2,3,7,8-tetrachlorodibenzo-p-dioxin increases the potency of androgens and estrogens as feedback inhibitors of luteinizing hormone secretion in male rats

Tetrachlorodibenzo-p-dioxin (TCDD) decreases plasma androgen concentrations in male rats, without increasing plasma luteinizing hormone (LH) concentrations. If plasma LH concentrations had increased appropriately, plasma androgen concentrations in these animals would have returned to normal. The mechanism by which TCDD prevents the compensatory increase in plasma LH concentrations was therefore investigated. TCDD was found to have no effect on the plasma disappearance of iv administered LH. Therefore, the failure of plasma LH concentrations to rise was not due to increased clearance of LH from the circulation, but rather to an effect of TCDD on LH synthesis and/or secretion by the pituitary. In the absence of gonadal steroids (i.e., in castrated rats) TCDD did not prevent the compensatory increase in plasma LH concentrations from occurring. This was shown by 20-fold increases in plasma LH concentrations in both control and TCDD-treated rats 1 week after castration. Thus, (1) the presence of gonadal steroids is required for TCDD to prevent the compensatory increase in plasma LH concentrations, and (2) TCDD does not impair LH secretion by acting, itself, as an androgen or estrogen. TCDD treatment also did not affect pituitary LH content in castrated, testosterone-implanted rats. The above findings demonstrate that TCDD does not decrease the maximum rate at which the pituitary can synthesize and secrete LH. Rather, TCDD alters the feedback regulation of LH secretion when gonadal steroids are present. To determine if TCDD affects the potency of testosterone and its metabolites 5 alpha-dihydrotestosterone and 17 beta-estradiol as feedback inhibitors of LH secretion, rats were dosed with TCDD, castrated, and implanted with sustained-release capsules containing graded amounts of each steroid. Seven days later, the potencies of all three hormones as feedback inhibitors of LH secretion were increased by TCDD, with little effect on their plasma concentrations. The TCDD dose dependence for the increased effectiveness of testosterone as a feedback inhibitor of LH secretion (ED50 10 micrograms/kg) was similar to that reported for the imbalance between plasma LH and androgen concentrations (ED50 15 micrograms/kg). Also, time courses for both responses were similar; each was detected within 1 day of TCDD dosing and each was fully developed after 7 days. We conclude that the mechanism by which TCDD prevents the compensatory increase in plasma LH concentrations in male rats is by increasing the potencies of androgens (and estrogens) as feedback inhibitors of LH secretion.     

Population pharmacokinetic/pharmacodynamic (PK/PD) modelling of the hypothalamic-pituitary-gonadal axis following treatment with GnRH analogues

AIMS: To develop a population pharmacokinetic/pharmacodynamic (PK/PD) model of the hypothalamic-pituitary-gonadal (HPG) axis describing the changes in luteinizing hormone (LH) and testosterone concentrations following treatment with the gonadotropin-releasing hormone (GnRH) agonist triptorelin and the GnRH receptor blocker degarelix. METHODS: Fifty-eight healthy subjects received single subcutaneous or intramuscular injections of 3.75 mg of triptorelin and 170 prostate cancer patients received multiple subcutaneous doses of degarelix of between 120 and 320 mg. All subjects were pooled for the population PK/PD data analysis. A systematic population PK/PD model-building framework using stochastic differential equations was applied to the data to identify nonlinear dynamic dependencies and to deconvolve the functional feedback interactions of the HPG axis. RESULTS: In our final PK/PD model of the HPG axis, the half-life of LH was estimated to be 1.3 h and that of testosterone 7.69 h, which corresponds well with literature values. The estimated potency of LH with respect to testosterone secretion was 5.18 IU l(-1), with a maximal stimulation of 77.5 times basal testosterone production. The estimated maximal triptorelin stimulation of the basal LH pool release was 1330 times above basal concentrations, with a potency of 0.047 ng ml(-1). The LH pool release was decreased by a maximum of 94.2% by degarelix with an estimated potency of 1.49 ng ml(-1). CONCLUSIONS: Our model of the HPG axis was able to account for the different dynamic responses observed after administration of both GnRH agonists and GnRH receptor blockers, suggesting that the model adequately characterizes the underlying physiology of the endocrine system.     

GnRH agonists and antagonists in cancer therapy

Gonadotropin releasing hormone (GnRH) is a hypothalamic decapeptide that binds to GnRH receptors on pituitary gonadotrope cells to modulate the synthesis and secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins in turn regulate gonadal steroidogenesis and gametogenesis. Chemical characterization and structure-activity analysis of GnRH variants containing systematic amino acid substitutions led to the discovery of GnRH superagonists and antagonists. These peptides are widely used for the treatment of clinical conditions in which modulation of or interference with sex hormone production is beneficial to prevent development or progression of benign conditions (e.g. endometriosis, uterine fibroids) or malignant tumors (e.g. breast, ovarian, endometrial and prostate carcinoma). When compared to native GnRH, GnRH superagonists have increased potency for the short-term release of gonadotropins. However, they show paradoxical action in that chronic treatment with superagonists results in inhibition of gonadotropin production as a result of desensitization of the gonadotropes and down regulation of its receptor. In contrast, GnRH antagonists produce a rapid and dose-dependent suppression of gonadotropin release by competitive blockade of the GnRH receptors without any initial stimulatory effect as seen with superagonists. In recent years, a search for peptidomimetic compounds to replace peptides as therapeutic agents has been undertaken to find compounds with higher affinity for the GnRH receptor but do not have the disadvantages of peptides. Such efforts have resulted in the identification and development of small-molecule non-peptide compounds that are sufficiently stable in vivo and possess favorable pharmacological parameters comparable to peptide antagonists. Some of these compounds are being tested in human volunteers and the preliminary results are very encouraging.     

Non-peptidic GnRH receptor antagonists

Gonadotropin-releasing hormone (GnRH) or luteinizing hormone-releasing hormone (LHRH) is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) hypothalamic hormone that acts upon 7-trans membrane spanning GnRH receptors in the pituitary. This action leads to the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that in turn act on the reproductive organs regulating gonadal steroid production, spermatogenesis and follicular development. Peptidic agonists of the GnRH receptor have been known for many years and are currently employed therapeutically in the treatment of prostate and breast tumours, uterine fibroids, precocious puberty, endometriosis, premenstrual syndrome, contraception and infertility. Peptidic antagonists to date have only been employed commercially in the treatment of infertility during assisted reproductive therapy; however, many peptidic antagonists are currently in late stage development for many of the aforementioned indications. Whilst peptidic agonists and antagonists of the GnRH receptor have been discovered and exploited clinically, they are limited to predominantly parenteral administration due to their poor oral bioavailability. Recently, several small molecule GnRH antagonist series have been discovered offering the prospect of orally active therapeutics based on GnRH receptor antagonism. This article will review the current medicinal chemistry literature and structure activity relationships known for non-peptidic GnRH receptor antagonists.     

Preparation and characterization of photoreactive derivatives of GnRH

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys⁶-GnRH(1–9)-ethylamide, was prepared by selective modification of the e-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]⁶-GnRH-(1–9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion whch could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

Androgenic deficiency in male rats treated with perfluorodecanoic acid

Effects of perfluorodecanoic acid (PFDA, 20-80 mg/kg, ip) on the androgenic status of sexually mature male rats were investigated 7 days after treatment. PFDA decreased plasma androgen concentrations in a dose-dependent fashion with an ED50 of approximately 30 mg/kg. The highest dose of PFDA decreased plasma testosterone and 5 alpha-dihydrotestosterone concentrations to 12 and 18%, respectively, of ad libitum-fed control (ALC) values. Secondary to the decreased plasma androgen concentrations were dose-related decreases in the weights and epithelial heights of accessory sex organs. Results from pair-fed control (PFC) rats show that hypophagia in PFDA-treated rats was not a major cause of the low plasma androgen concentrations. When rats were castrated and implanted with testosterone-containing capsules, PFDA-treated and ALC rats had similar plasma testosterone concentrations and secondary sex organ weights. Therefore, the androgenic deficiency in intact PFDA-treated rats does not result from increased plasma clearance of androgens. Rather, PFDA must cause the androgenic deficiency by decreasing the secretion of testosterone from the testis. The decrease in testosterone secretion does not appear to result from a decrease in plasma luteinizing hormone (LH) concentrations, because plasma LH concentrations were not significantly altered by PFDA treatment. This finding suggests that PFDA treatment decreases testicular responsiveness to LH stimulation. The observation that PFDA treatment reduced the secretion of testosterone by testes stimulated in vitro with the LH analog human chorionic gonadotropin demonstrates that this is the case. In addition, since plasma LH concentrations did not increase in response to the low plasma androgen concentrations in PFDA-treated rats, we suggest that PFDA disrupts the normal feedback relationship which exists between plasma androgen and LH concentrations.     

Effects of 5-Day Styrene Inhalation on Serum LH and Testosterone Levels and on Hypothalamic and Striatal Amino Acid Neurotransmitter Concentrations in Male Rats

The volatile chemical styrene may impair male fertility. Testicular testosterone (T) production is controlled by the hypothalamic/pituitary/gonadal axis. From the mediobasal hypothalamus (MBH), gonadotropin-releasing hormone (GnRH) is released, which stimulates luteinizing hormone (LH) secretion from the pituitary, which in turn enhances T production. GnRH release is controlled by glutamate (GLU) and gamma-aminobutyric acid (GABA). GLU and GABA neurons are regulated by T. Thus, reduced fertility of styrene-exposed male workers may result from altered GLU/GABA neurotransmission, causing insufficient GnRH, LH, and T secretion. Therefore, we compared LH and T levels of male rats that have inhaled styrene (0, 150, 500, 1500 ppm for 6 h on 5 consecutive days) to GLU and GABA concentrations in the MBH and striatum. Animals were killed directly following the last exposure (immediate group) or after 24 h (recovery group). No suppression of LH or T levels was observed after styrene inhalation. LH levels of the immediate groups with 500 or 1500 ppm exposure were slightly but signficantly elevated. Hypothalamic GLU and GABA concentrations remained unchanged. Increased striatal GABA concentrations were determined in recovery groups with 500 or 1500 ppm exposure. Striatal GLU concentrations remained unaffected. Thus, we demonstrate slightly increased LH and T levels in styrene-exposed male rats after inhalation of the two higher doses. This effect did not correlate with hypothalamic GLU and GABA concentrations. With the limitations inherent to any animal model, these data obtained from a 5-day exposure study with rats suggest, but do not unequivocally prove, that styrene may have also no reproductive toxicity effects in men chronically exposed to this chemical.     

Effects of 5-day styrene inhalation on serum LH and testosterone levels and on hypothalamic and striatal amino acid neurotransmitter concentrations in male rats

The volatile chemical styrene may impair male fertility. Testicular testosterone (T) production is controlled by the hypothalamic/pituitary/gonadal axis. From the mediobasal hypothalamus (MBH), gonadotropin-releasing hormone (GnRH) is released, which stimulates luteinizing hormone (LH) secretion from the pituitary, which in turn enhances T production. GnRH release is controlled by glutamate (GLU) and gamma-aminobutyric acid (GABA). GLU and GABA neurons are regulated by T. Thus, reduced fertility of styrene-exposed male workers may result from altered GLU/GABA neurotransmission, causing insufficient GnRH, LH, and T secretion. Therefore, we compared LH and T levels of male rats that have inhaled styrene (0, 150, 500, 1500 ppm for 6 h on 5 consecutive days) to GLU and GABA concentrations in the MBH and striatum. Animals were killed directly following the last exposure (immediate group) or after 24 h (recovery group). No suppression of LH or T levels was observed after styrene inhalation. LH levels of the immediate groups with 500 or 1500 ppm exposure were slightly but significantly elevated. Hypothalamic GLU and GABA concentrations remained unchanged. Increased striatal GABA concentrations were determined in recovery groups with 500 or 1500 ppm exposure. Striatal GLU concentrations remained unaffected. Thus, we demonstrate slightly increased LH and T levels in styrene-exposed male rats after inhalation of the two higher doses. This effect did not correlate with hypothalamic GLU and GABA concentrations. With the limitations inherent to any animal model, these data obtained from a 5-day exposure study with rats suggest, but do not unequivocally prove, that styrene may have also no reproductive toxicity effects in men chronically exposed to this chemical.     

The effect of neonatal exposure to DES and o,p'-DDT on pituitary responsiveness to GnRH in adult castrated rats.

While exposure of vertebrates to estrogens during early development has been shown to alter adult reproductive behavior, neuroanatomy, and neurophysiology, effects on gonadotropin secretion have not been studied. We conducted the present studies to assess the effects of neonatal exposure to xenobiotic estrogens on luteinizing hormone secretion in castrated adult rats. Rat pups were injected with either corn oil, 1 micrograms diethylstilbestrol (DES), or 0.5 mg o,p'-DDT on postnatal days 1 to 10, and castration was performed on day 21. On day 42 of life, GnRH (50 ng/kg) was administered via right heart catheters, and blood was sampled for LH at 0, 5, 10, 15, and 30 min. Neonatal exposure to DES in both males and females significantly decreased basal and GnRH-induced LH secretion throughout the sampling period in castrated adults. o,p'-DDT significantly suppressed initial LH levels and blunted GnRH-induced release in males at the 5 min interval, while in females it had no effect. These data show that early exposure to environmental estrogens alters adult pituitary response to GnRH. Our results suggest that sexually distinct effects of environmental estrogens occur and can be readily demonstrated in this experimental model.     

Inhibition of testicular steroidogenesis in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats: evidence that the key lesion occurs prior to or during pregnenolone formation

The mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment decreases testosterone (T) secretion without significantly altering plasma luteinizing hormone (LH) concentrations was investigated. Testes from sexually mature Sprague-Dawley rats dosed 7 days earlier with 100 micrograms TCDD/kg secreted 30-75% less T than did testes from control rats when perfused in vitro with the LH analog human chorionic gonadotropin (hCG). This decrease confirms that testicular responsiveness to LH, the hormone which regulates T secretion in vivo, is impaired by TCDD treatment. Because TCDD also reduced intratesticular T content, the decrease in T secretion is due to an inhibition of T synthesis rather than to a failure of the secretion process. These effects of TCDD are not secondary to undernutrition, because perfused testes from feed-restricted control rats were fully hCG responsive. TCDD treatment neither increased the hCG-stimulated secretion of any T precursor nor significantly decreased the efficiency with which testes converted the pregnenolone (PREG) they synthesized into T (PREG is the initial steroidogenic intermediate). In addition, TCDD did not inhibit T secretion when steroidogenesis was supported by exogenous PREG at approximately the in vivo rate. We conclude that TCDD does not inhibit the conversion of PREG to T. The inhibition of T biosynthesis must instead result from an inhibition of PREG formation. The finding that TCDD treatment substantially decreased the rate at which hCG-perfused testes secreted PREG and its metabolites (a decrease seen across all hCG concentrations) confirms this conclusion. This inhibition of LH/hCG-stimulated PREG formation by TCDD must be due to a reduction in the activity of the enzyme which converts cholesterol to PREG (cytochrome P450scc), and/or an impairment in the multistep process responsible for mobilizing cholesterol to this enzyme.     

Evidence for Ah receptor mediation of increased ACTH concentrations in primary cultures of rat anterior pituitary cells exposed to TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to increase plasma ACTH concentrations in male Sprague-Dawley rats and in male rat primary anterior pituitary cell cultures. The present study examined whether the anterior pituitary effects observed after TCDD exposure are mediated via the Ah receptor (AhR). Primary anterior pituitary cell cultures were prepared from normal 180- to 220-g male rats and the cultures treated with alpha-naphthoflavone (ANF), an antagonist; beta-naphthoflavone (BNF), an agonist; BNF + TCDD; 3,3',4,4',5-pentachlorobiphenyl (PCB), which is known to bind to the AhR; and 2,2',4,4',5,5'-hexachlorobiphenyl (HCB), which does not bind the AhR. Support for the TCDD-AhR-mediated increases in ACTH concentrations is provided by the following observations: (1) ANF inhibited both the 1.3- to 2-fold TCDD-induced increase in basal medium and intracellular ACTH concentrations and the 30% TCDD-induced decrease in medium ACTH levels and the 1.2-fold increase in intracellular ACTH levels in corticotropin-releasing hormone (CRH)-stimulated cells, (2) BNF increased basal medium (1.7-fold) and intracellular (1.3-fold) ACTH concentrations, (3) BNF + TCDD demonstrated additivity by increasing basal medium (2.4-fold) and intracellular (1.7-fold) ACTH concentrations, (4) PCB increased basal medium (1.8- to 2.1-fold) and intracellular (1.3- to 1.8-fold) ACTH concentrations and inhibited medium ACTH secretion in CRH stimulated cells by 24-43%, and (5) HCB did not effect basal or CRH stimulated medium and intracellular ACTH concentrations. From this study it appears that TCDD-induced changes in ACTH secretion and synthesis by cultured anterior pituitary cells is mediated through the Ah receptor.     

Evidence that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague-Dawley rats without changing estrogen receptor action.

High oral doses of atrazine (ATRA) disrupt normal neuroendocrine function, resulting in suppression of the luteinizing hormone (LH) surge in adult, ovariectomized (OVX) estrogen-primed female rats. While the mechanism by which ATRA inhibits LH secretion is not known, current data indicate that ATRA does have anti-estrogenic properties in vitro and in vivo. In the body, ATRA is rapidly converted to diaminochlorotriazine (DACT). The present study was conducted to investigate the effects of ATRA and DACT on the estradiol benzoate (EB)/progesterone (P) induced LH surge and to determine if such changes correlate with impaired estrogen receptor (ER) function. ATRA, administered by gavage for five consecutive days to adult OVX, female Sprague-Dawley rats, caused a dose-dependent suppression of the EB/P induced LH surge. Although to a lesser degree than ATRA, DACT significantly suppressed total plasma LH and peak LH surge levels in EB/P primed animals by 60 and 58%, respectively. DACT treatment also decreased release of LH from the pituitary in response to exogenous gonadotropin releasing hormone (GnRH) by 47% compared to control. Total plasma LH secretion was reduced by 37% compared to control, suggesting that in addition to potential hypothalamic dysfunction, pituitary function is altered. To further investigate the mechanism by which hypothalamic function might be altered, potential anti-estrogenicity of ATRA and DACT were assessed by evaluating ER function treated rats. Using an in vitro receptor binding assay, ATRA, but not DACT, inhibited binding of [(3)H]-estradiol to ER. In contrast, ATRA, administered to female rats under dosing conditions which suppressed the LH surge, neither changed the levels of unoccupied ER nor altered the estrogen induced up-regulation of progesterone receptor mRNA. Collectively, these results indicate that although ATRA is capable of binding ER in vitro, the suppression of LH after treatment with high doses of ATRA is not due to alterations of hypothalamic ER function.     

Therapeutic potential of GnRH antagonists in the treatment of precocious puberty

Pituitary-gonadal axis activation depends upon pulsatile hypothalamic gonadotropin-releasing hormone (GnRH) secretion. This phenomenon has led to clinical use of GnRH agonists in the treatment of central precocious puberty. GnRH analogues contain substitutions of the native decapeptide. Depending upon the substitutions, the analogues have GnRH agonistic or antagonistic properties. The pharmacokinetics of GnRH agonists, the established treatment of precocious puberty, includes an initial 'flare-up' of the pituitary-gonadal axis, followed by a reduced luteinising hormone secretion by desensitisation of pituitary GnRH receptors. Antagonistic GnRH analogues act by competitive binding to the pituitary GnRH receptors, thereby preventing the action of endogenous GnRH - theoretically offering a more direct and dose-dependent treatment alternative. The antagonist available today in Germany is a concomitant in assisted reproduction with only 1 - 3 days duration. However, long-acting depot preparations of other GnRH antagonists are in primate-testing phase. Our animal tests indicate strong potential for the development and testing of long-acting depot preparations of GnRH antagonists in treating precocious puberty.     

Therapeutic potential of GnRH antagonists in the treatment of precocious puberty

Pituitary-gonadal axis activation depends upon pulsatile hypothalamic gonadotropin-releasing hormone (GnRH) secretion. This phenomenon has led to clinical use of GnRH agonists in the treatment of central precocious puberty. GnRH analogues contain substitutions of the native decapeptide. Depending upon the substitutions, the analogues have GnRH agonistic or antagonistic properties. The pharmacokinetics of GnRH agonists, the established treatment of precocious puberty, includes an initial ‘flare-up’ of the pituitary-gonadal axis, followed by a reduced luteinising hormone secretion by desensitisation of pituitary GnRH receptors. Antagonistic GnRH analogues act by competitive binding to the pituitary GnRH receptors, thereby preventing the action of endogenous GnRH – theoretically offering a more direct and dose-dependent treatment alternative. The antagonist available today in Germany is a concomitant in assisted reproduction with only 1 – 3 days duration. However, long-acting depot preparations of other GnRH antagonists are in primate-testing phase. Our animal tests indicate strong potential for the development and testing of long-acting depot preparations of GnRH antagonists in treating precocious puberty.     

Testicular toxicity induced by a triple neurokinin receptor antagonist in male dogs

Mechanism mediating the testicular toxicity induced by CS-003, a triple neurokinin receptor antagonist, was investigated in male dogs. Daily CS-003 administrations showed testicular toxicity, such as a decrease in the sperm number, motility and prostate weight; and an increase in sperm abnormality, accompanying histopathological changes in the testis, epididymis and prostate. A single CS-003 administration suppressed plasma testosterone and LH levels in intact and castrated males. The suppressed LH release was restored by GnRH agonist injection, suggesting that pituitary sensitivity to GnRH is not impaired by CS-003. Treatment with SB223412, a neurokinin 3 receptor antagonist, caused a similar effect to CS-003, such as toxicity in the testis, prostate and epididymis and decreased plasma level of LH and testosterone. In conclusion, CS-003-induced testicular toxicity is caused by the inhibition of neurokinin B/neurokinin 3 receptor signaling probably at the hypothalamic level in male dogs.     

Progress towards the development of non-peptide orally-active gonadotropin-releasing hormone (GnRH) antagonists: therapeutic implications

Gonadotropin-releasing hormone (GnRH) is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) which is produced from a precursor polypeptide in hypothalamic neurons and secreted in a pulsatile fashion to stimulate the secretion of LH and FSH via its interaction with a cognate receptor on gonadotropes. Low doses of the native peptide delivered in a pulsatile manner to mimic that found in the hypothalamic portal vessels restore fertility in hypogonadal patients, and are also effective in treating cryptorchidism and delayed puberty. Administration of high doses of GnRH, or agonist analogues, causes desensitization of the gonadotrope with consequent decline in gonadal gametogenesis and steroid and peptide hormone synthesis. This phenomenon finds extensive therapeutic application in clinical medicine in a wide spectrum of disease (Table 1). In addition, GnRH analogues have promise as new generation male and female contraceptives in conjunction with steroid hormone replacement. GnRH antagonists inhibit the reproductive system through competition with endogenous GnRH for the receptor and, in view of their rapid effects, are being increasingly used for the above mentioned applications. The peptide agonists and antagonists currently available require parenteral administration, typically in the form of long-acting depots. A new generation of non-peptide GnRH antagonists are beginning to emerge which should allow oral administration and, therefore, may provide greater flexibility of dosing, lower costs and increased patient acceptance.     

Alarin stimulates food intake and gonadotrophin release in male rats

BACKGROUND AND PURPOSE

Alarin is a recently discovered member of the galanin peptide family encoded by a splice variant of galanin-like peptide (GALP) mRNA. Galanin and GALP regulate energy homeostasis and reproduction. We therefore investigated the effects of alarin on food intake and gonadotrophin release.

EXPERIMENTAL APPROACH

Alarin was administered into the third cerebral ventricle (i.c.v.) of rats, and food intake or circulating hormone levels were measured. The effect of alarin on the hypothalamo–pituitary–gonadal axis was investigated in vitro using hypothalamic and anterior pituitary explants, and immortalized cell lines. Receptor binding assays were used to determine whether alarin binds to galanin receptors.

KEY RESULTS

The i.c.v. administration of alarin (30 nmol) to ad libitum fed male rats significantly increased acute food intake to 500%, and plasma luteinizing hormone (LH) levels to 170% of responses to saline. In vitro, 100 nM alarin stimulated neuropeptide Y (NPY) and gonadotrophin-releasing hormone (GnRH) release from hypothalamic explants from male rats, and 1000 nM alarin increased GnRH release from GT1-7 cells. In vivo, pretreatment with the GnRH receptor antagonist cetrorelix prevented the increase in plasma LH levels observed following i.c.v. alarin administration. Receptor binding studies confirmed alarin did not bind to any known galanin receptor, or compete with radiolabelled galanin for hypothalamic binding sites.

CONCLUSIONS AND IMPLICATIONS

These results suggest alarin is a novel orexigenic peptide, and that it increases circulating LH levels via hypothalamic GnRH. Further work is required to identify the receptor(s) mediating the biological effects of alarin.     

Effects of psychoactive and nonpsychoactive cannabinoids on the hypothalamic-pituitary axis of the adult male rat

The acute dose-response effects of delta-9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) on gonadotropin and testosterone (T) secretion and on hypothalamic norepinephrine (NE) metabolism were tested in adult male rats. THC and CBN both produced an acute suppression of plasma-luteinizing hormone (LH) and T levels and median eminence NE turnover although a dose-response relationship could not be demonstrated. CBD had no significant effect on any of these parameters and none of these cannabinoids had any effect on plasma follicle-stimulating hormone levels or median eminence LH-releasing hormone (LHRH) content. Except for the highest dose of CBN, none of the in vivo cannabinoid treatments significantly altered in vitro LH secretion although there was a trend towards decreased LH secretion. These results suggest that the decrease in LH secretion in THC- or CBN-treated rats is due to reductions in NE stimulation of LHRH release rather than to changes in LHRH synthesis or pituitary LHRH response.