A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion1

A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion1

Molecular and Cellular Endocrinology 132 (1997) 53 – 59 A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of l...

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Molecular and Cellular Endocrinology 132 (1997) 53 – 59

A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion1 M.P. Cassina 2, K.D. Grantham, J.D. Neill * Department of Physiology and Biophysics, Schools of Medicine and Dentistry, Uni6ersity of Alabama at Birmingham, BHSB 812, 1918 Uni6ersity Boule6ard, Birmingham, AL 35294, USA Received 3 March 1997; accepted 11 June 1997

Abstract The classical mode of luteinizing hormone (LH) secretory desensitization in the rat appears after 3 – 6 h of continuous in vitro administration of gonadotropin (GnRH). A second mode has been reported to occur very rapidly ( B 2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal. Here, the existence of a third mode of desensitization is reported, occurring at 40–50 min after initiation of continuous GnRH administration. Rat pituitary cells were perifused with 10 − 8 M GnRH for 6 h; 10 min samples were collected for LH measurements by radioimmunoassay. As expected, the pattern of LH release was biphasic: LH levels peaked in the first phase at 30 min, decreased at 40 – 50 min, increased in the second phase to maximal levels at 90–110 min, and then decreased in the classical desensitization mode to near-baseline values by 300 – 360 min. Static incubations of pituitary cells in Petri dishes in the presence of high (10 − 8 M) or submaximal (10 − 9 M) GnRH concentrations confirmed the decrease in LH secretion at 40 – 50 min. Measurement of LH by reverse hemolytic plaque assay (RHPA) confirmed the existence of this new mode of desensitization; since 93% of all gonadotropes had become secretory at 40–50 min, the possibility of two subpopulations of gonadotropes accounting for the two phases of LH secretion appears to be ruled-out. GnRH receptor binding studies demonstrated a  50% decrease in cell-surface binding in association with the desensitization at 40–50 min. These studies suggest the existence of a third mode of GnRH-induced LH secretory desensitization that is not due to gonadotrope subpopulations but may be causally associated with decreased GnRH receptor binding. © 1997 Elsevier Science Ireland Ltd. Keywords: Gonadotropin releasing hormone; Luteinizing hormone; Desensitization; Pituitary (gonadotrope)

1. Introduction Gonadotropin (GnRH) administration can either stimulate or inhibit the secretion of luteinizing hormone

* Corresponding author. Tel.: +1 205 9342499; fax: + 1 205 9341445; e-mail: [email protected] 1 Presented in preliminary form at the 10th International Meeting of Endocrinology (Abstract P3–234, p. 813), San Francisco, CA, 1996. 2 Present address: Departamento de Histologia y Embriologia, Facultad de Medicina, Gral. Flores 2125 CP 11800, Montevideo, Uruguay.

(LH) and follicle stimulating hormone (FSH) from pituitary gonadotropes depending on its pattern, length of time and concentration, and whether the gonadotropes are under the influence of ovarian hormones (Conn et al., 1987). Constant infusion of GnRH to rat pituitary cells under the influence of estrogens results in a well known biphasic secretion of LH (De Koning et al., 1976; Baldwin et al., 1983): this pattern is characterized by an initial acute release of LH (lag phase response) followed later by a second phase with an augmented secretion rate (primed state response) (De Koning et al., 1976; Baldwin et al., 1983). After

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3 – 6 h, LH secretion declines gradually to a level at or slightly above, baseline despite the continued presence of the stimulus (Smith and Vale, 1981; Badger et al., 1983). This is the classical mode of LH secretory desensitization to GnRH, and can be quite profound. It constitutes the basis for many of the therapeutic applications of GnRH (Conn and Crowley, 1994). A second mode of LH secretory desensitization also has been reported to occur rapidly (B2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal (Weiss et al., 1995). In the present work, we investigated the possibility that the decrease in LH secretion rate occurring during the transition between the GnRH-induced lag phase response and the primed state response (De Koning et al., 1976; Baldwin et al., 1983) represented a third mode of GnRH-induced desensitization of gonadotropes.

seal the cover-slips forming the chamber onto the Oring; perifusions are performed through two hypodermic needles (23-gauge) connected to polyethylene tubing (PE50, Clay Adams) that penetrate the O-ring into the chamber through holes on opposite sides of the metal ring. The total volume of the assembled chamber was 0.75 ml. Two chambers were perifused simultaneously for 6 h at a rate of 0.2 ml/min either with DMEM–BSA or with DMEM–BSA containing 10 − 8 M GnRH. Samples of the perifusate were obtained every 10 min for LH measurements by radioimmunoassay using reagents supplied by the National Hormone and Pituitary Program of the NIDDK, NIH; the rat LH for radioiodination was I-9, the anti-rat LH antiserum was S-11, and the rat LH reference preparation was RP-3.

2.3. Static incubations 2. Materials and methods

2.1. Pituitary cell cultures Adult female Sprague – Dawley rats (Charles River) were housed in a controlled environment (21 9 1°C; lights on 06:00–18:00 h) with food and water supplied ad libitum. Female rats showing regular 4 day estrous cycles were used as the pituitary donors; stages of the estrous cycle were determined by daily vaginal cytology as well as by the presence or absence of uterine ballooning at euthanasia. The animals were euthanized between 9:30 and 10:30 h, the anterior pituitaries excised, and dispersed into single cells using 1 mg/ml trypsin (Difco 1:250, Detroit, MI) in Eagle’s minimal essential medium (MEM) containing 0.1% bovine serum albumin (BSA) and penicillin/streptomycin as described previously (Smith et al., 1986). The cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.1% BSA and antibiotics.

2.2. Pituitary perifusions Dispersed pituitary cells from rats at mixed stages of the estrous cycle were plated onto poly-L-lysine treated 25 mm round glass cover-slips (  2 ×106 cells/coverslip) (Frawley and Neill, 1984). In five of six experiments, the cells were placed in DMEM/10% horse serum for 24 h (one experiment) or for 48 h (four experiments) before being perifused. In one experiment, the cells were perifused immediately after enzymatic dispersion. Perifusions were performed in a Sykes– Moore chamber (Frawley and Neill, 1984) as described earlier; the chamber is formed by two round glass cover-slips (pituitary cells were attached to one of the two) sandwiched over a rubber O-ring; this assemblage is mounted between two interlocking metal rings which

Anterior pituitary cells from proestrus rats immediately after dispersion were placed into poly-L-lysine coated 24 multi-well plates (Falcon; Becton Dickinson and Company) (400 000 cells/well). The cells were incubated overnight in DMEM/10% gelding horse serum (1 ml/well). The following day the medium was aspirated, and the cells were incubated for approximately 2 h in DMEM–BSA before starting the experiment. Two wells each were simultaneously incubated with 0, 10 − 9 or 10 − 8 M GnRH for times ranging from 0 to 90 min. The media were assayed for LH content with the NIDDK radioimmunoassay kit described above.

2.4. Re6erse hemolytic plaque assay This assay has been described in detail previously (Smith et al., 1986). In brief, anterior pituitary cells from proestrus rats were plated in poly-L-lysine treated 35 mm Petri dishes (Falcon; Becton Dickinson and Company), and incubated overnight in DMEM/10% gelding horse serum (approximately 3 × 106 cells/3 ml of medium). The following day the cells were detached with 2.5 mg/ml trypsin (Difco) in MEM for five min at 37°C. The cells were resuspended in DMEM–BSA and mixed v/v with an 18% suspension of ovine red blood cells coupled to protein A. The final concentration of cells was 300 000 pituitary cells/ml. The cells were then loaded into Cunningham chambers (Smith et al., 1986). The cells were treated with GnRH (10 − 8 M) plus LH antibody (1:40) for times ranging from 0 to 90 min. Then they were incubated at 4°C for 30 min to allow diffusion of the hormone, followed by the addition of guinea pig complement (1:50) for 30 min at 37°C. The reaction was stopped by infusion of 2% glutaraldehyde in 0.1 M phosphate buffer. LH secretion results in the complement-mediated lysis of LH antibody-coated erythrocytes around the gonadotropes, so that clear areas

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of lysis (plaques) surround them. The presence of a plaque around a cell identifies it as a gonadotrope, and the area of a plaque is related to the amount of LH secreted. Areas of individual LH plaques were measured with a Bioquant video image-analysis system (Smith et al., 1986). Previous studies have shown this LH antibody to be specific for LH (Smith et al., 1986; Cassina et al., 1995) and not to cross-react with FSH or TSH (Smith et al., 1986).

2.5. [ 125I]GnRH-A binding studies Anterior pituitary cells from proestrus rats immediately after dispersion were plated into poly-L-lysine coated 24 multi-well plates (800 000 cells/well). The cells were incubated overnight in DMEM/10% gelding horse serum (1 ml/well). The following day the medium was aspirated, and the cells were incubated for approximately 2 h in DMEM – BSA before starting the experiment. Two wells were simultaneously incubated with 10 − 10 M [125I]D-Ala6-desGly10-GnRH ethyl amide (GnRH-A) for times ranging from 0 to 90 min; the specific radioactivity of the [125I]GnRH-A was approximately 700 mCi/mg (Neill et al., 1997). After incubation, the cells were washed with cold medium (4°C) and incubated for 10 min with 0.2 M acetic acid/0.5 M NaCl (pH 2.6) at 4°C, to elute surface-bound radioactivity as described by Mason et al. (1994). The cells then were solubilized with 1 M NaOH/0.1% SDS to determine internalized radioactivity (Mason et al., 1994). Nonspecific binding was determined by incubating the cells with 10 − 7 M unlabeled ligand.

2.6. Data analysis

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The decrease in LH secretion at 40–50 min (Fig. 1, hatched bar) appeared to represent GnRH-induced desensitization; that is, a decreased secretory response to unchanging GnRH concentrations. To rule-out the possibility that this observation was a peculiarity of the perifusion system, we incubated pituitary cells in Petri dishes at 10 min intervals over 90 min in the presence of GnRH (10 nM) (Fig. 2A). A pattern of LH secretion similar with that found in perifusions was observed except that net LH secretion at the 40–50 min interval was almost undetectable (Fig. 2B). A lower concentration of GnRH (1 nM) also induced desensitization at 40–50 min indicating that the decrease in LH secretion at that time probably was not due to depletion of LH stores and hence was unlikely to be a pharmacologic effect of GnRH (Fig. 2). Therefore, these results reinforce those from the perifusion experiments (Fig. 1) that desensitization occurs at 40–50 min after the initiation of GnRH treatment and, moreover, it can be observed with a dose of GnRH in the physiologic range. A potential explanation for the two phases of LH secretion was heterogeneity of cell function, a phenomenon reported previously for gonadotropes (Neill et al., 1987; Childs, 1995). To determine if separate gonadotrope subpopulations might account for the two phases of LH secretion, we performed the reverse hemolytic plaque assay at 10 min intervals for 90 min on separate samples of pituitary cells. An elaboration of the plaque assay procedure was required for measurement of LH secretion during the short time periods used in these experiments; previously, the plaque assay was performed by incubating the pituitary cells with

The results are presented as the mean 9 S.E.M. of at least three independent experiments. The data were converted to percentages of the maximum response where indicated. The statistical tests used are indicated in the figure legends, and were performed using SigmaStat™ Statistical Software for Windows (Jandel Scientific, San Rafael, CA). 3. Results The pattern of LH secretion from pituitary cells continuously perifused with 10 − 8 M GnRH for 6 h in Sykes–Moore chambers is shown as Fig. 1. As expected from previous studies (De Koning et al., 1976; Baldwin et al., 1983), the pattern of LH release was biphasic: LH levels in the first phase peaked at 20 – 30 min ( $ 5 ng/ml), decreased at 40 and 50 min (4.1 and 4.3 ng/ml), increased in the second phase to maximal levels at 90 – 110 min (13.2–13.9 ng/ml), and then decreased in the classical desensitization mode to near-baseline values by 300–360 min.

Fig. 1. GnRH-stimulated LH secretion from rat anterior pituitary cells perifused in Sykes/Moore chambers. LH levels were measured in the perifusion medium at 10 min intervals by radioimmunoassay. Administration of GnRH began at time 0. Results are presented as the mean9 S.E.M. of six independent experiments. The vertical, hatched bar at 40 – 50 min indicates where desensitization occurred.

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Fig. 2. GnRH-stimulated LH secretion from rat anterior pituitary cells incubated in Petri dishes; LH levels were measured by radioimmunoassay. The data are presented as the mean 9 S.E.M. of four independent experiments. (A) The vertical hatched bar at 40– 50 min indicates where desensitization occurred. The data are presented as % of maximum, the maximal value being the LH level at 90 min in the group treated with 10 nM GnRH. Two-way analysis of variance followed by Student-Newman-Keuls test revealed that the two GnRH-treated groups differed from each other (PB 0.05), and from the control group (P B0.05). (B) Net LH secretion per 10 min interval derived by subtracting each preceding value from the succeeding value; e.g. the LH concentrations in the 10 min samples were subtracted from the 20 min samples, and the resulting values plotted in the interval between 10 and 20 min in the figure. Note the near-zero secretion rate of LH during the 10 min interval from 40 – 50 min.

GnRH for 2–3 h before adding complement to form plaques (Smith et al., 1986; Neill et al., 1987). Instead of adding complement immediately after the end of GnRH treatment as usual, we incubated the slides at 4°C for 30 min to permit secreted LH surrounding gonadotropes time to diffuse radially so as to bind LH antibody on distant erythrocytes. Addition of this step resulted in more and larger plaques being formed at the 10 min intervals shown in Fig. 3, suggesting that con-

siderable secreted hormones would have been lost if this 30 min period for hormone diffusion had not been added. A second concern was whether LH antibody had sufficient time to bind the Protein A coupled erythrocytes before hormone secretion began. To test this possibility, we incubated erythrocytes with LH antibody for 30 min before addition of GnRH and additional LH antibody. However, the number or size of plaques was not changed by this maneuver. The total amount of LH secreted (total plaque area) as measured by the plaque assay showed a pause at 40–50 min (Fig. 3) as found in previous experiments (Figs. 1 and 2); however, 97% of all gonadotropes were secretory at 40–50 min. This suggests that all or nearly all the cells became desensitized rather than a separate subpopulation of cells beginning to secrete for the first time after 40–50 min (Fig. 3). Extensive studies with a number of receptors from the same family as the GnRH receptor (the so-called G protein-coupled receptors), have revealed a general scheme for homologous desensitization involving a rapid uncoupling of receptor from the G-protein, subsequent sequestration of receptors from the plasma membrane, and internalization followed by proteolytic degradation or recycling to the cell surface (Dohlman et al., 1991). Therefore, we performed a time course for GnRH-A binding during the first 90 min of agonist stimulation. [125I]GnRH-A binding studies to pituitary cells from proestrus rats showed that in the first 40–50 min of agonist stimulation, the cell surface GnRH binding decreased by 40–60%, and the internalized radioactivity increased by 20% (Fig. 4). A similar down-regulation of binding sites has been described before for GnRH (Mason et al., 1994; McArdle et al., 1987; Clayton, 1989; McArdle et al., 1995). The decrease in cell surface binding coincided with the pause in LH secretion observed in our experiments, but binding was not elevated when LH secretion increased in the second phase (Fig. 4).

4. Discussion The biphasic pattern of LH secretion in response to constant administration of GnRH has been described previously (De Koning et al., 1976; Baldwin et al., 1983; Pickering and Fink, 1976). It consists of an initial acute release of LH (lag phase response) which cannot be influenced by protein synthesis inhibitors and is succeeded by a second phase with an augmented secretion rate (primed state response) which can be suppressed by protein synthesis inhibitors; the second phase is equivalent to the self-priming effect of GnRH (De Koning et al., 1976; Baldwin et al., 1983; Pickering and Fink, 1976). In the present paper, we have shown that pituitary cells stimulated with GnRH, whether in a

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perifusion system, a static culture, or the reverse hemolytic plaque assay, display a pause in LH secretion after the first 40–50 min of GnRH stimulation. This pause fits the classical definition of desensitization (Hausdorf et al., 1990): the waning of a stimulated response in the face of continuous agonist exposure. Others have noted this pause but do not seem to have

Fig. 4. Cell surface vs. internalized [125I]GnRH-A during GnRH-induced desensitization of LH release at the 40 – 50 min period. The results are the mean9S.E.M of five independent experiments. The statistical test was analysis of variance followed by Student-NewmanKeuls test for pairwise comparisons. *P B 0.05 vs. the 10 min surface bound [125I]GnRH-A group; +P B0.05 vs. the corresponding surface group.

realized its significance relative to desensitization (De Koning et al., 1976; Baldwin et al., 1983; Bourne and Baldwin, 1980; De Koning et al., 1987). The reverse hemolytic plaque assay showed that most of the cells were secretory at 40 min, and therefore the pause likely was not due to one subpopulation of cells becoming desensitized and then a new subpopulation beginning to secrete; instead, it probably was due to GnRH-induced secretory desensitization of most of the cells. We postulate then that the brief decrease in LH secretion occurring at 40–50 min in the presence of constant, low concentrations of GnRH is a mode of desensitization that is intermediate in time to the classical, late form of desensitization (Smith and Vale, 1981; Badger et al., 1983) and the early form described by Weiss et al. (1995).

Fig. 3.

Fig. 3. GnRH-stimulated LH secretion measured by reverse hemolytic plaque assay (RHPA). Rat anterior pituitary cells were obtained from proestrous females, incubated overnight, and then subjected to RHPA. The results are the mean9 S.E.M. of five independent experiments. The grey bar at 40 – 50 min indicates where desensitization occurred. (A) Total amount of LH (total plaque area) secreted by all gonadotropes. According to analysis of variance followed by Student-Newman-Keuls test for all possible comparisons, the values at 40 min and at 50 min did not vary from each other (P \0.05) but both differed significantly (P B0.05) from the 30 min and from the 60 min values. (B) Mean amount of LH secreted (mean plaque area) per gonadotrope. Statistical differences at 30, 40, 50, and 60 min were the same as for total plaque area. (C) Number of secretory gonadotropes (plaque-forming cells). Using the statistical analysis identified above, we found that the values at the 10 min intervals from 30 – 100 min did not differ from each other (P\ 0.05) whereas the 10 and 20 min values were significantly lower (P B 0.05) than those at 40 – 90 min.

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The decrease in LH secretion rate occurring during the period from 40 – 50 min after the initiation of GnRH-treatment probably is the outcome of combined desensitization/GnRH self-priming processes. If GnRH is present continuously, this decrease lasts only about 10 min. Then, a GnRH self-priming process comprised of an augmented LH secretion rate overwhelms desensitization. On the basis of this finding, we speculate that desensitization lasts longer than 10 min but its true extent is hidden by GnRH self-priming. It is interesting that the two phases of LH secretion are not seen in pituitary cells derived from ovariectomized rats (De Koning et al., 1987). Moreover, the second phase appears not to occur or is very low in cells from estrous and diestrous 1 animals (Baldwin et al., 1983; Fowler and Templeton, 1996). It has been suggested that the initial lag phase of LH secretion (0 to 40 – 50 min) is due to the action of a gonadotropin surge attenuating/inhibiting factor (GnSAF/IF) that is produced by the ovary which maintains the pituitary in an initial state of lowered responsiveness to GnRH (De Koning et al., 1987; Fowler and Templeton, 1996). Therefore GnSAF/IF bioactivity may be necessary for the intermediate form of GnRH-induced desensitization to occur. The decrease in the number of receptors seen at 40 – 50 min may be involved in the transient desensitization occurring at 40 – 50 min after the initiation of GnRH administration; Zhou et al. (1995) have shown that GnRH receptor number is directly related to second messenger response, which, in turn would be expected to be related to amount of LH secreted. However, as suggested previously (Clayton, 1989), the quantitative relationship between receptor occupancy and LH responsiveness is not always parallel. This is shown by our experiments during the second phase of LH secretion: the number of binding sites remains low while LH secretion is rising at a rapid rate. This probably is due to the GnRH self-priming response mentioned above during which the LH secretory responsiveness has been increased probably at a postGnRH receptor site (Turgeon and Waring, 1994). The physiological significance of this brief, intermediate form of GnRH-induced desensitization observed in vitro is not entirely clear. Decreases or pauses in the rate of LH secretion lasting 10 min would be difficult to detect in rats showing spontaneous surges of LH secretion (Gallo, 1981; Fox and Smith, 1985) because of the intrinsic variability of LH levels perhaps due to pulsatile GnRH secretion. However, a precipitous decline in GnRH-receptor number has been reported to occur in association with the initiation of the LH surge at proestrus (Savoy-Moore et al., 1980) which is reminiscent of the 50% decline we observed in association with the intermediate form of desensitization. Thus, we suppose that under the carefully controlled experimental

conditions used in the current studies, we have uncovered a form of desensitization that also occurs in the intact animal. In addition to the classical, late form of GnRH-induced desensitization of LH secretion (Smith and Vale, 1981; Badger et al., 1983), that we previously have implicated in quenching the proestrous LH surge (Cassina and Neill, 1996), and the intermediate form described here, there exists an early form occurring rapidly (B2 min) after the onset of GnRH administration and reversing within 3 min after its withdrawal (Weiss et al., 1995). Catt’s group also has reported data that is consistent with this early desensitization (Stojilkovic et al., 1992; Izumi et al., 1989). In response to a 10–15 min infusion of GnRH, rat pituitary cells show an initial burst followed by a secondary sustained release of LH. In contrast, the aT3-1 cell line which is the major experimental surrogate for gonadotropes, does not show this rapid form of desensitization: aT3-1 cells treated with GnRH do not show rapid desensitization of the total inositol phosphate response (McArdle et al., 1995; Davidson et al., 1994; Anderson et al., 1995). However, GnRH treatment of aT3-1 cells caused receptor down-regulation and desensitization of the inositol phosphate response within 60 min (McArdle et al., 1995) which resembles the intermediate desensitization response we have described here. Therefore, aT3-1 cells may be more useful for characterizing the intermediate form of desensitization observed here in gonadotropes and less useful for the rapid form of gonadotrope desensitization (Weiss et al., 1995). As evidence favoring the latter, we recently have described the existence of a rapid form of desensitization in COS-1 cells bearing the GnRH receptor (Neill et al., 1997); i.e. a 5 min pretreatment of such cells with GnRH completely inhibited intracellular inositol 1,4,5-trisphosphate (IP3) increases in response to GnRH treatment (Neill et al., 1997). In summary, we have shown that rat pituitary cells become desensitized after 40–50 min of GnRH administration. However, this desensitization is transient because continuous exposure to the peptide overrides this effect. Our studies suggest the existence of a new, third mode of GnRH-induced LH secretory desensitization that is not causally associated with gonadotrope subpopulations but may be with decreases in GnRH receptor binding.

Acknowledgements We thank Cindy Urthaler for preparation of the manuscript, Lois C. Musgove for assistance with the [125I]GnRH-A binding experiments, and the National Hormone and Pituitary Program of the NIDDK, NIH, for the radioimmunoassay reagents.

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