Desensitization of pituitary gonadotropes by mediators of LH release

Vo1.153, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 919-924

June 30,1988

DESENSITIZATION OF PITUITARY GONADOTROPES BY MEDIATORS OF LH RELEASE

John P. Chang, Jauelle S. Graeter, and Kevin J. Cart

Endocrinology and Reproduction Research Branch, National Institute of Child Heath and Human Development, National Institutes of Health, Bethesda, MD 20892

Received May 13, 1988

SUMMARY: DesensifiT~ion of pituitary gonadou-upes by exposure to 10 nM gonadotrupin-releasing hormone (GnRH) for 6 h severely impairnd the luteinizinghormone (LH) response to a second 3-h treatment with GnRH, and reduced the seeretory resunses to 50 ~M arachidonic acid (AA), 100 nM tetrndecanoyl phorbol-13-acetate (TPA), and AA + TPA. Pretrenunent with AA blocknd subsequent responses to AA but not to other seeretagogues. Pretrcatment with TPA attentuated the LH response to TPA, but not to GnRH, AA, and AA + TPA. After exposure to AA + TPA, all subsequent responses were abolished. Each of the secretagogues reduced GnRH receptor binding, but only GnRH-inducedreceptor loss and desensitization were ~ e r s e d by simultaueons incubationwith a GnRH antagonist. Similar results wexe obtained when 16-h pretreatment periods were used, or when the data were normalized for the concomitant reduction of cellular LH contenL These findings indicate that GnRH-receptor loss and depletion ofLH contentare not the sole causes of GnRH-indnceddesensiti~,~don. Receptor uncoupling and impairment of AA- and protein kinase C-dependent pathways may also be involved in this process.

The observa6ons (1,2) that gonadotrupin-releasing hormone (OnRH) stimulation of LH release involves both extracellular Ca2+ -dependent and -independent mechanisms have been substantiated (3-6). Besides changes in intracellularcalcium concentration, the mobilizationand metabolism of arachidonic acid (AA), as well as activation of protein klnase C, also appear to mediate GnRH action (7,8). GnRH rapidly stimulates the hydrolysis of polyphosphoinositides to produce inositol phosphates that mediate Ca2+ mobilization, and diacylglycerol which activates protein kinase C (3,10,11) and serves as a source of AA (11). GnRH treatment has been shown to desensitize gonadotrupes to subsequent exposure to GnRH, as shown by decreased LH responses to the releasing peptide (12). Desensitization is usually accompanied by a decrease in GnRH receptor number as measured by a reduction in specific GnRH binding sites (13,14). However, loss of GnRH receptors and depletion of cellular LH content do not alone account for the decreased LH response, which could also result from impairment of second messenger coupling or function (15). It has been reported that activation of protein kinase C Coy phorbol esters) or phospholipase A2 (by melittin) does not induce refractoriness to GnRH (16,17). To analyze the involvement of second messenger systems in the desensitization process, LH responses and GnRH-receptor binding were measured after pretreatment of cultured pituitary cells with GnRH, AA, tetradecanoyl phorbol- 13-acetate (TPA), and AA plus TPA. The effects of GnRH antagonist treatment on secretagogue-Jnducexldesensitization and GnRH-receptor loss werc also evaluated.

Abbreviations: AA, arachidonic acid; GnRH, gonadouopi,-releasing hormone; GnRHa. [D-Lys6JGnRH-Nethylamide; GnRH-ant, OnRH antagonist; LH, luteinizinghormone; TPA, teWadecanoylphorbol acetate. 0006-291X/88

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MATERIALS AND METHODS

G.eneral Antcxior pituitary glands from adult female SIxague-Dawley rats were dispersed into single cells by controlled trypsinization as previously described (18). GnRI-I, [D-Lys6]GnRH-N-cthylamide (GnRHa), and [I3Phe2,pro3,D-Phe6]GnRH (OnRH-antagonist, GnRH-ant; Sigma or Peninsula Lab.) were dissolved in distilled deionized water. AA (Nu-chck) and TPA (Sigma) were dissolved in ethanol and dimethylsulfoxide, respectively. All drugs and hormones were diluted with test medium to give final concentrations of cartier solvents (less than 0.1%, v:v) that did not alter LH release. LH Release Studies Dispersed anterior pituitary cells (5 x 105 ceils/well in 24-well plates) were cultured in M199-E (medium 199 containing Earle's salts, sodium bicarbonate, 10% horse serum, 10 Ixg/ml streptomycin, and 100 U/ml penicillin) at 37°C under 5% CO2 and satm'ated humidity for 5-7 days. Cells were washed twice with M199 (medium 199 containing Hanks salts, sodium bicarbonate, 25 mM Hepes, and 0.01% bovine serum albumin) and incubated at 37°(2 for 1 h before addition of fresh media and drugs. After a pretreatment period of 6 or 16 h, media were collected and the cells washed for 2 rain with ice-cold M199 (pH 3). Secretagogues were then added to the cells for a further 3-h incubation, after which cells were lysed by the addition of distilled water, sodium carbonate, and freezing and thawing. LH in the pretreatment and second 3-h incubation media, as well as .that remaining in the cells, was measured by radioimmunoassay (19) using rLH-RP-2 standard provided by the National Pituitary Agency (Baltimore, MD). Four wells were used for each treatment in all experiments and all samples from each experiment were assayed together in duplicate. GnRH rec.eotor-bindin~ studies Ceils for GnRH-binding studies were plated in M199-E at 3 x 106 ceils per well in 6.weU plates and treated similarly to those used forLH release studies. Two to 4 wells were used for each treatment, and cells were then washed for 2 rain with ico-cold M199 (pH 3) to dissociate surface-bound GnRH or GnRH-ant from the receptors. Cells were scraped from each well, resuspended in ico-cold M199, and transferred to individual 12 x 75 mm glass tubes. Ceil pellets were harvested after contrifugetion at 1000 rpm for 20 rain, resuspended in 200 I.tl assay buffer (10 mM Tris, 0.1% BSA, 1 mM dithiothreitol), and subdivided into 2 x I00 Ixl aliquots. 125I-labelled [D-Lys6]GnRH-N-cthylamide (125I-GnRHa; Meloy labs. Inc., VA) was then added to all tubes (50,000 cpm/tube/100 p.l). One tube of each pair was used to measure total binding and received 100 I.d of assay buffer, while the other received 100 p.l of GnRHa (to 3 x 10.6 M) to indicate non-specific binding. After 2-h incubation on ice, bound and free radioactivity were separated by filtration using GF/C filters (Whatman; presoaked in I0 mM Tris containing 2% BSA) as previously described (20). Bound radioactivity collected on the filters was quantified in an automatic gamma spectrometer. RESULTS AND DISCUSSION Initial studies on the ldnefics of GnRH-induced desensidTmlon indicated that subsequent LH responses to the same concentration of GnRH (10 nM) were severely impaired (< 10% of control) after 6 h and were abolished after 16 h. Dose-dependent decreases in the magnitude and sensitivity (ED50) of LH responses to GnRH were observed (Fig. 1), suggesting that both 6- and 16-h pretrcatment protocols resulted in desensitization of the gonadou'opin response. Similar results were observed in a previous study following 12-h prctrcatment with the receptor agonist 05). Since results from both 6. and 16-h pretrcatmem protocols employed in subsequent e×pcriments in this study were similar, only observations employing a 6-h prior exposure are presented. Consistent with the idea that AA mexabolism and protein ldnase C activation are major pathways mediating LH release, the addition of AA, TPA, and AA plus TPA increased LH secretion during the preb'eatment period (Table 1). Exposure to 10 nM GnRH, 50 ~M AA, 100 nM TPA, and AA plus TPA attenuated the absolute LH responses to subsequent challenges with ~ secretagognes, reduced specific 125I-GnRHa binding, and lowered cellular LH content as shown by a decrea~ in total LH during the second 3-h incubmion (total -- secrcmd + cellular; Table 1, Fig. 2). To adjust for the decrease in LH content, gonadotropin release during the second challenge was also expressed in relative values, as a percentage of the total LH present during the final incubation, and as foldincrease over basal. The reductions in relative and absolute LI-Iresponses were similar.

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6-h pretreatment: control

28O 240

16-h pretreotment: 200 8

x

control

GnRHt0-11M

200

x

160

s

160

x/i

GnRH t0"10M

¢rl t-

/~

. / .

GnRHI0°ION

!~j

9,-'-----,

GnPJ-i10-8M GnRHIO'7M

120,

-1._1

80 GnRH 10"8M I

I

0

-11

,

,

I

I

I

.

40

GnRH10"7M I

I

-10 -9 -8 LOG GnRH (M}

0

0

-7

-11

-10 -9 -8 -7 LOG GnRH {M}

Fi[. 1. Dose-de~ndence of GnRH-induced desensitization of subsequeat LH responses to the releasing peptide. Resu|ts (Mean + SE, n=4) from one of four and two experiments using 16- and 6-h pretreatments are presented. Dotted lines indicate ED50s extrapolated graphically.

Table I. Desensitization of LH responses following 6.-hpreu'enUnentwith 10 nM OnRH, 50 p.M AA, 100 nM TPA, and AA + TPA 6h prctreatment

2nd 3 h incubation

LH release during 2rid incubation (ng/10%eUs)

Conu'ol

Conu'ol GnRH AA TPA AA + TPA

17.3 ± 1.1 171.0 ± 10.6" 143.3 ± 12.9" 124.7 + 5.7* 171.6 ± 9.3 *

(48.3 + 1.7)°

GnRH (283.8 + 5.7) b

AA (213.2 + 5.3)b

TPA (238.7 ± 4.5) b

AA +TPA (276.0 + 6.7)b

Total LH present during 2rid incubation (ng/10~cclls) 143.3 + 3.4 210.2 ± 14.3' 188.6 ± 11.2" 206.0 ± 7.8* 206.3 ± 10.5"

LH release as fold increase above basal

12.1 ± 0.9 81.4 ± 0.9* 75.1 + 3.7* 61.1 ± 5.3* 83.1 + 1.4"

..... 6.7 6.2 5.0 6.8

21.1 ± 3.1 47.8 ± 2.9* 58.7 ± 1.9" 53.3 ± 1.7" 66.5 ± 1.9'

..... 2.3 2.8 2.5 3.2

Control GnRH AA TPA AA +TPA

9.8 ± 21.7 ± 25.8 ± 26.2 + 32.7 ±

0.9 1,2" 1.0' 1.4" 0.4*

43.5 ± 45.7 + 44.2 ± 49.3 ± 46.9 ±

Comrol GnRH AA TPA AA + TPA

9.8 ± 44.4 + 26.3 ± 50.2 ± 50.6 +

0.6 3.5* 1.2' 3.8* 5.3*

23.9 ± 1.7 75.9 + 14.7" 66.2 ± 11.2" 60.2 ± 5.1" 64.5 ± 3.5*

40.9 ± 0.9 81.9 + 1.3" 40.2 ± 1.5 79.9 ± 2.4* 83.7 ± 2.2*

..... 2.0 1.0 1.9 2.0

Control GnRH AA TPA AA +TPA

32.2 ± 2.1 48.5 + 3.8 56.3 ± 3.4* 34.9 + 2.7* 61.5 + 2.9*

68.9 + 6.1 68.6 ± 5.7 80.7 ± 10.1 61.1 + 4.2 80.0+ 1.8"

48.6 ± 68.4 ± 71,2 ± 51.7 ± 76.1 +

..... 1.5 1.6 1.1 1.7

Control GnRH AA TPA AA + TPA

10.8 + 11.8 ± 11.7± 13.7 + 14.1 +

18.7 ± 1.6 21.1 ± 1.3 17.8±0.9 20.9 ± 3.8 23.9 ± 2.0

58.6 ± 4.5 58.8 ± 1.4 65.3±5.2 59.3 ± 7.3 64.3 ± 1.5

0.7 0.8 1,I 3.6 3.9

3.4 2.9 2.9 7.5 1.1

LI--Irelease as % of tom

3.4 1.9" 3.9* 2.1 0.9*

..... 1.0 1.1 1.0 1. l

a L H releaseduring preu~atment (ng/10~ cells;mean + SE; n=20) b Significandy greater than conuol during pmu'eatment period (p
Results (Mean + SE, n--4) are representative of four similar experiments. Results from three experiments using a 16-h prelreatment period are also similar.

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120

6-h oret~atmcnt

100

so g .~_

60

i~

4o

o

' R e~nN

GnRtt

AA

TPA

AA + TPA

PRETREA~ Fig..2. Decreases in specific t25I-GnRHabinding following6-h prctreatment with 10 nM GnRH, 50 gM AA, 100 nM TPA, and AA + TPA. Pool¢d results from four 6-h prctreatmentexperiments arc presented. (Averagetotal specific binding was 11.'7+ 1.7%.)

Prior exposure to GnRH desensitized the gonadotropcs against subsequent challenges by the releasing peptidc in this as in previous studies (12,15). Such a loss of response could be partly attributable to the concomitant decrease in GnRH receptors and depletion of cellular LH content, as previously proposed (15,21,22). Earlier studies have shown that GnRH-induced loss of CmRH binding sites is due to a decrease in receptor number and not to changes in binding affinity (15,23). However, partial loss of GnRH receptors cannot fully explain the attenuation of responses to GnRH following imm'eatmont with the releasing pcptide. Pretreatment with AA and/or TPA also ~

GnRH binding sites (Fig. 2) but there was no direct correlation between the level of GnRH-

receptor loss and the reduction of LH resIXmse. Furthermore, direct stimulation of the proposed second messenger systems by the addition of AA and TPA each elicited a greater LH response than that obtained with GnRH in GnRHdesensitized cells. These results suggest that GnRtt-induced desensitization involves uncoupling of receptors from activation of second messenger systems. Since the responses to AA and TPA were also attenuated in GnRHpmtreated cells when compared to controls, GnRH-induced desensitization probably also involves impairment of expression or effectors of AA metabolism and protein idnase C activation. The results of this study support the idea that activation of second messenger pathways by secretogogues can desensitize gonadotropes against subsequent stimulation. Pretreatment with AA or TPA severely compromised LH responses to subsequent challenges of these agents (Table 1). Although GnRH could still elicit LH release after treatment with AA or TPA, these responses were smaller that those observed without prior exposure to secretagogues. Furthermore, the LH responses to GnRH following TPA and AA treatments were similar to those to AA and TPA applications, respectively. In contrast, desensitization with AA plus TPA completely abolished all subsequent responses (Table 1). These observations suggest that when one of the major pathways mediating LH secretion is impaired, the reduced response to GnRH is equivalent to that expressed by the remaining functional pathway(s). Only when the functions of all major messenger systems were impaired did complete loss of response CW-.£Ur'.

Agonist activation of receptors and loss of receptors arc coupled events, and the GnRH-induccd decrease in spcci0c binding sims and subsequent LH responses were blocked by coincubation with 1 ~LMGnRH antagonis~ (Fig. 3) as in earlier studies (14). Phorbol esters may exert nonspccific membrane effects, including loss of hormone receptor sites (24,25). However, addition of a GnRH receptor antagonist did not impair TPA- and AAinduced LH release, loss of GnRH receptors, or desensitization of the LH response to subsequent GnRH challenges 922

Vol. 153, No. 3, 1988

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LH release in pretreutment (%of 6nRH-induced release)

12°'t

[125I] GnRH Binding_ (% control specific-binding)

6nRH-stimulated LH release after pretreatment [% of release following control pretreutment without antagonist}

z= -]without antagonist

10o

J with C-nRH antagonist

'°il 60

40

2O

:11

control GnRH

• AA

TPA AA*TPA

control GnRH

AA

TPA AA+TPA

control GnRH

AA

TPA AA+TPA

PRET-REATMENT

Fig. 3, Revczsalof GnRH-inducedreceptor loss and desensitizationby 1 p.M GnRH-ant. Pooled results from five experiments using a 6-h pretreatment with 10 nM GnRH, 50 p.M AA, I00 nM TPA, and AA + TPA are presented. (AverageGnRH-sfimulatedLH release during pretreatmentwas 273.8 _+39.7 rig/106ceils; total specificbinding in controls, 7.3 + 1.0%; and LH response to GnRH rechallengefollowingcontrol pretreatment without antagonist, 179.7 _+ 19.7 ng/106 cells.)

(Fig. 3). These observations suggest that the actions of AA and TPA on desensitization are not mediated through non-specific activafiorrof the GnRI-I receptor. The mechanism whereby AA and TPA cause loss of GnRH binding - sites-req~kesfurther clarification. The present results alsosnggest that rgcmitmentof radioimmunoassayabl¢ LH occurs daring stimulation by

GnRH, AA and TPA. Tic total LH during the second incubation was increased by GnRH, AA, TPA, and AA plus TPA in the control pretreated groups. Whether this increase in radioimmunoassayable LH represents new synthesis or new processing and assembly of pre-existing hormone subunits is not known, but it is interesting to note that pretreatraent with GnRH and AA plus TPA for 6 or 16 h abolished such recruitment or synthesis. Such abolition of synthetic or hormone assembly mechanisms may also play a part in hormone-induced desensitization. In summary, the results of rids study indicate that although desensitizafiou of LH responses may he partly due to loss of GnRH reCelXO~and depletion of LH content, uncoupling and impairment of AA and protein kinase C pathways are also involved in the inhibition of pituitary function by CmRH agouist. Complete or partial loss of pituitary function can also be induced by impairm~t of one or both of two of the major mechanisms that appear to mediate GnRH action, namely AA- and protein kinase C.
ACKNOWLEDGEMENT J.P.C. was supported by a fellowship from the Alberla Heritage Foundation for Medical Research.

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