0013-7227/90/1275-2393$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 127, No. 5 Printed in U.S.A.
Gonadotropin-Releasing Hormone Modulation of Protein Kinase-C Activity in Per ifused Anterior Pituitary Cell Cultures* WILLIAM V. ANDREWS, JAMES R. HANSEN, JO ANN JANOVICK, AND P. MICHAEL CONN Departments of Pharmacology (W. V.A., J.A.J., P.M.C.), Internal Medicine (W. V.A.), and Pediatrics (J.R.H.), University of Iowa College of Medicine, Iowa City, Iowa 52242-1109
ABSTRACT. The ability of GnRH to modulate protein kinase-C (PKC) activity was examined in perifused rat pituitary cell cultures. Under these conditions, LH release and GnRH receptor number remained unchanged after repeated pulses of 1 nM GnRM, whereas PKC (measured both enzymatically and by radioligand assay) showed an initial increase in kinase activity after the first pulse of GnRH (~2-fold), followed by downregulation of PKC activity with subsequent pulses of the releas-
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nRH is a hypothalamic decapeptide which regulates the release of pituitary gonadotropins, LH and FSH, from gonadotropes. These effects appear to be mediated by changes in intracellular calcium, which fulfills the requirements of a second messenger for gonadotropin release (1). In addition to the acute effects on gonadotropin release, GnRH regulates both the number of GnRH receptors and the responsiveness of gonadotropes; these processes have calcium-dependent and independent components (2-11). As with many hormonal systems that mobilize calcium in response to receptor occupancy, it is now well established that agonist occupancy of the GnRH receptor causes the rapid hydrolysis of polyphosphoinositides, leading to the accumulation of inositol phosphate and 1,2-diacylglycerols (12-15), putatively acting as endogenous mediators for calcium mobilization and protein kinase-C (PKC) activation, respectively. The hydrolysis of inositol phospholipids occurs through a phospholipase-C-type reaction which appears to be mediated by a guanine nucleotide-binding protein (16, 17). The demonstration that GnRH causes the redistribution of PKC from the cytosolic to the particulate fraction Received May 7, 1990. Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Department of Pharmacology, University of Iowa College of Medicine, Bowen Science Building, Iowa City, Iowa 52242-1109. * This work was supported by NIH Grants HD-19899 and Physician Scientist Award AM-01295.
ing hormone. It was also observed that the GnRH-stimulated down-regulation of PKC was dependent on the presence of extracellular calcium, which was not the case for the initial upregulation of PKC. These findings are consistent with a modulating role of the GnRH receptor on PKC activity through a Ca2+-dependent process. This study also provides further evidence that GnRH-stimulated LH release and PKC activity can be uncoupled. (Endocrinology 127: 2393-2399, 1990)
of gonadotropes both in vitro (18) and in vivo (19) and that exogenous PKC activators (20, 21) stimulate LH release from gonadotropes and act synergistically with mobilized calcium (22) suggested a possible role for PKC in mediating gonadotropin release in response to GnRH. However, studies in our laboratory and others have shown that cellular LH released in response to GnRH is not altered by the depletion of PKC from pituitary cells or by inhibitors of this enzyme (23-26). It has also been shown that homologous down-regulation of cell surface receptor number is unaltered in PKC-depleted cells (27). These observations raise doubts about a role for PKC in GnRH-regulated LH release or GnRH receptor loss, even though the possibility of the former has been proposed (18). A role for GnRH as a mediator of changes in LH biosynthesis at translational and transcriptional levels in gonadotropes has been demonstrated (28-35), and PKC has been implicated in this process (28, 32). It has also been shown that GnRH can stimulate increases in PKC activity with a similar dose response and time course as the effects on LH/3 mRNA levels (28). In the present study we investigated the possibility that activation of the GnRH receptor modulates the activity of PKC. We have measured cell surface GnRH receptors, PKC activity, and phorbol ester binding in perifused rat anterior pituitary cells under conditions similar to those used for assessment of ligand-stimulated
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LH release. We also evaluated the requirement for extracellular calcium on GnRH-mediated regulation of PKC. Materials and Methods Cell culture and perifusion Female weanling Sprague-Dawley (Sasco, Omaha, NE) rats were used as a source of pituitary tissue in these experiments and were killed by cervical dislocation. Primary pituitary cells were prepared by enzymatic dispersion, as previously described (36), and suspended (2 x 106 cells/0.55 ml) in medium 199 (M199; Gibco, Grand Island, NY, or the media core of the DERC, University of Iowa College of Medicine, Iowa City, IA) containing 0.3% BSA (fraction V, Sigma, St. Louis, MO), 2.5% fetal bovine serum, 10% horse serum (M. A. Bioproducts, Walkersville, MD), 20 ng/ml gentamicin sulfate (Sigma), and 10 mM HEPES (pH 7.4; plating medium). Cell culture and perifusion were performed as a modification of previously described methods (20, 37). Briefly, autoclaved Cytodex beads (16.7 mg/column; Pharmacia Fine Chemicals, Piscataway, NJ) were swollen in PBS (Gibco) and drained; 0.55 ml cell suspension was added to each column along with an additional 0.45 ml plating medium. Columns were sealed and incubated in a water-saturated atmosphere at 37 C for 24 h. The plating medium (1 ml) was replaced after 24 h, and columns were incubated for an additional 24 h. The columns were then submerged in a 37 C water bath and connected to a peristaltic pump via polypropylene tubing and perifused with M199-BSA at a flow rate of 0.25 ml/min. The cells were allowed to adjust to the perifusion environment for 30-60 min before releasing factors were added to the medium. The effluent containing the secreted hormone was collected every 5 min and maintained at 4 C. For experiments assessing the dependence of extracellular calcium, 3 mM EGTA (Sigma) was added to the perifusion medium. Cells were pulsed with GnRH (1 nM) for 5 min at 15min intervals. This pulse frequency was chosen because maximal effects on PKC were observed without significant alteration of GnRH receptor numbers or total cellular LH release. In several experiments for determination of cellular LH, cells were solubilized by one cycle of freeze-thawing and then sonication in 1 ml M199-BSA containing 0.1% Triton X-100 (Sigma). Binding Dispersed pituitary cells were prepared, suspended in plating medium, maintained in culture, and perifused as described above. GnRH receptor binding was assessed by a modification of previously described methods (27), using buserelin ([DSer(tBu)6,Pro9NHEt]GnRH; Hoechst-Roussel Pharmaceuticals, Sommerville, NJ), a metabolically stable GnRH agonist analog (36). The columns were washed with 1 ml M199-BSA at 23 C and equilibrated at this temperature for 20 min. The M199-BSA was decanted and replaced with 500 y\ M199-BSA at 23 C containing 50-2000 pM [125I]buserelin (0.5-1.5 Ci/mg) and 1 mM bacitracin (Sigma). Binding was measured for 20 min at 23 C; nonspecific binding was defined as that observed in the presence of 10 ^M GnRH. The binding incubation was terminated by removal of the radioligand-containing medium and washing cells with 1 ml M199-BSA at 4 C. The cells were then collected by transfer of beads to a Microfuge tube with 1
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ml M199-BSA containing 2.5 mM EGTA. The beads were layered over 1 ml M199-BSA containing 0.3 M sucrose and were collected as a pellet by centrifugation (10 min; 2000 X g; 4 C). The radioactivity in the pellet was determined using a Beckman 5500 7-counter (Fullerton, CA). Phorbol ester binding in intact cell cultures was measured by a procedure analogous to that employed for GnRH receptor binding (38), using [20(iV)-3H]phorbol-12,13-dibutyrate ([3H] PDBu; Amersham, Arlington Heights, IL). Pituitary cells were dispersed, maintained in culture for 2 days, and perifused as described above. The columns were then washed with 1 ml cold PBS (4 C) and equilibrated at this temperature for 20 min. The binding incubation was initiated by the addition of 500 ^1 cold PBS containing 7.25-100 nM (0.07-1.0 /xCi) [3H]PDBu and incubated for 3 h at 4 C. The binding period was terminated by removal of the radioligand-containing medium and washing the cells with 1 ml cold PBS. The cells were collected by transfer of Cytodex beads to a Microfuge tube with 1 ml PBS. The beads were layered over 0.5 ml PBS containing 0.3 mM sucrose and were collected as a pellet by centrifugation (10 min; 2000 X g at 4 C). The radioactivity of the pellet was determined by liquid scintillation spectroscopy using a Beckman LS38O1 counter. Specific binding was determined by subtracting nonspecific binding (incubation containing 1 nM cold phorbol myristate acetate; Sigma) from total binding. PKC assay To assess the effect of GnRH on PKC activity, cells were prepared, cultured on Cytodex beads, and perifused as described above, and the Triton X-100-extractable PKC activity was measured. After perifusion, the columns were washed with 1 ml PBS (37 C), and beads with adherent cells were removed from the columns for determination of PKC activity. Cells were homogenized in 3 ml 25 mM Tris-HCl (pH 7.5), containing 0.25 M sucrose, 2.55 mM MgCl2, 2.5 mM EGTA, 2 mM EDTA, 50 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonylfluoride with 0.3% Triton X-100. The suspension was shaken gently for 30 min at 4 C and applied to a DEAE-Bio-Gel-A column (0.5 ml; Bio-Rad, Richmond, CA) that had been equilibrated with buffer. The columns were then washed with 1 ml buffer containing 20 mM NaCl. PKC was eluted with 5 ml buffer containing 100 mM NaCl and concentrated to 1 ml in a Centricell Ultrafilter (Polysciences, Warrington, PA). We have previously found that 95% of the PKC activity elutriates in the 20- to 100-mM fraction (39). Kinase activity in this fraction was assayed by determing the transfer rate of 32P from [7-'2P] ATP to histone, as previously described (40). The standard assay solution contained in 250 /*1: 5 /umol Tris-HCl (pH 7.5), 1.25 Atmol MgCl2, 50 ng histone (Sigma, type III-S), 2.5 Mmol [T-32P]ATP (Amersham; 6000 Ci/mmol), and 50 ^tl of the concentrated column eluate. The solution was supplemented with EGTA (1 mM), CaCl2 (1 mM), or CaCl2and lipids (10 ng phosphatidylserine and 1 /xg 1,2-diolein; Sigma). The assay reaction was started by the addition of enzyme and was continued for 5 min at 30 C. The reaction was stopped by precipitation of protein on 3 MM filter paper (Whatman, Clifton, NJ) in 10% trichloroacetic acid. After washing, 32P incorporation into precipitated protein was determined by liq-
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GnRH MODULATION OF PKC IN PITUITARY CELLS uid scintillation spectroscopy on a Beckman LS3801 counter. Kinase activity was assessed as counts per min of 32P incorporated/Vg protein from the 20- to 100-mM NaCl column eluate. The Ca'2+- and lipid-dependent kinase activity less the Ca2+dependent kinase activity of this fraction was used as a measure of cellular PKC (39). Protein was assayed according to the Bradford method (41), using BSA (fatty acid free) as a standard. LH determination by RIA LH was measured by RIA, using antiserum (C-102) prepared and characterized in our laboratory (42). Highly purified rat LH (LH 1-7, NIDDK) was iodinated by a modification of the method of Hunter and Greenwood (43), and a second preparation of rat LH (RP-2, NIDDK) was used as the reference protein. Immobilized protein-A was used to separate free and bound hormone (44). When the LH content of Triton X-100solubilized cells was measured, the standard curve samples contained the same final concentration of Triton X-100 as the unknown samples. Data analysis Unless otherwise indicated, the data shown are the mean ± SE of at least three separate experiments. Differences between control and treatment groups were assessed using paired Student's t test; P < 0.05 was considered significant. The number and affinity of GnRH and phorbol ester receptors were assessed by Scatchard analysis (45) of [125I]buserelin and [3H]PDBu binding data, respectively. The line of best fit for the binding data was determined by the least squares method, assuming single site kinetics. Results
Effects of pulsatile GnRH on LH release Repeated pulses of 1 nM GnRH provoked the pulsatile release of LH from perifused pituitary cell cultures, with a diminishing absolute release when pulses were given for 5 min every 15 min (Fig. 1). Since the reduction in
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cellular LH may reduce the subsequent LH-releasing action of GnRH, we have expressed LH release as a percentage of the total cellular LH and have considered the LH-releasing effect of GnRH to be reduced only when the proportion of cellular LH released in response to a subsequent challenge with GnRH is reduced. When the pattern of LH release in response to pulsatile GnRH (1 nM) was evaluated in terms of the percentage of total cellular LH released with each pulse of GnRH, there was an initial release of 12.8% of the total cellular LH, with approximately 7% release with subsequent pulses of GnRH (Fig. 2). GnRH receptor binding The specific binding of [125I]buserelin to pituitary cells was found to be saturable by Scatchard analysis, indicating a single class of high affinity receptors. The pooled data revealed a Kd of 0.58 ± 0.06 nM and a binding capacity (Bmax) of 15 fmol/106 cells, which was not different from that in control cells treated with medium alone (data not shown). Evaluation of GnRH receptor binding after multiple pulses of GnRH (1 nM) showed no significant change in Bmax or Kd after the first and sixth pulses (Fig. 3). Assessment of PKC PKC activity was evaluated by two independent measures: enzymatically, by the ability to transfer 32P from [7-32P]ATP to histone, and by [3H]PDBu binding. GnRH stimulated a marked increase in PKC activity, as measured by 32P incorporation into histone, after the first pulse, with diminishing levels of activity after subsequent pulses of GnRH (Fig. 4). = 16
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FIG. 1. LH release in response to intermittent pulses of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone where indicated by • at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA for GnRH-treated (•) and control (O) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
FIG. 2. Fractional secretion of LH in response to intermittent pulses of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA and is expressed as a percentage of the total cellular LH. Total cellular LH was determined by the LH released in response to GnRH divided by the Triton X-100-extractable LH plus the LH released in response to GnRH (1 nM; • ) and in control (•) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
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FIG. 3. Saturation analysis of [125I]buserelin binding after pulsatile GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. GnRH receptor binding was assessed after the first and sixth pulses of 1 nM GnRH. The cells were washed and incubated for 20 min at 23 C in 500 Atl M199-BSA containing 50-2000 pM [125I]buserelin and 1 mM bacitracin. Specific binding (pulse 1, O; pulse 6, • ) was calculated by subtraction of nonspecific binding (in the presence of 10 nM GnRH) from the total binding. Scatchard analysis (inset) of these data demonstrated no differences in Bmax (~15 fmol/106 cells) or Kd (~0.58 nM) after the first (O) or sixth (•) pulses. The values shown are the mean ± SEM from three separate experiments.
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FlG. 4. PKC activity in response to intermittent pulse of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals (•) or M199-BSA alone (•) at a perifusion rate of 0.25 ml/min. Cellular PKC activity was determined by Triton X-100 extraction, DEAE-cellulose chromatography, and transfer of 32P from [7-32P]ATP to histone, as described in Materials and Methods. The values shown are the mean ± SEM from four separate experiments. *, P < 0.05 compared to control.
As an independent measure of PKC, [3H]PDBu binding was measured. Under control conditions, the Bmax of phorbol binding was approximately 0.09-0.1 pmol/106 cells (Fig. 5A). After the first pulse of 1 nM GnRH, phorbol binding increased 2-fold (Bmax, ~0.19 pmol/106 cells; Fig. 5B). After the sixth pulse of GnRH, phorbol binding returned to control levels (Fig. 5C).
H-PDBu. BOUND (pmol/10 6 cells)
FlG. 5. [3H]PDBu binding after pulsatile GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. Phorbol binding was assessed under control conditions (A) and after the first (B) and sixth (C) pulses of 1 nM GnRH. The cells were washed and then incubated for 3 h at 4 C in 500 ^1 PBS containing 7.25-100 nM [3H]PDBu. Specific binding was determined by subtraction of nonspecific binding (in the presence of 1 fiM phorbol mystrate acetate) from total binding. Data are expressed in Scatchard form; the Bma, was approximately 0.9 pmol/106 cells under control conditions (A), increased 2-fold in response to the initial pulse of GnRH (~0.19 pmol/106 cells; B), and returned to control levels after the sixth pulse of GnRH (C). The values shown are the mean ± SEM from four separate experiments.
Dependence on calcium of GnRH-mediated PKC activity We also evaluated the calcium dependence of GnRHmediated changes in PKC activity. In these experiments 3 mM EGTA was added to the perifusion medium to chelate extracellular Ca2+. Under these conditions, no measurable LH release was observed in response to 1 nM GnRH (Fig. 6). PKC activity was determined under these conditions by [3H]PDBu binding. In these experiments there was an increase in phorbol binding after the first pulse of GnRH (Fig. 7B) compared to that in basal conditions (Fig. 7A). No subsequent down-regulation of
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FIG. 6. LH release in response to intermittent pulses of GnRH in the absence of extracellular calcium. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone in perifusion medium containing 3 mM EGTA (•) at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA for GnRH-treated (•) and control (O) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
PKC activity was observed after the sixth pulse of GnRH in the absence of extracellular Ca2+ (Fig. 7C).
Discussion In addition to stimulation of gonadotropin release, GnRH regulates both GnRH receptor number (2, 3) and the responsiveness of gonadotropes to the releasing hormone (4-7). These effects are of importance, as it is thought that the increase in gonadotrope responsiveness at the time of proestrus is necessary for the LH surge to occur at ovulation (46, 47). In addition, the reduction in gonadotropin secretion produced by prolonged exposure to stable agonist analogs of GnRH has provided the rationale for the clinical use of these compounds (48). Until recently, little was known of the molecular mechanisms by which gonadotrope responsiveness is altered by GnRH or other compounds. The ability of the pituitary to secrete gonadotropins in response to GnRH is dependent on the availability of cell surface receptors, the efficiency of receptor-effector coupling, and the functional state of the effector. Early studies in this system suggested that gonadotrope responsiveness may be regulated by changes in GnRH receptor number (6). However, subsequent studies have revealed that the number of GnRH receptors is not necessarily predictive of gonadotrope responsiveness, suggesting that postreceptor regulation is also involved (23,25,49). Multiple lines of evidence have demonstrated that the GnRH receptor is linked to the hydrolysis of inositol phospholipids and the production of conditions that favor activation of PKC (12-16, 18, 19). It has also been shown that GnRH-stimulated PKC activation and LH release can be uncoupled in vitro and in vivo (12, 24), but the GnRH receptor (29-31, 33-35) and PKC activa-
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FIG. 7. [3H]PDBu binding after pulsatile GnRH in the absence of extracellular calcium. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. Phorbol binding was assessed under control conditions (A) and after the first (B) and sixth (C) pulses of 1 nM GnRH. The cells were washed and then incubated for 3 h at 4 C in 500 fi\ PBS containing 7.25-100 nM [3H]PDBu. Specific binding was determined by subtraction of nonspecific binding (in the presence of 1 pM phorbol mystrate acetate) from total binding. Data are expressed in Scatchard form; the Bmax was approximately 0.1 pmol/106 cells under control condtions (A), increased 2-fold in response to the initial pulse of GnRH (~0.19 pmol/106 cells; B), and remained increased after the sixth pulse of GnRH (C). The values shown are the mean ± SEM from three separate experiments.
tion appear to be linked to LH biosynthesis (28, 32). The present data demonstrate the ability of the agonist-bound GnRH receptor to regulate the activity of PKC in the rat pituitary gonadotrope. In perifused rat pituitary cell cultures, the data show differential regulation of LH release and PKC activity in response to pulsatile GnRH. Whereas PKC activity was increased after the initial pulse of GnRH and then down-regulated to control levels after multiple pulses of GnRH, measured both enzymatically and by radioligand assay, total cellular LH release and GnRH receptor number remained
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essentially unchanged with repeated pulses of the releasing hormone under these same conditions. In addition, the process of GnRH-stimulated PKC down-regulation is dependent on the presence of extracellular calcium in perifused pituitary cell cultures. It has been shown that the activation (50-52) and down-regulation (53, 54) of PKC involve the activation of calcium-dependent neutral proteases (calpains) in multiple cell types (55). Further, calpains redistribute to the plasma membrane in response to phorbol esters and millimolar levels of calcium, and calcium is required for full proteolytic activity of these proteases toward PKC (51, 52, 54). It has also been shown in a rat pituitary cell line that PKC turnover is enhanced by conditions that favor the association of PKC with the plasma membrane (56). In addition, immunohistochemical methods have demonstrated that calpains are present in relatively high quantities in the anterior pituitary in cells that produce glycoprotein hormones (57). These observations together with the previous findings that gonadotropes mobilize calcium (1) and redistribute PKC to the plasma membrane in response to GnRH (18, 39) are consistent with the findings in this study that agonist occupancy of the GnRH receptor modulates PKC activity, and that this occurs in a calcium-dependent manner. The findings that PKC is up-regulated after the initial GnRH pulse, and that this increase in PKC activity is not affected by the removal of extracellular calcium suggest that this process may be more dependent on the generation of endogenous activators of PKC through the phosphatidyl inositol cycle, which can occur in the absence of mobilization of extracellular calcium (49). By contrast, the down-regulation of PKC in response to pulsatile GnRH required the presence of mobilized extracellular calcium, suggesting involvement of different proteases in this process. This type of differential regulation of isotypes of both PKC and calcium-dependent neutral proteases has been reported in other cell systems (50-55). Although GnRH stimulates relatively small changes in total PKC activity in perifused primary pituitary cell cultures, the changes are significant and specific to GnRH stimulation. The amplitude of the observed changes in PKC activity are probably masked by the contribution of other cell types within the pituitary. The data presented in this report are consistent with the view that GnRH mediates PKC activity. While regulation of PKC in these cells may contribute to the level of expression of gonadotropin synthesis and release during different phases of the estrous cycle by varying the pulse frequency of GnRH stimulation, it appears that measurable levels of PKC activity are not predictive of the LH response to GnRH. This system provides a model for further evaluation of the PKC and protease isotypes involved in these processes.
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Note Added in Proof Since acceptance of this manuscript, another manuscript (58) has appeared questioning the role of PKC in GnRH stimulated LH release.
Acknowledgment We thank Sue Birely for her aid in preparing this manuscript.
References 1. Conn PM, Huckle WR, Andrews WV, McArdle CA 1987 The molecular mechanism of action of gonadotropin releasing hormone (GnRH) in the pituitary. Recent Prog Horm Res 43:29-68 2. Clayton RN, Catt KJ 1981 Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev 2:186-209 3. Conn PM, Rogers DC, Seay SG 1984 Biphasic regulation of the
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gonadotropin-releasing hormone receptor by microaggregation and intracellular calcium levels. Mol Pharmacol 25:51-5 Smith WA, Conn PM 1984 Microaggregation of the gonadotropinreleasing hormone-receptor: relation to gonadotrope desensitization. Endocrinology 114:553-9 DeKonig J, vanDieten JAMJ, vanRees GP 1978 Refractoriness of the pituitary gland after exposure to luteinizing hormone releasing hormone. J Endocrinol 79:311-8 Zilberstein M, Rakut H, Naor Z 1983 Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone. Life Sci 32:663-9 Badger TM, Loughlin JS, Naddaff PG 1983 The luteinizing hormone-releasing hormone (LHRH)-desensitized rat pituitary: luteinizing hormone responsiveness to LHRH in vitro. Endocrinology 112:793-9 Jinnah HA, Conn PM 1986 GnRH-mediated desensitization of cultured rat anterior pituitary cells can be uncoupled from LH release. Endocrinology 112:2599-603 Andries M, Denef C 1986 Characterization of luteinizing-hormonereleasing hormone receptor binding in rat pituitary cell monolayer cultures: influence of intracellular communication. Mol Cell Endocrinol 44:147-58 Marian J, Conn PM 1980 The requirement in GnRH-stimulated LH release is not mediated through a specific action on receptor binding. Life Sci 27:87-92 Smith WA, Conn PM 1979 GnRH-mediated desensitization of the pituitary gonadotrope is not calcium dependent. Endocrinology 112:408-10 Snyder GD, Bleasdale JE 1982 Effects of LHRH on incorporation of [32P]orthophosphate into phosphatidyl-inositol by dispersed anterior pituitary cells. Mol Cell Endocrinol 28:55-63 Andrews WV, Conn PM 1986 Gonadotropin-releasing hormone stimulates mass changes in phosphoinositides and diacylglycerol accumulation in purified gonadotrope cell cultures. Endocrinology 118:1148-58 Schrey MP 1985 Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue. Biochem J 226:563-9 Huckle WR, Conn PM 1987 The relationship between gonadotropin-releasing hormone stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators. Endocrinology 120:160-9 Andrews WV, Staley DD, Huckle WR, Conn PM 1986 Stimulation of luteinizing hormone (LH) release and phospholipid breakdown by guanosine triphosphate in permeabilized pituitary gonadotropes: antagonist action suggests association of a G protein and gonadotropin-releasing hormone receptor. Endocrinology 119:2537-46 Waters SB, Hawes BE, Conn PM 1990 Stimulation of luteinizing hormone release by sodium fluoride is independent of protein kinase C activity and not affected by desensitization to gonadotro-
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GnRH MODULATION OF PKC IN PITUITARY CELLS pin-releasing hormone. Endocrinology 126:2583-91 18. Hirota K, Hirota T, Aguilera G, Catt KJ 1985 Hormone-induced redistribution of calcium-activated phospholipid-dependent protein kinase in pituitary gonadotrophs. J Biol Chem 260:3243-6 19. McArdle CA, Conn PM 1986 Hormone stimulated redistribution of gonadotrope protein kinase C in vivo: dependence on calcium influx. Mol Pharmacol 29:570-6 20. Badger TM 1986 Perifusion of anterior pituitary cells: release of gonadotropins and somatotropins. In: Conn PM (ed) Methods in Enzymology: Hormone Action. Academic Press, Orlando, vol 124:79-90 21. Conn PM, Ganong BR, Ebeling J, Staley D, Neidel JE, Bell RM 1985 Diacylglycerols release LH: structure-activity relations and protein kinase C. Biochem Biophys Res Commun 126:532 22. Harris CE, Staley D, Conn PM 1985 Diacylglycerols and protein kinase C: potential amplifying mechanism for Ca2+-mediated GnRH-stimulated LH release. Mol Pharmacol 27:532-6 23. McArdle CA, Huckle WR, Conn PM 1987 Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not to Ca2+-mobilizing secretagogues: does protein kinase C mediate gonadotropin releasing hormone action? J Biol Chem 262:5028-35 24. Drouva SV, Laplante GE, Rerat E, Enjalbert A, Kordon C 1990 Estradiol modulates protein kinase C in vivo and in vitro. Endocrinology 126:536-44 25. Johnson MS, Mitchell R, Fink G 1987 The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro. J Endocrinol 116:231-8 26. Miller WL, Beggs MJ 1989 GnRH-stimulated LH release from ovine gonadotrophs in culture is separate from phorbol esterstimulated LH release. Endocrinology 124:667-74 27. McArdle CA, Gorospe WC, Huckle WR, Conn PM 1987 Homologous down-regulation of gonadotropin releasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C. Mol Endocrinol 1:420-9 28. Andrews WV, Maurer RA, Conn PM 1988 Stimulation of rat luteinizing hormone-/? messenger RNA levels by gonadotropin releasing hormone. J Biol Chem 263:13755-61 29. Papavasiliou SS, Zmeici S, Khoury S, Landefeld TD, Chin WW, Marshall JC 1986 Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone a and /3 subunits in the male rat. Proc Natl Acad Sci USA 83:4026-9 30. Leung K, Kaynard AH, Negrini BP, Kim KE, Maurer RA, Landefeld TD 1987 Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes. Mol Endocrinol 1:724-8 31. Barkan AL, Reame NE, Kelch RP, Marshall JC 1985 Idiopathic hypogonadotropic hypogonadism in men: dependence of hormone responses to gonadotropin-releasing hormone (GnRH) on the magintude of the endogenous GnRH secretory defect. J Clin Endocrinol Metab 61:1118-24 32. Counis GB, Hoffman AR, Jutisz M, Gonadotropin-releasing hormone, cyclic AMP, and phorbol esters stimulate the biosynthesis of luteinizing hormone polypeptide chains. 68th Annual Meeting of The Endocrine Society, Anaheim CA, 1986, p 148 (Abstract) 33. Brostrom MA, Chin K-V, Cade C, Gmitter D, Brostrom CO 1987 Stimulation of protein synthesis in pituitary cells by phorbol esters and cyclic AMP. Evidence for rapid induction of a component of translational initiation. J Biol Chem 262:16515-23 34. Liu T-C, Jackson GL 1978 Modification of luteinizing hormone biosynthesis and release by gonadotropin-releasing hormone, cycloheximide, and actinomycin D. Endocrinology 103:1253-9 35. Liu T-C, Jackson GL 1987 Stimulation by phorbol esters and diacyglycerol of luteinizing horomone glycosylation and release by rat anterior pituitary cells. Endocrinology 121:1589-95 36. Marian J, Conn PM 1979 Gonadotropin releasing hormone stimulation of cultured pituitary cells requires calcium. Mol Pharmacol 6:196-201 37. Hansen JR, Conn PM 1990 Measurement of pulsatile hormone release from perifused pituitary cells immobilized on microcarriers.
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In: Conn PM (ed) Methods in Neurosciences. Academic Press, New York, vol 2:181-94 McArdle CA, Conn PM 1988 The use of protein kinase C-depleted cells for investigation of the role of protein kinase C in stimulusresponse coupling in the pituitary. In: Conn PM (ed) Methods in Enzymology. Academic Press, New York, vol 168:287-301 McArdle CA, Conn PM 1986 Hormone stimulated redistribution of gonadotrope protein kinase C in vivo: dependence on calcium influx. Mol Pharmacol 29:570-6 Castagna M, Takai Y, Kaibucki K, Sano K, Kikkawa U, Nishizuka Y 1982 Direct activation of calcium reactivated, phospholipiddependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257:7847-51 Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54 Smith WA, Cooper RL, Conn PM 1982 Altered pituitary responsiveness to gonadotropin-releasing hormone in middle-aged rats with 4-day estrous cycles. Endocrinology 111:1843-8 Hunter WM, Greenwood FC 1962 Preparation of iodine-131 labeled growth hormone of high specific activity. Nature 194:495-6 Gupta R, Morton DC 1979 Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay. Clin Chem 25:752-6 Scatchard G 1949 The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660-72 Conn PM (ed) 1986 Endocr Rev 7:1-125 Marian J, Cooper RL, Conn PM 1981 Regulation of the rat pituitary gonadotropin-releasing hormone receptor. Mol Pharmacol 19:319-405 Cutler GB, Hoffman AR, Swerdloff RS, Santen RJ, Meldrum DR, Comite F 1985 Therapeutic applications of luteinizing-hormonereleasing hormone and its analogs. Ann Intern Med 102:643-57 Huckle WR, Hawes BE, Conn PM 1989 Protein kinase C-mediated gonadotropin-releasing hormone receptor sequestration is assoicated with uncoupling of phosphoinositide hydrolysis. J Biol Chem 264:8619-26 Suzuki K, Imajoh S, Emori Y, Kawasaki H, Minami Y, Ohno S 1987 Calcium-activated neutral protease and its endogenous inhibitor: activation at the cell membrane and biological function. FEBS Lett 220:271-7 Melloni E, Pontremoli S, Michetti M, Sacco O, Sparatore B, Horecker BL 1986 The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid. J Biol Chem 261:4101-5 Melloni E, Pontremoli S, Michetti M, Sacco O, Sparatore B, Salamino F, Horecker BL 1985 Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring protease. Proc Natl Acad Sci USA 82:6435-9 Huang FL, Yoshida Y, Cunha-Melo JR, Beaven MA, Huang K-P 1989 Differential down-regulation of protein kinase C isozymes. J Biol Chem 264:4238-43 Kishimoto A, Mikawa K, Hashimoto K, Yasuda I, Tanaka S-i, Tominaga M, Kuroda T, Nishizuka Y 1989 Limited proteolysis of protein kinase C subspecies by calcium dependent neutral protease (calpain). J Biol Chem 264:4088-92 Murachi T 1985 The proteolytic system involving calpains. Biochem Soc Trans 13:1015-8 Ballester R, Rosen OM 1985 Fate of immunoprecipitable protein kinase C in GH3 cells treated with phorbol 12-myristate 13-acetate. J Biol Chem 260:15194-9 Kitahara A, Takano E, Ohtsuki H, Kirihata Y, Yamagata Y, Kannagi R, Murachi T 1986 Reversed distribution of calpains and calpastatin in human pituitary gland and selective localization of calpastatin in adrenocorticotropin-producing cells as demonstrated by immunohistchemistry. J Clin Endocrinol Metab 63:343-7 Van de Merwe PA, Millar RP, Davidson N 1990 Calcium stimulates LH exocytosis by a mechanism independent of PKC. Biochem J 268:493-498
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Vol. 127, No. 5 Printed in U.S.A.
Gonadotropin-Releasing Hormone Modulation of Protein Kinase-C Activity in Per ifused Anterior Pituitary Cell Cultures* WILLIAM V. ANDREWS, JAMES R. HANSEN, JO ANN JANOVICK, AND P. MICHAEL CONN Departments of Pharmacology (W. V.A., J.A.J., P.M.C.), Internal Medicine (W. V.A.), and Pediatrics (J.R.H.), University of Iowa College of Medicine, Iowa City, Iowa 52242-1109
ABSTRACT. The ability of GnRH to modulate protein kinase-C (PKC) activity was examined in perifused rat pituitary cell cultures. Under these conditions, LH release and GnRH receptor number remained unchanged after repeated pulses of 1 nM GnRM, whereas PKC (measured both enzymatically and by radioligand assay) showed an initial increase in kinase activity after the first pulse of GnRH (~2-fold), followed by downregulation of PKC activity with subsequent pulses of the releas-
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nRH is a hypothalamic decapeptide which regulates the release of pituitary gonadotropins, LH and FSH, from gonadotropes. These effects appear to be mediated by changes in intracellular calcium, which fulfills the requirements of a second messenger for gonadotropin release (1). In addition to the acute effects on gonadotropin release, GnRH regulates both the number of GnRH receptors and the responsiveness of gonadotropes; these processes have calcium-dependent and independent components (2-11). As with many hormonal systems that mobilize calcium in response to receptor occupancy, it is now well established that agonist occupancy of the GnRH receptor causes the rapid hydrolysis of polyphosphoinositides, leading to the accumulation of inositol phosphate and 1,2-diacylglycerols (12-15), putatively acting as endogenous mediators for calcium mobilization and protein kinase-C (PKC) activation, respectively. The hydrolysis of inositol phospholipids occurs through a phospholipase-C-type reaction which appears to be mediated by a guanine nucleotide-binding protein (16, 17). The demonstration that GnRH causes the redistribution of PKC from the cytosolic to the particulate fraction Received May 7, 1990. Address all correspondence and requests for reprints to: Dr. P. Michael Conn, Department of Pharmacology, University of Iowa College of Medicine, Bowen Science Building, Iowa City, Iowa 52242-1109. * This work was supported by NIH Grants HD-19899 and Physician Scientist Award AM-01295.
ing hormone. It was also observed that the GnRH-stimulated down-regulation of PKC was dependent on the presence of extracellular calcium, which was not the case for the initial upregulation of PKC. These findings are consistent with a modulating role of the GnRH receptor on PKC activity through a Ca2+-dependent process. This study also provides further evidence that GnRH-stimulated LH release and PKC activity can be uncoupled. (Endocrinology 127: 2393-2399, 1990)
of gonadotropes both in vitro (18) and in vivo (19) and that exogenous PKC activators (20, 21) stimulate LH release from gonadotropes and act synergistically with mobilized calcium (22) suggested a possible role for PKC in mediating gonadotropin release in response to GnRH. However, studies in our laboratory and others have shown that cellular LH released in response to GnRH is not altered by the depletion of PKC from pituitary cells or by inhibitors of this enzyme (23-26). It has also been shown that homologous down-regulation of cell surface receptor number is unaltered in PKC-depleted cells (27). These observations raise doubts about a role for PKC in GnRH-regulated LH release or GnRH receptor loss, even though the possibility of the former has been proposed (18). A role for GnRH as a mediator of changes in LH biosynthesis at translational and transcriptional levels in gonadotropes has been demonstrated (28-35), and PKC has been implicated in this process (28, 32). It has also been shown that GnRH can stimulate increases in PKC activity with a similar dose response and time course as the effects on LH/3 mRNA levels (28). In the present study we investigated the possibility that activation of the GnRH receptor modulates the activity of PKC. We have measured cell surface GnRH receptors, PKC activity, and phorbol ester binding in perifused rat anterior pituitary cells under conditions similar to those used for assessment of ligand-stimulated
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LH release. We also evaluated the requirement for extracellular calcium on GnRH-mediated regulation of PKC. Materials and Methods Cell culture and perifusion Female weanling Sprague-Dawley (Sasco, Omaha, NE) rats were used as a source of pituitary tissue in these experiments and were killed by cervical dislocation. Primary pituitary cells were prepared by enzymatic dispersion, as previously described (36), and suspended (2 x 106 cells/0.55 ml) in medium 199 (M199; Gibco, Grand Island, NY, or the media core of the DERC, University of Iowa College of Medicine, Iowa City, IA) containing 0.3% BSA (fraction V, Sigma, St. Louis, MO), 2.5% fetal bovine serum, 10% horse serum (M. A. Bioproducts, Walkersville, MD), 20 ng/ml gentamicin sulfate (Sigma), and 10 mM HEPES (pH 7.4; plating medium). Cell culture and perifusion were performed as a modification of previously described methods (20, 37). Briefly, autoclaved Cytodex beads (16.7 mg/column; Pharmacia Fine Chemicals, Piscataway, NJ) were swollen in PBS (Gibco) and drained; 0.55 ml cell suspension was added to each column along with an additional 0.45 ml plating medium. Columns were sealed and incubated in a water-saturated atmosphere at 37 C for 24 h. The plating medium (1 ml) was replaced after 24 h, and columns were incubated for an additional 24 h. The columns were then submerged in a 37 C water bath and connected to a peristaltic pump via polypropylene tubing and perifused with M199-BSA at a flow rate of 0.25 ml/min. The cells were allowed to adjust to the perifusion environment for 30-60 min before releasing factors were added to the medium. The effluent containing the secreted hormone was collected every 5 min and maintained at 4 C. For experiments assessing the dependence of extracellular calcium, 3 mM EGTA (Sigma) was added to the perifusion medium. Cells were pulsed with GnRH (1 nM) for 5 min at 15min intervals. This pulse frequency was chosen because maximal effects on PKC were observed without significant alteration of GnRH receptor numbers or total cellular LH release. In several experiments for determination of cellular LH, cells were solubilized by one cycle of freeze-thawing and then sonication in 1 ml M199-BSA containing 0.1% Triton X-100 (Sigma). Binding Dispersed pituitary cells were prepared, suspended in plating medium, maintained in culture, and perifused as described above. GnRH receptor binding was assessed by a modification of previously described methods (27), using buserelin ([DSer(tBu)6,Pro9NHEt]GnRH; Hoechst-Roussel Pharmaceuticals, Sommerville, NJ), a metabolically stable GnRH agonist analog (36). The columns were washed with 1 ml M199-BSA at 23 C and equilibrated at this temperature for 20 min. The M199-BSA was decanted and replaced with 500 y\ M199-BSA at 23 C containing 50-2000 pM [125I]buserelin (0.5-1.5 Ci/mg) and 1 mM bacitracin (Sigma). Binding was measured for 20 min at 23 C; nonspecific binding was defined as that observed in the presence of 10 ^M GnRH. The binding incubation was terminated by removal of the radioligand-containing medium and washing cells with 1 ml M199-BSA at 4 C. The cells were then collected by transfer of beads to a Microfuge tube with 1
Endo«1990 Voll27-No5
ml M199-BSA containing 2.5 mM EGTA. The beads were layered over 1 ml M199-BSA containing 0.3 M sucrose and were collected as a pellet by centrifugation (10 min; 2000 X g; 4 C). The radioactivity in the pellet was determined using a Beckman 5500 7-counter (Fullerton, CA). Phorbol ester binding in intact cell cultures was measured by a procedure analogous to that employed for GnRH receptor binding (38), using [20(iV)-3H]phorbol-12,13-dibutyrate ([3H] PDBu; Amersham, Arlington Heights, IL). Pituitary cells were dispersed, maintained in culture for 2 days, and perifused as described above. The columns were then washed with 1 ml cold PBS (4 C) and equilibrated at this temperature for 20 min. The binding incubation was initiated by the addition of 500 ^1 cold PBS containing 7.25-100 nM (0.07-1.0 /xCi) [3H]PDBu and incubated for 3 h at 4 C. The binding period was terminated by removal of the radioligand-containing medium and washing the cells with 1 ml cold PBS. The cells were collected by transfer of Cytodex beads to a Microfuge tube with 1 ml PBS. The beads were layered over 0.5 ml PBS containing 0.3 mM sucrose and were collected as a pellet by centrifugation (10 min; 2000 X g at 4 C). The radioactivity of the pellet was determined by liquid scintillation spectroscopy using a Beckman LS38O1 counter. Specific binding was determined by subtracting nonspecific binding (incubation containing 1 nM cold phorbol myristate acetate; Sigma) from total binding. PKC assay To assess the effect of GnRH on PKC activity, cells were prepared, cultured on Cytodex beads, and perifused as described above, and the Triton X-100-extractable PKC activity was measured. After perifusion, the columns were washed with 1 ml PBS (37 C), and beads with adherent cells were removed from the columns for determination of PKC activity. Cells were homogenized in 3 ml 25 mM Tris-HCl (pH 7.5), containing 0.25 M sucrose, 2.55 mM MgCl2, 2.5 mM EGTA, 2 mM EDTA, 50 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonylfluoride with 0.3% Triton X-100. The suspension was shaken gently for 30 min at 4 C and applied to a DEAE-Bio-Gel-A column (0.5 ml; Bio-Rad, Richmond, CA) that had been equilibrated with buffer. The columns were then washed with 1 ml buffer containing 20 mM NaCl. PKC was eluted with 5 ml buffer containing 100 mM NaCl and concentrated to 1 ml in a Centricell Ultrafilter (Polysciences, Warrington, PA). We have previously found that 95% of the PKC activity elutriates in the 20- to 100-mM fraction (39). Kinase activity in this fraction was assayed by determing the transfer rate of 32P from [7-'2P] ATP to histone, as previously described (40). The standard assay solution contained in 250 /*1: 5 /umol Tris-HCl (pH 7.5), 1.25 Atmol MgCl2, 50 ng histone (Sigma, type III-S), 2.5 Mmol [T-32P]ATP (Amersham; 6000 Ci/mmol), and 50 ^tl of the concentrated column eluate. The solution was supplemented with EGTA (1 mM), CaCl2 (1 mM), or CaCl2and lipids (10 ng phosphatidylserine and 1 /xg 1,2-diolein; Sigma). The assay reaction was started by the addition of enzyme and was continued for 5 min at 30 C. The reaction was stopped by precipitation of protein on 3 MM filter paper (Whatman, Clifton, NJ) in 10% trichloroacetic acid. After washing, 32P incorporation into precipitated protein was determined by liq-
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GnRH MODULATION OF PKC IN PITUITARY CELLS uid scintillation spectroscopy on a Beckman LS3801 counter. Kinase activity was assessed as counts per min of 32P incorporated/Vg protein from the 20- to 100-mM NaCl column eluate. The Ca'2+- and lipid-dependent kinase activity less the Ca2+dependent kinase activity of this fraction was used as a measure of cellular PKC (39). Protein was assayed according to the Bradford method (41), using BSA (fatty acid free) as a standard. LH determination by RIA LH was measured by RIA, using antiserum (C-102) prepared and characterized in our laboratory (42). Highly purified rat LH (LH 1-7, NIDDK) was iodinated by a modification of the method of Hunter and Greenwood (43), and a second preparation of rat LH (RP-2, NIDDK) was used as the reference protein. Immobilized protein-A was used to separate free and bound hormone (44). When the LH content of Triton X-100solubilized cells was measured, the standard curve samples contained the same final concentration of Triton X-100 as the unknown samples. Data analysis Unless otherwise indicated, the data shown are the mean ± SE of at least three separate experiments. Differences between control and treatment groups were assessed using paired Student's t test; P < 0.05 was considered significant. The number and affinity of GnRH and phorbol ester receptors were assessed by Scatchard analysis (45) of [125I]buserelin and [3H]PDBu binding data, respectively. The line of best fit for the binding data was determined by the least squares method, assuming single site kinetics. Results
Effects of pulsatile GnRH on LH release Repeated pulses of 1 nM GnRH provoked the pulsatile release of LH from perifused pituitary cell cultures, with a diminishing absolute release when pulses were given for 5 min every 15 min (Fig. 1). Since the reduction in
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cellular LH may reduce the subsequent LH-releasing action of GnRH, we have expressed LH release as a percentage of the total cellular LH and have considered the LH-releasing effect of GnRH to be reduced only when the proportion of cellular LH released in response to a subsequent challenge with GnRH is reduced. When the pattern of LH release in response to pulsatile GnRH (1 nM) was evaluated in terms of the percentage of total cellular LH released with each pulse of GnRH, there was an initial release of 12.8% of the total cellular LH, with approximately 7% release with subsequent pulses of GnRH (Fig. 2). GnRH receptor binding The specific binding of [125I]buserelin to pituitary cells was found to be saturable by Scatchard analysis, indicating a single class of high affinity receptors. The pooled data revealed a Kd of 0.58 ± 0.06 nM and a binding capacity (Bmax) of 15 fmol/106 cells, which was not different from that in control cells treated with medium alone (data not shown). Evaluation of GnRH receptor binding after multiple pulses of GnRH (1 nM) showed no significant change in Bmax or Kd after the first and sixth pulses (Fig. 3). Assessment of PKC PKC activity was evaluated by two independent measures: enzymatically, by the ability to transfer 32P from [7-32P]ATP to histone, and by [3H]PDBu binding. GnRH stimulated a marked increase in PKC activity, as measured by 32P incorporation into histone, after the first pulse, with diminishing levels of activity after subsequent pulses of GnRH (Fig. 4). = 16
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FIG. 1. LH release in response to intermittent pulses of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone where indicated by • at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA for GnRH-treated (•) and control (O) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
FIG. 2. Fractional secretion of LH in response to intermittent pulses of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA and is expressed as a percentage of the total cellular LH. Total cellular LH was determined by the LH released in response to GnRH divided by the Triton X-100-extractable LH plus the LH released in response to GnRH (1 nM; • ) and in control (•) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
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FIG. 3. Saturation analysis of [125I]buserelin binding after pulsatile GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. GnRH receptor binding was assessed after the first and sixth pulses of 1 nM GnRH. The cells were washed and incubated for 20 min at 23 C in 500 Atl M199-BSA containing 50-2000 pM [125I]buserelin and 1 mM bacitracin. Specific binding (pulse 1, O; pulse 6, • ) was calculated by subtraction of nonspecific binding (in the presence of 10 nM GnRH) from the total binding. Scatchard analysis (inset) of these data demonstrated no differences in Bmax (~15 fmol/106 cells) or Kd (~0.58 nM) after the first (O) or sixth (•) pulses. The values shown are the mean ± SEM from three separate experiments.
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FlG. 4. PKC activity in response to intermittent pulse of GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals (•) or M199-BSA alone (•) at a perifusion rate of 0.25 ml/min. Cellular PKC activity was determined by Triton X-100 extraction, DEAE-cellulose chromatography, and transfer of 32P from [7-32P]ATP to histone, as described in Materials and Methods. The values shown are the mean ± SEM from four separate experiments. *, P < 0.05 compared to control.
As an independent measure of PKC, [3H]PDBu binding was measured. Under control conditions, the Bmax of phorbol binding was approximately 0.09-0.1 pmol/106 cells (Fig. 5A). After the first pulse of 1 nM GnRH, phorbol binding increased 2-fold (Bmax, ~0.19 pmol/106 cells; Fig. 5B). After the sixth pulse of GnRH, phorbol binding returned to control levels (Fig. 5C).
H-PDBu. BOUND (pmol/10 6 cells)
FlG. 5. [3H]PDBu binding after pulsatile GnRH. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. Phorbol binding was assessed under control conditions (A) and after the first (B) and sixth (C) pulses of 1 nM GnRH. The cells were washed and then incubated for 3 h at 4 C in 500 ^1 PBS containing 7.25-100 nM [3H]PDBu. Specific binding was determined by subtraction of nonspecific binding (in the presence of 1 fiM phorbol mystrate acetate) from total binding. Data are expressed in Scatchard form; the Bma, was approximately 0.9 pmol/106 cells under control conditions (A), increased 2-fold in response to the initial pulse of GnRH (~0.19 pmol/106 cells; B), and returned to control levels after the sixth pulse of GnRH (C). The values shown are the mean ± SEM from four separate experiments.
Dependence on calcium of GnRH-mediated PKC activity We also evaluated the calcium dependence of GnRHmediated changes in PKC activity. In these experiments 3 mM EGTA was added to the perifusion medium to chelate extracellular Ca2+. Under these conditions, no measurable LH release was observed in response to 1 nM GnRH (Fig. 6). PKC activity was determined under these conditions by [3H]PDBu binding. In these experiments there was an increase in phorbol binding after the first pulse of GnRH (Fig. 7B) compared to that in basal conditions (Fig. 7A). No subsequent down-regulation of
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FIG. 6. LH release in response to intermittent pulses of GnRH in the absence of extracellular calcium. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone in perifusion medium containing 3 mM EGTA (•) at a perifusion rate of 0.25 ml/min. LH release was assessed by RIA for GnRH-treated (•) and control (O) pituitary cell cultures. The values shown are the mean ± SEM from three separate experiments.
PKC activity was observed after the sixth pulse of GnRH in the absence of extracellular Ca2+ (Fig. 7C).
Discussion In addition to stimulation of gonadotropin release, GnRH regulates both GnRH receptor number (2, 3) and the responsiveness of gonadotropes to the releasing hormone (4-7). These effects are of importance, as it is thought that the increase in gonadotrope responsiveness at the time of proestrus is necessary for the LH surge to occur at ovulation (46, 47). In addition, the reduction in gonadotropin secretion produced by prolonged exposure to stable agonist analogs of GnRH has provided the rationale for the clinical use of these compounds (48). Until recently, little was known of the molecular mechanisms by which gonadotrope responsiveness is altered by GnRH or other compounds. The ability of the pituitary to secrete gonadotropins in response to GnRH is dependent on the availability of cell surface receptors, the efficiency of receptor-effector coupling, and the functional state of the effector. Early studies in this system suggested that gonadotrope responsiveness may be regulated by changes in GnRH receptor number (6). However, subsequent studies have revealed that the number of GnRH receptors is not necessarily predictive of gonadotrope responsiveness, suggesting that postreceptor regulation is also involved (23,25,49). Multiple lines of evidence have demonstrated that the GnRH receptor is linked to the hydrolysis of inositol phospholipids and the production of conditions that favor activation of PKC (12-16, 18, 19). It has also been shown that GnRH-stimulated PKC activation and LH release can be uncoupled in vitro and in vivo (12, 24), but the GnRH receptor (29-31, 33-35) and PKC activa-
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0.04 3
0.0B
0.12
0.16
0.20
H-PDBu, BOUND (pmol/10 6 cells)
FIG. 7. [3H]PDBu binding after pulsatile GnRH in the absence of extracellular calcium. After 48 h in culture on Cytodex microcarriers, pituitary cells received either a 5-min pulse of 1 nM GnRH at 15-min intervals or medium alone (control) at a perifusion rate of 0.25 ml/min. Phorbol binding was assessed under control conditions (A) and after the first (B) and sixth (C) pulses of 1 nM GnRH. The cells were washed and then incubated for 3 h at 4 C in 500 fi\ PBS containing 7.25-100 nM [3H]PDBu. Specific binding was determined by subtraction of nonspecific binding (in the presence of 1 pM phorbol mystrate acetate) from total binding. Data are expressed in Scatchard form; the Bmax was approximately 0.1 pmol/106 cells under control condtions (A), increased 2-fold in response to the initial pulse of GnRH (~0.19 pmol/106 cells; B), and remained increased after the sixth pulse of GnRH (C). The values shown are the mean ± SEM from three separate experiments.
tion appear to be linked to LH biosynthesis (28, 32). The present data demonstrate the ability of the agonist-bound GnRH receptor to regulate the activity of PKC in the rat pituitary gonadotrope. In perifused rat pituitary cell cultures, the data show differential regulation of LH release and PKC activity in response to pulsatile GnRH. Whereas PKC activity was increased after the initial pulse of GnRH and then down-regulated to control levels after multiple pulses of GnRH, measured both enzymatically and by radioligand assay, total cellular LH release and GnRH receptor number remained
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GnRH MODULATION OF PKC IN PITUITARY CELLS
essentially unchanged with repeated pulses of the releasing hormone under these same conditions. In addition, the process of GnRH-stimulated PKC down-regulation is dependent on the presence of extracellular calcium in perifused pituitary cell cultures. It has been shown that the activation (50-52) and down-regulation (53, 54) of PKC involve the activation of calcium-dependent neutral proteases (calpains) in multiple cell types (55). Further, calpains redistribute to the plasma membrane in response to phorbol esters and millimolar levels of calcium, and calcium is required for full proteolytic activity of these proteases toward PKC (51, 52, 54). It has also been shown in a rat pituitary cell line that PKC turnover is enhanced by conditions that favor the association of PKC with the plasma membrane (56). In addition, immunohistochemical methods have demonstrated that calpains are present in relatively high quantities in the anterior pituitary in cells that produce glycoprotein hormones (57). These observations together with the previous findings that gonadotropes mobilize calcium (1) and redistribute PKC to the plasma membrane in response to GnRH (18, 39) are consistent with the findings in this study that agonist occupancy of the GnRH receptor modulates PKC activity, and that this occurs in a calcium-dependent manner. The findings that PKC is up-regulated after the initial GnRH pulse, and that this increase in PKC activity is not affected by the removal of extracellular calcium suggest that this process may be more dependent on the generation of endogenous activators of PKC through the phosphatidyl inositol cycle, which can occur in the absence of mobilization of extracellular calcium (49). By contrast, the down-regulation of PKC in response to pulsatile GnRH required the presence of mobilized extracellular calcium, suggesting involvement of different proteases in this process. This type of differential regulation of isotypes of both PKC and calcium-dependent neutral proteases has been reported in other cell systems (50-55). Although GnRH stimulates relatively small changes in total PKC activity in perifused primary pituitary cell cultures, the changes are significant and specific to GnRH stimulation. The amplitude of the observed changes in PKC activity are probably masked by the contribution of other cell types within the pituitary. The data presented in this report are consistent with the view that GnRH mediates PKC activity. While regulation of PKC in these cells may contribute to the level of expression of gonadotropin synthesis and release during different phases of the estrous cycle by varying the pulse frequency of GnRH stimulation, it appears that measurable levels of PKC activity are not predictive of the LH response to GnRH. This system provides a model for further evaluation of the PKC and protease isotypes involved in these processes.
Endo • 1990 Vol 127 • No 5
Note Added in Proof Since acceptance of this manuscript, another manuscript (58) has appeared questioning the role of PKC in GnRH stimulated LH release.
Acknowledgment We thank Sue Birely for her aid in preparing this manuscript.
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