Mammary hypertrophy is not associated with increased estrogen receptors.

Estrogen promotes secondary female sex characteristics, including breast enlargement. Since excessive breast hypertrophy is unrelated to elevated serum estrogen levels, it has been postulated that the enlarged breast is a hypersensitive "target organ." At the cellular level, estrogen crosses the cell membrane, is bound to a cytoplasmic estrogen receptor (ER), and induces the formation of specific anabolic proteins. In breast cancer, this estrogen receptor is regarded as a measure of the sensitivity of the cell to estrogen. To determine if mammary hypertrophy is related to an increase in the number of estrogen receptors, we assayed breast tissue, not fat, from 25 consecutive breast reductions. The median age of patients was 26 years (17 to 77 years), with 752 gm per breast removed on average. Twenty-four percent of the patients were taking estrogens, primarily birth control bills. Cellular estrogen-receptor status was measured by a standardized cytosol extraction radioactive estradiol technique. Estrogen receptors were undetectable (less than 3 fmol/mg cytosol protein) in all patients. We conclude that estrogen receptors alone, and hence estrogen, are not a determinant in mammary hypertrophy. If the enlarged breast is a "target organ," it is by another mechanism.

Amniotic fluid prolactin levels in rhesus isoimmunized patients.

The concentration of prolactin in the amniotic fluid (AFPRL) was measured in 75 samples obtained in the third trimester of 14 isoimmunized women. There was a uniform decline in prolactin levels with advancing gestation in each pregnancy (r = -0.89 to -0.99). The decline in AFPRL was similar in uncomplicated pregnancies. AFPRL levels were not predictive of umbilical cord hemoglobin or bilirubin levels and amniotic fluid lecithin/sphingomyelin ratio.

Dopaminergic regulation of alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin secretion in the fetal lamb.

alpha MSH is present in high concentrations in the intermediate lobe of the fetal pituitary and has been implicated as a regulator of fetal adrenal steroidogenesis and fetal growth. However, there are few data regarding alpha MSH levels in fetal plasma or the control of fetal alpha MSH secretion. We measured alpha MSH immunoactivity in the plasma of chronically catheterized fetal lambs (gestational age, 116-138 days), newborn lambs, and adult sheep both in the baseline state and after dopamine receptor blockade with metoclopramide. The effect of metoclopramide on the release of another proopiomelanocortin-derived peptide, N-acetyl-beta-endorphin (N-acetyl-beta EP), which is synthesized together with alpha MSH in the intermediate lobe, was also studied. Baseline fetal plasma alpha MSH was significantly greater than maternal alpha MSH [35.6 +/- 2.2 (+/- SEM) vs. 10.0 +/- 1.0 pg/ml]. In eight studies in five fetal lambs, alpha MSH rose to a peak level of 121 +/- 23 pg/ml 15 min after metoclopramide administration to the fetus. Simultaneous maternal alpha MSH levels did not change, suggesting that the alpha MSH in fetal plasma was of fetal pituitary origin. Gel filtration of pooled fetal plasma extracts revealed that the alpha MSH immunoactivity eluted in the same position as the alpha MSH standard. Metoclopramide caused the secretion of nearly equimolar amounts of alpha MSH and N-acetyl-beta EP into fetal plasma. In four fetal lambs, basal N-acetyl-beta EP levels of 156 +/- 34 pg/ml rose to 305 +/- 65 pg/ml 15 min after metoclopramide treatment. Metoclopramide also stimulated plasma alpha MSH in newborn and adult sheep. In six newborn lambs, alpha MSH rose from 45.2 +/- 13 to 211 +/- 38 pg/ml 15 min after metoclopramide treatment, whereas in four adult sheep, a basal alpha MSH level of 11.1 +/- 2.2 pg/ml rose to 20.1 +/- 2.7 pg/ml 15 min after metoclopramide. In addition, metoclopramide stimulated fetal and neonatal PRL secretion, but had no effect on plasma vasopressin concentrations or acid-base and blood gas values. These studies indicate that immunoreactive alpha MSH and N-acetyl-beta EP are secreted into ovine fetal plasma and that the secretion of these peptides in the fetus appears to be under tonic dopamine inhibition, as is the case in the adult sheep and newborn lamb.

Effects of betamethasone on maternal plasma corticotropin releasing factor, ACTH and cortisol during pregnancy.

Corticotropin releasing factor immunoactivity (CRFi) has been identified in the plasma of women in the second half of gestation. There are several lines of evidence supporting a placental source for this hormone. Regulation of placental CRFi is poorly understood. In this study, the effect of a long-acting glucorticoid on the release of placental CRFi was investigated. Eleven women in the third trimester of pregnancy had plasma samples measured for CRFi, ACTH and cortisol before and after receiving 12 mg betamethasone. There was a significant decrease in ACTH (p less than 0.05) and cortisol levels (p less than 0.01) but no change in CRFi. It is concluded that the secretion of CRFi by the placenta is not inhibited by the administration of betamethasone while maternal levels of cortisol and ACTH are lowered. These results suggest that the acute regulation of placental CRFi is distinct from the regulation of hypothalamic CRF.

High levels of corticotropin-releasing hormone immunoactivity in maternal and fetal plasma during pregnancy.

Corticotropin-releasing hormone immunoactivity (CRHi) was measured in the plasma of 31 pregnant women and 6 nonpregnant women as well as in the umbilical cord plasma of 40 term fetuses. CRHi was not detectable (less than 44 pg/ml) in the plasma of 6 nonpregnant women or in 6 women in the first trimester of pregnancy. Mean plasma CRHi rose progressively to 58 +/- 18 and 270 +/- 68 pg/ml during the second and third trimesters, respectively, and again became undetectable within 24 h after delivery. Mean CRHi in 40 umbilical cord plasma samples was 136 +/- 16 pg/ml. Gel filtration of both fetal and maternal plasma showed that the majority of the CRHi eluted in the same position as synthetic human CRH. There was no significant correlation between CRHi and either beta-endorphin or ACTH in umbilical cord plasma, suggesting that this CRHi may not be primarily responsible for the release of beta-endorphin and ACTH into fetal plasma at delivery. A close correlation (r = 0.82) was found between simultaneously obtained maternal and umbilical cord plasma CRHi in 10 maternal-fetal pairs, supporting a common source for this peptide in maternal and fetal circulation. A placental source for fetal and maternal CRHi was suggested by the finding of a higher CRHi concentration in the umbilical vein than in the umbilical artery and by the disappearance of this peptide from maternal plasma after delivery. We conclude that a large amount of CRHi is secreted by the placenta into both the maternal and fetal circulation during pregnancy and suggest that this may be an important modulator of the maternal and fetal hypothalamic-pituitary-adrenal axis during gestation.

Regulation of beta-endorphin and ACTH in brain by estradiol.

The effect of estradiol on the brain concentration of immunoreactive beta-endorphin (beta-EP) and C-terminal ACTH (CLIP) was studied in ovariectomized rats. Dopamine, a known inhibitor of pituitary intermediate lobe pro-opiomelanocortin (POMC), was examined as a possible mediator of the estradiol induced changes in brain POMC. Animals were treated for 1 or 3 weeks with either 1) saline; 2) silastic estradiol implants; or 3) estradiol implants plus haloperidol 1 mg/kg/day. After one week of treatment no significant change in hypothalamic beta-EP content was noted in any group compared to the control level of 4.13 +/- .33 (SEM) pmoles although in the neurointermediate lobe beta-EP increased from 566 +/- 72 to 942 +/- 73 pmoles after haloperidol (p less than .005). After 3 weeks, however, hypothalamic beta-EP decreased from 3.96 +/- .28 to 2.74 +/- .19 pmoles (p less than .005) and C-terminal ACTH decreased from 3.78 +/- .33 to 2.82 +/- .18 pmoles (p less than .02) in the estradiol treated rats. This estradiol induced decrease in the hypothalamic content of beta-EP and C-terminal ACTH was not blocked by haloperidol. We conclude that estradiol lowers the hypothalamic content of beta-EP and CLIP and that this effect does not appear to be mediated by dopamine.

Suppression of basal and stress-induced prolactin release and stimulation of luteinizing hormone secretion by alpha-melanocyte-stimulating hormone.

alpha-MSH and beta-endorphin, both synthesized from a common precursor, have opposite behavioral actions. In order to determine if these peptides have opposite effects on pituitary function, basal LH secretion and basal and stress-induced prolactin release were studied in adult male rats after intraventricular injection of alpha-MSH. Each rat also received intraventricular saline in order to serve as its own control. 18 micrograms alpha-MSH stimulated plasma LH from 16.5 +/- 2.5 (SEM) ng/ml to a peak of 27.2 +/- 4.0 and 26.0 +/- 4.9 ng/ml at 5 and 10 min, and suppressed prolactin from 3.5 +/- 0.7 ng/ml to 1.3 +/- 0.1 and 1.2 +/- 0.1 ng/ml at 15 and 30 min. Intraventricular alpha-MSH also significantly blunted the prolactin rise associated with the stress of swimming. 10 and 20 min after the onset of swimming, prolactin levels in rats pretreated with alpha-MSH were significantly diminished: 7.4 +/- 1.5 and 6.5 +/- 2.0 ng/ml vs 23.8 +/- 3.6 and 15.2 +/- 2.8 after normal saline. Similarly, des-acetyl alpha-MSH which is the predominant form of alpha-MSH in the hypothalamus, diminished the stress-induced prolactin rise from 18.4 +/- 5.3 and 11.2 +/- 3.4 ng/ml at 10 and 20 min to 10.0 +/- 2.4 and 5.5 +/- 1.6 ng/ml. We conclude that centrally administered alpha-MSH stimulates LH and suppresses basal and stress-induced prolactin release in male rats. These actions are opposite to those previously shown for beta-endorphin and suggest that alpha-MSH may antagonize the effects of beta-endorphin on pituitary function.

Prolactin response to breast stimulation in lactating women is not mediated by endogenous opioids.

Several reports have shown that the prolactin response to suckling in rats can be blunted by administration of the opiate antagonist naloxone. In order to investigate whether the prolactin response to breast stimulation in women is similarly affected by naloxone, nine healthy lactating women participated in 10 studies. Each woman served as her own control and was studied on two occasions, receiving pretreatment with either saline solution or naloxone. Prolactin was measured in the baseline state and for 60 minutes after the onset of a 20-minute period of nipple stimulation by use of the Egnell mechanical breast pump. Neither baseline nor stimulated prolactin values were different by paired t test. Thus, in contrast to rats, an opioid pathway does not appear to be involved in the prolactin response to suckling in humans.

Adrenocorticotropin immunoactivity in monkey hypophyseal portal blood.

ACTH was measured with both C-terminal and midportion antibodies in monkey hypophyseal portal plasma, and compared to levels in monkey peripheral plasma, medial basal hypothalamus, and anterior pituitary. In nine female monkeys, mean hypophyseal portal blood C-terminal ACTH immunoactivity was 5290 +/- 2010 (SEM) pg/ml, whereas the mean midportion ACTH level was 949 +/- 178 pg/ml. These immunoactivities were not lower in two monkeys that were completely hypophysectomized 30 min before portal blood collection. The ratio of C-terminal to midportion ACTH immunoactivity was 4:1 in two monkey medial basal hypothalami, and 1:1 in four monkey anterior pituitary glands. Gel filtration of hypophyseal portal plasma extract and of medial basal hypothalamus showed that the C-terminal ACTH immunoactivity eluted in the same position as the corticotropin-like-intermediate lobe peptide standard. The similarity of the C-terminal to midportion ACTH ratios in monkey medial basal hypothalamus and portal blood, and the observation that ACTH immunoactivity was not significantly lower in two hypophysectomized monkeys suggests that portal blood C-terminal ACTH immunoactivity is of hypothalamic rather than pituitary origin. We conclude that monkey hypophyseal portal blood contains high levels of a C-terminal fragment of ACTH, which coelutes with corticotropin-like intermediate lobe peptide on gel filtration, and which is secreted from the brain directly into the portal circulation.

Brain beta-endorphin during pregnancy, parturition, and the postpartum period.

Brain beta-endorphin (beta-EP) was measured in the rat during pregnancy, parturition, and the postpartum period. beta-EP increased in the hypothalamus, midbrain, and amygdala during gestation and remained elevated through delivery until 1-2 days postpartum. The concentration of beta-EP increased in the hypothalamus from 31.8 +/- 1.4 (+/- SE) ng/mg protein in nonpregnant controls to 41.4 +/- 1.8 and 39.2 +/- 1.9 during early (8-10 days) and late (18-20 days) pregnancy, respectively, and in the midbrain from 3.20 +/- 0.17 to 5.21 +/- 0.30 and 5.25 +/- 0.64 ng/mg protein (P less than 0.01). In another experiment, the brain content of beta-EP expressed as nanograms per region, increased from 12.6 +/- 0.29 to 14.7 +/- 0.33 in the hypothalamus, from 4.09 +/- 0.44 to 6.03 +/- 0.34 in the midbrain, and from 0.93 +/- 0.11 to 1.32 +/- 0.06 ng in the amygdala at 16-17 days of gestation compared with that in nonpregnant controls (P less than 0.01). When hypothalamic beta-EP was measured 1 week postpartum in lactating and nonlactating rats, a significant decline in the beta-EP concentration of both groups was noted compared with that measured during pregnancy; beta-EP levels were similar in the lactating and nonlactating rats. We conclude that pregnancy and parturition are associated with significant changes in brain beta-EP and suggest that beta-EP of central rather than peripheral origin may mediate changes in pain perception and maternal behavior during pregnancy.

ACTH-beta-endorphin in pregnancy.

Following a discussion of recent findings in the physiology of opioid peptides, this article discusses the actions of beta-endorphin and its related peptides in the pregnant woman and in the fetus.

Effect of chronic naltrexone and methadone administration on brain immunoreactive beta-endorphin in the rat.

The effects of chronic treatment with methadone, a long-acting opiate agonist, and naltrexone, a long-acting opiate antagonist on brain immunoreactive beta-endorphin (IR-beta-EP) concentrations were studied in the rat. Male rats were treated for 30 days with either methadone, 2.5 mg/kg/day; naltrexone 2 mg/kg/day, or saline. In a repeat experiment, rats were treated for 36 days with either methadone 2.5 mg/kg/day; naltrexone 4 mg/kg/day, or saline. Brain regions were homogenized in 0.2 N HCl and assayed for IR-beta-EP by RIA. No change in the IR-beta-EP content of the hypothalamus, thalamus, midbrain, or amygdala was measured in either experiment after methadone treatment. Naltrexone, however, significantly lowered brain IR-beta-EP in both experiments. In the first study hypothalamic IR-beta-EP fell from 189 +/- 17 (SEM) to 132 +/- 7.0 ng/g wet weight of tissue after naltrexone treatment (p less than 0.01). In the second experiment naltrexone lowered IR-beta-EP in the hypothalamus from 23.4 +/- 3.6 to 15.5 +/- 1.2 ng/mg protein (p less than 0.005). Similar decreases in the IR-beta-EP content of the thalamus (from 6.74 +/- 0.59 to 4.59 +/- 0.38 ng/mg protein) and amygdala (from 1.31 +/- 0.08 to 0.90 +/- 0.10) were also measured (p less than 0.01). We conclude that occupancy of opiate receptors by an opiate antagonist reduces brain levels of IR-beta-EP and suggests that chronic opiate receptor blockade may result in a compensatory increase in brain beta-EP release.

Pergolide for the treatment of pituitary tumors secreting prolactin or growth hormone.

We gave pergolide mesylate, a new long-acting ergot derivative with dopaminergic properties, to 47 patients with hypersecretion of prolactin or growth hormone. Single doses produced long-lasting reductions of serum prolactin levels; after 24 hours, the values remained depressed at a mean of 28.8 per cent of the base-line value. Among 41 patients (22 women and 19 men) with hyperprolactinemia who took pergolide for three months or more, prolactin levels fell to normal in 37 and remained slightly elevated in 2. In the two patients in whom the levels fell to only 38 to 52 per cent of base line, treatment was regarded as a failure. The level of growth hormone fell to a mean of 52.8 per cent of base line in patients with acromegaly who were taking 100 micrograms of pergolide per day. Among patients for whom adequate CT scans were available, definite tumor shrinkage occurred in 10 of 13 with macroadenomas and definite or probable shrinkage in 5 of 9 with microadenomas. Menses returned in 76 per cent of treated women and testosterone levels rose in 10 of 14 men. We conclude that pergolide reduces hypersecretion and shrinks most prolactin-secreting macroadenomas. In some patients long-term pergolide therapy may be superior to surgery and x-ray treatment.

Persistence of beta-endorphin in human cerebrospinal fluid after hypophysectomy.

beta-Endorphin immunoactivity was measured in the plasma and cerebrospinal fluid (CSF) of 13 patients with metastatic cancer 1 day before and 5 days after complete transsphenoidal hypophysectomy. Preoperatively, mean beta-endorphin-like immunoactivity in plasma was 18.2 +/- 3.5 pg/ml (SEM) and in CSF 32.3 +/- 6.3 pg/ml. No correlation was noted between the concentration of beta-endorphin in plasma and CSF. Postoperatively, plasma beta-endorphin was undetectable (less than 7 pg/ml) in 12 patients and was low (9.6 pg/ml) in 1 patient. In CSF, however, beta-endorphin was detectable in 10 of the 13 patients postoperatively, with a mean of 14.0 +/- 2.2 pg/ml. Chromatography on Sephadex G-50 of CSF extracts pooled from 3 patients after hypophysectomy showed that the majority of beta-endorphin immunoactivity eluted in the same position as synthetic human beta-endorphin. We conclude that beta-endorphin becomes undetectable in plasma after hypophysectomy in patients receiving exogenous glucocorticoid replacement but remains detectable in significant amounts in CSF. It appears, therefore, that a considerable portion of the beta-endorphin in CSF is of nonpituitary origin, most likely resulting from synthesis and secretion of this peptide by brain directly into the CSF.

Circadian rhythms and levels of beta-endorphin, ACTH, and cortisol during chronic methadone maintenance treatment in humans.

Previous studies have shown alterations of neuroendocrine function in humans receiving any narcotic on short-term basis or short-acting narcotics on a chronic basis. In this study of 16 well-stabilized patients, it was demonstrated that normalization of hypothalamic-pituitary-adrenal axis function, as reflected by normal levels and normal circadian rhythm of levels of beta-endorphin, ACTH and cortisol, is achieved during long-term steady state methadone maintenance treatment of former heroin addicts.

Effect of sex steroids on beta-endorphin in hypophyseal portal blood.

Previous studies in female monkeys have shown that beta-endorphin (beta-EP) of hypothalamic origin is present in high concentrations in the hypophyseal portal blood and declines at the time of menses and after ovariectomy. In this study we have examined the effects of estradiol and progesterone replacement on portal blood beta-EP in ovariectomized monkeys. After acute iv administration of estradiol (2 micrograms), beta-EP did not rise from previously low levels (less than 133 pg/ml) over the ensuing 3 h. After chronic estradiol replacement for 3 weeks, portal beta-EP was detectable in 2 of 4 ovariectomized monkeys, with peak values of 341 and 733 pg/ml, respectively. When progesterone as well as estradiol were replaced chronically, high levels of beta-EP, [1610 +/- 192 (SE) pg/ml] were measured in all 13 portal blood samples collected from 4 ovariectomized monkeys. The majority of the beta-EP immunoactivity in these samples eluted from a Sephadex G-50 column in the same position as synthetic human beta-EP. Cation exchange chromatography showed that the majority of immunoactive beta-EP in portal plasma appeared to be nonacetylated beta-EP (1-31). We conclude that ovarian steroids are necessary for the release of hypothalamic beta-EP into portal blood and suggest that cyclic changes in sex steroids may affect anterior pituitary function in part via a mechanism involving hypothalamic beta-EP.

beta-Endorphin in hypophyseal portal blood: variations throughout the menstrual cycle.

Concentrations of beta-endorphin were measured in the venous effluent of the hypothalamus (hypophyseal portal blood) at various phases of the menstrual cycle and after ovariectomy in rhesus and pigtailed monkeys. In the rhesus, beta-endorphin concentrations were high during the mid- to late follicular phase [737 +/- 256 pg/ml (mean +/- SE)] and the luteal phase (1675 +/- 1108) of the menstrual cycle, but were undetectable (less than 133) at menstruation. Concentrations were also high in pigtailed monkeys during stages of the menstrual cycle other than at menstruation (4870 +/- 1090 pg/ml), but undetectable (less than 133) 4--12 months after ovariectomy. These results indicate that beta-endorphin concentrations in hypophyseal portal blood are related to menstrual cycle events, probably changes in ovarian steroids; this in turn suggests that beta-endorphin may participate in the ovarian feedback regulation of gonadotropin secretion.

Intracellular localization of prolactin receptor and prolactin in the rat ovary by immunocytochemistry.

The localization of the PRL receptor as well as of PRL has been studied by immunoperoxidase techniques in the ovaries of cycling, pregnant, and lactating rats. Specific antisera to the receptor and to the hormone were used. By light microscopy, immunostaining for the PRL receptor coincided with that for the hormone. Staining was found intracellularly in most components of the ovary, except the theca, and was most striking in the luteal cells. Both PRL and its receptor were concentrated heavily in the ovum. Beginning 24-36 h postpartum, there was a change in the pattern of luteal cell staining, with a shift in the intensity of staining products to the periphery of the luteal cell, giving a ring appearance to these cells. The results suggest roles for PRL in ovarian function involving both maintenance of the corpus luteum and maturation of the ovum. This study also demonstrates the intracellular localization of a polypeptide hormone in association with its specific receptor.