GnRH Ethylamide Research Articles

Estradiol Alters the Effectiveness of Gonadotropin- Releasing Hormone (GnRH) in Ovine Pituitary Cultures: GnRH Receptors Versus Responsiveness to GnRH*

17 beta-Estradiol (E) rapidly (5-12 h) induced ovine pituitary cultures to increase their binding of des-Gly10-[D-Ala6] GnRH ethylamide by 2.5- to 4.5-fold. The ED50 for E was near 0.1 nM. Scatchard analysis indicated that E increased binding by increasing receptors for GnRH. GnRH-stimulated release of LH increased 2-fold along with the increase in GnRH receptors, but heightened responsiveness to GnRH disappeared after 27 h of E treatment even though GnRH receptors remained elevated. These data are consistent with the concepts that 1) E increased GnRH receptors, which initially enhanced gonadotroph responsiveness to GnRH, and 2) E subsequently nullified the increased response to GnRH by blocking a mechanism of LH secretion not associated with GnRH receptor number. Further support for this hypothesis came from studies with porcine follicular inhibin. Inhibin in ovine pituitary culture concomitantly increased GnRH receptors by 5-fold and GnRH-stimulated LH secretion by 2-fold. Inhibin maintained both effects long term (48 h). When E was added along with inhibin, the increase in GnRH receptor number became greater than with either hormone alone (7-fold increase at 48 h), but the initial increase in GnRH-stimulated LH secretion was fully inhibited by E at 48 h. The increase in GnRH receptors caused by E may be important physiologically to help create the preovulatory LH surge; the delayed inhibitory action of E on GnRH-stimulated LH secretion may limit the LH surge.

Urinary excretion of [D-Ser(t-Bu)6, Des-Gly10]GnRH ethylamide (buserelin) during therapy of central precocious puberty: a multicentre study

In order to compare the clinical effects of buserelin on central precocious puberty to its excretion in urine, as a parameter of metabolism or compliance, we have studied 52 patients treated either sc or intranasally. In girls with good control, urinary buserelin excretion represented 0.5 +/- 0.1% of the daily intranasal dose vs 12.5 +/- 2.3% of the daily sc dose. In boys, it represented 0.8 +/- 0.2 and 9.9 +/- 2.7%, respectively. In the sc treated group, 3 patients (2 girls and 1 boy) with poor control who exhibited excretion levels similar to those with good control were classified as resistant to therapy. Clinical control was poor in 4 intranasally treated girls: 2 had low excretion values suggesting poor compliance or failure of absorption by the nasal mucosa, and 2 appeared resistant to therapy, as urinary excretion levels of buserelin were similar to those of well-controlled patients. In addition, these data suggest that the small amount of buserelin absorbed by the nasal mucosa, as expressed by urinary excretion, is sufficient to desensitize the pituitary gonadotropes without any significant first-pass effect in the systemic circulation. This may explain the clinical effectiveness of the intranasal route for administration of small hormonal peptides acting on the pituitary gland.

Evidence for coupling of different receptors for gonadotropin-releasing hormone to phospholipases C and A 2 in cultured rat luteal cells

Effects of [D-Ala 6,Des-Gly 10]gonadotropin-releasing hormone (GnRH), ethylamide (GnRHa), and prostaglandin F 2α (PGF 2α) on inositol phosphate (IPs) formation and arachidonic acid (AA) release were studied in rat luteal cells of primary culture. In the cells obtained from one-day-old corpora lutea, PGF 2α (100 nM) and GnRHa (100 nM) significantly increased the IPs formation and the AA release. Antagonists of GnRH added solely or with GnRHa did not stimulate the IPs formation but did stimulate the AA release. In the cells obtained from 5-day-old corpora lutea, GnRHa failed to stimulate the IPs formation but significantly stimulated the AA release. The stimulation of both IPs formation and AA release by PGF 2α was consistently found in cells of two different luteal ages. These results suggest that GnRH receptor independently couples to both phospholipases C and A 2 through different classes of GnRH receptors.

Model of the Distribution and Metabolism of the Gonadotropin-Releasing Hormone (GnRH) Agonist [d-Trp6,Des-Gly-NH210]GnRH Ethylamide in Man

The kinetics of plasma immunoreactive [D-Trp6,Des-Gly-NH2(10)]GnRH ethylamide, a potent GnRH agonist, was assessed in nine normal adult men over a period of 24 h after a rapid sc injection of 1 or 10 micrograms peptide/kg BW. The specific RIA of the GnRH agonist is characterized by a minimum detectable dose of 5.0 +/- 0.5 pg peptide, while the midrange effective dose is 73 +/- 8 pg. The within- and between-assay coefficients of variation range from 2-7% and from 10-15%, respectively. When the volume of the central compartment is set proportional to the logarithm of the plasma concentration of the GnRH agonist, the time courses of both doses are statistically well represented by a two-compartment model to which a source compartment is added to represent the sc route of administration. The sc diffusion to plasma is 5 times faster than any other fractional transport rate of the model (5%/min). Disposal occurs at a maximum rate of 0.55%/min from the central compartment, which includes plasma, and 0.25%/min from the peripheral compartment. As simulated with the model, 90% of the steady state is reached 13 h and 45 min after the onset of a sc continuous infusion. A molecule spends, on the average, from 6 h to 9 h and 45 min in the whole organism; 30-50% of this period is accounted for by residence in the central compartment. The volume of the central compartment reaches 208 mL/kg BW, and the MCR is estimated at 1.18 mL/min.kg BW for a steady state of 1 ng/mL plasma. The plasma dynamics of [D-Trp6,Des-Gly-NH2(10)]GnRH ethylamide in men are at least 10 times slower than those reported for the natural GnRH, thus indicating the relevance of its clinical use in suppressive therapy of LH secretion.

Model of the distribution and metabolism of a GnRH superagonist in dogs

The plasma kinetics of [D-Trp6, des-Gly-NH2(10)]gonadotropin-releasing hormone (GnRH) ethylamide was assessed in eight dogs over a period of 8 h after rapid intravenous or subcutaneous injection. Each animal received doses of 0.2, 2, and 20 micrograms/kg body wt iv and 1 and 10 micrograms/kg body wt sc. A two-compartment structure, to which a source compartment was added to represent the subcutaneous route, adequately fits the five kinetics when the apparent volume of distribution follows a plasma concentration-dependent sigmoid function. Despite the nonlinearity, the apparent volume of distribution can be approximated by a constant value of 280 ml/kg body wt for the dynamics corresponding to the three lowest and more physiological doses. The metabolic clearance rate is 4.63 ml.min-1.kg body wt-1. The two exponential components that characterize the two-compartment structure are equal to 0.0348 +/- 0.0053 and 0.00470 +/- 0.00060 min-1, respectively. The agonist injected subcutaneously diffuses to plasma at a fractional rate of 0.0265 +/- 0.0029 min-1. Disposal occurs at a maximal rate of 0.017 and 0.0055 min-1 of the amount of agonist present in the central and peripheral compartments, respectively. The highest fractional exchange rate between compartments reaches 0.01 min-1. As simulated with the model, a continuous infusion of 4.63 ng.min-1.kg body wt-1 leads to a steady state of 1 ng/ml plasma; 90% of that level is reached 7 h after the onset of the subcutaneous input signal. The kinetics of plasma [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide is many times slower than that of the native hormone and of the other GnRH agonists.

Ageing does not influence the ultrashort feedback control of GnRH secretion in vitro

Evidence indicates that long and short feedback systems are altered in the aged male rat. Data also indicate the existence of an ultrashort feedback mechanism controlling GnRH secretion. The present experiments were performed to test whether the ultrashort feedback control of GnRH is operating also in old male rats. Mediobasal hypothalami of 18-month-old male rats were perifused in vitro either in the presence or in the absence of a GnRH agonistic analogue (Buserelin: [D-Ser(TBU)6,Des-Gly10]GnRH ethylamide) and stimulated with 5-min pulses of K+ (for a total of six pulses) in order to test their ability to release GnRH. The hypothalamic fragment was exposed to the GnRH analogue either for a part of the experimental period (at the beginning or at the end) or for the whole duration of the perifusion. In both cases, the presence of the analogue diminished or totally abolished the responses to K+ stimulation. This is in line with the results obtained in young animals. The data suggest that the ultrashort feedback mechanism controlling GnRH release is normally functioning also in aged male rats despite the fact that other types of feedback mechanisms (long and short loop) are substantially altered.

Endocrinological Studies on TAP-144-SR, a Sustained-Release Formulation of a Potent GnRH Agonist (D-Leu6-(des-Gly10-NH2)-GnRH Ethylamide), in male Rats.

The paradoxical effects of TAP-144-SR, a biodegradable sustained-release formulation of a potent GnRH agonist (TAP-144, leuprolide acetate) were evaluated in male rats by comparing its potency with that of TAP-144 solution. A single sc injection of TAP-144-SR (equivalent to 0.1 mg/kg/day as TAP-144), prepared by encapsulating the agonist in microcapsules of copoly (DL-lactic/glycolic acid), suppressed serum levels of androgens, and the levels remained suppressed for 4 weeks. The potency of the paradoxical effects of TAP-144-SR was evaluated 4 weeks after treatment by comparing it with that of TAP-144 solution administered daily for 4 weeks. Both daily injections of TAP-144 solution and a single injection of TAP-144-SR (equivalent to 0.02, 0.2 or 2 mg/kg/day as TAP-144) decreased the weight of the testes, prostates and seminal vesicles in a dose-dependent manner in a 4-week assay in male rats. TAP-144-SR was more effective than TAP-144 solution in reducing these organ weights. Serum and pituitary concentrations of LH and FSH and serum testosterone levels were also lower in TAP-144-SR-treated than in TAP-144 solution-treated rats. These results indicate that the paradoxical effects were more extensive upon TAP-144-SR treatment, suggesting that maintaining constant serum TAP-144 levels results in more extensive desensitization of the pituitary and testes. These results also suggest advantages of TAP-144-SR over TAP-144 solution in both efficacy and convenience as an anti-prostatic tumor agent.

Normal gonadal functions and fertility after 23 months of treatment of prepubertal male and female dogs with the GnRh agonist [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide.

The GnRH agonist [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide was administered to prepubertal male and female dogs daily for 23 months by subcutaneous injection. During GnRH agonist treatment, plasma steroid levels, namely dehydroepiandrosterone, androst-5-ene-3 beta-17 beta-diol, androstenedione, testosterone, dihydrotestosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane, 3 beta, 17 beta-diol were markedly inhibited in male animals, whereas in female animals, the plasma concentration of DHEA and delta 5-diol were decreased. Within 2 months following cessation of therapy, all steroids increased to normal adult levels. Morphological studies reveal that treatment of male animals with the GnRH agonist is accompanied by a small volume of seminiferous tubules, Leydig cells, and prostate gland, whereas in the ovaries of female animals, there is a large number of primordial follicles, a few primary follicles, but no secondary follicles. In the pituitary gland of animals of both sexes, LH-secreting cells have high levels of glycogen particles in their cytoplasm and tend to be either of normal appearance with dilated rough endoplasmic reticulum (RER) or strongly atrophied with a dark-stained cytoplasm, a contraction of RER, and a decrease in the number of secretory granules. Reticular cells of the connective tissue also show high levels of glycogen particles. After the 14 month recovery period, spermatogenesis has a normal adult appearance, the prostate gland shows a normal secretory epithelium, and secondary follicles are easily observed in the ovary. Gonadotrophs are free of glycogen accumulation, but reticular cells continue to show an accumulation of glycogen particles in their cytoplasm. Two male and two female animals were mated after the recovery period and produced normal offspring with normal fertility. The present results indicate that GnRH agonist treatment achieves a blockade of sexual maturation and that following cessation of treatment, normal pituitary-gonadal functions resume, with apparently normal fertility and normal offspring.

Chronic administration of a gonadotropin-releasing hormone (GnRH) agonist affects testicular microvasculature.

Three months of daily sc injections of adult male dogs with the gonadotropin-releasing hormone agonist (GnRH-A) [D-Trp6, des-Gly-NH2(10)]GnRH ethylamide produced significant decrease in the diameter of seminiferous tubules and conspicuously altered the ultrastructure of testicular microvasculature. In contrast to capillaries and venules in untreated controls, which had typical continuous endothelial layers surrounded by a basal lamina, in testes of dogs chronically treated with GnRH-A, 14.3% of the capillaries and 21.2% of the venules showed wide (30-500 nm) endothelial gaps. In a few capillaries (1.7%) and venules (4%) endothelial fenestrations were found. A high percentage of capillaries (59.3%) and venules (32.8%), with endothelial gaps or continuous endothelium were surrounded by multiple layers of basal lamina. All arterioles, 24.7% of the capillaries and 42.6% of the venules showed the normal features as found in the controls. Superfluous basal laminae, not associated with cells were present in the testes of the chronically treated dogs, but were also found after 4 months of recovery from the GnRH-A treatment. However, within 4 months after cessation of the GnRH-A treatment, the diameters of the seminiferous tubules were comparable to those in untreated controls. Capillaries and venules with endothelial gaps or fenestrations were completely absent. All arterioles, 43.6% of the capillaries and 65.6% of the venules revealed the normal features of continuous endothelium. However, 56.4% of the capillaries and 34.4% of the venules were characterized by superfluous layers of basal lamina.(ABSTRACT TRUNCATED AT 250 WORDS)

Induced spawning of maturing milkfish ( Chanos chanos) using human chorionic gonadotropin and mammalian and salmon gonadotropin releasing hormone analogues

The response of maturing female milkfish to D-Ala 6-des Gly 10 mammalian GnRH ethylamide (mGnRH-A), D-Arg 6-des Gly 10 salmon GnRH ethylamide (sGnRH-A) and human chorionic gonadotropin (hCG) was investigated. The GnRH analogues and hCG were equally effective when administered by intramuscular injection at doses of 10 μg/kg and 100 μg GnRH-A/fish or 1000 IU hCG/fish. All of the females injected with HCG and 87.5% ( 7 8 ) of females injected with GnRH-A spawned. Pellet implantation of the GnRH analogues, however, was less effective based on 100 μg of pellet per fish, which provided from 20 to 36 μg of analogue per kg fish. Fish implanted with mGnRH-A or sGnRH-A showed responses which varied from oocyte hydration to spawning. Only 3 7 implanted with mGnRH-A and 1 7 implanted with sGnRH-A spawned; in the latter group, the average egg diameter was 11–17% smaller at the time of treatment compared with the other treated groups. Except for one, all fish with egg diameters above 0.65 mm had hydrated/ovulated oocytes or spawned. Females which spawned had egg diameters above 0.71 mm.

Effects of Prostaglandin F2αand a Gonadotropin-Releasing Hormone Agonist on Inositol Phospholipid Metabolism in Isolated Rat Corpora Lutea of Various Ages*

The sensitivity of rat corpora lutea to luteolytic agents increases with luteal age. We examined the effect of prostaglandin F2 alpha (PGF2 alpha) and [D-Ala6,Des-Gly10]GnRH ethylamide (GnRHa) on inositol phospholipid metabolism in day 2 and day 7 corpora lutea from PMSG-treated rats. Isolated corpora lutea were incubated with 32PO4 or [3H]inositol and were treated with LH, PGF2 alpha, or GnRHa. Phospholipids were purified by TLC, and the water-soluble products of phospholipase-C activity (inositol phosphates) were isolated by ion exchange chromatography. In day 2 corpora lutea, PGF2 alpha, (10 microM) and GnRHa (100 ng/ml) significantly increased 32PO4 incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI), but not into other fractions. LH provoked slight increases in PA. Results were similar with 30 min of prelabeling or simultaneous addition of 32PO4 and stimulants. In other experiments, PGF2 alpha and GnRHa provoked rapid increases (1-5 min) in the accumulation of inositol mono-, bis-, and trisphosphates. LH did not significantly increase inositol phosphate accumulation, but stimulated cAMP accumulation in 2-day-old corpora lutea. Inositol phospholipid metabolism was increased in day 7 corpora lutea compared to that in day 2 corpora lutea. This increase was associated with increased incorporation of 32PO4 into PA and PI and increased accumulation of [3H]inositol phosphates. In day 7 corpora lutea, which are very sensitive to the luteolytic effect of PGF2 alpha, the PG-induced increase in PA labeling was small and inconsistent, whereas PI labeling was unaffected in 30-min incubations. GnRHa was without effect in such corpora lutea. LH, PGF2 alpha, or GnRHa did not increase inositol phosphate accumulation in 7-day-old corpora lutea. These studies demonstrate that the transformation of young (day 2) to mature (day 7) corpora lutea is associated with an increase in luteal inositol phospholipid metabolism. The results also show that PGF2 alpha and GnRHa stimulate phospholipase-C activity in young corpora lutea, but are ineffective in mature corpora lutea, and suggest that an increase in inositol phospholipid metabolism by itself is not sufficient to explain the acute luteolytic action of PGF2 alpha and GnRH in vitro. However, phospholipase-C-derived second messengers may be involved in the action of hormones that control luteal function.

Induced sexual maturation of herring using GnRH ‘quick-release’ cholesterol pellets

An analogue of gonadotropin-releasing hormone, [D-Ala 6, des Gly 10] mammalian GnRH ethylamide (mGnRH-A) was effective in advancing maturation of Pacific herring ( Clupea harengus pallasi) provided the fish had matured to Hay's modified Hjort stage IV (gonads are two-thirds of mature size; germinal vesicles have not migrated). The analogue was administered in new ‘quick-release’ cholesterol pellets made of cholesterol and cellulose (4:1, w w ). In vitro perfusion studies indicated 94% of the 100 μg of mGnRH was released from such pellets during 12 h. Implanted ‘quick-release’ mGnRH-A pellets resulted in more mature fish; the advancement over control fish was maintained for at least 3 weeks. Also, a higher percentage of fish reached a more mature Hjort's stage in response to mGnRH in pellets containing 100 μg (dose approximately 800–1000 μg/kg) compared with 20 μg (dose approximately 170–220 μg/kg). The response to different GnRH analogues may be species specific because [D-Ala 6, des Gly 10] salmon GnRH ethylamide was not effective in herring, but is known to be as effective as mGnRH-A in milkfish. A potential application of mGnRH-A is to advance maturation of stage IV herring kept in captivity (impounded) for commercial production of spawn-on-kelp.

In vivo regulation of granulosa cell somatomedin-C/insulin-like growth factor I receptors.

The characteristics and regulation of the murine granulosa cell type I insulin-like growth factor (IGF) receptor under in vivo conditions were studied. In vivo treatment of immature hypophysectomized diethylstilbestrol-treated rats with increasing doses (0.3-30 microgram/rat, twice daily) of FSH for 72 h resulted in dose-dependent increments in specific granulosa cell somatomedin-C (Sm-C)/IGF-I binding, peaking (5150 +/- 350 cpm/3 x 10(5) cells) at the 10 micrograms/rat (twice daily) dose level to yield a 2.6-fold increase relative to that in untreated controls. This FSH (10 micrograms/rat, twice daily) effect proved time dependent; the first significant (P less than 0.05) increase in binding (3670 +/- 150 cpm/3 x 10(5) cells) was noted after 48 h of treatment (1.6-fold increase). Significantly, little or no variation was observed for basal Sm-C/IGF-I binding over the course of the experiment, suggesting that this component of Sm-C/IGF-I receptor complement may be independent of the trophic influence(s) of the pituitary gland. Equilibrium competition studies carried out with granulosa cells derived from both control and FSH-treated rats revealed linear Scatchard plots consistent with a single class of noninteracting binding sites, a 2.8-fold increase in FSH-associated Sm-C/IGF-I-binding capacity, but not affinity (Kd control, 1.9 +/- 0.3 nM; kd FSH, 2.6 +/- 0.9 nM). Limited specificity studies of the FSH-induced receptor revealed related peptides to compete for Sm-C/IGF-I binding with a relative rank order of potency of Sm-C/IGF-I much greater than multiplication-stimulating activity greater than insulin, a pattern compatible with a type I IGF receptor. A series of other polypeptides, including porcine relaxin, porcine proinsulin, epidermal growth factor, basic fibroblast growth factor as well as transforming growth factor-alpha and -beta (TGF beta) were nonreactive. Significantly, the induced type I IGF receptor proved functionally coupled to granulosa cell proteoglycan biosynthesis. The ability of FSH (10 micrograms/rat, twice daily) to enhance granulosa cell Sm-C/IGF-I binding was significantly (P less than 0.05) up-regulated (1.53-fold amplification) by ovine GH (100 micrograms/rat, twice daily); a down-regulatory effect (64% inhibition) was observed for a potent GnRH agonist [( D-Ala6,Des-Gly10]GnRH ethyl amide; 25 micrograms/rat, twice daily). Once induced, the Sm-C/IGF-I receptor of the granulosa cell required the continued presence of either FSH or LH for its maintenance; the lactogenic receptor agonist PRL had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and distribution of receptors for gonadotropin-releasing hormone in the rat hippocampus.

Distribution and properties of receptors for gonadotropin-releasing hormone (GnRH) were analyzed in the brain of adult male rats. Binding of the iodinated GnRH agonist Des-Gly10-(D-Ala6)-GnRH ethylamide was studied in hippocampus and anterior pituitary using three convergent approaches: quantitative autoradiography on frozen tissue, binding to fresh slices, and binding to crude membrane preparations. In all cases, binding was specific, saturable, and time, pH, and temperature dependent. Quantitative autoradiography revealed that the density of binding sites was high in the stratum oriens and stratum radiatum of the CA1-CA4 regions of Ammon's horn. The pyramidal cell layer was faintly labelled. Binding was almost undetectable in the dentate gyrus. The highest density of sites (Bmax = 11.6 +/- 1.0 fmol/mg protein) was observed in the stratum radiatum of the CA3 region. Under the same conditions the value obtained for pituitary tissues was 20.7 +/- 2.8 fmol/mg protein. Analysis of saturation curves indicated only one class of high-affinity sites for the hippocampus (CA3; Kd = 0.28 +/- 0.03 nM) and for the pituitary (Kd = 0.29 +/- 0.08 nM). Both native GnRH and GnRH antagonist were potent competitors of binding. Fresh slices and membrane preparations from whole hippocampus confirmed these autoradiographic data and yielded affinity constants of 0.28 +/- 0.01 and 0.52 +/- 0.08 nM, respectively. In addition, a very high binding density was present in the amygdaloid complex, while binding was barely detectable in the hypothalamus. These results demonstrate that high densities of specific GnRH receptors are present in areas concerned with the regulation of behavioral functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological study of the effects of an GnRH agonist on the canine testis after 4 months of treatment and recovery.

After 4 months of treatment of adult male dogs with the GnRH agonist (GnRH-A) [D-Trp6]GnRH ethylamide, the seminiferous tubules contained only type A and B spermatogonia, Sertoli cells, and rare primary spermatocytes, thus causing a 64% decrease in testis weight. At the electron microscope level, Sertoli cells showed an increase in phagosomes and lipid droplets. Leydig cells were markedly atrophied with the accumulation of lipid droplets and showed a predominance of mitochondria with lamellar instead of vesicular cristae. Four months after cessation of treatment with GnRH-A, a complete return to normal spermatogenesis and Leydig cell morphology was observed. The full reversibility of spermatogenesis in the dog after chronic GnRH-A treatment suggests that this well-tolerated peptide could be used as a reversible method of male contraception.

Effect of a GnRH-analogue and of the phorbol ester PMA on rat luteal cell steroidogenesis

In the rat, the hypothalamic peptide GnRH acts not only at the pituitary but can also modulate ovarian function directly [1, Review]. Recently, a role for a phospholipid- and Ca2+-dependent protein kinase (PK-C) has been suggested in the action of GnRH on the pituitary [2]. GnRH is known to reduce gonadotrophin-stimulated progesterone (P) production by rat luteal cells through the inhibition of hormone-responsive adenylate cyclase [3]. The tumor-promoting phorbol-ester, phorbol 12-myristate 13-acetate (PMA), a well-known activator of PK-C, has been shown to reduce steroidogenesis by mouse Leydig cells through inhibition of hormone-stimulated cAMP formation [4]. In order to determine whether the inhibitory action of GnRH on luteal steroidogenesis is effected through activation of PK-C, we compared the effects of a GnRH agonist, Des-10-Gly-(6Trp)-GnRH-ethylamide (GnRH) on steroidogenesis and cAMP formation by rat luteal cells with those of PMA.

Pharmacodynamics of [D-Ser (t-Bu), Des-Gly]GnRH ethylamide (Buserelin®) in 6 normal volunteers.

The disappearance rate of [D-Ser(t-bu)6,des-Gly10]GnRH ethylamide (Buserelin, HOE 766) was studied in plasma and urine after intranasal (300 micrograms) or sc (10 micrograms/kg) administration. A radioimmunoassay for HOE 766 was developed using 125I[D-Trp6,Des-Gly10]GnRH ethylamide as tracer and an antiserum raised against HOE 766. Cross-reaction with native GnRH was only 1.7%. Sensitivity was 1 pg/tube. In 6 male adolescents, the mean plasma HOE 766 concentration (+/- SEM) was 0.46 +/- 0.08, 0.50 +/- 0.10, 0.28 +/- 0.04, 0.24 +/- 0.04, 0.13 +/- 0.03, and 0.08 +/- 0.02 micrograms/l 30, 60, 90, 120 and 180 min after the intranasal administration, respectively. Concomitant urinary excretion of HOE 766-like material was 9.43 +/- 1.96 micrograms/4 h. There was a good correlation between integrated plasma levels and urinary excretion (r = 0.92). In the same 6 volunteers, the plasma HOE 766 levels were 21.2 +/- 3.0, 25.9 +/- 0.8, 21.2 +/- 0.9, 17.1 +/- 0.7, 12.8 +/- 1.1, 8.9 +/- 0.4, and 5.9 +/- 0.8 micrograms/l 20, 40, 60, 90, 120, 180 and 240 min after sc injection, respectively. The mean urinary excretion was 543 +/- 61 micrograms/4 h. In two girls with precocious puberty treated during 12 to 15 months with intranasal administration of HOE 766, urinary excretion of HOE 766-like material was shown to correlate well with the degree of inhibition of plasma 17 beta-E2 and of plasma LH and FSH responses to a GnRH challenge. Thus, monitoring of HOE 766 in urine appears to be helpful for evaluating of intranasal therapy with a GnRH analog in precocious puberty.

Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3',5'-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone-releasing activity of certain ANFs.

The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph-enriched elutriated pituitary cells. The same ANFs also displaced [125I-D-Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist.

Mechanisms of Action of Gonadotropin Releasing Hormone on Rat Granulosa Cells

To elucidate the mechanisms of stimulatory actions of GnRH on rat granulosa cells (GC), we have compared the actions of a GnRH agonist with those of a tumor-promoting phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA) and Ca+2 ionophore, A23187. GC were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG. Following prelabeling with 3[H]arachidonic acid (AA), the cells were incubated with the test substances for 10 min and AA release determined. A GnRH agonist, [D-Ala6, des-Gly-NH2(10)] GnRH ethylamide (GnRHa; 10 ng/ml) increased AA release 175% compared to the control value. AA release in the presence of GnRHa was larger than that due to 1 microM A23187 or 40 nM TPA alone. A23187 or TPA increased GnRHa-stimulated AA release further. GC were incubated with the test substances for longer time periods, i.e., up to 5 h. GnRHa caused a 4-fold increase in prostaglandin (PG) synthase activity at 5 h. GnRHa increased PGE accumulation to the same extent as TPA, but only increased PG synthase activity about half as much. In combination with TPA, GnRHa had no influence on TPA-stimulated PG synthase activity, but increased PGE accumulation to levels comparable to those with A23187 plus TPA. GnRHa caused a 2.5 fold increase in progesterone (P) accumulation, which was the same as TPA. P accumulation in the presence of GnRHa was affected by neither A23187 nor TPA. These data indicate that the combination of TPA and A23187 can substitute for GnRH action on PGE and P accumulation in rat GC.

Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors.

The present study examined the question of whether modulation of estradiol-induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28-day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor.