Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of the gonadotrope lineage.

Intracellular Responses to Gonadotropin-Releasing Hormone in a Clonal Cell Line of the Gonadotrope Lineage

Friedemann Horn*, Louise M. Bilezikjian, Marilyn H. Perrin, Martha M. Bosmat, Jolene J. WindleJ, Kathleen S. Huber, Amy L. Blount, Bertil Hille, Wylie Vale, and Pamela L. Mellon Regulatory Biology Laboratory (F.H., J.J.W., K.S.H., P.L.M.) Peptide Biology Laboratory (L.M.B., M.H.P., A.L.B., W.V.) The Salk Institute for Biological Studies La Jolla, California 92037 Department of Physiology and Biophysics (M.M.B., B.H.) University of Washington School of Medicine Seattle, Washington 98195

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (aT3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in «T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of a-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of aT3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses. (Molecular Endocrinology 5: 347-355, 1991)

portal system in a pulsatile pattern in response to neural and neuroendocrine signals in the brain and steroid and peptide hormone signals from various endocrine organs. It regulates the synthesis and release of the gonadotropin hormones from the gonadotrope cells of the anterior pituitary through binding to a specific receptor present on these cells. The gonadotropin hormones LH and FSH are heterodimeric glycoprotein hormones composed of a common a-subunit and specific LH or FSH /3-subunits, and stimulate gametogenesis and steroidogenesis in the gonads (2). The second messenger pathways involved in the regulation of gonadotropin synthesis and release by GnRH have been the subject of intensive research over the past two decades. The results have often been contradictory, suggesting a role in GnRH signal transduction for cAMP (3, 4), cGMP (5), calcium and 1,2diacylglycerols (6-8), and arachidonic acid (9,10). More recently, it has been demonstrated that a crucial event of GnRH action in gonadotrope cells is the stimulation of phosphoinositide turnover leading to diacylglycerol accumulation and raised intracellular free calcium levels (11, 12). Moreover, diacylglycerols as well as agents elevating intracellular calcium levels were shown to stimulate gonadotropin release (13,14). Based on these results, an important role of the calcium- and diacylglycerol-activated protein kinase-C was proposed (15, 16). In fact, the stimulation of LH /3-subunit mRNA levels by GnRH was shown to depend on the presence of protein kinase-C (17), whereas the requirement for this enzyme for the release of gonadotropins is still controversial (15, 18, 19). In spite of some early reports that GnRH treatment elevated pituitary cAMP levels (3, 5), it is now widely accepted that this second messenger does not transmit the GnRH signal in gonadotrope cells, but, rather, facilitates GnRH-induced gonadotropin release (20, 21). However, many details of the regulatory

INTRODUCTION

Reproductive function is controlled through a hormonal cascade that originates with the hypothalamic decapeptide GnRH (1). GnRH is released into the hypophysial0888-8809/91 /0347-0355$03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society

347

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 August 2015. at 00:47 For personal use only. No other uses without permission. . All rights reserved.

Vol 5 No. 3

MOL ENDO-1991 348

mechanisms of GnRH signal transduction in gonadotrope cells await further investigation. Studying the second messenger pathways involved in GnRH action has been difficult due to the lack of stable gonadotrope cell lines. Experiments were performed using either whole pituitaries or primary pituitary cell cultures that are quite heterogeneous, such that gonadotropes comprise only a small percentage of the cell population (22). To circumvent this difficulty, we recently produced transgenic mice bearing a chimeric gene consisting of the coding sequence of the SV40 large T-antigen linked to the promoter region of the glycoprotein hormone a-subunit gene. This promoter targeted expression of the oncogene SV40 T-antigen to the glycoprotein hormone-producing pituitary cells. From the pituitary tumors that developed in these mice, we established several cell lines, some of which express the glycoprotein hormone a-subunit gene in a GnRHregulated manner, indicating derivation from the gonadotrope lineage (23). Here we present detailed studies of the actions of GnRH on the clonal cell line «T3-1. We show that «T31 cells express specific GnRH receptors and represent an excellent model system for the study of GnRH signal transduction pathways and GnRH-regulated expression of the glycoprotein hormone a-subunit gene. Our results confirm the roles of stimulation of the protein kinase-C, phosphoinositide, and calcium pathways in GnRH signal transduction.

RESULTS Identification of GnRH Receptors The addition of a GnRH agonist to «T3-1 cells induces a-subunit mRNA in a dose- and time-dependent manner. This activation can be inhibited by the inclusion of a specific antagonist (23). To further establish the suitability of the «T3-1 cell line for studying GnRH signal transduction and regulation of gonadotropin expression, we first examined the cells for the presence of membrane receptors specific for GnRH. Using the GnRH analog [DAIa6,Me-Leu7,Pro9-NEt]-GnRH (24) for binding in vitro to cell membrane preparations, we demonstrated the existence of specific high affinity binding sites in «T3-1 cells. As shown in Table 1, binding of the GnRH analog to these sites is characterized by a dissociation constant similar to that measured in normal mouse and rat anterior pituitary. The GnRH

receptors from aT3-1 cells also show characteristics similar to those of receptors from rat pituitary with respect to the relative potencies of different GnRH analogs (Table 2). The number of GnRH receptors (per mg protein) was about 5 times higher in «T3-1 cells than in normal anterior pituitary. Although a direct comparison of «T31 and gonadotrope cells is not possible from these data, taking into account that gonadotropes represent only approximately 10% of all pituitary cells (25, 26), the number of GnRH receptors in oT3-1 cells is not significantly lower than in gonadotropes. Phosphoinositide Turnover Despite early results indicating that cAMP was a second messenger for GnRH in pituitary (3, 4), more recent experiments supported a mechanism by which GnRH activates the phosphoinositide-specific phospholipase C, giving rise to elevated intracellular concentrations of inositol polyphosphates and 1,2-diacylglycerols (6,11). To analyze the second messenger pathways for GnRH in «T3-1 cells, we first asked whether phosphoinositide turnover is increased by GnRH treatment of these cells. For that purpose, the concentrations of several inositol phosphates were measured after the addition of GnRH to the culture medium. A dramatic increase in the inositol 1,4-bisphosphate (Ins-1,4-P2) concentration was detectable as early as 30 sec after the application of GnRH (Fig. 1). The concentrations of inositol 1,3,4trisphosphate (lns-1,3,4-P3), inositol 1,4,5-trisphosphate (lns-1,4,5-P3), and inositol 1-phosphate (lns-1-P) also increased, but more slowly. Since phosphatidylinositols are the only known source of inositol phosphates (27), these data demonstrate the activation of phospholipase C by GnRH in «T3-1 cells, lns-1,4,5-P3 is the immediate product of the cleavage of phosphatidylinositol 4,5-bisphosphate, the major substrate of phospholipase C (27), and is known to cause mobilization of calcium from intracellular pools (28). It can, therefore, be expected that a quick rise of cytosolic calcium levels in «T3-1 cells will occur after GnRH administration, as has been reported for primary gonadotropes (7, 29-31). lns-1,4,5-P3 is catabolized intracellularly via two alternative pathways; it is either dephosphorylated to lns-1,4-P2, lns-4-P, and inositol, or it is phosphorylated to lns-1,3,4,5-P4 and subsequently dephosphorylated to lns-1,3,4-P3) which itself is further converted to inositol by different phosphatases (27). Both pathways seem to be used in «T3-1 cells, as both

Table 1. Comparison of the GnRH Receptor on Mouse, «T3-1, and Rat Anterior Pituitary Membrane Homogenates

R° (pmol/mg)

Mouse

«T3-1

Rat

0.51 (0.22-1.2) 0.33(0.19-0.67)

0.50 (0.38-0.67) 1.6(1.3-1.9)

0.20(0.14-0.30) 0.31 (0.24-0.41)

Kd, Dissociation constant of [DAIa6,Me-Leu7,Pro9-NEt]-GnRH; R°, total number of binding sites. The 95% confidence limits are given in parentheses (47). Pituitary membranes were prepared from intact male rats or mice. The data for mouse anterior pituitary represent one experiment. The «T3-1 and rat data represent six experiments.


GnRH Response in a Gonadotrope Cell Line

349

Table 2. Relative Potencies of GnRH Analogs for Binding to c*T3-1 Membranes Relative EC50 ± SEM

[DAIa6,Me-Leu7,Pro9-NEt]-GnRH GnRH [Ac-APro 1 ,D4FPhe2,DTrp3'6]-GnRH [Ac-D2Nal1,D4CIPhe2,D3Pal3,DLys6,Lys8,DAIa10]-GnRH

Rat pituitary

«T3-1

1 21 ± 4 0.55 ± 0.09 0.72 ± 0.07

1 45 ± 17 0.28 ± 0.10 0.68 + 0.15

The rat pituitary membranes were prepared from intact male rats. Data represent averages ± SEM for three experiments, with the exception of those for [Ac-APro1,D4FPhe2,DTrp36]-GnRH treatment of rat pituitary, which are from six experiments.

12000 Cytosol 20000 -

-PS +PS

15000 -

10000 •

5000 -

E in

E 13000

1 Membrane

12000\

Fig. 1. GnRH Stimulates Phospohinositide Turnover in «T3-1 Cells [3H]lnositol phosphate levels were measured at the indicated times after stimulation with 20 nivi GnRH. The polyphosphates were resolved by chromatography on a Mono Q HR5/5 anion exchange column. Values are normalized to those in untreated cells and represent the mean ± SEM of triplicate determinations.

Ins-1,4-P2 and Ins-1,3,4-P3 accumulate after GnRH treatment. Morgan et al. (32) found little, if any, accumulation of lns-1,3,4-P3 in primary gonadotropes, which might be due to slow formation of this compound, its rapid turnover, or the heterogeneity of primary cultures. Translocation of Protein Kinase-C The cleavage of phosphoinositides by phospholipase C also produces 1,2-diacylglycerols, which are known to activate protein kinase-C (33). Therefore, we were interested in whether protein kinase-C is activated by GnRH. Intracellular activation of this enzyme is accompanied by an apparent translocation of the cytosolic form to the plasma membrane (34). By in vitro measurements of protein kinase-C activities in the soluble and particulate fractions from «T3-1 cell homogenates, we could demonstrate the translocation of a portion of the enzyme after treatment with GnRH (Fig. 2). This effect was significant at the 15 min point (78 ± 6% for cytosol and 190 ± 30% for membrane; n = 3; P < 0.05), but may be transient, since the effect was decreased at 60 min (107 ± 6% for cytosol and 162 ±

o CL

7500 -

5000 -

2500 -

Fig. 2. Protein Kinase-C Activity Is Affected by GnRH in «T31 Cells Protein kinase-C activities of cytosolic and membrane fractions were measured in the presence or absence of phophatidylserine (PS) in untreated «T3-1 cells or after treatment with either 50 nM GnRH or 20 nivi TPA for the indicated times. The data presented in the figure are from a representative experiment and are the mean ± SEM of six determinations (three each on duplicate dishes). This experiment was repeated two additional times with consistant results.

37% for membrane; n = 3). The tumor promoter 12-Otetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase-C, caused an even more pronounced redistribution of the enzyme when applied to «T3-1 cells. This greater extent of protein kinase-C mobilization by TPA probably reflects the higher intracellular stability of this agent compared to the 1,2diacylglycerols, which are rapidly catabolized and, therefore, might not reach comparably high concentra-


Vol 5 No. 3

MOL ENDO-1991 350

tions within the cells. Redistribution of protein kinaseC has also been reported to occur after GnRH treatment of pituitaries in vivo (35) and primary gonadotrope cells in vitro (36). In the latter case, the extent of protein kinase-C translocation was in the same range as that reported here for «T3-1 cells.

and were carried in medium that contains approximately 0.4 ng/ml androgen. In accordance with our results, Conn et al. (37) were not able to detect significant changes in cAMP levels after GnRH treatment of primary gonadotrope cells. Voltage-Sensitive Calcium Channels

Cyclic AMP Levels It was also important to establish whether GnRH affects the intracellular concentration of cAMP in «T3-1 cells. As shown in Fig. 3, no significant change in cAMP levels could be detected in these cells after treatment with the GnRH analog nafarelin. Since small or temporary increases in cAMP concentration might not be detected due to the fast turnover of this second messenger, we treated the cells with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) in addition to nafarelin. This inhibitor blocks the degradation of cAMP, and therefore, the accumulation rather than the steady state concentration of cAMP is measured. Again, there was no significant difference between the cAMP levels of GnRH-treated or untreated cells (Fig. 3). However, treatment with an activator of adenylate cyclase, forskolin, increased the intracellular cAMP concentration about 200-fold, proving that this signal transduction pathway can be stimulated in «T3-1 cells. In contrast to these results, a rise in cAMP levels after GnRH treatment has been reported in whole pituitaries (3). Naor et al. (5) reported that this response occurs only in male, but not in castrated male or female rats, and, therefore, requires the presence of testosterone. The lack of any response of the cAMP system to GnRH in «T3-1 cells might be due to this requirement; however, «T3-1 cells were isolated from a tumor in a male mouse

50,000 i

Fsk + IBMX

g Fsk 25,000 -

S GnRH-A + IBMX $ IBMX $ GnRH-A

200 -

g Control

150 100 -

50

0

1

2

3

4

5

6

Primary gonadotropes have at least two types of voltage-sensitive calcium channels, giving transient and sustained currents, respectively (25), that resemble currents in T- and L-type calcium channels of other cell types. The sustained secretory response to GnRH in primary gonadotropes uses a GTP-binding protein (38), requires extracellular calcium (39), and is blocked by dihydropyridine blockers of L-type channels (40). Using the whole cell voltage clamp technique, we observed two components of calcium channel current in «T3-1 cells, functionally similar to those reported in primary gonadotropes. Cells were bathed in 15 ITIM barium chloride to enhance currents in calcium channels, and external sodium was replaced by A/-methyl-Dglucamine, a large inert cation, to eliminate sodium currents. Cesium chloride was included in the pipette to suppress potassium currents. Depolarizing voltage steps to 20 mV from a holding potential of - 8 0 mV elicited transient and sustained inward barium currents (Fig. 4A). The peak current was 63 ± 8 pamp (mean ± SEM; n = 17), and the sustained current was 38 ± 7 pamp in the same cells. The transient component was absent when these cells were held at - 4 0 mV (Fig. 4B), and the sustained current was 39 ± 9 pamp. The relative size of the two components varied among cells. Like L-type current of other cells, the sustained current was high voltage activated and dihydropyridine sensitive, and like T-type current, the transient current was low voltage activated and rapidly inactivated (Bosma, M. M., and B. Hille, in preparation). To preserve responses involving GTP-binding proteins, our pipette solution included Mg, GTP, ATP, leupeptin, and no fluorides. Addition of 100 nM GnRH to the bathing medium enhanced the barium current in six of seven cells (Fig. 4). The increase, determined from maxima of complete current-voltage relations in controls and after GnRH treatment in these six cells, was 36 ± 12% for the sustained current and 13 ± 3% for the peak (n = 5). In one cell, a lower concentration (4 nM GnRH) caused no increase in the peak and a 9% increase in the sustained current. These results show that GnRH augments voltage-sensitive calcium currents in «T3-1 cells.

time (h) Fig. 3. Cyclic AMP Levels Are not Changed by GnRH in «T31 Cells Intracellular levels of cAMP were analyzed by RIA in aT3-1 cells treated with the GnRH agonist, nafarelin (10~7 M); the activator of adenylate cyclase, forskolin (Fsk; 10~5 M); and the phosphodiesterase inhibitor, IBMX (5 x 10~4 M). Assays were performed in triplicate in two different experiments. Error bars represent the SEM.

Regulation of Glycoprotein Hormone a-Subunit mRNA Levels We have previously shown that c*T3-1 cells respond to GnRH (but not TRH) by elevating glycoprotein hormone a-subunit gene expression (23). Here, we determined the a-subunit mRNA levels after treatment of oT3-1 cells with a GnRH analog, forskolin, or TPA using


351


A

B / control

control

GnRH Fig. 4. GnRH Augments Currents in Voltage-Gated Calcium Channels in aT3-1 Cells Time course of barium currents through calcium channels elicited by 120-msec depolarizing steps. After the control current was recorded, the bath was perfused with 100 nM GnRH. A, A cell was held at - 8 0 mV and stepped to 20 mV, eliciting both transient and sustained current components. B, A different cell was held at - 4 0 mV and stepped to 20 mV, eliciting only the sustained current component. The horizontal scale bar is 20 msec; the vertical scale bar is 15 pamp. Dashed lines indicate zero current.

Control GnRH-A TPA

Fig. 5. Second Messenger Regulation of «-Subunit mRNA in «T3-1 Cells The «T3-1 cells were treated with 10 ^M forskolin (Fsk), 0.1 HM GnRH analog [olmBzl-His6,Pro6-NEt]-GnRH, 162 nM TPA, and pairwise combinations for 16 h. Northern blots were hybridized with mouse a-subunit cDNA (55), then densitometry was performed on the autoradiograms. The mean ± SEM are presented, with densitometry values set at 1 for controls. • , P < 0.05 compared to controls (n = 3). Values for TPA and the pairwise combinations are not significantly different from those for the GnRH analog, as determined by the StudentNewman-Keuls test with analysis of variance.

Northern blotting (Fig. 5). GnRH and TPA increased the cv-subunit mRNA level significantly by more than 4-fold (P < 0.05), while forskolin increased RNA levels by 2fold, although this proved not to be statistically significant. The pairwise combinations of GnRH, forskolin,

and TPA were not significantly greater than GnRH or TPA alone. Stimulation of glycoprotein hormone gene expression by GnRH has been demonstrated in pituitary in vivo (41) and in primary gonadotropes in vitro (17). Andrews et al. (17) reported that depleting gonadotrope cells of protein kinase-C by high doses of TPA abolished the ability of GnRH to stimulate LH /?-subunit gene mRNA levels. Protein kinase-C, therefore, seems to play a central role in transducing the GnRH signal to the nucleus, in accordance with our finding that TPA strongly elevates the glycoprotein hormone a-subunit mRNA level in «T3-1 cells. The weak activation by forskolin may indicate that cAMP-elevating factors might augment not only the release, as has been reported (20, 21), but also perhaps the synthesis of gonadotropins in the pituitary.

DISCUSSION

We have characterized the intracellular responses to the neuroendocrine releasing hormone GnRH in a clonal cell line of the gonadotrope lineage. This cell line was derived by targeted oncogenesis in transgenic mice (23) and is, therefore, a transformed cell dependent on the expression of SV40 T-antigen for cell division. Although the «T3-1 cell line does not express the j3-subunit genes for the gonadotropins, it does express the «subunit gene and maintain responsiveness to GnRH (though not to TRH) (23), indicating that it represents a precursor of the gonadotrope lineage.


Vol 5 No. 3

MOL ENDO-1991 352

To test whether the aT3-1 cell line would be an advantageous model system for the study of GnRH action, we characterized the receptors present in these cells and the intracellular responses to GnRH treatment. The GnRH receptors have binding characteristics very similar to those found in normal mouse and rat pituitary. They show parallel specificity for different GnRH analogs and similar dissociation constants. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists causes phosphoinositide turnover and protein kinaseC translocation to membranes, and augments currents in voltage-sensitive calcium channels, but fails to affect cAMP levels. These responses mirror those found in primary pituitary cultures, with some minor exceptions in detail. Such differences might be expected, due to contrasting medium conditions, animal models, and the use here of a clonal continuous culture of transformed cells. However, insofar as we have investigated, the response of «T3-1 cells to GnRH appears to have characteristics equivalent to those of primary pituitary gonadotropes. Our results demonstrate that the «T31 cell line is an excellent model system for the analysis of GnRH signal transduction. The mRNA for the a-subunit gene is significantly induced by GnRH and phorbol esters, and weakly by forskolin (which increases cAMP levels). However, pairwise combinations of these agents failed to induce mRNA levels more significantly than GnRH or TPA alone. The mouse a-subunit gene may show only a modest response to forskolin in these pituitary gonadotrope cells. The response of this gene to forskolin in the clonal tumor cell line of thyrotrope origin, «TSH (42), was also found to be modest. This stands in contrast to the dramatic increases (8- to 10-fold) in transcription of the human cv-subunit gene observed upon treatment of placental cell lines with agents that increase intracellular levels of cAMP (43, 44). The mouse a-subunit gene carries a single base mutation in the region homologous to the human a-subunit gene cAMP response element (45), which may explain the marginal response. Since GnRH fails to increase cAMP in these cells, cAMP may act as the second messenger of another hormonal modulator involved in the regulation of the a-subunit gene in pituitary gonadotropes. Two important issues in the pituitary response to GnRH remain to be investigated. First, we have not investigated the results of pulsatile administration of GnRH on the second messenger or mRNA regulatory responses. In vivo, GnRH is released from the hypothalamus in a pulsatile pattern, with an approximate half-hourly cycle. The second messenger responses we have measured take place within a very short time, but may be affected by the pulsatile nature of GnRH release in the long term. Weiss et al. (46) have shown that pulsatile administration of GnRH has differential effects on the mRNAs for the individual subunits. The a-subunit mRNA is increased independent of the mode of administration, the LH /3-subunit mRNA did not respond, and the FSH /3-subunit mRNA was increased by pulsatile

and decreased by continuous administration. Secondly, we have not investigated GnRH-mediated receptor down-regulation. Again, the second messenger responses we have measured occur in a short enough time frame that this down-regulation will not play a role. However, the mRNA regulation occurs in a much longer time frame, in which both receptor down-regulation and the absence of pulsatility could have important consequences. Our results demonstrate that «T3-1 cells offer an excellent opportunity to study not only the second messenger pathways of GnRH action, but also the regulation of gonadotropin gene expression. Using this cell line, we have recently mapped the promoter regions required for basal and GnRH-induced transcription of the glycoprotein hormone a-subunit gene, and isolated and characterized a new cell-specific transcription factor from aT3-1 cells (manuscripts in preparation). However, as a model system for the study of gonadotropes, this cell lacks an important feature, expression of the /3-subunit genes for LH and FSH. We have recently established long term cell cultures that express both the a- and /3-subunit genes of LH (though not as yet FSH) by targeted oncogenesis using the 5' flanking region of the rat LH /3-subunit gene (manuscript in preparation). These cultures display responsiveness to GnRH and may ultimately serve as a more differentiated gonadotrope cell model for the study of GnRH responsiveness and gonadotropin regulation, allowing study of the processing and release of intact LH heterodimers and the regulation of the LH /3-subunit gene.

MATERIALS AND METHODS GnRH Receptor Assay «T3-1 cells grown to confluence in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) in 15-cm tissue culture dishes were harvested by washing with 0.3 M sucrose and freezing on dry ice in the presence of 10 ml 0.3 M sucrose. After thawing, the dishes were scraped, and the sucrose solution was centrifuged at 40,000 x g for 20 min. The resulting pellet was homogenized in 0.3 M sucrose (2-3 ml/dish) and centrifuged at 600 x g for 5 min. The supernatant was removed, and the pellet was washed once with 30 ml sucrose. The supernatants were combined and centrifuged at 100,000 x g for 40 min. The resulting pellet, which was used for all receptor assays, was resuspended in 0.3 M sucrose (10-20 mg/ml) and stored frozen at - 2 0 C. Membrane fractions from rat and mouse pituitary glands were prepared as described previously for the rat (24). lodination of [DAIa6,Me-Leu7,Pro9-NEt]-GnRH and binding to the rat membrane fractions were carried out as described previously (24). For the oT3-1 membranes and the mouse membranes, 10 and 30 ^g protein, respectively, were used per assay tube. The dissociation constants and number of binding sites were determined by computer analysis (Ligand) (47) of competitive displacement experiments using increasing (six or more) doses of unlabeled agonist. The relative potencies of other GnRH analogs were determined from computer analysis (Allfit) (48) of competitive displacement assays using [DAIa6,Me-Leu7,Pro9-NEt]-GnRH as the standard reference compound. All receptor assays were performed at least three times; each assay contained triplicate tubes for each point.



Protein was determined with the Bio-Rad protein kit (Richmond, CA). Phosphatidylinositol Turnover Measurements The cells were plated in six-well culture plates (Costar, Cambridge, MA) and allowed to reach 60-70% confluency in DMEM supplemented with 10% FBS. The cells were washed twice and labeled for 72 h with 15 /^Ci [3H]inositol in 1.5 ml inositol-free DMEM supplemented with 1 % FBS/well. Just before initiating treatment with GnRH, the cells were washed twice (2 ml) with the same medium and, after the addition of 2 ml medium/well, allowed to equilibrate for 15 min in the presence of 5 mw LiCI at 25 C. The cells were stimulated for the indicated periods of time with 20 nivi GnRH, and the incubation was terminated by aspirating the medium and adding 2 ml 0.6 N HCI. The chloroform-methanol extraction was performed as previously described (49). The 3H-labeled inositides recovered from the aqueous phase were resolved on a Mono Q HR 5/5 anion exchange column (Pharmacia, Piscataway, NJ), as previously described (49). The radioactivity of the eluate was monitored using a /? flow-through radioisotope detector (Beckman, Palo Alto, CA), and the data are based on integrated peak areas. Protein Kinase-C Assay Cells were plated in 100-mm tissue culture dishes in DMEM supplemented with 10% FBS and allowed to reach confluency. One day before the experiment, the cells were washed and transferred to DMEM containing 1 % FBS. The cells were treated with either 50 nM GnRH or 20 nM TPA for the indicated times, and the incubation was terminated by aspirating the medium. The procedure was essentially as described previously (50). After lysis, cytosolic and particulate fractions were prepared by centrifugation at 100,000 x g for 1 h. The particulate fraction was solubilized with 1 % Nonidet P-40 for 1 h, and both fractions were loaded on DEAE-cellulose. Protein kinase-C activity associated with each fraction was batcheluted from the columns and monitored using lysine-rich histone as a substrate. Three independent experiments were performed, using duplicate dishes for each experiment, and statistical significance was determined by the Student-Newman-Keuls test with analysis of variance. Cyclic AMP RIA Confluent «T3-1 cell cultures were split 1:10 and grown for 2 days in 24-well cell culture plates in DMEM with 10% FBS. The cells were preincubated for 90 min in DMEM containing 1 % FBS with or without 0.5 ITIM IBMX. Then, forskolin (10 HM) or nafarelin (100 nM) was added, and the incubation was continued for the indicated periods. The medium was removed, and the cells were washed once with PBS. Cyclic AMP was extracted by adding 0.5 ml 95% ethanol-0.1 N hydrochloric acid to each well. The cells were scraped off using a rubber policeman, and the suspensions were transferred to 1.5-ml reaction tubes. The cellular debris was spun down in a minifuge, and the supernatants were transferred to fresh tubes and evaporated. The samples were acetylated, and the cAMP content was measured using a RIA kit (Amersham, Arlington Heights, IL) with [125l]succinyl cAMP-tyrosine methyl ester as tracer, following the instructions given by the supplier. Measurement of Calcium Currents For electrophysiological work, cells were grown at low density in 35-mm tissue culture dishes and usually recorded within 2 3 days. Whole cell recording pipettes were pulled from 75 n\ micropipette glass (VWR) using a two-stage process. Cells were bathed initially in normal Ringer's (containing 150 mM NaCI, 2.5 mM KCI, 3 mM CaCl2, 1 mM MgCb, 8 mM glucose,

353

and 10 mM HEPES, pH 7.4), and after the pipette was sealed on the cell, the bathing solution was replaced by a high barium solution containing 132 mM A/-methyl-D-glucamine, 15 mM BaCI2, 2.5 mM KCI, 1 mM MgCI2, 8 mM glucose, 3 mM 4aminopyridine, and 10 mM HEPES, pH 7.4. The pipettes were filled with 140 mM CsCI, 10 mM EGTA, 5 mM HEPES, 2 mM MgCI2, 0.2 mM GTP, 2.5 mM ATP, and 0.1 mM leupeptin, pH 7.4, with CsOH. Currents were recorded using a List EPC-7 patch clamp amplifier (Medical Systems Corp., Great Neck, NY). Voltage pulses were given and currents recorded online, using the BASIC-FASTLAB package software (Indec Systems, Sunnyvale, CA). Cells were voltage clamped to the potentials indicated, and pulses were given every 4 sec to the indicated potentials. Agonist was applied by perfusing 15-25 vol GnRH-containing solution through the bath in 1 min. The peak transient current was measured during the first 30 msec of the pulse, and sustained currents as the average of the last 30 msec of the pulse. The records were not leak subtracted. Northern Blotting The aT3-1 cells were plated in DMEM containing 5% FBS and 5% equine serum (23). When cells reached approximately 50-70% confluency, hormones and agents were added for 16 h. The GnRH analog was [DlmBzl-His6,Pro9-NEt]-GnRH and was kindly provided by J. Rivier. RNA was prepared by the method of Chirgwin et al. (51), and Northern blotting was carried out as previously described (52), using 10 »g total RNA in each lane. The probes were generated by random priming (53, 54) from plasmids containing the cDNA for the mouse «subunit of the glycoprotein hormones (55). The nitrocellulose filters were washed twice (5 min/wash) in 0.1 x SSPE-0.1% sodium dodecyl sulfate (0.1 x SSPE = 18 mM NaCI, 1.0 mM NaH2PO4, and 0.1 mM EDTA) at 100 C for rehybridization to the histone control probe (56). Autoradiograms were scanned with a densitometer, and data were analyzed using the Student-Newman-Keuls test with analysis of variance.

Acknowledgments The authors wish to thank Richard I. Weiner for helpful discussions; Jesse Marron, Yaira Haas, and John Porter for technical assistance, and Jean Rivier for GnRH analogs. Received August 9,1990. Revision received December 21, 1990. Accepted December 26,1990. Address requests for reprints to: Pamela L. Mellon, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. This work was supported by NIH Grant HD-20377 and MOD 1-1182 (to P.L.M.), NIH Grant NS-08174 and McKnight Neuroscience Research Award (to B.H.), and NIH Grant HD13527 (to W.V.). * Supported by a Deutsche Forschungsgemeinschaft Training Fellowship (HO 1133/1-1). Present address: Institute Fur Biochemie an der RWTH, Aachen, Neuklinikum Pauwelsstrasse, D-5100 Aachen, Germany. $ Supported by an American Cancer Society Fellowship, California Division (S-60-89). Present address: Division of Cellular Biology, Cancer Therapy and Research Center, 4450 Medical Drive, San Antonio, Texas 78229. t Supported by NIH Training Grant NS-07097.

REFERENCES 1. Vale W, Rivier C, Brown M 1977 Regulatory peptides of the hypothalamus. Annu Rev Physiol 39:473-527


Vol 5 No. 3

MOL ENDO-1991 354

2. Pierce JG 1988 Gonadotropin chemistry and biosynthesis. In: Knobil E, Neill JD (eds) The Physiology of Reproduction. Raven Press, New York, pp 1335-1348 3. Borgeat P, Chavancy G, Dupont A, Labrie F, Arimura A, Schally AV 1972 Stimulation of adenosine 3':5'-cyclic monophosphate accumulation in anterior pituitary gland in vitro by synthetic luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 69:2677-2681 4. Adams TE, Wagner TOF, Sawyer HR, Nett TM 1979 GnRH-interaction with anterior pituitary. II. Cyclic AMP as an intracellular mediator of the GnRH-activated gonadotroph. Biol Reprod 21:735-747 5. Naor Z, Fawcett CP, McCann SM 1979 Differential effects of castration and testosterone replacement on basal and LHRH-stimulated cAMP and cGMP accumulation and on gonadotropin release from the pituitary of the male rat. Mol Cell Endocrinol 14:191-198 6. Andrews WV, Conn PM 1986 Gonadotropin-releasing hormone stimulates mass changes in phosphoinositides and diacylglycerol accumulation in purified gonadotrope cell cultures. Endocrinology 118:1148-1158 7. Tasaka K, Stojilkovic SS, Izumi S, Catt KJ 1988 Biphasic activation of cytosolic free calcium and LH responses by gonadotropin-releasing hormone. Biochem Biophys Res Commun 154:398-403 8. Chang JP, McCoy EE, Graeter J, Tasaka K, Catt KJ 1986 Participation of voltage-dependent calcium channels in the action of gonadotropin-releasing hormone. J Biol Chem 261:9105 9. Chang JP, Graeter J, Catt KJ 1986 Coordinate actions of arachidonic acid and protein kinase C in pituitary stimulation by gonadotropin-releasing hormone. Biochem Biophys Res Commun 134:134-139 10. Chang JP, Morgan RO, Catt KJ 1988 Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267. J Biol Chem 263:1861418620 11. Huckle WR, Conn PM 1987 The relationship between gonadotropin releasing hormone-stimulated luteinizung hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators. Endocrinology 120:160-169 12. Andrews WV, Staley DD, Huckle WR, Conn PM 1986 Stimulation of LH release and phospholipid breakdown by GTP in permeabilized pituitary cell gonadotropes: antagonist action suggests association of a G-protein and GnRH receptor. Endocrinology 119:2537-2546 13. Conn PM 1989 Does protein kinase C mediate pituitary actions of gonadotropin-releasing hormone? Mol Endocrinol 3:755-757 14. Conn PM, Ganong BR, Ebeling J, Staley D, Neidel J, Bell RM 1985 Diacylglycerols release LH: structure-activity relations reveal a role for protein kinase C. Biochem Biophys Res Commun 126:532-539 15. Stojilkovic SS, Chang JP, Ngo D, Catt KJ 1988 Evidence for a role of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells. J Biol Chem 263:17307-17311 16. Stojilkovic SS, Chang JP, Izumi S, Tasaka K, Catt KJ 1988 Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization. J Biol Chem 263:1730117306 17. Andrews WV, Maurer RA, Conn PM 1988 Stimulation of rat luteinizing hormone-beta messenger RNA levels by gonadotropin releasing hormone. Apparent role for protein kinase C. J Biol Chem 263:13755-13761 18. McArdle CA, Huckle WR, Conn PM 1987 Phorbol esters reduce gonadotrope responsiveness to protein kinase C activators but not Ca++-mobilizing secretagogues: does

19.

20.

21.

22. 23.

24.

25.

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

protein kinase C mediate gonadotropin releasing hormone action. J Biol Chem 262:5028-5035 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 116:231-238 Turgeon JL, Waring DW 1987 Adenosine 3',5'-monophosphate primes the secretion of follicle-stimulating hormone. Endocrinology 120:1602-1607 Cronin MJ, Evans WS, Hewlett EL, Thorner MO 1984 LH release is facilitated by agents that alter cyclic AMPgenerating system. Am J Physiol 246:E44-E51 Childs GV 1986 Functional ultrastructure of gonadotropes: a review. Curr Top Neuroendocrinol 7:49-97 Windle JJ, Weiner Rl, Mellon PM 1990 Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. Mol Endocrinol 4:597-603 Perrin MH, Haas Y, Rivier JE, Vale WW 1982 Gonadotropin-releasing hormone binding to rat anterior pituitary membrane homogenates. Comparison of antagonists and agonists using radiolabeled antagonists and agonists. Mol Pharmacol 23:44-51 Stutzin A, Stojilkovic SS, Catt KJ, Rojas E 1989 Characteristics of two types of calcium channels in rat pituitary gonadotrophs. Am J Physiol 257:C865-C874 Farquhar MG, Skutelsky EH, Hopkins CR 1975 Structure and function of pituitary cells. In: Tixier-Vidal A, Farquhar MG (eds) The Anterior Pituitary. Academic Press, New York, pp 83-135 Berridge MJ, Irvine RF 1989 Inositol phosphates and cell signalling. Nature 341:197-205 Berridge MJ, Irvine RF 1984 Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312:315-321 Limor R, Ayalon D, Capponi AM, Childs GV, Naor Z 1987 Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone. Endocrinology 120:497 Naor Z, Capponi AM, Rossier MF, Ayalon D, Limor R 1988 Gonadotropin-releasing hormone-induced rise in cytosolic free Ca2+ levels: mobilization of cellular and extracellular Ca2+ pools and relationship to gonadotropin secretion. Mol Endocrinol 2:512-520 Shangold G, Murphy S, Miller R 1988 Gonadotropinreleasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx. Proc Natl Acad Sci USA 85:65666570 Morgan RO, Chang JP, Catt KJ 1987 Novel aspects of gonadotropin-releasing hormone action on inositol polyphosphate metabolism in cultured pituitary gonadotrophs. J Biol Chem 262:1166-1171 Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y 1982 Distribution activation of calcium-activated, phospholipid-dependent protein kinase by tumorpromoting phorbol esters. J Biol Chem 257:7847 Farrar WL, Anderson WB 1985 lnterleukin-2 stimulates association of protein kinase C with plasma membrane. Nature 315:233-235 Naor Z, Zer J, Zakut H, Hermon J 1985 Characterization of pituitary calcium-activated, phospholipid-dependent protein kinase: redistribution by gonadotropin-releasing hormone. Proc Natl Acad Sci USA 82:8203-8207 Hirota K, Hirota T, Aguilera G, Catt KJ 1985 Hormoneinduced redistribution of calcium-activated phospholipiddependent protein kinase in pituitary gonadotrophs. J Biol Chem 260:3243-3246 Conn PM, Morrell DV, Dufau ML, Catt KJ 1979 Gonadotropin-releasing hormone action in cultured pituicytes: independence of luteinizing hormone release and adenosine 3',5'-monophosphate production. Endocrinology 104:448-453 Perrin M, Haas Y, Porter J, Rivier J, Vale W 1989 The gonadotropin-releasing hormone pituitary receptor inter-


355


39.

40.

41.

42.

43.

44.

45.

46.

acts with a guanosine triphosphate binding protein: differential effects of guanyl nucleotides on agonist and antagonist binding. Endocrinology 124:798-804 Chang JP, Stojilkovic SS, Graeter JS, Catt KJ 1988 Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms. Endocrinology 122:87-97 Stojilkovic SS, Izumi S, Catt KJ 1988 Participation of voltage-sensitive calcium channels in pituitary hormone release. J Biol Chem 263:13054-13061 Papavasiliou S, Zmeili S, Khoury S, Landefeld T, Chin W, Marshall J 1986 Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone alpha and beta subunits in male rats. Proc Natl Acad Sci USA 83:4026-4029 Akerblom IA, Ridgway EC, Mellon PL 1990 An a-secreting cell line derived from a mouse thyrotrope tumor. Mol Endocrinol 4:589-596 Jameson JL, Jaffe RC, Gleason SL, Habener JF 1986 Transcriptional regulation of chorionic gonadotropin a and /3-subunit gene expression by 8-bromo-adenosine 3',5'monophosphate. Endocrinology 119:2560-2567 Darnell RB, Boime I 1985 Differential expression of the human gonadotropin alpha gene in ectopic and eutopic cells. Mol Cell Biol 5:3157-3167 Ocran K, Sarapura V, Wood W, Gordon D, GutierrezHartmann A, Ridgway E 1990 Identification of c/s-acting promoter elements important for expression of the mouse glycoprotein hormone «-subunit gene in thyrotropes. Mol Endocrinol 4:766-772 Weiss J, Jameson JL, Burrin JM, Crowley WFJ 1990 Divergent responses of gonadotropin subunit messenger RNAs to continuous versus pulsatile gonadotropin-releasing hormone in vitro. Mol Endorinol 4:557-564

47. Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239 48. DeLean A, Nunson P, Rodbard D 1978 Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am J Physiol 235:E97-E102 49. Bilezikjian LM, Blount AL, Vale WW 1987 The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action. Mol Endocrinol 1:451-458 50. Bilezikjian LM, Woodgett JR, Hunter T, Vale WW 1987 Phorbol ester-induced down-regulation of protein kinase C abolishes vasopressin-mediated responses in rat anterior pituitary cells. Mol Endocrinol 1:555-560 51. Chirgwin JM, Prezybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:52945299 52. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning-A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 53. Feinberg AP, Vogelstein B 1983 A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13 54. Feinberg AP, Vogelstein B 1984 Addendum. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266-267 55. Chin WW, Kronenberg HM, Dee PC, Maloof F, Habener JF 1981 Nucleotide sequence of the mRNA encoding the pre-a-subunit of mouse thyrotropin. Proc Natl Acad Sci USA 78:5329-5333 56. Delegeane AM 1984 Use of Temperature Sensitive Mammalian Cell Line in Cell Cycle Studies. PhD Thesis, University of Southern California, pp 1 -151


Electrophysiological properties of a cell line of the gonadotrope lineage.

Delayed activation of phospholipase D by gonadotropin-releasing hormone in a clonal pituitary gonadotrope cell line (alpha T3-1).

Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice.

Glutamate-induced inward current in a clonal neuroblastoma cell line.

Sodium and calcium fluxes in a clonal nerve cell line.

Purine and pyrimidine salvage in a clonal Drosophila cell line.

Storage and release of acetylcholine by a clonal cell line.

Intracellular inactivation of thyroid hormone is a survival mechanism for muscle stem cell proliferation and lineage progression.

The sensitivity of a malignant cell line to hyperthermia (42 degrees C) at low intracellular pH.

Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors.

Analyzing responses to serotonin in glial cell line.

CD8+ T Cell Responses to Plasmodium and Intracellular Parasites.

Sodium fluoride provokes gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and gonadotrope sensitization to A23187: evidence for multiple G proteins in GnRH action.

Thyroid hormone transport in a human glioma cell line.

Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation.

Clonal Epstein-Barr virus genome in T-cell-rich lymphomas of B or probable B lineage.

Nerve growth factor-mediated induction of tyrosine hydroxylase in a clonal pheochromocytoma cell line.

Clonal variation of DNA repair in a human glioma cell line.

progenitor cell progeny.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue.

Systemic Mastocytosis with Associated Clonal Hematological Non-Mast Cell Lineage Disorder (MDS-RCMD): A Difficult Disease to Diagnose and Treat.

Anti-Müllerian hormone: a new actor of sexual dimorphism in pituitary gonadotrope activity before puberty.

Genetic mosaics and the germ line lineage.

Changes in protein synthesis during myogenesis in a clonal cell line.