An inhibitory effects of interleukin-1a on basal gonadotropin release in the ovariectomized rhesus monkey: reversal by a corticotropin-releasing factor antagonist

Interleukin-1 (IL-1), an important component of the immune system, has recently been shown to influence the release of several hormones in the rodent. In this paper, the effectiveness of IL-1a in modulating basal gonadotropin secretion as well as cortisol release in the primate has been investigated. Eight adult ovariectomized rhesus monkeys were given a 30-min intracerebroventricular infusion of physiological saline (n = 5), various doses of IL-1a (17 micrograms n = 5; 8.5 micrograms; n = 3; 4.2 micrograms n = 5; and 2.1 micrograms n = 4) or IL-1a plus a CRF antagonist (n = 5). LH and FSH concentrations were measured at 15-min intervals during the 3-h preinfusion baseline control and the 5-h postinfusion period, while cortisol concentrations were determined at 45-min intervals. While LH concentrations remained unchanged in the monkeys receiving saline only, they decreased significantly after the 30-min IL-1a infusion. By hour 5 after IL-1a administration, mean (+/- SE) hourly areas under the LH curves (expressed as a percentage of preinfusion baseline) were 27.7% +/- 7.3 (17 micrograms IL-1a), 31.9% +/- 8.4 (8.5 micrograms), 33.3% +/- 5.5 (4.2 micrograms), and 39% +/- 4.0 (2.1 micrograms) (P less than 0.05 vs. morning control). FSH concentrations were also significantly decreased after IL-1a, 17 micrograms: by hour 5, they were 67.4% +/- 5.0 of baseline control. While cortisol concentrations decreased thoughout the experiment in the animals receiving saline, they increased with all IL-1a doses: overall mean (+/- SE) postinfusion concentrations were 21.8 +/- 1.1 (saline), 49.5 +/- 2.2 (IL-1a, 17 micrograms), 35.1 +/- 1.9 (8.5 micrograms), 45.7 +/- 1.5 (4.2 micrograms), and 39.5 +/- 1.5 (2.1 micrograms) micrograms/dl (P less than 0.05 IL-1a vs. saline). Concomitant infusion of the CRF antagonist, [D-Phe12, NLE 21,38caMe LEU37] CRF (12-41), (120-360 micrograms), prevented the IL-1a induced LH inhibition. By hour 5, areas under LH curves were 33.5% +/- 1.7 for IL-1a alone and 99.2% +/- 4.2 (NS vs. saline) for IL-1a + CRF antagonist. The CRF antagonist also blocked the ability of IL-1a to increase cortisol secretion: mean cortisol concentrations were 28.6 +/- 1.4 micrograms/dl (NS vs. saline). The results clearly indicate that the cytokine IL-1a inhibits pulsatile LH and FSH secretion in the ovariectomized rhesus monkey and demonstrate that this inhibition is causally related to the activation of CRF by this cytokine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasopressin mediates the interleukin-1 alpha-induced decrease in luteinizing hormone secretion in the ovariectomized rhesus monkey.

Arginine vasopressin (AVP) has previously been shown to participate in the neuroendocrine control of the adrenal axis. In this study we investigated the role of AVP in the mechanisms linking stress and decreased gonadotropin secretion and evaluated the action of an AVP antagonist on interleukin-1 alpha (IL-1 alpha)-induced changes in gonadotropin and cortisol release in the primate. Adult ovariectomized rhesus monkeys were given a 30-min intracerebroventricular infusion of IL-1 alpha (2.1 micrograms/30 min; n = 5) or IL-1 alpha plus an AVP antagonist (240 micrograms/120 min; [deamino-Pen1,O-Me-Tyr2,Arg8] vasopressin; n = 7); the AVP antagonist infusion was started 30 min before IL-1 alpha and continued for 2 h. Controls included intracerebroventricular infusions of physiological saline (n = 5) or AVP antagonist alone (n = 3). LH concentrations were measured at 15-min intervals during a 3-h preinfusion morning baseline control period and a 5-h postinfusion period. Cortisol concentrations were determined at 45-min intervals. Pulsatile LH release remained unchanged after a control saline or AVP antagonist infusion. Overall LH concentrations decreased significantly after IL-1 alpha infusion, from a morning control baseline of 109.9 +/- 8.8 to 53.7 +/- 3.2 ng/ml after the infusion (P less than 0.05). Concomitant infusion of the AVP antagonist prevented the IL-1 alpha-induced LH inhibition (morning control baseline, 144.5 +/- 6.8; postinfusion, 132.3 +/- 5.8; P = NS vs. saline; P less than 0.0001 vs. IL-1 alpha). While cortisol concentrations decreased throughout the experimental period in the animals receiving saline, they increased after IL-1 alpha infusion: mean +/- SE postinfusion cortisol concentrations were 29.6 +/- 1.9 micrograms/dl (saline) vs. 44.0 +/- 1.7 micrograms/dl (IL-1 alpha; P less than 0.0001). Coinfusion of AVP antagonist and IL-1 alpha did not block the IL-induced cortisol increase (46.8 +/- 1.5 micrograms/dl; P less than 0.0001 vs. morning). After the infusion of AVP antagonist alone, cortisol concentrations significantly decreased from a morning control value of 40.2 +/- 1.6 to 34.9 +/- 1.6 micrograms/dl (P less than 0.05). The results confirm our previous demonstration of an inhibitory effect of IL-1 alpha on gonadotropin secretion in the ovariectomized rhesus monkey and indicate for the first time an important inhibitory role for AVP in the control of gonadotropin secretion during stress. The data also suggest that in this species, the adrenocortical response to IL-1 does not require AVP.     

Endogenous opioid peptides modulate the effect of corticotropin-releasing factor on gonadotropin release in the primate

Stress can induce endocrine abnormalities and menstrual dysfunction in the primate. Here, we examine the effects that CRF, the principal neurohormone in control of the hypothalamic-pituitary-adrenal axis, exerts on pulsatile gonadotropin secretion and the role that the endogenous opioid peptides may play in this phenomenon. Ovariectomized rhesus monkeys were given a 5-h continuous iv infusion of physiological saline (2 ml/h), human CRF (100 micrograms/2 ml . h), or hCRF plus the opiate receptor antagonist naloxone (2 mg/2 ml/h; 5 mg in two experiments; n = 7 experiments/group). LH and FSH concentrations were measured at 15-min intervals for a 3-h preinfusion baseline control, during the 5-h infusion, and during a 2-h postinfusion observation period, while cortisol concentrations were measured at frequent intervals during the entire experiment. CRF infusion produced a progressive and significant decrease in both LH and FSH. Mean areas (+/- SE) under the LH and FSH curves during the 5-h CRF infusion, expressed as a percentage of preinfusion baseline, were 59.9 +/- 4.6% and 83.0 +/- 3.1% (+/- SE), respectively (P less than 0.001 and P less than 0.01 vs. saline controls). Large amplitude LH pulses were abolished during the CRF infusion. However, after cessation of CRF infusion, there was a rapid resumption of LH pulsatile release in four of the seven experiments. Addition of naloxone to CRF prevented the CRF-mediated suppression of LH and FSH release. Mean areas for LH and FSH during the 5-h combined infusion were 100.3 +/- 6.6% and 99.6 +/- 4.3% of the preinfusion baseline, respectively (P less than 0.001 and P less than 0.05 vs. CRH alone; NS vs. saline), and pulsatile LH secretion was maintained. Regardless of whether naloxone was administered, CRF increased cortisol levels significantly. Mean cortisol levels at the end of the CRF and CRF plus naloxone infusions were 48.2 +/- 10.4 and 52.9 +/- 7.4 micrograms/dl (+/- SE), respectively, compared to 21.0 +/- 3.0 with saline (P less than 0.05). These results demonstrate that in the ovariectomized rhesus monkey, CRF suppresses the secretion of both LH and FSH, and this effect can be sustained. They also indicate that the CRF inhibitory action on gonadotropin is primarily mediated by endogenous opioid peptides, independent of glucocorticoid levels.     

Interaction between beta-endorphin and alpha-melanocyte-stimulating hormone in the control of prolactin and luteinizing hormone secretion in the primate

The ability of alpha MSH, a POMC-derived peptide, to antagonize the effects of beta-endorphin (beta EP) on PRL and LH secretion was studied in the primate. Seven ovariectomized rhesus monkeys bearing chronic indwelling third ventricular catheters for peptide infusion were used for these studies. Peripheral blood samples for PRL and LH RIA were obtained every 15 min during a 3-h control period when saline was infused into the ventricle, followed by a 5-h period of peptide infusion at a rate of 25 microliters/h. When beta EP was infused at a dose of 5 micrograms/h, plasma PRL rose from a mean baseline of 3.5 +/- 0.7 ng/ml to a peak of 21.3 +/- 2.2 ng/ml. When the same animals were infused with 20 micrograms alpha MSH together with 5 micrograms beta EP, the peak concentration of PRL was reduced to 8.2 +/- 1.7 ng/ml (P less than 0.001). When a higher dose of beta EP (20 micrograms/h) was infused, PRL rose to a peak of 38.2 +/- 1.8 ng/ml. This response was again markedly blunted, and the peak PRL response was reduced to 7.3 +/- 2.2 ng/ml when 20 micrograms beta EP were infused together with 80 micrograms alpha MSH (P less than 0.001). Analysis of the area under the plasma PRL concentration curves demonstrated a significant reduction in area during the 5-h infusion with beta EP plus alpha MSH compared to that during infusion of beta EP alone. The mean area was reduced from 3480 +/- 570 ng min/ml after 5 micrograms beta EP alone to 1030 +/- 200 after 5 micrograms beta EP plus 20 micrograms alpha MSH and from 6230 +/- 990 after 20 micrograms beta EP to 1020 +/- 320 ng min/ml after 20 micrograms beta EP plus 80 micrograms alpha MSH (P less than 0.01). Des-acetyl alpha MSH (80 micrograms) was also effective in reducing the PRL response to 830 +/- 380 ng min/ml (P less than 0.05). The suppression of pulsatile LH release by beta EP was also attenuated by alpha MSH. During the 5-h infusion of beta EP, total LH secretion was reduced to 65.9 +/- 3.8% of that measured during the 3-h saline infusion compared to 87.2 +/- 2.7% after infusion of beta EP plus alpha MSH (P less than 0.001) or 91.7 +/- 4.1% after beta EP plus des-acetyl alpha MSH (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Agouti-related protein stimulates the hypothalamic-pituitary-adrenal (HPA) axis and enhances the HPA response to interleukin-1 in the primate

alpha-MSH antagonizes many of the immune and neuroendocrine effects induced by inflammatory cytokines. Studies have shown that alpha-MSH attenuates the stimulatory effect of IL-1 on the hypothalamic-pituitary-adrenal (HPA) axis and plays a physiological role in limiting the HPA response to IL-1. Recently an alpha-MSH antagonist, agouti-related protein (AGRP), has been identified in the hypothalamus, which stimulates food intake by antagonizing the effects of alpha-MSH at specific melanocortin receptors. It is unknown whether AGRP can also modulate neuroendocrine responses to inflammatory cytokines. We have therefore examined the effects of AGRP on the HPA axis and on prolactin (PRL) at baseline and in response to stimulation by IL-1 beta in nine ovariectomized rhesus monkeys. In the first study, the effects of intracerebroventricular (i.c.v) infusion of 20 microg (n = 6) and 50 micro g (n = 4) of human AGRP (83-132)-NH(2) were compared with icv saline infusion. There was a significant stimulatory effect of 20 microg AGRP on cortisol release over time (P < 0.001). The area under the hormone response curve (AUC) for cortisol increased by 29% after 20 microg AGRP vs. saline; the AUC for ACTH increased by 166% (P = 0.028); the AUC for PRL increased by 108% (P = 0.046). There was a significant stimulatory effect of 50 microg AGRP on ACTH (P < 0.001), cortisol (P < 0.001), and PRL (P < 0.001) release over time. The AUC for ACTH after 50 microg AGRP increased by 98%; the AUC for cortisol increased by 37%; the AUC for PRL increased by 161%. The effects of AGRP on ACTH, cortisol, and PRL release were prevented by alpha-MSH infusion. In the second study, animals received icv either 50 ng of human IL-1 beta or 20 microg of AGRP followed by 50 ng IL-1 beta. AGRP significantly enhanced the ACTH (P < 0.05) response to IL-1 beta. The peak ACTH response to IL-1 beta alone was 124 +/- 55 pg/ml vs. 430 +/- 198 pg/ml after IL-1 beta plus AGRP; the peak cortisol response was 70 +/- 8.2 microg/dl vs. 77 +/- 6.2 microg/dl, but this was not significantly different. In conclusion, AGRP stimulated ACTH, cortisol, and PRL release in the monkey and enhanced the ACTH response to IL-1 beta. These studies suggest that, in addition to its known orexigenic effects, AGRP may play a role in neuroendocrine regulation and specifically that AGRP may interact with alpha-MSH to modulate neuroendocrine responses to inflammation.     

Unexpected inhibitory action of N-methyl-D,L-aspartate or luteinizing hormone release in adult ovariectomized rhesus monkeys: a role of the hypothalamic-adrenal axis

N-methyl-D,L,-aspartate (NMA), an analog of the excitatory neurotransmitter aspartate, has been previously shown to acutely stimulate LH release in the rodent and primate. In this study, we examine the effect of NMA on LH secretion in the long term ovariectomized adult rhesus monkey. After a 3-h control period, three successive iv bolus injections of NMA (10 or 45 mg) were administered at hourly intervals, and LH and cortisol responses were compared with those after iv administration of physiological saline. LH concentrations remained unchanged throughout the saline infusion (n = 6) and during the 10-mg NMA injection regimen (n = 5). Unexpectedly, LH decreased during NMA injections at a dose of 45 mg (n = 10): areas under the LH curve, expressed as percentage of baseline control, were: hour 1, -16.0% (+/- 2.7 SE); hour 2, -28.4 (+/- 3.2 SE); hour 3; -30.9 (+/- 3.2 SE), P less than 0.005 vs. saline or 10 mg NMA. This inhibitory effect of NMA was prevented by the coadministration of GnRH (3 micrograms) (n = 5), suggesting that NMA acts at a suprapituitary level. Cortisol secretion was significantly increased by 45 mg of NMA; Total areas under the cortisol curve, expressed as percentage of baseline control, were: saline, -24.2% (+/- 4.2 SE); NMA (10 mg), -24.2 (+/- 2.0); NMA (45 mg), +22.2 (+/- 6.2); P less than 0.001 vs. NMA (10 mg) and saline, suggesting that NMA at the higher dose may activate the adrenal axis. To examine a possible role of the adrenal axis on NMA-induced LH inhibition, we next examined the effects of intraventricular administration of antiserum to CRF. Pretreatment with CRF antiserum prevented the decrease in LH levels seen during NMA (45 mg) in 4 of 8 monkeys (hour 2, -8.5% (+/- 6.5); hour 3, -10.3% (+/- 4.3); P less than 0.01 vs. NMA). The NMA-induced cortisol increase was prevented in the antiserum responsive but not in the nonresponsive animals. A similar preventive action on LH was seen after administration of the endogenous opiate receptor antagonist naloxone (2 or 5 mg/h), most notably in caged animals (n = 4: hour 1, 6.2% (+/- 3.8); hour 2, -2.8 (+/- 4.0); hour 3, -9.9 (+/- 5.0); P less than 0.005 vs. NMA, 45 mg, for hour 1 and hour 2). We conclude that the unexpected inhibitory effects of NMA on LH secretion in the adult ovariectomized monkey are the result of the activation of the hypothalamic-pituitary-adrenal axis by NMA and specifically of the release of CRF and endogenous opioid peptides.     

The interaction of gonadotropin-releasing hormone and estradiol on luteinizing hormone and prolactin gene expression in female hypogonadal (hpg) mice

We have investigated the interaction of estrogen with GnRH on the regulation of LH subunit mRNA in female hypogonadal (hpg) mice receiving constant frequency and amplitude pulsatile GnRH treatment for up to 18 days. The level of cytosolic common alpha mRNA in female hpg mouse pituitaries was 45 +/- 6% of normal female littermate values, and treatment with pulsatile GnRH increased alpha mRNA to 40% above the normal value at 24 h and 2-4 times normal at 7 and 12 days (P less than 0.001); by 18 days levels had returned to those of untreated hpg controls. Concurrent treatment with estradiol (E2) did not affect those changes. However, in ovariectomized hpg mice the 2- to 4-fold rise in alpha mRNA was sustained for 18 days with GnRH treatment. E2 treatment alone for 7 and 12 days doubled alpha mRNA. LH beta mRNA levels in untreated female hpg mice were between 5-10% of normal values. Levels increased significantly (77 +/- 6.4%) 24 h after GnRH treatment and were normal at 7, 12, and 18 days. E2 together with GnRH did not affect the LH beta mRNA increase at 12 days, but reduced it to 45% of normal at 18 days. Ovariectomy did not alter the LH beta mRNA response to GnRH treatment, and E2 treatment alone did not increase LH beta mRNA. Serum LH concentrations were normalized by GnRH treatment at all times and did not increase in ovariectomized animals. LH release was prevented when E2 was combined with GnRH. Pituitary LH content in hpg mice was 20% of normal and increased gradually with GnRH treatment. Neither concurrent treatment with E2 nor ovariectomy affected the GnRH-induced synthesis of LH. PRL mRNA levels were 30-40% of normal littermate values in untreated female hpg mice, and pulsatile GnRH increased these to 70-80% of normal. E2 alone raised PRL mRNA slightly above normal values, although together with GnRH this rise was attenuated by about 40%. Pulsatile GnRH treatment of ovariectomized hpg mice did not increase PRL mRNA. E2 increased pituitary PRL content, and GnRH did not attenuate this aspect of E2 action. Serum PRL levels rose with E2 treatment at 7 and 12 days, and concurrent GnRH treatment prevented the rise at 12 days. We conclude the following: 1) The stimulatory action of pulsatile GnRH on the expression of both common alpha and LH beta mRNA is rapid (less than 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Corticotropin-releasing factor and gonadotropin-releasing hormone pulse generator activity in the rhesus monkey. Electrophysiological studies

The effect of corticotropin-releasing factor (CRF) on the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, the central neuronal system governing pulsatile pituitary luteinizing hormone (LH) secretion, was studied electro-physiologically in 6 ovariectomized rhesus monkeys bearing bilateral arrays of recording electrodes implanted in the mediobasal hypothalamus. 'Volleys' of increased multiunit activity (MUA) were recorded for 6-10 h in animals placed in primate chairs. The circulating concentrations of LH and cortisol were determined by radioimmunoassay in blood samples taken every 10 min for 3-4 h prior to the administration of CRF (200 micrograms, i.v.) and for 3-6 h thereafter. CRF resulted in a significant decrease in the frequency of pulse generator activity in 4 of 6 animals, a significant decrease in the duration of MUA volleys and a rise in circulating cortisol levels in all 6 monkeys. Treatment with metyrapone (30 mg/kg, i.m.), an inhibitor of adrenal steroidogenesis that prevented the CRF-induced rise in serum cortisol levels, did not reverse the inhibitory effects of CRF on the frequency or duration of MUA volleys. The opiate antagonist naloxone (0.8 mg/kg, i.v., 10 min prior to CRF followed by 0.8 mg/kg/h infusion) blocked the effects of CRF on MUA volley frequency in 2 of 3 animals, but failed to block the effect of CRF on MUA volley duration, suggesting that endogenous opioids may mediate the action of CRF on pulse generator frequency but not on duration.     

Inhibition of endogenous catecholamine synthesis augments early follicular phase luteinizing hormone secretion.

The physiological role of catecholamines, particularly dopamine and norepinephrine, in the regulation of gonadotropin secretion in humans is unclear. We administered the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine (AMPT, 500 mg at 800 and 1000 h) to five women in the early follicular phase of the menstrual cycle and compared LH secretion patterns to those in five untreated controls. Commencing at 800 h, blood was drawn every 15 min for LH and PRL measurements until 1600 h. AMPT elevated PRL concentrations (mean +/- SEM) from a baseline of 14.72 +/- 2.51 micrograms/L to a peak of 102.2 +/- 24 micrograms/L. LH concentrations [21.97 +/- 0.56 (AMPT) vs. 13.51 +/- 0.16 IU/L (control), P less than 0.0001], LH area under the curve [11014 +/- 1815 (AMPT) vs. 7009 +/- 404 IU.min/L (control), P = 0.05] and LH pulse amplitude [9.99 +/- 2.38 (AMPT) vs. 4.03 +/- 0.61 IU/L (control), P = 0.04] were all greater in the group in which catecholamine synthesis was inhibited. There was no difference in pulse frequency between the groups (7.4 +/- 0.51 vs. 6.6 +/- 0.24 pulses/8 h, P greater than 0.05). We conclude 1) inhibition of endogenous catecholamine synthesis augments LH levels in the early follicular phase, and 2) increased LH secretion during catecholamine synthesis inhibition is due, at least in part, to increased LH pulse amplitude but not increased LH pulse frequency.     

Upregulation of interleukin-1 receptors in mouse AtT-20 pituitary tumor cells following treatment with corticotropin-releasing factor.

Treatment of AtT-20 cell cultures with increasing concentrations of rat/human corticotropin-releasing factor (r/hCRF) for 24 h resulted in a dose-dependent 2-3 fold increase in specific 125I-labelled recombinant human IL-1 alpha (125I-IL-1 alpha) binding that was paralleled by a 70-80% decrease in 125I-Tyro-ovine CRF binding. Saturation analysis of 125I-IL-1 alpha binding in control and CRF-treated cultures indicated that CRF produced an increase in the density (Bmax) of IL-1 receptors without altering their affinity (KD). The CRF-induced upregulation of IL-1 receptors appears to be mediated through specific membrane receptors for CRF since the CRF receptor antagonist, alpha-helical oCRF (9-41), blocked the CRF-induced upregulation of IL-1 receptors without producing any effect on 125I-IL-1 alpha binding by itself. In summary, these data demonstrate complex interactions between CRF and IL-1 at the pituitary level and identify potential novel mechanisms for cytokines to alter neuroendocrine function.     

Plasma adrenocorticotropin, cortisol, and aldosterone responses to corticotropin-releasing factor: modulatory effect of basal cortisol levels

Two hundred micrograms of corticotropin-releasing factor (CRF) were administered as an iv bolus injection to 10 normal subjects (5 men and 5 women). Mean plasma ACTH levels rose significantly (P less than 0.0005, by Friedman's nonparametric analysis of variance) from a basal value of 27 +/- 5 pg/ml (mean +/- SEM) to a peak value of 63 +/- 8 pg/ml 30 min after CRF administration. This ACTH response was followed by a rise in plasma mean cortisol levels (P less than 0.0005, by Friedman's test) from a baseline value of 12.3 +/- 1.4 micrograms/100 ml to a peak value of 21.0 +/- 0.7 micrograms/100 ml 60 min after CRF and a rise in mean plasma aldosterone levels from a basal value of 13 +/- 2 ng/100 ml to a peak value of 23 +/- 2 ng/100 ml. There was no significant difference between men and women in the responsiveness of ACTH, cortisol, and aldosterone to CRF administration. The individual basal cortisol levels were highly significantly and negatively correlated with the areas under the individual ACTH curves (r = -0.76; P less than 0.005, by Pearson's correlation test) and cortisol curves (r = -0.91; P less than 0.001, by Pearson's test). These data suggest a modulatory effect of physiological cortisol levels on the response of the pituitary-adrenal axis to CRF.     

Naloxone increases the frequency of pulsatile luteinizing hormone secretion in women with hyperprolactinemia

The ability to change the frequency and amplitude of pulsatile GnRH secretion may be an important mechanism in maintaining regular ovulatory cycles. Hyperprolactinemia is associated with anovulation and slow frequency LH (GnRH) secretion in women. To assess whether the slow frequency of LH (GnRH) secretion is due to increased opioid activity, we examined the effect of naloxone infusions in eight amenorrheic hyperprolactinemic women (mean +/- SE, serum PRL, 160 +/- 59 micrograms/L). After a baseline period, either saline or naloxone was infused for 8 h on separate days, and LH was measured in blood obtained at 15-min intervals. Additional samples were obtained for plasma FSH, PRL, estradiol, and progesterone. Responses to exogenous GnRH were assessed at the end of the infusions. LH pulse frequency increased in all subjects from a mean of 4.0 +/- 0.5 pulses/10 h (mean +/- SE) during saline infusion to 8.0 +/- 1.0 pulses/10 h during naloxone infusion (P less than 0.01). LH pulse amplitude did not change, and mean plasma LH increased from 7.4 +/- 0.8 IU/L (+/- SE) to 11.2 +/- 1.5 IU/L during naloxone (P less than 0.01). A small but significant increase was seen in mean plasma FSH. Plasma PRL, estradiol, and progesterone were unchanged by naloxone infusion. These data suggest that elevated serum PRL reduces the frequency of LH (GnRH) secretion by increasing hypothalamic opioid activity and suggest that the anovulation in hyperprolactinemia is consequent upon persistent slow frequency LH (GnRH) secretion.     

Operating characteristics of the hypothalamo-pituitary-gonadal axis in men: circadian, ultradian, and pulsatile release of prolactin and its temporal coupling with luteinizing hormone

To investigate the circadian, ultradian, and pulsatile nature of PRL release in the human, we sampled blood at 10-min intervals for 24 h in each of 12 normal young men. The subsequent serum PRL time series were subjected to contemporary techniques of rhythmic and episodic peak detection. Fourier analysis revealed a significant circadian rhythm in serum PRL concentrations in all 12 men. The mean circadian amplitude was 2.1 +/- 0.3 ng/mL (micrograms/L), which accounted for an average of 30 +/- 4% of the 24-h mean PRL concentration. In addition, multiple ultradian PRL rhythms were found with periodicities ranging from 22-242 min. Spectral analysis disclosed ultradian cycles with periodicities of 30-32, 51-59, 90-98, and 234 min. Assessment of episodic PRL pulsatility by Cluster analysis revealed 14 +/- 1 PRL peaks/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 95 +/- 6 min. The average duration of a serum PRL peak was 67 +/- 5 min, and its incremental amplitude was 4.0 +/- 0.3 ng/ml (micrograms/L), which represented a 58 +/- 6% increase above the preceding nadir. Discrete PRL peaks were separated by nonpulsatile valleys, with a mean duration of 27 +/- 1 min. Analysis of the temporal coupling between LH and PRL release by bivariate autoregressive modeling in six men revealed significant cross-correlations between LH and simultaneous PRL concentrations as well as between LH and PRL concentrations that lagged LH by 10 or 20 min. Cross-spectral analysis demonstrated significantly correlated PRL and LH cycles with periodicities of 33-37, 47-52, and 84-106 min. In summary, PRL release in normal young men is characterized by significant circadian and ultradian periodicities, by discrete episodic pulsations that occur approximately every 95 min, and by a close temporal coupling with LH (temporal lag between LH and PRL of 0-20 min).     

Pituitary stimulation by combined administration of four hypothalamic releasing hormones in normal men and patients

Ten normal young men (22-28 yr of age), within 10% of their ideal body weight, were given the four releasing hormones (TRH, 200 micrograms; GnRH, 100 micrograms; ovine corticotropin-releasing hormone, 50 micrograms; GH-releasing hormone, 80 micrograms) iv on separate days and then in combination on the same day. Plasma TSH, PRL, FSH, LH, cortisol, ACTH, and GH were measured by RIA in samples collected from 20 min before to 120 min after injection. There were no significant differences in responses to the separate and combined tests for FSH, LH, cortisol, ACTH, and GH. The plasma TSH (0.001 less than P less than 0.01) and PRL (P less than 0.001) responses were significantly higher after the combined test. The tolerance was identical to that of TRH alone. In eight patients studied after pituitary surgery, combined administration provided results comparable to those obtained after separate administration of TRH, GnRH, and insulin.     

Interactions of galanin and arginine on growth hormone, prolactin, and insulin secretion in man.

Galanin (GAL), a 29 amino acid neuropeptide, is known to increase both basal and growth hormone-releasing hormone (GHRH)-induced growth hormone (GH) secretion while not significantly increasing prolactin (PRL) secretion in man. GAL is also endowed with an inhibiting effect on glucose-stimulated insulin release in animals, but not in man. We studied the effect of GAL (80 pmol/kg/min infused over 60 minutes) on the arginine- (ARG, 30 g infused over 30 minutes) stimulated GH, PRL, insulin, and C-peptide secretion in eight healthy volunteers (age, 20 to 30 years). GAL induced an increase of GH (GAL v saline, area under curve [AUC], mean +/- SEM: 316.5 +/- 73.9 v 93.2 +/- 20.9 micrograms/L/h, P less than .05), but failed to modify both PRL and insulin secretion. GAL enhanced the ARG-induced stimulation of both GH (1,634.1 +/- 293.1 v 566.9 +/- 144.0 micrograms/L/h, P less than .02) and PRL secretion (1,541.9 +/- 248.8 v 1,023.8 +/- 158.7 micrograms/L/h, P less than .02). On the contrary, GAL blunted the ARG-stimulated insulin (816.3 +/- 87.7 v 1,322.7 +/- 240.9 mU/L/h, P less than .05), as well as C-peptide secretion (105.1 +/- 9.8 v 132.8 +/- 17.3 micrograms/L/h, P less than .02). ARG administration induced a transient increase of glucose levels (P less than .01 v baseline) followed by a significant decrease (P less than .05 v baseline). This latter effect was prevented by the coadministration of GAL. In conclusion, these results show that in man GAL potentiates the GH response to ARG, suggesting that these drugs act at the hypothalamic level, at least in part, via different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined pituitary stimulation test: interactions of hypothalamic releasing hormones in man

The use of hypothalamic releasing hormones for the clinical assessment of anterior pituitary function is both simple and free of severe side effects. Tests with the recently discovered substances GRF and CRF as well as with combinations of several releasing hormones are therefore used in many clinics. A reliable interpretation of such combined tests, however, is only possible when positive or negative interactions between these releasing hormones are known. After a rest of 2 h to reach basal cortisol levels, 7 groups of 5 male volunteers each received an iv bolus injection consisting of either: A): GRF (100 micrograms) + CRF (50 micrograms) + TRH (200 micrograms) + LHRH (100 micrograms), B): CRF + TRH, C): GRF + TRH, D): LHRH + TRH, E): TRH, F): GRF, G): CRF. During the following 2 h, GH, TSH, cortisol, LH, FSH and prolactin were measured every 15 min. The TSH response after the injection of all 4 releasing hormones was significantly higher (delta TSH = 16.5 +/- 2.0 microU/ml, x +/- SE) compared to the injection of TRH alone (delta TSH = 9.3 +/- 1.4 microU/ml; p less than 0.025). This increment in TSH secretion was confirmed when 2 groups of 5 female volunteers were studied with the TRH-test (delta TSH = 9.9 +/- 1.8 microU/ml) or the combination of all four releasing hormones (delta TSH = 16.8 +/- 2.9 microU/ml; p less than 0.05). This exaggerated TSH-response to TRH was demonstrated to be entirely due to simultaneous administration of GRF, whereas CRF and LHRH in combination with TRH had no additional effect on TSH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

DIFFERENTIAL EFFECTS OF OVINE AND HUMAN CORTICOTROPHIN-RELEASING FACTOR IN HUMAN SUBJECTS

Ten healthy subjects received 200 micrograms of human CRF (hCRF) and 200 micrograms of ovine CRF (oCRF) as an intravenous bolus injection on two different occasions. After hCRF plasma ACTH levels rose significantly (P less than 0.0005, by Friedman's nonparametric analysis of variance) from a basal value of 35 +/- 3 pg/ml (mean +/- SEM) to a peak value of 80 +/- 7 pg/ml 30 min after hCRF administration. This ACTH response was followed by a rise in plasma cortisol levels (P less than 0.0005, by Friedman's test) from a baseline value of 0.32 +/- 0.03 mumol/l to a peak value of 0.56 +/- 0.02 mumol/l 60 min after hCRF. Ovine CRF elicited similar rises in the plasma ACTH and cortisol levels. However, as derived from the faster rate of decline of ACTH and cortisol after hCRF than after oCRF, human CRF had a significantly shorter duration of action than ovine CRF in humans. Human CRF not only stimulated ACTH release by the human pituitary gland but also prolactin release. After hCRF administration prolactin levels rose significantly (P less than 0.005, by Friedman's test) from a basal value of 179 +/- 18 mU/l to a peak value of 288 +/- 34 mU/l at 10 min.     

Estradiol regulation of proopiomelanocortin gene expression and peptide content in the hypothalamus.

Previous studies have shown that the hypothalamic concentrations of beta-endorphin (beta-EP) and other proopiomelanocortin (POMC)-derived peptides change in the female rat following castration and gonadal steroid replacement. In this study we have measured POMC mRNA by solution hybridization assay in the medial basal hypothalamus (MBH) of ovariectomized rats treated with a regimen of estradiol (E2) that we have previously shown alters brain beta-EP peptide content. In addition the effect of progesterone (P) was also studied. In the first experiment the concentration of beta-EP and alpha-melanocyte-stimulating hormone (alpha-MSH) in the MBH of castrated rats decreased significantly after 3 weeks of E2 treatment compared to castrated unreplaced rats: beta-EP decreased from 6.00 +/- 0.46 to 4.32 +/- 0.38 ng/mg protein and alpha-MSH decreased from 3.00 +/- 0.23 to 2.35 +/- 0.15 ng/mg protein (p less than 0.05). A similar decrease in peptide content was noted in the anterior hypothalamus/preoptic area. A parallel reduction in the concentration of POMC mRNA was measured in the MBH of the E2-replaced animals: 1.17 +/- 0.14 vs. 0.72 +/- 0.08 pg/microgram RNA (p less than 0.02). In a second study castrated rats were studied after 2 weeks of E2 or E2 plus P treatment. After 2 weeks, POMC peptide levels did not change significantly in the MBH of either the E2- or E2 plus P-treated rats. POMC mRNA, however, was significantly reduced from 1.10 +/- 0.10 pg/micrograms RNA in the unreplaced rats to 0.58 +/- 0.05 and 0.61 +/- 0.06 pg/microgram RNA after E2 or E2 plus P, respectively (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of estrogen and progestin on endogenous opioid activity in oophorectomized women

Sex steroids may modulate the secretion of beta-endorphin (beta-EP). Naloxone (Nal), an opioid antagonist, has been used as a probe of central opioid activity. Nal-evoked responses of PRL and LH were evaluated in the midluteal (ML) and late follicular (LF) phases of ovulatory women (Pre) and compared to responses of oophorectomized women before and after the administration of conjugated estrogens (CE) and again after CE and progestin administration. In the ML and LF phases, serum LH increased significantly (P less than 0.05 and P less than 0.01, respectively) during Nal infusion for 4 h, while PRL did not change. In oophorectomized women, there were no significant changes in LH or PRL during Nal infusion. After 3 weeks of CE treatment (1.25 mg daily), LH increased during Nal infusion (P less than 0.05), as did PRL (P less than 0.01). After treatment with CE and medroxyprogesterone acetate (MPA), LH and PRL both increased (P less than 0.05 and P less than 0.01, respectively). The area under the LH curve during Nal infusion after CE and MPA treatment was greater than that after CE alone. Both of these responses were comparable to those of the LF and ML phases of Pre women. During Nal infusion, LH pulse frequency increased in the ML compared to the LF phase of the cycle and, in oophorectomized women, was greater after CE and CE with MPA treatment compared to pretreatment values (P less than 0.05). LH amplitudes during Nal infusion were highest in the ML phase and after CE and MPA treatment in oophorectomized women, and these LH amplitudes were similar. No correlation was found between peripheral plasma beta-EP and Nal-evoked LH responses. No differences were evident in plasma beta-EP levels between Pre and oophorectomized women. In conclusion, 1) endogenous opioid activity is low in oophorectomized women; 2) treatment with estrogen increases opioid activity, and the addition of a progestin increases this activity further; and 3) these data support the contention that sex steroids exert a profound influence on endogenous opioid activity.     

Regulation of pituitary glycoprotein alpha-subunit secretion after administration of a luteinizing hormone-releasing hormone antagonist in normal men.

Pituitary glycoprotein hormones are composed of two different subunits, the alpha- and beta-subunits. The alpha-subunit is common to all FSH, LH, and TSH, while the beta-subunit is specific for each of these hormones. We studied the effects of a potent LHRH antagonist on alpha-subunit and LH secretion in normal men. The LHRH antagonist Nal-Glu, ([Ac-D2Nal1,D4ClPhe2,D3Pal3,Arg5,DGlu6(AA) ,Ala10]LHRH), was given (10 mg daily) as one injection of 5 mg every 12 h. Blood samples were drawn every 24 h, and on days 1 and 7 a day curve was established by drawing hourly blood samples for 26 h. Mean serum alpha-subunit levels decreased progressively (P less than 0.001) from 2.9 +/- 0.49 micrograms/L at baseline to a nadir of 1.4 +/- 0.27 micrograms/L on day 8. In contrast, mean immunoreactive LH (IR-LH) levels decreased rapidly from 3.2 +/- 0.6 U/L at baseline to 0.9 +/- 0.08 U/L on day 2 and remained suppressed (P less than 0.001) throughout the treatment period. On day 1 after the administration of Nal-Glu mean alpha-subunit levels decreased, although not significantly (P = 0.054), from 3.0 +/- 0.6 micrograms/L at baseline to a nadir of 2.0 +/- 0.3 micrograms/L at 17 h. alpha-Subunit remained at this level for the remainder of day 1. On day 7, however, the baseline serum alpha-subunit level was 1.5 +/- 0.3 micrograms/L, significantly suppressed (P less than 0.01) compared to the level on day 1, and no further decrease was seen after administration of Nal-Glu. IR-LH on day 1 before the first injection of 5 mg Nal-Glu was 3.5 +/- 0.8 U/L. Then, IR-LH levels decreased significantly (P less than 0.001) to a nadir of 0.9 +/- 0.1 U/L and remained at this level throughout day 1. IR-LH levels on day 7 were at or below 1.0 U/L throughout the sampling period. These results indicate that alpha-subunit secretion can be partially suppressed after chronic administration of a LHRH antagonist. Furthermore, LH serum levels dissociate from those of pituitary glycoprotein alpha-subunit after administration of LHRH antagonist analogs.