Influence of leptin on luteinizing hormone and follicle stimulating hormone secreted from cultured rat anterior pituitary cells☆

BACKGROUND： Leptin may regulate reproductive function via release of hypothalamic neuropeptide Y. However, it is unknown whether this regulatory effect is limited to the hypothalamus. OBJECTIVE： To detect the effect of different dosages of leptin on luteinizing hormone （LH） and follicle stimulating hormone （FSH） secretion from in vitro cultured rat anterior pituitary cells. DESIGN： Contrast study based on cells. SETTING： This study was performed in the Basic Institute of Chengde Medical College, Chengde City, Hebei Province, China from March to June 2007. MATERIALS： Eighteen female Wistar rats of three months of age, weighing 200-220 g, and of clean grade were used. Leptin was provided by Peprotech Company, DMEM culture medium by Invitrogen Company, and the radioimmunological kit by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. METHODS： Three glandular organs were regarded as one group for culture of anterior pituitary cells. In the control group, saline was added to the culture medium instead of leptin. In the leptin group, leptin was prepared into different concentrations of 1×10^-12, 1×10^-11, 1×10^-9, 1×10^-7, and 1×10^-6 mol/L for stimulation of cultured cells. The culture supernatant was obtained at three hours after additional of saline/leptin. MAIN OUTCOME MEASURES： Contents of LH and FSH were detected by radioimmunology. RESULTS： Following leptin stimulation, LH release increased with increasing concentrations of leptin up to 1×10^-9 mol/L, where LH release peaked. LH release then progressively decreased with increasing leptin concentrations （P 〈 0.01）. LH release in the leptin （1×10^-12, 1×10^-11, 1×10^-7, and 1×10^-6 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. FSH content in the leptin （1×10^-11, 1×10^-9, and 1×10^-7 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. CONCLUSION： Leptin can directly affect pituitary tissue to promote the secretion of LH and FSH in a d     

Effect of phenylethanolamine N-methyltransferase inhibition on serum concentrations of luteinizing hormone, follicle stimulating hormone and prolactin in pro-oestrous and ovariectomized rats

In a dose dependent manner, LY 87130, an inhibitor of adrenaline synthesis in the rat brain, either shifts the pro-oestrous surges of luteinizing hormone and follicle stimulating hormone from the late afternoon to the middle of the night or blocks any occurrence of the surges and prevents ovulation. Because the pulsatile release of LH is also blocked by LY 87130, it is proposed that the role of adrenaline in the production of the preovulatory surge is concerned with the regulation of the LH pulses which constitute the surge.     

Modelling the pituitary response to luteinizing hormone-releasing hormone

The pituitary response to luteinizing hormone-releasing hormone (LHRH) is steroid-dependent and varies throughout the reproductive cycle, but the rapid rise in pituitary sensitivity on the day of the ovulation-inducing LH surge is due to a 'self-priming' effect of exposure to LHRH that results in a potentiation of pituitary responsiveness 35-40 min later. The expression of this effect is itself steroid-dependent, and is most marked on pro-oestrus. Here, a model of LHRH-induced LH release was developed to incorporate the changes in pituitary sensitivity observed throughout the reproductive cycle. LH release is based on the Law of Mass Action, and a component related to self-priming is included in the model, incorporating the delay between initial exposure and potentiation of responsiveness and an upper maximum to the achievable level of priming. Where possible, model parameters were obtained from biological values, otherwise they were optimized to fit an experiment performed in vivo. These parameters were then used to test the model against other experimental data obtained both in vivo and in vitro. The model provided a good fit to the in vivo data but the in vitro experimental data required a change in one parameter, the upper limit of priming. We conclude that this model of the pituitary release mechanism can simulate the changes in pituitary responsiveness throughout the reproductive cycle. We suggest that substitution of this model in a previous model of the LHRH pulse generator could allow more appropriate tests of the LHRH pulse generator model.     

Dopaminergic neuronal function, anterior pituitary dopamine content, and serum concentrations of prolactin, luteinizing hormone and progesterone in the aged female rat

The serum concentrations of prolactin (PRL), progesterone and luteinizing hormone (LH), and the content and rate of synthesis of dopamine (DA) in selected brain regions were determined in young (3–6 months), intermediate (13–15 months) and aged (24–25 months) female Long-Evans rats. Young rats were examined on the days of diestrus 2 and estrus. Intermediate rats were divided into 2 groups, a group which was cycling regularly (examined on the day of estrus) and a group which exhibited constant estrus. Aged rats were divided into 3 groups one which cycled regularly (examined on day of estrus), one which exhibited constant estrus, and one which exhibited repetitive pseudopregnancies.Serum PRL was increased in all intermediate and aged rats when compared to values in young animals. Serum LH was increased and progesterone decreased in those intermediate and aged rats which exhibited constant estrous reproductive patterns. The DA content was generally decreased in the median eminence, posterior pituitary, striatum, nucleus accumbens and olfactory tubercle of all aged rats, while the rate of DA synthesis was decreased only in the median eminence of aged, non-cycling rats. This suggests that all DA neuronal systems except those in the tuberoinfundibular system are able to compensate for the age-related loss. Despite the apparent reduction of tuberinfundibular DA neuronal function the concentration of DA in the anterior pituitary, which is believed to represent amine released from the neurons, is dramatically increased in intermediate age rats in constant estrus, and in all groups of aged rats. The maintenance of high PRL secretion despite the elevated content of DA in the anterior pituitary suggests in age-related defect in the dynamics of DA in this gland; this defect may contribute to the loss of reproductive function in the aging rat.     

Short-Term Regulation of Gonadotropin Subunit mRNA Levels by Estrogen: Studies in the Hypothalamo-Pituitary Intact and Hypothalamo-Pituitary Disconnected Ewe

In this study the levels of mRNA for the pituitary gonadotropin hormone subunits luteinizing hormone β (LHβ), follicle stimulating hormone β (FSHβ) and the common α-subunit were assessed during the acute feedback stages of estradiol benzoate (EB) action in ovariectomized (OVX) ewes with and without hypothalamo-pituitary disconnection (HPD). In OVX/HPD ewes maintained on hourly pulses of 250 μg of gonadotropin-releasing hormone (GnRH) a single i.m. injection of EB in oil caused a biphasic (decrease and then increase) change in plasma LH levels and a monophasic decrease in FSH levels. There was a decrease in pituitary α-subunit and FSHβ mRNA levels during the acute negative (8 h post EB) and through the positive feedback (20 h post EB) stages of the response. No significant change was seen in LHβ mRNA levels following treatment with EB. In hypothalamic-pituitary intact OVX ewes the same EB treatment as above caused a biphasic change in LH secretion with the positive feedback component being much greater than in GnRH-pulsed OVX-HPD ewes. The levels of mRNA for all three gonadotropin subunits were reduced by 8 h after EB injection and remained low throughout the positive feedback period. These data suggest that the LH surge in this experimental model does not require an increase in LHB mRNA levels. Furthermore, the fall in LHβ subunit mRNA seen after estrogen injection to OVX ewes is most likely due to an effect of estrogen to decrease GnRH secretion, since pulsatile GnRH replacement prevents this effect. These data also show that estrogen feedback can effect rapid alterations in pituitary gonadotropin subunit mRNA levels. Short-term changes in FSHβ mRNA are reflected in changes in FSH secretion; the same is not true for LH.     

Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells

Ghrelin, a novel growth hormone (GH)-releasing peptide, was recently isolated from the rat stomach as an endogenous ligand to growth hormone secretagogue receptor (GHS-R). Ghrelin specifically stimulates the release of GH from the rat anterior pituitary gland, but the regulational effect of ghrelin on GH secretion has not yet been clarified. We used a perifusion system to examine the single effect and combined effects of ghrelin with growth hormone-releasing hormone (GHRH) and somatostatin on GH secretion from rat anterior pituitary cells. The increase in GH concentration due to ghrelin stimulation showed a transitory peak that was almost the same as that previously reported for GHS, but apparently distinct from that of GHRH. Ghrelin (10(-10) M to 10(-8) M) stimulated GH secretion from the rat anterior pituitary cells in a dose-dependent manner. Serial ghrelin stimulation of the dispersed cells at 1-h intervals decreased the GH response, but the response recovered with stimulation at 3-h intervals, indicating that ghrelin strongly desensitized cells. Costimulation with ghrelin and GHRH elicited neither a synergistic nor an additive GH response from the rat pituitary cells. Furthermore, pretreatment to anterior pituitary cells with somatostatin strongly abolished ghrelin- and/or GHRH-stimulated GH secretion. In this study, we demonstrated that ghrelin caused weaker GH secretion than that caused by GHRH, and we also showed that costimulation with GHRH had no additive or synergistic effect on GH secretion, suggesting that ghrelin indirectly affects coordinated GH release from pituitary gland, as found in vivo.     

卵巢切除后大鼠垂体前叶生长相关蛋白-43免疫反应性及其与促性腺激素细胞的关系

目的：探讨卵巢切除后大鼠垂体前叶生长相关蛋白-43(growth—associated protein43，GAP-43)免疫反应神经纤维的数量变化及其与腺细胞的关系。方法：大鼠经双侧卵巢切除，分别于术后4d和15d取垂体，应用免疫荧光双重标记技术，进行GAP-43和卵泡刺激素(follicle stimulating hormone，FSH)，GAP-43和P物质(substance P，SP)免疫荧光双标，激光共聚焦显微镜观察垂体前叶GAP-43免疫反应神经纤维的数量变化、与促性腺激素细胞及SP免疫反应神经纤维的关系。结果：正常大鼠垂体前叶很少有GAP-43免疫反应神经纤维。卵巢切除后4d垂体前叶出现了数量较多的GAP-43免疫反应神经纤维，术后15d神经纤维明显增多，多走行于腺细胞间，少量神经纤维与FSH细胞密切接触。GAP-43免疫反应神经纤维与SP共存。结论：外周内分泌干预后，垂体前叶的神经纤维可通过芽生增加纤维数量，以对机体内分泌状态改变作出积极的应答。神经纤维可能直接作用于腺细胞，但更大的可能性是通过旁分泌方式实现对腺细胞的调节。     

Studies on a high molecular weight luteinizing hormone release stimulating factor of the ovine pineal gland

Summary Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high M_r (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in shortterm culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226±23 ug LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high M_r forms, slightly heavier than authentic pituitary LH (M_r 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.Supported in part by the Netherlands Foundation for Medical Research (FUNGO) with the financial aid from the Netherlands Organisation for the Advancement of Pure Research (ZWO, grant No 13-35-33).     

Release of immunoreactive luteinizing hormone (LH) from rat hypothalamus-pars tuberalis explants

A luteinizing hormone (LH)-like molecule has been described in the rodent central nervous system and has been shown to have immunologic, chromatographic, and biologic activity similar to that of pituitary LH. In this communication we report that a depolarizing stimulus, high potassium concentration in the presence of calcium, causes in vitro release of LH from hypothalamic explants containing the pars tuberalis. Release from other brain areas containing LH was not seen nor was release of LH, in vitro, significantly provoked from dorsal segments of the hypothalamus, thus suggesting that most, if not all, of the releasable LH from intact hypothalamic explants may come from cells of the pars tuberalis.     

Acute effects of N-methyl-DL-aspartate on the release of pituitary gonadotropins and prolactin in the adult female rhesus monkey

The acute effects of an aspartic acid analogue, N-methyl-dl-aspartate (NMA), upon the release of several anterior pituitary hormones were examined in adult female rhesus monkeys. Intravenous injection of NMA (15 mg/kg body weight) induced a large and rapid rise in plasma levels of luteinizing hormone, follicle stimulating hormone, and prolactin. Thyroid stimulating hormone concentrations were not altered. Thus, in primates, as in rodents, systemically administered NMA is capable of eliciting pituitary hormone secretion, although the site of its action in the rhesus monkey remains to be established.     

Ghrelin-induced growth hormone release from isolated rat anterior pituitary cells depends on intracellullar and extracellular Ca2+ sources

Ghrelin, a novel growth hormone (GH)-releasing peptide, was isolated from the rat stomach as an endogenous ligand to the growth hormone secretagogues receptor. It is known that ghrelin stimulates the release of GH from the rat anterior pituitary gland, but the intracellular signal cascade in somatotrophs has not yet been clarified. In this study, using an isolated cell perifusion system, we examined whether ghrelin- and growth hormone-releasing hormone (GHRH)-induced GH secretion from rat pituitary cells depends on intra- and extracellular Ca(2+) and voltage-gated Ca(2+) channels. For this purpose, we first measured ghrelin- or GHRH-stimulated GH concentration following treatment with reduced extracellular Ca(2+) and/or thapsigargin, an endoplasmic reticulum Ca(2+) ATPase inhibitor. Reductions in the extracellular Ca(2+) concentration to 0.25 mM and to 0 mM resulted in decreases in ghrelin-stimulated GH secretion to 81% and 39% and decreases in GHRH-induced GH secretion to 83% and 13%, respectively, compared to the levels in the case of 2.5 mM Ca(2+) concentration, suggesting that extracellular Ca(2+) is essential for both ghrelin- and GHRH-induced GH secretion. Pretreatment with thapsigargin resulted in a reduction in ghrelin-induced GH secretion to approximately 60% of the control level, but GHRH treatment had not effect on the GH secretion. Moreover, preincubation with thapsigargin and 0 mM extracellular Ca(2+) concentration resulted in significant inhibition of GHRH- and ghrelin-induced GH secretion. Subsequently, to determine whether ghrelin-stimulated GH secretion was induced through voltage-gated Ca(2+) channels, we measured the ghrelin-stimulated GH concentration following treatment with nifedipine, an L-type Ca(2+) channel inhibitor, and found that the amount of GH secretion was reduced to 44% of the control level. Furthermore, by replacement of extracellular Na(2+) in the medium with N-methyl-D(-)-glucamine, an impermeable molecule, GH secretion was reduced to 47%. In this study, we demonstrated that the GH-stimulatory effect of ghrelin, unlike that of GHRH, is achieved through both intracellular and extracellular Ca(2+) sources and that ghrelin-induced extracellular Ca(2+) influx involves an L-type voltage-gated Ca(2+) channel and Na(+) influx.     

Changes in anterior pituitary hormone secretion and hypothalamic catecholamine metabolism during morphine withdrawal in the female rat

Studies were undertaken to evaluate the acute responses of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated morphine (MOR) withdrawal in the rat. Ovariectomized female rats were rendered MOR-dependent and injected with NAL (1 mg/kg b.w., s.c.). During precipitated MOR withdrawal, a decline in norepinephrine (NE) concentrations was preceded by an increase in the level of its metabolite normetanephrine (NME) in the medial basal hypothalamus (MBH) as well as the preoptic area-anterior hypothalamus (POA-AH). Both dopamine (DA) and its major acid metabolite, dihydroxyphenylacetic acid (DOPAC), showed increased concentrations in these two hypothalamic regions within 30 min of NAL administration. Elevated luteinizing hormone (LH) and beta-endorphin secretion was evident within 5 min of NAL injection to MOR-dependent rats, while serum prolactin (PRL) increased 15 min into MOR withdrawal. Both growth hormone (GH) and thyroid-stimulating hormone (TSH) were depressed over the course of MOR withdrawal. Although a cause and effect relationship cannot be established, during NAL-precipitated MOR withdrawal, a heightened hypothalamic monoaminergic neuronal activity is accompanied by a differential response of anterior pituitary hormones. The observed responses, which are similar to those seen during acute stress, indicate that MOR withdrawal may activate the same mechanisms which mediate the neuroendocrine response to stress.     

Evidence from phospholipid metabolism changes for muscarinic cholinergic receptors on rat anterior pituitary cells

In incubations of dissociated adult rat anterior pituitary cells with [32P]orthophosphate, carbamylcholine causes an increase in phosphatidic acid labeling accompanied by a small reduction in phosphatidylinositol labeling. Carbamylcholine and oxotremorine produce about the same maximum change while that caused by pilocarpine is smaller. Low concentrations of atropine, quinuclidinyl benzilate, and scopolamine completely inhibit the effect caused by carbamylcholine, whereas d-tubocurarine does not decrease the stimulation, even at higher concentrations. The muscarinic antagonists alone produce a rise in phosphatidylinositol labeling and a drop in phosphatidic acid labeling, an effect opposite from that produced by the agonists, but d-tubocurarine alone has no effect. Thus changes in phospholipid metabolism are mediated through muscarinic cholinergic receptors in dissociated rat anterior pituitary cells, confirming the presence of functional binding sites. The present studies also demonstrate the utility of experiments on precursor incorporation into phospholipids in identifying the existence on cells or tissues of certain receptor classes.     

Oestrogen regulates neurofilament expression in a subset of anterior pituitary cells of the adult female rat

It is the prevailing view that the neurohypophysis derives from neural crest while the pituitary's anterior lobe is of ectodermal origin. However, it has been recently suggested that anterior pituitary cells could have in part neuro-ectodermal origin, and thus should express specific neuronal markers. This issue was examined previously with conflicting results. The present study attempts to clarify the question of whether or not neuronal markers are expressed in the adenohypophysis. Using quantitative immunofluorescence, we have positively identified a subset of anterior pituitary cells, which express immunoreactivity for neuronal markers, including 68 kDa neurofilament (NF68). Interestingly, we noticed that the expression of NF68 is sexually dimorphic (i.e. neurofilament-positive cells are more abundant in sexually mature female rats). In addition, NF68 expression in female rats increases during ontogenic development and reaches a plateau level after puberty. Thereafter, it displays plastic changes along the oestrous cycle, with the maximum of neurofilament expression at oestrus and the minimum at proestrus. NF68 immunoreactivity was examined after ovariectomy, oestradiol replacement and treatment with an specific oestrogen receptor antagonist. Bilateral ovariectomy induced a significant reduction in the number of NF68-positive cells. This effect was completely prevented by treatment of ovariectomized rats with oestradiol. When intact female rats were treated with the anti-oestrogen tamoxifen, a drastic decrease in NF68 expression in anterior pituitary cells was observed. Furthermore, oestradiol administration in castrated male rats increased NF68 immunoreactivity. Double-immunolabelling experiments provided evidence that pituitary cells expressing neuronal traits correspond to subsets of lactotrophs, somatotrophs, thyrotrophs and gonadotrophs. It remains to be established if NF68 induction in the pituitary is due to direct and/or indirect effects of oestrogens. Also, the possible functional role of this subset of NF68-positive anterior pituitary cells in the female rat remains to be examined.     

Colocalization of NGF and TSH-like immunoreactivity in cultures of adult rat anterior pituitary cells

Nerve growth factor (NGF) has been well-characterized with respect to its role as a trophic agent for various peripheral nervous system (PNS) and central nervous system (CNS) neuronal populations. Recent evidence indicates that NGF may also play a functional role in endocrine systems, although investigations in this field are only beginning to define sites of action and molecular mechanisms involved in NGF endocrine interactions. A potential site for such an interaction to occur is within the pituitary. Previous investigations have demonstrated the presence of NGF and NGF receptors in the pituitary and our group has recently reported the presence of NGF-like immunoreactivity exclusively within the thyrotrophic cells of the anterior pituitary of the adult rat. Since many questions regarding how NGF interacts in the anterior pituitary will be more efficiently addressed using an in vitro system, it was necessary to first determine if cultured adult anterior pituitary cells retain the NGF-like staining and unique association of NGF with thyroid-stimulating hormone-producing cells seen in vivo. Results of the present investigation confirm that cultured anterior pituitary cells retain the characteristics previously observed in vivo and further demonstrate the stability of these cells and their specific NGF and pituitary hormone contents in culture for as long as 6 days. © 1995 Wiley-iss, Inc.     

Pituitary Gonadotrophic Hormone Synthesis,Secretion, Subunit Gene Expression and Cell Structure in Normal and Follicle‐Stimulating Hormone β Knockout,Follicle‐Stimulating Hormone Receptor Knockout,Luteinising Hormone Receptor Knockout,Hypogonadal and Ovariectomised Female Mice

To investigate the relationship between gonadotroph function and ultrastructure, we have compared, in parallel in female mice, the effects of several different mutations that perturb the hypothalamic‐pituitary‐gonadal axis. Specifically, serum and pituitary gonadotrophin concentrations, gonadotrophin gene expression, gonadotroph structure and number were measured. Follicle‐stimulating hormone β knockout (FSHβKO), follicle‐stimulating hormone receptor knockout (FSHRKO), luteinising hormone receptor knockout (LuRKO), hypogonadal (hpg) and ovariectomised mice were compared with control wild‐type or heterozygote female mice. Serum levels of LH were elevated in FSHβKO and FSHRKO compared to heterozygote females, reflecting the likely decreased oestrogen production in KO females, as demonstrated by the threadlike uteri and acyclicity. As expected, there was no detectable FSH in the serum or pituitary and an absence of expression of the FSHβ subunit gene in FSHβKO mice. However, there was a significant increase in expression of the FSHβ and LHβ subunit genes in FSHRKO female mice. The morphology of FSHβKO and FSHRKO gonadotrophs was not significantly different from the control, except that secretory granules in FSHRKO gonadotrophs were larger in diameter. In LuRKO and ovariectomised mice, stimulation of LHβ and FSHβ mRNA, as well as serum protein concentrations, were reflected in subcellular changes in gonadotroph morphology, including more dilated rough endoplasmic reticula and fewer, larger secretory granules. In the gonadotophin‐releasing hormone deficient hpg mouse, gonadotrophin mRNA and protein levels were significantly lower than in control mice and gonadotrophs were correspondingly smaller with less abundant endoplasmic reticula and reduced numbers of secretory granules. In summary, major differences in pituitary content and serum concentrations of the gonadotrophins LH and FSH were found between control and mutant female mice. These changes were associated with changes in expression of the gonadotrophin subunit genes and were reflected in the cellular structure and secretory granule appearance within the gonadotroph cells.     

Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat

Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i. c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 microg/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly lower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 microg of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen. Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.     

Effect of manipulating central catecholamines on puberty and the surge of luteinizing hormone and gonadotropin releasing hormone induced by pregnant mare serum gonadotropin in female rats

We have investigated the effect of manipulating central catecholamines on the timing of puberty (as assessed by vaginal opening) in female rats and the surge of luteinizing hormone (LH) and gonadotropin releasing hormone (GnRH) induced by pregnant mare serum gonadotropin (PMSG) in immature female rats. Manipulation of the catecholamines was carried out with either 6-hydroxydopamine (6-OHDA) administered with or without either desipramine (DMI) or pargyline, orα-methyl-p-tyrosine (α-MPT).The neonatal administration of 6-OHDA delayed puberty, an effect which was potentiated by pretreatment with DMI and was associated with a reduction in the rate of body growth. Catecholamine fluorescence in animals aged 60–65 days that had been treated with DMI followed by 6-OHDA was diminished only in the caudatus-putamen; treatment with 6-OHDA alone resulted in diminished fluorescence in the hypothalamus and in the intermediate but not the external layer of the median eminence. The neonatal administration of α-MPT had no significant effect on either the growth rate or the timing of puberty. Regular oestrous cycles occured after puberty in animals treated with either 6-OHDA or α-MPT.The PMSG-induced LH surge was significantly enhanced by 6-OHDA (administered i.v.) plus DMI, and reduced by 6-OHDA injected into the lateral ventricle (v). The inhibitory effect of 6-OHDA (v) wasreduced by DMI, but in animals given 6-OHDA (i.v.) after pargyline there was a marked reduction in the height of the LH surge. There was a good correlation between the changes in the concentrations of LH in peripheral plasma and the concentrations of GnRH in pituitary stalk plasma in that the PMSG-induced surge of GnRH was significantly increased by 6-OHDA (i.v.) plus DMI and reduced by 6-OHDA (v). In animals treated with 6-OHDA (i.v.) plus DMI catecholamine fluorescence was reduced only in the external layer of the median eminence, while after 6-OHDA (v) plus DMI degeneration was seen in the medial forebrain bundle.These results demonstrate a marked difference between the long-term and acute effects of 6-OHDA on the gonadotropin control system. Neonatal treatment with 6-OHDA plus DMI significantly delays puberty and the rate of body growth, but does not affect cyclical gonadotropin release and has no persistent effect on the hypothalamic catecholaminergic systems. The acute administration of 6-OHDA, depending upon the route of administration and whether it is given after DMI, can either potentiate or inhibit the PMSG-induced surge of GnRH and consequently LH by mechanisms which involve destruction, respectively, of either dopaminergic terminals in the median eminence or catecholaminergic fibres in the dorsal hypothalamus.     

Glucocorticoid effects on the molecular forms of adrenocorticotrophic hormone secreted by cultures of rat anterior pituitary cell monolayers

The effects of glucocorticoids and corticotrophin-releasing factor (CRF) on the release of various molecular forms of adrenocorticotrophic hormone (ACTH) have been investigated in primary cultures of rat anterior pituitary. The rat cells responded to a 30 min challenge with CRF by secreting increased amounts of ACTH, as assessed both by bioassay, using rat adrenocortical cells, and by radioimmunoassay. Inclusion of a synthetic glucocorticoid, such as dexamethasone (DEX), in the incubation for 5 min prior to, and during the CRF challenge, had no effect on the response as measured by radioimmunoassay. Bioassay, however, indicated profound suppression of the response to CRF. This discrepancy between ACTH immuno- and bioactivity was investigated by fractionating the immunoreactive ACTH species using high-performance liquid chromatography. The lower molecular weight (<15kd) forms (ACTH(1-39) , phosphorylated ACTH(1-39) and glycosylated ACTH(1±39) ) were separated from higher molecular weight (>15kd) forms (i.e. ACTH biosynthetic intermediate and proopiomelanocortin) using C-18 Sep-Pak. The lower molecular weight molecules were further resolved into glycosylated and non-glycosylated ACTH, using an acetonitrile gradient high-performance liquid chromatography with trifluoroacetic acid as an ion-pairer. Neither the proportion of low molecular weight forms of ACTH, nor that of glycosylated ACTH(1-39) secreted in response to CRF, were affected by DEX. Further fractionation of non-glycosylated ACTH, also using acetonitrile gradient high-performance liquid chromatography but with heptafluorobutyric acid as the ion-pairer, yielded peaks corresponding to phosphorylated and non-phosphorylated ACTH(1-39) . DEX significantly increased the proportion of phosphorylated ACTH secreted in response to CRF by 18%. An additional effect of DEX was revealed when Sep-Pak extracts were treated with alkaline phosphatase, prior to analysis. After dephosphorylation, it became clear that the peptides released by CRF-stimulated cells were different if DEX was present in the medium. The peptide released in the presence of DEX (ACTH-S) had a slightly, but consistently, different retention time on high-performance liquid chromatography and very little biological activity. Antibody cross-reactivity studies suggested that ACTH-S was modified in the 1-24 region of the peptide. It is concluded that challenge of anterior pituitary cells in primary culture with CRF, following 5 min previous exposure to DEX, results in a molecular change. The consequence of this is that ACTH immunoreactivity is released, but the molecule has reduced biological activity. This may be part of the mechanism by which fast feedback inhibition of ACTH secretion is effected.     

Effect of urocortin on ACTH secretion from rat anterior pituitary in vitro and in vivo: comparison with corticotropin-releasing hormone

Both urocortin (UCN) and corticotropin-releaing hormone (CRH) are known to stimulate secretion of adrenocorticotropic hormone (ACTH) by corticotroph cells via type-1 corticotropin-releasing hormone receptor (CRHR-1). We extensively examined UCN effects on the anterior pituitary (AP), particularly on proopiomelanocortin (POMC) mRNA and CRHR-1 mRNA as well as ACTH secretion in vivo. Moreover, signal transduction with UCN exposure was assessed in AP cell cultures in comparison with transduction following CRH exposure. Intravenously administered of UCN (5 μg/kg) increased ACTH and corticosterone secretion. Similarly, intravenous administration of UCN increased POMC mRNA and decreased CRHR-1 mRNA in the AP. These UCN effects were more potent and long-lasting than those of CRH. The prominent effect of UCN on ACTH secretion in vivo was confirmed in AP cell cultures, where application of UCN stimulated ACTH release approximately 7 times more strongly than CRH. The effect of UCN on ACTH release was enhanced by phorbol esters which activate protein kinase C, but was reduced by the selective cAMP-dependent protein kinase inhibitor, H-89. These results suggest that, as with CRH, UCN stimulates ACTH production and/or release through cAMP-dependent mechanisms, and that protein kinase C-dependent mechanism has a synergistic effect upon UCN-induced ACTH release. The more potent effects of UCN relative to CRH may be attributable to UCN's higher affinity for CRHR-1.