ACTH Antiserum Research Articles

An immunocytochemical study of the pituitary gland of the white seabream (Diplodus sargus).

The adenohypophysis of the white seabream (Diplodus sargus) was studied using histochemical and immunocytochemical techniques. The adenohypophysis was composed of rostral pars distalis, proximal pars distalis and pars intermedia. Prolactin (anti-chum salmon prolactin positive) and adrenocorticotropic (anti-human ACTH positive) cells were found in the rostral pars distalis. Prolactin cells were organized into follicles, while ACTH cells were arranged in cords around neurohypophyseal tissue branches that penetrated the rostral pars distalis. In the proximal pars distalis, somatotropic (anti-chum salmon and anti-gilthead seabream growth hormone positive), gonadotropic (anti-chum salmon beta-gonadotrophin II and anti-carp beta-gonadotrophin II positive, but anti-chum salmon beta-gonadotrophin I negative) and thyrotropic (anti-human beta-thyrotropin positive) cells were observed. Growth hormone cells were restricted to the dorsal and ventral part of the proximal pars distalis. They were clustered or surrounded the neurohypophyseal branches. Only one type of gonadotrophin cell was identified and they were clustered or isolated in the proximal pars distalis. Scattered groups of thyrotropin cells were located throughout the proximal pars distalis. In the pars intermedia somatolactin (anti-chum salmon and anti-gilthead seabream somatolactin positive) and melanotropic (anti-alpha-melanotropic hormone positive) cells were localized. In addition, gonadotrophin cells surrounded the pars intermedia or distributed evenly between somatolactin and melanotropic hormone cells. Somatolactin cells were periodic acid-Schiff negative and surrounded the neurohypophyseal branches intermingled with melanotropic cells. These cells were also immunoreactive to anti-human ACTH antiserum.

Leukemia Inhibitory Factor as an Intrapituitary Mediator of ACTH Secretion

In the normal anterior pituitary, intercellular interactions are important for the expression of hormones including adrenocorticotropin (ACTH). Leukemia inhibitory factor (LIF) is secreted by anterior pituitary cells and stimulates both basal and corticotropin-releasing hormone (CRH)-stimulated ACTH secretion in AtT₂₀ cells. To determine the effects of LIF on normal pituitary cells, we measured the effects of LIF and an immunoneutralizing antiserum against LIF on ACTH secretion by cultured sheep anterior pituitary cells. In intact populations of anterior pituitary cells, LIF (10 nM) stimulated ACTH secretion (from 0.30 ± 0.06 to 0.77 ± 0.01 ng/well per 3 h) and antiserum to LIF (by itself) had no effect (0.29 ± 0.05 ng/well per 3 h). In marked contrast, following pharmacological elimination of CRH-target cells, a condition known to disinhibit ACTH secretion, basal ACTH secretion was elevated (0.74 ± 0.13 ng/well per 3 h); LIF produced no further stimulation (0.73 ± 0.22 ng/well per 3 h) but immunoneutralization of LIF significantly reduced secretion to 0.50 ± 0.10 ng/well per 3 h. Medium, conditioned by exposure to CRH-target-depleted cultures of anterior pituitary cells, increased net ACTH secretion (from 0.29 ± 0.03 to 6.54 ± 0.71 ng/well per 3 h), when added as a challenge to naive, cultured anterior pituitary cells. Inclusion of antiserum to LIF significantly attenuated (5.29 ± 0.62 ng/well per 3 h) this response. The presence in and secretion of LIF by normal individual pituitary cells was detected using immunocytochemical methods. Seven to 8% of all cells stained positively for LIF, with 66 ± 11% of those secreting detectable amounts of LIF under unstimulated conditions. LIF colocalized with TSH and LH in pituitary cells. Taken together, these data suggest that LIF can stimulate ACTH secretion by normal anterior pituitary cells, and potentially plays a role as an intrapituitary stimulator of ACTH secretion under certain conditions.

Corticosterone increase inhibits stress-induced gastric erosions in rats.

The role of glucocorticoids released in response to stress in the pathogenesis of stress-induced gastric erosions has been reevaluated. Gastric erosions elicited in male rats by 3-h cold-restraint or water-restraint stresses were studied after acute reduction of corticosterone release or occupation of glucocorticoid receptors by the antagonist RU-38486 during stress. Stress-induced corticosterone production was reduced by creating a lesion on the hypothalamic paraventricular nucleus (PVN) 4 days before stress as well as by pretreatment with a rabbit antiserum to adrenocorticotropin (ACTH) 30 min before stress. RU-38486 (10 mg/kg po) was administered 20 min before and 60 min after the onset of stress. Corticosterone for replacement was injected 15 min before the onset of stress to mimic stress-induced corticosterone response. Plasma corticosterone levels were measured by fluorometry or RIA. Gastric erosions were quantitated by measuring the area of damage. Four days after PVN lesion, stress-induced corticosterone release was decreased and gastric erosions were increased. Injecting corticosterone significantly attenuated the effect of PVN lesion on gastric erosions. The ACTH antiserum inhibited corticosteroid secretion in response to stress and markedly increased gastric erosions. The administration of the glucocorticoid/progesterone antagonist RU-38486 significantly potentiated the formation of stress-induced gastric erosions. These observations support the suggestion that glucocorticoids released during stress have a gastroprotective action rather than an ulcerogenic effect as was generally accepted.

Immunocytochemical localization of adrenocorticotropic hormone-immunoreactive cells of the pars intermedia in Thoroughbreds

Abstract Objective To analyze and characterize adenoma and hyperplasia of the pars intermedia (PI) of Thoroughbred mares. Animals 165 Thoroughbred mares, without clinical signs of hyperadrenocorticism that had been euthanatized or had died, of causes such as sudden death, colic, pneumonia, or trauma, and were necropsied. Five of those horses, 17 to 25 years old, had a large pituitary gland at necropsy. Eight mares, 5 to 15 years old with normal-size pituitary gland, were selected at random for comparison. Procedure A morphologic comparison of the pituitary gland between horses with and without tumors of the PI was conducted by use of immunocytochemistry and morphometry. Results In horses with normal pituitary gland, the PI was supplied by a vast capillary or sinusoidal plexus, which connected that in the pars distalis (PD) with that in the pars nervosa (PN). Cells of the PI stained slightly with ACTH antiserum, but some cells in the border region, which is contiguous to the PD, were strongly ACTH immunoreactive. At necrospy, horses with an enlarged pituitary gland also had adenoma of the pituitary gland involving the PI. Cells of the border region were hypertrophied and stained strongly with ACTH antiserum. The area and number of individual tumor cells of the border region of the PI of horses with adenoma were significantly increased, compared with those in horses with normal pituitary gland. Conclusions Cells of the PI-PD border region may secrete substantial quantities of ACTH, owing to stimulation by corticotropin-releasing factor. Adenoma and hyperplasia of the PI in Thoroughbred mares may be associated with hyperadrenocorticism. (Am J Vet Res 1997;58:920–924)

Immunological identification and localization of yeast aspartic protease 3-like prohormone-processing enzymes in mammalian brain and pituitary.

The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

Adrenocorticotropin causes vasodilatation in the human fetal-placental circulation.

During human pregnancy, ACTH is produced by both the placenta and fetal pituitary. ACTH has been shown to cause vasodilatation in the adrenal cortex in vitro. In this context we have investigated the vasoactive effects of ACTH in the human fetal-placental circulation. Single lobules of term human placentas were bilaterally perfused in vitro with Krebs solution (maternal and fetal, 5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3), and changes in fetal placental arterial perfusion pressure (FAP) were measured. ACTH (40-4000 pmol/L; n = 5) caused a dose-dependent reduction of both KC1 and PGF2alpha-induced increases in FAP in the fetal placental circulation. The reductions were of a similar magnitude in the presence of either constrictor agent. ACTH was 187.4 (95% confidence limits, 162.7-215.9) times more potent than prostacyclin (PGI2; 1.2-1180 nmol/L; n = 6), which is a known vasodilator of the fetal-placental circulation. The threshold concentrations for ACTH and PGI2 were 40 pmol/L and 1.2 nmol/L, respectively. ACTH-induced reductions in PGF2alpha-induced increases in FAP in the fetal placental circulation were not inhibited by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (100 micromol/L; n = 5), the cyclooxygenase inhibitor indomethacin (3 micromol/L; n = 5), or a guanylate cyclase inhibitor LY83583(1 micromol/L; n = 5). The inhibitory effect of ACTH was attenuated by the antagonist, ACTH-(7-38) (240 pmol/L; n = 4), and a polyclonal ACTH antiserum (1:1000 dilution; n = 4). We have demonstrated that ACTH causes a reduction in fetal placental vascular resistance in the human fetal-placental circulation in vitro. The mechanism by which it exerts these effects has not been defined, but neither nitric oxide nor PG-mediated pathways appear to be involved.

Influence of Environmental Salinity on Prolactin and Corticotropic Cells in the Gilthead Sea Bream (Sparus aurata L.)

The influence of the environmental salinity on the prolactin (PRL) and corticotropic (ACTH) cells of the rostral pars distalis of the adenohypophysis of the gilthead sea bream (Sparus aurata) adapted to sea water (SW, 980 mOsm/kg) and brackish water (BW, 200 mOsm/kg) has been studied by immunocytochemical, morphometric, and electron-microscopic techniques. Prolactin (PRL) cells of fish adapted to BW occupied a greater hypophysial volume (about 24% of the total hypophysial volume in BW, 10% in SW) and had larger nuclear areas than those of SW-adapted fish (about 21 μm2 in BW. 12 μm2 in SW). Conversely, immunoreactivity against PRL antiserum was lower (mean optical density 117 in BW, 157 in SW). Characteristic ultrastructural features of PRL cells of BW-adapted fishes included a distended rough endoplasmic reticulum and large granules. Together the volumetric, densitometric, and ultrastructural evidences suggest an activation of synthesis and release of PRL in S. aurata adapted to hypoosmotic environments. ACTH cells occupied similar hypophysial volumes in both SW- and BW-adapted fishes (about 6.5%), but nuclear areas were higher (16 μm2 in BW, 13 μm2 in SW) and immunoreactivity against ACTH antiserum was lower in BW fishes (mean optical density 117 in BW, 139 in Sw). Their ultrastructure suggested cell activation in BW-adapted fishes. Plasma levels of cortisol were eventually threefold greater in BW (about 142 ng/ml) than in SW fish (54 ng/ml). These data suggest activation of ACTH cells of S. aurata adapted to BW.

Release of bioactive ACTH by perifused human placenta at early and late gestation.

This study assessed whether bioactive ACTH is released by the human placenta during perifusion in vitro at early and late gestation. Human placental villous fragments from early (8-12 weeks) and late (38-40 weeks) gestation were perifused at a constant rate for 6.5 h. To assess ACTH-like bioactivity released by this tissue, the perifusion effluent was redirected through adjacent chambers containing freshly dispersed adrenocortical cells obtained from adult rats. Baseline secretion of corticosterone by these adrenocortical cells averaged 95 +/- 26 (S.E.M.) fmol/min, and this increased at least fivefold (P < 0.01, two-way ANOVA) in response to placental effluent at early and late gestation. The magnitude of this increase, expressed as a percentage of the maximal response to a subsequent stimulus with ACTH(1-24), was similar for placentas obtained at early (41 +/- 12% of maximal response) and late (42 +/- 17%) gestation. Immunoreactive (I)-ACTH was readily detectable in placental effluent from all preparations (5.5 +/- 2.3 fmol/min per g tissue), and there was no apparent difference with stage of gestation. To determine whether all of the ACTH-like bioactivity released by the placenta was attributable to I-ACTH, a second series of placental/adrenal perfusions was conducted. In these, I-ACTH was selectively removed from placental effluent by immunoneutralization, and the residual bioactivity measured. Immunoneutralization involved preincubation of placental effluent with ACTH antiserum (1:100), and preincubation with normal rabbit serum (NRS) served as a control.(ABSTRACT TRUNCATED AT 250 WORDS)

Passive immunization with antiserum to adrenocorticotropin increases weight gain in normal female rats.

Passive immunization against ACTH was used to test the hypothesis that growth in female rats is constrained by physiological concentrations of glucocorticoids. When animals were stressed by 15-min exposure to ether before blood sampling by cardiac puncture, serum concentrations of corticosterone were lower (P less than .05) in immunized rats than in stressed controls. The maximum effect was apparent 2 h after injection of ACTH antiserum, and no effect was apparent 6 h after injection. To examine the effects of ACTH immunization on growth, rats received daily injections of either saline, sheep immunoglobulin G, or ACTH antiserum, 2 h before the afternoon peak in plasma concentrations of corticosterone. After 7 d of treatment, rats treated with ACTH antiserum had gained 37% more body weight than saline-injected controls, and this effect was accompanied by a 59% reduction in peak plasma concentrations of corticosterone. Immunoglobulin G purified from normal sheep serum had no effect on weight gain. It is concluded that growth rate in normal female rats can be stimulated through the suppression of adrenal activity.

Brain acth prevents stress gastric lesions in rats

This study evaluated the effect of ACTH and several ACTH fragments on the development of gastric glandular lesions induced by cold-restraint stress in rats. Intracerebroventricular administration of ACTH 1–39 dose-dependently (0.1–10 μg) inhibited stress gastric lesion formation. Studies with smaller molecular weight forms of ACTH (in a dose equimolar to 10 μg of ACTH 1–39) revealed that ACTH 1–13 and ACTH 1–10 were also protective. The ACTH fragments ACTH 5–10, ACTH 34–39 and ACTH 1–17 were without effect. Immunoneutralization of endogenous brain ACTH 1–39 significantly increased stress gastric lesion severity. Antisera raised against synthetic somatostatin, gonadotropin-releasing hormone, and L-enkephalin were ineffective. These results with ACTH coupled with our previous demonstration of a protective effect of β-endorphin suggest that specific brain pro-opiomelanocortin gene products modulate gastric mucosal integrity in response to stress. Brain ACTH ACTH fragments Stress ulcers ACTH antiserum Male rats

Immunocytochemical location of pituitary cells containing ACTH, α-MSH, and β-endorphin in Acipenser transmontanus, Lepisosteus spatula, and Amia calva

This immunocytochemical study of the pituitaries of the primitive actinopterygians, Acipenser transmontanus, Lepisosteus spatula, and Amia calva, showed a strict delineation between the hormonal fragments of proopiomelanocorticotropin (POMC) produced by corticotropes of the pars distalis and the melanotropes of the pars intermedia. Corticotropes were immunoreactive only for ACTH and not to either of the further degradation products, α-MSH or β-endorphin. Melanotropes were shown to be immunoreactive to all three antisera but it is argued that immunoreactivity of melanotropes to ACTH antiserum is due to that antiserum's cross-reactivity with the cleavage product corticotropin-like intermediate peptide. The PAS positivity of both the corticotropes and the melanotropes of all three primitive fish argues for an ancient origin of a carbohydrate component of POMC and for its loss or reduction in teleosts where these cells are PAS negative.

Beta-Endorphin Mediates Clonidine Stimulated Growth Hormone Release

The role of beta-endorphin in the stimulation of growth hormone secretion elicited by administration of clonidine (Clon), an alpha2-adrenergic agonist, was investigated in awake, freely moving male rats. Animals were infused slowly with either 1 ml of normal rabbit serum (NRS), beta-endorphin antiserum (beta-end-AS) or ACTH antiserum (ACTH-AS) 2 h before the administration of Clon (100 microgram/kg body weight, intravenously). In addition, naloxone (Nal) (2.5 mg/kg body weight, intravenously) was given 15 min prior to Clon in some experiments. Blood samples were taken at 15-min intervals prior to and following Clon administration. Clon caused plasma GH levels to rise 15-fold to peak levels of 177 +/- 38 ng/ml (p less than 0.01) at 30 min. Pretreatment of both Nal or beta-end-AS significantly reduced Clon-stimulated GH secretion to 72 +/- 19 ng/ml (p less than 0.05) and 87 +/- 30 ng/ml (p less than 0.05) respectively. In contrast, the infusion of ACTH antiserum did not affect Clon-stimulated GH release. Our data suggest that beta-endorphin or a related opioid peptide is an important mediator of GH secretion induced by alpha2-adrenergic stimulation. Since blockade of opioid receptors blunted Clon-induced GH release only partially (approximately 50%), other mediators are most likely activated following alpha 2-adrenergic stimulation.

Quantitative changes of CRF-like immunoreactivity in eels treated with reserpine and cortisol.

Immunocytochemical techniques were applied to brain and pituitary sections of European eels after experimental manipulation of the pituitary-interrenal activity. A corticotropin-releasing factor (CRF) antiserum allowed the identification of a CRF-like peptide in the preoptic nucleus (PON) and rostral and caudal neurohypophysis (NH). CRF-immunoreactivity (ir) was not affected in solvent-injected eels compared to noninjected eels. Reserpine induced a stimulation of the pituitary interrenal axis, decreased ir-CRF in the rostral NH, but did not affect hypothalamic ir-CRF. Cortisol reduced the immunostaining of hypothalamic CRF-ir perikarya and perikarya cross-sectional area. In the rostral NH, CRF-ir fibers decreased in number and almost disappeared in long-term treated eels. The immunostaining of ACTH cells with ACTH antiserum was greatly reduced. These data suggest that cortisol induces a marked reduction in the activity of the CRF-corticotrop axis. The intensity of the ir-CRF staining observed in the caudal NH, close to the intermediate lobe (IL) was not significantly affected in reserpine-treated eels, and only slightly reduced in long-term cortisol-treated eels. The intensity of ir-CRF staining in the caudal NH did not correlate with melanocorticotropic activity or plasma cortisol level. These data suggest that immunoreactive CRF fibers in the rostral and caudal NH are differently regulated.

Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary.

In order to investigate the role of N-terminal proopiomelanocortin (N-POMC) in adrenal regeneration after bilateral adrenal enucleation (hereafter referred to as enucleation) rats 13 days after enucleation were injected (200 microliters s.c. plus 200 microliters i.p.) at 08.00 and 20.00 h with normal rabbit serum (NRS), an ACTH antiserum or an N-POMC antiserum. On the next day the animals were injected with colchicine, killed and mitotic figures in adrenal histological sections counted. The same treatment was given to rats 20 days after enucleation. Only the N-POMC antiserum significantly diminished adrenal mitotic activity 14 and 21 days after enucleation (P less than 0.01 and 0.05 respectively) when compared with NRS-treated enucleated rats, whereas plasma corticosterone levels in rats 14 days after enucleation were significantly (P less than 0.005) decreased only by treatment with ACTH antiserum. To determine whether the mitogenic N-POMC peptides involved in adrenal regeneration originated from the pituitary intermediate lobe, 0.9% (w/v) NaCl or ergocryptine (10 mg/kg body weight) was administered s.c. twice daily to rats between 7 and 13 days after enucleation. On day 14, adrenal mitotic activity and plasma levels of ACTH and N-POMC were not significantly different between ergocryptine and saline-treated enucleated rats, whereas alpha-MSH levels in ergocryptine-treated enucleated rats were significantly (P less than 0.02) decreased. Increases in N-POMC content of the pituitary lobe accompanied those of ACTH in animals 7, 10, 14 and 21 days after enucleation (P less than 0.01 compared with sham-treatment). Anterior lobes from rats 10 days after enucleation or from adrenalectomized rats showed raised ACTH and N-POMC levels compared with sham-treated animals, whereas alpha-MSH content in the anterior lobe of enucleated rats was significantly (P less than 0.005) decreased. Adrenalectomized animals had raised (P less than 0.005) amounts of alpha-MSH compared with sham-treated animals. Plasma levels of ACTH and N-POMC were significantly (P less than 0.01) raised in rats 10 days after enucleation or in adrenalectomized rats compared with those in sham-treated animals, whereas alpha-MSH levels were raised (P less than 0.005) only in adrenalectomized rats. Sephadex G-75 chromatography of anterior lobe extracts obtained 10 days after surgery from sham-treated, enucleated and adrenalectomized rats was performed.(ABSTRACT TRUNCATED AT 400 WORDS)

25. Restoration of the vasopressin innervation in the senescent rat brain by testosterone supplementation

Immunocytochemical techniques were applied to brain and pituitary sections of European eels after experimental manipulation of the pituitary-interrenal activity. A corticotropin-releasing factor (CRF) antiserum allowed the identification of a CRF-like peptide in the preoptic nucleus (PON) and rostral and caudal neurohypophysis (NH). CRF-immunoreactivity (ir) was not affected in solvent-injected eels compared to noninjected eels. Reserpine induced a stimulation of the pituitary interrenal axis, decreased ir-CRF in the rostral NH, but did not affect hypothalamic ir-CRF. Cortisol reduced the immunostaining of hypothalamic CRF-ir perikarya and perikarya cross-sectional area. In the rostral NH, CRF-ir fibers decreased in number and almost disappeared in long-term treated eels. The immunostaining of ACTH cells with ACTH antiserum was greatly reduced. These data suggest that cortisol induces a marked reduction in the activity of the CRF-corticotrop axis. The intensity of the ir-CRF staining observed in the caudal NH, close to the intermediate lobe (IL) was not significantly affected in reserpine-treated eels, and only slightly reduced in long-term cortisol-treated eels. The intensity of ir-CRF staining in the caudal NH did not correlate with melanocorticotropic activity or plasma cortisol level. These data suggest that immunoreactive CRF fibers in the rostral and caudal NH are differently regulated.

Ontogenesis of corticotropes and lactotropes in situ in the pituitary gland of the hamster. An immunohistochemical study.

The development of corticotropes and lactotropes was investigated in the golden Syrian hamster using an anti-porcine ACTH antiserum and a homologous anti-hamster PRL antiserum. Oval corticotropes were first visible in the ventral region of the pars distalis at 13 days of gestation. By the end of gestation, corticotropes were found throughout the pars distalis and in the pars intermedia. Corticotropes in the pars distalis of postnatal hamsters were either round or irregularly-shaped, often appearing in clusters. Throughout development, corticotropes often appeared to be surrounding other cells. Scarce, very small lactotropes were first observed in the pars distalis of hamsters on the first postnatal day. The number of these cells, which were either round or polyhedral, increased dramatically between 4 and 20 days of postnatal life. These observations indicate that the sequence of appearance of corticotropes and lactotropes in the hamster is similar to that in other species and that lactotropes are confined to the pars distalis of postnatal hamsters.

In vitro synthesis of ACTH- and β-endorphin-related substances in the pars distalis of Anolis carolinensis

In order to investigate the biosynthesis of ACTH- and β-endorphin-related substances in the pars distalis of Anolis carolinensis, explants of pars distali were incubated for 24 hr in a complete medium which contained [ 3H]tyrosine. Acid extracts of the incubates were immunoprecipitated with either an affinity-purified ACTH antiserum or an affinity-purified β-endorphin antiserum and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Three distinct peaks of ACTH-related material were detected. The major peak comigrated with human ACTH(1–39), while two minor peaks corresponded in apparent molecular weight to ACTH biosynthetic intermediate and precursor-sized material. Three peaks of β-endorphin-related material were also detected. The major peak comigrated with β-endorphin(1–31), while two minor peaks corresponded to β-lipotropin (LPH) and precursor-sized material. Sequential immunoprecipitation experiments indicated that the precursor-sized material had antigenic determinants for both ACTH and β-endorphin. In addition this peak was identical in apparent molecular weight to the common precursor for α-melanocyte-stimulating hormone (α-MSH) and β-endorphin in the pars intermedia of A. carolinensis (R. M. Dores, Peptides 3, 925–935). Analysis of extracts of reptile pars distalis by gel-filtration chromatography revealed a single peak of naloxone-reversible opiate bioactivity which coeluted with the peak of β-endorphin-sized immunoreactivity. On a molar basis there is tenfold more opiate bioactivity in the reptile pars distalis than in the reptile pars intermedia.

Selective loss of alpha-melanotropin-amidating activity in primary cultures of rat intermediate pituitary cells.

The production of alpha-melanotropin (alpha-N-acetyl-ACTH(1-13)NH2 (alpha MSH) in rat intermediate pituitary was investigated using reverse phase high performance liquid chromatography, immunoassays, and biosynthetic labeling. Extracts of rat intermediate pituitary contain primarily alpha-N,O-diacetyl-ACTH(1-13)NH2, with smaller amounts of des- and monoacetyl-ACTH(1-13)NH2. No significant amount of ACTH(1-14)OH- or ACTH(1-13)OH-related material was observed. Analysis of the newly synthesized alpha MSH-sized peptides produced by freshly prepared rat intermediate pituitary cell suspensions revealed labeled peptides co-migrating primarily with des-, mono-, and diacetyl-ACTH(1-13)NH2. In contrast, when extracts of 2-week-old intermediate pituitary cultures were analyzed, the major peak of material detected with the general NH2-terminal ACTH antiserum eluted at the position expected for alpha-N,O-diacetyl-ACTH(1-14)OH; smaller amounts of material co-migrating with des- and monoacetyl-ACTH(1-14)OH were also detected. Analysis of the newly synthesized alpha MSH-sized peptides produced by 16-day-old cultures of intermediate pituitary cells revealed labeled peptides co-migrating with des-, mono-, and diacetyl-ACTH(1-14)OH. Thus, rat intermediate pituitary cells in culture maintain the ability to perform several major proteolytic cleavages of pro-ACTH/endorphin as well as the ability to alpha-N-acetylate alpha MSH and beta-endorphin, but selectively lose the ability to alpha-amidate the carboxyl terminus of alpha MSH.

Corticotropin (ACTH) and the N-terminal fragment of pro-opiomelanocortin are located in the same granules in cells of rat pituitary gland.

The localization of corticotropin (ACTH) and of the N-terminal fragment (NTF) of pro-opiomelanocortin were assessed by light and electron microscope immunochemistry. Using the unlabeled technique of Sternberger at the light microscope level, a strongly positive reaction for both NTF and ACTH antisera was observed in all secretory cells of the pars intermedia. In the pars distalis, immunostaining with ACTH antiserum was localized in stellate cells dispersed throughout the lobe. As observed in serial, consecutive sections, the NTF antiserum stained exactly the same cells. At the electron microscope level, using the protein A-gold technique, all the secretory granules of the cells of the pars intermedia showed a positive reaction with both antisera. In the pars distalis, after exposure to either of the antisera, gold particles were found over secretory granules of typical stellate corticotrophs. Making use of the possibility of reacting both faces of a fine section with gold particles of different sizes, it was found that the same secretory granules in all the cells of the pars intermedia and in corticotrophs of the pars distalis contained particles of both sizes. These results indicate that ACTH and NTF are contained in the same secretory granules and released together in the circulation where NTF is found in high amounts.

Evidence that the insulin secretagogue, β-cell-tropin, is ACTH22–39

The pituitary neurointermediate lobe of genetically obese (ob/ob) mice contains a hormone which stimulates insulin release and which cross-reacts with a -COOH-terminal ACTH antiserum, suggesting that it is related to corticotropin-like intermediate lobe peptide (CLIP), the 18-39 fragment of ACTH. The hormone, which we have called beta-cell-tropin, has been shown to be present in the plasma of the ob/ob mouse and to potentiate glucose induced insulin secretion. We have now shown that ACTH22-39 prepared by tryptic digestion of human synthetic CLIP behaves similarly on Biogel chromatography and on reverse-phase HPLC to the naturally occurring beta-cell-tropin. Furthermore, beta-cell-tropin and ACTH22-39 have indistinguishable antigenic and insulin releasing properties.