Anthony S. Liotta

Presence of corticotropin in brain of normal and hypophysectomized rats

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.

ACTH, beta-lipotropin, and related peptides in brain, pituitary, and blood.

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.

Presence of immunoassayable β-lipotropin in bovine brain and spinal cord: Lack of concordance with ACTH concentrations

Abstract 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.

β-lipotropin in brain: Localization in hypothalamic neurons by immunoperoxidase technique

Summaryβ-Lipotropin (β-LPH) has been localized in hypothalamus and pituitary of sheep and ox by the immunoperoxidase technique. In both species β-LPH was found in perikarya of arcuate neurons as well as in cells of the anterior and intermediate lobes of the pituitary. A large number of immunoreactive axons were found in the arcuate region; some appeared to innervate other neurons and others projected to portal capillaries. Stained fiber segments were also scattered throughout the hypothalamus. The presence of β-LPH in hypothalamic neurons supports the possibility that brain β-LPH may be a precursor for opiate-like or other peptides which may be involved in neuromodulation or neurohormonal activities.

Immunocytochemical distribution of corticotropin (ACTH) in monkey brain

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.

Pituitary ACTH responsiveness in the vasopressin deficient rat.

Abstract 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 bioassay for ACTH determination.

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.

Plasma β-lipotropin in the human

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.