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We previously reported that immunoreactive corticotropin-releasing hormone (CRH) is present in human placenta and third trimester maternal plasma, and that such material is very similar to rat CRH and the predicted structure of human CRH. We suggested that maternal plasma immunoreactive CRH may be of placental origin. To further investigate this possibility, we measured plasma immunoreactive CRH in women during pregnancy, labor, and delivery and 1 and 2 h postpartum, and in nonpregnant women. Umbilical cord plasma and placental CRH concentrations were also measured. In the first trimester of pregnancy, the mean maternal plasma level was 5.9 +/- 1.0 pg (+/- SEM)/ml (n = 24), not significantly different from that in 10 nonpregnant women (5.8 +/- 0.8 pg/ml). Plasma CRH concentrations progressively increased during pregnancy (second trimester, 35.4 +/- 5.9 pg/ml (n = 39); early third trimester (28-34 weeks), 263 +/- 41 pg/ml (n = 14); late third trimester (35-40 weeks), 800 +/- 163 pg/ml (n = 20)]. There was a significant correlation between maternal plasma CRH levels and weeks of pregnancy. Plasma CRH concentrations were further elevated (2215 +/- 329 pg/ml; n = 9). During early labor, peaked at delivery (4409 +/- 591 pg/ml; n = 28), and declined rapidly after delivery [1 h postpartum, 1042 +/- (353 pg/ml (n = 13); 2 h postpartum, 346 +/- 91 pg/ml (n = 13)]. There was a significant correlation (r = 0.562; P less than 0.01) between matched maternal plasma and placental CRH concentrations. The mean umbilical cord plasma CRH level (50.6 +/- 6.1 pg/ml; n = 28) was much lower than that in the mother at the time of delivery. Umbilical venous plasma CRH levels were significantly greater than those in simultaneously obtained umbilical arterial plasma (70.8 +/- 11.3 and 41.8 +/- 4.9 pg/ml, respectively; n = 11). There was a significant correlation (r = 0.384; P less than 0.05) between maternal and fetal CRH concentrations. Gel filtration of plasma obtained from women during the third trimester, at delivery, and early postpartum and placental extracts revealed two major peaks of immunoreactive CRH: a high mol wt peak and one at the elution position of rat CRH. In contrast, only rat CRH-sized material was detected in plasma from nonpregnant women and umbilical cord plasma. Maternal plasma immunoreactive CRH-sized material stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with rat CRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Summary Sertoli cells synthesize and secrete a number of cell-specific products including androgen binding protein (ABP), as well as “serum proteins” such as transferrin. The secretion of these proteins is regulated by extra-testicular hormones such as FSH and insulin; Leydig cell-produced steroids and proopiomelanocortin-derived peptides; and the presence of peritubular myoid cells and/or factors secreted by these cells. Many of the Sertoli cell proteins are secreted in a cyclic fashion during the different stages of the spermatogenic cycle suggesting communication between Sertoli cells and developing germ cells. The availability of quantitative measurements for Sertoli cell-specific proteins such as ABP make it feasible to follow Sertoli cell function in vivo by measuring these products in serum. A bidirectional secretion of proteins by Sertoli cells is proposed to explain the presence of specific peptides in the male reproductive tract and blood.

Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.

An antiserum to βh-lipotropin (LPH) which does not cross react with βh-endorphin has been obtained utilizing two different methods of affinity chromatography. This was employed in studies of three normal human subjects in whom the metabolic clearance rate (MCR) apparent volume of distribution (Vd) and fractional rate of disappearance (Kd) of ACTH and βh-LPH were determined following bolus simultaneous injection of 270 μg highly purified βh-LPH and 230 μg of synthetic human ACTH. A biphasic disappearance curve was noted for both hormones. β h -LPH : MCR-0.571, 0.519, and 0.461 L/minute; Vd-30.7, 27.7, 25.0 liters, representing 49, 46 and 35% of body weight; Kd-0.0186, 0.0187, 0.0185 min−1. ACTH : MCR-0.274, 0.266, and 0.332 L/minute; Vd-6.5, 6.5, 14.5 liters, representing 10.4, 10.8 and 20.4% of body weight; Kd-0.0418, 0.0409, 0.0229 min−1. The observed larger MCR of βh-LPH can account for previous observations of basal plasma ACTH/LPH ratios greater than unity, even though these peptides are present in the pituitary in equimolar concentrations.

Despite variations in plasma and/or pituitary ACTH concentrations, no changes in median eminence or medial basal hypothalamic immunoreactive ACTH-like concentrations were seen in rats studied 6 weeks posthypophysectomy, 12 h or 28 days after bilateral adrenalectomy, 6 days after implantation of corticosterone pellets (75 mg), after dexamethasone administration (300 micrograms/100 g BW, ip, twice daily for 4 days), or after chronic immobilization stress. The lack of concordant variation of brain, pituitary, and plasma ACTH concentrations further supports the suggestion of a nonpituitary, central nervous system origin of some portion of brain immunoreactive ACTH-like activity.

Normal human pituitary cells and human placental cells derived from the trophoblastic shell and adhering decidua incorporate [3H]lysine and [35S]methionine during a 30- min incubation period into high molecular weight glycoproteins containing the antigenic determinants of both ACTH and β- endorphin, demonstrating for the first time actual placental synthesis of such material and synthesis by the human pituitary. Such high molecular weight ACTH/β-endorphin was resolved into at least two molecular species upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, with apparent molecular weights of 34,000 ± 2,600 and 46,000 ± 3,900. These high molecular weight glycoproteins were retained on a Concanavalin-A column and were eluted with excess D-mannose but not with synthetic ACTH or β-endorphin. Partial tryptic mapping of high molecular weight material revealed two fragments physicochemically similar to ACTH-(9-15) and β-endorphin-(l–9) [Mipotropin-(61–69)]. Additional immunoreactive species of lower mol...

Derivatives of proopiomelanocortin (POMC), physicochemically similar to beta-endorphin and desacetyl alpha MSH, have been identified in adult testes, where these peptides were localized to Leydig cells. In the present study, the presence of immunostainable derivatives of POMC was established in fetal, neonatal, and pubertal testes with the unlabeled antibody peroxidase-antiperoxidase method. Specificity of staining was established by absorption of primary antisera with excess antigen. In the mouse, immunoreactive beta-endorphin was detectable in a few primitive interstitial cells on day 14 of gestation, the day after testicular differentiation. Thereafter, the number of immunostainable cells progressively increased throughout fetal life, so that at birth, they comprised 55% of the total interstitial cells. After birth, the number of immunostaining cells declined, so that they were only 12% of interstitial cells by 5 days of age. After 10 days of age, the number of immunopositive cells progressively increased, and by 40 days, interstitial cells showed intense staining comparable to that in adult mice. At 10 days of age, when the number of immunostainable cells was low, hCG treatment increased both the number and staining intensity of beta-endorphin-positive cells to those seen in adult testes. Antibodies directed against gamma MSH, a peptide within the N-terminal segment of POMC, also produced specific staining of fetal and adult interstitial cells in the mouse. In the hamster, the pattern of staining with anti-beta-endorphin in fetal, neonatal, and pubertal interstitial cells was similar to that observed in mice; the number and staining intensity of immunostainable cells increased during fetal life, declined after birth, and rose again at puberty.1) the number and staining intensity of immunostainable interstitial cells have two peaks in mouse and hamster, at birth and after puberty; 2) the number and staining intensity of mouse interstitial cells can be increased by hCG; and 3) the development of immunostainable beta-endorphin activity correlates with the previously reported spontaneous and hCG-induced maturation of morphology and enzyme activities of Leydig cells.

Immunoreactive and bioreactive corticotropin (ACTH-like) activities have been detected in the median eminence and remaining medial basal hypothalamus of both normal and hypophysectomized adult male rats: bioreactive ACTH (pg/100 mug of protein) 1028 in median eminence and 1289 in medial basal hypothalamus; immunoreactive ACTH (midportion ACTH antibody), 1554 in median eminence and 1887 in medial basal hypothalamus. By use of appropriate antibodies and bioassay, it was demonstrated that immunoreactivity was not due solely to alpha-melanotropin, which has previously been reported to be present in the brain of hypophysectomized animals. The Sephadex G-50 gel filtration patterns determined by immunoassay of column eluates obtained from hypothalamic extracts of normal or hypophysectomized animals were similar but were not identical to the pattern derived from whole pituitary. Immunoreactive (midportion ACTH antibody) ACTH concentrations (pg/100 mug of protein) of other central nervous system areas in normal animals were: cerebellum 34.3, cortex 46.3, thalamus 23.8, and hippocampus 116.3. The total amount of bioreactive ACTH present in the median eminence and medial basal hypothalamus is approximately 1% of that present in the pituitary. The present data suggest that such ACTH may have a diencephalic rather than pituitary origin and raise the question of the functional significance of such ACTH.

Since plasma ACTH, beta-lipotropin [beta-LPH-(1-91)], gamma-lipotropin (gamma-LPH: [beta-LPD-(1-58)]), and beta-endorphin (beta-EP: [beta-LPH-(61-91)]) are all derived from a common precursor molecule, their quantification in the same plasma under basal and stimulatory conditions should help to elucidate factors involved in their secretion and regulation. A sequential immune affinity chromatographic procedure was used to separate immunoreactive beta-LPH, gamma-LPH, and beta-EP on individual patient samples. Basal morning plasma concentrations [femtomoles per ml (to convert values to picograms per ml, femtomoles per ml values are multiplied by 10 for beta-LPH, by 5.8 for beta-LPH, by 4.5 for ACTH, and by 3.4 for beta-EP; n = 19; mean +/- SEM)] were: beta-LPH, 6.1 +/- 0.8; gamma-LPH, 4.4 +/- 0.5; and ACTH, 11.1 +/- 1.3. beta-EP was undetectable (1.5 fmol ml-1) in 7 of the 19 basal samples. The mean +/- SEM for the 12 remaining samples was 2.3 +/- 0.2. Insulin-induced hypoglycemia and Pitressin administration were associated with nearly equivalent increments of immunoreactive ACTH and beta-LPH concentrations. The resolving power of the technique was tested by separately applying the immunoreactive beta-LPH, gamma-LPH, and beta-EP fractions obtained from plasma pools to Sephadex G-50 gel filtration for molecular weight estimation. Greater than 88% of all immunoreactive material eluted with a Kav similar to the appropriate standard peptide markers. This immune affinity chromatographic system, therefore, permits simultaneous quantification of these peptides on small plasma volumes more rapidly and with greater resolution than when molecular sieve chromatography is used as an adjunct to RIA.

Adrenocorticotropin and β-lipotropin (β-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-μm sections were immunostained first for either ACTH or β-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and β-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and β-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained β-LPH, but β-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. β-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and β-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.

Peptide and protein hormones usually considered as being of pituitary origin have been detected within the central nervous system by means of radioimmunoassay, bioassay, and immunocytochemical techniques. Intracerebral administration of some of these hormones or fragments thereof elicit behavioral responses, suggesting that they may have a physiological role similar to that described for other peptidergic neurotransmitter or neuromodulator substances. Evidence available for some of these hormones indicates that they are synthesized within the central nervous system and that their regulation may differ from that of their pituitary counterparts.

A sensitive radioimmunoassay for human β-lipotropin (LPH) has been developed utilizing an N-terminal antibody which exhibits no cross-reactivity with β h -MSH and appears to be species specific, with less than 10% crossreactivity with rat, ovine or bovine LPH. 0800-0900 mean plasma LPH concentrations were 47.9±5.7 pg/ml (5 normal subects), 100.5±13.2 pg/ml (Cushing's Disease (CD) n=6), 769.3±390.4 pg/ml (Nelson's Syndrome (NS) n=5). Mean plasma ACTH/plasma LPH ratios were: 1.96±0.13 (normal subjects), 1.69±0.11 (CD) and 1.16±0.07 (NS) Plasma ACTH and LPH rose in parallel in response to insulin-induced hypoglycemia in 4 normal subjects. There was a 375% increase in plasma ACTH concentration, a 474% increase in plasma LPH concentration. Plasma ACTH/LPH ratios in specimens obtained following attainment of peak concentrations were significantly lower than those in either control or peak specimens.

Studies performed on Zucker fa fa rats revealed the absence of circadian periodicity of plasma corticosterone concentrations, food and water intake and activity in the genetically obese rats while such periodicity was present in their lean littermates. Plasma corticosterone levels after 30 minutes immobilization stress did not differ between groups. Immunoreactive (IR) β-endorphin concentrations were significantly higher in brains and neurointermediate lobes of the fa fa rats while there was not a significant difference between fa fa and lean rats in anterior pituitary or plasma concentrations. Ion exchange chromatography revealed that fa fa and lean neurointermediate lobe IR β-endorphin (which elutes with a Kav similar to synthetic camel β-endorphin) was separable into multiple molecular species, one of which corresponded to the authentic molecule; in contrast the majority of anterior pituitary IR β-endorphin corresponded to authentic β-endorphin. The proportions of the various forms of β-endorphin were qualitatively similar in fa fa and lean when comparing their respective profiles in brain and pituitary.

Antisera against proopiomelanocortin (POMC)-derived peptides have previously been employed to demonstrate immunostainable materials in the male reproductive tract and in the corpus luteum of rat ovary. The present study was designed to determine how the distribution of such stainable materials varies in mouse ovary as a function of the reproductive status of the animal. Peptide-like activities were localized with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique in ovaries removed from mice during fetal and neonatal development, during different stages of estrous cycle, and during pregnancy, with antisera against beta-endorphin, gamma 3MSH, and an extended N-terminal portion of POMC (16 K). beta-endorphin-like activity was also quantified in ovarian extracts by RIA. Immunostainable beta-endorphin, gamma 3MSH, and 16 K fragment-like activities were present in ovaries of pregnant and normally cycling (but not immature) mice. Intense staining was found predominantly in the corpora lutea. Less intense staining was observed in the interstitium and in the following parts of large follicles: parietal granulosa, corona radiata, and cumulus oophorus. When neonatal mice were injected with hCG, immunostainable beta-endorphin-like material in the ovarian interstitial area increased. Treatment with PMSG increased staining in both secondary follicles and the interstitium. Immunoassayable beta-endorphin-like activity was twice as high (per g wet wt) at pregnancy as during the cycle. We conclude that peptides similar or identical to POMC and/or its components are present in ovarian cells and that the concentration of such material appears to be regulated by gonadotropins.

alpha-Endorphin, beta-endorphin, gamma-endorphin, and N-terminal ACTH immunoreactivity were detectable in acid extracts of rat testes with concentrations of 0.07 +/- 0.01, 0.18 +/- 0.03, 0.06 +/- 0.01, and 0.33 +/- 0.08 (+/- SD) pmol/g wet wt, respectively. The forms of these immunoreactive peptides were characterized by reverse phase high performance liquid chromatography. Immunoreactive beta-endorphin was also analyzed by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results indicated that the major form of immunoreactive beta-endorphin present appears to be beta-endorphin-(1-31). No alpha-N-acetylated forms of beta-endorphin or beta-lipotropin-sized material were detected. Immunoreactive alpha- and gamma-endorphin appear to be present as alpha-endorphin and des-Tyr1-gamma-endorphin, respectively. Immunoreactive alpha MSH was present as its desacetylated form. No immunoreactive ACTH fractionating with ACTH-(1-39) or its glycosylated forms was detected. This peptide profile is most similar to that seen for proopiomelanocortin-derived peptides in the brain. The low concentrations of these peptides in rat testes suggest a paracrine function.

beta-Endorphin is not detectable in plasma from normal human subjects when measured under baseline conditions or after the subjects have received vasopressin, an agent that elevates beta-lipotropin and adrenocorticotropic hormone (ACTH). Significant amounts of beta-endorphin are present in plasma of patients with endocrine disorders associated with increased ACTH and beta-lipotropin production. Highly purified, natural beta-lipotropin is not peripherally converted to beta-endorphin in vivo in normal subjects.

Immunoreactive (IR) corticotropin-releasing factor (CRF)-like activity was detectable in the majority of plasma samples obtained from women in the third trimester of pregnancy (68.7 +/- 23.6 pg/ml (14.4 +/- 4.9 fmol/ml); mean +/- SE, n = 15), but not in plasma (less than 10 pg/ml) from first (n = 9) or second (n = 11) trimester of pregnancy, 1 day post partum (n = 7), non-pregnant women (n = 10), or in plasma obtained from patients with Cushing's disease (n = 2) or Nelson's syndrome (n = 1), or in basal (n = 6) or ether-stressed (n = 6) rat plasma. Gel filtration of third trimester pooled plasma revealed that the majority of such material eluted with Kav of rat CRF (1-41). The IR CRF (1-41)-sized material eluted with the identical retention time as rat CRF in a reverse phase high performance liquid chromatography (HPLC) system. The detection of IR CRF exclusively in third trimester maternal plasma, together with our previous demonstration that material physicochemically indistinguishable from it is present in human term placental extracts, suggests that the placenta may be the source of plasma IR CRF.

Synthesis and release of pro-opiomelanocortin-derived peptides are under differential regulation in the anterior and intermediate lobes of the pituitary. Glucocorticoids inhibit synthesis of pro-opiomelanocortin-related peptides in the anterior lobe but not in the intermediate lobe. These two lobes are also characterized by differences in neural innervation and blood flow, both of which may represent routes of access for regulatory factors (the intermediate lobe is avascular). Immunoreactive glucocorticoid receptor, which can be demonstrated in many tissues, is absent from the intermediate lobe. Immunocytochemistry was used to demonstrate the presence of immunoreactive glucocorticoid receptor in the intermediate lobe after pituitary stalk transection, neurointermediate lobe grafts to kidney capsule, or monolayer culture of neurointermediate pituitary cells. This appearance of the glucocorticoid receptor is presumably a consequence of removal of intermediate pituitary cells from neural influences that may be responsible for inhibiting their expression under normal conditions in vivo.

The pituitary intermediate lobe of most species is cytologically monotonous, but that of the dog is composed of two immunocytochemically distinct cell types. The predominant A cells are typical pars intermedia cells: they stain immunocytochemically for alpha-melanotropin and, more weakly, for adrenocorticotropin and beta-lipotropin. The B cells are like the corticotrophs of the anterior lobe: they stain intensely for adrenocorticotropin and beta-lipotropin but not for alpha-melanotropin. The B cells may account for the high concentration of bioactive adrenocorticotropin measured in the canine pars intermedia, and may explain why in dogs adenomas causing Cushing's disease through hypersecretion of adrenocorticotropin can arise from the intermediate as well as the anterior pituitary lobe.

Corticotropin-Releasing Factor (CRF) activity was determined (dispersed pituitary cell assay) in rat median eminence (ME), various hypothalamic nuclei, as well as in entire median basal hypothalamus (MBH) and extra-hypothalamic areas. Highest concentrations were seen in ME, with decreased concentrations noted proceeding dorsally and cephalad from ME. Potency (NIAMDD HE-RP-1, ME reference extract, equivalent to 1.0) estimates were: ME-2.2; arcuate n.-0.88; dorsomedial n.-041; ventromedial n.-0.35; periventricular n.-0.24; hypothalamus-0.05; thalamus-0.01; cortex-0.005. Measurable, but lesser amounts, than in the above cited nuclei, were present in supraoptic and paraventricular nuclei. CRF activity was not measurable in preoptic area, septum, olfactory bulb, striatum, mesencephalon, pons, medulla or cerebellum. Complete hypothalamic deafferentation was accompanied by an increase in CRF activity/mug protein in ME and MBH, associated with decreased AM plasma ACTH and corticosterone concentrations. CRF-like activity in ME and MBH increased following hypophysectomy and after dexamethasone pretreatment. These findings indicate that CRF is mainly synthesized in the ME and surrounding area, and this source of CRF is sensitive to feedback effects and that extrahypothalamic inputs affect CRF release. Female animals had higher ME CRF content than did male animals. Homozygous and heterozygous Brattleboro rats had significantly less CRF in ME and MBH than did control animals, with significant differences also noted between homozygous and heterozygous animals.

The distribution of adrenocorticotropin (ACTH) in monkey brain was examined by immunoperoxidase immunohistochemistry. An antiserum to ACTH that recognized the C-terminal portion of the molecule was used. Immunoreactive ACTH was visualized as an intraneuronal constituent with a widespread distribution throughout the brain. Reactive cell bodies were seen only in the region of the arcuate nucleus of the hypothalamus. Dense axonal networks were seen in the hypothalamus, mesencephalic gray, and in the region around the anterior commissure. No staining was seen in the cerebral cortex, cerebellum, hippocampus, or striatum. ACTH or fragments of ACTH may function as neurotransmitters or neuromodulators in primate brain.

Publisher Summary This chapter presents the demonstration of a precursor molecule containing adrenocorticotropic hormone (ACTH) and β -lipotropin in multiple tissues, which has the potential to be processed into products both similar to and different from those elaborated by the anterior and intermediate pituitary lobes. This has raised a number of fundamental questions with regard to its biosynthetic pathways and regulation and to the functions of the peptide products. The chapter discusses the nature and regulation of extrapituitary form of ACTH family of peptides and the forms secreted by human pituitary, both in normal subjects and in diseases characterized by disorders of ACTH regulation. Both ACTH and β -lipotropin can serve as precursors for other biologically active peptides. In humans, ACTH and β -melanotropin ( β -MSH) are secreted concomitantly and occur within the same cell of the anterior pituitary. The distribution of ACTH and β -lipotropin cells is identical. Within the arcuate nucleus, these cells in colchicine-treated rats are distributed throughout its rostrocaudal extent as for α -MSH in the vinblastine-treated rat. The concept of a hypothalamic center distributing ACTH, α -MSH, or β -endorphin via axons to regulate many other brain regions is comparable to the oxytocin and vasopressin fibers in many extra hypothalamic sites that originate in the magnocellular paraventricular nucleus of the hypothalamus. The ACTH- β -lipotropin and vasopressin-oxytocin systems are unique among known brain peptides as their perikarya is restricted to the hypothalamus. The hypothalamus is the center of the ACTH- β -lipotropin brain.

Stress-induced suppression of mitogen-induced lymphocyte proliferation was demonstrated in hypophysectomized rats. Stress effects on the numbers of peripheral blood lymphocytes and lymphocyte subsets and on splenic natural killer cell activity require the presence of pituitary. A pituitary-dependent restraining influence on stress-induced alteration of immunity is described. These results indicate that stress-induced modulation of immunity is complex and includes a range of enhancing and inhibitory mechanisms.

The concentration of epidermal growth factor (EGF) in mouse submaxillary gland displays a circadian variation, which can be phase-shifted by light-dark reversal, and which is abolished by superior cervical ganglionectomy (SCG). Such circadian variation persists in fasted animals. Peak concentrations occur during light hours, trough concentrations during dark, although peak concentrations in female animals occur 8 h in advance of the time when they are seen in male animals. The nocturnal fall can be reversed by a one hour exposure to light presented during the normal dark period. SCG does not abolish the increase in submaxillary gland EGF concentrations to levels characteristic of the normal male that is seen following testosterone administration to female mice (who normally exhibit much lower EGF concentrations). Plasma EGF concentrations do not exhibit a ciradian variation.

Discrete areas of freshly obtained adult bovine brain were assayed for their content of immunoreactive β-lipotropin (β-LPH), ACTH and β-endorphin. Highest concentrations (pg/100ug protein) of β-LPH were present in hypothalamus (517 ± 81), hippocampus (218 ± 60), central grey rostral mesencephalic level, pons, striatum, and spinal cord (163–258). Lesser concentrations (49–138) were present in other parts of the limbic system, brain stem, cortex and thalamus. Immunoreactive ACTH concentrations were highest in hypothalamus (1702 ± 487) and hippocampus (210 ± 40), with markedly lesser concentrations (5–24) being present in all the other aforementioned areas. Immunoreactive β-endorphin concentrations in hypothalamus were 1990 ± 510, in hippocampus 280 ± 50.

Publisher Summary Proopiomelanocortin (POMC)-derived peptides act as paracrine and autocrine regulators of Sertoli and Leydig cells, respectively. Proopiomelanocortin is a precursor molecule that gives rise to multiple peptides that appear to have been used throughout phylogeny for various purposes. In prokaryotes, these peptides may act as chemotactic agents whereas in eukaryotes the same peptides may act by multiple actions. For example, in the pituitary gland, these peptides are secreted as classic hormones; in the central nervous system, they act as neurotransmitters; and in the testis as autocrine and paracrine regulators. The fact that immune-stainable β -endorphin and other POMC-derived peptides in Leydig cells appear to increase during periods of testosterone synthesis in fetal life and again at puberty suggests that the expression of these peptides might be dependent upon gonadotropin secretion. The chapter also explains the effect of human chorionic gonadotropin (hCG) treatment on testicular POMC-like mRNA in hypo-physectomized animals. It has been found that treatment with hCG resulted in an increase in total content of POMC-like mRNA in the testis.

Using antibodies against peptides derived from different portions of the POMC molecule, immunocytochemical evidence suggests that this precursor and/or the peptides present within it are localized in testicular Leydig cells of at least five species. There is no evidence for the localization of these peptides or their precursor in any other cell type in this organ. Examination of testicular extracts by gel filtration, SDS-PAGE, and RP-HPLC indicate that the testis contains low concentrations of POMC-derived peptides relative to brain. Further analysis indicates that POMC is processed to alpha-MSH and beta-endorphin similar to its processing in intermediate pituitary lobe and brain. The relative mobilities of immunoreactive alpha-MSH and beta-endorphin on RP-HPLC columns indicate that they are in the unacetylated state as in brain and in contrast to the acetylated forms in the intermediate pituitary lobe. The potential for Leydig cells to synthesize POMC and its peptides was suggested by the demonstration of POMC-like mRNA in total testis and Leydig cell cultures. The size of the POMC-like mRNA is approximately 150 base pairs shorter than anterior or intermediate pituitary POMC mRNA. POMC-like mRNA activity has also been localized to Leydig cells in sections of testes using in situ hybridization. Immunostainable beta-endorphin and other POMC-derived peptides are present in testicular Leydig cells during fetal life and following puberty at times when testosterone secretion is maximal. The accumulation of immunostainable POMC-derived peptides in Leydig cells is dramatically increased by LH and hCG. A variety of observations suggests that testicular cells can respond to POMC-derived peptides. ACTH and the MSHs stimulate growth and cAMP accumulation in Sertoli cells. By contrast, studies using antagonists suggested that beta-endorphin and/or another testicular opioid inhibit Sertoli cell proliferation and ABP secretion. These observations are consistent with the postulate that different portions of the POMC molecule may have opposite effects on Sertoli cell function and suggest a mechanism by which Leydig cells could modulate Sertoli cell activity. Intratesticular administration of opiate antagonists inhibits testosterone secretion both in vivo and in vitro. These observations suggest that Leydig cell-derived beta-endorphin may facilitate testosterone secretion either directly or indirectly. The finding of POMC and its derivative peptides in testis, ovary, adrenal, and placenta suggests that all steroid hormone-secreting organs in mammals may utilize this peptidergic system.

It is well known that adrenocorticotropin (ACTH) and β-endorphin, the 31-amino-acid C-terminal end of β-lipotropin (β-LPH), are derived from a common precursor (Mains et al., 1977; Nakanishi et al., 1979). Immunoreactive β-endorphin is present in plasma (Suda et al., 1978; Krieger et al., 1979), pituitary (Liotta et al., 1978; Pelletier et al., 1977; Li and Chung, 1976; Li et al., 1976), brain (Krieger et al., 1977a; Rossier et al., 1977), and cerebrospinal fluid (CSF) (Terenius and Wahlstrom, 1975; Shickmanter et al., 1978; Hosobuchi et al., 1979; Akil et al., 1978a). The origin of immunoreactive β-endorphin in brain and CSF is still an arguable point.

Immunostainable β-endorphin and ACTH are present in the cytoplasm of Leydig cells and in the epithelium of the epididymis, seminal vesicle., and yas deferens of rats,. The intensity of immunostaining for β- endorphin is not altered by hypophysectomy, and epididymal staining persists after (Castration, β-endorphin, γ-endorphin, and N- and C-terminal ACTH activities are also detected by xadioimmunoassay of male tissue extracts, and their concentrations do not decrease following hypophysectomy. These peptides therefore appear to be of local rather than pituitary origin and may serve a paracrine function within the reproductive tract.

In the pituitary, adrenocorticotropic hormone (ACTH)- and β-endorphin-related peptides are post-translationally derived from a common precursor glycoprotein1,2. ACTH- and β-endorphin-like peptides have been demonstrated in human placental extracts by radioimmunoassay (RIA), bioassay and opioid radioreceptor assay3–7. Recently, we have shown that short-term cultured human placental trophoblastic cells synthesize a high molecular weight glycoprotein(s) physicochemically similar to the pituitary common precursor molecule; ACTH(9–15)- and β-endorphin(1–9)-like fragments were detected in a tryptic digest of this precursor. Pulse-chase experiments revealed that radiolabel disappeared from the molecule and accumulated in smaller molecular species having either ACTH or β-endorphin antigenic determinants8. We now report the use of re verse-phase HPLC to resolve the tryptic fragments of one component of β-endorphin-like peptide synthesized in placental cells, and show that it is comparable to synthetic human β-endorphin. The two reverse-phase HPLC systems used resolved all tryptic fragments of human β-endorphin (βh-endorphin) with differing selectivities, thus providing two unique ‘fingerprints’, in analogy to conventional two-dimensional peptide analysis. The fact that identical maps were obtained for βh-endorphin and the placental peptide in both systems, provides strong evidence that they are structurally identical.

Immunoassayable (I) and bioassayable (B) ACTH activity is present in extracts of extensively washed human placental tissue and dispersed viable trophoblasts (Tr). Similar concentrations (I) (1.7-2.7 ng/g) are present in term placentas from elective cesarian section or vaginal delivery and from plancentas obtained by prostaglandin-induced abortion at 12, 15 and 18 weeks of gestation. Oral dexamethasone (8-12 mg) administered over an 8-48 h period prior to delivery does not significantly alter placental ACTH content. B-ACTH concentrations are 31-40% of I-ACTH. Sephadex G-50 filtration of term placental homogenates demonstrates two I-ACTH peaks, one eluting in the void volume and the other eluting in the region of synthetic hACTH1-39. When Tr are incubated in tissue culture medium, ACTH content of cells and of medium is significantly greater than pre-incubation levels.

In normal human subjects under basal conditions, we have reported that molar concentrations of immunoreactive β-lipotropin (IR-β-LPH) are approximately threefold greater than those of IR-β-endorphin (β-Ep). Following acute stimulation, there is a further two- to threefold disproportionate rise in plasma concentrations of IR-β-LPH as compared to those of IR-β-Ep. To begin to assess the possible factors involved in such altered IR-β-LPH/IR-β-Ep ratios in plasma, the metabolic clearance rate (MCR), volume of distribution (Vd), fractional rate of disappearance (Kd), and half-life ( t 1 2 ) of these peptides were determined by means of bolus injection of highly purified human β-LPH and synthetic human β-Ep in normal human subjects. β-Ep was found to have an MCR and a Kd greater than that of β-LPH, and a shorter t 1 2 . These differences, however, although they may in part be contributory, cannot solely account for the greater ratio of IR-β-LPH to IR-β-Ep in plasma, or for the disproportionate rise in plasma concentrations of these peptides after acute stimulation.

IMMUNOASSAY and immunocytochemical techniques, in some instances with additional characterisation by bioassay and gel filtration, have demonstrated the presence of ACTH-like1–10, β-lipotropin (LPH)-like6,8,9,11–13 and beta-endorphin-like6,14,15 activity within the central nervous system. However, the source of these peptides in the central nervous system is uncertain3. Hypophysectomy is not associated with any change in hypothalamic content of ACTH1,5,10 or of beta-endorphin-like material14, suggesting a non-pituitary, presumably central nervous system, origin. Immunoreactive perikarya containing ACTH-like8–10,16,17, β-LPH-like8,9,11,13,16,17 and β-endorphin-like15–17 material have thus far only been demonstrated in the arcuate nucleus and adjacent lateral areas of the mediobasal hypothalamus. We considered that if the arcuate nucleus were the source of ACTH-like activity within other areas of the central nervous system, its destruction would result in a decrease of such activity. Neonatal administration of sodium glutamate produces central nervous system lesions, restricted largely to neurones of the arcuate nucleus and to the retina18, while sparing fibres of passage19. We report here that, using neonatal sodium glutamate administration to produce such arcuate nucleus destruction in rats, the content of ACTH and β-endorphin-like material in the hypothalamus and other brain regions is markedly reduced in the absence of any change in whole pituitary, anterior lobe or plasma ACTH concentrations.

Immunoreactive ACTH and beta-lipotropin (beta-LPH) plasma concentrations are elevated in clinically stable chronic renal failure patients on hemodialysis (LPH: patients, 271.8 +/- 35.7 fmol ml-1; normal subjects; 6.6 +/- 0.5; ACTH: patients, 56.4 +/- 15.3; normal subjects, 19.4 +/- 1.7). To begin to study the etiology of such elevated levels, the MCR, apparent volume of distribution, and fractional rate of disappearance of synthetic human ACTH and highly purified human beta-LPH were determined in two clinically stable chronic renal failure patients on hemodialysis, after bolus simultaneous injection of both peptides. Biphasic disappearance curves were obtained for beta-LPH; triphasic for ACTH. The MCR of ACTH was within the range seen in normal subjects, whereas the MCR of beta-LPH was less than one half the normal rate. The data indicate that a decrease in MCR (rather than an increase in pituitary secretory rate) may account for the higher plasma levels of beta-LPH in uremic patients.

In view of current evidence indicating that ACTH and lipotropin (LPH) are synthesized in the form of a common precursor molecule, plasma ACTH and LPH concentrations were measured simultaneously to determine if such concentrations vary concomitantly. Studies were performed in normal subjects under basal conditions and those of pituitary-adrenal stimulation (insulin-induced hypoglycemia and Pitressin administration) and suppression (dexamethasone administration). Patients with pituitary-adrenal disease (Cushing's disease, Nelson's syndrome, and Addison's disease) were studied under basal and circadian conditions and during pituitary suppression. In normal subjects, basal plasma LPH concentrations at 0800-0900 h were 6.6 ± 0.5 fmol/ml; those of ACTH were 19.4 ± 1.7 fmol/ml. Effective hypoglycemia or Pitressin administration resulted in parallel rises in plasma ACTH and LPH concentrations. Plasma ACTH and LPH concentrations at 0900 h declined significantly after midnight dexamethasone administration....

ACTH concentration and the responsiveness of dispersed anterior pituitary cells to hypothalamic extract and to vasopressin were studied in homozygous (DI), heterozygous (HTZ), DI-pitressin treated (DIP) Brattleboro rats, and in control Long-Evans rats. Absolute, but not relative, anterior pituitary weights of HTZ, DI, and DIP animals were significantly smaller than those of controls. The concentration of immunoreactive (I) and bioreactive (B) ACTH in dispersed anterior pituitary cells was greater in DI and DIP than in HTZ or in control animals, although the total amount of ACTH was greater in control than DI or HTZ animals. Media from incubates of pituitary cells derived from DI and DIP animals contained less I and B ACTH than those from HTZ or control animals. Pituitary cells derived from DI animals secreted markedly less ACTH following incubation with hypothalamic extract (NIH-HE-RP-1) than did cells from HTZ animals. The response in DIP animals was intermediate between that of DI and HTZ animals. In contrast, pituitary cells derived from DI and DIP animals were significantly more responsive to vasopressin than those from control or HTZ animals.

An improved method for the bioassay of ACTH has been developed using short-term culture of adrenal cell suspensions derived from intact rats. Six separate pools of adrenals were used and cells derived from the same pool were compared after acute dispersion and overnight culture. The ED50, defined as the concentration of ACTH required to produce half maximal steroidogenesis, for cultured cells was 35.4+/-2 pg/ml compared to 240+/-60 pg/ml for cells used immediately after dispersion. The minimum concentrations of ACTH necessary to produce a response significantly above basal levels were 0.98+/-.10 pg/ml and 12.1+/-3 pg/ml respectively. Response characteristics of the cultured cells were highly reproducible. The incubation volume employed in this study was 0.25 ml, so the ED50 expressed as dose of ACTH per tube was actually 8.8 pg for the cultured cells. Such sensitivity provides requisite methodology for the measurement of ACTH under varying physiological conditions.

To date there has been no specific immunoassay for human lipotropin (LPH). The importance of such an assay is evident with the realization that [β-LPH, not β-MSH, is present in human pituitary and plasma. There is no evidence for the presence of a human s-MSH. This has been shown to be a breakdown product derived by cleavage from β-LPH during in vitro extraction procedures. Previous reports of the measurement of human LPH have relied on indirect evidence obtained from data derived by RIA for ‘βh-MSH’. These assays have utilized antibodies, raised either against ACTH or β-MSH. In some of these reports, but not all, it has been demonstrated that such antibodies partially or completely cross-react with β-LPH or γ-LPH and react with a material in plasma which has an elution pattern on gel filtration identical or closely related to that of the lipotropins. The studies to be reported are based on the development of a homologous RIA for human LPH which has no cross-reactivity with β-MSH. Data will be presented on the measurement of basal and stimulated plasma LPH concentrations in normal subjects, and in patients with abnormalities in ACTH and cortisol secretion, and correlation of these concentrations with those of plasma ACTH.

Enzymatically dispersed hypothalamic cells derived from 15-day-old female rats were maintained in tissue culture for 4 days and then incubated for 2 hr in the presence of [35S]methionine. After such incubation, cell extracts contained multiple forms of 35S-labeled products that were specifically bound by immobilized affinity-purified antisera to corticotropin and beta-endorphin. Sequential use of these immobilized antisera revealed two molecular species (apparent Mr of 33,000 and 36,500) that contained both corticotropin and beta-endorphin antigenic determinants within the same molecule. Substances containing only one of these determinants were also present and co-eluted with corticotropin, alpha-melanotropin, beta-lipotropin, or beta-endorphin upon Sephadex G-50 gel filtration. Extracts of similarly labeled rat anterior and intermediate pituitary lobe cells contained two forms of the common precursor molecule corresponding to the same molecular weights estimated for the hypothalamic material and similar to it with respect to other physicochemical parameters. These data suggest that rat hypothalamus synthesizes corticotropin-related and beta-endorphin-related products via sequential cleavage of a larger common precursor molecule in a manner similar to the processing pathway demonstrated for the intermediate lobe of the pituitary.

beta-Lipotropin is the predominant opioid peptide of the human pituitary and rat pars distalis and is present in concentrations essentially equimolar with corticotropin. When freshly, obtained nonfrozen rat anterior pituitaries were homogenized with 0.2 M HCl, approximately 98% of the immunoreactivity detected utilizing an antiserum that crossreacts equally with beta-lipotropin and beta-endorphin coeluted with 125I-labeled human beta-lipotropin upon molecular sieve chromatography. The remainder of the activity eluted with synthetic human beta-endorphin. Similar results were obtained for human pituitary. HCl homogenization of thawed tissue or homogenization of fresh tissue with acetic acid yielded substantially greater concentrations of beta-endorphin and decreased concentrations of beta-lipotropin. In human subjects, acute anterior pituitary stimulation using either insulin-induced hypoglycemia or vasopressin administration was associated with increased plasma beta-lipotropin and corticotropin levels. At the time of peak concentrations, no significant levels of beta-endorphin were detectable. These data indicate the lack of significant amounts of beta-endorphin in human pituitary. Additionally, there appears to be no specific intrapituitary conversion of beta-lipotropin to beta-endorphin.