) plays
2
GAbdilnour, G.A. Bourne / Molecular und Cellulur Endocrinology 107 (1995) l-7
a pivotal but indirect role as a mediator of the protein synthesis-dependent, phase II secretion of the hormone (Bourne and Baldwin, 1987a; Bourne, 1988). If the phase II secretion of LH and the self-priming effect of GnRH are indeed the result of common cellular mechanisms, CAMP should be involved (indirectly) in the self-priming effect of GnRH. The present study was undertaken to explore this possibility. Flufenamate inhibits GnRH-stimulated CAMP production (Naor et al., 1975a; Naor et al., 1975b: Bourne and Baldwin, 1987a,b) apparently by disrupting G, protein function (Bourne, 1992). Forskolin which increases CAMP production in anterior pituitaries (Kolp et al., 1991; Bourne, 1992) by directly affecting the catalytic protein (Downs and Aurbach, 1982), bypasses the inhibitory effects of flufenamate on CAMP production (Bourne, 1992). Consequently, these two compounds were used as pharmacological probes to delineate potential roles of CAMP as a mediator of GnRH self-potentiation. A previous study evaluating the practicability of utilizing forskolin to restore intracellular CAMP in the presence of flufenamate (Das and Bourne, 1992) had demonstrated that forskolin offered distinct advantages over the use of exogenously administered CAMP analogues. 2. Materials and methods 2.1. Materials The following materials were purchased from Sigma Chemical Co. (St. Louis, MO): bovine serum albumin (BSA); cycloheximide; dimethyl sulfoxide (DMSO); flufenamic acid and forskolin. GnRH was obtained from Calbiochem Corporation (La Jolla, CA). Flufenamic acid (10 g/l) was dissolved in 0.1 mol/l NaOH and added to the perifusion medium prior to adjusting the pH. Forskolin was solubilized in DMSO. The concentrations of cycloheximide, flufenamate and forskolin used in this study were based on results from previous reports (Bourne and Baldwin, 1980, 1987a; Das and Bourne, 1992). 2.2. Animals Adult (75-85 days), female Sprague-Dawley rats (bred from a departmental colony) were maintained in a controlled environment (20 + 1°C; lights on 06001800 h). Food and water were supplied ad libitum. Female rats exhibiting at least two consecutive 4-day estrous cycles (determined by daily vaginal cytology) were selected as pituitary donors on the second day of diestrous. The animals were killed by decapitation between 1200 and 1230 h and the anterior pituitaries rapidly excised and placed in pre-warmed (37°C) perifusion medium (Bourne and Baldwin, 1980). 2.3. Perifusion system The in vitro perifusion
system
was described
previ-
ously (Fahmy and Bourne, 1993). Briefly, two quartered pituitary glands were placed in a perifusion chamber (Millipore Swinnex-13 filter holder) and superfused with Krebs Improved Ringer I medium (McKenzie and Dawson, 1969) supplemented with essential amino acids and vitamins (Flow Laboratories, Rockville, MD) at a flow rate of 0.25 ml/min. The medium was maintained at 37°C and continuously equilibrated with 95% 0Z/5% C02. The perifusate from the first hour was discarded, at which time stable, basal LH levels were established. Then, sequential effluent samples were collected every 10 min in tubes precoated with 2% BSA. The pituitaries were challenged with 20 nmol/l GnRH and/or 1 pmol/l forskolin for 10 min. The first pulse of secretagogue(s) was administered 60 min after sample collection was initiated, with the second pulse following 150 min later. At the end of an experiment, the samples were stored frozen at -20°C until assayed for LH. 2.4. Radioimmunoassay The samples were assayed for LH in duplicate using the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) rat radioimmunoassay kit. Sample estimates were calculated using the log-logit transformation and weighted least squares regression (Robard et al., 1969). Values for LH were compared with the NDDK reference preparation of rat LH-RP-3, which is equipotent to LH-RP-2 and -61 times more potent than LH-RP-1. The intra-assay and interassay coefficients of variation were 4.8% and 5.4%, respectively, at 159pgll. 2.5. Data analysis The total amounts of LH secreted in response to the IO-min pulses of sectretagogues were calculated as follows: three values of basal secretion (obtained just before the IO-min pulses of secretagogue(s)) were used to calculate a mean value for basal secretion/l0 min. This value was subtracted from each of the IO-min fractions. The values corrected for basal secretion were summed to obtain the total amounts of LH secreted in response to the secretagogues. The duration of the response was from the time of infusion of the secretagogue(s) up to the time when LH secretion had returned to stable, baseline levels. Differences between the total amounts of LH secreted in response to the sequential pulses of secretagogues were analyzed by Student’s t-test for paired data to determine whether the priming effect was apparent. The amounts of LH secreted in response to secretagogues in different experimental (treatment) groups were compared using analysis of variance, with differences between the means being determined by Duncan’s new multiple range test. A value of P < 0.05 was considered significant. All statistical tests were performed by the Number Cruncher Statistical System software (NCSS, Kaysville, UT).
3
G.Abdilnour, G.A. Bowne I Molecular and Cellular Endocrinology 107 (1995) l-7
110 0
100
2
GnRH
l GuRH+
600
-
500
-
CYCLQHFXIMIDE
90
80 1
70
z 2
60
Fig. 2. Total amounts of LH released in response to IO-min pulses of secretagogues administered 150 min apart. Values are the means f SEM (n =4 chambers). The abbreviations are: cycloheximide (cycle), forskolin (forsk) and flufenamate (flu). *P < 0.01 and **P < 0.001 for comparisons of 1st and 2nd pulses by paired t-test. Other statistical data appear in the text.
!!I a 50
to GnRH. The results are shown in Fig. 3. Ten-minute pulses of 1 pmol/l forskolin infused 150 min apart were ineffective in stimulating LH secretion (Fig. 3). When the diterpene was administered in conjunction with GnRH, it did not significantly affect the magnitude of the first response to GnRH (Figs. 2 and 3) but did establish a new 01
0
I
I
60
120
It
180
240
110
1
300
Cl
360
TIME (minuteil)
heximide was present in the medium from the beginning of the experiment. Results are the means i SEM (n = 4 chambers). Absence of standard error bars is indicative of the fact that SEMs are smaller than the size of the symbol. The data reported in the figures are the results from one experiment. However, the experiments weE repeated at least twice to confirm their reproducibility.
3.Results The data from the first series of experiments illustrate the characteristics of the self-priming effect of GnRH. The concentration of GnRH (20 nmol/l) used in this study was submaximal and was chosen from a dose-response curve (data not shown). As shown in Fig. 1, a IO-min pulse of GnRH elicited an increase in LH secretion. The second LH response to a similar GnRH challenge administered 150 min later was significantly greater than the first response (P < 0.01; Figs. 1 and 2). Cycloheximide (S,~mol/l) which did not significantly affect the first response to GnRH (Figs. 1 and 2) inhibited the enhanced LH secretion of the second response, reducing it to the level of the first response (Figs. 1 and 2). The second series of experiments were undertaken to determine whether increases in endogenous CAMP (stimulated by forskolin) would affect the LH responses
0 GnRIi + FOR8KOLlN
100
Fig. 1. LH responses from diestrous rats to IO-min pulses of 20 nmol/l GnRH administered 150 min apart (indicated by the black square on the abscissa) in the presence or absence of 5 pmol/l cycloheximide. Cyclo-
MR8KOLlN
0 GnRH + FORSKOW + CYCLOHKKMDE
90
80 fl
7c
z 2
6C
SC
10
I
0
60
I,
I
120 FLM
180
240
4
300
360
(minutes)
Fig. 3. LH responses to IO-min pulses of 1 PmoUl forskolin or GnRH + forskolin administered 150 min apart (indicated by the black squares on the abscissa) in the presence or absence of cycloheximide (n = 4 chambers). For further details see legend for Fig. I.
4
GAbdilnour.
1st PULSE
0
G.A. Bourne I Moleculur
2nd
PULSE
l
CnRH
-
FORSKOLJN
o
DYSO
-
GnEN
0
FORSKOLJN -
CnRH
+
FORSKOLJN -
CnRH + FORSKOIJN
60
120 TIME
180
240
300
360
(minutes)
Fig 4. LH responses to IO-mm pulses of various combinations of forskolin and/or GnRH 150 min apart (indicated by the black squares on the abscissa). For further details see legend for Fig. I (n = 4 chambers).
baseline (70 min after administration) which was approximately twice the secretory rate of basal secretion occurring prior to the administration of the secretagogues (Fig. 3). Additionally, the second response to GnRH was greatly potentiated by forskolin when compared with the second response to infusions of GnRH alone (P < 0.05; Fig. 2). Cycloheximide reduced the potentiated, second response to GnRH + forskolin to the same level as the first response (Figs. 2 and 3). These results suggest that CAMP does not participate in the initial LH responses to GnRH but can sensitize gonadotrophs (through protein synthesis dependent mechanisms) to subsequent exposures of the decapeptide. However, it is still unknown whether subsequent increases in CAMP are involved in the ‘immediate’ responses to subsequent administrations of GnRH. This possibility was explored in the following series of experiments. GnRH elicited a characteristic LH response when administered at 60 min (Fig. 4; Table 1). Forskolin did not significantly affect LH secretion when infused 150 min later (Fig. 4; Table 1). DMSO (the vehicle for forskolin) did not stimulate LH secretion when administered at
and Cellulur Endocrinology
IO7 (1995) 1-7
60 min, nor did it affect the GnRH-stimulated response 150 min later; i.e. the response to GnRH at 220 min was similar to the GnRH-stimulated response obtained at 60 min (Fig. 4; Table 1). Forskolin also did not stimulate LH secretion when administered at 60 min, but did potentiate the response to GnRH 150 min later (P < 0.05; Fig. 4; Table I), an effect which was blocked by cycloheximide (data not shown). Interestingly, the LH response to a pulse of GnRH + forskolin infused 150 min after the administration of forskolin was not significantly different from the response to GnRH given 150 min after forskolin (Fig. 4; Table 1). These results suggest that CAMP only participates in sensitizing gonadotrophs to subsequent exposures of GnRH and is not otherwise involved in the ‘immediate’ responses to subsequent applications of the decapeptide. In the final series of experiments, flufenamate which inhibits GnRH-stimulated CAMP production in female rat pituitaries (Bourne and Baldwin, 1987a; Bourne, 1988) was used as a pharmacological probe to evaluate the effects of an absence of increases in CAMP concentrations on the self-priming effect of GnRH. Since the inhibition of GnRH-stimulated CAMP production by flufenamate was documented previously (Bourne and Baldwin, 1987a; Bourne, 1988), similar experiments were not repeated for this study. As shown in Fig. 5a, flufenamate (1O~molll) reduced the magnitude of the second LH response to GnRH to the level of the first response without significantly affecting the first response; i.e. the compound only inhibited the self-priming effect of GnRH (Figs. 2 and 5a). As a result, the responses to GnRH obtained in the presence of flufenamate were similar to those obtained in the presence of cycloheximide (Fig. 2). Forskolin which bypasses the inhibitory effects of flufenamate on CAMP production (Bourne, 1992) was efficacious in reversing the flufenamate-induced inhibition of the self-priming
Table
1
Total amounts of LH secreted in response secretagogues administered 150 min apart Secretagogues
to
IO-min
pulses
of
LH secreted (ng)
1st pulse
2nd pulse
I st pulse
2nd pulse
GnRH DMSO Forskolin Forskolin
Forskolin GnRH GnRH GnRH + forskolin
95.0 f. 18.8”
9.3 f 102.5 240.5 250.6
_ _
3.3c + 17.5” + 15.6b f 12.4b
Values represent the mean * SEM (n = 4). The ‘-’ indicates the secretagogue was ineffective in stimulating LH secretion above basal levels. The superscript letters indicate statistical significance or insignificance. Values with the same superscript letters are statistically similar, whereas values denoted by different superscripts are significantly different from each other. The P values and definitions of abbreviations appear in the text.
G.Abdilnour, G.A. Boume I Molecular and Cellular Endocrinology 107 (1995) 1-7 110 OGaRH+l’WFENAHATZ
l GnRH+hLRIcNuLITc + ml?sKoLIN
100
-
90
-
80
-
-
B 0
IwRsKoLtN
+ ITulmNAMAm
l GaR?I+~RSKGbtN+~~~ + cYclmxMnt
A
70
1
70
$
60
5
BO-
E !I
50
ii
40
!I 30
OL 0
60
120 TM!
160
240
300
360
(ldnu~)
0
60
120 lmE
160
240
300
360
(miautm)
Fig. 5. LH responses to IO-mitt pulses of GnRH f forskolin in the presence of lO~mol/l flufenamate (A) and forskolin + GnRH in the presence of flufenamate + cycloheximide (B). The secretagogues were administered 150 min apart (indicated by the black squares on the abscissa), while flufenamate and cycloheximide were present in the medium from the beginning of the experiment (n = 4 chambers). For further details see legend for Fig. 1.
effect of GnRH (P c 0.001; Figs. 2 and 5a). The inhibitory effects of flufenamate were also reversed when a pulse of GnRH was administered 150 min after GnRH + forskolin (data not shown). The reversal of the inhibition of the self-priming effect of GnRH could not be attributed interactions between forskolin and to synergistic flufenamate, since these compounds did not affect LH secretion (Fig. 5b). Finally, cycloheximide prevented the forskolin-induced restoration of the self-priming effect of GnRH (Figs. 2 and 5B), indicating that this response was also dependent on protein synthesis. 4. Discussion Flufenamate was previously utilized in studies demonstrating a pivotal role for CAMP in the biphasic secretion of LH from pituitaries of diestrous II rats (Bourne and Baldwin, 1987a). However, the application of this pharmacological agent to directly assess the potential involvement of CAMP in the self-priming effect of GnRH was not possible until recently. Crucial experiments in such studies are the demonstration that the replacement of CAMP results in the restoration of the flufenamateinduced inhibition of GnRH-stimulated LH secretion. In previous studies, CAMP replacement was achieved by the exogenous administration of dibutylyl CAMP
(dbcAMP). Unfortunately, this CAMP analogue (and others) compounded data interpretation by potentiating the initial (acute) GnRH-stimulated LH responses by protein synthesis-independent mechanisms (Bourne and Baldwin, 1987a; Das and Bourne, 1992). Recent demonstrations that forskolin bypasses flufenamate-induced inhibition of CAMP production in anterior pituitaries (Bourne, 1992) and does not affect the protein synthesisindependent components of GnRH-stimulated LH secretion (Das and Bourne, 1992) effectively negated this problem, facilitating the successful completion of the present study. For decades the role of CAMP in GnRH-stimulated LH secretion remained enigmatic. The demonstration that the nucleotide has little or no direct effect on LH secretion (Liu and Jackson, 1981; Liu et al., 1981; Turgeon and Waring, 1986; Bourne and Baldwin, 1987a; Conn et al., 1987; Kolp et al., 1991), formed the basis of the concept that CAMP does not participate in the acute release of LH. The current study supports this concept. The present findings that (i) CAMP (stimulated by forskolin) does not affect the LH response to the first pulse of GnRH, and (ii) these initial responses were not affected by the absence of GnRH-stimulated increases in CAMP production, provide the first direct proof that CAMP does not affect LH secretion during initial exposures to GnRH.
6
GAbdilnnur,
G.A. Bourne / Molecular
Although CAMP does not appear to be involved in mediating the initial LH responses to GnRH, the nucleotide does sensitize gonadotrophs to subsequent exposures of the decapeptide by activating mechanisms that involve de novo protein synthesis. These observations coupled with the demonstration that the flufenamate-induced inhibition of the self-priming effect of GnRH was reversed by CAMP replacement, provide persuasive evidence that CAMP plays a pivotal role in the genesis of the selfpriming effect of LH. Further indirect evidence that is consistent with CAMP playing a role in the genesis of GnRH self-potentiation is the demonstration that GnRH ‘priming’ of pituitaries (Pickering and Fink, 1979; Dekoning et al., 1982) and GnRH-stimulated increases in CAMP production (Bourne, 1988) both occur in the absence of extracellular Ca2+. It should be noted, however, that although GnRH can increase CAMP production in anterior pituitaries, it is still unclear whether this represents a direct or indirect (resulting from paracrine interactions) effect of GnRH. Using a totally different experimental approach, Waring and Turgeon (1992) also suggested an involvement of CAMP in GnRH self-potentiation. These investigators further demonstrated that CAMP’S involvement appears to include a protein kinase A (PKA) cascade which involves transcriptional activation as a result of cross-talk between PK.4 and the progesterone receptor (Waring and Turgeon. 1992). The demonstration that CAMP does not mediate LH responses to initial exposures of GnRH does not preclude the nucleotide from being involved (in addition to stimulating de novo protein synthesis) in mediating the enhanced LH responses to subsequent administrations of GnRH. However, this apparently is not the case. The similarity of the responses to GnRH, and GnRH + forskolin, 150 min after a single pulse of forskolin, coupled with the ability of a pulse of forskolin + GnRH (at 60 min) to cause similar restorations of the flufenamateinhibited self-priming effect when subsequently challenged with GnRH or GnRH + forskolin, strongly suggest that the immediate presence of the cyclic nucleotide during the second response is not required for the expression of the self-priming effect. In other words, once the CAMP-stimulated, newly synthesized proteins are available, the nucleotide is no longer required for the manifestation of GnRH self-potentiation. The fact that the role of CAMP in gonadotrophs appears to be limited to the production of specific proteins raises some intriguing possibilities. Other reports have demonstrated that cholera toxin, forskolin and/or dbcAMP can potentiate GnRH-stimulated LH secretion from dispersed pituitary cells (Janovick and Conn, 1993) and pituitaries of ovariectomized rats by protein synthesis-dependent mechanisms (Das and Bourne, 1992). However, the dispersed cells and gonadotrophs from ovariectomized animals do not exhibit the self-priming
and Cellular
Endocrinology
107 (1995) 1-7
effect of GnRH (Aiyer et al., 1974; Beck et al., 1978, Cover and Buckingham, 1989; Janovick and Conn, 1993). These observations highlight two important points. First, they indicate the need to make a distinction between the ability of CAMP to ‘potentiate’ submaximal GnRH responses (as in the case of ovariectomized animals) and its ability to mediate the self-priming effect of GnRH. In this regard, it is interesting to note that non-specific, receptorindependent secretagogues (such as elevated extracellular K+ and the calcium ionophores) can elicit a greater release of LH after GnRH exposure (Pickering and Fink, I976b, 1979; Turgeon and Waring, 1983; Johnson and Mitchell, 1991). We believe that these responses are a manifestation of CAMP’S ability to cause a general facilitation of LH secretion by making available the proteins whose synthesis is regulated by the cyclic nucleotide. Secondly, the observations make it tempting to speculate that the manifestation of the self-priming effect of GnRH in intact females might be due to the fact that CAMP produces different subsets of proteins in pituitaries from ovariectomized and intact animals, or at the very least, the nucleotide produces additional proteins in pituitaries from intact females. Alternatively, the nucleotide may produce identical proteins in pituitaries from both animal models, thereby requiring an additional, unique steroid-dependent mechanism to generate the self-priming effect of GnRH in pituitaries from intact animals. Since these suggestions are not mutually exclusive, the differences in the responses of gonadotrophs from ovariectomized and intact animals may be due to any combination of these possibilities. With respect to the possible requirement for an additional unique steroid-dependent mechanism to generate the self-priming effect of GnRH, our unpublished results suggest that the cellular mechanisms involved in mediating the GnRH-stimulated, extracellular Ca2*-independent component of LH secretion are apparent only under those steroidal conditions required for the expression of GnRH self-potentiation, suggesting their involvement in the phenomenon. Consequently, we believe that the mechanisms involved in this component of LH release and the proteins produced by CAMP are both necessary for the manifestation of the self-priming effect of GnRH. In summary, CAMP does not mediate the LH responses to initial administrations of GnRH and the immediate presence of the nucleotide is not required for the expression of the self-priming effect during subsequent exposures to the decapeptide. However, CAMP does play a pivotal but indirect role in the genesis of GnRH selfpotentiation, an involvement that is dependent on the stimulation of de novo protein synthesis. Acknowledgments This work was supported by grants from the Saskatchewan Health Research Board and the NSERC Presi-
GAbdilnour, G.A. Boume / Molecular and Cellular Endocrinology 107 (1995) 1-7
dential Award (University of Saskatchewan). The materials for the radioimmunoassay of LH were kindly provided by Dr. A.F. Parlow and the National Hormone and Pituitary Program, NIDDK, NIH (Bethesda MD, USA). References Aiyer. MS.. Chiappa, S.A. and Fink, G. (1974) J. Endocrinol. 62, 573588. Baldwin, D.M., Ramey, J.W. and Wiltinger, W.W. (1983) Biol. Reprod. 29,99-111. Beck, L.V., Bay, M., Smith, A.F., King, D. and Long, R. (1978) J. Endocrinol. 77,293-299. Boume, GA. (1988) Mol. Cell. Endocrinol. 58, 155-160. Bourne, GA. (1992) Pharmacol. Toxicol. 71,391-394. Boume, GA. and Baldwin, D.M. (1980) Endocrinology 107,780-788. Boume, G.A. and Baldwin, D.M. (1987a) Am. J. Physiol. 253, E29& E295. Boume, G.A. and Baldwin, D.M. (1987b) Am. J. Physiol. 253, E296E299. Buckingham, J.C. and Cover. P.O. (1986) Acta Endocrinol. 113, 479486. Conn, PM., McArdle, C.A., Andrews, W.V. and Huckle, W.R. (1987) Biol. Reprod. 36, 17-35. Cover, PO. and Buckingham, J.C. (1989) Acta Endocrinol. 121, 365373. Das, S. and Bourne, G.A. (1992) Pharmacol. Toxicol. 71, 395400. Das, S., Fahmy, N.W. and Boume, G.A. (1989) Mol. Cell. Endocrinol. 66, l-8. DeKoning, J., Tijssen, A.M.I., van Dieten, J.A.M.J. and van Rees, G.P. (1982) J. Endocrinol. 94, 11-20. Downs Jr., R.W. and Auxbach, G.D. (1982) J. Cyclic Nucleotide Res. 8, 235-242. Edwardson, J.A. and Gilbert D. (1975) Nature 255.71. Edwardson. J.A. and Gilbert, D. (1976) J. Endocrinol. 68,197-207.
7
Evans, W.S., Uskavitch, D.R, Kaiser, D.L., Hellmann, P., Borges, L.C. and Thomer, M.O. (1984) Endocrinology 114,861-867. Fahmy, N.W. and Boume, G.A. (1993) Cell Calcium 14.25-32. Fink, G., Chiappa, S.A. and Aiyer, M.S. (1976) J. Endocrinol. 69, 359372. Janovick, J.A. and Conn, P.M. (1993) Endocrinology 132,2131-2135. Johnson, M.S. and Mitchell, R (1991) J. Endocrinol. 129, 351-355. Kolp, L.A., Krieg, R.J. and Evans, W.S. (1991) Neuroendocrinology 54.399-404. Liu, T. and Jackson, G.L. (1981) Am. J. Physiol. 241, E6-E13. Liu, T., Wang, P.S. and Jackson, G.L. (1981) Am. J. Physiol. 241, E14E21. McKenzie, H.A. and Dawson, R.M.C. (1969) in Data for Biochemical Research (Dawson, R.M., Elliot, D.C., Elliot, W.H. and Jones, K.M., eds.), p. 507. Oxford University Press, New York. Naor, Z., Koch, Y., Bauminger, S. and Zor. U. (1975a) Prostaglandins 9, 211-219. Naor, Z., Koch, Y., Chobsieng, P and Zor, U. (1975b) FEBS Lett. 58, 318-321. Pickering, A J.M.C. and Fink, G. (1976a) J. Endocrinol. 69.373-379. Pickering, A.J.M.C. and Fink, G. (1976b) J. Endocrinol. 69.453454. Pickering, A J.M.C. and Fink, G. (1979) I. Endocrinol. 81,223-234. Rodbard. D., Bridson, W. and Rayford, PL. (1969) J. Lab. Clin. Med. 74,77&780. Turgeon, J.L. and Waring, D.W. (1983) Am. J. Physiol. 224, E170E176. Turgeon, J.L. and Waring, D.W. (1986) Am J. Physiol. 250, E62-E68. Turgeon, J.L. and Waring, D.W. (1990) Endocrinology 127,773-780. Turgeon, J.L. and Wa&g, D.W. (1991) Endocri&logy 129, 32343239. Waring, D.W. and Turgeon, J.L. (1980) Endocrinology 106, 14301436. Waring, D.W. and Turgeon, J.L. (1983) Am. J. Physiol. 244, C410C418. Waring, D.W. and Turgeon, J.L. (1992) Endocrinology 130, 32753282.
Molecular
and Cellular Endocrinology
107 (1995) l-7
Adenosine 3 ’ ,S-cyclic monophosphate and the self-priming effect of gonadotrophin-releasing hormone George Abdilnour, Gregory A. Bourne* Department
of Physiology
Vniversity
of Saskatchewan,
Saskutoon, Saskatchewan,
Received 6 May 1994; 6 October
S7N OWO, Canada
1994
Abstract An in vitro perifusion system was used to ascertain the role of CAMP in the genesis of the self-priming effect of gonadotrophinreleasing hormone (GnRH) in rat pituitaries. Ten-minute pulses of 20 nmol/l GnRH administered 150 min apart resulted in the manifestation of the self-priming effect, an effect which was inhibited by 5 pmol/l cycloheximide. Forskolin (1 PmoYl) which does not stimulate luteinizing hormone (LH) secretion or affect the initial LH response to GnRH significantly potentiated the second response through protein synthesis-dependent mechanisms. Additionally, an initial IO-min pulse of forskolin alone was sufficient to prime the pituitary to a subsequent pulse of GnRH 150 min later. Interestingly, similar amounts of LH were secreted in response to forskohn + GnRH or GnRH administered 150 min after forskolin. Flufenamate, an inhibitor of GnRH-stimulated increases in CAMP production prevented the manifestation of the self-priming effect of GnRH. Forskolin which bypasses the inhibitory effects of flufenamate on CAMP production reversed the flufenamate-induced inhibition of the self-priming effect of GnRH through protein synthesis-dependent processes. These results suggest that CAMP does not mediate the LH response to an initial exposure of GnRH, but does play a pivotal role in the genesis of the self-priming effect of GnRH through the stimulation of de novo protein synthesis. Once the newly synthesized proteins are available, the nucleotide is not required for the manifestation of the phenomenon. Keywords:
Cycloheximide; Flufenamate; Forskolin; Luteinizing hormone; Perifusion system; Pituitary (rat)
1. Introduction Studies on the secretory actions of gonadotrophinreleasing hormone (GnRH) have established that repeated pulsatile administration of equal amounts of GnRH result in the secretion of progressively greater amounts of luteinizing hormone (LH) (Aiyer et al., 1974; Edwardson and Gilbert, 1976; Waring and Turgeon, 1980, Evans et al., 1984; Buckingham and Cover, 1986). This phenomenon, known as the self-priming effect of GnRH, is dependent on estradiol, progesterone (Aiyer et al., 1974; Beck et al., 1978; Cover and Buckingham, 1989; Turgeon 1990, 1991) and protein synthesis and Waring, (Edwardson and Gilbert, 1975; Pickering and Fink, 1976a, 1979; Turgeon and Waring, 1991) and requires a
*
Corresponding
author, Department
of Livestock
Science, Faculty of
Agriculture, University of the West Indies, St Augustine, Trinidad. Tel.: (809) 6622002;
Fax: (809) 6639686.
0 1995 Elsevier Science Ireland Ltd. All rights reserved 0303-7207/95/$09.50 SSDl 0303-7207(94)03418-8
minimum of 30-60 min to develop (Aiyer et al., 1974; Waring and Turgeon, 1983). Continuous infusions of GnRH result in a biphasic secretion of LH (Fink et al., 1976; Bourne and Baldwin, 1980; Waring and Turgeon, 1983; Das et al., 1989). The response is characterized by an initial acute release of the hormone (phase I), followed approximately 30-60 min later by a greatly augmented secretion rate (phase II). The initial release occurs independently of de novo protein synthesis, while the secondary phase is dependent on protein synthesis (Bourne and Baldwin, 1980; Das et al., 1989) and estradiol (Baldwin et al., 1983). The similarities in the time lag required for the manifestation of the biphasic response and the self-priming effect, coupled with the mutual dependency on de novo protein synthesis and estradiol, strongly suggest that these two events result from common underlying cellular mechanisms. Studies designed to elucidate potential second messengers of the biphasic secretion of LH have demonstrated that adenosine 3’S’-cyclic monophosphate (CAMP) plays
2
GAbdilnour, G.A. Bourne / Molecular und Cellulur Endocrinology 107 (1995) l-7
a pivotal but indirect role as a mediator of the protein synthesis-dependent, phase II secretion of the hormone (Bourne and Baldwin, 1987a; Bourne, 1988). If the phase II secretion of LH and the self-priming effect of GnRH are indeed the result of common cellular mechanisms, CAMP should be involved (indirectly) in the self-priming effect of GnRH. The present study was undertaken to explore this possibility. Flufenamate inhibits GnRH-stimulated CAMP production (Naor et al., 1975a; Naor et al., 1975b: Bourne and Baldwin, 1987a,b) apparently by disrupting G, protein function (Bourne, 1992). Forskolin which increases CAMP production in anterior pituitaries (Kolp et al., 1991; Bourne, 1992) by directly affecting the catalytic protein (Downs and Aurbach, 1982), bypasses the inhibitory effects of flufenamate on CAMP production (Bourne, 1992). Consequently, these two compounds were used as pharmacological probes to delineate potential roles of CAMP as a mediator of GnRH self-potentiation. A previous study evaluating the practicability of utilizing forskolin to restore intracellular CAMP in the presence of flufenamate (Das and Bourne, 1992) had demonstrated that forskolin offered distinct advantages over the use of exogenously administered CAMP analogues. 2. Materials and methods 2.1. Materials The following materials were purchased from Sigma Chemical Co. (St. Louis, MO): bovine serum albumin (BSA); cycloheximide; dimethyl sulfoxide (DMSO); flufenamic acid and forskolin. GnRH was obtained from Calbiochem Corporation (La Jolla, CA). Flufenamic acid (10 g/l) was dissolved in 0.1 mol/l NaOH and added to the perifusion medium prior to adjusting the pH. Forskolin was solubilized in DMSO. The concentrations of cycloheximide, flufenamate and forskolin used in this study were based on results from previous reports (Bourne and Baldwin, 1980, 1987a; Das and Bourne, 1992). 2.2. Animals Adult (75-85 days), female Sprague-Dawley rats (bred from a departmental colony) were maintained in a controlled environment (20 + 1°C; lights on 06001800 h). Food and water were supplied ad libitum. Female rats exhibiting at least two consecutive 4-day estrous cycles (determined by daily vaginal cytology) were selected as pituitary donors on the second day of diestrous. The animals were killed by decapitation between 1200 and 1230 h and the anterior pituitaries rapidly excised and placed in pre-warmed (37°C) perifusion medium (Bourne and Baldwin, 1980). 2.3. Perifusion system The in vitro perifusion
system
was described
previ-
ously (Fahmy and Bourne, 1993). Briefly, two quartered pituitary glands were placed in a perifusion chamber (Millipore Swinnex-13 filter holder) and superfused with Krebs Improved Ringer I medium (McKenzie and Dawson, 1969) supplemented with essential amino acids and vitamins (Flow Laboratories, Rockville, MD) at a flow rate of 0.25 ml/min. The medium was maintained at 37°C and continuously equilibrated with 95% 0Z/5% C02. The perifusate from the first hour was discarded, at which time stable, basal LH levels were established. Then, sequential effluent samples were collected every 10 min in tubes precoated with 2% BSA. The pituitaries were challenged with 20 nmol/l GnRH and/or 1 pmol/l forskolin for 10 min. The first pulse of secretagogue(s) was administered 60 min after sample collection was initiated, with the second pulse following 150 min later. At the end of an experiment, the samples were stored frozen at -20°C until assayed for LH. 2.4. Radioimmunoassay The samples were assayed for LH in duplicate using the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) rat radioimmunoassay kit. Sample estimates were calculated using the log-logit transformation and weighted least squares regression (Robard et al., 1969). Values for LH were compared with the NDDK reference preparation of rat LH-RP-3, which is equipotent to LH-RP-2 and -61 times more potent than LH-RP-1. The intra-assay and interassay coefficients of variation were 4.8% and 5.4%, respectively, at 159pgll. 2.5. Data analysis The total amounts of LH secreted in response to the IO-min pulses of sectretagogues were calculated as follows: three values of basal secretion (obtained just before the IO-min pulses of secretagogue(s)) were used to calculate a mean value for basal secretion/l0 min. This value was subtracted from each of the IO-min fractions. The values corrected for basal secretion were summed to obtain the total amounts of LH secreted in response to the secretagogues. The duration of the response was from the time of infusion of the secretagogue(s) up to the time when LH secretion had returned to stable, baseline levels. Differences between the total amounts of LH secreted in response to the sequential pulses of secretagogues were analyzed by Student’s t-test for paired data to determine whether the priming effect was apparent. The amounts of LH secreted in response to secretagogues in different experimental (treatment) groups were compared using analysis of variance, with differences between the means being determined by Duncan’s new multiple range test. A value of P < 0.05 was considered significant. All statistical tests were performed by the Number Cruncher Statistical System software (NCSS, Kaysville, UT).
3
G.Abdilnour, G.A. Bowne I Molecular and Cellular Endocrinology 107 (1995) l-7
110 0
100
2
GnRH
l GuRH+
600
-
500
-
CYCLQHFXIMIDE
90
80 1
70
z 2
60
Fig. 2. Total amounts of LH released in response to IO-min pulses of secretagogues administered 150 min apart. Values are the means f SEM (n =4 chambers). The abbreviations are: cycloheximide (cycle), forskolin (forsk) and flufenamate (flu). *P < 0.01 and **P < 0.001 for comparisons of 1st and 2nd pulses by paired t-test. Other statistical data appear in the text.
!!I a 50
to GnRH. The results are shown in Fig. 3. Ten-minute pulses of 1 pmol/l forskolin infused 150 min apart were ineffective in stimulating LH secretion (Fig. 3). When the diterpene was administered in conjunction with GnRH, it did not significantly affect the magnitude of the first response to GnRH (Figs. 2 and 3) but did establish a new 01
0
I
I
60
120
It
180
240
110
1
300
Cl
360
TIME (minuteil)
heximide was present in the medium from the beginning of the experiment. Results are the means i SEM (n = 4 chambers). Absence of standard error bars is indicative of the fact that SEMs are smaller than the size of the symbol. The data reported in the figures are the results from one experiment. However, the experiments weE repeated at least twice to confirm their reproducibility.
3.Results The data from the first series of experiments illustrate the characteristics of the self-priming effect of GnRH. The concentration of GnRH (20 nmol/l) used in this study was submaximal and was chosen from a dose-response curve (data not shown). As shown in Fig. 1, a IO-min pulse of GnRH elicited an increase in LH secretion. The second LH response to a similar GnRH challenge administered 150 min later was significantly greater than the first response (P < 0.01; Figs. 1 and 2). Cycloheximide (S,~mol/l) which did not significantly affect the first response to GnRH (Figs. 1 and 2) inhibited the enhanced LH secretion of the second response, reducing it to the level of the first response (Figs. 1 and 2). The second series of experiments were undertaken to determine whether increases in endogenous CAMP (stimulated by forskolin) would affect the LH responses
0 GnRIi + FOR8KOLlN
100
Fig. 1. LH responses from diestrous rats to IO-min pulses of 20 nmol/l GnRH administered 150 min apart (indicated by the black square on the abscissa) in the presence or absence of 5 pmol/l cycloheximide. Cyclo-
MR8KOLlN
0 GnRH + FORSKOW + CYCLOHKKMDE
90
80 fl
7c
z 2
6C
SC
10
I
0
60
I,
I
120 FLM
180
240
4
300
360
(minutes)
Fig. 3. LH responses to IO-min pulses of 1 PmoUl forskolin or GnRH + forskolin administered 150 min apart (indicated by the black squares on the abscissa) in the presence or absence of cycloheximide (n = 4 chambers). For further details see legend for Fig. I.
4
GAbdilnour.
1st PULSE
0
G.A. Bourne I Moleculur
2nd
PULSE
l
CnRH
-
FORSKOLJN
o
DYSO
-
GnEN
0
FORSKOLJN -
CnRH
+
FORSKOLJN -
CnRH + FORSKOIJN
60
120 TIME
180
240
300
360
(minutes)
Fig 4. LH responses to IO-mm pulses of various combinations of forskolin and/or GnRH 150 min apart (indicated by the black squares on the abscissa). For further details see legend for Fig. I (n = 4 chambers).
baseline (70 min after administration) which was approximately twice the secretory rate of basal secretion occurring prior to the administration of the secretagogues (Fig. 3). Additionally, the second response to GnRH was greatly potentiated by forskolin when compared with the second response to infusions of GnRH alone (P < 0.05; Fig. 2). Cycloheximide reduced the potentiated, second response to GnRH + forskolin to the same level as the first response (Figs. 2 and 3). These results suggest that CAMP does not participate in the initial LH responses to GnRH but can sensitize gonadotrophs (through protein synthesis dependent mechanisms) to subsequent exposures of the decapeptide. However, it is still unknown whether subsequent increases in CAMP are involved in the ‘immediate’ responses to subsequent administrations of GnRH. This possibility was explored in the following series of experiments. GnRH elicited a characteristic LH response when administered at 60 min (Fig. 4; Table 1). Forskolin did not significantly affect LH secretion when infused 150 min later (Fig. 4; Table 1). DMSO (the vehicle for forskolin) did not stimulate LH secretion when administered at
and Cellulur Endocrinology
IO7 (1995) 1-7
60 min, nor did it affect the GnRH-stimulated response 150 min later; i.e. the response to GnRH at 220 min was similar to the GnRH-stimulated response obtained at 60 min (Fig. 4; Table 1). Forskolin also did not stimulate LH secretion when administered at 60 min, but did potentiate the response to GnRH 150 min later (P < 0.05; Fig. 4; Table I), an effect which was blocked by cycloheximide (data not shown). Interestingly, the LH response to a pulse of GnRH + forskolin infused 150 min after the administration of forskolin was not significantly different from the response to GnRH given 150 min after forskolin (Fig. 4; Table 1). These results suggest that CAMP only participates in sensitizing gonadotrophs to subsequent exposures of GnRH and is not otherwise involved in the ‘immediate’ responses to subsequent applications of the decapeptide. In the final series of experiments, flufenamate which inhibits GnRH-stimulated CAMP production in female rat pituitaries (Bourne and Baldwin, 1987a; Bourne, 1988) was used as a pharmacological probe to evaluate the effects of an absence of increases in CAMP concentrations on the self-priming effect of GnRH. Since the inhibition of GnRH-stimulated CAMP production by flufenamate was documented previously (Bourne and Baldwin, 1987a; Bourne, 1988), similar experiments were not repeated for this study. As shown in Fig. 5a, flufenamate (1O~molll) reduced the magnitude of the second LH response to GnRH to the level of the first response without significantly affecting the first response; i.e. the compound only inhibited the self-priming effect of GnRH (Figs. 2 and 5a). As a result, the responses to GnRH obtained in the presence of flufenamate were similar to those obtained in the presence of cycloheximide (Fig. 2). Forskolin which bypasses the inhibitory effects of flufenamate on CAMP production (Bourne, 1992) was efficacious in reversing the flufenamate-induced inhibition of the self-priming
Table
1
Total amounts of LH secreted in response secretagogues administered 150 min apart Secretagogues
to
IO-min
pulses
of
LH secreted (ng)
1st pulse
2nd pulse
I st pulse
2nd pulse
GnRH DMSO Forskolin Forskolin
Forskolin GnRH GnRH GnRH + forskolin
95.0 f. 18.8”
9.3 f 102.5 240.5 250.6
_ _
3.3c + 17.5” + 15.6b f 12.4b
Values represent the mean * SEM (n = 4). The ‘-’ indicates the secretagogue was ineffective in stimulating LH secretion above basal levels. The superscript letters indicate statistical significance or insignificance. Values with the same superscript letters are statistically similar, whereas values denoted by different superscripts are significantly different from each other. The P values and definitions of abbreviations appear in the text.
G.Abdilnour, G.A. Boume I Molecular and Cellular Endocrinology 107 (1995) 1-7 110 OGaRH+l’WFENAHATZ
l GnRH+hLRIcNuLITc + ml?sKoLIN
100
-
90
-
80
-
-
B 0
IwRsKoLtN
+ ITulmNAMAm
l GaR?I+~RSKGbtN+~~~ + cYclmxMnt
A
70
1
70
$
60
5
BO-
E !I
50
ii
40
!I 30
OL 0
60
120 TM!
160
240
300
360
(ldnu~)
0
60
120 lmE
160
240
300
360
(miautm)
Fig. 5. LH responses to IO-mitt pulses of GnRH f forskolin in the presence of lO~mol/l flufenamate (A) and forskolin + GnRH in the presence of flufenamate + cycloheximide (B). The secretagogues were administered 150 min apart (indicated by the black squares on the abscissa), while flufenamate and cycloheximide were present in the medium from the beginning of the experiment (n = 4 chambers). For further details see legend for Fig. 1.
effect of GnRH (P c 0.001; Figs. 2 and 5a). The inhibitory effects of flufenamate were also reversed when a pulse of GnRH was administered 150 min after GnRH + forskolin (data not shown). The reversal of the inhibition of the self-priming effect of GnRH could not be attributed interactions between forskolin and to synergistic flufenamate, since these compounds did not affect LH secretion (Fig. 5b). Finally, cycloheximide prevented the forskolin-induced restoration of the self-priming effect of GnRH (Figs. 2 and 5B), indicating that this response was also dependent on protein synthesis. 4. Discussion Flufenamate was previously utilized in studies demonstrating a pivotal role for CAMP in the biphasic secretion of LH from pituitaries of diestrous II rats (Bourne and Baldwin, 1987a). However, the application of this pharmacological agent to directly assess the potential involvement of CAMP in the self-priming effect of GnRH was not possible until recently. Crucial experiments in such studies are the demonstration that the replacement of CAMP results in the restoration of the flufenamateinduced inhibition of GnRH-stimulated LH secretion. In previous studies, CAMP replacement was achieved by the exogenous administration of dibutylyl CAMP
(dbcAMP). Unfortunately, this CAMP analogue (and others) compounded data interpretation by potentiating the initial (acute) GnRH-stimulated LH responses by protein synthesis-independent mechanisms (Bourne and Baldwin, 1987a; Das and Bourne, 1992). Recent demonstrations that forskolin bypasses flufenamate-induced inhibition of CAMP production in anterior pituitaries (Bourne, 1992) and does not affect the protein synthesisindependent components of GnRH-stimulated LH secretion (Das and Bourne, 1992) effectively negated this problem, facilitating the successful completion of the present study. For decades the role of CAMP in GnRH-stimulated LH secretion remained enigmatic. The demonstration that the nucleotide has little or no direct effect on LH secretion (Liu and Jackson, 1981; Liu et al., 1981; Turgeon and Waring, 1986; Bourne and Baldwin, 1987a; Conn et al., 1987; Kolp et al., 1991), formed the basis of the concept that CAMP does not participate in the acute release of LH. The current study supports this concept. The present findings that (i) CAMP (stimulated by forskolin) does not affect the LH response to the first pulse of GnRH, and (ii) these initial responses were not affected by the absence of GnRH-stimulated increases in CAMP production, provide the first direct proof that CAMP does not affect LH secretion during initial exposures to GnRH.
6
GAbdilnnur,
G.A. Bourne / Molecular
Although CAMP does not appear to be involved in mediating the initial LH responses to GnRH, the nucleotide does sensitize gonadotrophs to subsequent exposures of the decapeptide by activating mechanisms that involve de novo protein synthesis. These observations coupled with the demonstration that the flufenamate-induced inhibition of the self-priming effect of GnRH was reversed by CAMP replacement, provide persuasive evidence that CAMP plays a pivotal role in the genesis of the selfpriming effect of LH. Further indirect evidence that is consistent with CAMP playing a role in the genesis of GnRH self-potentiation is the demonstration that GnRH ‘priming’ of pituitaries (Pickering and Fink, 1979; Dekoning et al., 1982) and GnRH-stimulated increases in CAMP production (Bourne, 1988) both occur in the absence of extracellular Ca2+. It should be noted, however, that although GnRH can increase CAMP production in anterior pituitaries, it is still unclear whether this represents a direct or indirect (resulting from paracrine interactions) effect of GnRH. Using a totally different experimental approach, Waring and Turgeon (1992) also suggested an involvement of CAMP in GnRH self-potentiation. These investigators further demonstrated that CAMP’S involvement appears to include a protein kinase A (PKA) cascade which involves transcriptional activation as a result of cross-talk between PK.4 and the progesterone receptor (Waring and Turgeon. 1992). The demonstration that CAMP does not mediate LH responses to initial exposures of GnRH does not preclude the nucleotide from being involved (in addition to stimulating de novo protein synthesis) in mediating the enhanced LH responses to subsequent administrations of GnRH. However, this apparently is not the case. The similarity of the responses to GnRH, and GnRH + forskolin, 150 min after a single pulse of forskolin, coupled with the ability of a pulse of forskolin + GnRH (at 60 min) to cause similar restorations of the flufenamateinhibited self-priming effect when subsequently challenged with GnRH or GnRH + forskolin, strongly suggest that the immediate presence of the cyclic nucleotide during the second response is not required for the expression of the self-priming effect. In other words, once the CAMP-stimulated, newly synthesized proteins are available, the nucleotide is no longer required for the manifestation of GnRH self-potentiation. The fact that the role of CAMP in gonadotrophs appears to be limited to the production of specific proteins raises some intriguing possibilities. Other reports have demonstrated that cholera toxin, forskolin and/or dbcAMP can potentiate GnRH-stimulated LH secretion from dispersed pituitary cells (Janovick and Conn, 1993) and pituitaries of ovariectomized rats by protein synthesis-dependent mechanisms (Das and Bourne, 1992). However, the dispersed cells and gonadotrophs from ovariectomized animals do not exhibit the self-priming
and Cellular
Endocrinology
107 (1995) 1-7
effect of GnRH (Aiyer et al., 1974; Beck et al., 1978, Cover and Buckingham, 1989; Janovick and Conn, 1993). These observations highlight two important points. First, they indicate the need to make a distinction between the ability of CAMP to ‘potentiate’ submaximal GnRH responses (as in the case of ovariectomized animals) and its ability to mediate the self-priming effect of GnRH. In this regard, it is interesting to note that non-specific, receptorindependent secretagogues (such as elevated extracellular K+ and the calcium ionophores) can elicit a greater release of LH after GnRH exposure (Pickering and Fink, I976b, 1979; Turgeon and Waring, 1983; Johnson and Mitchell, 1991). We believe that these responses are a manifestation of CAMP’S ability to cause a general facilitation of LH secretion by making available the proteins whose synthesis is regulated by the cyclic nucleotide. Secondly, the observations make it tempting to speculate that the manifestation of the self-priming effect of GnRH in intact females might be due to the fact that CAMP produces different subsets of proteins in pituitaries from ovariectomized and intact animals, or at the very least, the nucleotide produces additional proteins in pituitaries from intact females. Alternatively, the nucleotide may produce identical proteins in pituitaries from both animal models, thereby requiring an additional, unique steroid-dependent mechanism to generate the self-priming effect of GnRH in pituitaries from intact animals. Since these suggestions are not mutually exclusive, the differences in the responses of gonadotrophs from ovariectomized and intact animals may be due to any combination of these possibilities. With respect to the possible requirement for an additional unique steroid-dependent mechanism to generate the self-priming effect of GnRH, our unpublished results suggest that the cellular mechanisms involved in mediating the GnRH-stimulated, extracellular Ca2*-independent component of LH secretion are apparent only under those steroidal conditions required for the expression of GnRH self-potentiation, suggesting their involvement in the phenomenon. Consequently, we believe that the mechanisms involved in this component of LH release and the proteins produced by CAMP are both necessary for the manifestation of the self-priming effect of GnRH. In summary, CAMP does not mediate the LH responses to initial administrations of GnRH and the immediate presence of the nucleotide is not required for the expression of the self-priming effect during subsequent exposures to the decapeptide. However, CAMP does play a pivotal but indirect role in the genesis of GnRH selfpotentiation, an involvement that is dependent on the stimulation of de novo protein synthesis. Acknowledgments This work was supported by grants from the Saskatchewan Health Research Board and the NSERC Presi-
GAbdilnour, G.A. Boume / Molecular and Cellular Endocrinology 107 (1995) 1-7
dential Award (University of Saskatchewan). The materials for the radioimmunoassay of LH were kindly provided by Dr. A.F. Parlow and the National Hormone and Pituitary Program, NIDDK, NIH (Bethesda MD, USA). References Aiyer. MS.. Chiappa, S.A. and Fink, G. (1974) J. Endocrinol. 62, 573588. Baldwin, D.M., Ramey, J.W. and Wiltinger, W.W. (1983) Biol. Reprod. 29,99-111. Beck, L.V., Bay, M., Smith, A.F., King, D. and Long, R. (1978) J. Endocrinol. 77,293-299. Boume, GA. (1988) Mol. Cell. Endocrinol. 58, 155-160. Bourne, GA. (1992) Pharmacol. Toxicol. 71,391-394. Boume, GA. and Baldwin, D.M. (1980) Endocrinology 107,780-788. Boume, G.A. and Baldwin, D.M. (1987a) Am. J. Physiol. 253, E29& E295. Boume, G.A. and Baldwin, D.M. (1987b) Am. J. Physiol. 253, E296E299. Buckingham, J.C. and Cover. P.O. (1986) Acta Endocrinol. 113, 479486. Conn, PM., McArdle, C.A., Andrews, W.V. and Huckle, W.R. (1987) Biol. Reprod. 36, 17-35. Cover, PO. and Buckingham, J.C. (1989) Acta Endocrinol. 121, 365373. Das, S. and Bourne, G.A. (1992) Pharmacol. Toxicol. 71, 395400. Das, S., Fahmy, N.W. and Boume, G.A. (1989) Mol. Cell. Endocrinol. 66, l-8. DeKoning, J., Tijssen, A.M.I., van Dieten, J.A.M.J. and van Rees, G.P. (1982) J. Endocrinol. 94, 11-20. Downs Jr., R.W. and Auxbach, G.D. (1982) J. Cyclic Nucleotide Res. 8, 235-242. Edwardson, J.A. and Gilbert D. (1975) Nature 255.71. Edwardson. J.A. and Gilbert, D. (1976) J. Endocrinol. 68,197-207.
7
Evans, W.S., Uskavitch, D.R, Kaiser, D.L., Hellmann, P., Borges, L.C. and Thomer, M.O. (1984) Endocrinology 114,861-867. Fahmy, N.W. and Boume, G.A. (1993) Cell Calcium 14.25-32. Fink, G., Chiappa, S.A. and Aiyer, M.S. (1976) J. Endocrinol. 69, 359372. Janovick, J.A. and Conn, P.M. (1993) Endocrinology 132,2131-2135. Johnson, M.S. and Mitchell, R (1991) J. Endocrinol. 129, 351-355. Kolp, L.A., Krieg, R.J. and Evans, W.S. (1991) Neuroendocrinology 54.399-404. Liu, T. and Jackson, G.L. (1981) Am. J. Physiol. 241, E6-E13. Liu, T., Wang, P.S. and Jackson, G.L. (1981) Am. J. Physiol. 241, E14E21. McKenzie, H.A. and Dawson, R.M.C. (1969) in Data for Biochemical Research (Dawson, R.M., Elliot, D.C., Elliot, W.H. and Jones, K.M., eds.), p. 507. Oxford University Press, New York. Naor, Z., Koch, Y., Bauminger, S. and Zor. U. (1975a) Prostaglandins 9, 211-219. Naor, Z., Koch, Y., Chobsieng, P and Zor, U. (1975b) FEBS Lett. 58, 318-321. Pickering, A J.M.C. and Fink, G. (1976a) J. Endocrinol. 69.373-379. Pickering, A.J.M.C. and Fink, G. (1976b) J. Endocrinol. 69.453454. Pickering, A J.M.C. and Fink, G. (1979) I. Endocrinol. 81,223-234. Rodbard. D., Bridson, W. and Rayford, PL. (1969) J. Lab. Clin. Med. 74,77&780. Turgeon, J.L. and Waring, D.W. (1983) Am. J. Physiol. 224, E170E176. Turgeon, J.L. and Waring, D.W. (1986) Am J. Physiol. 250, E62-E68. Turgeon, J.L. and Waring, D.W. (1990) Endocrinology 127,773-780. Turgeon, J.L. and Wa&g, D.W. (1991) Endocri&logy 129, 32343239. Waring, D.W. and Turgeon, J.L. (1980) Endocrinology 106, 14301436. Waring, D.W. and Turgeon, J.L. (1983) Am. J. Physiol. 244, C410C418. Waring, D.W. and Turgeon, J.L. (1992) Endocrinology 130, 32753282.